C3H10T1/2成软骨分化过程中miR-19a与CCND1的表达及调控

Mir-19a and CCND1 Gene Expression in Mechanism Exploration of C3H10T1/2 Chondrogenic Differentiation

【目的】在间充质干细胞诱导成软骨分化过程中,探讨mi R-19a与细胞周期基因CCND1之间的关系,为揭示mi R-19a参与调控成软骨分化的可能机制提供基础。【方法】选取小鼠间充质干细胞C3H10T1/2为研究对象,运用离心沉淀法进行pellet细胞微球培养,用含有TGF-β3及2%FBS的高糖DMEM进行成软骨诱导并作为实验组,用含2%FBS的高糖DMEM培养(替换)导作为对照组。分别在1、2、3周进行定量PCR检测成软骨分化相关基因表达以及在第3周进行甲苯胺蓝染色。【结果】与对照组比较,细胞功能基因Bax与Klf-4表达均下调,差异具有统计学意义(P<0.05)。成软骨分化关键启动因子SOX-9及相关基因aggrecan,collagen II,Collagen Xa1的表达均在2周时显著升高,差异具有统计学意义(P<0.05)。通过互联网生物信息学预测CCND1可能是mi R-19a的靶基因;在成软骨分化过程中,mi R-19a表达逐渐上升(P<0.05),而CCND1的表达与对照组相比逐渐下降(P<0.05)。甲苯胺蓝染色pellet细胞微球呈蓝紫色,细胞核呈深蓝色,诱导组中细胞外基质含量明显增多。【结论】在C3H10T1/2成软骨分化过程中,mi R-19a参与维持细胞干性及增殖基因表达降低,分化能力得到增强;且mi R-19a参与该分化调控过程。

【Objective】 To explore mi R-19 a functions that may inhibit CCND1 expression in mesenchymal stem cells,we applied C3H10T1/2 stem cell of chondrogenic differentiation model to illustrate the regulation of mechanism.【Method】 In C3H10T1/2chondrogenic differentiation,we apply high cell density pellet culture systems formed by centrifugation and evaluate the chondrogenic capacity.Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3（10 ng/m L）,dexamethasone（100nmo L/L）,ascorbic acid（50ug/m L）,1：100 dilution ITS＋Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum.And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group.Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of mi R-19 a,Bax,klf-4,CCND1,SOX9,collagen IIa1/Xa1 and aggrecan for the 1st,2nd and 3rd week respectively.【Result】 In pellet model for3 weeks chondrogenic differentiation,the expression of Bax and Klf-4 were both down-regulated through the differentiation（P〈0.05）.Moreover,master transcription factor SOX9 as well as the marker genes of collagen IIa1/Xa1 and aggrecan were all peaked on the second week（P〈0.05）.In addition,the expression of mi R-19 a was up-regulated in compared with the control group（P〈0.05）while the CCND1 was down-regulated in both the second and third week of the differentiation（P〈0.05）.Histochemistry staining of the pellet for 3 week revealed extracellular matrix expression and more obviously in compared with the control group.【Conclusion】Our data suggest mi R-19 a was involved in the chondrogenic differentiation,which the CCND1,Bax,Klf-4 were all depressed for the differentiation period.