电穿孔法介导siRNA高效转染小鼠原代软骨细胞  

Highly Efficient Transfection of Small Interfering RNA into Murine Primary Chondrocytes via Optimized Electroporation

作　　者：刘安军[1,2] 麻献华[3] 杨瑞[3] 高琳[3] 陈李斌佶[3] 章卫平[3] 谢志芳[3] 

机构地区：[1]中国人民解放军第二军医大学基础部,上海200433 [2]济南军区总医院麻醉科,250031 [3]上海中国人民解放军第二军医大学基础部,上海200433

出　　处：《医学研究杂志》2015年第7期23-25,共3页Journal of Medical Research

基　　金：国家自然科学基金资助项目(31171395;31270814)

摘　　要：目的建立小鼠原代软骨细胞高效电转si RNA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p CMV-EGFP表达质粒或si RNA,用台盼蓝检测细胞存活率;转染后48h分析转染效率和si RNA靶分子的m RNA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%;si RNA靶分子的m RNA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si RNA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.

关 键 词：原代软骨细胞 RNA干扰 SIRNA 电穿孔 

分 类 号：R34[医药卫生—基础医学]