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

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5.5mmol/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、40mmol/L),FGF-21干预组(Glu 5.5mmol/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。

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 5.5retool/ 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 of ALP, 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 OhBMSCs differentiated into osteoblast cells and lipocyte. The hBMSCs were identified by flow cytometry. ①Compared with control group, significant increases of ALP mRNA, OCN mRNA and Runx2 mRNA levels, as well as phosphorylation of ERK, P38 and JNK were observed in Glu A, B, C groups. ②Compared 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 . ③Compared 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.