Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during devel...Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during development. Researchers can add moieties like peptide nucleic acid or methoxyethyl-modified ribose sugars to enhance specificity and reduce toxicity. Current research suggests that challenges such as nonspecific action, interference at various stages, adverse reactions, and nuclease degradation may soon be manageable with advanced technologies. ASOs show particular promise in treating rare conditions like Duchenne Muscular Dystrophy (DMD) and Timothy syndrome. Stereopure ASOs with repeated left-right patterns offer increased potency and half-life due to their resistance to nuclease activity and improved cellular uptake. This review explores how technological advancements can enhance the use of ASOs to manage various rare disease conditions effectively. Despite challenges in development and application, ASO therapy holds the potential to become a viable treatment option for a wide range of rare diseases. Advances in technology offer the possibility of increasing specificity and reducing toxicity, making ASO therapy a more effective and safe treatment option for patients with rare diseases.展开更多
Functionalized carbon nanotubes （CNTs） were made for the delivery of genes and drugs and CNT-based biosensors. The basis of CNTs is for binding with biomolecules in biomedical applications. The binding tendency with...Functionalized carbon nanotubes （CNTs） were made for the delivery of genes and drugs and CNT-based biosensors. The basis of CNTs is for binding with biomolecules in biomedical applications. The binding tendency with small interfering RNA oligonucleotides and cytotoxicity of cetyltrimethyl ammonium bromide （CTAB）-coated single-walled carbon nanotubes （SWNTs） were studied. The field emission scanning electron microscopy and transmission electron microscopy results show that a SWNT suspension in CTAB solution was well-dispersed and stable. CTAB is the cross-linker between SWNTs and oligonucleotides. The CTAB-coated SWNTs have less cytotoxicity to human umbilical vein endothelial cells than single SWNTs and the cytotoxicity of CTAB-coated SWNTs depended on the concentration of CTAB-coated SWNTs.展开更多
Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in a...Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.展开更多
Objective To examine antisense and decoy oligonucleotides of nuclear factor kappa B in vivo effects on intima proliferation and balloon-injured monocytes chemotactic protein-1 （ MCP-1 ） and extraceUular signal regul...Objective To examine antisense and decoy oligonucleotides of nuclear factor kappa B in vivo effects on intima proliferation and balloon-injured monocytes chemotactic protein-1 （ MCP-1 ） and extraceUular signal regulated kinase-2 （ERK2） κexpression in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. Rats are divided into 7 groups （ n = 18 ） and each group includes6 time points （6 h, 1, 3, 5, 7, 14 d） （n =3）. Uninjured right carotid artery of the same rat was used as controls. Results In model group, sense group and scramble group, vessel intima area , media area and intima/media ratio increased after 5 d and reached the maximum after 7 d. The effect of antisense plus decoy group on intimal hyperplasia was more obvious than that of antisense group and decoy group alone. MCP-1 mRNA expression was increased expression continuously at 3, 5 and 7 d and decreased at 14 d. Compared with model group, sense group and scramble group, antisense group, decoy group and antisense plus decoy group had lowered MCP-1 mRNA expression in each time point （ P 〈 0. 05 ）. NF-KB p65 was dispersed positive stain 6 h after injury and increased after 1 d and peaked at 7 d, but the protein expression was weak at 14 d. ERK2 protein synthesis increased at I d and reached the peak at 7 d, while protein expression after 14 d was similar to that at 7 d. Treatment of antisense group, decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group, sense group and scramble group（ P 〈 0. 05）. Conclusion NF-KB modulates genes expression and protein synthesis of MCP-1 and ERK2. Celluar proliferation in vessel wall was dynamically changed after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-KB by local lipofectaraine transfer inhibit NF-KB activating gene modulation and neointimal hyperplasia.展开更多
AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of dir...AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.展开更多
AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer ...AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.展开更多
AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell prolifer...AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.展开更多
AIM： To detect the common intestinal pathogenic bacteria quickly and accurately.METHODS： A rapid （〈3 h） experimental procedure was set up based upon the gene chip technology, Target genes were amplified and hybri...AIM： To detect the common intestinal pathogenic bacteria quickly and accurately.METHODS： A rapid （〈3 h） experimental procedure was set up based upon the gene chip technology, Target genes were amplified and hybridized by oligonucleotide microarrays.RESULTS： One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 （96.2%） were identified.CONCLUSION： Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus , Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonudeotides to the arrays without any significant increase of complexity or cost.展开更多
Synthetic oligonucleotides including antisense oligonucleotides and siRNA have shown promising therapeutic potential.However,to realize the therapeutic potential of synthetic oligonucleotides,many obstacles have to be...Synthetic oligonucleotides including antisense oligonucleotides and siRNA have shown promising therapeutic potential.However,to realize the therapeutic potential of synthetic oligonucleotides,many obstacles have to be overcome,such as their poor biological stability,non-specific activity and inadequate cell membrane permeability.In this paper,the achievements by Lihe Zhang＇s group in the study of isonucleotide modified oligonucleotides and oligonucleotides conjugated with cell penetrating peptides are summarized.展开更多
AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human he...AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.展开更多
Two photolabile amphiphilic supramolecules were designed and synthesized with mono-dendrite and tri-dendrite, which can reversibly self-assemble to spheroid and wedge-shaped nanoparticles. With multiple branches of te...Two photolabile amphiphilic supramolecules were designed and synthesized with mono-dendrite and tri-dendrite, which can reversibly self-assemble to spheroid and wedge-shaped nanoparticles. With multiple branches of terminal amine labeled PEG, these nanoparticles can associate with a negatively charged oligonucleotide and their usage for oligonucleotide delivery was evaluated. Oligonucleotide/nanoparticle complex containing tri-dendrite can efficiently deliver oligonucleotide into cells via endocytosis, while the complex containing mono-dendrite almost lost their ability to deliver oligonucleotide. Further light activation triggered the dissociation of tri-dendrite supramolecular assembly via 1,4- and 1,6-quinone-methide rearrangement, leading to the efficient unpacking of the oligonucleotide in cells.展开更多
AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomera...AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.展开更多
Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DN...Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray Results Of the 102 rifampin-resistant strains 98 （96.1%） had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.展开更多
Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use o...Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure： reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide （random hexamer or non-ribosomal hexanucleotide） primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease （HFMD） and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.展开更多
Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge.Bacteria and the extracellular polysaccharides(EPS)cause biofilms to become adherent,toxic,resistant to antibi...Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge.Bacteria and the extracellular polysaccharides(EPS)cause biofilms to become adherent,toxic,resistant to antibiotics,and ultimately difficult to remove.Inhibition of EPS synthesis can prevent the formation of bacterial biofilms,reduce their robustness,and promote removal.Here,we have developed a framework nucleic acid delivery system with a tetrahedral configuration.It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes.We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site.Once delivered to bacterial cells,they significantly decreased EPS synthesis and biofilm thickness.Compared to existing approaches,this system is highly efficacious because it simultaneously reduces the expression of all targeted genes(gtfBCD,gbpB,ftf).We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.展开更多
AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed fo...AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS： HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% （n = 33）, 83.2% （n = 333）, and 8.5% （n = 34）, respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION： The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.展开更多
Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the...Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.展开更多
AIM: To investigate the molecular pathways involved in human cholangiocarcinogenesis by gene expression profiling. METHODS: Oligonucleotide arrays (Affymetrix U133A) were used to establish a specific gene expression p...AIM: To investigate the molecular pathways involved in human cholangiocarcinogenesis by gene expression profiling. METHODS: Oligonucleotide arrays (Affymetrix U133A) were used to establish a specific gene expression profile of intrahepatic CCC in comparison to corresponding non- malignant liver tissue. To validate the expression values of the most overexpressed genes, RT-PCR experiments were performed. RESULTS: Five hundred and fifty-two statistically differentially expressed genes/ESTs (221 probes significantly up-regulated, 331 probes down-regulated; P < 0.05; fold change > 2; ≥ 70%) were identified. Using these data and two-dimensional cluster analysis,a specific gene expression profile was obtained allowing fast and reproducible differentiation of CCC, which was confirmed by supervised neuronal network modelling. The most consistently overexpressed gene (median fold change 33.5, significantly overexpressed in 100%) encoded osteopontin. Furthermore, an association of various genes with the histopathological grading could be demonstrated. CONCLUSION: A highly specific gene expression profile for intrahepatic CCC was identified, allowing for its fast and reproducible discrimination against non- malignant liver tissue and other liver masses. The most overexpressed gene in intrahepatic CCC was the gene encoding osteopontin. These data may lead to a better understanding of human cholangiocarcinogenesis.展开更多
AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepa...AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.展开更多
The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were stu...The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.展开更多
文摘Gene therapy and antisense oligonucleotides (ASOs) are promising approaches to treating rare diseases by targeting specific genes. However, ASOs can have off-target effects that need careful consideration during development. Researchers can add moieties like peptide nucleic acid or methoxyethyl-modified ribose sugars to enhance specificity and reduce toxicity. Current research suggests that challenges such as nonspecific action, interference at various stages, adverse reactions, and nuclease degradation may soon be manageable with advanced technologies. ASOs show particular promise in treating rare conditions like Duchenne Muscular Dystrophy (DMD) and Timothy syndrome. Stereopure ASOs with repeated left-right patterns offer increased potency and half-life due to their resistance to nuclease activity and improved cellular uptake. This review explores how technological advancements can enhance the use of ASOs to manage various rare disease conditions effectively. Despite challenges in development and application, ASO therapy holds the potential to become a viable treatment option for a wide range of rare diseases. Advances in technology offer the possibility of increasing specificity and reducing toxicity, making ASO therapy a more effective and safe treatment option for patients with rare diseases.
基金Project (30770838) supported by the National Natural Science Foundation of China Project (2008WK2003) supported by Hunan Science and Technology Foundation, China+1 种基金 Project (2010QZZD006) supported by the Key Program of Central South University Advancing Front Foundation, China Project (200806) supported by Opening Foundation of State Key Laboratory of Powder Metallurgy, Central South University, China
文摘Functionalized carbon nanotubes （CNTs） were made for the delivery of genes and drugs and CNT-based biosensors. The basis of CNTs is for binding with biomolecules in biomedical applications. The binding tendency with small interfering RNA oligonucleotides and cytotoxicity of cetyltrimethyl ammonium bromide （CTAB）-coated single-walled carbon nanotubes （SWNTs） were studied. The field emission scanning electron microscopy and transmission electron microscopy results show that a SWNT suspension in CTAB solution was well-dispersed and stable. CTAB is the cross-linker between SWNTs and oligonucleotides. The CTAB-coated SWNTs have less cytotoxicity to human umbilical vein endothelial cells than single SWNTs and the cytotoxicity of CTAB-coated SWNTs depended on the concentration of CTAB-coated SWNTs.
基金This work was supported by the National Natural Sciences Foundation of China (No. 3017111) and National Project "863" (No. 2001AA234041)
文摘Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.
文摘Objective To examine antisense and decoy oligonucleotides of nuclear factor kappa B in vivo effects on intima proliferation and balloon-injured monocytes chemotactic protein-1 （ MCP-1 ） and extraceUular signal regulated kinase-2 （ERK2） κexpression in the carotid artery of rats. Methods Sprague-Dawley rats underwent balloon-dilation injury of the left carotid artery. Rats are divided into 7 groups （ n = 18 ） and each group includes6 time points （6 h, 1, 3, 5, 7, 14 d） （n =3）. Uninjured right carotid artery of the same rat was used as controls. Results In model group, sense group and scramble group, vessel intima area , media area and intima/media ratio increased after 5 d and reached the maximum after 7 d. The effect of antisense plus decoy group on intimal hyperplasia was more obvious than that of antisense group and decoy group alone. MCP-1 mRNA expression was increased expression continuously at 3, 5 and 7 d and decreased at 14 d. Compared with model group, sense group and scramble group, antisense group, decoy group and antisense plus decoy group had lowered MCP-1 mRNA expression in each time point （ P 〈 0. 05 ）. NF-KB p65 was dispersed positive stain 6 h after injury and increased after 1 d and peaked at 7 d, but the protein expression was weak at 14 d. ERK2 protein synthesis increased at I d and reached the peak at 7 d, while protein expression after 14 d was similar to that at 7 d. Treatment of antisense group, decoy group and antisense plus decoy group inhibited protein synthesis more significantly than those of model group, sense group and scramble group（ P 〈 0. 05）. Conclusion NF-KB modulates genes expression and protein synthesis of MCP-1 and ERK2. Celluar proliferation in vessel wall was dynamically changed after balloon angioplasty injury. Antisense and decoy oligonucleotide of NF-KB by local lipofectaraine transfer inhibit NF-KB activating gene modulation and neointimal hyperplasia.
文摘AIM To study the specific inhibition of HBV gene expression by liver-targeting antisense oligonucleotide (ASON) directed against pre-c and c regious in a sequence-specific manner.METHODS According to the result of direct sequencing of PCR amplified products, a 16-mer phosphorothioate analogue of the antisense oligonucleotide (PS-ASOn) directed against the HBV U5-like region was synthesized and then linked with one live-targeting ligand, the galactosylated poly-L-lysine. Their effect on the expression of HBV gene was observed using the 2.2.15 cells.RESULTS HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw1 by sequencing so that antisense oligonucleotides could bind specifically to the target sequence through base piring. Under the same experimental conditions, the inhibitory rates of PS-ASON to HBsAg and HBeAg were 70% and 58% at a concentration of 10μmol/L, while by ligand-PS-ASON they were 96% and 82%, the amount of HBV DNA in cultured supernatant and cells was reduced significantly. An unrelated sequence oligonucleotide showed no effectiveness. All the oligonucleotides had no cytotoxicity.CONCLUSION Antisense oligonucleotides complexed by the liver-targeting ligand can be targeted to cells via asialoglycoprotein receptors, resulting in supecific inhibition of HBV gene expression and replication.
文摘AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers can abrogate HSP70 expression in SGC-7901 cells, which may in turn induce apoptosis and inhibit cell proliferation, conversely suggesting that HSP70 is required for the proliferation and survival of human gastric cancer cells under normal conditions.
基金Supported by grants from the Zhejiang Province Medicine and Health Research Fund, No. 2003A077Huzhou Natural Science Foundation, No. 2004SZX07-11, China
文摘AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.
基金Supported by the National High Technology ResearchDevelopment Program of China (863 Program), No.2002AA2Z2011
文摘AIM： To detect the common intestinal pathogenic bacteria quickly and accurately.METHODS： A rapid （〈3 h） experimental procedure was set up based upon the gene chip technology, Target genes were amplified and hybridized by oligonucleotide microarrays.RESULTS： One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 （96.2%） were identified.CONCLUSION： Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus , Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonudeotides to the arrays without any significant increase of complexity or cost.
基金National Natural Science Foundation (Grant No.20932001)the Ministry of Science and Technology (Grant No.2012CB720604)
文摘Synthetic oligonucleotides including antisense oligonucleotides and siRNA have shown promising therapeutic potential.However,to realize the therapeutic potential of synthetic oligonucleotides,many obstacles have to be overcome,such as their poor biological stability,non-specific activity and inadequate cell membrane permeability.In this paper,the achievements by Lihe Zhang＇s group in the study of isonucleotide modified oligonucleotides and oligonucleotides conjugated with cell penetrating peptides are summarized.
基金grants from Medical and Sanitary Research Foundation of Zhejiang Province, (No. 2003A077)Huzhou Natural Science Foundation, (No. 2004SZX07-11)
文摘AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) and in situ human hepatocellular carcinoma (HCC). METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylin- eosin (HE) staining. RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues. CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.
基金National Natural Science Foundation of China(Grant No.21372018)the National Basic Research Program of China(973 Program,Grant No.2013CB933800)+1 种基金Program for New Century Excellent Talents in University(Grant No.NCET-10-0203)the State Key Laboratory of Pharmaceutical Biotechnology(Grant No.KF-GN-201305)
文摘Two photolabile amphiphilic supramolecules were designed and synthesized with mono-dendrite and tri-dendrite, which can reversibly self-assemble to spheroid and wedge-shaped nanoparticles. With multiple branches of terminal amine labeled PEG, these nanoparticles can associate with a negatively charged oligonucleotide and their usage for oligonucleotide delivery was evaluated. Oligonucleotide/nanoparticle complex containing tri-dendrite can efficiently deliver oligonucleotide into cells via endocytosis, while the complex containing mono-dendrite almost lost their ability to deliver oligonucleotide. Further light activation triggered the dissociation of tri-dendrite supramolecular assembly via 1,4- and 1,6-quinone-methide rearrangement, leading to the efficient unpacking of the oligonucleotide in cells.
基金Supported by the Science and Research Foundation of Bureau of Health, Hunan Province, China, No. Y02-083
文摘AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.
基金supported by the grant from the National Natural Science Foundation of China (No. 30400018)
文摘Objective To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray. Methods Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray Results Of the 102 rifampin-resistant strains 98 （96.1%） had mutations in the rpoB genes. Conclusion Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.
基金supported by the China Mega-Project for Infectious Disease(2016ZX10004-101,2016ZX10004-215)Beijing Municipal Science&Technology Commission Project(D151100002115003)Guangzhou Municipal Science&Technology Commission Project(2015B2150820)
文摘Objective To provide a feasible and cost-effective next-generation sequencing （NGS） method for accurate identification of viral pathogens in clinical specimens, because enormous limitations impede the clinical use of common NGS, such as high cost, complicated procedures, tremendous data analysis, and high background noise in clinical samples. Methods Viruses from cell culture materials or clinical specimens were identified following an improved NGS procedure： reduction of background noise by sample preprocessing, viral enrichment by barcoded oligonucleotide （random hexamer or non-ribosomal hexanucleotide） primer-based amplification, fragmentation-free library construction and sequencing of one-tube mixtures, as well as rapid data analysis using an in-house pipeline. Results NGS data demonstrated that both barcoded primer sets were useful to simultaneously capture multiple viral pathogens in cell culture materials or clinical specimens and verified that hexanucleotide primers captured as many viral sequences as hexamers did. Moreover, direct testing of clinical specimens using this improved hexanucleotide primer-based NGS approach provided further detailed genotypes of enteroviruses causing hand, foot, and mouth disease （HFMD） and identified other potential viruses or differentiated misdiagnosis events. Conclusion The improved barcoded oligonucleotide primer-based NGS approach is simplified, time saving, cost effective, and appropriate for direct identification of viral pathogens in clinical practice.
基金the National Key R&D Program of China(2019YFA0110600)National Natural Science Foundation of China(81970916,81671031).
文摘Biofilm formation is responsible for numerous chronic infections and represents a serious health challenge.Bacteria and the extracellular polysaccharides(EPS)cause biofilms to become adherent,toxic,resistant to antibiotics,and ultimately difficult to remove.Inhibition of EPS synthesis can prevent the formation of bacterial biofilms,reduce their robustness,and promote removal.Here,we have developed a framework nucleic acid delivery system with a tetrahedral configuration.It can easily access bacterial cells and functions by delivering antisense oligonucleotides that target specific genes.We designed antisense oligonucleotide sequences with multiple targets based on conserved regions of the VicK protein-binding site.Once delivered to bacterial cells,they significantly decreased EPS synthesis and biofilm thickness.Compared to existing approaches,this system is highly efficacious because it simultaneously reduces the expression of all targeted genes(gtfBCD,gbpB,ftf).We demonstrate a novel nucleic acid-based nanomaterial with multi-targeted inhibition that has great potential for the treatment of chronic infections caused by biofilms.
文摘AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS： HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% （n = 33）, 83.2% （n = 333）, and 8.5% （n = 34）, respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION： The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.
基金supported by the National Key Research and Development Program of China(2016YFD0102001)the National Natural Science Foundation of China(31571653,31771782,31201204,31501305)+3 种基金International Cooperation and Exchange Programme of the National Natural Science Foundation of China(31661143005)Introducing the Technique to Exploring the Genetic Germplasm Based on the Chromosome Sorting and Sequencing(2015-Z41)the Special Fund of Jiangsu Province for the Transformation of Scientific and Technological Achievements(BA2017138)supported by European Regional Development Fund Project“Plants as a Tool for Sustainable Global Development”(CZ.02.1.01/0.0/0.0/16_019/0000827)。
文摘Haynaldia villosa is a wild relative of wheat and a valuable gene resource for wheat improvement.Owing to the limited number of probes available for fluorescence in situ hybridization(FISH),the resolution at which the karyotype of H.villosa can be characterized is poor,hampering accurate characterization of small segmental alien introgressions.We designed ten oligonucleotide probes using tandem repeats in DNA sequences derived from the short arm of H.villosa chromosome 6 V(6 VS).FISH with seven of them resulted in clear signals on H.villosa chromosomes.Using these,we constructed FISH karyotypes for H.villosa using oligo-6 VS-1 and oligo-6 VS-35 oligonucleotides and characterized the distribution of the two probes in five different H.villosa accessions.The new FISH probes can efficiently characterize H.villosa introgressions into wheat.
基金Supported by The fortüne-program of the University of Tuebingen, No. F1281305
文摘AIM: To investigate the molecular pathways involved in human cholangiocarcinogenesis by gene expression profiling. METHODS: Oligonucleotide arrays (Affymetrix U133A) were used to establish a specific gene expression profile of intrahepatic CCC in comparison to corresponding non- malignant liver tissue. To validate the expression values of the most overexpressed genes, RT-PCR experiments were performed. RESULTS: Five hundred and fifty-two statistically differentially expressed genes/ESTs (221 probes significantly up-regulated, 331 probes down-regulated; P < 0.05; fold change > 2; ≥ 70%) were identified. Using these data and two-dimensional cluster analysis,a specific gene expression profile was obtained allowing fast and reproducible differentiation of CCC, which was confirmed by supervised neuronal network modelling. The most consistently overexpressed gene (median fold change 33.5, significantly overexpressed in 100%) encoded osteopontin. Furthermore, an association of various genes with the histopathological grading could be demonstrated. CONCLUSION: A highly specific gene expression profile for intrahepatic CCC was identified, allowing for its fast and reproducible discrimination against non- malignant liver tissue and other liver masses. The most overexpressed gene in intrahepatic CCC was the gene encoding osteopontin. These data may lead to a better understanding of human cholangiocarcinogenesis.
基金Supported by the Postdoctoral Science Foundation of China(No.1999-10 State Postdoctoral Foundation Commission)
文摘AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.
文摘The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.
