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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 To study the specific killing effect of the herpes simplex virus thymidine kinase (HSV-TK) on human hepatocellular carcinoma cells, and advance the feasibility of the suicide gene therapy for primary hepatocellular carcinoma, so as to lay a foundation for targeted hepatoma gene therapy. Methods The pc DNA3.1-p Survivin-TK plasmid was transfected into human hepatoma Hep G2 cells. The suicide gene m RNA level was detected by RT-PCR and the expression of suicide gene protein was detected by Western blot. The plasmid was transfected into hepatoma again. In cell Hep G2, the cytotoxic effect of pcDNA3.1-p Survivin-TK recombinant vector on the cells was monitored as the dose of ganciclovir (GCV) was increased. CCK-8 assay was used to detect the toxic effects of GCV on hepatoma cells. Flow cytometry was used to detect apoptosis. Results RT-PCR and Western blot showed that Hep G2 cells transfected with the recombinant plasmid could express TK gene successfully. In addition, specific killing of hepatoma cells was also achieved under the action of GCV. The CCK-8 method showed that With the increase of drug concentration, the cell survival rate of the plasmid transfection group gradually decreased. Flow cytometry showed that the cells were treated with GCV for 48 h and then transfected with the pcDNA3.1-p Survivin-TK recombinant plasmid. The apoptotic rate was (37.37±4.02)%, but the apoptotic rate in the untransfected plasmid group was (1.00±0.62)%. There was a statistically significant difference between the two groups (P<0.01). Conclusion The toxic effect of HSV-TK/GCV suicide gene increases with the increase of GCV concentration, and HSV-TK/GCV system has specific targeted killing effect on human hepatocellular carcinoma cells.