OPN影响小鼠MSCs迁移及其相关分子机制研究

Study on the effects of OPN on migration of MSCs and its molecular mechanism in mice

目的探讨外源骨桥蛋白(OPN)对小鼠骨髓间充质干细胞(MSCs)迁移能力的影响。方法采用OPN-/-小鼠和野生型C57小鼠,进行MSCs的原代分离培养,流式细胞术鉴定分拣MSCs细胞传代培养;Transwell迁移实验检测OPN是否能够诱导MSCs定向迁移;蛋白免疫印迹法(Western blot)检测OPN、CD44和Integrinβ1相关蛋白的表达变化。结果传代后的细胞形态符合MSCs特征,细胞表达CD44和CD105,但不表达CD34,符合MSCs表面标记抗原的一般规律。0.5μg/mL的OPN能增加体外MSCs的细胞迁移,而野生型C57小鼠MSCs细胞迁移为最多。同时,这一趋势与OPN作用时间正相关,与OPN-/-小鼠相比,差异均有统计学意义(P<0.05)。0.5μg/mL重组OPN蛋白量明显增加,但仍低于野生型C57小鼠,差异有统计学意义(P<0.01)。若以OPN-/-小鼠为基数100,0.5μg/mL重组OPN组CD44、Integrinβ1蛋白表达增多,差异有统计学意义(P<0.01);而野生型C57小鼠细胞CD44、Integrinβ1蛋白表达最多,差异有统计学意义(P<0.01)。结论 OPN可以通过上调CD44、Integrinβ1的表达,促进MSCs定向迁移。

Objective To investigate the effects of exogenous osteopontin （OPN ） the migration of bone marrow mesenchymal stem cells（MSCs） in mice .Methods Using the OPN -/- and wild-type C57 mice ,the MSCs were isolated and cultured .These cells were analysed and sorted by flow cytometry .Then ,Transwell migration assay detected whether OPN can induce the migration of these MSCs .Finally ,the method of Western blot was used to detect the changes of expression of OPN ,CD44 and Integrinβ1 pro-teins .Results The morphology and features of MSCs were proved correctly on 3 passages ,which these cells expressed the proteins of CD44 and CD105 ,but not CD34 .The surface marker antigens were accorded with the general features of MSCs .Compared with OPN -/- mice ,the MSCs significantly increased cell migration in groups of 0 .5 μg/mL OPN and wild type C57 mice ,which was positively related to the times using OPN .Using recombinant OPN protein（0 .5μg/mL） ,expression of related proteins was signifi-cantly increased ,but still lower than the group of wild type mice （P〈0 .01） .Compared with OPN -/- mice ,expression of OPN , CD44 ,and Integrinβ1 protein increased significantly different （P〈0 .01） in groups of 0 .5 μg/mL OPN and wild type C57 mice . Conclusion OPN can up-regulate the expression of CD44 and Integrinβ1 proteins and promote MSCs migration .