BMP7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 BMP7 and EGFP reconbinant adenovirus transfection on rabbit bone marrow derived mesenchymal stem cells in vitro.

目的观测体外培养的兔骨髓间充质干细胞生物学特性,并转染重组腺病毒Ad5-EGFP-BMP7,观察BMP7与EGFP的表达情况。方法通过密度梯度离心联合贴壁法,分离、纯化间充质干细胞;流式细胞仪检测细胞周期和表面标记;体外诱导间充质干细胞向成骨细胞、成脂肪细胞分化并鉴定。转染后,荧光显微镜及免疫细胞化学染色检测外源基因的表达。结果原代及传代间充质干细胞为成纤维细胞样细胞;87%以上处于G0/G1期;CD44表达阳性,CD45表达阴性;经成骨细胞诱导后,碱性磷酸酶染色阳性,并有矿化结节形成;经成脂肪细胞诱导后,细胞内出现大量脂滴。转染Ad5-EGFP-BMP7后,荧光显微镜下可见EGFP表达阳性;免疫细胞化学染色显示细胞内BMP7表达阳性。结论成功分离、纯化间充质干细胞,并鉴定其具有成体干细胞特性;重组腺病毒介导BMP7和EGFP体外成功转染间充质干细胞,并稳定表达BMP7、EGFP,为下一步实验奠定了基础。

Objective To observe biological characteristics of rabbit bone marrow derived mesenchymal stem cells cultured in vitro,and expression of BMP7 and EGFP after Ad5-ECFP-BMP7 infected on them.Methods To isolate , purify the cells by density gradient centrifugation and adherent culture in vitro. Detected cell membrane＇s marks and cell cycle by flow cytometer. Mesenchymal stem cells were induced into csteogenetic cells in the osteogenesis supplement medium or adipose cells in adipogenesis supplement medium, and then they were verified. Detected expression of exogenous gene in the cells by fluorescence microscope and Immunocytochemical stain.Results The morpheus of the primary cells and passage cells is similar to fibroblast cell. More than 87% cells were in G0/G1 phase;CD44 is positive, CD45 is negative. After mescnchymal stem cells were induced, alkaline phosphatasc is positive in osteogenetic cells, and they formed mineralization nodus;lipid droplets existed in adipose cells. After adenovirus infected mesenchymal stem cells, dectected the expression of BMP7 and EGFP. Conclusion Mesenchymal stem cells cultured have the characteristics of adult stem cells; Ad5-EGFP-BMP7 infected successfully mesenchymal stem cells,and gained the stable expression of BMP7 and EGFP.