成纤维细胞生长因子18对骨髓间充质干细胞增殖、成骨分化的影响及其机制

Effects of fibroblast growth factor 18 on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and its mechanism

目的探讨成纤维细胞生长因子18(FGF18)对骨髓间充质干细胞(BMSCs)增殖、成骨分化的影响及其机制。方法SD大鼠长骨提取BMSCs,用含不同浓度FGF18的成骨诱导分化培养基培养,采用细胞试剂盒(CCK-8)法检测细胞增殖。设定空白对照组,碱性磷酸酶(ALP)染色、茜素红染色和ALP活性检测评估BMSCs的成骨分化能力;采用荧光定量聚合酶链反应(PCR)检测核心结合蛋白因子2(Runx2)、骨形态发生蛋白4(BMP4)和Ⅰ型胶原蛋白(COLⅠ)基因的mRNA表达;采用蛋白质印迹法(Western blot)检测成骨相关蛋白(Runx2、COLⅠ、OCN)和丝裂原活化蛋白激酶(MAPK)通路相关蛋白[细胞外信号调节激酶(ERK1/2)、磷酸化(p)-ERK1/2、p38、p-p38]的表达。两组间比较采用t检验。结果CCK结果显示,FGF18对BMSCs的促增殖效应随浓度升高和时间延长而递增。干预7 d和14 d后,FGF18组ALP染色和茜素红染色的着色程度均明显高于对照组。ALP活性随FGF18浓度升高和时间延长而递增。干预3 d时,FGF18组Runx2、BMP4基因的mRNA表达量高于对照组,差异有统计学意义(1.891±0.327比1.103±0.331,2.115±0.403比1.134±0.327,t=2.933、3.274,P<0.05)。干预7 d时,FGF18组Runx2、BMP4和COLⅠ基因的mRNA表达均较对照组明显上升,差异均有统计学意义(5.852±0.525比1.903±0.451,5.115±0.633比1.734±0.527,4.451±0.553比1.525±0.483,t=9.883、7.110、6.902,P<0.05)。干预3 d和7 d时,FGF18组Runx2、COLⅠ、p-ERK1/2和p-p38蛋白表达量均高于对照组,差异有统计学意义(Runx2为0.927±0.108比0.647±0.087,1.257±0.188比0.825±0.103,t=3.497、3.490,P<0.05;COLⅠ为0.538±0.062比0.405±0.047,0.729±0.106比0.513±0.074,t=2.961、2.894,P<0.05;p-ERK1/2为0.908±0.113比0.614±0.072,0.968±0.142比0.686±0.086,t=3.800、2.942,P<0.05;p-p38为1.203±0.194比0.826±0.082,1.135±0.186比0.803±0.076,t=3.100、2.862,P<0.05)。结论FGF18可以通过MAPK信号通路来实现对BMSCs细胞增殖以及早期成骨分化的调控,且具有良好的诱导成骨分化的生物活性。

Objective To explore the effects of fibroblast growth factor 18(FGF18)on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and its mechanism.Methods BMSCs were extracted from long bones of SD rats and cultured in osteogenic differentiation medium containing different concentrations of FGF18.Cell proliferation was detected by cell count kit-8(CCK-8).The blank control group was set.Alkaline phosphatase(ALP)staining,alizarin red staining and ALP activity were used to evaluate the osteogenic differentiation ability of BMSCs.The mRNA expressions of related transcription factor-2(Runx2),bone morphogenetic protein 4(BMP4)and typeⅠcollagen(COLⅠ)genes were detected by fluorescence quantitative polymerase chain reaction(PCR).Western blotting was used to detect the expression of osteogenesis related proteins(Runx2,COLⅠ,OCN)and mitogen-activated protein kinase(MAPK)pathway related proteins[extracellular signal regulated kinase(ERK1/2),phosphorylation(p-)ERK1/2,p38,p-p38].T-test was used to compare the two groups.Results CCK results showed that the proliferative effect of FGF18 on BMSCs increased with the increase of concentration and the prolongation of culture time.After 7 and 14 days of intervention,the staining degree of ALP staining and alizarin red staining in FGF18 group was higher than those in the control group at the same time point.ALP activity increased with the increase of FGF18 concentration and the prolongation of culture time.On the 3rd day of intervention,the mRNA expression of Runx2 and BMP4 genes in FGF18 group was significantly higher than those in the control group(1.891±0.327 vs.1.103±0.331,2.115±0.403 vs.1.134±0.327,t=2.933,3.274,P<0.05).On the 7th day of intervention,the mRNA expression of Runx2,BMP4 and COLⅠgenes in FGF18 group was significantly higher than those in the control group(5.852±0.525 vs.1.903±0.451,5.115±0.633 vs.1.734±0.527,4.451±0.553 vs.1.525±0.483,t=9.883,7.110,6.902,P<0.05).On the 3rd and 7th day of intervention,the expression of Runx2,COLⅠ,p-ERK1/2 and p-p38 protein in FGF18 group were significantly higher than those in the control group(Runx20.927±0.108 vs.0.647±0.087,1.257±0.188 vs.0.825±0.103,t=3.497,3.490,P<0.05;COLⅠ0.538±0.062 vs.0.405±0.047,0.729±0.106 vs.0.513±0.074,t=2.961,2.894,P<0.05;p-ERK1/20.908±0.113 vs.0.614±0.072,0.968±0.142 vs.0.686±0.086,t=3.800,2.942,P<0.05;p-p381.203±0.194 vs.0.826±0.082,1.135±0.186 vs.0.803±0.076,t=3.100,2.862,P<0.05).Conclusion FGF18 can regulate BMSCs cell proliferation and early osteogenic differentiation through MAPK signaling pathway,and has good biological activity to induce osteogenic differentiation.