牵张力对小鼠骨髓间充质干细胞成骨分化能力的作用及机制研究

Effects of tension force on osteogenic differentiation of mouse bone marrow mesenchymal stem cells and its mechanism

目的:探讨牵张力作用对小鼠骨髓间充质干细胞(BMSCs)成骨分化能力的影响及作用机制。方法:原代分离并培养小鼠BMSCs,并将BMSCs分为对照组、牵张力作用6 h组、牵张力作用12 h组和牵张力作用12 h+雷帕霉素(Rap)组,采用多单元细胞拉伸装置施加动态牵张力进行干预。生化法检测细胞中碱性磷酸酶(ALP)活性;ELISA法检测细胞培养液上清中骨膜蛋白(POSTN)含量;RT-qPCR检测细胞中Runt相关转录因子2(RUNX2)、骨钙素(OCN)、Osterix和骨桥蛋白(OPN)mRNA表达水平;Western blot检测细胞中RUNX2、OCN、Osterix、OPN、POSTN、哺乳动物雷帕霉素靶蛋白(mTOR)和磷酸化mTOR(p-mTOR)蛋白表达水平。结果:与对照组比较,牵张力作用6 h组和12 h组细胞中ALP活性及RUNX2、Osterix、OCN和OPN mRNA表达水平均显著升高(均P<0.05),上清液POSTN含量、细胞中POSTN蛋白及p-mTOR/mTOR蛋白比值也显著升高(均P<0.05),且牵张力作用12 h组更明显。与牵张力作用12 h组比较,牵张力作用12 h+Rap组细胞中ALP活性和RUNX2、OCN、Osterix、OPN mRNA及蛋白表达水平均显著降低(均P<0.05),而细胞培养液上清中的POSTN含量无明显差异(P>0.05)。结论:牵张力通过介导POSTN表达调控mTOR信号通路激活,从而促进BMSCs成骨分化。

Objective:To explore the effect and mechanism of tension on osteogenic differentiation ability of mouse bone marrow mesenchymal stem cells(BMSCs).Methods:The primary mouse BMSCs were isolated and cultured,and the BMSCs were divided into control group,tension group for 6 h,12 h,and 12 h+rapamycin(Rap).Dynamic tension was applied using a multiple-unit cell stretching device for intervention.Biochemical method was used to detect alkaline phosphatase(ALP)activity in cells.ELISA method was used to detect the content of periostin(POSTN)in the supernatant of cell culture medium.RT-qPCR was used to detect the mRNA expression levels of runt related transcription factor 2(RUNX2),osteocalcin(OCN),osterix and osteopontin(OPN)in cells.Western blot was used to detect the protein expression levels of RUNX2,OCN,Osterix,OPN,POSTN,mammalian target of rapamycin(mTOR)and phospho mTOR(p-mTOR)in cells.Results:Compared with the control group,the activity of ALP and the mRNA expression levels of RUNX2,Osterix,OCN and OPN in the 6 h tension and 12 h tension groups were significantly increased(P<0.05),while the POSTN content in supernatant liquid,the expression level of POSTN protein and p-mTOR/mTOR protein ratio were also significantly increased(P<0.05),and the 12 h tension group being more pronounced.Compared with the 12 h tension group,the ALP activity and the expression levels of RUNX2,OCN,Osterix and OPN mRNA and protein in the 12 h tension+Rap group cells were significantly reduced(P<0.05),while the POSTN content in the supernatant of the cell culture medium showed no significant difference(P>0.05).Conclusion:Tension regulates the activation of the mTOR signaling pathway by mediating POSTN expression,thereby promoting osteogenic differentiation in BMSCs.