大鼠骨髓间充质干细胞成软骨过程中微小RNA的表达  

The expression of miRNAs of rat bone marrow mesenchymal stem cells during chondrogenic differentiation

作　　者：陈松 符培亮 吴海山 许震宇[2] 

机构地区：[1]长征医院关节外科,上海200003 [2]第二军医大学组织胚胎学教研室

出　　处：《中华实验外科杂志》2014年第7期1406-1409,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金青年科学基金资助项目（81000798）

摘　　要：目的 探讨转化生长因子-β3（TGF-β3）诱导骨髓间充质干细胞（BMSCs）向软骨分化过程中微小RNA（miRNAs）的表达,并观察过表达miR-90a对BMSCs成软骨分化的影响。方法 体外分离培养大鼠BMSCs,取第3代BMSCs,在全培养基基础上将其分成两组：实验组加入TGF-β3诱导成软骨分化,对照组不加TGF-β3。在诱导BMSCs向软骨分化第7、14、21天,采用微阵列基因芯片技术分析分化过程中miRNAs的表达,并采用实时定量逆转录聚合酶链反应（RT-qPCR）技术对差异表达的miRNAs进行验证。在实验组的基础上,将成软骨诱导的BMSCs分为两组：实验组转染miR-90a mimics使其过表达,对照组转染mimics-NC,从而验证过表达miR-90a对BMSCs成软骨分化的影响。结果 体外成功分离培养大鼠BMSCs。微阵列基因芯片技术筛选出了BMSCs向软骨分化过程中相对于对照组表达差异在2倍以上的miRNAs共16个,其中表达上调的有9个,表达下调的有7个。采用RT-qPCR方法对表达上调的miR-340和miR-140以及下调的miR-21和miR-132进行验证,结果显示RT-qPCR与微阵列芯片的结果一致,说明本研究芯片结果真实可靠。实验组转染miR-90a mimics使其过表达结果显示,软骨细胞特异性细胞质成分糖胺多糖（GAG）较对照组明显减少。结论 BMSCs向软骨分化过程中有诸多miRNAs参与调节,过表达miR-90a可以明显抑制BMSCs的成软骨过程。Objective To explore the expression of miRNAs of rat bone marrow mesenchymal stem cells （BMSCs） during ehondrogenic differentiation induced by Transforming growth faetor-β3 （TGF-[33） , and study the effect of over-expression of miR-90a on the differentiation of BMSCs toward chondrocytes. Methods The rat BMSCs were isolated and cultured invitro and were divided into two groups： treated with and without TGF-β3. The express of miRNAs were analyzed by mieroarray gene chip at 7 , 14, 21 d, and the results were verified by real-time reverse transeriptase-polymerase chain reaction （RT-qPCR）. Mean- while the BMSCs treated with TGF-β3 were also divided into two groups, transfected with miR-90a mimics or mimics-NC, to explore the effect of over-expression of miR-90a on the chondrogenesis of BMSCs. Re- suits The BMSCs were isolated from the rat marrow successful. Mieroarray gene chip screening resulted in 16 miRNAs whose expression differences were more than 2 folds compared with the controls. Of them, 9 were up-regulated and 7 down-regulated. At the same time, the miR-340, miR-140, miR-21 and miR-β2 were determined by RT-qPCR, and the results showed that RT-qPCR and mieroarray gene chip results are consistent. The group transfeeted with miR-90a mimics express fewer glycosaminoglycans （GAG） than that of transfected with mimics-NC. Conclusion Many miRNAs were involved in the process of BMSCs differentiation into chondroeytes, and over-expression of miR-90a could inhibit the process.

关 键 词：骨髓间充质干细胞 成软骨分化 微小RNA 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]