颌骨骨髓间充质干细胞成骨分化过程中线粒体功能变化的研究

Study on mitochondrial function changes during osteogenic differentiation of jaw bone marrow mesenchymal stem cells

背景线粒体对成骨分化起着关键调控作用,但颌骨骨髓间充质干细胞(jaw bone marrow mesenchymal stem cells,JBMMSCs)成骨分化过程中线粒体功能的变化尚不明确。目的探究JBMMSCs成骨分化过程中线粒体功能的变化。方法提取小鼠JBMMSCs进行培养并进行鉴定。采用第3代JBMMSCs接种于孔板内,设置为对照组、成骨诱导3 d组、成骨诱导7 d组;检测ATP含量和柠檬酸合酶活性;RT-qPCR检测线粒体功能相关标志物MFN1、MFN2、NRF1、PGC-1α的mRNA表达;Western blot分析线粒体功能相关标志物MFN1、MFN2、FIS1、COXⅣ的蛋白表达;JC-1染色评估线粒体膜电位。结果提取的JBMMSCs形态呈长梭形,具有三向分化潜能。与对照组相比,成骨诱导7 d时,ATP含量和柠檬酸合酶活性增加(P均<0.05);RT-qPCR结果表明,线粒体功能相关标志物MFN1、MFN2、NRF1、PGC-1α的mRNA表达上调(P<0.05);Western blot结果显示,MFN1、MFN2、FIS1、COXⅣ的蛋白表达上调(P均<0.05);JC-1染色结果证实,线粒体膜电位升高(P<0.05)。结论JBMMSCs成骨分化过程中线粒体功能相关标志物均表达上调,提示线粒体功能增强。

Background Mitochondrion plays a critical regulatory role in osteogenesis,but the changes in mitochondrial function during the osteogenic differentiation of jaw bone marrow mesenchymal stem cells(JBMMSCs)are not yet clear.Objective To investigate the changes in mitochondrial function during the osteogenic differentiation of JBMMSCs.Methods Mouse JBMMSCs were extracted,cultured,and identified.The 3rd generation JBMMSCs were inoculated into the orifice plate and were set as control group(Ctrl),osteogenic induction 3d group(Ost 3d)and osteogenic induction 7d group(Ost 7d),ATP content and citrate synthase activity were measured;RT-qPCR was used to detect the expression of mitochondrial function-related markers MFN1,MFN2,NRF1,PGC-1αmRNA;Western blot analysis was used to examine the expression of mitochondrial function-related markers MFN1,MFN2,FIS1,COXⅣproteins;JC-1 staining was used to assess mitochondrial membrane potential.Results The extracted JBMMSCs were fusiform in shape and had tri-lineage differentiation potential.Compared with the control group,on the 7th day of osteogenic induction,ATP content and citrate synthase activity increased(P<0.05);RT-qPCR results showed that the expression of mitochondrial function-related markers MFN1,MFN2,NRF1,PGC-1αmRNA was upregulated(P<0.05);Western blot results indicated that the expression of MFN1,MFN2,FIS1,COXⅣproteins was increased(P<0.05);JC-1 staining results confirmed an increase in mitochondrial membrane potential(P<0.05).Conclusion During the osteogenic differentiation of JBMMSCs,the expression of mitochondrial function-related markers is upregulated,suggesting an enhancement of mitochondrial function.