Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading...Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.展开更多
Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord ...Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.展开更多
Gene spectrum analysis has shown that gene expression and signaling pathways change dramatically after spinal cord injury,which may affect the microenvironment of the damaged site.Microarray analysis provides a new op...Gene spectrum analysis has shown that gene expression and signaling pathways change dramatically after spinal cord injury,which may affect the microenvironment of the damaged site.Microarray analysis provides a new opportunity for investigating diagnosis,treatment,and prognosis of spinal cord injury.However,differentially expressed genes are not consistent among studies,and many key genes and signaling pathways have not yet been accurately studied.GSE5296 was retrieved from the Gene Expression Omnibus DataSet.Differentially expressed genes were obtained using R/Bioconductor software(expression changed at least two-fold;P < 0.05).Database for Annotation,Visualization and Integrated Discovery was used for functional annotation of differentially expressed genes and Animal Transcription Factor Database for predicting potential transcription factors.The resulting transcription regulatory protein interaction network was mapped to screen representative genes and investigate their diagnostic and therapeutic value for disease.In total,this study identified 109 genes that were upregulated and 30 that were downregulated at 0.5,4,and 24 hours,and 3,7,and 28 days after spinal cord injury.The number of downregulated genes was smaller than the number of upregulated genes at each time point.Database for Annotation,Visualization and Integrated Discovery analysis found that many inflammation-related pathways were upregulated in injured spinal cord.Additionally,expression levels of these inflammation-related genes were maintained for at least 28 days.Moreover,399 regulation modes and 77 nodes were shown in the protein-protein interaction network of upregulated differentially expressed genes.Among the 10 upregulated differentially expressed genes with the highest degrees of distribution,six genes were transcription factors.Among these transcription factors,ATF3 showed the greatest change.ATF3 was upregulated within 30 minutes,and its expression levels remained high at28 days after spinal cord injury.These key genes screened by bioinformatics tools can be used as biological markers to diagnose diseases and provide a reference for identifying therapeutic targets.展开更多
Regulatory T cells,a subset of CD4^(+)T cells,play a critical role in maintaining immune tolerance and tissue homeostasis due to their potent immunosuppressive properties.Recent advances in research have highlighted t...Regulatory T cells,a subset of CD4^(+)T cells,play a critical role in maintaining immune tolerance and tissue homeostasis due to their potent immunosuppressive properties.Recent advances in research have highlighted the important therapeutic potential of Tregs in neurological diseases and tissue repair,emphasizing their multifaceted roles in immune regulation.This review aims to summarize and analyze the mechanisms of action and therapeutic potential of Tregs in relation to neurological diseases and neural regeneration.Beyond their classical immune-regulatory functions,emerging evidence points to non-immune mechanisms of regulatory T cells,particularly their interactions with stem cells and other non-immune cells.These interactions contribute to optimizing the repair microenvironment and promoting tissue repair and nerve regeneration,positioning non-immune pathways as a promising direction for future research.By modulating immune and non-immune cells,including neurons and glia within neural tissues,Tregs have demonstrated remarkable efficacy in enhancing regeneration in the central and peripheral nervous systems.Preclinical studies have revealed that Treg cells interact with neurons,glial cells,and other neural components to mitigate inflammatory damage and support functional recovery.Current mechanistic studies show that Tregs can significantly promote neural repair and functional recovery by regulating inflammatory responses and the local immune microenvironment.However,research on the mechanistic roles of regulatory T cells in other diseases remains limited,highlighting substantial gaps and opportunities for exploration in this field.Laboratory and clinical studies have further advanced the application of regulatory T cells.Technical advances have enabled efficient isolation,ex vivo expansion and functionalization,and adoptive transfer of regulatory T cells,with efficacy validated in animal models.Innovative strategies,including gene editing,cell-free technologies,biomaterial-based recruitment,and in situ delivery have expanded the therapeutic potential of regulatory T cells.Gene editing enables precise functional optimization,while biomaterial and in situ delivery technologies enhance their accumulation and efficacy at target sites.These advancements not only improve the immune-regulatory capacity of regulatory T cells but also significantly enhance their role in tissue repair.By leveraging the pivotal and diverse functions of Tregs in immune modulation and tissue repair,regulatory T cells–based therapies may lead to transformative breakthroughs in the treatment of neurological diseases.展开更多
AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing rec...AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.展开更多
AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the im...AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.展开更多
Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or ...Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.展开更多
BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely ...BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.展开更多
AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cass...AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.展开更多
The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel d...The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.展开更多
AIM:To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene tran-scription. METHODS:Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus ...AIM:To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene tran-scription. METHODS:Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation. RESULTS:Lpcat1 translocates into the nucleus from thecytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli , two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overex-pressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.CONCLUSION:These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.展开更多
The human retina,a complex and highly specialized structure,includes multiple cell types that work synergistically to generate and transmit visual signals.However,genetic predisposition or age-related degeneration can...The human retina,a complex and highly specialized structure,includes multiple cell types that work synergistically to generate and transmit visual signals.However,genetic predisposition or age-related degeneration can lead to retinal damage that severely impairs vision or causes blindness.Treatment options for retinal diseases are limited,and there is an urgent need for innovative therapeutic strategies.Cell and gene therapies are promising because of the efficacy of delivery systems that transport therapeutic genes to targeted retinal cells.Gene delivery systems hold great promise for treating retinal diseases by enabling the targeted delivery of therapeutic genes to affected cells or by converting endogenous cells into functional ones to facilitate nerve regeneration,potentially restoring vision.This review focuses on two principal categories of gene delivery vectors used in the treatment of retinal diseases:viral and non-viral systems.Viral vectors,including lentiviruses and adeno-associated viruses,exploit the innate ability of viruses to infiltrate cells,which is followed by the introduction of therapeutic genetic material into target cells for gene correction.Lentiviruses can accommodate exogenous genes up to 8 kb in length,but their mechanism of integration into the host genome presents insertion mutation risks.Conversely,adeno-associated viruses are safer,as they exist as episomes in the nucleus,yet their limited packaging capacity constrains their application to a narrower spectrum of diseases,which necessitates the exploration of alternative delivery methods.In parallel,progress has also occurred in the development of novel non-viral delivery systems,particularly those based on liposomal technology.Manipulation of the ratios of hydrophilic and hydrophobic molecules within liposomes and the development of new lipid formulations have led to the creation of advanced non-viral vectors.These innovative systems include solid lipid nanoparticles,polymer nanoparticles,dendrimers,polymeric micelles,and polymeric nanoparticles.Compared with their viral counterparts,non-viral delivery systems offer markedly enhanced loading capacities that enable the direct delivery of nucleic acids,mRNA,or protein molecules into cells.This bypasses the need for DNA transcription and processing,which significantly enhances therapeutic efficiency.Nevertheless,the immunogenic potential and accumulation toxicity associated with non-viral particulate systems necessitates continued optimization to reduce adverse effects in vivo.This review explores the various delivery systems for retinal therapies and retinal nerve regeneration,and details the characteristics,advantages,limitations,and clinical applications of each vector type.By systematically outlining these factors,our goal is to guide the selection of the optimal delivery tool for a specific retinal disease,which will enhance treatment efficacy and improve patient outcomes while paving the way for more effective and targeted therapeutic interventions.展开更多
Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a label...Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the differences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China(approval No. 2016-x9-07) in September 2016.展开更多
Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable disease...Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable diseases also affect at least one-third of the population in low and middle-income countries and lead to hypertension, elevated cholesterol, malignancy, and neurodegenerative disorders such as Alzheimer's disease and stroke. With the climbing lifespan of the world's population, increased prevalence of these disorders is expected requiring the development of new therapeutic strategies against these disabling disease entities. Targeting stem cellproliferation for cardiac disease, vascular disorders, cancer, and neurodegenerative disorders is receiving great enthusiasm, especially those that focus upon SIRT1, a mammalian homologue of the yeast silent information regulator-2. Modulation of the cellular activity of SIRT1 can involve oversight by nicotinamide/nicotinic acid mononucleotide adenylyltransferase, mammalian forkhead transcription factors, mechanistic of rapamycin pathways, and cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma over-expressed gene family members that can impact cytoprotective outcomes. Ultimately, the ability of SIRT1 to control the programmed cell death pathways of apoptosis and autophagy can determine not only cardiac, vascular, and neuronal stem cell development and longevity, but also the onset of tumorigenesis and the resistance against chemotherapy. SIRT1 therefore has a critical role and holds exciting prospects for new therapeutic strategies that can offer reparative processes for cardiac, vascular, and nervous system degenerative disorders as well as targeted control of aberrant cell growth during cancer.展开更多
The etiopathogenesis of gastrointestinal diseases is varied in nature.Various etiogenic factors described are infective,inflammatory,viral,bacterial,parasitic,dietary and lifestyle change.Rare causative agents are imm...The etiopathogenesis of gastrointestinal diseases is varied in nature.Various etiogenic factors described are infective,inflammatory,viral,bacterial,parasitic,dietary and lifestyle change.Rare causative agents are immunological,and others associated as idiopathic,are undiagnosed by all possible means.Some of the rare diseases are congenital in nature,passing from the parent to the child.Many of the undiagnosed diseases are now being diagnosed as genetic and the genes have been implicated as a causative agent.There is a search for newer treatments for such diseases,which is called genomic medicine.Genomic medicine is an emerging medical discipline that involves the use of genomic information about an individual.This is used both for diagnostic as well as therapeutic decisions to improve the current health domain and pave the way for policymakers for its clinical use.In the developing era of precision medicine,genomics,epigenomics,environmental exposure,and other data would be used to more accurately guide individual diagnosis and treatment.Genomic medicine is already making an impact in the fields of oncology,pharmacology,rare,infectious and many undiagnosed diseases.It is beginning to fuel new approaches in certain medical specialties.Oncology is at the leading edge of incorporating genomics,as diagnostics for genetic and genomic markers are increasingly included in cancer screening,and to guide tailored treatment strategies.Genetics and genetic medicine have been reported to play a role in gastroenterology in several ways,including genetic testing(hereditary pancreatitis and hereditary gastrointestinal cancer syndromes).Genetic testing can also help subtype diseases,such as classifying pancreatitis as idiopathic or hereditary.Gene therapy is a promising approach for treating gastrointestinal diseases that are not effectively treated by conventional pharmaceuticals and surgeries.Gene therapy strategies include gene addition,gene editing,messenger RNA therapy,and gene silencing.Understanding genetic determinants,advances in genetics,have led to a better understanding of the genetic factors that contribute to human disease.Family-member risk stratification and genetic diagnosis can help identify family members who are at risk,which can lead to preventive treatments,lifestyle recommendations,and routine follow ups.Selecting target genes helps identify the gene targets associated with each gastrointestinal disease.Common gastrointestinal diseases associated with genetic abnormalities include-inflammatory bowel disease,gastroesophageal reflux disease,non-alcoholic fatty liver disease,and irritable bowel syndrome.With advancing tools and technology,research in the search of newer and individualized treatment,genes and genetic medicines are expected to play a significant role in human health and gastroenterology.展开更多
Neurite outgrowth and synaptogenesis are critical steps for functional recovery following ischemic stroke.Damaged axons of the central nervous system in adult mammals exhibit limited regenerative capacity,resulting in...Neurite outgrowth and synaptogenesis are critical steps for functional recovery following ischemic stroke.Damaged axons of the central nervous system in adult mammals exhibit limited regenerative capacity,resulting in enduring neurological deficits.Recent findings from our research indicate that inhibition of Rho-associated kinase(ROCK)2 facilitates neuroprotection in different models of central nervous system diseases.In addition,our prior studies have demonstrated that axonal protection enhances the regeneration of injured axons.However,it remains unclear whether the axonal protection mediated by ROCK2 inhibition also facilitates synaptogenesis.In this study,we aimed to investigate the effects of inhibiting ROCK2 expression on synaptogenesis and neurogenesis in ischemic stroke using an shRNA-expressing adeno-associated virus(AAV)vector(AAV-sh.ROCK2).We demonstrated that AAV-sh.ROCK2 increased neurite outgrowth and facilitated synaptogenesis in vivo.Furthermore,AAV-sh.ROCK2 increased neuronal survival and promoted neurogenesis following middle cerebral artery occlusion surgery as well as long-term motor functional recovery after ischemia/reperfusion injury.Notably,AAV-sh.ROCK2 also stimulated serotonergic and dopaminergic axon sprouting after ischemia/reperfusion injury.Mechanistically,AAV-sh.ROCK2 activity resulted in increased anti-collapsin response mediator protein 2 activation and reductions in RhoA and ROCK2 expression.Our study identified ROCK2 as a critical regulator of synaptogenesis and neurogenesis,highlighting it as a promising target to facilitate neuroprotection and regeneration in ischemic stroke.展开更多
Breast cancer is the leading cause of death in women. Prognosis of breast cancer is often pessimistic because the tumors are prone to metastasizing to the bone, brain, and lung. The estrogen signaling receptor (ESR) p...Breast cancer is the leading cause of death in women. Prognosis of breast cancer is often pessimistic because the tumors are prone to metastasizing to the bone, brain, and lung. The estrogen signaling receptor (ESR) pathway contains 39 main genes and proteins which makes it one of the larger signaling pathways. Predominately this pathway and the proteins within are involved in breast growth and development, making it a prospective area of study for breast cancer. While the healthy ESR pathway has been constructed and is well established, a mechanistic model of mutated genes of ESR pathway has not been delved upon. Such mutated models could be utilized for selecting combinational targets for drug therapies, as well as elucidating crosstalk between other pathways and feedback mechanisms. To construct the mutated models of the ESR pathway it is imperative to assess what is currently understood in the literature and what inconsistencies exist in order to resolve them. Without this information, a model of the ESR pathway will be unreliable and likely unproductive. This review is the detailed literature survey of the biological studies performed on ESR pathways genes, and their respective roles in breast cancer. Furthermore, the details mentioned in the review can be beneficial for the integrated study of the ESR pathway genes, which includes, structural and dynamics study of the genes products, to have a holistic understanding of the cancer mechanism.展开更多
AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human...AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.展开更多
AAV-PHP.eB is an artificial adeno-associated virus(AAV)that crosses the blood-brain barrier and targets neurons more efficiently than other AAVs when administered systematically.While AAV-PHP.eB has been used in vario...AAV-PHP.eB is an artificial adeno-associated virus(AAV)that crosses the blood-brain barrier and targets neurons more efficiently than other AAVs when administered systematically.While AAV-PHP.eB has been used in various disease models,its cellular tropism in cerebrovascular diseases remains unclear.In the present study,we aimed to elucidate the tropism of AAV-PHP.eB for different cell types in the brain in a mouse model of ischemic stroke and evaluate its effectiveness in mediating basic fibroblast growth factor(bFGF)gene therapy.Mice were injected intravenously with AAV-PHP.eB either 14 days prior to(pre-stroke)or 1 day following(post-stroke)transient middle cerebral artery occlusion.Notably,we observed a shift in tropism from neurons to endothelial cells with post-stroke administration of AAV-PHP.eB-mNeonGreen(mNG).This endothelial cell tropism correlated strongly with expression of the endothelial membrane receptor lymphocyte antigen 6 family member A(Ly6A).Furthermore,AAV-PHP.eB-mediated overexpression of bFGF markedly improved neurobehavioral outcomes and promoted long-term neurogenesis and angiogenesis post-ischemic stroke.Our findings underscore the significance of considering potential tropism shifts when utilizing AAV-PHP.eB-mediated gene therapy in neurological diseases and suggest a promising new strategy for bFGF gene therapy in stroke treatment.展开更多
The prognosis for patients who experience intracerebral hemorrhage is poor because of a lack of effective treatments.Tumor necrosis factor-α-stimulated gene 6(TSG6)is a secreted glycoprotein that exerts anti-inflamma...The prognosis for patients who experience intracerebral hemorrhage is poor because of a lack of effective treatments.Tumor necrosis factor-α-stimulated gene 6(TSG6)is a secreted glycoprotein that exerts anti-inflammatory effects in various inflammatory diseases.We previously showed that adipose-derived stem cells can inhibit inflammation by upregulating TSG6 secretion in an in vitro model of intracerebral hemorrhage.However,the direct effects of TSG6 on hematoma clearance in vivo remain largely unknown.The aim of this study was to determine how TSG6 affects hematoma absorption in mice subjected to intracerebral hemorrhage and to explore the potential underlying mechanisms.We first analyzed the gene profiles of patients with intracerebral hemorrhage from the GEO database and examined changes in TSG6 expression in the brain tissues of mice subjected to intracerebral hemorrhage.We found that TSG6 expression exhibited a transient increase following intracerebral hemorrhage,and that there was a negative correlation between the initial hematoma volume and TSG6 levels.Immunofluorescence analysis showed that TSG6 was primarily expressed in microglia and macrophages.Furthermore,we found that TSG6 promoted functional recovery in mice subjected to intracerebral hemorrhage by accelerating hematoma clearance,reducing the number of apoptotic cells and degenerated neurons,increasing the proportion of phagocytic microglia/macrophages,and decreasing iron deposition.Western blotting and immunofluorescence analysis indicated that TSG6 promoted M2 polarization of microglia/macrophages.In vitro phagocytosis experiments confirmed that TSG6 enhanced the ability of microglia to phagocytize red blood cells.Finally,we identified the signal transducer and activator of transcription 6/growth arrest-specific protein 6 signaling pathway as playing a critical role in TSG6-mediated hematoma absorption.In summary,our results demonstrate an essential role for TSG6 in promoting hematoma absorption in a mouse model of intracerebral hemorrhage.These findings suggest that TSG6 accelerates hematoma clearance and improves neurological function by promoting microglia/macrophage polarization to the M2 phenotype,activating the STAT6/GAS6 signaling pathway,and increasing phagocytic receptor expression on the surface of phagocytes,thereby enhancing their ability to phagocytize red blood cells.展开更多
文摘Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.
基金supported by the State Key Program of National Natural Science Foundation of China,No.81330042(to SQF)the International Cooperation Program of the National Natural Science Foundation of China,No.81620108018(to SQF)
文摘Zebrafish and human genomes are highly homologous;however,despite this genomic similarity,adult zebrafish can achieve neuronal proliferation,regeneration and functional restoration within 6–8 weeks after spinal cord injury,whereas humans cannot.To analyze differentially expressed zebrafish genes between axon-regenerated neurons and axon-non-regenerated neurons after spinal cord injury,and to explore the key genes and pathways of axonal regeneration after spinal cord injury,microarray GSE56842 was analyzed using the online tool,GEO2R,in the Gene Expression Omnibus database.Gene ontology and protein-protein interaction networks were used to analyze the identified differentially expressed genes.Finally,we screened for genes and pathways that may play a role in spinal cord injury repair in zebrafish and mammals.A total of 636 differentially expressed genes were obtained,including 255 up-regulated and 381 down-regulated differentially expressed genes in axon-regenerated neurons.Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment results were also obtained.A protein-protein interaction network contained 480 node genes and 1976 node connections.We also obtained the 10 hub genes with the highest correlation and the two modules with the highest score.The results showed that spectrin may promote axonal regeneration after spinal cord injury in zebrafish.Transforming growth factor beta signaling may inhibit repair after spinal cord injury in zebrafish.Focal adhesion or tight junctions may play an important role in the migration and proliferation of some cells,such as Schwann cells or neural progenitor cells,after spinal cord injury in zebrafish.Bioinformatic analysis identified key candidate genes and pathways in axonal regeneration after spinal cord injury in zebrafish,providing targets for treatment of spinal cord injury in mammals.
基金supported by the Natural Science Foundation of Shaanxi Province of China,No.2018JQ8029(to LG)
文摘Gene spectrum analysis has shown that gene expression and signaling pathways change dramatically after spinal cord injury,which may affect the microenvironment of the damaged site.Microarray analysis provides a new opportunity for investigating diagnosis,treatment,and prognosis of spinal cord injury.However,differentially expressed genes are not consistent among studies,and many key genes and signaling pathways have not yet been accurately studied.GSE5296 was retrieved from the Gene Expression Omnibus DataSet.Differentially expressed genes were obtained using R/Bioconductor software(expression changed at least two-fold;P < 0.05).Database for Annotation,Visualization and Integrated Discovery was used for functional annotation of differentially expressed genes and Animal Transcription Factor Database for predicting potential transcription factors.The resulting transcription regulatory protein interaction network was mapped to screen representative genes and investigate their diagnostic and therapeutic value for disease.In total,this study identified 109 genes that were upregulated and 30 that were downregulated at 0.5,4,and 24 hours,and 3,7,and 28 days after spinal cord injury.The number of downregulated genes was smaller than the number of upregulated genes at each time point.Database for Annotation,Visualization and Integrated Discovery analysis found that many inflammation-related pathways were upregulated in injured spinal cord.Additionally,expression levels of these inflammation-related genes were maintained for at least 28 days.Moreover,399 regulation modes and 77 nodes were shown in the protein-protein interaction network of upregulated differentially expressed genes.Among the 10 upregulated differentially expressed genes with the highest degrees of distribution,six genes were transcription factors.Among these transcription factors,ATF3 showed the greatest change.ATF3 was upregulated within 30 minutes,and its expression levels remained high at28 days after spinal cord injury.These key genes screened by bioinformatics tools can be used as biological markers to diagnose diseases and provide a reference for identifying therapeutic targets.
基金supported by the National Natural Science Foundation of China,Nos.32271389,31900987(both to PY)the Natural Science Foundation of Jiangsu Province,No.BK20230608(to JJ)。
文摘Regulatory T cells,a subset of CD4^(+)T cells,play a critical role in maintaining immune tolerance and tissue homeostasis due to their potent immunosuppressive properties.Recent advances in research have highlighted the important therapeutic potential of Tregs in neurological diseases and tissue repair,emphasizing their multifaceted roles in immune regulation.This review aims to summarize and analyze the mechanisms of action and therapeutic potential of Tregs in relation to neurological diseases and neural regeneration.Beyond their classical immune-regulatory functions,emerging evidence points to non-immune mechanisms of regulatory T cells,particularly their interactions with stem cells and other non-immune cells.These interactions contribute to optimizing the repair microenvironment and promoting tissue repair and nerve regeneration,positioning non-immune pathways as a promising direction for future research.By modulating immune and non-immune cells,including neurons and glia within neural tissues,Tregs have demonstrated remarkable efficacy in enhancing regeneration in the central and peripheral nervous systems.Preclinical studies have revealed that Treg cells interact with neurons,glial cells,and other neural components to mitigate inflammatory damage and support functional recovery.Current mechanistic studies show that Tregs can significantly promote neural repair and functional recovery by regulating inflammatory responses and the local immune microenvironment.However,research on the mechanistic roles of regulatory T cells in other diseases remains limited,highlighting substantial gaps and opportunities for exploration in this field.Laboratory and clinical studies have further advanced the application of regulatory T cells.Technical advances have enabled efficient isolation,ex vivo expansion and functionalization,and adoptive transfer of regulatory T cells,with efficacy validated in animal models.Innovative strategies,including gene editing,cell-free technologies,biomaterial-based recruitment,and in situ delivery have expanded the therapeutic potential of regulatory T cells.Gene editing enables precise functional optimization,while biomaterial and in situ delivery technologies enhance their accumulation and efficacy at target sites.These advancements not only improve the immune-regulatory capacity of regulatory T cells but also significantly enhance their role in tissue repair.By leveraging the pivotal and diverse functions of Tregs in immune modulation and tissue repair,regulatory T cells–based therapies may lead to transformative breakthroughs in the treatment of neurological diseases.
文摘AIM: To establish the role of vascular endothelial growth factor (VEGF) in the oncogenesis of human gastric carcinoma more directly. METHODS: The expression of VEGF and its receptor kinase-domain insert containing receptor (KDR) in human gastric cancer tissue were observed by immunohistochemical staining. VEGF levels were manipulated in human gastric cancer cell using eukaryotic expression constructs designed to express the complete VEGF(165) complimentary DNA in either the sense or antisense orientation. The biological changes of the cells were observed in which VEGF was up-regulated or down-regulated. RESULTS: VEGF-positive rate was 50%, and VEGF was mainly localized in the cytoplasm and membrane of the tumor cells, while KDR was mainly located in the membrane of vascular endothelial cells in gastric cancer tissues and peri-cancerous tissue. In 2 cases of 50 specimens, the gastric cancer cells expressed KDR, localized in both the cytoplasm and membrane. Introduction of VEGF(165) antisense into human gastric cancer cells (SGC-7901, immunofluorescence intensity, 31.6%)) resulted in a significant reduction in VEGF-specific messenger RNA and total and cell surface VEGF protein (immunofluorescence intensity, 8.9%) (P【0.05). Conversely, stable integration of VEGF(165) in the sense orientation resulted in an increase in cellular and cell surface VEGF (immunofluorescence intensity, 75.4%) (P【0.05). Lowered VEGF levels were associated with a marked decrease in the growth of nude mouse xenografted tumor (at 33 days postimplantation, tumor volume: 345.40 +/- 136.31 mm3)(P【0.05 vs control SGC-7901 group: 1534.40 +/- 362.88 mm3), whereas up-regulation of VEGF resulted in increased xenografted tumor size (at 33 days postimplantation, tumor volume: 2350.50 +/- 637.70 mm3) (P【0.05 vs control SGC-7901 group). CONCLUSION: This study provides direct evidence that VEGF plays an important role in the oncogenesis of human gastric cancer.
基金Supported by a faculty research grant of Yonsei University College of Medicine for 2002,No.2002-06
文摘AIM:rAAV mediated endostatin gene therapy has been examined as a new method for treating cancer.However, a sustained and high protein delivery is required to achieve the desired therapeutic effects.We evaluated the impact of topoisomerase inhibitors in rAAV delivered endostatin gene therapy in a liver tumor model. METHODS:rAAV containing endostatin expression cassettes were transduced into hepatoma cell lines.To test whether the topoisomerase inhibitor pretreatment increased the expression of endostatin,Western blotting and ELISA were performed.The biologic activity of endostatin was confirmed by endothelial cell proliferation and tube formation assays. The anti-tumor effects of the rAAV-endostatin vector combined with a topoisomerase inhibitor,etoposide,were evaluated in a mouse liver tumor model. RESULTS:Topoisomerase inhibitors,including camptothecin and etoposide,were found to increase the endostatin exPression level in vitro.The over-expressed endostatin, as a result of pretreatment with a topoisomerase inhibitor, was also biologically active.In animal experiments,the combined therapy of topoisomerase inhibitor,etoposide with the rAAV-endostatin vector had the best tumor- suppressive effect and tumor foci were barely observed in livers of the treated mice.Pretreatment with an etoposide increased the level of endostatin in the liver and serum of rAAV-endostatin treated mice.Finally,the mice treated With rAAV-endostatin in combination with etoposide showed the longest survival among the experimental models. CONCLUSION:rAAV delivered endostatin gene therapy in combination with a topoisomerase inhibitor pretreatment is an effective modality for anticancer gene therapy.
文摘Common neurodegenerative diseases of the central nervous system are characterized by progressive damage to the function of neurons, even leading to the permanent loss of function. Gene therapy via gene replacement or gene correction provides the potential for transformative therapies to delay or possibly stop further progression of the neurodegenerative disease in affected patients. Adeno-associated virus has been the vector of choice in recent clinical trials of therapies for neurodegenerative diseases due to its safety and efficiency in mediating gene transfer to the central nervous system. This review aims to discuss and summarize the progress and clinical applications of adeno-associated virus in neurodegenerative disease in central nervous system. Results from some clinical trials and successful cases of central neurodegenerative diseases deserve further study and exploration.
基金Supported by the National Natural Science Foundation of China,No.U1504815 and No.U1504808
文摘BACKGROUND Pancreatic cancer is a highly invasive malignant tumor. Expression levels of the autophagy-related protein microtubule-associated protein 1 A/1 B-light chain 3(LC3) and perineural invasion(PNI) are closely related to its occurrence and development. Our previous results showed that the high expression of LC3 was positively correlated with PNI in the patients with pancreatic cancer. In this study, we further searched for differential genes involved in autophagy of pancreatic cancer by gene expression profiling and analyzed their biological functions in pancreatic cancer, which provides a theoretical basis for elucidating the pathophysiological mechanism of autophagy in pancreatic cancer and PNI.AIM To identify differentially expressed genes involved in pancreatic cancer autophagy and explore the pathogenesis at the molecular level.METHODS Two sets of gene expression profiles of pancreatic cancer/normal tissue(GSE16515 and GSE15471) were collected from the Gene Expression Omnibus.Significance analysis of microarrays algorithm was used to screen differentially expressed genes related to pancreatic cancer. Gene Ontology(GO) analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis were used to analyze the functional enrichment of the differentially expressed genes. Protein interaction data containing only differentially expressed genes was downloaded from String database and screened. Module mining was carried out by Cytoscape software and ClusterOne plug-in. The interaction relationship between the modules was analyzed and the pivot nodes between the functional modules were determined according to the information of the functional modules and the data of reliable protein interaction network.RESULTS Based on the above two data sets of pancreatic tissue total gene expression, 6098 and 12928 differentially expressed genes were obtained by analysis of genes with higher phenotypic correlation. After extracting the intersection of the two differential gene sets, 4870 genes were determined. GO analysis showed that 14 significant functional items including negative regulation of protein ubiquitination were closely related to autophagy. A total of 986 differentially expressed genes were enriched in these functional items. After eliminating the autophagy related genes of human cancer cells which had been defined, 347 differentially expressed genes were obtained. KEGG pathway analysis showed that the pathways hsa04144 and hsa04020 were related to autophagy. In addition,65 clustering modules were screened after the protein interaction network was constructed based on String database, and module 32 contains the LC3 gene,which interacts with multiple autophagy-related genes. Moreover, ubiquitin C acts as a pivot node in functional modules to connect multiple modules related to pancreatic cancer and autophagy.CONCLUSION Three hundred and forty-seven genes associated with autophagy in human pancreatic cancer were concentrated, and a key gene ubiquitin C which is closely related to the occurrence of PNI was determined, suggesting that LC3 may influence the PNI and prognosis of pancreatic cancer through ubiquitin C.
基金Project supported by National Natural Science Foundation of China,No.863 Z2001-04
文摘AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.
文摘The molecular scalpel of clustered regularly interspersed short palindromic repeats/CRISPR associated protein 9(CRISPR/Cas9) technology may be sharp enough to begin cutting the genes implicated in inflammatory bowel disease(IBD) and consequently decrease the 6.3 billion dollar annual financial healthcare burden in the treatment of IBD. For the past few years CRISPR technology has drastically revolutionized DNA engineering and biomedical research field. We are beginning to see its application in gene manipulation of sickle cell disease,human immunodeficiency virus resistant embryologic twin gene modification and IBD genes such as Gatm(Glycine amidinotransferase, mitochondrial),nucleotide-binding oligomerization domain-containing protein 2, KRT12 and other genes implicated in adaptive immune convergence pathways have been subjected to gene editing, however there are very few publications. Furthermore,since Crohn's disease and ulcerative colitis have shared disease susceptibility and share genetic gene profile, it is paramount and is more advantageous to use CRISPR technology to maximize impact. Although, currently CRISPR does have its limitations due to limited number of specific Cas enzymes, off-target activity,protospacer adjacent motifs and crossfire between different target sites. However,these limitations have given researchers further insight on how to augment and manipulate enzymes to enable precise gene excision and limit crossfire between target sites.
基金Supported by A United States National Institutes of Health R01 grant HL091916 to Zhao Yan American Heart Association grant 12SDG12040330 to Zou C, in part
文摘AIM:To report that Lpcat1 plays an important role in regulating lipopolysaccharide (LPS) inducible gene tran-scription. METHODS:Gene expression in Murine Lung Epithelial MLE-12 cells with LPS treatment or Haemophilus influenza and Escherichia coli infection was analyzed by employing quantitative Reverse Transcription Polymerase Chain Reaction techniques. Nucleofection was used to deliver Lenti-viral system to express or knock down Lpcat1 in MLE cells. Subcellular protein fractionation and Western blotting were utilized to study Lpcat1 nuclear relocation. RESULTS:Lpcat1 translocates into the nucleus from thecytoplasm in murine lung epithelia (MLE) after LPS treatment. Haemophilus influenza and Escherichia coli , two LPS-containing pathogens that cause pneumonia, triggered Lpcat1 nuclear translocation from the cytoplasm. The LPS inducible gene expression profile was determined by quantitative reverse transcription polymerase chain reaction after silencing Lpcat1 or overexpression of the enzyme in MLE cells. We detected that 17 out of a total 38 screened genes were upregulated, 14 genes were suppressed, and 7 genes remained unchanged in LPS treated cells in comparison to controls. Knockdown of Lpcat1 by shRNA dramatically changed the spectrum of the LPS inducible gene transcription, as 18 genes out of 38 genes were upregulated, of which 20 genes were suppressed or unchanged. Notably, in Lpcat1 overex-pressed cells, 25 genes out of 38 genes were reduced in the setting of LPS treatment.CONCLUSION:These observations suggest that Lpcat1 relocates into the nucleus in response to bacterial infection to differentially regulate gene transcriptional repression.
基金Hongguang Wu,Both authors contributed equally to this work and share first authorshipLing Dong,Both authors contributed equally to this work and share first authorship。
文摘The human retina,a complex and highly specialized structure,includes multiple cell types that work synergistically to generate and transmit visual signals.However,genetic predisposition or age-related degeneration can lead to retinal damage that severely impairs vision or causes blindness.Treatment options for retinal diseases are limited,and there is an urgent need for innovative therapeutic strategies.Cell and gene therapies are promising because of the efficacy of delivery systems that transport therapeutic genes to targeted retinal cells.Gene delivery systems hold great promise for treating retinal diseases by enabling the targeted delivery of therapeutic genes to affected cells or by converting endogenous cells into functional ones to facilitate nerve regeneration,potentially restoring vision.This review focuses on two principal categories of gene delivery vectors used in the treatment of retinal diseases:viral and non-viral systems.Viral vectors,including lentiviruses and adeno-associated viruses,exploit the innate ability of viruses to infiltrate cells,which is followed by the introduction of therapeutic genetic material into target cells for gene correction.Lentiviruses can accommodate exogenous genes up to 8 kb in length,but their mechanism of integration into the host genome presents insertion mutation risks.Conversely,adeno-associated viruses are safer,as they exist as episomes in the nucleus,yet their limited packaging capacity constrains their application to a narrower spectrum of diseases,which necessitates the exploration of alternative delivery methods.In parallel,progress has also occurred in the development of novel non-viral delivery systems,particularly those based on liposomal technology.Manipulation of the ratios of hydrophilic and hydrophobic molecules within liposomes and the development of new lipid formulations have led to the creation of advanced non-viral vectors.These innovative systems include solid lipid nanoparticles,polymer nanoparticles,dendrimers,polymeric micelles,and polymeric nanoparticles.Compared with their viral counterparts,non-viral delivery systems offer markedly enhanced loading capacities that enable the direct delivery of nucleic acids,mRNA,or protein molecules into cells.This bypasses the need for DNA transcription and processing,which significantly enhances therapeutic efficiency.Nevertheless,the immunogenic potential and accumulation toxicity associated with non-viral particulate systems necessitates continued optimization to reduce adverse effects in vivo.This review explores the various delivery systems for retinal therapies and retinal nerve regeneration,and details the characteristics,advantages,limitations,and clinical applications of each vector type.By systematically outlining these factors,our goal is to guide the selection of the optimal delivery tool for a specific retinal disease,which will enhance treatment efficacy and improve patient outcomes while paving the way for more effective and targeted therapeutic interventions.
基金supported by National Key Research&Development Program of China,No.2016YFC11011601,2017YFA0104701the Youth Cultivation Project of Military Medical Science,China,No.15QNP091(to YW)People’s Liberation Army Youth Training Project for Medical Science of China,No.16QNP144(to YW)
文摘Sensory and motor nerve fibers of peripheral nerves have different anatomies and regeneration functions after injury. To gain a clear understanding of the biological processes behind these differences, we used a labeling technique termed isobaric tags for relative and absolute quantitation to investigate the protein profiles of spinal nerve tissues from Sprague-Dawley rats. In response to Wallerian degeneration, a total of 626 proteins were screened in sensory nerves, of which 368 were upregulated and 258 were downregulated. In addition, 637 proteins were screened in motor nerves, of which 372 were upregulated and 265 were downregulated. All identified proteins were analyzed using the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of bioinformatics, and the presence of several key proteins closely related to Wallerian degeneration were tested and verified using quantitative real-time polymerase chain reaction analyses. The differentially expressed proteins only identified in the sensory nerves were mainly relevant to various biological processes that included cell-cell adhesion, carbohydrate metabolic processes and cell adhesion, whereas differentially expressed proteins only identified in the motor nerves were mainly relevant to biological processes associated with the glycolytic process, cell redox homeostasis, and protein folding. In the aspect of the cellular component, the differentially expressed proteins in the sensory and motor nerves were commonly related to extracellular exosomes, the myelin sheath, and focal adhesion. According to the Kyoto Encyclopedia of Genes and Genomes, the differentially expressed proteins identified are primarily related to various types of metabolic pathways. In conclusion, the present study screened differentially expressed proteins to reveal more about the differences and similarities between sensory and motor nerves during Wallerian degeneration. The present findings could provide a reference point for a future investigation into the differences between sensory and motor nerves in Wallerian degeneration and the characteristics of peripheral nerve regeneration. The study was approved by the Ethics Committee of the Chinese PLA General Hospital, China(approval No. 2016-x9-07) in September 2016.
基金American Diabetes AssociationAmerican Heart Association+3 种基金NIH NIEHSNIH NIANIH NINDSNIH ARRA
文摘Cardiovascular disease, nervous system disorders, and cancer in association with other diseases such as diabetes mellitus result in greater than sixty percent of the global annual deaths. These noncommunicable diseases also affect at least one-third of the population in low and middle-income countries and lead to hypertension, elevated cholesterol, malignancy, and neurodegenerative disorders such as Alzheimer's disease and stroke. With the climbing lifespan of the world's population, increased prevalence of these disorders is expected requiring the development of new therapeutic strategies against these disabling disease entities. Targeting stem cellproliferation for cardiac disease, vascular disorders, cancer, and neurodegenerative disorders is receiving great enthusiasm, especially those that focus upon SIRT1, a mammalian homologue of the yeast silent information regulator-2. Modulation of the cellular activity of SIRT1 can involve oversight by nicotinamide/nicotinic acid mononucleotide adenylyltransferase, mammalian forkhead transcription factors, mechanistic of rapamycin pathways, and cysteine-rich protein 61, connective tissue growth factor, and nephroblastoma over-expressed gene family members that can impact cytoprotective outcomes. Ultimately, the ability of SIRT1 to control the programmed cell death pathways of apoptosis and autophagy can determine not only cardiac, vascular, and neuronal stem cell development and longevity, but also the onset of tumorigenesis and the resistance against chemotherapy. SIRT1 therefore has a critical role and holds exciting prospects for new therapeutic strategies that can offer reparative processes for cardiac, vascular, and nervous system degenerative disorders as well as targeted control of aberrant cell growth during cancer.
文摘The etiopathogenesis of gastrointestinal diseases is varied in nature.Various etiogenic factors described are infective,inflammatory,viral,bacterial,parasitic,dietary and lifestyle change.Rare causative agents are immunological,and others associated as idiopathic,are undiagnosed by all possible means.Some of the rare diseases are congenital in nature,passing from the parent to the child.Many of the undiagnosed diseases are now being diagnosed as genetic and the genes have been implicated as a causative agent.There is a search for newer treatments for such diseases,which is called genomic medicine.Genomic medicine is an emerging medical discipline that involves the use of genomic information about an individual.This is used both for diagnostic as well as therapeutic decisions to improve the current health domain and pave the way for policymakers for its clinical use.In the developing era of precision medicine,genomics,epigenomics,environmental exposure,and other data would be used to more accurately guide individual diagnosis and treatment.Genomic medicine is already making an impact in the fields of oncology,pharmacology,rare,infectious and many undiagnosed diseases.It is beginning to fuel new approaches in certain medical specialties.Oncology is at the leading edge of incorporating genomics,as diagnostics for genetic and genomic markers are increasingly included in cancer screening,and to guide tailored treatment strategies.Genetics and genetic medicine have been reported to play a role in gastroenterology in several ways,including genetic testing(hereditary pancreatitis and hereditary gastrointestinal cancer syndromes).Genetic testing can also help subtype diseases,such as classifying pancreatitis as idiopathic or hereditary.Gene therapy is a promising approach for treating gastrointestinal diseases that are not effectively treated by conventional pharmaceuticals and surgeries.Gene therapy strategies include gene addition,gene editing,messenger RNA therapy,and gene silencing.Understanding genetic determinants,advances in genetics,have led to a better understanding of the genetic factors that contribute to human disease.Family-member risk stratification and genetic diagnosis can help identify family members who are at risk,which can lead to preventive treatments,lifestyle recommendations,and routine follow ups.Selecting target genes helps identify the gene targets associated with each gastrointestinal disease.Common gastrointestinal diseases associated with genetic abnormalities include-inflammatory bowel disease,gastroesophageal reflux disease,non-alcoholic fatty liver disease,and irritable bowel syndrome.With advancing tools and technology,research in the search of newer and individualized treatment,genes and genetic medicines are expected to play a significant role in human health and gastroenterology.
基金supported by the National Natural Science Foundation of China,No.82471327the Natural Science Foundation of ShandongProvince,No.ZR2024MH200(both to SL).
文摘Neurite outgrowth and synaptogenesis are critical steps for functional recovery following ischemic stroke.Damaged axons of the central nervous system in adult mammals exhibit limited regenerative capacity,resulting in enduring neurological deficits.Recent findings from our research indicate that inhibition of Rho-associated kinase(ROCK)2 facilitates neuroprotection in different models of central nervous system diseases.In addition,our prior studies have demonstrated that axonal protection enhances the regeneration of injured axons.However,it remains unclear whether the axonal protection mediated by ROCK2 inhibition also facilitates synaptogenesis.In this study,we aimed to investigate the effects of inhibiting ROCK2 expression on synaptogenesis and neurogenesis in ischemic stroke using an shRNA-expressing adeno-associated virus(AAV)vector(AAV-sh.ROCK2).We demonstrated that AAV-sh.ROCK2 increased neurite outgrowth and facilitated synaptogenesis in vivo.Furthermore,AAV-sh.ROCK2 increased neuronal survival and promoted neurogenesis following middle cerebral artery occlusion surgery as well as long-term motor functional recovery after ischemia/reperfusion injury.Notably,AAV-sh.ROCK2 also stimulated serotonergic and dopaminergic axon sprouting after ischemia/reperfusion injury.Mechanistically,AAV-sh.ROCK2 activity resulted in increased anti-collapsin response mediator protein 2 activation and reductions in RhoA and ROCK2 expression.Our study identified ROCK2 as a critical regulator of synaptogenesis and neurogenesis,highlighting it as a promising target to facilitate neuroprotection and regeneration in ischemic stroke.
文摘Breast cancer is the leading cause of death in women. Prognosis of breast cancer is often pessimistic because the tumors are prone to metastasizing to the bone, brain, and lung. The estrogen signaling receptor (ESR) pathway contains 39 main genes and proteins which makes it one of the larger signaling pathways. Predominately this pathway and the proteins within are involved in breast growth and development, making it a prospective area of study for breast cancer. While the healthy ESR pathway has been constructed and is well established, a mechanistic model of mutated genes of ESR pathway has not been delved upon. Such mutated models could be utilized for selecting combinational targets for drug therapies, as well as elucidating crosstalk between other pathways and feedback mechanisms. To construct the mutated models of the ESR pathway it is imperative to assess what is currently understood in the literature and what inconsistencies exist in order to resolve them. Without this information, a model of the ESR pathway will be unreliable and likely unproductive. This review is the detailed literature survey of the biological studies performed on ESR pathways genes, and their respective roles in breast cancer. Furthermore, the details mentioned in the review can be beneficial for the integrated study of the ESR pathway genes, which includes, structural and dynamics study of the genes products, to have a holistic understanding of the cancer mechanism.
文摘AIM: To determine whether normal genetically immunocompetent rodent hosts could be manipulated to accept human hepatocyte transplants with long term survival without immunosuppression. METHODS: Tolerance towards human hepatocytes was established by injection of primary human hepatocytes or Huh7 human hepatoma cells into the peritoneal cavities of fetal rats. Corresponding cells were subsequently transplanted into newborn rats via intrasplenic injection within 24h after birth. RESULTS: Mixed lymphocyte assays showed that spleen cells from non-tolerized rats were stimulated to proliferate when exposed to human hepatocytes, while cells from tolerized rats were not. Injections made between 15 d and 17 d of gestation produced optimal tolerization. Transplanted human hepatocytes in rat livers were visualized by immunohistochemical staining of human albumin. By dot blotting of genomic DNA in livers of tolerized rats 16 weeks after hepatocyte transplantation, it was found that approximately 2.5 X 10(5) human hepatocytes survived per rat liver. Human albumin mRNA was detected in rat livers by RT-PCR for 15 wk, and human albumin protein was also detectable in rat serum. CONCLUSION: Tolerization of an immuno-competent rat can permit transplantation, and survival of functional human hepatocytes.
基金supported by the National Natural Science Foundation of China,Nos.81870921(to YW),81974179(to ZZ),82271320(to ZZ),82071284(to YT)National Key R&D Program of China,No.2022YFA1603600(to ZZ),2019YFA0112000(to YT)+1 种基金Scientific Research and Innovation Program of Shanghai Education Commission,No.2019-01-07-00-02-E00064(to GYY)Scientific and Technological Innovation Act Program of Shanghai Science and Technology Commission,No.20JC1411900(to GYY).
文摘AAV-PHP.eB is an artificial adeno-associated virus(AAV)that crosses the blood-brain barrier and targets neurons more efficiently than other AAVs when administered systematically.While AAV-PHP.eB has been used in various disease models,its cellular tropism in cerebrovascular diseases remains unclear.In the present study,we aimed to elucidate the tropism of AAV-PHP.eB for different cell types in the brain in a mouse model of ischemic stroke and evaluate its effectiveness in mediating basic fibroblast growth factor(bFGF)gene therapy.Mice were injected intravenously with AAV-PHP.eB either 14 days prior to(pre-stroke)or 1 day following(post-stroke)transient middle cerebral artery occlusion.Notably,we observed a shift in tropism from neurons to endothelial cells with post-stroke administration of AAV-PHP.eB-mNeonGreen(mNG).This endothelial cell tropism correlated strongly with expression of the endothelial membrane receptor lymphocyte antigen 6 family member A(Ly6A).Furthermore,AAV-PHP.eB-mediated overexpression of bFGF markedly improved neurobehavioral outcomes and promoted long-term neurogenesis and angiogenesis post-ischemic stroke.Our findings underscore the significance of considering potential tropism shifts when utilizing AAV-PHP.eB-mediated gene therapy in neurological diseases and suggest a promising new strategy for bFGF gene therapy in stroke treatment.
基金supported by the National Natural Science Foundation of China,Nos.92148206,82071330(both to ZT),82201474(to GL)a grant from Tongji Hospital,No.2022ZHFY01(to ZT).
文摘The prognosis for patients who experience intracerebral hemorrhage is poor because of a lack of effective treatments.Tumor necrosis factor-α-stimulated gene 6(TSG6)is a secreted glycoprotein that exerts anti-inflammatory effects in various inflammatory diseases.We previously showed that adipose-derived stem cells can inhibit inflammation by upregulating TSG6 secretion in an in vitro model of intracerebral hemorrhage.However,the direct effects of TSG6 on hematoma clearance in vivo remain largely unknown.The aim of this study was to determine how TSG6 affects hematoma absorption in mice subjected to intracerebral hemorrhage and to explore the potential underlying mechanisms.We first analyzed the gene profiles of patients with intracerebral hemorrhage from the GEO database and examined changes in TSG6 expression in the brain tissues of mice subjected to intracerebral hemorrhage.We found that TSG6 expression exhibited a transient increase following intracerebral hemorrhage,and that there was a negative correlation between the initial hematoma volume and TSG6 levels.Immunofluorescence analysis showed that TSG6 was primarily expressed in microglia and macrophages.Furthermore,we found that TSG6 promoted functional recovery in mice subjected to intracerebral hemorrhage by accelerating hematoma clearance,reducing the number of apoptotic cells and degenerated neurons,increasing the proportion of phagocytic microglia/macrophages,and decreasing iron deposition.Western blotting and immunofluorescence analysis indicated that TSG6 promoted M2 polarization of microglia/macrophages.In vitro phagocytosis experiments confirmed that TSG6 enhanced the ability of microglia to phagocytize red blood cells.Finally,we identified the signal transducer and activator of transcription 6/growth arrest-specific protein 6 signaling pathway as playing a critical role in TSG6-mediated hematoma absorption.In summary,our results demonstrate an essential role for TSG6 in promoting hematoma absorption in a mouse model of intracerebral hemorrhage.These findings suggest that TSG6 accelerates hematoma clearance and improves neurological function by promoting microglia/macrophage polarization to the M2 phenotype,activating the STAT6/GAS6 signaling pathway,and increasing phagocytic receptor expression on the surface of phagocytes,thereby enhancing their ability to phagocytize red blood cells.
