【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。更多还原

【Abstract】 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.更多还原