摘要
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6h组、牵张力作用12h组和牵张力作用12h +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 6h组和 12h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12h组更明显.与牵张力作用12h组比较,牵张力作用12h + 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.
Abstract
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 con-tent of periostin(POSTN)in the supernatant of cell culture medium.RT-qPCR was used to detect the mRNA ex-pression 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 con-trol group,the activity of ALP and the mRNA expression levels of RUNX2,Osterix,OCN and OPN in the 6 h ten-sion and 12 h tension groups were significantly increased(P<0.05),while the POSTN content in supernatant liq-uid,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 activi-ty 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.
基金项目
湖南省自然科学基金资助项目(2022JJ30090)
NETL
NSTL
维普
万方数据