AF0.3启动子调控的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异杀伤作用  

Killing Effect of PNP/MeP-dR Suicide Gene System Driven by an AFP Promoter AF0.3 on AFP-positive Hepatoma Cells

作　　者：蔡晓坤[1] 周俊立[2] 周鹤俊[1] 张玲[2] 吴健鸿[3] 林菊生[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院消化内科肝病研究所,湖北武汉430030 [2]华中科技大学同济医学院微生物教研室,湖北武汉430030 [3]华中科技大学同济医学院基础部,湖北武汉430030

出　　处：《癌症》2006年第11期1334-1339,共6页Chinese Journal of Cancer

基　　金：国家自然科学基金重点项目(No.30330680)~~

摘　　要：背景与目的:甲胎蛋白(α-fetoprotein,AFP)启动子调控下的目的基因能在AFP阳性肝癌组织中特异性表达;嘌呤核苷磷酸化酶(Escherichiacolipurinenucleosidephosphorylase,PNP)/6-甲基嘌呤-2′-脱氧核糖核苷(6-methylpurine-2-deoxyriboside,MeP-dR)自杀基因系统具有强杀瘤效应。本研究旨在探讨AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性肝癌细胞的特异性杀伤作用。方法:构建甲胎蛋白启动子AF0.3调控下PNP基因表达载体pAF0.3/PNP,导入AFP阳性肝癌细胞HepG2和阴性的肝癌细胞株SMMC7721,利用G418筛选获得稳定转染PNP基因的HepG2/0.3-PNP及SMMC7721/0.3-PNP细胞。RT-PCR检测二者在细胞中的表达。台盼蓝拒染法检测细胞增殖效应,MTT法和流式细胞仪检测两株细胞对MeP-dR的敏感性及旁观者效应,高效液相色谱法(highperformanceliquidchromatography,HPLC)检测PNP基因产物活性。结果:不论有氧还是缺氧条件,HepG2/0.3-PNP对MeP-dR均较为敏感,SMMC7721/0.3-PNP则对MeP-dR完全不敏感。在任何一种条件下,HepG2/AF0.3-PNP在混合细胞中比例达25%后,就可致明显旁观者效应;而在同样条件下,SMMC7721/0.3-PNP不导致明显的旁观者效应。HPLC结果显示,pAF0.3/PNP在HepG2细胞中可以将MeP-dR转化为6-MP,但其在SMMC7721中则不具有转化活性。结论:AF0.3启动子调控下的PNP/MeP-dR系统对AFP阳性HepG2细胞有较好的杀伤作用。BACKGROUND ＆ OBJECTIVE： α-fetoprotein （AFP） promoter- driven target gene could be specifically expressed in AFP-positive hepatoma, Escherichia coil purine nucleoside phosphorylase/6-methylpurine-2- deoxyriboside （PNP/MeP-dR） suicide gene system has powerful killing effects on tumor cells. This study was to investigate the specific killing effect of PNP/MeP-dR suicide gene system driven by an AFP promoter, AF0.3, on AFP-positive hepatoma cells. METHODS. Inserting PNP gene into pAF0.3, a eukaryotic expression vector containing PNP gene, pAF0.3/PNP, was constructed. Then it was transfected into AFP-positive HepG2 and AFP- negative SMMC7721 hepatoma cell lines, respectively. Two cell lines HepG2/AF0.3-PNP and SMMC7721/AF0.3-PNP, stably transfected with PNP gene, were obtained with G418 selection. The expression of PNP gene was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The proliferation of HepG2/AF0.3-PNP and SMMC7721/AF0.3-PNP cells was determined with trypan blue exclusion. The sensitivity of the cells to MeP-dR and the bystander effects were assessed with MTT assay and flow cytometry （FCM）, The enzymatic activities of PNP gene products were determined with high performance liquid chromatography （HPLC）. RESULTS： Whether hypoxia or normoxia, HepG2/AF0.3-PNP cells were sensitive to MeP-dR, whereas SMMC7721/AF0.3-PNP cells were not, Under both conditions, obvious cytotoxic effects on HepG2 cells were observed when the proportion of HepG2/AF0.3-PNP cells in the mixture reached 25%. But there were no similar effects on SMMC7721 cells under the same conditions. HPLC assay showed that the product of PNP gene driven by AF0.3 promoter could convert a spot of MeP-dR into 6-MP in HepG2 cells, but not in SMMC7721 cells, CONCLUSION： PNP/MeP-dR system, driven by AF0.3 promoter, has powerful killing effect on AFP-positive hepatoma HepG2 cells.

关 键 词：AF0.3启动子 PNP/MeP-dR自杀基因系统 AFP阳性 肝肿瘤 HEPG2细胞 基因治疗 

分 类 号：R735.7[医药卫生—肿瘤]