人骨形成蛋白4基因腺相关病毒及绿色荧光蛋白基因腺相关病毒体外诱导成骨的比较

AAV-hBMP-4 versus AAV-EGFP for osteogenic induction of rabbit bone marrow mesenchymal stem cells in vitro

背景:由于骨形成蛋白的释放速度与新骨生长速度不匹配,单纯应用骨形成蛋白的效果尚不理想,因此,应有合适的载体来调节骨形成蛋白的释放速度。目的:观察重组人骨形成蛋白4基因腺相关病毒及绿色荧光蛋白基因腺相关病毒诱导兔骨髓间充质干细胞向成骨方向分化的作用。方法:全骨髓法培养兔骨髓间充质干细胞,分别应用人骨形态形成蛋白4基因腺相关病毒载体及绿色荧光蛋白基因腺相关病毒转染兔骨髓间充质干细胞,设定感染复数值为5×104,观察两组细胞形态改变,分别行碱性磷酸酶染色、Von Kossa染色、茜素红染色及碱性磷酸酶含量测定,比较两组成骨活性差异。结果与结论:人骨形态形成蛋白4基因腺相关病毒组转染骨髓间充质干细胞后,细胞形态呈现典型的成骨改变,碱性磷酸酶染色及Von Kossa染色、茜素红染色均出现成骨的特征性改变。绿色荧光蛋白基因腺相关病毒组未观察到上述改变。人骨形态形成蛋白4基因腺相关病毒组碱性磷酸酶含量高于绿色荧光蛋白基因腺相关病毒组(P<0.01)。提示人骨形态形成蛋白4基因腺相关病毒转染骨髓间充质干细胞后,骨髓间充质干细胞表现出更加明显的成骨活性改变。

BACKGROUND： The releasing speed of human bone morphogenetic protein （hBMP） is not consistent with the new bone formation, thus, it is necessary to adjust the BMP release speed by suitable vector. OBJECTIVE： To determine osteogenic effects of transfection of adeno-associated virus （AAV）-hBMP-4 and AAV-enhanced green fluorescent proteins （EGFP） on rabbit bone marrow mesenchymal stem cells （BMSCs） in vitro. METHODS： The BMSCs were cultured using total bone marrow method, and then the cells were transfected with AAV-hBMP-4 and AAV-EGFP, respectively, with 5×104 infection value. After that, the differences of osteogenic effects of 2 groups were compared by observing cell morphological changes, alkali phosphatase staining, Von Kossa staining, alizarin bordeaux staiing and alkali phosphatase content. RESULTS AND CONCLUSION： The ossification of BMSCs was obviously observed after transfected with AAV-hBMP4, which was identified by alkali phosphatase staining, Von Kossa staining and alizarin bordeaux staining. No similar changes were observed in the AAV-EGFP group. The content of alkali phosphatase was greater in the AAV-hBMP4 group than that of the AAV-EGFP group （P 0.01）. The results demonstrated that AAV-EGFP transfected BMSCs presented notably osteogenic activity.