肝细胞特异性杀伤载体的构建和应用  

Construction and application of a recombinant plasmid inducing cell death in hepatocytes after transfection

作　　者：高春辰[1] 赵俊龙[1] 韩俊[1] 李东东[1] 赵高炀 韩骅[1] 秦鸿雁[1] 

机构地区：[1]第四军医大学基础部医学遗传与发育生物学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2015年第4期491-495,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金重大研究计划培育项目(91029731);国家自然科学基金重点项目(81030010)

摘　　要：目的间充质干细胞(MSC)有重要的免疫调节和组织再生作用,但其治疗肝损伤的机制尚不完全清楚,既可能通过旁分泌因子调节炎症,也可直接转分化为肝细胞。为明确这两种机制,拟构建巨细胞病毒(CMV)启动子驱动的膜定位绿色荧光蛋白(GFP)与白蛋白启动子调控的PE40毒素的共表达质粒,以期杀伤转染MSC中向肝细胞转分化的细胞。方法用PCR获得GFP以及DLL1(Delta-like 1)跨膜区基因片段,融合成膜定位GFP基因并插入pFlag-CMV-1载体。将白蛋白基因启动子和PE40基因片段插入已构建好的膜定位GFP序列下游,构建真核表达载体,荧光显微镜检测GFP的表达。应用可表达白蛋白的人正常肝脏细胞检测该载体对肝细胞的杀伤。结果成功构建肝细胞特异性杀伤载体,该载体转染后对正常肝脏细胞具有杀伤作用,提示该载体可用于探索MSC治疗肝损伤的机制。结论成功构建能特异性杀伤肝细胞的载体。Objective The mechanisms of mesenchymal stromal cells（MSCs）-mediated treatment of liver damage have been unclear.Two major mechanisms,which involve paracrine effects and / or direct trans-differentiation,have been proposed.To clarify which mechanism is more important,we planned to construct a recombinant plasmid expressing green fluorescent protein（GFP） driven by the cytomegalovirus（CMV） promoter and Pseudomonas aeruginosa exotoxin 40（PE40） driven by the albumin（alb） promoter,which can induce cell death as soon as MSCs differentiate into hepatocytes.Methods To construct the recombinant eukaryotic expression vector p Flag-CMV-GFP-TM-albp-PE40,GFP,transmembrane domain of DLL1,alb promoter and PE40 were obtained by PCR and were inserted into p Flag-CMV-1.The expression of GFP was observed under a fluorescence microscope and the killing effect on hepatocytes was analyzed by flow cytometry.Results The recombinant plasmid inducing cell death in hepatocytes was successfully constructed,suggesting that the plasmid could be employed to study the mechanism of MSCs-mediated treatment on liver damage.Conclusion This study might provide a promising tool for revealing the mechanism of MSCs-mediated treatment on liver diseases.

关 键 词：肝细胞 绿色荧光蛋白 PE40毒素 载体构建 

分 类 号：Q255[生物学—细胞生物学] R575.29[医药卫生—消化系统]