肝癌特异性多靶点慢病毒的体外实验研究  

Antibody-directed lentiviral vector-mediated hepatocarcinoma cells targeting IGF1R-siRNA/P53 genes therapy

作　　者：牛坚[1] 赵何伟[2] 王文[3] 于彬[4] 刘斌[1] 王学浩[5] 张业伟[2] 

机构地区：[1]徐州医学院附属医院普外科,221004 [2]南京医科大学附属江苏省肿瘤医院普外科 [3]宁夏医科大学附属医院影像科 [4]上海交通大学肿瘤研究所癌基因及相关基因国家重点实验室 [5]南京医科大学附属第一医院普外科

出　　处：《中华肝胆外科杂志》2012年第5期372-376,共5页Chinese Journal of Hepatobiliary Surgery

基　　金：国家自然科学基金专项基金

摘　　要：目的研究anti—AFPscFv（抗AFP单链抗体）介导的慢病毒（1entivirus）载体对表达AFP肝癌细胞特异性基因转移以及双靶点基因系统对肝癌细胞生长的抑制作用。方法用脂质体Lipofectamine 2000将目的基因转移载体、包装结构及包膜结构质粒共转染包装细胞293T，大量收集病毒上清，过滤、浓缩。用携带AFP—WtP53-pPRIME—miR30-shRNA-IGF1R融合基因的慢病毒体外转染培养的肝癌细胞HEP3B。荧光显微镜下观察感染效果。用PCR、Western blotting鉴定WtP53、miR30-shRNA—IGF1R在细胞中的整合转录结果。CCK8观察重组慢病毒对肝癌细胞生长的影响，TUNEL检测细胞凋亡。结果成功构建anti—AFPscFv介导的慢病毒；滴度为4．58×10^9 PFU／ml；PCR、Western blotting均显示阳性条带，证明anti—AFP scFv介导的慢病毒在靶细胞中整合且转录表达。重组慢病毒对肝癌细胞的生长有明显的抑制作用且有促进细胞凋亡的作用。结论anti—AFPscFv介导的慢病毒可将双靶点基因系统高效靶向性转染、杀伤肝癌细胞。Objective To investigate the targeting infection of single chain antibody against AFP （scFv anti-AFP） directed lentivirus and the inhibitory effects of a dual-growth inhibition system on hepatocarcinoma cells. Methods Plasmids WtP53-pPRIME-miR30-shRNA-IGFIR, pMD2G-Anti-AFP, and psPAX2 have previously been constructed to cotransfect to the packaging cell line 293T using Lipofectamine2000. The infection results were observed through fluorescence microscopy. PCR and Western blotting were used to demonstrate the successful transduction and transcription of the WtP53-pPRIME-miR30-shRNA-IGFIR gene. The effects of reconstructed lentivirus infected liver cell growth were assessed by the cell growth curve of CCK8 cells. Apoptosis was evaluated by the TUNEL assay. Results Recombined lentivirus was successfully constructed with the functional PFU titers of recombined lentivirus at 4.58 × 10^9 PFU/ml. This positive result was confirmed by PCR and Western blotting. Conclusions The targeted therapy mediated by anti-AFP scFv could significantly inhibit the proliferation of HEP3B cells and promote the apoptosis.

关 键 词：肝癌细胞 人胰岛素样生长因子1类受体 野生型P53 慢病毒 

分 类 号：R735[医药卫生—肿瘤]