目的:采用RNA干扰技术(siRNA)阻断5-LOX基因的表达,观察其抑制人胰腺癌细胞增殖及诱导细胞凋亡的作用.方法:构建靶向5-LOX的siRNA质粒表达载体,采用Lipofectamine-2000转染人胰腺癌细胞株SW1990,采用RT-PCR检测RNA干扰后5-LOX mRNA表达,...目的:采用RNA干扰技术(siRNA)阻断5-LOX基因的表达,观察其抑制人胰腺癌细胞增殖及诱导细胞凋亡的作用.方法:构建靶向5-LOX的siRNA质粒表达载体,采用Lipofectamine-2000转染人胰腺癌细胞株SW1990,采用RT-PCR检测RNA干扰后5-LOX mRNA表达,MTT法检测细胞的增殖抑制率,流式细胞仪检测细胞凋亡率.结果:靶向5-L O X序列特异性的三条s i R N A(组1、组2、组3)可以有效地抑制SW1990细胞5-LOX基因表达,其表达抑制率分别为19.6%±1.9%、55.4%±2.6%和55.2%±2.7%.转染靶向5-LOX siRNA的三个质粒表达载体可以显著抑制SW1990细胞的增殖,细胞接种24h后,三组增殖抑制率分别为5.37%±1.19%、11.63%±1.25%和13.67%±1.04%;48h后其增殖抑制率分别为16.13%±1.5%、26.63%±1.22%和25.47%±1.67%,各siRNA组增殖抑制率高于空白组和阴性对照组(均P<0.05),转染后24h和48h三组细胞凋亡率分别为5.56%±1.05%、11.45%±1.44%、12.13%±1.36%和7.37%±1.23%、18.75%±1.5%和22.02%±1.45%,均高于空白对照组和阴性对照组(P<0.05).结论:所构建的靶向5-LOX的siRNA质粒表达载体可以有效阻断SW1990细胞5-LOX基因表达,显著地抑制SW1990细胞增殖,并在一定程度上诱导其凋亡.展开更多
Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well a...Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P<0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P<0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P<0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genome(NS5B and NS3) also decreased HCV viral load by 85%(P< 0.05) as compared to siRNA inhibitory effects alone(70% and 64% respectively, P<0.05). Conclusions: siRNAs directed against NS3 gene significantly decreased m RNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.展开更多
文摘目的:采用RNA干扰技术(siRNA)阻断5-LOX基因的表达,观察其抑制人胰腺癌细胞增殖及诱导细胞凋亡的作用.方法:构建靶向5-LOX的siRNA质粒表达载体,采用Lipofectamine-2000转染人胰腺癌细胞株SW1990,采用RT-PCR检测RNA干扰后5-LOX mRNA表达,MTT法检测细胞的增殖抑制率,流式细胞仪检测细胞凋亡率.结果:靶向5-L O X序列特异性的三条s i R N A(组1、组2、组3)可以有效地抑制SW1990细胞5-LOX基因表达,其表达抑制率分别为19.6%±1.9%、55.4%±2.6%和55.2%±2.7%.转染靶向5-LOX siRNA的三个质粒表达载体可以显著抑制SW1990细胞的增殖,细胞接种24h后,三组增殖抑制率分别为5.37%±1.19%、11.63%±1.25%和13.67%±1.04%;48h后其增殖抑制率分别为16.13%±1.5%、26.63%±1.22%和25.47%±1.67%,各siRNA组增殖抑制率高于空白组和阴性对照组(均P<0.05),转染后24h和48h三组细胞凋亡率分别为5.56%±1.05%、11.45%±1.44%、12.13%±1.36%和7.37%±1.23%、18.75%±1.5%和22.02%±1.45%,均高于空白对照组和阴性对照组(P<0.05).结论:所构建的靶向5-LOX的siRNA质粒表达载体可以有效阻断SW1990细胞5-LOX基因表达,显著地抑制SW1990细胞增殖,并在一定程度上诱导其凋亡.
文摘Objective: To explore inhibitory effects of genome-specific, chemically synthesized siRNAs(small interference RNA) against NS3 gene of hepatitis C virus(HCV) 1a genotype in stable Huh-7(human hepatoma) cells as well as against viral replication in serum-inoculated Huh-7 cells. Methods: Stable Huh-7 cells persistently expressing NS3 gene were produced under antibiotic gentamycin(G418) selection. The cell clones resistant to 1 000 μg antibiotic concentration(G418) were picked as stable cell clones. The NS3 gene expression in stable cell clone was confirmed by RT-PCR and Western blotting. siRNA cell cytotoxicity was determined by MTT cell proliferation assay. Stable cell lines were transfected with sequence specific siRNAs and their inhibitory effects were determined by RT-PCR, real-time PCR and Western blotting. The viral replication inhibition by siRNAs in serum inoculated Huh-7 cells was determined by real-time PCR. Results: RT-PCR and Western blot analysis confirmed NS3 gene and protein expression in stable cell lines on day 10, 20 and 30 post transfection. MTT cell proliferation assay revealed that at most concentrated dose tested(50 nmol/L), siRNA had no cytotoxic effects on Huh-7 cells and cell proliferation remained unaffected. As demonstrated by the siRNA time-dependent inhibitory analysis, siRNA NS3-is44 showed maximum inhibition of NS3 gene in stable Huh-7 cell clones at 24(80%, P=0.013) and 48 h(75%, P=0.002) post transfection. The impact of siRNAs on virus replication in serum inoculated Huh-7 cells also demonstrated significant decrease in viral copy number, where siRNA NS3-is44 exhibited 70%(P<0.05) viral RNA reduction as compared to NS3-is33, which showed a 64%(P<0.05) decrease in viral copy number. siRNA synergism(NS3-is33 + NS3-is44) decreased viral load by 84%(P<0.05) as compared to individual inhibition by each siRNA(i.e., 64%–70%(P<0.05) in serum-inoculated cells. Synthetic siRNAs mixture(NS5Bis88 + NS3-is33) targeting different region of HCV genome(NS5B and NS3) also decreased HCV viral load by 85%(P< 0.05) as compared to siRNA inhibitory effects alone(70% and 64% respectively, P<0.05). Conclusions: siRNAs directed against NS3 gene significantly decreased m RNA and protein expression in stable cell clones. Viral replication was also vividly decreased in serum infected Huh-7 cells. Stable Huh-7 cells expressing NS3 gene is helpful to develop anti-hepatitis C drug screening assays. siRNA therapeutic potential along with other anti-HCV agents can be considered against hepatitis C.
