pSiRNA-Shh逆转录病毒载体的构建及对恶性脑胶质瘤细胞作用的离体实验研究  

Construction of pSiRNA-Shh recombinant retroviral vector and its application in malignant glioma cells in vitro

作　　者：尹昌林[1] 吕胜青[1] 黄轶[2] 李光辉[3] 唐莉[4] 杨辉[1] 

机构地区：[1]第三军医大学新桥医院神经外科,重庆400037 [2]重庆医科大学生化与分子生物学教研室 [3]第三军医大学新桥医院肿瘤研究所 [4]第三军医大学西南医院乳腺疾病中心

出　　处：《中国实验诊断学》2007年第5期598-602,共5页Chinese Journal of Laboratory Diagnosis

基　　金：国家自然科学基金项目(30500527);重庆市自然科学基金资助项目(CSTC;2006BB5092)

摘　　要：目的建立逆转录病毒介导的人Shh基因RNA干扰体外表达体系并观察对恶性脑胶质瘤细胞的作用。方法将人Shh基因RNA干扰双链转录DNA片段重组到逆转录病毒质粒Psilencer 5.1-H1 Retro中,构建成携带人Shh基因RNA干扰逆转录病毒载体pSiRNA-Shh,经PT67细胞包装后,产生的重组逆转录病毒感染恶性脑胶质瘤细胞株U251和CHG-5细胞,用WST-8、RT-PCR和Western Blotting分别检测对转染细胞活性、人Shh mRNA和蛋白表达的影响。结果重组pSiRNA-Shh质粒经测序鉴定正确。重组逆转录病毒滴度可达210×104CFU/ml,感染U251和CHG-5恶性脑胶质瘤细胞株后3 d能明显抑制细胞生长,RT-PCR和Western Blotting检测人Shh mRNA和蛋白表达水平明显低于阴性对照组和未干扰组。结论携带人Shh基因RNA干扰双链转录DNA片段的逆转录病毒体现出明显的抑制恶性脑胶质瘤细胞生长作用,为下一步开展基因治疗恶性脑胶质瘤奠定了基础。Objective To construct a retroviral-mediated expression system containing double strands DNA for RNA interference on human sonic hedgehog （Shh） and study its inhibitory effect on U251 and CHG-5 human glioma cell lines in vitro.Methods A recombinant retroviral vector pSiRNA-Shh was generated by cloning a double strands DNA for RNA interference on human Shh into a retroviral vector Psilencer 5.1-H1 Retro.U251 and CHG-5 human glioma cell lines were infected with the viral supematant from the PT67 clones.After 3d, viabiliity, Shh mRNA and protein of transfected cells were examined by WST-8 assay, RT-PCR and Western Blotting.Results The pSiRNA-Shh recombinant retroviral vector had been constructed correctly. The liter assayed on NIH3T3 cells was up to 210 × 10^4 CFU/ml.3 d after transfecfion, viabiliity,Shh mRNA and protein of transfected cells decreased significantly. Conclusion The constructed pSiRNA-Shh retroviral vector shows effective inhibition of viabiliity in human malignant glioma cells,with potential utility in the gene therapy for human malignant glioma.

关 键 词：pSiRNA-Shh逆转录病毒载体 恶性脑胶质瘤 基因治疗 

分 类 号：R739.41[医药卫生—肿瘤]