摘要
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.
Osteocytes are the major mechanosensors in bone tissue.It is important for explaining the mechanism of bone loss to investigate the regulating role of osteocytes on its effector cells under weightlessness.To investigate the regulating effects of conditioned medium of osteocytes on functions of osteoblasts,the 3-D rotation was used to simulate weightlessness.After mouse osteocyte-like cell line MLO-Y4 subjected 3-D rotation for 72 hours,the conditioned medium of rotation（RCM） and conditioned medium of control（CCM） were collected.The effects of RCM and CCM on proliferation,cell cycle,ALP activity and osteogenesis regulated gene expression of MC3T3-E1 cells were analyzed by MTT（methyl thiazolyl tetrazolium）,pNPP（p-nitrophenyl phosphate）,flow cytometry and RT-PCR,respectively.The results showed that the proliferation of MC3T3-E1 was significantly stimulated by RCM of MLO-Y4 after subjected 3-D rotation for 72 hours.After MC3T3-E1 cells were cultured for 24 and 48 hours,the rates of proliferation in 50% RCM were 1.62 and 1.60-fold than in CCM,respectively.（P0.05）.After 72 hours,the rate of proliferation in 100% RCM was 1.69-fold than in CCM.The cell cycle assay indicated that the cell cycle arrest of MC3T3-E1 induced by 100% CCM were partly restored by 100% RCM.There was no difference between RCM and CCM on ALP activity of MC3T3-E1 cells.RT-PCR showed that 100% RCM significantly decreased the mRNA expression of ALP,Runx2,OPN and OC（＊P0.05,＊＊P0.01,＊＊＊P 0.00 1）.In summary,the proliferation of osteoblasts were stimulated by conditioned medium of MLO-Y4 after subjected 3-D rotation for 72 hours.However,the osteogenesis regulated gene expression were significantly decreased.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2010年第2期181-188,共8页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金面上项目(No.30970689)~~
关键词
骨细胞条件培养基
成骨细胞
三维回转
成骨功能
conditioned medium of osteocytes
osteoblasts
3-D rotaiton
functions of osteoblasts
