电穿孔法介导 siRNA 高效转染小鼠原代软骨细胞

摘要

目的：建立小鼠原代软骨细胞高效电转siRNA的方法。方法常规分离得到的小鼠原代软骨细胞继续用链丝菌蛋白酶消化3h，然后应用高效电转缓冲液和优化的电转参数转染pCMV－EGFP表达质粒或siRNA，用台盼蓝检测细胞存活率；转染后48h分析转染效率和siRNA靶分子的mRNA和蛋白表达水平。结果电穿孔后原代软骨细胞的存活率＞80％，质粒的转染效率达到37．3％±5．2％；siRNA靶分子的mRNA和蛋白表达水平分别下调了75％和66％。结论成功建立了通过电穿孔介导siRNA转染小鼠原代软骨细胞的方法，达到了很好的基因沉默效果且保持了较高的细胞存活率。%Objective To develop an efficient and reliable method to transfect murine primary chondrocytes with small interfering RNA ( siRNA) by electroporation.Methods Murine primary chondrocytes were isolated and treated with pronase for 3 hours.The cells were then electroporated with either pCMV-EGFP plasmid or siRNA using a high performance electroporation buffer and optimized condi-tions.Cell viability was determined by trypan blue.The transfection efficiency and expression levels of siRNA-targeted gene were evalua-ted 48 hours post-electroporation.Results By using proper electroporation condition, 37.3%±5.2%of cells were transfected by the plasmid with high cellular viability (>80%) .Transfection of siRNA using the same electroporation resulted in effective down-regulation of its targeted gene expression at both mRNA and protein levels (75% and 66% decrease, respectively).Conclusion Transfection of murine primary chondrocytes with siRNA in this optimized electroporation condition was successful and resulted in effective gene silencing and high cellular viability.