Chinese Journal of Tissue Engineering Research ›› 2020, Vol. 24 ›› Issue (13): 2039-2046.doi: 10.3969/j.issn.2095-4344.2032
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Liu Zhongsheng, Yang Jianhong
Received:
2019-07-11
Revised:
2019-07-13
Accepted:
2019-07-31
Online:
2020-05-08
Published:
2020-03-07
Contact:
Yang Jianhong, PhD, Professor, Medical School, University of Chinese Academy of Sciences, Beijing 101400, China
About author:
Liu Zhongsheng, Medical School, University of Chinese Academy of Sciences, Beijing 101400, China
Supported by:
CLC Number:
Liu Zhongsheng, Yang Jianhong. Regulatory effect of uncarboxylated osteocalcin on osteogenic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells under high-glucose conditions[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(13): 2039-2046.
2.3 未羧化骨钙素抑制高糖条件下骨髓间充质干细胞的成脂分化 油红染色显示,与对照组相比,高糖促进骨髓间充质干细胞产生脂滴,经过未羧化骨钙素处理后,骨髓间充质干细胞产生脂滴的量显著减少。对油红染色进行定量分析,高糖处理组吸光度值明显高于对照组,差异有显著性意义(P < 0.05);未羧化骨钙素处理组吸光度值明显低于对照组,差异有非常显著性意义(P < 0.01);高糖+未羧化骨钙素处理组吸光度值明显低于高糖处理组,差异有显著性意义(P < 0.05),见图3A-E。 qRT-PCR检测结果显示,高糖处理组成脂分化标志基因的表达明显高于对照组(PFabp4 < 0.01;PPPARγ < 0.01;PAdipsin < 0.01;PFAS < 0.01);未羧化骨钙素处理组成脂分化基因的表达明显低于对照组(PFabp4 < 0.01;PPPARγ < 0.01;PAdipsin < 0.01;PFAS < 0.01)。高糖+未羧化骨钙素处理组成脂分化基因的表达明显低于高糖处理组(PFabp4 < 0.01;PPPARγ < 0.05;PAdipsin < 0.01;PFAS < 0.01),见图3F。 "
2.4 未羧化骨钙素促进高糖条件下骨髓间充质干细胞的成骨分化 茜素红染色显示,与对照组相比,高糖明显抑制骨髓间充质干细胞形成钙结节,而未羧化骨钙素促进骨髓间充质干细胞形成钙结节。对茜素红染色进行定量分析,高糖处理组吸光度值明显低于对照组,差异有显著性意义(P < 0.05);未羧化骨钙素处理组吸光度值明显高于对照组,差异有非常显著性意义(P < 0.01);高糖+未羧化骨钙素处理组吸光度值明显高于高糖处理组,差异有显著性意义(P < 0.05),见图4A-E。 qRT-PCR检测结果显示,高糖处理组成骨分化标志基因的表达明显低于对照组(PRunx2 < 0.05;POsx < 0.05; PALP < 0.01;PCOLⅠ < 0.01);未羧化骨钙素处理组成骨分化基因的表达明显高于对照组(PRunx2 < 0.01;POsx < 0.01;PALP < 0.01;PCOLⅠ < 0.01);高糖+未羧化骨钙素处理组成骨分化基因的表达明显高于高糖处理组(PRunx2 < 0.05;POsx < 0.05;PALP < 0.01;PCOLⅠ < 0.01),见图4F。 高糖处理组碱性磷酸酶活性以及Ⅰ型胶原蛋白水平明显低于对照组(P < 0.05);未羧化骨钙素处理组碱性磷酸酶活性以及Ⅰ型胶原蛋白水平明显高于对照组(碱性磷酸酶活性,P < 0.01;Ⅰ型胶原蛋白水平,P < 0.05);高糖+未羧化骨钙素处理组成骨分化基因的表达明显高于高糖处理组(碱性磷酸酶活性,P < 0.01;Ⅰ型胶原蛋白水平,P < 0.05),见图4G,H。 "
2.5 未羧化骨钙素通过Erk/AMPKα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 Western blot表明,未羧化骨钙素可上调P-Erk(P < 0.01)和P-AMPKα (P < 0.01)的表达;P-Erk抑制剂PD98059使P-Erk、P-AMPKα下调(P < 0.01),这种作用被未羧化骨钙素所逆转(P < 0.05);当使用AMPKα抑制剂BML时,P-AMPKα下调(P < 0.01),这种作用被未羧化骨钙素所逆转(P < 0.01),而P-Erk水平未发生明显变化(P > 0.05)。上述结果说明P-AMPKα处于P-Erk下游,见图5A-H。 为验证未羧化骨钙素是否通过Erk/AMPKα通路调控骨髓间充质干细胞的分化,应用10 μmol/L BML预处理细胞1 h,未羧化骨钙素处理细胞7 d。qRT-PCR结果显示,抑制剂BML抑制AMPKα后,成脂分化基因表达上调,而成骨分化基因表达下调(高糖+BML抑制剂组与高糖处理组相比,PFabp4 < 0.01;PPPARγ < 0.01;PRunx2 < 0.01,POsx < 0.01)。然而,未羧化骨钙素逆转了抑制剂对分化的影响(高糖+BML抑制剂+未羧化骨钙素组与高糖+BML抑制剂组相比,PRunx2 < 0.01,POsx < 0.01),见图5I,J。上述结果说明,未羧化骨钙素是通过Erk/AMPKα通路抑制高糖条件下骨髓间充质干细胞向脂肪细胞分化而促进成骨分化。 "
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