Wnt3a联合骨形态发生蛋白2对骨髓间充质干细胞增殖及成骨分化的影响  

Effect of Wnt3a factor and bone morphogenetic protein-2 on bone marrow mesenchymal stem cell proliferation and osteogenic differentiation

作　　者：叶劲[1] 赵利波[1] 田志超[1] 李亮宇[1] 肖骏[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中华实验外科杂志》2011年第10期1780-1782,共3页Chinese Journal of Experimental Surgery

基　　金：湖北省青年科技人才基金资助项目（QJX2008-5）;武汉市科技攻关计划资助项目（200960223069）

摘　　要：目的观察经典Wnt／β-catenin以及骨形态发生蛋白（BMP）信号通路对大鼠骨髓来源的间充质干细胞（BMSCs）体外增殖以及向成骨方向分化的影响。方法应用密度梯度离心联合贴壁筛选法分离培养骨髓间充质干细胞并分4组：对照组、Wnt组、BMP组、Wnt3a和BMP-2联合组，在不同时间点用细胞计数试剂盒培（CCK-8）法检测细胞增殖活性，碱性磷酸酶（ALP）活性定量测定、VonKossa染色观察细胞向成骨分化和基质矿化程度。逆转录-聚合酶链反应（RT—PCR）检测成骨特异性标志物表达。结果培养第7天，CCK-8测吸光度值联合组为0．845，Wnt组为0．738，明显高于对照组0．409（P〈0．05）。培养第6天和第12天，ALP活性吸光度值联合组为63．8和144．3，BMP-2组为40．8和104．1，均明显高于对照组7．3和18．9（P〈0．05）。培养14d后，联合组骨钙素mRNA表达量高于对照组，而Runx2及Osterix表达量与其他组比较增高不显著。培养3周后，VonKossa染色显示联合组钙结节数量及大小均高于对照组。结论Wnt3a因子和骨形态发生蛋白-2联合诱导能有效地促进骨髓间充质干细胞增殖及向成骨方向分化。Objective To observe the impact of classical Wnt/β-catenin and bone morphogenetic protein （BMP） signaling pathway on rat bone marrow-derived mesenehymal stem cell （BMSCs） proliferation and osteogenic differentiation. Methods Bone marrow mesenchymal stern cells were isolated by densi- ty gradient centrifugation combined with adherence separation methods and divided into four groups： control group, Wnt group, BMP group, Wnt3a and BMP-2 combination group. Cell proliferation activity and alka- line phosphatase （ALP） activity were analyzed quantitatively by CCK8 protocol. Cellular osteogenic differ- entiation and matrix mineralization were decided by Von Kossa staining. The expression of osteoblast-spe- cific markers was detected by reverse transcription-polymerase chain reaction （RT-PCR）. Results At culture day 7, CKK-8 activity was 0. 845 in combination group, 0. 738 in Wnt3a group, which was significantly higher than 0. 409 in control group （P 〈 0.05 ）. At culture day 6 and 12, cellular ALP activity was 63.8 and 144. 3 in combination group, 40. 8 and 104. 1 in BMP-2 group, which was higher than 7.3 and 18.9 in control group （ P 〈 0. 05 ）. After culture for 14 days, osteocalcin mRNA expression was significantly higher in combination group, but there was no significant difference in the expression of Runx2 and Osterix among all groups. After Culture for 3 weeks, Von Kossa staining showed the number and size of calcium nodules in combination group were increased as compare with control group. Conclusion Combined induction of Wnt3a factor and BMP-2 can effectively promote mesenchymal stem cell proliferation and osteogenic differentiation.

关 键 词：骨髓间充质干细胞 WNT3A 骨形态发生蛋白-2 骨分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]