流式细胞仪快速优化大鼠原代培养肝细胞基因转染

Rapid Optimization of Gene Transfection in Rat Primary Cultured Hepatocyte by Using Flow Cytometry

【目的】 用流式细胞仪(FCM)快速检测外源性血管内皮生长因子基因(VEGF基因)在大鼠原代培养肝细胞转染表达,根据结果优化其转染表达条件?【方法】 以加强型黄色荧光素蛋白(EYFP)为标记,用FCM快速检测重组质粒pIRES-EYFP/VEGF121在大鼠原代培养肝细胞中转染表达,根据结果优化pIRES-EYFP/VEGF121转染表达条件?【结果】 pIRES-EYFP/VEGF121得以成功构建,并转染大鼠原代培养肝细胞;优化的转染表达条件:在细胞数密度0.1×106/mL,质粒孵育时间30 min,质粒与脂质体混合物孵育时间15 min,质粒与脂质体比例为1∶10,转染时间2 h,NAIR-1作转染培养液时,转染效率达17.5%? 【结论】以EYFP为标记, FCM可简便快捷地检测并优化外源性VEGF基因在大鼠原代培养肝细胞转染表达,可为研究VEGF基因修饰大鼠原代培养肝细胞移植和肝基因治疗打基础?

Objective To rapidly examine exogeous VEGF genes transfection and expression in rat primary culture hepatocyte by using flow cytometry, and optimize the condition of exogeoukxs VEGF genes transfection and expression according to the results. Methods Recombinant plasmid pIRES- EYFP/VEGF121 was transfected into rat primary cultured hepatocyte. The transfection and expres-sion of which was rapidly examined by using flow cytometer (FCM) with enhanced yellow fluoresent protein (EYFP) as a marker. The condition of pIRES-EYFP/VEGF121 transfection and expression were optimized on the based results. Results pIRES-EYFP/VEGF121 plasmid was constructed suc-cessfully and transfected into rat primary cultured hepatocyte. The transfection efficiency was 17.5% in condition as flow cell density 0.1×106 /mL, plasmid incubation time 30 min, plasmid and lipo-some incubation 15 min, plasmid and liposome ratio as 1 ∶10, transfection time 2 h, NAIR-1 as trans-fection culture medium. Conclusion Exogenous VEGF genes transfection and expression in rat prima-ry cultured hepatocyte can be examined and optimized conveniently and quickly by FCM with EYFP as a marker. Based on the results, the condition of transfection and expression can be optimized. The back-ground for rat primary cultured hepatocyte transplantation and liver-derived gene therapy would be estabil-shed.