骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化  

Synergistic effect of bone morphogenetic protein 2 and transforming growth factor beta 2 on osteogenic differentiation of bone marrow mesenchymal stem cells

作　　者：常晓朋 陈涛 赵寅 梁明 Chang Xiaopeng;Chen Tao;Zhao Yin;Liang Ming(Zhengzhou Central Hospital,Zhengzhou University,Zhengzhou 450000,Henan Province,China)

机构地区：[1]郑州大学附属郑州中心医院,河南省郑州市450000

出　　处：《中国组织工程研究》2019年第1期1-6,共6页Chinese Journal of Tissue Engineering Research

摘　　要：背景:从细胞及基因水平揭示骨形态发生蛋白2和转化生长因子β2具有协同促进骨髓间充质干细胞成骨分化的机制,为骨细胞的再生提供了实验和理论基础。目的:探讨过表达骨形态发生蛋白2和转化生长因子β2协同促进骨髓间充质干细胞成骨分化的作用。方法:通过酶消化法原代培养获取大鼠骨髓间充质干细胞,慢病毒包装骨形态发生蛋白2及转化生长因子β2的重组质粒。将携带目的基因重组质粒的慢病毒转染至第3代骨髓间充质干细胞,MTS实验检测骨髓间充质干细胞的增殖情况,定量PCR检测骨形态发生蛋白2和转化生长因子β2的m RNA表达水平,细胞爬片免疫组化检测二维混合培养条件下成骨分化标志物RUNX2在蛋白水平的表达量,体外三维培养获得的细胞团块经石蜡切片做Vonkossa染色(银染)定量分析成骨分化的阳性区域及表达强度。结果与结论:(1)转染骨形态发生蛋白2及转化生长因子β2的骨髓间充质干细胞的增殖能力与未转染的骨髓间充质干细胞相比差异无显著性意义(P> 0.05);(2)转染后骨髓间充质干细胞的骨形态发生蛋白2及转化生长因子β2 mRNA水平高于未转染的骨髓间充质干细胞,差异有显著性意义(P <0.05);(3)体外二维培养条件下细胞爬片RUNX2免疫组化及体外三维培养石蜡切片Vonkossa染色均发现共转染组骨髓间充质干细胞的成骨分化能力强于单独转染组(P <0.05);(4)结果表明,慢病毒携带骨形态发生蛋白2和转化生长因子β2的重组质粒过表达具有协同促进大鼠骨髓间充质干细胞成骨分化的作用。BACKGROUND:It is revealed from the cell and gene level that bone morphogenetic protein 2(BMP2)and transforming growth factorβ2(TGFβ2)synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells,providing an experimental and theoretical basis for the bone regeneration.OBJECTIVE:To investigate the overexpression of BMP2 and TGFβ2 in vitro to synergistically promote the osteogenic differentiation of bone marrow mesenchymal stem cells.METHODS:Primary rat bone marrow mesenchymal stem cells were obtained by enzymatic digestion.Recombinant plasmids of BMP2 and TGFβ2 were packaged by lentivirus.The lentivirus carrying the recombinant plasmids of the target genes BMP2 and TGFβ2 was transfected into the third generation of bone marrow mesenchymal stem cells.MTS(cell proliferation assay)was used to detect the proliferation of bone marrow mesenchymal stem cells in different treatment groups at different time points.Quantitative PCR was used to detect the overexpression of BMP2 and TGFβ2 at mRNA levels.Immunohistochemistry of cell slides was performed to detect the expression level of osteogenic differentiation marker RUNX2 at the protein level under two-dimensional mixed culture conditions.The cell pellet obtained by three-dimensional culture in vitro was subjected to paraffin sectioning for Vonkossa staining(silver staining)to determine the positive region and expression intensity of osteogenic differentiation,and quantitative analysis was performed.RESULTS AND CONCLUSION:(1)The proliferation of bone marrow mesenchymal stem cells transfected with BMP2 and TGFβ2 was not statistically different from that of untransfected cells(P>0.05).(2)The BMP2 and TGFβ2 mRNA levels were significantly higher in the transfected cells than untransfected ones(P<0.05).(3)RUNX2 immunohistochemistry under the in vitro two-dimensional culture conditions and Vonkossa staining under the in vitro three-dimensional culture showed that the BMP2 and TGFβ2 co-transfected bone marrow mesenchymal stem cells had stro

关 键 词：骨髓间充质干细胞 骨形态发生蛋白2 转化生长因子Β2 慢病毒 RUNX2 成骨分化 干细胞 骨髓 间质干细胞 骨形态发生蛋白质类 转化生长因子Β2 组织工程 

分 类 号：R459.9[医药卫生—治疗学] R394.2[医药卫生—临床医学]