PNP—CD融合自杀基因系统对肝癌细胞杀伤作用的实验研究  

Killing effects of PNP-CD chimeric suicide gene vector on HCC cells

作　　者：彭友缘[1] 严武[1] 蔡晓坤[1] 尹震宇[1] 

机构地区：[1]厦门大学附属中山医院肝胆外科,361004

出　　处：《中华肝胆外科杂志》2010年第8期624-627,共4页Chinese Journal of Hepatobiliary Surgery

摘　　要：目的分析PNP—CD融合自杀基因系统对肝癌细胞HepG2的杀伤作用并探讨其可能的机制。方法利用重组PCR定点诱变法制备融合基因PNP—CD。将PNP—CD插入真核表达载体pcDNA3．O中，构建融合自杀基因表达载体pcDNA3．0／PNP—CD。经酶切、PCR及测序鉴定重组体，G418筛选获得稳定转染pcDNA3．0／PNP—CD的抗性细胞克隆。用RTPCR和Western Blotting法检测PNPCD基因在HepG2细胞中的表达。用台盼蓝排斥法检测细胞生长曲线，用MTT法检测细胞细胞克隆对相应前药敏感性及所导致的旁观者效应。结果融合基因片段PNP—CD正确插入了pcDNA3．0中，pcDNA3．0／PNPCD在HepG2细胞中实现了表达。细胞抗性克隆对特定的前药高度敏感。在两种前药联合作用下，pcDNA3．0／PNP—CD所致旁观者效应明显强于只给予MeP—dR一种前药所致旁观者效应。结论具有双自杀基因功能的PNPCD融合基因系统是肝癌基因治疗中一种高效治疗载体，对肝癌细胞有着良好的杀伤作用。Objective To investigate the cytotoxic effects and mechanism of PNP-CD chimeric gene vector originated from PNP/MeP dR system on HCC cells. Methods The fusion suicide gene PNP-CD obtained by site directed mutagenesis technique was subeloned into pcDNA3.0 to construct a eukaryotic expression vector containing a chimeric gene, pcDNA3.0/ PNP-CD. After being identified by recombinant enzyme, PCR and subsequent sequencing, it was transfected into HepG2 cells by lipo- some-mediation method. The G418-resistant cellular clone with stable transfection of pcDNA3.0/ PNP CD, HepG2/PNP-CD was established by selection. The expression of PNP-CD gene was also verified by RT PCR and Western blotting. The curve of cellular growth was assayed by Trypan blue exclusion. The cellular sensitivity of HepG2/PNP-CD to its specific prodrugs and its bystander effects were also assayed by MTT method. Results The chimeric gene, PNP CD, was inserted into pcDNA3.0 correctly, and the stable expression of pcDNA3.0/PNP CD in HepG2 cells was confirmed. This cellular clone was highly sensitive to its corresponding prodrugs. It was indicated that its bystander effects with the synergetic treatment of its specific prodrugs were substantially higher than those caused by the same vector with the administration of only a single prodrug, MeP-dR. Conclusion The bi-functional fusion suicide gene vector, pcDNA3.0/PNP-CD, yields powerful cytotoxic effects on HCC cells in the presence of the synergetic treatment of its specific prodrugs, which would be a high-performance therapeutic vector in gene therapy for liver cancer.

关 键 词：癌 肝细胞 PNP/MeP—dR系统 CD/5 FC系统 融合自杀基因 基因治疗 

分 类 号：R686[医药卫生—骨科学]