脂质体介导化学合成siRNA转染原代心肌细胞:筛选理想浓度

Liposomes-mediated chemosynthesis siRNA transfection to primary cardiomyocytes: Selection of an ideal concentration

背景:siRNA转染是完成RNA干扰的关键一步。目前用于心肌细胞的RNA干扰技术多以质粒为载体转染长链shRNA,其步骤复杂。脂质体转染化学合成短链siRNA是一种操作简便,低毒高效的转染方法,将其应用于心肌细胞转染,有利于RNA干扰技术的推广应用。目的:筛选脂质体介导化学合成siRNA转染原代心肌细胞的最佳浓度,以及简单高效的RNA干扰操作方法。方法:应用脂质体siPORTTMNeoFXTM转染试剂介导5,10,20,30nmol/L的CY3-NegativesiRNA转染心肌细胞,同时设立空白对照组,转染24h后分别应用荧光显微镜,流式细胞仪检测转染效率和细胞凋亡率,筛选最优浓度。根据优化浓度转染PHBsiRNA,48h后检测蛋白表达验证转染效果。结果与结论:随CY3-NegativesiRNA浓度的增加,在荧光显微镜下观察到激发出红色荧光的细胞比例随之增加,流式细胞仪检测出各组平均转染效率也依次增高(P<0.05),其中30nmol/L组转染效率最高(P<0.05),而各实验组与空白对照组之间的细胞凋亡率差异无显著性意义(P>0.05)。将心肌细胞转染30nmol/L的PHBsiRNA,48h后PHB蛋白表达平均下降74.11%(P<0.05)。实验结果表明,脂质体转染试剂siPORTTMNeoFXTM介导化学合成siRNA转染原代心肌细胞的理想浓度是30nmol/L。

BACKGROUND： siRNA transfection is a key step in RNA interference. The methods of cardiomyocytes transfection were most use of plasmids as vector to transfect long-chain shRNA. However, the processes were complex. It was a simple efficient and low-toxic method that liposomes-mediated chemosynthesis siRNA transfection. It was useful for expanding RNA interference application. OBJECTIVE： To choice the optimal concentration of liposomes-mediated chemosynthesis siRNA transfection, and to discover a simple efficient RNA interference application. METHODS： CY3-Negative siRNA was mediated by lipid-based agent siPORTTM NeoFXTM to transfect cardiomyocytes. A blank control group was set. After 24 hours, the transfection efficiency and apoptotic rate were evaluated by fluorescent microscope and flow cytometer to select an optimal concentration. Based the best concentration, siRNA PHB was transfected to cardiomyocytes. 48 hours later, the expression of PHB was tested. RESULTS AND CONCLUSION： With the increased concentration of CY3-Negative siRNA, the number of cells emitted red fluorescence grew under fluorescence microscope, and the transfection efficiency was also increased （P 0.05）. The best concentration was 30 nmol/L （P 0.05）. There was no significant difference in apoptotic rate between transfected groups and the control group （P 0.05）. The PHB expression of cardiomyocytes transfected siRNA PHB was dropped by 74.11% on average （P 0.05）. The results indicated that lipid based agent siPORTTM NeoFXTM was suitable to transfect chemosynthesis siRNA to cardiomyocytes,and the best transfection concentration of siRNA was 30 nmol/L.