CAJ | 학술논문

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pcDNA3.0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RT-PCR和Western Blotting法检测PNP-CD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/PNP-CD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PNP-CD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.
목적 분석PNP-CD융합자살기인계통대간암세포HepG2적살상작용병탐토기가능적궤제.방법 이용중조PCR정점유변법제비융합기인PNP-CD.장PNP-CD삽입진핵표체재체pcDNA3.0중,구건융합자살기인표체재체pcDNA3.0/PNP-CD.경매절、PCR급측서감정중조체,G418사선획득은정전염pcDNA3.0/PNP-CD적항성세포극륭.용RT-PCR화Western Blotting법검측PNP-CD기인재HepG2세포중적표체.용태반람배척법검측세포생장곡선,용MTT법검측세포세포극륭대상응전약민감성급소도치적방관자효응.결과 융합기인편단PNP-CD정학삽입료pcDNA3.0중,pcDNA3.0/PNP-CD재HepG2세포중실현료표체.세포항성극륭대특정적전약고도민감.재량충전약연합작용하,pcDNA3.0/PNP-CD소치방관자효응명현강우지급여MeP-dR일충전약소치방관자효응.결론 구유쌍자살기인공능적PNP-CD융합기인계통시간암기인치료중일충고효치료재체,대간암세포유착량호적살상작용.
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 subcloned 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 liposome-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.