磷酸钙法转染瘤细胞效率的优化

The enhancement of the cancer cell transfection efficiency by an optimized calcium phosphate transfection method

为了探索磷酸钙法转染瘤细胞的最佳转染条件,以磷酸钙法将ANXA2特异性重组干扰质粒pU6H1-GFP-siANXA2导入人肝癌细胞SMMC-7721并优化了以下转染参数：转染前细胞汇合率、质粒剂量、质粒复合物形成时间、孵育时间、血清、抗生素有无及细胞传代次数等.以荧光显微镜观察GFP表达、半定量RT-PCR和western blotting评估转染效率.结果显示,转染前细胞50%~60%汇合率、8μg质粒用量（直径30 mm培养皿）、20~30 min沉淀形成时间及6 h孵育时间转染效率较高;细胞传代次数及抗生素有无对转染效率影响不显著,但血清在实验条件下是必不可少的.转染48 h后,GFP表达进入高峰期、ANXA2 mRNA和蛋白水平下调（P〈0.05）.

To enhance the cancer cell transfection efficiency by the optimized calcium phosphate method,the ANXA2 specific inhibition recombinant plasmid（pU6H1-GFP-siANXA2） was introduced into the human hepatocarcinoma cell,SMMC-7721.The following conditions were optimized： Cultured cell cover ratio before transfection,plasmid dose,DNA precipitation formation time,incubation time,cell growth status,and the absence/attendance of serum and antibiotics.The results indicated the optimal transfection conditions as the follows： 50%~60% cultured cell cover ratio before transfection,8 μg plasmid per 30 mm dish,20~30 min complex formation time,6 h incubation time.The absence/attendance of antibiotics showed no significant influence.Serum was indispensable for this method.ANXA2 mRNA and protein level detection results indicated that the target gene expression was significantly inhibited at 48 h post transfection（P0.05）.