VEGF_(165)在恒河猴骨髓间充质干细胞中的表达及鉴定

Expression and identification of VEGF_(165) in bone marrow mesenchymal stem cells of rhesus

目的探讨血管内皮生长因子(VEGF_(165))转染恒河猴间充质干细胞(MSCs)后的表达情况,以及转染后对MSCs功能的影响。方法通过Ficoll法分离并培养恒河猴骨髓MSCs,进行细胞表型鉴定。转染pc DNA-e GFP-VEGF_(165)至MSCs,荧光显微镜观察增强型绿色荧光蛋白(e GFP)表达情况,同时流式细胞技术对转染成功的细胞进行细胞表型及e GFP表达鉴定,RT-PCR法检测转染后VEGF_(165)表达情况。结果成功分离到纯度较高的MSCs,转染后的MSCs及子代细胞均有e GFP和VEGF_(165)的表达,且保留了MSCs的特性。结论 VEGF_(165)基因转染MSCs后可以稳定表达外源基因,且可以维持MSCs的特性。

Objective To detect the transferred vascular endothelial growth factor(VEGF)_(165) gene expression in rhesus autologous bone marrow mesenchymal stem cells(MSCs),and to explore the functional viability of transgenic MSCs.Methods MSCs from rhesus bone were isolated by Ficoll,which were used to detect the phenotype.After the culturing,the expression vector pcDNA- eGFP- VEGF_(165) was transfected into bone marrow MSCs.Fluorescence microscope and flow cytometry were used to detect the enhanced green fluorescent protein(eGFP) expression.At the same time,the phenotype in transfected MSCs was also indentified.The VEGF_(165) expression level was detected by RT- PCR.Results The highly purified MSCs were collected successfully.The transfected MSCs and daughter cells showed expressions of eGFP and VEGF_(165),which also remained the characteristics of MSCs.Conclusion The VEGF_(165) gene that is transfected into MSCs can maintain characteristics of MSCs,and stably express foreign genes.