逆转录病毒介导的HSV-TK基因对人骨肉瘤细胞的杀伤作用

Study on killing effect of retrovirus-mediated HSV-TK gene therapy for osteosarcoma

目的 探讨骨肉瘤基因治疗的可行性。方法 采用逆转录病毒介导的HSV -TK基因转移和GCV治疗的方法进行研究。结果 成功构建含HyTK基因的重组逆转录病毒载体DORHyTK ;采用DOTAP将其转入PA3 17细胞中进行包装 ,筛选 ,并进行病毒滴度测定、收获病毒上清体外感染骨肉瘤细胞株OS73 2和SAOS -2。分别用PCR和RNA斑点杂交的方法证明假病毒中不含有复制活性病毒 ,HSV -TK基因已整合到宿主细胞基因组中 ,并获得表达。MTT比色法测定结果显示GCV对OS73 2TK和SAOS -2TK细胞均有明显的杀伤作用 ,且前者强于后者。旁观者效应在高细胞密度接种条件下强于低细胞密度 ,在SAOS -2细胞中的作用强于OS73 2。结论 HSV

Objective To study the gene therapy for osteosarcoma in vitro.Methods Retroviral vector and HSV-TK/GCV system were used.Results Recombinant retroviral vector (DORHyTK) containing Hygromycin phosphotransferase-thymidine Kinase fusion gene(HyTK) was constructed and introduced into PA317 retrovirus packaging cell by DOTAP. After selective culturing and measuring of retroviral titers, the osteosarcoma cells OS732 and SAOS-2 were infected by pseudotype retroviral supernatant, which did not contain replication-competent retrovirus(RCR). The HSVTK gene existence and expression in OS732TK and SAOS-2TK cells were examined by PCR and RNA dot blot analysis. HSV-TK/GCV gene therapy system showed its strong killing effects in osteosarcoma cells OS732 and SAOS-2 by means of MTT colorimetric assay, with the former stronger than the latter. However the “bystander effect” was weeker in OS732 than in SAOS-2 and was stronger in the high-density population group. Conclusion HSV-TK/GCV gene therapy system may provide a new therapeutic approach for treatment of osteosarcoma.