嘌呤受体P2Y2在骨髓间充质干细胞成骨和成脂分化中的作用  

Roles of purine receptor P2Y2 in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells

作　　者：李文凯 张英驰 杨勇 吴华[1] LI Wen-kai;ZHANG Ying-chi;YANG Yong;WU Hua(Department of Orthopedics,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院附属同济医院骨科,武汉430030

出　　处：《中华骨质疏松和骨矿盐疾病杂志》2021年第1期41-48,共8页Chinese Journal Of Osteoporosis And Bone Mineral Research

基　　金：国家自然科学基金(51807078)。

摘　　要：目的探讨嘌呤受体P2Y2在骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成骨和成脂分化中的作用及其分子机制。方法体外分离培养大鼠BMSCs,取第3代细胞进行实验。使用实时定量聚合酶链反应(reverse transcription polymerase chain reaction,RT-qPCR)检测BMSCs成骨和成脂分化基因的表达,茜素红染色和油红O染色分别检测钙结节和脂滴的生成。使用三磷酸尿苷(uridine triphosphate,UTP)作为P2Y2受体激动剂,使用P2Y2和P2Y4受体小干扰RNA(small interfering RNA,siRNA)、P2Y6受体拮抗剂评估P2Y2受体在UTP对BMSCs成骨和成脂分化影响中的作用。蛋白免疫印迹(Western blot)法检测UTP及P2Y2受体对丝裂原活化蛋白激酶(mitogen activated protein kinase,MAPK)通路的影响。结果与对照组相比较,25和125μmol/L浓度的UTP可以使BMSCs细胞成骨特异性转录因子(RUNX2)的表达分别下降19%和62%、碱性磷酸酶(alkaline phosphatase,ALP)的表达分别下降56%和55%、骨桥蛋白(osteopontin,OPN)的表达分别下降26%和70%,使细胞过氧化物酶体增生物激活受体γ(peroxisome proliferator activated receptorγ,PPARγ)分别增加38%和52%、脂肪酸结合蛋白4(fatty acid binding protein 4,FABP4)分别增加17%和29%、降脂素(Adipsin)分别增加54%和93%,且不影响BMSCs细胞增生。此外,与对照组相比较,125μmol/L浓度的UTP可以使BMSCs细胞钙结节生成减少64%,脂滴形成增加40%。沉默P2Y2受体后UTP对BMSCs分化的影响明显减弱,而沉默P2Y4受体或抑制P2Y6受体则没有影响。UTP可激活BMSCs的ERK1/2信号通路,沉默P2Y2受体后UTP对ERK1/2信号通路的激活作用明显减弱。ERK1/2信号通路的阻滞剂U0126可以明显减弱UTP对BMSCs抑制成骨和促进成脂的作用。结论UTP通过激活P2Y2受体及ERK1/2信号通路促进BMSCs成脂分化、抑制其成骨分化。Objective To investigate the roles of purine receptor P2Y2 in osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)and the underlying mechanism.Methods BMSCs were isolated from rats and cultured in vitro,and the third passage cells were taken for experiment.Real time quantitative polymerase chain reaction(RT-qPCR)was used to detect the gene expression of osteogenic and adipogenic differentiation of BMSCs.Alizarin red staining and oil red O staining were used to detect the formation of calcium nodules and lipid droplets.Uridine triphosphate(UTP)was used as P2Y2 receptor agonist.Small interfering RNA(siRNA)of P2Y2 and P2Y4 receptor,antagonist of P2Y6 receptor were used to evaluate the role of P2Y2 receptor in differentiation of BMSCs induced by UTP.Western-blot was used to detect the effects of UTP and P2Y2 receptor on mitogen activated protein kinase(MAPK)pathway.Results Compared with the control group,25 and 125μmol/L UTP decreased the expression of osteogenic specific transcription factor(Runx2),alkaline phosphatase(ALP)and osteopontin(OPN)in BMSCs by 19%and 62%,56%and 55%,26%and 70%,respectively.25 and 125μmol/L UTP increased the expression of peroxisome proliferator activated receptorγ(PPARγ),fatty acid binding protein 4(FABP4),adiposin in BMSCs by 38%and 52%,17%and 29%,54%and 93%,respectively.In addition,125μmol/L UTP reduced the formation of calcium nodules by 64%and increased the formation of lipid droplets by 40%compared with the control group.Silencing P2Y2 receptor significantly weakened the effect of UTP on differentiation of BMSCs,while silencing P2Y4 receptor or inhibiting P2Y6 receptor had no effect.UTP activated ERK1/2 signaling pathway of BMSCs,and the effect was significantly weaken after P2Y2 receptor was silenced.U0126,a blocker of ERK1/2 signaling pathway,significantly attenuated the effect of UTP on osteogenic and adipogenic differentiation of BMSCs.Conclusion UTP promotes adipogenic differentiation and inhibits osteogenic differentiation of BMSCs by activ

关 键 词：P2Y2受体 三磷酸尿苷 骨髓间充质干细胞 成骨分化 成脂分化 

分 类 号：R681[医药卫生—骨科学]