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目的将外源基因p Survivin-HSV-TK导入质粒PBI-CMV2中,将外源基因p AFP-HSV-TK导入质粒PBI-CMV2中,构建两种特异性表达的质粒,比较两种启动子调停的优越性。方法由阳离子脂质体lipofectamine2000TM,运载两种质粒转染肝癌细胞Hu H-7,流式细胞术测定转染效率,半定量PCR检测TK基因在肝癌细胞中的表达水平。转染肝癌细胞Hu H-7,联合更昔洛韦杀伤细胞,流式细胞术检测细胞凋亡率,CCK-8检测加药(GCV)后细胞的增殖情况。结果阳离子脂质体lipofectamine2000TM介导Survivin-TK-p BI-CMV2质粒转染的Hu H-7细胞的转染率与介导AFP-TK-p BI-CMV2质粒转染的Hu H-7细胞的转染率分别为(14.43±0.88)%和(12.51±1.74)%(P=0.38),无统计学差异。半定量PCR检测HSVtk在转染的Hu H-7细胞中有表达,且转染Survivin-TK-p BI-CMV2质粒的细胞中TK基因的表达更高(P=0.001),在未转染细胞内未检测到表达。细胞凋亡实验表明GCV对转染的Hu H-7细胞有显著的杀伤作用,且转染Survivin-TK-p BI-CMV2质粒的Hu H-7细胞凋亡作用更明显(P=0.001)。结论肝细胞靶向基因运输载体PBI-CMV2介导的由AFP和Survivin启动子调控的HSVtk/GCV自杀基因系统对Hu H-7肝癌细胞均具有显著杀伤作用,且Survivin启动子调控作用相对明显,这为肝癌的靶向基因治疗策略的发展提供了新思路。
Objective To introduce the exogenous gene p Survivin-HSV-TK into plasmid PBI-CMV2 and introduce the foreign gene p AFP-HSV-TK into the plasmid PBI-CMV2 to construct two plasmids with specific expression. The superiority. Methods Lipofectamine 2000TM was used to transfect liver cancer cells Hu H-7 by two plasmids. The transfection efficiency was determined by flow cytometry. The expression of TK gene in hepatocellular carcinoma cells was detected by semi-quantitative PCR. Transfection of hepatocellular carcinoma cells Hu H-7, combined with ganciclovir killer cells, flow cytometry detection of apoptosis, CCK-8 detection of drug (GCV) after the proliferation of cells. Results The transfection efficiency of cationic liposome lipofectamine2000TM transfected Hu H-7 cells transfected with Survivin-TK-p BI-CMV2 plasmid was similar to that of Hu H-7 cells transfected with AFP-TK-p BI-CMV2 plasmid The transfection rates were (14.43 ± 0.88)% and (12.51 ± 1.74)%, respectively (P = 0.38), with no significant difference. The expression of HSVtk in transfected Hu H-7 cells was detected by semi-quantitative PCR, and the expression of TK gene was higher in cells transfected with Survivin-TK-p BI-CMV2 plasmid (P = 0.001). In untransfected cells No expression was detected. Apoptosis experiments showed that GCV had a significant killing effect on transfected Hu H-7 cells, and the apoptosis of Hu H-7 cells transfected with Survivin-TK-p BI-CMV2 plasmid was more obvious (P = 0.001). Conclusion The HSVtk / GCV suicide gene system mediated by the hepatocyte targeting gene delivery vector PBI-CMV2 mediated by AFP and Survivin promoters has a significant killing effect on Hu H-7 hepatoma cells, and the regulatory effect of Survivin promoter is relatively obvious. This provides a new idea for the development of targeted gene therapy strategies for liver cancer.