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目的建立对肝癌细胞具有靶向调控的高感染性和高分泌性产病毒细胞系。方法将重组反义甲胎蛋白增强子/单纯疱疹病毒胸苷激酶基因逆转录病毒载体(pL/TK/AFE/SN)转染至PA317包装细胞,经G418(400mg/L)筛选、病毒滴度测定。以病毒上清感染培养的Hep3B肝癌细胞和Hela宫颈癌细胞,并抽提细胞基因组DNA作Southernblot分析;观察GCV(100mg/L)对细胞生长的影响。结果转染pL/TK/AFE/SN的PA317细胞经G418筛选2周获阳性克隆;病毒滴度为5.4×104～3.8×105CFU/ml。Southern杂交证实前病毒已完整整合于PA317细胞,并稳定整合于Hep3B肝癌细胞和Hela宫颈癌细胞中。导入AFE/TK基因的Hep3B肝癌细胞经GCV作用后的存活率在2、4、6d时分别降低35%、87%、95%,生长明显受抑制(F=7.23,P<0.01)。结论携带AFE/TK基因的产病毒细胞系能够成功地将AFE/TK基因转移到感染细胞中。
Objective To establish a highly infectious and highly secreted virus-producing cell line with targeted regulation on hepatoma cells. Methods Recombinant antisense AFP / herpes simplex virus thymidine kinase gene retroviral vector (pL / TK / AFE / SN) was transfected into PA317 packaging cells and screened by G418 (400mg / L) Determination. The infected Hep3B cells and Hela cervical carcinoma cells were infected with virus supernatant. Southern blotting was used to analyze the effect of GCV (100 mg / L) on cell growth. Results PA317 cells transfected with pL / TK / AFE / SN were positive for 2 weeks after G418 selection. The titer of virus was 5.4 × 104 ~ 3.8 × 105CFU / ml. Southern hybridization confirmed that the provirus has been completely integrated into PA317 cells and stably integrated into Hep3B hepatocarcinoma cells and Hela cervical carcinoma cells. The survival rate of Hep3B hepatocarcinoma cells transfected with AFE / TK gene decreased by 35%, 87% and 95% respectively at 2, 4 and 6 days after treatment with GCV. The growth was obviously inhibited (F = 7.23, P <0.01). Conclusion The AFE / TK gene-producing virus-producing cell line can successfully transfer AFE / TK gene into infected cells.