【摘要】 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。更多还原

【Abstract】 Objective To compare the superiority of two expression plasmid vetors constructed by introducing exogenous gene, p Survivin-HSVtk and p AFP-HSVtk to plasmid p BI-CMV2, respectively. Methods The two plasmids were constructed by Biovector NTCC, and then were transfected to Hu H-7 hepatoma cells with lipofectamine2000 TM. The transfection efficiency was detected by flow cytometry. The expression level of HSV-TK was detected by Sq RT-PCR, flow cytometry was to detect the cell apoptosis. And cytotoxicity of HSVtk suicide gene on hepatoma cells was detected by CCK-8 reagents. Results The two plasmid vectors AFP-TK-p BI-CMV2 and Survivin-TK-p BI-CMV2 had been constructed successfully. However, the transfection efficiency of Survivin-TK-p BI-CMV2 and AFP-TK-p BI-CMV2 were(14.43±0.88)% and(12.51±1.74)%(P=0.38, P>0.05), there had no significant difference. HSVtk gene expression could be detected in hepatoma cells. Sq RT-PCR showed HSVtk gene expression in Hu H-7 transfected by the plasmid, and the efficiency of Survivin-TK-p BI-CMV2 was higher than AFP-TK-p BICMV2(P=0.000 7). After treatment with GCV, the apoptosis rate of Survivin-positive was higher than AFP-positive(P=0.000 3). Also increasing the dose of ganciclovir, the cytotoxicity of HSVtk suicide gene gradually increased. Conclusions The HSVtk suicide gene was expressed successfully in hepatoma cell under the regulation of AFP promoter and Survivin promoter, and the expression of Survivin is higher. Our experiments might provide a theory of targeted gene therapy for liver cancer.更多还原