miRNA-206介导BMSCs分化为软骨细胞及对骨关节炎模型的影响  

The miRNA-206 mediated the differentiation of bone mesenchymal stem cells into chondrocytes and its effect in osteoarthritis models

作　　者：杨震[1] 佘荣峰[1] 李博[1] 李洋 陈彪 陈克州 李波[1] 田晓滨[1] Yang Zhen;She Rongfeng;Li Bo;Li Yang;Chen Biao;Chen Kezhou;Li Bo;Tian Xiaobin(Department of Orthopaedics,Guizhou Provincial People's Hospital,Guiyang 550002,China)

机构地区：[1]贵州省人民医院骨科,贵阳550002

出　　处：《中华显微外科杂志》2019年第5期467-472,共6页Chinese Journal of Microsurgery

基　　金：国家自然科学基金项目(31660265,81060145,81560356);贵州省人民医院国家自然科学基金补助基金项目(黔科合平台人才[2017]5724-5);贵州省人民医院青年基金项目(GZSYQN[2015]06)。

摘　　要：目的探讨miRNA-206介导骨髓间充质干细胞(BMSCs)分化为软骨细胞及其在骨关节炎(OA)模型中的可能作用。方法2017年1月至2018年7月,分离大鼠BMSCs,流式细胞术检测CD90、CD45,鉴定BMSCs的纯度。使用慢病毒载体将miRNA-206或miRNA-206抑制剂转染到BMSCs,地塞米松诱导14 d,阿利新蓝染色和II型胶原免疫染色检测成软骨分化的能力。MTT法检测BMSCs的增殖能力,Western blot检测成软骨细胞中标志蛋白Aggrecan、Col II、Sox9、Runx2的表达水平。RT-PCR检测成软骨细胞中标志基因Sox9 mRNA的表达水平。在OA大鼠模型中尾静脉注射慢病毒载体转染miRNA-206或miRNA-206抑制剂,Western blot检测软骨形成的标志蛋白Aggrecan、Col II、Sox9、Runx2。结果分离的BMSCs的纯度为(80.7±3.9)%;miRNA-206转染BMSCs可以促进细胞增殖能力,增加其成软骨分化;Western blot结果表明miRNA-206转染组可以增加软骨细胞中标志性蛋白Aggrecan、Col II、Sox9的表达,下调Runx2的表达。同时RT-PCR结果表明miRNA-206上调BMSCs成软骨细胞标志基因Sox9 mRNA的表达。与OA组比较,miRNA-206可以增加软骨组织中信号蛋白Aggrecan、Col II、Sox9的表达(P<0.05),下调Runx2的表达水平(P<0.05)。结论miRNA-206可以正调控BMSCs分化为软骨细胞,增加细胞增殖能力,上调软骨标志蛋白Aggrecan、Col II、Sox9的表达,下调Runx2,并可能增加OA大鼠模型中成软骨能力。Objective To investigate the differentiation of bone mesenchymal stem cells(BMSCs)into chondrocytes by miRNA-206 and its mechanism in osteoarthritis(OA).Methods From January,2017 to July,2018,rat BMSCs were isolated,and their CD90 and CD45 were detected by flow cytometry.Transfection of miRNA-206 or miRNA-206 inhibitors into BMSCs using lentiviral vectors,dexamethasone induction for 14 d,then use alician blue staining and type II collagen immunostaining to detect chondrogenic differentiation.MTT assay was used to detect the proliferation of mesenchymal stem cells.Western blot analysis was used to detect the Aggrecan,Col II,Sox9 and Runx2 markers in chondroblast cells.The expression level of the marker gene of Sox9 mRNA in chondroblasts were detected by RT-PCR.OA rat models were treated with lentiviral vectors transfected with miRNA-206 or miRNA-206 inhibitors,and Aggrecan,Col II,Sox9,Runx2 which were the markers of chondrogenesis were detected by Western blot.Results The purity of isolated BMSCs was(80.7±3.9)%.BMSCs transfected with miRNA-206 could promote cell proliferation and increase chondrogenic differentiation.Western blot results showed that the expression of Aggrecan,Col II and Sox9 was increased in the miRNA-206 transfection group,and the expression of Runx2 was down-regulate.Meanwhile,RT-PCR results showed that miRNA-206 can up-regulate the expression of the chondroblast marker gene Sox9 mRNA in BMSCs.Compared with the OA group,miRNA-206 could increase the expression of Aggrecan,Col II and Sox9 signaling proteins in cartilage tissue(P<0.05),and down-regulate the expression level of Runx2(P<0.05).Conclusion The miRNA-206 can positively regulate the differentiation of BMSCs into chondrocytes,increase the ability of cell proliferation,up-regulate the expression of Aggrecan,Col II and Sox9,and down-regulate Runx2.The miRNA-206 increase chondrogenic capacity in rat models of osteoarthritis.

关 键 词：骨髓间充质干细胞 骨关节炎 成软骨分化 miRNA-206 大鼠 

分 类 号：R32[医药卫生—人体解剖和组织胚胎学]