高糖损害小鼠胚胎成骨前体细胞糖代谢并通过原纤毛/Hedgehog信号通路抑制成骨分化的研究  

High glucose impairs glucose metabolism and inhibits osteoblast differentiation via primary cilia/Hedgehog pathway in mouse embryonic osteoblast precursor cells

作　　者：刘杰 许洁 陈茜[1] 汪长东[1] LIU Jie;XU Jie;CHEN Qian(Department of College of Basic Medicine,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学基础医学院,400016

出　　处：《中国糖尿病杂志》2021年第11期836-842,共7页Chinese Journal of Diabetes

基　　金：重庆市自然科学基金(cstc2014jcyjA10024);重庆市教委(CY170402);重庆市渝中区科委科研项目(20200112);教育部博士点基金项目(20125503120015);重庆市研究生科研创新项目(CYS19204)。

摘　　要：目的研究高糖对小鼠胚胎成骨前体细胞(MC3T3-E1)增殖、成骨分化和糖代谢影响。方法 MC3T3-E1细胞予5.6 mmol/L(Con组)、25.0 mmol/L(HG组)葡萄糖浓度的α-MEM培养。CCK-8、Ki67免疫荧光检测细胞增殖,免疫荧光检测原纤毛;ELISA检测碱性磷酸酶(ALP)活力;Western blot检测ALP、骨桥蛋白(OPN)、乙酰化α-tubulin(Ac-α-tubulin)、γ-tubulin、印度/音/沙漠刺猬信号分子(IHH,SHH,DHH)、神经胶质瘤相关癌基因(Gli1/Gli2)、糖原合酶1(GYS1)、GluT4、磷酸果糖激酶(PFKM)、乙酰-CoA羧化酶α(ACACA)、糖原磷酸化酶(PYGL)、丙酮酸脱氢酶磷酸酶1(PDP1)蛋白表达;茜素红染色检测矿化结节。结果与Con组比较,HG组Ki67阳性率升高[(13.85±2.28)%vs(30.23±3.83)%,P<0.01],矿化结节形成受抑制(P<0.01),ALP酶活力降低(P<0.01),ALP、OPN、Gli2、IHH、SHH、GYS1、ACACA、PFKM、GluT4、Ac-α-tubulin、γ-tubulin蛋白表达减低(P<0.05或P<0.01),PYGL、PDP1表达升高(P<0.05或P<0.01),原纤毛Ac-α-tubulin和γ-tubulin平均荧光强度降低(P<0.05或P<0.01)。结论高糖促进MC3T3-E1细胞增殖,损害糖代谢机制,破坏原纤毛,阻断Hedgehog信号,抑制成骨分化和矿化。Objective To study the effects of high glucose on the proliferation,osteoblast differentiation and glucose metabolism of mouse embryonic osteoblast precursor cells(MC3 T3-E1).Methods MC3 T3-E1 cells were cultured with glucose α-MEM at concentrations of 5. 6 mmol/L(Con group)and 25. 0 mmol/L(HG group). Cell proliferation was detected by CCK-8 and Ki67 immunofluorescence. Primary cilia were detected by immunofluorescence. ELISA assayed alkaline phosphatase(ALP)activity. Western blot determined protein expression of ALP,osteopontin(OPN),γ-tubulin,acetylated α-tubulin(Ac-α-tubulin),Indian/Sonic/Desert hedgehog signaling molecule(IHH,SHH,DHH),glioma-associated oncogene(Gli1,Gli2),glycogen synthase 1(GYS1),glucose transporter 4(Glu T4),phosphofructokinase(PFKM),acetyl-Co A carboxylase alpha(ACACA),glycogen phosphorylase(PYGL)and pyruvate dehydrogenase phosphatase 1(PDP1). Alizarin red staining detected extracellular matrix mineralized nodules.Results Compared with Con group,the positive rate of Ki67 in HG group increased[(13. 85±2. 28)% vs(30. 23±3. 83)%,P<0. 01],the formation of mineralized nodules was inhibited(P<0. 01),and ALP activity decreased(P<0. 01).The expression of ALP,OPN,Gli2,IHH,SHH,GYS1,ACACA,PFKM,GluT4,Ac-α-tubulin,γ-tubulin decreased(P<0. 05 or P<0. 01),the expression of PYGL and PDP1 increased(P<0. 05 or P<0. 01),and the average fluorescence intensity of Ac-α-tubulin and γ-tubulin of primary cilia decreased(P<0. 05 or P<0. 01).Conclusion High glucose promotes the proliferation of MC3 T3-E1 cells,but disorders glucose metabolism,impairs primary cilia,blocks Hedgehog signaland inhibits osteoblast differentiation and mineralization.

关 键 词：高糖 成骨分化 原纤毛 糖代谢 

分 类 号：R587.1[医药卫生—内分泌]