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.
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)
