不同浓度葡萄糖对大鼠颌骨骨髓基质细胞成骨分化的影响

摘要

目的:体外观察不同葡萄糖浓度对大鼠颌骨骨髓基质细胞(BMSCs)向成骨细胞分化能力的影响.方法:无菌条件下从4周龄SD大鼠下颌骨获取BMSCs,采用全骨髓贴壁培养法对细胞进行纯化及培养,流式细胞仪检测干细胞表型及进行成骨分化检测,随后选取第4代细胞在不同糖浓度(5.5,16.5,25,35mM)干预下向成骨细胞定向诱导分化,其后对各组进行茜素红染色,碱性磷酸酶染色和活性测定,培养基钙、磷含量测定,并采用Western blot测定骨形成蛋白2(BMP-2)水平以及real-time PCR测定Runx2基因表达变化.组间比较采用单因素方差分析法(SPSS 11.0).结果:随着葡萄糖浓度的升高,茜素红染色钙结节形成逐渐减少,碱性磷酸酶(ALP)活性下降,细胞钙、磷分泌显著降低,差异有统计学意(P＜0.05);BMP-2水平以及Runx2 mRNA表达也明显降低(P＜0.05).结论:在高糖条件下,大鼠颌骨骨髓基质细胞成骨分化能力减弱,而BMP信号通路可能参与其中.%Objective:To observe the effects of different concentration of glucose on osteogenic differentiation of bone marrow stromal cells (BMSCs) obtained from rats' mandibular.Methods:The in vitro technique of cell culture was employed to harvest,purify and cultivate BMSCs from the mandibular of rats.Flow cytometry analysis of cell phenotypes and osteogenic differentiation were performed.The passage 4 cells were incubated with osteogenic DMEM containing 5.5,16.5,25,and 35 mM glucose,respectively.The cells were then stained with 2％ Alizarin red S and the Alizarin red S-stained nodules were observed microscopically and quantified.ALP staining and ALP activity assay were performed.The calcium and phosphonium content were assayed.Then the expression of bone morphogenetic protein-2 (BMP-2) was analysed by western blot and the expression of RUNX2 mRNA was analysed by real-time PCR.The data were subjected to one-way ANOVA using SPSS 11.0.Results:With the increasing of glucose concentrations,the number of red calcium nodules obviously decreased,ALP activity was inhibited,and the calcium and phosphonium content in medium were decreasd.The difference was significant (P＜0.05).Furthermore,BMP-2 levels and RUNX2 mRNA expression were decreased (P＜0.05).Conclusions:In high glucose microenvironment,osteogenic capacity of marrow stromal cells from rat mandibular was inhibited,and the BMP signaling pathway may be involved in this process.