论文部分内容阅读
骨细胞是骨组织中主要的力学感受器.研究失重条件下骨细胞对效应细胞的调控作用对于揭示失重引起的骨丢失机制具有重要意义.本研究拟采用三维回转器模拟失重,探讨模拟失重骨细胞条件培养基(RCM)对成骨功能的调节作用.小鼠骨细胞系MLO-Y4三维回转培养72h后,收集回转条件培养基(RCM)和未回转对照组的条件培养基(CCM),用四甲基偶氮唑盐比色(MTT)法、对硝基苯磷酸(pNPP)法和流式细胞术(FCM)分别检测RCM对小鼠成骨样细胞系MC3T3-E1增殖、周期及细胞分泌碱性磷酸酶(ALP)活性的影响.采用RT-PCR方法检测RCM对MC3T3-E1成骨相关基因表达的影响.结果显示,三维随机回转72h后的MLO-Y4RCM可促进MC3T3-E1增殖:条件培养基培养MC3T3-E124h和48h后,50%RCM组比CCM组分别增加了1.62和1.60倍,差异显著(*P<0.05),培养72h后,100%RCM组比CCM组增加了1.69倍,差异显著(*P<0.05);细胞周期检测结果表明,条件培养基培养24、48和72h后,RCM组部分恢复CCM引起的MC3T3-E1细胞周期阻滞;MC3T3-E1的ALP活性在RCM组和CCM组之间无差异;RT-PCR检测结果表明,100%MLO-Y4条件培养基培养MC3T3-E148h后,降低了成骨相关基因ALP、Runx2、OPN、OC的表达.差异显著(*P<0.05,**P<0.01,***P<0.001).实验结果表明,三维随机回转模拟失重培养骨细胞72h后的条件培养基促进了成骨细胞增殖,抑制了成骨相关基因表达.
Osteoblasts are the main mechanoreceptors in bone tissue.Studying the regulatory effect of osteocytes on effector cells under conditions of weightlessness is of great significance to reveal the mechanism of bone loss caused by weightlessness.In this study, we will use three-dimensional gyroscope to simulate weightlessness, Conditioned medium (RCM) on osteoblast function.After 72 hours of three-dimensional mouse osteoblast cell line MLO-Y4, Conditioned Medium (CCM) in Rotation Condition Medium (RCM) and Non-Rotation Control Group MTT assay, pNPP assay and flow cytometry (FCM) were used to detect the effects of RCM on the proliferation, cell cycle and cell cycle of mouse osteoblast-like cell line MC3T3-E1 The effect of RCM on the gene expression of osteoblast-related genes in MC3T3-E1 was detected by RT-PCR.The results showed that MLO-Y4RCM promoted the proliferation of MC3T3-E1 by 72 hours after three-dimensional random rotation: Conditioned medium incubated with MC3T3-E124h and 48h, 50% RCM increased by 1.62 and 1.60 times respectively compared with CCM, the difference was significant (* P <0.05). After cultured for 72h, 100% RCM increased 1.69 times than CCM , Significant difference (* P <0.05); cell cycle test results show that the conditions of culture The cell cycle arrest of MC3T3-E1 cells induced by CCM was partly restored in RCM group after 24, 48 and 72 hours of culture. The ALP activity of MC3T3-E1 was no difference between RCM group and CCM group. RT-PCR results showed that 100% MLO The expression of osteoblast-related genes ALP, Runx2, OPN and OC were decreased after MC3T3-E148h culture in -Y4 conditioned medium (P <0.05, ** P <0.01, *** P <0.001) The results showed that conditioned media cultured in three-dimensional random revolution simulated weightlessness for 72 hours promoted osteoblast proliferation and inhibited osteogenesis-related gene expression.