携带hHCN4基因重组腺病毒载体体外转染大鼠BMSCs的实验研究.pdfVIP

382 解放军医学院学报 Acad J Chinese PLA Med Sch Apr 2013，34（4） 携带hHCN4 基因重组腺病毒载体体外转染大鼠BMSCs 的实验研究 佟玉娜，王高频 辽宁医学院附属第一医院 心内科，辽宁锦州　121000 摘要 ：目的 构建重组腺病毒载体 Ad-hHCN4-IRES/eGFP 在体外转染大鼠骨髓间充质干细胞 (bone marrow mesenchymal stem cells，BMSCs)，检测目的基因在 BMSCs 中 mRNA 和蛋白水平是否有表达。方法 采用贴壁培养法分离纯化大鼠 BMSCs ； 将携带 hHCN4 基因的重组腺病毒转染大鼠 BMSCs，用荧光显微镜观察转染后 BMSCs 绿色荧光蛋白的表达，用 RT-PCR 和 Western Blot 法检测转染后 hHCN4 基因 mRNA 和蛋白水平的表达情况。结果 BMSCs 成功贴壁分离传代。转染目的基因 24 h 后荧光显微镜下即观察到绿色荧光蛋白的表达 ；RT-PCR 法检测到有 hHCN4 mRNA 的表达，Western blot 法检测到有其蛋 白水平的表达。结论 重组腺病毒载体转染的 BMSCs 能够表达 hHCN4 基因。 关键词 ：hHCN4 基因 ；重组腺病毒 ；骨髓间充质干细胞 ；生物起搏 DOI ： 中图分类号 ：R 34　文献标志码 ：A　文章编号 ：2095-5227(2013)04-0382-04　 10.3969/j.issn.2095-5227.2013.04.025 网络出版时间：2013-01-21 15:38　　　 网络出版地址 ：/kcms/detail/11.3275.R1538.004.html In vitro transfection of rat bone marrow mesenchymal stem cells with recombinant adenovirus vector containing human HCN4 gene: An experimental study TONG Yu-na, WANG Gao-pin Department of Cardiology, First Affiliated Hospital of Liaoning Medical College, Jinzhou 121000, Liaoning Province, China Corresponding author: WANG Gao-pin. Email: wgpwzy @ Abstract: Objective To transfect the rat bone marrow mesenchymal stem cells (BMSCs) with recombinant adenovirus vector Ad-hHCN4-IRES/eGFP and detect the expression of target gene in hHCN4 mRNA and protein of BMSCs. Methods Rat BMSCs were isolated and purified by adherent culture and transfected with recombinant adenovirus vector containing human HCN4 gene. Expressions of green fluorescent protein and transfected hHCN4 mRNA and protein in transfected rat BMSCs were detected by fluorescence microscopy, RT-PCR and Western blot, respectively. Results The BMSCs were successfully isolated after adherent culture. Expressions of green fluorescent protein and transfected hHCN4 mRNA and protein in transfected rat BMSCs could be detected by fluorescence microscopy, PCR