腺病毒介导的人骨形态发生蛋白2基因转染骨髓间充质干细胞

背景：骨髓间充质干细胞作为骨、软骨创伤缺损及退变修复的种子细胞越来越受到关注。目的：分析人骨形态发生蛋白2基因转染对白色封闭群大鼠(SD大鼠)骨髓间充质干细胞的影响。方法：分离纯化SD大鼠骨髓间充质干细胞并体外扩增，通过腺病毒载体介导人骨形态发生蛋白2基因转染骨髓间充质干细胞，分别通过荧光显微镜观察荧光表达情况及蛋白质水平来测定转染后人骨形态发生蛋白2的表达，碱性磷酸酶定量测定鉴定成骨活性及MTT法评估人骨形态发生蛋白2转染对骨髓间充质干细胞的影响。结果与结论：从SD大鼠骨髓提取物中分离培养的细胞形态为梭形，呈铺路石状、漩涡状生长，经流式细胞仪检测及多项分化能力鉴定符合骨髓间充质干细胞的特征；经转染人骨形态发生蛋白2基因后，骨髓间充质干细胞表达人骨形态发生蛋白2、碱性磷酸酶；MTT法检测转染人骨形态发生蛋白2基因后，骨髓间充质干细胞增殖能力明显增强(P < 0.05)。说明人骨形态发生蛋白2基因转染骨髓间充质干细胞后可以持续、高效表达人骨形态发生蛋白2和碱性磷酸酶，在体外明显促进骨髓间充质干细胞的增殖。

Adenovirus-mediated human bone morphogenetic protein 2 gene transfects bone marrow mesenchymal stem cells

BACKGROUND:Bone marrow mesenchymal stem cells as the seed cells for repair of bone and cartilage trauma and degeneration have been paid increasing attention.OBJECTIVE:To investigative the effects of human bone morphogenetic protein 2 gene transfection on Sprague-Dawley rat bone marrow mesenchymal stem cells. METHODS:Sprague-Dawley rat bone marrow mesenchyal stem cells were in vitro isolated, purified and amplified. Adenovirus-mediated human bone morphogenetic protein 2 was transfected into bone marrow mesenchymal stem cells. CD90 and CD45 expression levels were tested by flow cytometry. The successfully packaged virus was transfected into bone marrow mesenchymal stem cells and expression of human bone morphogenetic protein 2 gene was confirmed by enhanced green fluorescent protein expression under the fluorescence microscope. Enzyme linked immunosorbent assay was performed to monitor the expression levels of human bone morphogenetic protein 2 and alkaline phosphatase in mesenchymal stem cells. The effect of human bone morphogenetic protein 2 on the proliferation of bone marrow mesenchymal stem cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells were successfully harvested from bone marrow of sprague-dawley rats and identified by flow cytometry. After primary culture for 7-10 days, cultured cells displayed typical fusiform shape and the growth status was like “cobblestones” or “whirlpool” under light microscope and could be differentiated into osteoblast-, adipocyte- and neuron-like cells in vitro. After transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells expressed human bone morphogenetic protein 2 and alkaline phosphatase. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that after transfection by human bone morphogenetic protein 2 gene, bone marrow mesenchymal stem cells exhibited a stronger proliferation capacity (P < 0.05). These findings suggest that human bone morphogenetic protein 2 gene-transfected bone marrow mesenchymal stem cells can successively express high level of human bone morphogenetic protein 2 and alkaline phosphatase, indicating that human bone morphogenetic protein 2 gene can significantly promote the proliferation of bone marrow mesenchymal stem cells.