摘要
目的 探究胰岛素样生长因子1(IGF-1)介导丝裂原活化蛋白激酶(MAPKs)信号通路在C3H10T1/2细胞成骨分化中的调控作用及机制.方法 不同浓度IGF-1(0、5、10、20 ng/mL)培养C3H10T1/2细胞,碱性磷酸酶(ALP)与茜素红(ARS)染色检测ALP活性、钙盐沉积情况,qRT-PCR法检测成骨特性因子核心结合因子α-1(RUNX2)、成骨分化特异性因子骨桥蛋白(OPN)、骨钙蛋白(OCN)mRNA表达水平,Western Blot法检测MAPK通路蛋白磷酸化表达水平.对数期细胞分为空白组、IGF-1组、ERK通路抑制剂(PD98059)组、PD+IGF-1组、p38通路抑制剂(SB202192)组、SB+IGF-1组,qRT-PCR法检测成骨特性因子RUNX2、成骨分化特异性因子骨桥蛋白(OPN)、骨钙蛋白(OCN)mRNA表达水平.结果 不同浓度IGF-1组ALP显色加深,ALP活性升高,钙盐结节形成增多,RUNX2、OPN、OCN mRNA表达水平升高,磷酸化ERK、p38、JNK蛋白表达增加,具有剂量效应(P＜0.05).与空白组比较,PD组、SB组C3H10T1/2细胞RUNX2、OPN、OCN mRNA表达水平明显降低(P＜0.05),PD+IGF-1 组、SB+IGF-1 组 C3H10T1/2 细胞 RUNX2、OPN、OCN mRNA 表达水平明显升高(P＜0.05);但与 IGF-1 组比较,PD+IGF-1 组、SB+IGF-1 组 C3H10T1/2 细胞 RUNX2、OPN、OCN mRNA 表达水平明显降低(P＜0.05).结论 IGF-1 促进 C3H10T1/2细胞成骨分化,其作用机制可能与激活ERK信号通路和p38 MAPK信号通路有关.
Abstract
Objective To investigate the regulatory role and mechanism of insulin-like growth factor 1(IGF-1)mediated mitogen-activated protein kinase(MAPKs)signaling pathway in osteogenic differentiation of C3H10T1/2 cells.Methods C3H10T1/2 cells were cultured with different concentrations of IGF-1(0,5,10,20 ng/mL).ALP activityand calcium salt deposition were detected by alkaline phosphatase(ALP)and alizarin red(ARS)staining.mRNA expression levels of core binding factor α-1(RUNX2),osteopontin(OPN)and osteocalcin(OCN)were detected by qRT-PCR,and phosphorylated expression levels of MAPK pathway proteins were detected by Western Blot.Logarithmic cells were divided into blank group,IGF-1 group,ERK pathway inhibitor(PD98059)group,PD+IGF-1 group,p38 pathway inhibitor(SB202192)group and SB+IGF-1 group.mRNA expression levels of osteogenic characteristic factor RUNX2,osteopontin(OPN)and osteocalcin(OCN)were detected by qRT-PCR.Results In different concentrations of IGF-1 groups,ALP color rendering was deepened,ALP activity was increased,the formation of calcium salt nodules was increased,mRNA expression levels of RUNX2,OPN and OCN were increased,and protein expressions of phosphorylated ERK,p38 and JNK were increased,with dose effect(P＜0.05).Compared with blank group,the mRNA expression levels of RUNX2,OPN and OCN in C3H1OT1/2 cells in PD group and SB group were significantly decreased(P＜0.05).The mRNA expression levels of RUNX2,OPN and OCN in C3H10T1/2 cells of PD+IGF-1 group and SB+IGF-1 group were significantly increased(P＜0.05).However,compared with IGF-1 group,the mRNA expression levels of RUNX2,OPN and OCN in C3H10T1/2 cells in PD+IGF-1 group and SB+IGF-1 group were significantly decreased(P＜0.05).Conclusion IGF-1 promotes osteogenic differentiation of C3H10T1/2 cells,and its mechanism may be related to the activation of ERK signaling pathway and p38 MAPK signaling pathway
基金项目
武汉市卫生健康委医学科研项目(2020)(WZ20D09)
NETL
NSTL
万方数据