不同日龄大鼠颈上交感神经节神经元电转染方法

摘要

目的 建立一种高效电转染不同日龄大鼠颈上交感神经节(superior cervical sympathetic ganglion,SCG)神经元细胞的方法.提高转染后细胞的成活率、转染效率和干扰效率.方法 用传统的及经改良的神经元培养液分别培养电转染后的7日龄、14日龄和40日龄SD大鼠SCG细胞,24 h后用台盼蓝染色方法观察并计算细胞成活率；通过改变质粒DNA和siRNA与转染液比例,优化转染条件,于转染24h后在共聚焦显微镜下观察并计算转染效率或干扰效率.结果 改良培养液可使14日龄以上SD大鼠SCG细胞转染后成活率达到75％以上,明显高于传统培养液转染后的成活率(P＜0.01),且结果稳定,细胞状态良好,能够满足后续实验研究的要求；优化转染条件后,DNA 的转染率及siRNA的干扰率显著提高,当DNA与转染液比例为1∶100(μg∶μL)时,细胞转染率最高；当siRNA与转染液比例为1∶50(μg∶ μL)时干扰率最高.结论 通过改良神经元培养液及优化转染条件,成功提高了电转染后细胞的成活率、转染效率和干扰效率,利用电转染方法可成功转染不同日龄SD大鼠SCG神经元.%Objective To establish an efficient electrotransfection method of superior cervical sympathetic ganglion ( SCG) neurons from SD rats of different ages and improve the survival rate of transfected cells and the method to enhance the transfection efficiency and interference efficiency. Methods 7, 14 and 40 days old SD rat SCG neurons were cultured in common or modified culture medium, respectively, and observed at 24 hours after transfection, to compare cell survival rate determined on the basis of trypan blue dye exclusion. Optimum transfection conditions of SCG neurons were found by changing the proportion of plasmid DNA/siRNA and nucleofector solution, using confocal microscope to calculate the transfection efficiency or interference efficiency at 24 hours after transfection. SCG neurons obtained from 7-, 14- and 40-day old SD rats were cultured in common or modified culture medium, respectively. The cell survival rate was determined by trypan blue dye exclusion at 24 hours after transfection. Optimal conditions for transfection of SCG neurons were found by changing the proportion of plasmid DNA/siRNA and nucleofector solution. Confocal microscopy was used to calculate the transfection efficiency or interference efficiency at 24 hours after transfection. Results The survival rate of SCG neurons taken from SD rats aged over 14 days old cultured in the modified medium was higher than 75% at 24 hours after transfection, significantly higher than that of culture in the unmodified medium (P < 0. 01) , and the transfected cells were in good conditions and the results were stable. Those results indicated that the modified culture condition can meet the requirements of the following experiments. A higher transfection efficiency or siRNA interference efficiency was obtained after using the optimal transfect conditions: When the ratio of DNA and nucleofector solution was 1: 100 ( μg:μL) , the cells had a highest transfectionefficiency and the highest interference efficiency was obtained when the ratio of siRNA and nucleofector solution was 1: 50 μg:μL). Conclusions By using the modified culture medium and improved transfection conditions, SCG neurons from rats of different ages (7 d, 14 d and 40 d) can be successfully and efficiently electrotransfected.