Sclerostin对骨形态发生蛋白2诱导的人骨髓间充质干细胞成骨分化的影响

Effect of Sclerostin on BMP-2-induced osteogenesis of human mesenchymal stem cells

目的探讨Sclerostin对骨形态发生蛋白2(BMP-2)诱导的人骨髓间充质干细胞(BMSCs)成骨分化的影响。方法取两位男性车祸伤致截肢患者的骨髓组织进行体外人BMSCs培养。将培养细胞随机分为三组,1组未做转染(Mock),2组转染对照siRNA,3组转染Sclerostin(骨形态发生蛋白抑制剂)siRNA,均采用含有0.1μg/m L BMP-2的成骨诱导培养基培养。分别在诱导培养的第0、3、7天,采用CCK-8法检测各组细胞活性,采用Cy QUANT细胞增殖检测试剂盒检测DNA含量。在培养的第14天,采用RT-PCR方法检测骨相关基因骨钙素(OCN)、骨钙蛋白(OC)及骨桥蛋白(OPN),采用碱性磷酸酶(ALP)分析试剂盒进行ALP活性分析,采用BCA蛋白检测试剂盒检测细胞蛋白含量。结果在诱导培养的第3、7天,3组细胞增殖活性、DNA含量均高于1、2组(P均<0.05)。在诱导培养的第14天,3组OCN、OC、OPN mRNA相对表达量及蛋白含量均高于1、2组(P均<0.05);1、2、3组ALP活性分别为1.00%±0.08%、1.12%±0.09%、2.50%±0.03%,3组高于1、2组(P均<0.05)。结论抑制Sclerostin表达可以促进BMP-2诱导的人BMSCs的活性及成骨分化能力。

Objective To investigate the influence of Sclerostin on bone morphogenetic protein 2( BMP-2)-induced osteogenic differentiation of human bone marrow-derived mesenchymal stem cells( BMSCs). Methods The bone marrow tissues taken from two males receiving amputation because of traffic accident were used to culture the human BMSCs in vitro. The cultured cells were randomly divided into three groups: groups 1,2 and 3. The cells in the group 1 did not receive transfection( Mock),the cells in the group 2 were transfected with control siRNA,and the cells in the group 3 were transfected with Sclerostin siRNA( BMPs inhibitor). Meanwhile,the osteoblasts were cultured with 0. 1 μg / m L BMP-2.CCK-8 assay was used to detect the activities of the cells on the 0,3rd and 7th day of induction,and the content of DNA was detected by Cy QUANT cell proliferation assay kit. On the 14 th day of culture,the expression levels of osteocalcin( OCN),osteocalcin( OC) and osteopontin( OPN) were detected by using RT-PCR,the alkaline phosphatase( ALP) analysis kit was used to analyze the activity of ALP,and the BCA protein assay reagent kit was used to analyze the cell protein content. Results On the 3rd and 7th day of culture,the proliferation activity and total DNA content of group 3 were higher than those of the groups 1 and 2( all P < 0. 05). On the 14 th day of culture,the relative expression levels of OCN,OC and OPN mRNA and total protein content were higher than those of the groups 1 and 2( all P < 0. 05). The activities of ALP in the groups 1,2 and 3 were respectively 1. 00% ± 0. 08%,1. 12% ± 0. 09% and 2. 50% ± 0. 03%,and group 3was higher than group 1 and group 2( all P < 0. 05). Conclusion Inhibiting the Sclerostin expression may increase the cell viability of human BMSCs and osteogenic differentiation.