RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequenc...RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.展开更多
Objective: To investigate the effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia after rat carotid artery balloon injury. Methods :AT2R gene was transferred into rat carotid arteries...Objective: To investigate the effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia after rat carotid artery balloon injury. Methods :AT2R gene was transferred into rat carotid arteries by recombinant adenovirus pAd-AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested on the 14th day after gene transfer. The efficiency of trans-gene delivery was measured by the expression of adenovirus-encoding green fluorescent protein (GFP) under fluorescent microscope. The expression of AT2R and PCNA (proliferating cell nuclear antigen) was e-valuated by RT-PCR, immunocytochemistry, immunofluorescence staining, confocal microscopy, respectively. The ratio of intimal to medial area (I/M) was quantified with images and determined by an image analysis system. Results: GFP-positive area in adventitia, media and the forming neointima was about 40%. Adenoviral delivery of rat AT2R gene up-regulated AT2R expression in balloon-injured rat carotid and reduced PCNA expression and I/M significantly in neointima(P<0. 01). Double immunofluorescence labeling of AT2R and PCNA also showed that AT2R gene transfer inhibited VSMCs proliferation in neointima. Conclusion: AT2R gene transfer may be a novel promising therapy to limit neointimal hyperplasia.展开更多
基金supported by the Grant No.2003AA208215 from the National High Technology Programs of Chinathe Grant No.30270311 from the National Natural Science Foundation of China.
文摘RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80％ at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0～60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
文摘Objective: To investigate the effect of adenoviral vector-mediated AT2R gene transfection on neointimal hyperplasia after rat carotid artery balloon injury. Methods :AT2R gene was transferred into rat carotid arteries by recombinant adenovirus pAd-AT2R after the establishment of rat carotid balloon injury restenosis model. The arteries were harvested on the 14th day after gene transfer. The efficiency of trans-gene delivery was measured by the expression of adenovirus-encoding green fluorescent protein (GFP) under fluorescent microscope. The expression of AT2R and PCNA (proliferating cell nuclear antigen) was e-valuated by RT-PCR, immunocytochemistry, immunofluorescence staining, confocal microscopy, respectively. The ratio of intimal to medial area (I/M) was quantified with images and determined by an image analysis system. Results: GFP-positive area in adventitia, media and the forming neointima was about 40%. Adenoviral delivery of rat AT2R gene up-regulated AT2R expression in balloon-injured rat carotid and reduced PCNA expression and I/M significantly in neointima(P<0. 01). Double immunofluorescence labeling of AT2R and PCNA also showed that AT2R gene transfer inhibited VSMCs proliferation in neointima. Conclusion: AT2R gene transfer may be a novel promising therapy to limit neointimal hyperplasia.
