BMP-2与Wnt/β-catenin信号通路对间充质干细胞分化为成骨细胞的作用

摘要

目的 探讨骨形成蛋白(BM P)2与Wnt/β-链蛋白(β-catenin)信号通路对间充质干细胞分化为成骨细胞的作用.方法 将间充质干细胞C3H10T1/2分为五组:A组用正常培养基培养,B组用成脂诱导液IFMD(含有吲哚美辛10 μg/ml、10% FBS、1-甲基-3-异丁基黄嘌呤0.5 mmol/L和地塞米松1 mmol/L)处理,C组在IFMD处理的同时加入BM P-2100 ng/ml,D组在IFMD处理的同时加入氯化锂(LiCl)20 mmol/L,E组在IFMD处理的同时加入BM P-2100 ng/ml和LiCl 20 mmol/L.处理第8天,用 ALP染色及油红O染色鉴定成骨细胞及脂肪细胞的分化情况;处理第2、4、8天,检测Ⅰ型胶原(Col1a1)、骨钙素(Ocn)以及CCAAT增强子结合蛋白(C/EBPα)、脂肪酸结合蛋白(aP2)和葡萄糖转运蛋白4(Glut4)的表达;重亚硫酸盐测序法检测C/EBPα在各组细胞中启动子区甲基化情况.结果 A组无ALP及油红O阳性细胞,B组有大量油红O染色阳性细胞,E组细胞ALP活性最高.与B组比较,C组和E组Runx2、Col1a1和Ocn mRNA表达较高(P<0.05或P<0.01),C组、D组和E组C/EBPα、aP2和Glut4 mRNA表达较低(P<0.05或P<0.01).E组C/EBPα启动子区出现较高的甲基化水平,经5-氮杂胞苷处理后,C/EBPα mRNA表达升高(P<0.01),大部分细胞分化为脂肪细胞.结论 在易成脂的病理微环境中,BMP-2和Wnt/β-catenin信号通路单独作用均不能有效促进成骨细胞分化,需要两者协同作用才能使间充质干细胞分化为成骨细胞,具体机制与C/EBPα启动子区甲基化有关.%Objective To investigate the effect of bone morphogenetic protein(BMP)-2 and Wnt/β-catenin signaling pathway on the differentiation of mesenchymal stem cells into osteoblasts. Methods The mesenchymal stem cell C3H10T1/2 was divided into five groups.Group A was cultured with normal culture medium.Group B was cultured with IFMD,which contained indometacin 10 μg/ml,10% FBS,1-methyl-3-isobutylxanthine 0.5 mmol/L and dexamethasone 1 mmol/L.Group C was treated with BMP-2 100 ng/ml in addition to IFMD.Group D was treated with lithium chloride (LiCl)20 mmol/L in addition to IFMD.Group E was treated with BMP-2 100 ng/ml and LiCl 20 mmol/L in addition to IFMD.The differentiation of osteoblast and adipocyte was identified by ALP staining and oil red O staining on the 8thday.The mRNA expressions of collagen type Ⅰ(Col1a1), osteocalcin(Ocn),CCAAT/enhancer-binding proteins(C/EBPα),fatty acid binding proteins(aP2)and glucose transporter 4(Glut4)were detected on the 2nd,4thand 8thday.The C/EBPα promoter methylation status was tested by bisulfate sequencing.Results Group A existed no ALP and oil red O positive cells.Group B appeared much oil red O positive cells and the ALP activity in group E was the highest.Compared to group B,the mRNA expressions of Runx2,Col1a1 and Ocn were higher in groups of C and E,whereas C/EBPα,aP2 and Glut4 were lower in groups of C,D and E(P<0.05 or P<0.01). The C/EBPα promoter methylation status was higher in group E.The mRNA was elevated in group E treated with 5-azacytidine(P<0.01),which was differentiated into adipocyte.Conclusion In a pathological microenvironment proned to adipogenesis,BMP-2 or Wnt signal fails to induce osteoblasts differentiation alone,but BMP-2 and Wnt signal can synergistically promote the mesenchymal stem cells to differentiate into osteoblasts,which may be related to C/EBPα promoter methylation.