目的:构建以增强型绿色荧光蛋白(EGFP)基因为标志基因、人脑源性神经营养因子(hBDNF)基因为目的基因的重组腺病毒Ad5-hBDNF-EGFP,体外转染大鼠骨髓间充质干细胞(rMSCs),探讨转染效率及转染基因的表达情况.方法:构建穿梭质粒pDC316- hBDNF-mCMV- EGFP；利用AdMax包装系统,穿梭质粒与骨架质粒共转染293细胞,同源重组产生复制缺陷型重组腺病毒Ad5-hBDNF- EGFP,经反复感染293细胞扩增病毒.SD大鼠骨髓,体外分离、培养rMSCs,流式细胞仪检测rMSCs表面标记物；不同感染复数( MOI)的重组腺病毒转染第3代rMSCs,检测转染率及hBDNF蛋白的表达变化.结果:PCR、酶切鉴定及测序证实穿梭质粒和重组腺病毒载体成功构建.rMSCs流式细胞仪检测CD34、CD45阴性表达,CD9、CD90阳性表达.腺病毒体外转染rMSCs后荧光显微镜下可见绿色荧光信号表达,免疫印迹法检测出hBDNF蛋白的表达；绿色荧光表达强度、转染率及hBDNF蛋白的表达水平均随着腺病毒MOI的增加而增加,最佳MOI为100.结论:体外成功构建Ad5-hBD-NF-EGFP,并能有效转染rMSCs,成功获取基因工程干细胞,hBDNF及EGFP基因均可在该细胞中得到有效表达.%Objective: To construct the recombinant adenovirus vector carrying human brain-derived neurotrophic factor (Hbdnf) marked enhanced green fluorescent protein (EGFP) and then, to discuss the efficiency on transfection and the expression of Hbdnf and EGFP gene after Ad5-Hbdnf-EGFP transfecting rat mesenchymal stem cells (rMSCs) in vitro. Methods: The Hbdnf and EGFP genes from Pdc316-BDNF and Pdc316-Mcmv-EGFP were inserted into shuttle plasmid to construct Pdc316-Hbdnf-Mcmv-EGFP, which was subsequently cotransfected with adenovirus skeleton plasmid Pbh-Glox_El, 3Cre into 293 cells using AdMax system, to produce Ad5-Hbdnf-EGFP. And then, this recombinant adenovirus vector was used to transfect the third generation of rMSCs in vitro. The fluorescence expression was observed and the rate of transfection was analyzed. And, the level of Hbdnf expression was detected by using Western blot. Results : Both of recombinant shuttle plasmid and recombinant adenovirus vector were confirmed by PCR amplification, restriction enzyme analysis and sequencing. The cultured rMSCs were exhibited as CD29+ CD90+ CD34-CD45-, and produced Hbdna after the recombinant adenovirus vector Ad5-Hbdnf-EGFP been transfected. And, the Hbdnf expression was increased following the concentration of multiple of infection (MOD. The best MOI was 100. Conclusion: The rMSCs can be successfully infected with recombinant adenovirus vector Ad5-Hbdnf-EGFP, and therefore, produce Hbdnf.
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