PNP—TK融合自杀基因及PNP单自杀基因对肝癌细胞杀伤效率

目的分析PNP—TK融合自杀基因系统及PNP单自杀基因系统对肝癌细胞的杀伤作用及二者对肝癌细胞潜在的杀伤机制。方法构建融合基因表达我体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HepG2细胞克隆。RT—PCR和Western Blotting检测融合基因在HepG2细胞中的表达。台盼兰排斥法检测细胞的生长曲线，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。抗性细胞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所导致的旁观者效应明显强于只给予MeP-dR一种前药以及pcDNA3．0／PNP单基因系统所致的旁观者效应。结论具有双自杀基因功能的表达载体pcDNA3．0／PNP-TK对肝癌细胞的杀伤效果优于PNP单自杀基因系统。

Cytotoxic killing effects of a PNP-TK chimeric suicide gene system and a single PNP suicide gene system on hepatoma cells

Objective To analyze the killing effects of a PNP-TK chimeras gene system on the basis of PNP suicide gene and a single PNP suicide gene system on hepatoma cells and explore the potential mechanism of their killing effects. Methods A vector harboring a chimeric gene,pcDNA3.0/PNP-TK was constructed. The recombinant was identified by recombinant enzyme, PCR and sequencing. Then it was transfected into HepG2 cells by liposome-mediated method. The G-418 resistant cell clone with the stable transfection of pcDNA3.0/PNP-TK was developed. The expression of PNP-TK gene was detected by RT-PCR and Western blotting method. The cellular growth curves were determined by trypan blue exclusion. The sensitivity of cell clones to their respective prodrugs and the bystander effects they resulted in were also assayed by MTT method. Results The PNP-TK fusion gene was inserted into pcDNA3.0 correctly,and the stable expression of PNP-TK gene in HepG2 cells was positive. Two clones stably expressing PNP-TK and PNP gene,respectively, were both sensitive to their corresponding prodrugs. It was obvious that the bystander effects caused by pcDNA3.0/PNP-TK after synergetic treatment of its corresponding prodrugs were more effective than those of pcDNA3.0/PNP with the treatment of a single prodrug,MeP-dR. Conclusion The killing effects of the bi-functional suicide gene vector,pcDNA3.0/PNP-TK,on hepatoma cells surpass those of a single PNP suicide gene vector.