PNP-TK融合自杀基因系统对肝癌细胞杀伤作用的实验研究

Experimental researches on the cytotoxic effects of a PNP-TK fusion suicide gene on hepatoma cells

目的分析PNP-TK融合自杀基因系统对HepG2肝癌细胞的杀伤作用及其对肝癌细胞的杀伤机制。方法利用重组PCR定点诱变法制备PNP-TK融合基因,将其插入真核表达载体pcDNA3.0中,构建融合基因表达载体pcDNA3.0/PNP-TK,经酶切、PCR及测序鉴定重组体,G418筛选获得稳定转染了pcDNA3.0/PNP-TK的抗性细胞克隆。RT-PCR和Western Blotting检测PNP-TK基因在HepG2细胞中的表达。台盼兰排斥法测定细胞生长曲线,MTT法检测细胞对相应前药的敏感性及分别在一种和两种前药作用下所导致的旁观者效应。结果融合基因片段PNP-TK正确插入了pcDNA3.0载体中,pcDNA3.0/PNP-TK在肝癌细胞株HepG2中实现了表达。细胞抗性克隆对相应的前药十分敏感。在两种前药的联合作用下,pcDNA3.0/PNP-TK对肝癌细胞产生了良好的旁观者效应。结论具有双自杀基因功能的表达载体pcDNA3.0/PNP-TK对肝癌细胞HepG2有着良好的杀伤作用,有望将其应用于肝癌体内治疗。

Objective To investigate the cytotoxic effects of a PNP-TK chimeric gene on the basis of PNP suicide gene on HepG2 hepatoma ceils and explore the potential mechanism of their killing effects. Methods A fusion suicide gene,PNP-TK,was produced by site-directed mutagenesis technique. And then it was inserted into pcDNA3.0 to construct a vector harboring a chimeric gene, pcDNA3.0/PNP-TK. The recombinant was identified by recombinant enzyme, PCR and sequencing. Then it was transfected into HepG2 cells by liposome-mediated method. The cellular clone stably transfected with pcDNA3.0/PNP-TK was＇ established with G418 selection. The expression of PNP-TK gene was also detected by RT-PCR and Western blotting method. The growth curve of cellular clone was determined by trypan blue exclusion test. The sensitivity of the cell clone to its corresponding prodrugs and the caused bystander effects were assayed by MTT method. Results The PNP-TK fusion gene was inserted into pcDNA3.0 successfully, and its stable expression in HepG2 cells was confirmed. The cellular clone with the stable expression of PNP-TK gene was sensitive to its corresponding prodrugs, MeP-dR and GCV. It was demonstrated that the bystander effects derived from pcDNA3.0/PNP-TK after the synergetic treatment of MeP-dR and GCV were mighty. Conclusion The killing effects of the bi-functional suicide gene vector, pcDNA3.0/PNP-TK, on hepatoma cells are satisfactory. It was promising to apply pcDNA3.0/PNP-TK to gene therapy for hepatoma in vivo.