论文部分内容阅读
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。
Objective To analyze the killing effect of PNP-TK fusion suicide gene system and PNP single suicide gene system on hepatocarcinoma cells and the potential mechanism of their killing on hepatoma cells. Methods The fusion gene pcDNA3.0 / PNP-TK was constructed. The recombinant was identified by restriction enzyme digestion, PCR and sequencing. The clones of HepG2 cells stably transfected with pcDNA3.0 / PNP-TK were obtained by G418 screening. The expression of fusion gene in HepG2 cells was detected by RT-PCR and Western Blotting. Trypan blue exclusion method was used to detect the cell growth curve. MTT assay was used to detect the cell sensitivity to the corresponding prodrugs and the bystander effect caused by one or two prodrugs respectively. Results The fusion gene PNP-TK was correctly inserted into pcDNA3.0 vector and pcDNA3.0 / PNP-TK was expressed in HepG2 hepatoma cell line. Resistant cell clones are sensitive to the corresponding prodrugs. The bystander effect of pcDNA3.0 / PNP-TK was significantly stronger than the bystander effect of a prodrug given only with MeP-dR and a single gene system of pcDNA3.0 / PNP under the combined action of the two prodrugs . Conclusion The double suicide gene expression vector pcDNA3.0 / PNP-TK is superior to PNP single suicide gene system in killing hepatoma cells.