未羧化骨钙素对高糖条件下小鼠骨髓间充质干细胞成骨与成脂分化的调控效应

Regulatory effect of uncarboxylated osteocalcin on osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions

背景:寻找高糖条件下促进骨髓间充质干细胞成骨分化而抑制其成脂分化的方法,可以为治疗骨代谢疾病如糖尿病性骨质疏松提供预防及治疗思路。目的:探讨未羧化骨钙素对高糖条件下小鼠骨髓间充质干细胞成脂分化和成骨分化的影响,揭示未羧化骨钙素对骨髓间充质干细胞分化的作用机制。方法:采用全骨髓细胞培养及贴壁纯化小鼠骨髓间充质干细胞,不同质量浓度(0,1,3,10,30μg/L)未羧化骨钙素处理细胞,CCK-8试剂盒检测细胞增殖情况,确定最佳作用质量浓度。第3代骨髓间充质干细胞加入成脂(或成骨)分化诱导培养基并分成4组:对照组、高糖处理组、未羧化骨钙素处理组、高糖+未羧化骨钙素处理组,分别添加25.5 mmol/L外源葡萄糖,3μg/L未羧化骨钙素进行处理。采用油红和茜素红染色检测脂滴和钙结节的形成,q RT-PCR检测成脂分化标志基因(Fabp4、PPARγ、Adipsin和FAS)和成骨分化标志基因(Runx2、Osx、ALP和COLⅠ)的相对表达水平,试剂盒检测碱性磷酸酶活性和Ⅰ型胶原蛋白水平。另外,结合MEK和AMPK的特异性抑制剂(PD98059和BML),Western blot检测P-Erk和P-AMPKα的相对表达水平。结果与结论:①3μg/L未羧化骨钙素可显著促进细胞增殖(P<0.01);②未羧化骨钙素促进高糖条件下骨髓间充质干细胞产生钙结节(P<0.01)而抑制脂滴的形成(P<0.05),下调成脂分化标志性基因(PFabp4<0.01;PPPARγ<0.05;PAdipsin<0.01;PFAS<0.01)而上调成骨分化标志性基因(PRunx2<0.05;POsx<0.05;PALP<0.01;PCOLⅠ<0.01)的表达,增加碱性磷酸酶活性(P<0.01)和Ⅰ型胶原蛋白水平(P<0.05);③高糖条件下,未羧化骨钙素上调P-Erk(P<0.01)和P-AMPKα(P<0.01)表达水平;④结果表明,未羧化骨钙素通过Erk/AMPKα信号通路促进高糖条件下骨髓间充质干细胞的成骨分化而抑制成脂分化。

BACKGROUND:The method of promoting osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions to inhibit adipogenic differentiation can provide prevention and treatment ideas for the treatment of bone metabolic diseases such as diabetic osteoporosis.OBJECTIVE:To explore the effects of uncarboxylated osteocalcin on adipogenic and osteogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions so as to reveal the action mechanism of uncarboxylated osteocalcin on the differentiation of bone marrow mesenchymal stem cells.METHODS:Mouse bone marrow mesenchymal stem cells were cultured by whole bone marrow culture and adherent purification.Cells were treated with uncarboxylated osteocalcin at different concentrations(0,1,3,10,and 30μg/L).Cell proliferation was detected by cell counting kit-8 to determine the best mass concentration.Passage 3 bone marrow mesenchymal stem cells were incubated with adipogenic(or osteogenic)differentiation medium,and assigned to four groups:control group,high glucose group,uncarboxylated osteocalcin group,and high glucose+uncarboxylated osteocalcin group.Corresponding groups received the addition of 25.5 mmol/L exogenous glucose and 3μg/L uncarboxylated osteocalcin.Lipid droplets and calcium nodules were detected by oil red and alizarin red staining.Quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of adipogenic marker genes(Fabp4,PPARγ,Adipsin and FAS)and osteogenic differentiation marker genes(Runx2,Osx,alkaline phosphatase,and type I collagen).Kits were used to detect alkaline phosphatase activity and type I collagen levels.The relative expression levels of P-Erk and P-AMPKαwere detected using signal pathway specific inhibitors(PD98059 and BML)and western blot assay.RESULTS AND CONCLUSION:(1)Uncarboxylated osteocalcin 3μg/L promoted cell proliferation(P<0.01).(2)Uncarboxylated osteocalcin promoted the formation of calcium nodules(P<0.01)in bone marrow mesenchymal stem cells under high-glucose conditions but inhibited the formation of lipid droplets(P<0.05),down-regulating the relative expression levels of adipogenic marker genes(PFabp4<0.01;PPPARγ<0.05;PAdipsin<0.01;PFAS<0.01),but increasing the relative expression levels of osteogenic differentiation marker genes(PRunx2<0.05;POsx<0.05;PALP<0.01;PCOLI<0.01).Uncarboxylated osteocalcin increased alkaline phosphatase activity(P<0.01)and type I collagen level(P<0.05).(3)Uncarboxylated osteocalcin up-regulated the expression levels of P-Erk(P<0.01)and P-AMPKα(P<0.01)under high-glucose conditions.(4)These results indicate that uncarboxylated osteocalcin promoted osteogenic differentiation of bone marrow mesenchymal stem cells under high-glucose conditions through Erk/AMPKαsignaling pathway and inhibited adipogenic differentiation.