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Experimental study on transfection of adenovirus vector encoding green fluorescent protein into human bone marrow-derived mesenchymal stem cells
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- Author:
- No author available
- Journal Title:
- CHINESE JOURNAL OF NEUROMEDICINE
- Issue:
- 11
- DOI:
- 10.3760/cma.j.issn.1671-8925.2007.11.004
- Key Word:
- 骨髓间充质干细胞;绿色荧光蛋白;腺病毒载体;细胞分化
Abstract： 目的 研究绿色荧光蛋白基因(Ad-GFP)腺病毒载体在人骨髓间充质干细胞(BMSCs)转染和表达的量效关系及对细胞生物学特性的影响,探讨用该载体构建基因修饰BMSCs的可行性.方法 在体外分离培养人BMSCs,流式细胞仪检测细胞免疫表型,293细胞包装制备病毒,以不同滴度的Ad-GFP(1×103～1×1010PFU/mL)转染BMSCs,细胞计数法分析转染率,倒置显微镜观察细胞形态学改变,CCK8法检测细胞增殖活性,用血清撤离加入β-巯基乙醇诱导转染Ad-GFP的BMSCs向神经元样细胞定向分化. 结果 3～6代BMSCs表面标志CD34、CD45呈阴性而CD29、CD44呈阳性;当病毒滴度为1×107PFU/mL时转染率为55%,1×109及1×1010PFU/mL滴度时转染率均为85%,但1×1010PFU/mL滴度时出现细胞病理现象;7d荧光表达最强,28 d仍可见荧光表达.荧光显微镜下可见表达Ad-GFP的BMSCs有三种亚群结构;滴度≥1×106PFU/mL时,BMSCs增殖在早期受到抑制,且呈剂量依赖;转染Ad-GFP的BMSCs经β-巯基乙醇诱导可分化为神经元样细胞,神经元特异性烯醇化酶(NSE)阳性.结论 合适滴度的Ad-GFP可以高效转染BMSCs,对细胞的生物学特性影响较小,不影响诱导分化功能,BMSCs可以作为用Ad-GFP载体系统进行基因治疗研究的种子细胞.
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