重组腺相关病毒介导人血管内皮生长因子165基因在大鼠骨髓间充质干细胞中的表达

目的探讨以腺相关病毒(rAAV)为血管内皮细胞生长因子165基因(VEGIF165)载体,在体外转染大鼠骨髓间充质干细胞(MSCs),并研究其相关特性。方法 (1)全骨髓培养法提取培养MSCs,利用免疫组化法检测MSCs表面标志CD34、CD44;流式细胞分析法检测CD90。(2)rAAV-VEGF165转染MSCs,采用ELISA及PCR检测VEGF的表达,比较转染前后细胞的变化情况,观察VEGF165基因转染对MSCs的影响。结果 (1)成功培养出MSCs,CD44阳性表达,CD34阴性表达,CD90阳性表达。(2)在转染rAAV-VEGF165后转染组上清中VFGF165分泌水平明显高于未转染组(p<0.05),5 d时达到高峰,此后表达开始下降。琼脂糖凝胶电泳可见高亮度条带。表明rAAv-VEGF165成功转染进MSCs细胞中,绘制生长曲线,显示rAAV-VEGF165基因转染后对MSC生长无影响。结论rAAV-VEGF表达载体可有效感染MSCs,并在体外高效表达,为MSCs联合基因治疗提供了实验依据。

Expression of rAAV-VEGF in rat mesenchymal stem cells

Objective To evaluate the angiogenic effect of the bone marrow mesenchymal stem cells （ MSCs ） transfected by adeno-associated virus vector carrying human vascular endothelial growth factor （VEGF165） in vitro, and to detect the expression and bioactivity of VEGF in the MSCs. Methods （ 1 ） Bone marrow was cultured for extraction of MSCs. CD34 and CD44 on the cell surface were detected by immunohistochemical method, and CD90 by floweytomix. （2） The expression of VEGF in the transfected MSCs was detected by ELISA and PCR. Results （ 1 ） MSCs were successfully cultured with CD44 and CD90 positive, CD34 negative expression. （2） The VEGF level was significantly higher in transfected group than in non-transfected group （ P 〈0. 05 ）. Conclusions Adeno-associated virus as vectors carrying rAAV-VEGF gene can effectively transfect the bone marrow mesenchymal stem cells in vitro, which may provide an experimental evidence for gene therapy.