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目的 探讨利用同一逆转录病毒载体将单纯疱疹病毒胸苷激酶 (HSV TK)自杀基因与细胞因子GM CSF(粒细胞巨噬细胞集落刺激因子 )基因同时转入卵巢癌细胞后目的基因的表达状况 ,为卵巢癌的免疫基因治疗提供实验基础。方法 利用IRES(内部核糖体进入位点 )序列连接HSV TK与GM CSF基因 ,并将其置于逆转录病毒载体的LTR控制之下 ;通过乒乓转染法获得产病毒包装细胞PA317/TK GM ,并利用收集的病毒上清转染人卵巢癌细胞系SKOV3,经筛选获得阳性克隆SKOV3/TK GM ;经PCR及RT PCR检测目的基因在细胞基因组中的整合及转录后 ,利用MTT法测定GCV对SKOV3/TK GM细胞的杀伤活性并对TK GCV系统中的旁观者效应进行观察 ,同时对转基因细胞分泌的GM CSF进行ELISA分析。结果 构建的pLGM I TK双顺反子逆转录病毒载体可通过乒乓转染法导入包装细胞PA317中 ,且所分泌病毒滴度达 8 6× 10 5cfu/ml;逆转录病毒感染SKOV3细胞后目的基因能够整合到基因组中并正常启动转录 ;SKOV3/TK GM细胞可被GCV有效杀伤 ,IC50 值为 0 7μg/ml,并存在旁观者效应 ;GM CSF有蛋白水平的表达 ,ELISA法测定其分泌量为 6 0 4ng·ml 1·10 6 细胞 1·2天 1。结论 IRES序列可用于逆转录病毒载体介导的HSV TK和GM CSF双基因对卵巢癌细胞的共转染 ,在自杀基
Objective To investigate the expression of the target gene of HSV TK suicide gene and cytokine GM CSF (granulocyte-macrophage colony-stimulating factor) gene simultaneously in ovarian cancer cells using the same retroviral vector, Provide experimental basis for the immunotherapy of ovarian cancer. METHODS: The HSV TK and GM CSF genes were ligated with the IRES (internal ribosome entry site) and placed under the control of LTR retroviral vector. PA317 / TK GM was obtained by the ping-pong transfection method. And the transfected human ovarian cancer cell line SKOV3 was harvested with the collected virus supernatant to obtain the positive clone SKOV3 / TK GM. After the integration and transcription of the target gene in the cell genome were detected by PCR and RT PCR, the GCV pair SKOV3 / TK GM cells and observed the bystander effect in the TK GCV system, while ELISA analysis of GM CSF secreted by the transgenic cells was performed. Results The constructed pLGM I TK bicistronic retroviral vector was transfected into the packaging cell PA317 by the ping-pong transfection method and the secreted virus titer was 86 × 10 5 cfu / ml. After the retrovirus infected SKOV3 cells, the target gene Can integrate into the genome and start transcription normally; SKOV3 / TK GM cells can be effectively killed by GCV, the IC50 value is 0 7μg / ml, and there is a bystander effect; GM CSF protein expression, ELISA assay secretion 6 0 4ng · ml 1 · 10 6 cells 1 · 2 days 1. Conclusion IRES sequence can be used for retroviral vector-mediated HSV TK and GM CSF double gene co-transfection of ovarian cancer cells in suicide