| 电穿孔法介导siRNA高效转染小鼠原代软骨细胞 |
| 投稿时间：2014-11-29 修订日期：2014-12-12 点此下载全文 |
| 引用本文：刘安军,麻献华,杨瑞,高琳,陈李斌佶,章卫平,谢志芳.电穿孔法介导siRNA高效转染小鼠原代软骨细胞[J].医学研究杂志,2015,44(7):23-25 |
| DOI：
10.11969/j.issn.1673-548X.2015.07.007 |
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| 基金项目:国家自然科学基金资助项目(31171395,31270814) |
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| 中文摘要: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。 |
| 中文关键词:原代软骨细胞 RNA干扰 siRNA 电穿孔 |
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| Highly Efficient Transfection of Small Interfering RNA into Murine Primary Chondrocytes via Optimized Electroporation |
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| Abstract: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 conditions. Cell viability was determined by trypan blue. The transfection efficiency and expression levels of siRNA-targeted gene were evaluated 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. |
| keywords:Primary chondrocyte RNA interference Small interference RNA Electroporation |
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