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

doi:10.3969/j.issn.2095-5227.2013.04.025

携带hHCN4基因重组腺病毒载体体外转染大鼠BMSCs的实验研究

In vitro transfection of rat bone marrow mesenchymal stem cells with recombinant adenovirus vector containing human HCN4 gene: An experimental study

摘要

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。

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 and Western blot, respectively. Conclusion Recombinant adenovirus vector-transfected BMSC can express the hHCN4 gene.

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