高糖环境下FGF-21对人骨髓间充质干细胞成骨分化的影响

Effects of FGF-21 on Osteogenic Differentiation of Human Bone Marrow Mesenchymal Stem Cells in High Glucose Environment

中文关键词:  成纤维细胞生长因子21 人骨髓间充质干细胞 成骨诱导 高糖环境 MAPK信号通路

英文关键词:FGF-21 Human bone mesenchymal stem cells Osteogenic differentiation High glucose MAPK signaling

中文摘要:

      目的 研究高糖环境下成纤维细胞生长因子21(FGF-21)对人骨髓间充质干细胞（hBMSCs）骨向分化的作用及其机制。方法 取健康成人骨髓，分离培养hBMSCs，成骨、成脂诱导分化后，茜素红、油红O染色鉴定，流式细胞仪检测第3代细胞表面标志物CD105、CD90、 CD73和CD44水平。将hBMSCs分为对照组〔以葡萄糖（Glu）浓度5.5 mmol/L模拟正常生理状态糖浓度〕，Glu A、B、C高糖浓度梯度组（Glu 16.5、25、40 mmol/ L），FGF-21干预组(Glu 5.5 mmol/ L+ FGF-21)，高糖环境FGF-21干预组(Glu B+FGF-21)和Glu B+FGF-21干预+细胞丝裂原活化蛋白激酶(MAPK) 信号转导阻断剂（包括PD98059、SP600125、SB203580）组。成骨诱导第14天，检测碱性磷酸酶（ALP）活性；成骨诱导第21天，以RT-PCR检测骨向分化的相关因子ALP、骨钙素（OCN）、Runx2的mRNA水平，以Western blot检测MAPK通路中细胞外信号调节激酶(ERK1/ 2)、P38丝裂原活化蛋白激酶(P38)、c-Jun 氨基末端激酶(JNK)蛋白磷酸化水平。结果 ①分离培养得到的hBMSCs成骨、成脂诱导后茜红素、油红O染色阳性，hBMSCs被成功地诱导向成骨细胞（OB）和脂肪细胞分化。流式细胞仪鉴定为hBMSCs。②与对照组相比，高糖组的骨向分化标志物ALP、OCN、Runx2的mRNA水平明显增加，MAPK通路中ERK、P38、JNK磷酸化水平表达增加。③与Glu B组相比，Glu B+FGF-21组的ALP、OCN、Runx2的mRNA水平降低，ERK、P38和JNK的磷酸化水平降低。④与Glu B+FGF-21组相比，Glu B+FGF-21+MAPK信号转导阻断剂组的ALP、Runx2的mRNA水平进一步降低，且相应的ERK、JNK和P38及其磷酸化水平进一步降低。结论 高糖能促进hBMSCs的骨向分化。高糖环境下FGF-21具有抑制hBMSCs的成骨分化或矿化作用。

英文摘要:

      Objective To determine the effect of fibroblast growth factor-21（FGF-21）on the osteogenic differention of human bone mesenchymal stem cells (hBMSCs) exposed to a hyperglycemia condition in vitro. Methods hBMSCs were isolated from adult bone marrows, and identified by Alizarin red and oil red O staining. The expressions of immunophenotype were analysed using flow cytometry (CD105, CD90, CD73, CD44).HBMSCs were divided into control group 〔glucose (Glu) concentration of 5.5 mmol/L〕, Glu A, B, C groups (Glu 16.5, 25, 40 mmol/L), FGF-21 group (Glu 5.5mmol/ L+ FGF-21 ),Glu B+ FGF-21 group, and Glu B +FGF-21+cell mitogen activated protein kinase (MAPK) blocker (PD98059, SP600125 ,and SB203580) groups. The effect of FGF-21 on the differentiation of hBMSCs was detected using indicators as follows: alkaline phosphatase （ALP） on day 14, mRNA expressions ofALP, osteocalcin（OCN）and Runx2, protein expressions and phosphorylation of extracellular signal regulated kinase （ERK）, mitogen-activated protein kinase（P38） and c-Jun N-terminal kinases（JNK） on day 21. Results ①hBMSCs differentiated into osteoblast cells and lipocyte. The hBMSCs were identified by flow cytometry. ②Compared with control group, significant increases of ALP mRNA, OCN mRNA and Runx2mRNA levels, as well as phosphorylation of ERK, P38 and JNK were observed in Glu A, B, C groups. ③Compared with Glu B group, ALP, OCN and Runx2 mRNA levels, and phosphorylation of ERK, P38 and JNK were decreased in Glu B+FGF-21 group . ④Compared with Glu B+FGF-21 group, ALP and Runx2 mRNA levels, and phosphorylation of ERK, JNK and P38 were decreased in Glu B +FGF-21 +MAPK blocker groups. Conclusion High glucose could promote the biomineralization of hBMSCs. FGF-21 in high glucose environment could inhibit the osteogenic differentiation of hBMSCs.