Ad-hBMP-2/GFP转染兔骨髓间充质干细胞体外骨生成的实验研究  

The Experimental study on the osteogenesis of rabbit bone marrow mesenchymal stem cells transfected by Ad-hBMP-2/GFP in vitro

作　　者：宁寅宽[1] 李强[1] 蔡伟良[1] 武成聪[1] 陈佳滨[1] 石正松[1] 

机构地区：[1]桂林医学院附属医院四肢创伤手外科,广西桂林541001

出　　处：《中国骨质疏松杂志》2015年第3期264-268,共5页Chinese Journal of Osteoporosis

基　　金：国家自然科学基金资助项目(31160199)

摘　　要：目的观察Ad-h BMP-2/GFP转染兔骨髓间充质干细胞(BMSCs)体外骨生成中钙化斑形成,并对钙化斑元素构成进行研究。方法用携带h BMP2和GFP基因的腺病毒转染细胞,通过RT-PCR检测转染后BMSCs h BMP-2基因的表达,通过倒置荧光显微镜、钙结节茜素红染色观察转染后细胞形态改变和钙化斑的形成,结合扫描电镜和X线能谱分析(SEM/EDS)技术观测钙化斑表面微观形貌及其元素构成。结果 RT-PCR检测转染后BMSCs表达h BMP-2目的基因,细胞形态向成骨方向分化,钙结节茜素红染色见红色矿化结节,电镜下见钙化灶点状散布于细胞中,细胞重叠生长,分泌基质旺盛。X射线能谱分析显示其表面为钙、磷沉积物,其钙磷比(Ca/P)为1.53。结论 Ad-h BMP-2/GFP能成功转染兔BMSCs,体外诱导BMSCs向成骨方向分化,并具备较好的骨生成能力。Objective To observe the calcified plaque formation of rabbit bone marrow mesenchymal stem ceils （BMSCs） transfected by Ad-hBMP-2/GFP in vitro and to study the element composition of calcified plaque. Methods Rabbit BMSCs were transferred by adenovirus carrying hBMP-2/GFP gene. RT-PCR was used to detect the expression of hBMP-2 gene. Morphological change of the cells after transfection and calcified plaque formation were observed using inverted fluorescence microscope and Alizarin red staining, respectively. Surface microstructure and element composition of calcified plaque were studied using SEM and X-Ray spectroscopy （ SEM/EDS）. Results RT-PCR showed hBMP-2 target gene expression in BMSCs transfected by Ad-hBMP- 2/GFP. The cell morphology showed osteogenic differentiation. Alizarin red staining showed red mineralized nodules. SEM image demonstrated that mineralized calcium nodules were scattered in cells, and the growth of cells overlapped with strong matrix secretion, x-ray spectroscopy showed that the surface of calcified plaque consisted of calcium and phosphorus sediment with a calcium to phosphorus ratio （Ca/P） of 1.53. Conclusion Rabbit BMSCs can be successfully transfected by Ad-BMP-2/GFP which can induce osteogenic differentiation, with a preferable osteogenic capability.

关 键 词：重组腺病毒 骨形态生发蛋白 骨髓间充质干细胞 扫描电镜 能谱分析 

分 类 号：Q7[生物学—分子生物学] R68[医药卫生—骨科学]