MMP-2促进肌源性间充质干细胞成骨分化的实验研究

Experimental Study on MMP-2 Promoting Osteogenic Differentiation of Myogenic Mesenchymal Stem Cells

目的从大鼠肌肉组织中分离出肌源性间充质干细胞,过表达或干扰干细胞中基质金属蛋白酶(matrix metalloproteinase,MMP)-2的表达,观察MMP-2对肌源性间充质干细胞成骨分化的影响。方法取大鼠大腿肌肉组织进行原代培养,分离出肌源性间充质干细胞并进行流式细胞鉴定。构建Plvx-IRES-ZsGreen1-MMP-2过表达载体及Plvx-shRNA2-sh-MMP-2干扰载体,分别对间充质干细胞进行MMP-2过表达及抑制表达处理。分别在质粒转染肌源性间充质干细胞的第1、7、14天后,采取免疫蛋白印迹(western blot,WB)检测肌源性间充质干细胞中成骨基因Runt相关转录因子2(Runt related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)和碱性磷酸酶(alkaline phosphatase,ALP)的表达水平。在第7、14天用茜素红染色检测间充质干细胞成骨分化情况。结果原代培养的肌源性间充质干细胞,经流式细胞鉴定为大鼠肌肉干细胞;构建Plvx-IRES-ZsGreen1-MMP-2过表达载体及Plvx-shRNA2-sh-MMP-2干扰载体,能够有效干预肌源性间充质干细胞中MMP-2的表达;WB检测结果显示MMP-2过表达组的成骨基因RUNX2、OCN和ALP在第7天和第14天表达明显升高,且第14天表达更高,而培养第7天时茜素红染色差异不大,在培养第14天时MMP-2过表达组的茜素红染色钙结节最多;MMP-2干扰组中结果则与之相反。结论MMP-2能够促进肌源性间质干细胞成骨分化并形成钙结节。

Objective To observe the effect of MMP-2 on the osteogenic differentiation of muscle-derived mesenchymal stem cells.Methods The rat thigh muscle tissue was taken for primary culture,and mesenchymal stem cells were isolated and identified by flow cytometry.The Plvx-IRES-ZsGreen1-MMP-2overexpression vector and Plvx-shRNA2-sh-MMP-2interference vector were constructed,and the mesenchymal stem cells were treated with MMP-2 overexpression and suppression.Western blotting was used to detect the expression of osteogenic related genes RUNX2,OCN and ALP in the muscle-derived mesenchymal stem cells on the first,7th,and 14th days after plasmid transfection.Alizarin red staining was further performed to detect the osteogenic differentiation of mesenchymal stem cells on the 7th and 14th days.Results The primary cultured muscle-derived mesenchymal stem cells were identified as rat muscle stem cells by flow cytometry;the construction of Plvx-IRES-ZsGreen1-MMP-2overexpression vector and Plvx-shRNA2-sh-MMP-2interference vector could effectively interfere the expression of MMP-2in mesenchymal stem cells.Western blot detection results showed that the expression of osteogenic genes RUNX2,OCN and ALP in the MMP-2-upregulating group were significantly increased on the 7th and 14th days,and were significantly decreased on the 7th and 14th days in the MMP-2-downregulating group.However,on the 7th day of culture,there was little difference in Alizarin Red staining both in the MMP-2-upregulating group and MMP-2-downregulating group.On the 14th day of culture,the Alizarin Red staining of the MMP-2overexpression group had the most calcium nodules.Conclusion MMP-2can promote the osteogenic differentiation of myogenic mesenchymal stem cells and the formation of calcium nodules.