【摘要】 背景与目的:胃癌是全人类常见恶性肿瘤之一。在我国,胃癌居各种癌症死亡之首,其总体五年生存率仅15%~20%。在目前尚无有效一级预防措施的情况下,积极探讨有效防治胃癌的方法成为必然趋势。本研究利用RNA干扰稳定筛选-抑制技术,抑制人端粒酶逆转录酶(hTERT)基因表达,探讨靶向hTERT基因RNA i对胃癌细胞增殖的抑制效应。方法:设计靶向hTERT基因的小干扰RNA,构建重组表达质粒pGenesil-shRNA-hTERT并导入人胃癌细胞系SGC7901细胞株,经G418筛选,建立稳定表达siRNA-hTERT的细胞株。采用real tim e RT-PCR、MTT和PCR-TRAP法同时检测pGenesil-shRNA-hTERT稳定抑制组和未处理SGC7901细胞组hTERT基因表达、端粒酶活性及细胞增殖变化。结果:在稳定表达pGenesil-shRNA-hTERT的SGC7901细胞株中,RNA i效力持续、稳定存在,hTERT mRNA表达、端粒酶活性明显降低,瘤细胞增殖被抑制。结论:RNA干扰能持续、稳定地抑制靶基因hTERT mRNA表达及肿瘤细胞增殖,是潜在的肿瘤基因治疗新方法。更多还原

【Abstract】 Background and purpose:Stomach cancer is one of the most common malignancy of human being.In our country,it is one of leading cancer death,5-yr survival rate for the patients is just 15%-20%.At present,we do not have effective first line prevent measure for the disease,it becomes more and more important to pursue a effective method to prevent and treat gastric cancer.In our in vitro study,the inhibition of gastric-carcinoma cell SGC7901 proliferation by RNA interference could be observed.Methods:The stable screening-inhibition technique of RNAi was adopted to stably inhibit the expression of hTERT.Small hairpin interfering RNA(shRNA) targeting hTERT gene was designed,recombinant plasmid pGenesil-shRNA-hTERT was constructed,and the plasmid was transfected into gastric-carcinoma SGC7901 cells that clones were selected by G418 in order to establish gastric-carcinoma cell lines with stable expression of pGenesil-shRNA-hTERT.Real-time RT-PCR,MTT and PCR-TRAP were utilized to detect the alterations of hTERT mRNA expressions,telomerase activity and cell proliferation.Results:The effectiveness of RNAi could be observed in gastric-carcinoma SGC7901 cells with stable expression of pGenesil-shRNA-hTERT,in these cell lines,the expression of hTERT mRNA was obviously down-regulated.Meanwhile,the telomerase activity was significantly decreased.gastric-carcinoma SGC7901 cell proliferation was significantly inhibited in pGenesil-shRNA-hTERT group compared to negative control group.Conclusions:RNAi may continually and stably suppress hTERT mRNA expression and cell proliferation,which is a potential new approach for gene therapy of neoplasm.更多还原