Survivin启动子控制自杀基因HSV-TK真核表达载体对肝癌细胞杀伤活性的研究  

Anti-hepatoma effect of suicide gene therapy using HSV/TK combined with ganciclovir in hepatoma cells

作　　者：李勇芳 张英敏[1] 赵娜[1] 孟凡秀[1] 张琪[1] 高然朋[1] 张悦红[1] 于保锋[1] 郭睿[1] 王海龙[1] 解军[1] 徐钧[2] 

机构地区：[1]山西医科大学基础医学院生物化学与分子生物学教研室,太原030001 [2]山西医科大学附属山西省肿瘤医院普外科,太原030013

出　　处：《中华临床医师杂志（电子版）》2016年第2期229-233,共5页Chinese Journal of Clinicians(Electronic Edition)

基　　金：国家自然科学基金(30901821,81172136);山西省自然科学基金(2015011113)

摘　　要：目的研究自杀基因单纯疱疹病毒胸苷激酶(HSV-TK)对人肝癌细胞的特异性杀伤作用,推进该自杀基因治疗原发性肝癌的可行性,从而为肝癌靶向基因治疗奠定一定的基础。方法将pc DNA3.1-p Survivin-TK质粒转染人肝癌细胞Hep G2中,RT-PCR法检测自杀基因m RNA水平,Western blot法检测自杀基因蛋白量的表达;再次转染上述质粒到肝癌细胞Hep G2中,随着更昔洛韦(GCV)剂量的增加,来监测pc DNA3.1-p Survivin-TK重组载体对细胞的杀伤效果,CCK-8法检测GCV对肝癌细胞的毒性作用,流式细胞术检测细胞凋亡的情况。结果通过RT-PCR法、Western blot法表明了转染该重组质粒的Hep G2细胞可以成功表达TK基因;此外,在外源药物GCV作用下也实现了对肝癌细胞的特异杀伤,CCK-8法表明,随着药物浓度的增高质粒转染组细胞存活率逐渐下降;流式细胞术检测得知,细胞经GCV处理48 h后,转染pc DNA3.1-p Survivin-TK重组质粒的肝癌细胞组凋亡率约为(37.37±4.02)%,而未转染质粒组细胞凋亡率约为(1.00±0.62)%,两组间比较有统计学差异(P<0.01)。结论 HSV-TK/GCV自杀基因的毒性作用随着GCV浓度的增高而上升,并且HSV-TK/GCV系统对人肝癌细胞具有特异性靶向杀伤作用。Objective The present study was performed to investigate the selective cytotoxicity of pcDNA3.1-pSurvivin-TK suicide gene expression level in HepG2 hepatoma cells driven by the Survivin promoter.And to evaluate the cytotoxicity of pcDNA3.1-pSurvivin-TK suicide gene on hepatoma cells.Thus,it can lay a foundation for the liver cancer gene therapy.Methods The plasmid was transfected into the HepG2 hepatoma cells with liposomes,we detected the expression levels of pcDNA3.1-pSurvivin-TK suicide gene in liver cells and hepatoma cells by RT-PCR and Western blot method.In addition,transfecting pcDNA3.1-pSurvivin-TK plasmid vector into HepG2 hepatoma cells with lipoplexes vectors,with increasing of ganciclovir doses we explored the cytotoxicity of pcDNA3.1-pSurvivin-TK plasmid vector on hepatoma cells with CCK-8 reagents and flow cytometry.Results The pcDNA3.1-pSurvivin-TK plasmid vector expression level was higher in hepatoma cells than in the control group without transfecting the plasmid.Additionally,CCK-8 assay indicated,increasing the dose of ganciclovir into the cell with transfected the plasmid vector,the cytotoxicity of pcDNA3.1-pSurvivin-TK suicide gene gradually increased.With flow cytometry,the cytotoxicity of the transfected hepatoma cells with the vector and untransfected cells were（37.37＋4.02）%and（1.0＋0.62）%separately with the difference being significant among them（P〈0.01）.Conclusion The result verified the cytotoxicity of pcDNA3.1-pSurvivin-TK suicide gene on HepG2 hepatoma cells with different doses of GCV.What＇s more,our experiments laid a foundation for the liver-targeted gene therapy.

关 键 词：胸苷激酶 阳离子脂质体 肝癌细胞 SURVIVIN启动子 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]