BMP-2和EGFP重组腺病毒体外转染兔骨髓间充质干细胞的研究

Study of BMP2 and EGFP recombinant adenovirus transfection on rabbit bone marrow derived mesenchymal stem cells in vitro

目的体外培养兔骨髓间充质干细胞(BMSCs)并观察其生物学特性,通过腺病毒介导人骨形态发生蛋白-2(hBMP-2)和EGFP基因转染兔BMSCs后,观察BMP-2和EGFP的表达情况。方法通过密度梯度离心联合贴壁法,分离、培养兔BMSCs,流式细胞仪检测细胞周期和表面标记,体外诱导兔BMSCs向成骨细胞方向分化。转染后,免疫细胞化学染色及蛋白质印迹法(Western blot)检测外源基因的表达。结果兔BMSCs周期约有56.84%处于G1期;CD44表达阳性,CD45表达阴性;经成骨细胞诱导后,Ⅰ型胶原免疫组织化学染色阳性,茜素红染色阳性。转染后,Western blot及免疫细胞化学染色显示细胞内BMP-2有较强的表达。结论成功分离、培养兔BMSCs,并鉴定其具有成骨分化能力;构建的Ad-BMP-2/EGFP可高效转染兔BMSCs,并能稳定表达BMP-2目的基因。

Objective To culture the rabbit bone marrow derived mesenchymal stem cells（BMSCs） ,and to observe their biologi- cal characteristics in vitro and the expression of BMP2 and EGFP after Ad-EGFP-BMP2 infected on them. Methods To isolate and cultivate BMSCs by density gradient centrifugation and adherent culture in vitro;the cell cycle and the surface marks were detected by flow cytometer; rabbit BMSCs were differentiated into the direction of osteogenetic cells by in vitro induction. After transfeetion, the expression of exogenous gene in the cells was detected by the immunocytochemical staining and Western blot. Results About 56.84 ~ of rabbit BMSCs cell cycle was in the G1 phase;the D44 expression was positive and the CD45 expression was negative;af- ter induction by osteogenetic cells,the I type collagen immunohistoehemieal staining was positive and the alizarin red staining was positive. After transfection,the strong expression of BMP2 and EGFP in cells was showed by Western blot and the immunocyto- chemical staining. Conclusion rabbit BMSCs are successfully isolated,cultured and identified to have the ability of osteogenetic dif- ferentiation; the structured Ad-BMP-2/EGFP can efficiently transfected rabit BMSCs and stably express the target gene of BMP2.