糖化bFGF对HUVEC体外血管化的效应研究

Effect of Glycated b FGF on Angiogenesis of HUVEC in Vitro

目的体外观察糖化碱性成纤维细胞生长因子(Glycated basicfibroblast growth factor,Glycated bFGF)对人脐静脉内皮细胞(Human umbilical vein endothelial cells,HUVEC)体外血管化的影响。方法体外制备糖化bFGF并鉴定分析。bFGF和糖化bFGF干预后,分别用CCK-8法检测HUVEC的增殖水平,Matrigel胶观察其血管形成效应,流式细胞学检测其细胞凋亡水平。结果质谱显示bFGF的相对分子量为16 700 Da,糖化bFGF相对分子量为16 992 Da。高效液相显示,同H2O和bFGF相比,糖化bFGF在6~7 min出现差异色谱峰,随样品浓度增加,色谱峰呈增高趋势。细胞实验中,与正常培养的HUVEC相比,bFGF干预后第1、3、7天,细胞增殖显著增强;糖化bFGF干预后第1天细胞增殖显著增强,第3、7天未见明显差异。bFGF明显促进HUVEC血管形成,糖化bFGF明显抑制血管形成。干预后第7天,糖化bFGF组与bFGF组相比,细胞凋亡显著增加,其余时相点无差异。结论体外可成功制备糖化bFGF,糖化bFGF干预可抑制HUVEC形成血管。

Objective To observe the effect of glycated bFGF on angiogenesis of Human Umbilical Vein Endothelial Cells(HUVEC)in vitro.Methods The model of glycated bFGF in vitro was made.Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry(MALDI-TOF-MS)was used to identify the model and High Performance Liquid Chromatography(HPLC)was used to make analysis of the model.After treating HUVEC with bFGF and glycated bFGF,CCK-8 assay was used to detect the proliferation of HUVEC.Matrigel assay was used to detect the angiogenesis of HUVEC.Flow cytometry was used to detect the apoptosis of HUVEC.Results MALDI-TOF-MS showed that the relative molecular mass of bFGF was 16 700 Da,while glycated bFGF were 16 992 Da.By use of HPLC,glycated bFGF showed different chromatographic peaks at 6-7 minutes compared with H2O and bFGF as control.The value of the chromatographic peak was positively correlated with the concentration of the sample.Compared with the normal culture group,the proliferation of HUVEC increased on the 1st,3rd and 7th days in bFGF treating group.Compared with the normal culture group,the proliferation of HUVEC increased on the 1st day and presented no significant difference on other time points after glycated bFGF treating.bFGF promoted HUVEC tube formation while glycated bFGF inhibited it.The apoptosis increased in glycated bFGF treating group compared with bFGF treating group on the 7th day,and there was no difference on other time points.Conclusion The glycated bFGF model can be successfully prepared in vitro.The cell behavior of HUVEC with tubule formation is inhibited after treating with glycated bFGF.