BMP2重组慢病毒转染兔BMSCs黏附DBM支架的生物矿化研究  

BMP2 recombinant lentivirus transfected rabbit BMSC adhesive demineralized bone matrix scaffolds for biomineralization

作　　者：宁寅宽 刘林志 陈贤平 周次腊 NING Yinkuan;LIU Linzhi;CHEN Xianping;ZHOU Cila(Department of Vascular Surgery,Shaoyang Central Hospital,Shaoyang 422000,China;Department of Endocrinology,Shaoyang Central Hospital,Shaoyang 422000,China)

机构地区：[1]邵阳市中心医院血管外科,湖南邵阳422000 [2]邵阳市中心医院内分泌科,湖南邵阳422000

出　　处：《中国骨质疏松杂志》2023年第4期497-502,共6页Chinese Journal of Osteoporosis

基　　金：广西区自然科学基金资助项目(2014GXNSFAA118263)。

摘　　要：目的探究BMP2重组慢病毒转染兔骨髓间充质干细胞(BMSCs)黏附脱钙骨(DBM)支架体外构建转基因组织工程骨的生物矿化能力。方法密度梯度离心及贴壁培养法获取第5代兔BMSCs,用BMP2重组慢病毒转染细胞,通过RT-PCR检测目的基因的表达,利用倒置荧光显微镜观察细胞在DBM支架上的生长情况,借助扫描电镜和能谱分析观测细胞在DBM支架上的表面微观形貌及生物矿化能力。结果BMP2重组慢病毒成功转染兔BMSCs,RT-PCR显示转染后细胞能高效表达BMP2目的基因,在倒置荧光显微镜下观察见转染后细胞黏附在DBM支架上呈满天星样,并沿DBM支架孔隙内壁贴壁叠加生长、分裂增殖,扫描电镜下见细胞填满整个DBM支架孔隙,分泌大量细胞外基质,并在DBM支架表面形成凹凸不平的光泽结晶矿化物,能谱分析显示其钙磷比(Ca/P)为1.89±0.31,与正常骨组织成分相近(P>0.05)。结论BMP2重组慢病毒转染兔BMSCs黏附DBM支架构建的转基因组织工程骨成功完成了生物矿化过程,体现出较好的生物矿化能力。Objective Investigate the biomineralization ability of demineralized bone matrix attached with BMP2 recombinant lentivirus transfected rabbit bone marrow mesenchymal stem cells.Methods The 5 th generation rabbit BMSCs were obtained by density gradient centrifugation and adherent culture.The cells were transfected with BMP2 recombinant lentivirus,the expression of the target gene was detected by RT-PCR,and the growth of the cells adhered to the DBM scaffold after transfection was observed by an inverted fluorescence microscope.The surface morphology and biomineralization ability of the transfected cells on DBM scaffolds were observed by scanning electron microscopy(SEM)and energy dispersive analysis.Results BMP2 recombinant lentivirus was successfully transfected into rabbit BMSCs.RT-PCR showed that the transfected cells could express BMP2 target gene efficiently.Observed under an inverted fluorescence microscope,the transfected cells adhered to the DBM scaffold exhibited a gypsophila-like morphology,and superimposed grew and proliferated along the inner wall of DBM scaffold pores.Under the SEM,cells filled the entire DBM scaffold pore,secreted a large amount of extracellular matrix,and formed uneven luster crystalline mineralization on the surface of the DBM scaffold.Energy spectrum analysis showed that its calcium/phosphorus ratio(Ca/P)was 1.89±0.31,which was similar to that of normal bone tissue(P>0.05).Conclusion The transgenic tissue engineered bone constructed by BMP2 recombinant lentivirus transfected rabbit BMSCs adhered to DBM scaffolds successfully completed the biomineralization process,showing good biomineralization ability.

关 键 词：重组慢病毒载体 骨形态发生蛋白 骨髓间充质干细胞 基因治疗 生物矿化 

分 类 号：Q78[生物学—分子生物学]