摘要
Objectives To explore whether HSV-TK (herpes simplex virus thymidine kinase) and GM-CSF (granulocyte-macrophage colony-stimulating factor) genes could be linked by internal ribosome entry site (IRES) in one retroviral vector and expressed by ovarian cancer cells following transfection, and to observe the characteristics of the transduced cells.Methods Retroviral vector pLGM-I-TK was constructed by linking the HSV-TK gene and GM-CSF gene with the IRES sequence. By using the “ping-pong' technique, pLGM-I-TK was transfected into the packaging cell line, PA317, to produce a PA317/TK-GM cell line. Using the resulting viral supernatant to infect the ovarian cancer cell line SKOV3, PCR and RT-PCR were used to explore the integration and transcription of HSV-TK and GM-CSF genes. The cytotoxicity of GCV (gancyclovir) on SKOV3/TK-GM was determined by MTT assay and the bystander effect of the HSV-TK/GCV system was also assessed. ELISA was used to measure the expression of GM-CSF by the transgene cells.Results The bicistronic retroviral vector constructed could be successfully transduced into PA317 and the titer of the retroviral vector was about 8.6×105?cfu/ml. PCR and RT-PCR demonstrated the successful integration and transcription of HSV-TK and GM-CSF genes transduced into the SKOV3 cell. SKOV3/TK-GM cells could be killed by GCV, and the IC50 was 0.7?μg/ml. The bystander effect was demonstrated. The expression level of GM-CSF in SKOV3/TK-GM was 60.4?ng*ml-1*106 cells-1*2 days-1.Conclusion The IRES sequence can be used to construct retroviral vectors to facilitate co-transfection of two genes. SKOV3/TK-GM cells can simultaneously express the HSV-TK and GM-CSF genes with biological activities which could be useful for enhancing the function of immune cells on the basis of suicide gene therapy.
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 。
