Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse...Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.展开更多
Background:Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies.However,its application to solid tumors presents significant challenges due to the limited accessi...Background:Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies.However,its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity.Localized delivery of tumor-specific T-cells using biomaterials has shown promise,however,procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints.Methods:Polyethylene glycol(PEG)-based three-dimensional(3D)scaffolds were developed and conjugated with positively charged poly-L-lysine(PLL)using carbamide chemistry for efficient loading of lentiviruses(LVs)carrying tumor antigen-specific T-cell receptors(TCRs).The physical and biological properties of the scaffold were extensively characterized.Further,the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin(B16-OVA)tumor model to evaluate the anti-tumor response and the presence of transduced T-cells.Results:Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site.In B16-OVA melanoma tumor-bearing mice,the scaffolds efficiently transduce host T-cells with OVA-specific TCRs.These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs,resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines.Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds.Conclusions:Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors.This approach offers a viable alternative to in vitro manipulation of T-cells,circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells.It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells,thereby,overcoming a major hurdle.展开更多
Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration...Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration-approved Phase I clinical trial has been conducted to evaluate the safety of transplanted human autologous Schwann cells to treat patients with spinal cord injury.A major challenge for Schwann cell transplantation is that grafted Schwann cells are confined within the lesion cavity,and they do not migrate into the host environment due to the inhibitory barrier formed by injury-induced glial scar,thus limiting axonal reentry into the host spinal cord.Here we introduce a combinatorial strategy by suppressing the inhibitory extracellular environment with injection of lentivirus-mediated transfection of chondroitinase ABC gene at the rostral and caudal borders of the lesion site and simultaneously leveraging the repair capacity of transplanted Schwann cells in adult rats following a mid-thoracic contusive spinal cord injury.We report that when the glial scar was degraded by chondroitinase ABC at the rostral and caudal lesion borders,Schwann cells migrated for considerable distances in both rostral and caudal directions.Such Schwann cell migration led to enhanced axonal regrowth,including the serotonergic and dopaminergic axons originating from supraspinal regions,and promoted recovery of locomotor and urinary bladder functions.Importantly,the Schwann cell survival and axonal regrowth persisted up to 6 months after the injury,even when treatment was delayed for 3 months to mimic chronic spinal cord injury.These findings collectively show promising evidence for a combinatorial strategy with chondroitinase ABC and Schwann cells in promoting remodeling and recovery of function following spinal cord injury.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
Following the publication of Zeng et al.(2023),an inadvertent error was recently identified in Figure 1B and Supplementary Figure S3.To ensure the accuracy and integrity of our published work,we formally request a cor...Following the publication of Zeng et al.(2023),an inadvertent error was recently identified in Figure 1B and Supplementary Figure S3.To ensure the accuracy and integrity of our published work,we formally request a correction to address this issue and apologize for any confusion this error may have caused.For details,please refer to the modified Supplementary Materials.展开更多
AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiv...AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.展开更多
Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by...Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.展开更多
AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem c...AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.展开更多
Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGA...Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.展开更多
Post-stroke depression is associated with reduced expression of brain-derived neurotrophic factor （BDNF）. In this study, we evaluated whether BDNF overexpression affects depression-like behavior in a rat model of po...Post-stroke depression is associated with reduced expression of brain-derived neurotrophic factor （BDNF）. In this study, we evaluated whether BDNF overexpression affects depression-like behavior in a rat model of post-stroke depression. The middle cerebral artery was occluded to produce a model of focal cerebral ischemia. These rats were then subjected to isolation-housing combined with chronic unpredictable mild stress to generate a model of post-stroke depression. A BDNF gene lentiviral vector was injected into the hippocampus. At 7 days after injection, western blot assay and real-time quantitative PCR revealed that BDNF expression in the hippo- campus was increased in depressive rats injected with BDNF lentivirus compared with depressive rats injected with control vector. Furthermore, sucrose solution consumption was higher, and horizontal and vertical movement scores were increased in the open field test in these rats as well. These findings suggest that BDNF overexpression in the hippocampus of post-stroke depressive rats alleviates depression-like behaviors.展开更多
AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference tar...AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.展开更多
BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and em...BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.展开更多
AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The re...AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.展开更多
microRNAs （miRNAs） play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 （miR-124） overexpression in bone marrow-derived mesen...microRNAs （miRNAs） play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 （miR-124） overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesen- chymal stem cells, neural stem cells and neurons, miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We con- structed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers [3-III tu- bulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re- sults suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.展开更多
Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undou...Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.展开更多
AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mecha...AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.展开更多
Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineer...Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 （NT-3） and green fluorescence protein （GFP）, hemisection of spinal cord at the level of T10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups （ n = 14 ）, namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supematant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan （BBB） open-field locomotor test to assess the recovery of function. Results The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenie expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. Concision It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.展开更多
文摘Highlight: The present report reveals for the first time natural lentiviral infection of wild Indian NHPs, rhesus monkeys (Macaca mulatta) and langurs (Semnopithecus entellus) by SIVs that are phylogenetically diverse from all known SIVs, including “SIVmac”, which infects captive rhesus monkeys. The novel SIVs are intriguingly homologous to HIV-1, based on serology and partial lentiviral genomic sequence analyses. Diverse lenti-viruses infect human and nonhuman primates (NHPs). There are more than 45 different “species-specific” simian immunodeficiency viruses (SIVs) that infect their cognate NHP hosts in natural habitats in Africa. Indian NHPs are not known to be infected by SIVs in the wild. Conventionally SIVs are named after their natural hosts, except for SIVmac, which infects captive rather than wild rhesus macaques. SIVmac is therefore a misnomer. It is a genetic variant of the African SIVsmm, which infects wild African sooty mangabey monkeys. SIVsmm is the progenitor of human immunodeficiency virus (HIV-2), while SIVcpz that infects wild chimpanzees is the progenitor of HIV-1. Although natural infections cannot be easily studied in wild NHP populations, we have previously reported co-infection of wild Indian NHPs by other retroviruses: simian retroviruses (SRVs) and Simian Foamy viruses (SFV). Apart from zoonosis, transmission of pathogens from humans to animals: anthroponosis, has also been reported in literature.
基金Department of Biotechnology(DBT,Govt of India)(BT/PR31315/MED/32/667/2019)DBT along with Wadhwani Research Center for Bioengineering,IIT Bombay(BT/INF/22/SP23026/2017)Department of Biotechnology(DBT,Govt of India)(BT/INF/22/SP17358/2016).
文摘Background:Targeted T-cell therapy has emerged as a promising strategy for the treatment of hematological malignancies.However,its application to solid tumors presents significant challenges due to the limited accessibility and heterogeneity.Localized delivery of tumor-specific T-cells using biomaterials has shown promise,however,procedures required for genetic modification and generation of a sufficient number of tumor-specific T-cells ex vivo remain major obstacles due to cost and time constraints.Methods:Polyethylene glycol(PEG)-based three-dimensional(3D)scaffolds were developed and conjugated with positively charged poly-L-lysine(PLL)using carbamide chemistry for efficient loading of lentiviruses(LVs)carrying tumor antigen-specific T-cell receptors(TCRs).The physical and biological properties of the scaffold were extensively characterized.Further,the scaffold loaded with OVA-TCR LVs was implanted in B16F10 cells expressing ovalbumin(B16-OVA)tumor model to evaluate the anti-tumor response and the presence of transduced T-cells.Results:Our findings demonstrate that the scaffolds do not induce any systemic inflammation upon subcutaneous implantation and effectively recruit T-cells to the site.In B16-OVA melanoma tumor-bearing mice,the scaffolds efficiently transduce host T-cells with OVA-specific TCRs.These genetically modified T-cells exhibit homing capability towards the tumor and secondary lymphoid organs,resulting in a significant reduction of tumor size and systemic increase in anti-tumor cytokines.Immune cell profiling revealed a significantly high percentage of transduced T-cells and a notable reduction in suppressor immune cells within the tumors of mice implanted with these scaffolds.Conclusions:Our scaffold-based T-cell therapy presents an innovative in situ localized approach for programming T-cells to target solid tumors.This approach offers a viable alternative to in vitro manipulation of T-cells,circumventing the need for large-scale in vitro generation and culture of tumor-specific T-cells.It offers an off-the-shelf alternative that facilitates the use of host cells instead of allogeneic cells,thereby,overcoming a major hurdle.
基金supported in part by NIH R01 NS100531,R01 NS103481NIH R21NS130241(to LD)+3 种基金Merit Review Award I01 BX002356,I01 BX003705 from the U.S.Department of Veterans AffairsIndiana Spinal Cord and Brain Injury Research Foundation(No.19919)Mari Hulman George Endowment Funds(to XMX)Indiana Spinal Cord&Brain Injury Research Fund from ISDH(to NKL and LD)。
文摘Schwann cell transplantation is considered one of the most promising cell-based therapy to repair injured spinal cord due to its unique growth-promoting and myelin-forming properties.A the Food and Drug Administration-approved Phase I clinical trial has been conducted to evaluate the safety of transplanted human autologous Schwann cells to treat patients with spinal cord injury.A major challenge for Schwann cell transplantation is that grafted Schwann cells are confined within the lesion cavity,and they do not migrate into the host environment due to the inhibitory barrier formed by injury-induced glial scar,thus limiting axonal reentry into the host spinal cord.Here we introduce a combinatorial strategy by suppressing the inhibitory extracellular environment with injection of lentivirus-mediated transfection of chondroitinase ABC gene at the rostral and caudal borders of the lesion site and simultaneously leveraging the repair capacity of transplanted Schwann cells in adult rats following a mid-thoracic contusive spinal cord injury.We report that when the glial scar was degraded by chondroitinase ABC at the rostral and caudal lesion borders,Schwann cells migrated for considerable distances in both rostral and caudal directions.Such Schwann cell migration led to enhanced axonal regrowth,including the serotonergic and dopaminergic axons originating from supraspinal regions,and promoted recovery of locomotor and urinary bladder functions.Importantly,the Schwann cell survival and axonal regrowth persisted up to 6 months after the injury,even when treatment was delayed for 3 months to mimic chronic spinal cord injury.These findings collectively show promising evidence for a combinatorial strategy with chondroitinase ABC and Schwann cells in promoting remodeling and recovery of function following spinal cord injury.
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.
文摘Following the publication of Zeng et al.(2023),an inadvertent error was recently identified in Figure 1B and Supplementary Figure S3.To ensure the accuracy and integrity of our published work,we formally request a correction to address this issue and apologize for any confusion this error may have caused.For details,please refer to the modified Supplementary Materials.
基金Supported by Natural Science Foundation of Shanghai,No.11ZR1427100
文摘AIM： To investigate whether hepatitis B virus （HBV） could induce a hepatitis B virus core antigen （HBcAg）specific cytotoxic T lymphocyte （CTL） response in vitro by dendritic cells （DCs） transduced with lentiviral vector-encoding ubiquitinated hepatitis B virus core antigen （LV-Ub-HBcAg）.METHODS： Recombinant LV-Ub-HBcAg were transfected into highly susceptible 293 T cells to obtain high virus titres, Bone marrow-derived DCs isolated from BALB/c mice were cultured with recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin （IL）-4. LV-Ub-HBcAg, lentiviral vector-encoding hepatitis B virus core antigen （LV-HBcAg）, lentiviral vector （LV） or lipopolysaccharide were added to induce DC maturation, and the DC phenotypes were analyzed by flow cytometry. The level of IL-12 in the supernatant was detected by enzyme-linked immunosorbent assay （ELISA）. T lymphocytes were proliferated using Cell Counting Kit-8. DCs were cultured and induced to mature using different LVs, and co-cultured with allogeneic T cells to detect the secretion levels of IL-2, IL-4, IL-10and interferon-γ in the supernatants of T cells by ELISA. Intracellular cytokines of proliferative T cells were analyzed by flow cytometry, and specific CTL activity was measured by a lactate dehydrogenase release assay.RESULTS： LV-Ub-HBcAg-induced DCs secreted more IL-12 and upregulated the expression of CD80, CD86 and major histocompatibility class ]I, DCs sensitised by different LVs effectively promoted cytokine secretion; the levels of IL-2 and interferon-y induced by LV-Ub- HBcAg were higher than those induced by LV-HBcAg, Compared with LV-HBcAg-transduced DCs, LV-Ub- HBcAg-transduced DCs more efficiently stimulated the proliferation of T lymphocytes and generated HBcAgspecific cytotoxic T lymphocytes.CONCLUSION： LV-Ub-HBcAg effectively induced DC maturation. The mature DCs efficiently induced T cell polarisation to Thl and generated HBcAg-specific CTLs.
基金This work was supported by the Natural Science Foundation of Shanghai Municipality(No.03ZR14016).
文摘Objective To construct recombinant lentiviral vectors for gene delivery of the glial cell line-derived neurotropnic factor （GDNF）, and evaluate the neuroprotective effect of GDNF on lactacystin-damaged PC12 cells by transfecting it into bone marrow stromal cells （BMSCs）. Methods pLenti6/V5-GDNF plasmid was set up by double restriction enzyme digestion and ligation, and then the plasmid was transformed into Top10 cells. Purified pLenti6/V5-GDNF plasmids from the positive clones and the packaging mixture were cotransfected to the 293FT packaging cell line by Lipofectamine2000 to produce lentivirus, then the concentrated virus was transduced to BMSCs. Overexpression of GDNF in BMSCs was tested by RT-PCR, ELISA and immunocytochemistry, and its neuroprotection for lactacystin-damaged PC12 cells was evaluated by MTT assay. Results Virus stock of GDNF was harvested with the titer of 5.6×10^5 TU/mL. After tmnsduction, GDNF-BMSCs successfully secreted GDNF to supematant with nigher concentration （800 pg/mL） than BMSCs did （less than 100 pg/mL）. The supematant of GDNF-BMSCs could significantly alleviate the damage of PC12 cells induced by lactacystin （10 μmol/L）. Conclusion Overexpression of lentivirus-mediated GDNF in the BMSCs cells can effectively protect PC12 cells from the injury by the proteasome inhibitor.
基金Supported by National Institute of Genetic Engineering and Biotechnology,Ministry of Science Research and Technology,Tehran,Iran
文摘AIM: To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene.METHODS: In this study, human adipose tissue derived stem cells(hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis.Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1(Non-integrated LV-PDX1) were constructed using specific plasmids(pLV-HELP, pMD2G, LV-105-PDX1-1).Then, hADSCs were transduced with non-integrated LVPDX1. After transduction, ADSCsPDX1+were cultured in high glucose DMEM medium supplement by B27, nicotinamide and βFGF for 21 d. Expressions of PDX1 andinsulin were detected at protein level by immunofluorescence analysis. Expressions of PDX1, neurogenin3(Ngn3), glucagon, glucose transporter2(Glut2) and somatostatin as specific marker genes were investigated at mRNA level by quantitative RT-PCR. Insulin secretion of hADSCsPDX1+in the high-glucose medium was detected by electrochemiluminescence test. Human ADSCsPDX1+were implanted into hyperglycemic rats.RESULTS: Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture.Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs+PDX1became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1, Ngn3, glucagon, Glut2and somatostatin were detected by quantitative RTPCR. hADSCsPDX1+revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCsPDX1+in the high glucose medium was 2.32 μU/mL. hADSCsPDX1+implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level.CONCLUSION: Human ADSCs can differentiate into IPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.
文摘Objective Glutamic acid decarboxylase 2（GAD65） is a gamma-aminobutyric acid（GABA） synthetase.This study aimed to construct a recombinant lentivirus-rGAD65（rLV-rGAD65） vector containing the cDNA of rat GAD65（rGAD65） and assess its functional activity in vitro and in vivo.Methods cDNA of rGAD65 was amplified by RT-PCR and subcloned into the LV vector,forming the rLV-GFP-rGAD65 plasmid.The recombinant lentivirus particles（rLVrGAD65） were packaged by the LV Helper-Free System and the titer was measured.Primary rat lung fibroblasts were transfected with rLV-rGAD65.The expression of rGAD65 in fibroblasts was detected by immunocytochemistry and western blot and the level of GABA in the medium was assessed by high-performance liquid chromatograph（HPLC）.In vivo,rLV-rGAD65 was injected into the subthalamic nucleus（STN） of Sprague-Dawley rats using stereotaxic methods,and rGAD65 protein levels in the STN were assessed by immunohistochemistry and Western blot,while the GABA concentration in the substantia nigra pars reticulata（SNr） was assayed by HPLC.Results The sequence of rGAD65 cDNA was in accord with that in GenBank.The amino-acid sequence of rGAD65 had no mutations and the titer of rLVrGAD65 reached 6.8 × 108/mL.The efficiency of infection of fibroblasts was 80%,and the concentration of GABA in the medium was（48.14 ± 9.35） nmol/L.In vivo,rGAD65 expression was detected in the STN,and the concentration of GABA in the SNr increased from（5.95 ± 1.09） to（12.44 ± 3.79） nmol/g tissue.Conclusion The recombinant LVGFP-rGAD65 vector was successfully constructed.rLV-rGAD65-infected primary fibroblasts in vitro and the expressed rGAD65 catalyzed the formation of GABA from glutamic acid.In vivo,the concentration of GABA in the SNr was increased after rLV-rGAD65 injection into the STN.
基金supported by a grant from the Experimental Animal Science and Technology Project of Zhejiang Province in China,No.2012C37083
文摘Post-stroke depression is associated with reduced expression of brain-derived neurotrophic factor （BDNF）. In this study, we evaluated whether BDNF overexpression affects depression-like behavior in a rat model of post-stroke depression. The middle cerebral artery was occluded to produce a model of focal cerebral ischemia. These rats were then subjected to isolation-housing combined with chronic unpredictable mild stress to generate a model of post-stroke depression. A BDNF gene lentiviral vector was injected into the hippocampus. At 7 days after injection, western blot assay and real-time quantitative PCR revealed that BDNF expression in the hippo- campus was increased in depressive rats injected with BDNF lentivirus compared with depressive rats injected with control vector. Furthermore, sucrose solution consumption was higher, and horizontal and vertical movement scores were increased in the open field test in these rats as well. These findings suggest that BDNF overexpression in the hippocampus of post-stroke depressive rats alleviates depression-like behaviors.
基金Supported by The Affiliated First People’s Hospital, Shanghai Jiao Tong University and the Board of Education Fund for Scientific Research of Shanghai, China, No. 06BE067
文摘AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.
文摘BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.
基金Supported by National Natural Science Foundation of China,No.81101896
文摘AIM:To investigate the regulative effect of miRNA-3383p(miR-338-3p) on cell growth in colorectal carcinoma(CRC).METHODS:The lentiviral vector pLV-THM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed.The recombinant viral vector encoding the pre-miR338-3p or miR-338-3p-inhibitor and the two packaging plasmids psPAX2 and pMD2.G were cotransfected into human embryonic kidney 293T cells to package lentivirus.The supernatant containing the lentivirus particles was harvested to determine the viral titer,and this supernatant was then used to transduce CRCderived cell line,SW-620.Flow cytometry was utilized for sorting the green fluorescent protein(GFP) + cells to establish the SW-620 cell line stably expressing premiR-338-3p or miR-338-3p-inhibitor.Moreover,the expression of miR-338-3p was determined by real-time reverse transcriptase polymerase chain reaction,andWestern blotting was used to detect the expression of the smoothened(SMO,the possible target of miR-3383p) protein in SW-620 cells.Furthermore,the status of CRC cell proliferation and apoptosis were detected by 3-(4,5-dimethyl-2 thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide assay and flow cytometry,respectively.RESULTS:Restriction enzyme digestion and DNA sequencing demonstrated that the lentiviral vector pLVTHM-miR-338-3p and pLV-THM-miR-338-3p-inhibitor were constructed successfully.GFP was expressed after the SW-620 cells were transduced by the lentivirus.Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p was significantly increased(relative expression 3.91 ± 0.51 vs 2.36 ± 0.44,P < 0.01).Furthermore,overexpression of miR-338-3p inhibited the expression of SMO protein in SW-620 cells,which showed obviously suppressed proliferation ability [cellular proliferation inhibition rate(CPIR) 61.9% ± 5.2% vs 41.6% ± 4.8%,P < 0.01].Expression of miR-338-3p in SW-620 cells transduced with the lentivirus pLV-THM-miR-338-3p-inhibitor was significantly decreased(relative expression 0.92 ± 0.29 vs 2.36 ± 0.44,P < 0.01).Moreover,the downregulated expression of miR-338-3p caused upregulated expression of the SMO protein in SW-620 cells,which showed significantly enhanced proliferation ability(CPIR 19.2% ± 3.8% vs 41.6% ± 4.8%,P < 0.01).However,anti-SMO-siRNA largely,but not completely,reversed the effects induced by blockage of miR-338-3p,suggesting that the regulative effect of miR-338-3p on CRC cell growth was indeed mediated by SMO.CONCLUSION:miR-338-3p could suppress CRC growth by inhibiting SMO protein expression.
基金supported by the National Natural Science Foundation of China,No.81070971
文摘microRNAs （miRNAs） play an important regulatory role in the self-renewal and differentiation of stem cells. In this study, we examined the effects of miRNA-124 （miR-124） overexpression in bone marrow-derived mesenchymal stem cells. In particular, we focused on the effect of overexpression on the differentiation of bone marrow-derived mesenchymal stem cells into neurons. First, we used GeneChip technology to analyze the expression of miRNAs in bone marrow-derived mesen- chymal stem cells, neural stem cells and neurons, miR-124 expression was substantially reduced in bone marrow-derived mesenchymal stem cells compared with the other cell types. We con- structed a lentiviral vector overexpressing miR-124 and transfected it into bone marrow-derived mesenchymal stem cells. Intracellular expression levels of the neuronal early markers [3-III tu- bulin and microtubule-associated protein-2 were significantly increased, and apoptosis induced by oxygen and glucose deprivation was reduced in transfected cells. After miR-124-transfected bone marrow-derived mesenchymal stem cells were transplanted into the injured rat spinal cord, a large number of cells positive for the neuronal marker neurofilament-200 were observed in the transplanted region. The Basso-Beattie-Bresnahan locomotion scores showed that the motor function of the hind limb of rats with spinal cord injury was substantially improved. These re- sults suggest that miR-124 plays an important role in the differentiation of bone marrow-derived mesenchymal stem cells into neurons. Our findings should facilitate the development of novel strategies for enhancing the therapeutic efficacy of bone marrow-derived mesenchymal stem cell transplantation for spinal cord injury.
基金supported by grants from the National Basic Research Program of China (973 Program), No. 2012CB967900, 2012CB967904the National Natural Science Foundation of China, No. 81070782+2 种基金the Natural Science Foundation of Zhejiang Province, China, No. 30672308the Qianjiang Talent Project of Science and Technology Ministry in Zhejiang Province, No. 2011R10014the Natural Science Foundation of Ningbo, No. 2011A610042
文摘Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.
基金Supported by the National Natural Science Foundation of China(No.81070735)
文摘AIM: To evaluate the effects of lentivirus-mediated pigment epithelium-derived factor (PEDF) gene transfer performed in treatment of rats with established choroidal neovascularization (CNV), and investigates the mechanism by which PEDF inhibits CNV in rats. METHODS: Brown Norway (BN) rats (n=204) were induced by exposure to a laser, and then randomly assigned to 3 groups: no treatment; treatments with intravitreal injection of lentivirus-PEDF-green fluorescent protein (GFP) or lentivirus-control GFP (free fluorescent protein). Following induction and treatment, the CNV tissue was assessed for form, size and vessel leakage by fluorescein fundus angiography (FFA), optical coherence tomography (OCT), histopathology, and examination of choroidal flat mounts. VEGF, Flk-1, and PEDF expression were evaluated by real-time polymerase chain reaction (PCR) and Western blot. RESULTS: A stable laser-induced rat model of CNV was successfully established, and used to demonstrate lentivirus-mediated REDO gene transfer by intravitreal injection. Expression of green fluorescence labelled PEDF was observed in the retina up to 28d after injection. An intravitreal injection of lentivirus-PEDF-GFP at 7d led to a significant reduction in the size, thickness and area of CNV showed by FFA, OCT and choroidal flat mounts. PEDF was up-regulated while VEGF and Flk-1 were down-regulated in the lentivirus-PEDF-GFP group. The differences in VEGF and Flk-1 expression in the control and lentivirus-PEDF groups at 7, 14, 21 and 28d after laser induction were all statistically significant. CONCLUSION: Lentivirus-mediated PEDF gene transfer is effective for use in treatment of laser-induced CNV, and PEDF exerts its therapeutic effects by inhibiting expression of VEGF and Flk-1.
基金Supported by the Natural Science Foundation of Yunnan Province(2002C0070M).
文摘Objective To explore the feasibility for therapy of spinal cord injury （SCI） by genetic engineering neural stem cell （NSC） modified by lentiviral vector. Methods Following the construction of the genetic engineering NSC modified by lentivirus to secrete both neurotrophic factor-3 （NT-3） and green fluorescence protein （GFP）, hemisection of spinal cord at the level of T10 was performed in 56 adult Wistar rats that were randomly divided into 4 groups （ n = 14 ）, namely 3 therapeutic groups and 1 control group. The therapeutic groups were dealed with NSC, genetic engineering NSC, and concentrated lentiviral supematant which carries both GFP and NT-3, respectively. Then used fluorescence microscope to detect the transgenic expression in vitro and in vivo, migration of the grafted cells in vivo, and used the Basso, Beattie, and Bresnahan （BBB） open-field locomotor test to assess the recovery of function. Results The transplanted cells could survive for long time in vivo and migrate for long distance. The stable transgenie expression could be detected in vivo. The hindlimb function of the injured rats in 3 therapeutic groups, especially those dealed with genetic engineering NSC, improved obviously. Concision It is feasible to combine NSC with lentivirus for the repair of SCI. NSC modified by lentivirus to deliver NT-3, acting as a source of neurotrophic factors and function cell in vivo, has the potential to participate in spinal cord repair.
