激活BMP信号的骨细胞对骨髓基质细胞成骨及成脂分化的作用研究

The effect of osteocytes with activated BMP signal on osteogenesis and lipogenic differentiation of bone marrow stromal cells

目的激活骨细胞系MLO-Y4细胞中BMP信号检测培养上清对骨髓基质细胞系ST2成骨、成脂分化的影响,进一步探讨其机制。方法0.5‰DMSO,0.5μmol/L BMP激动剂FK506分别处理MLO-Y424 h后,使用CCK-8检测细胞活力变化,实时荧光定量PCR检测BMP下游靶基因ID1及ID2 mRNA表达变化;用20%MLO-Y4培养上清与80%新鲜培养基混合后培养ST2细胞,分为DMSO组、FK506组。碱性磷酸酶染色代表其成骨分化能力,通过实时荧光定量PCR检测碱性磷酸酶(ALP)、骨钙蛋白(OCN)、骨唾液酸蛋白(BSP)、Runx2等成骨细胞标志基因,过氧化物酶体增殖剂激活受体γ(PPARγ)和C/EBP成脂分化标志基因。免疫印迹试验(Western blotting)检测ST2细胞内Wnt信号下游β-catenin、BMP信号下游p-smad5蛋白表达水平。结果与DMSO作用的MLO-Y4细胞相比,FK506激动的MLOY4细胞内BMP信号靶基因ID1、ID2上调,但不影响细胞活力。FK506组ST2细胞同DMSO组对比,成骨分化相关标志物,包括ALP、OCN、BSP、Runx2(P<0.001)均显著升高;成脂分化标志物PPARγ及C/EBP表达则显著降低(P<0.001);ST2细胞内β-catenin蛋白表达量上调(P<0.05)。结论BMP信号激动后MLO-Y4细胞上清可以促进ST2细胞成骨分化、抑制成脂分化,其成骨能力增强与细胞内Wnt信号增强有关。

Objective To investigate the effect of activation of BMP signaling in MLO-Y4 cells on osteogenic and adipogenic differentiation by ST2 cells and its mechanism.Methods After MLO-Y4 cells were treated with 0.5‰DMSO and 0.5μmol/L BMP agonist(FK506)for 24 h,the cell activity was tested using CCK-8.The expression of target genes BMP,ID1,and ID2 was detected with quantitative real-time PCR.ST2 cells were cultured with 20%supernatant collected from MLO-Y4 culture and 80%fresh medium.The cells were divided into DMSO group and FK506 group.Alkaline phosphatase(ALP)staining represented the ability of osteogenic differentiation.The mRNA expression of osteogenic markers ALP,osteocalcin(OCN),bonesialoprotein(BSP),and Runx2,and the adipogenesis-related markers peroxisome proliferators-activated receptorsγ(PPARγ)and C/EBP were detected using qPCR.Western blotting was used to detect the protein expression levels ofβ-catenin and p-smad5,the downstream signal of Wnt and BMP in ST2 cells.Results Compared to those in DMSO group,BMP signal target genes ID1 and ID2 were upregulated in FK506-activited MLO-Y4 cells,with no effect on cell liability.The osteoblastic differentiation factors ALP,OCN,BSP,and Runx2 increased and expression of PPARγand C/EBP decreased(P<0.001)in ST2 cells of FK506 group,compared to those in DMSO group.The protein expression ofβ-catenin in ST2 cells was up-regulated(P<0.05).Conclusion After BMP signal in MLO-Y4 cells is stimulated,the supernatant promotes osteogenic differentiation and inhibits lipogenesis in ST2 cells.The enhancement of osteogenesis ability is related to the enhancement of intracellular Wnt signal.