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目的观察外源性糖化碱性成纤维细胞生长因子-2(glycated fibroblast growth factor-2,gFGF-2)对大鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCs)数量及体外生物学活性的影响。方法将10μg/ml FGF-2同250mmol/L浓度的D-果糖于37℃共同培养48h制备gFGF-2,用蛋白指纹图谱分析仪检测其糖化情况。密度梯度离心法获取大鼠骨髓单个核细胞,培养4d后,分组分别用FGF-2(150μg/ml)、gFGF-2(150μg/ml)继续干预培养3d。用免疫组织化学和免疫荧光染色作CD34、CD133、VEGFR-2、ac-LDL和UEA-1检测来鉴定EPC。胰酶消化培养7d的贴壁细胞,将细胞分别同FGF-2和gFGF-2于37℃下培养72h,用血管生成试剂盒检测其能力。收集培养7d的细胞,贴壁细胞作EPCs附能力测定;用改良的Boyden小室作EPCs迁移能力测定;MTT法测定EPCs增殖能力;用放射性免疫法测定细胞的培养液GM-CSF,EPO和IL-8的含量。结果经蛋白指纹图谱分析鉴定FGF-2成功糖化成gFGF-2。EPCs体外功能测定表明gFGF-2组骨髓EPCs比FGF-2组明显的黏附能力降低(P<0.05),迁移能力降低(P<0.01),增殖能力减弱(P<0.05),成血管能力降低(P<0.01);EPC分泌的EPO(P<0.05)、GM-CSF(P<0.01)和IL-8(P<0.01)比对照组都减少(P<0.01)。结论EPC在gFGF-2的作用下数量减少且体外生物学活性降低,并且血管新生能力也减弱,推断gFGF-2可能是糖尿病患者体内血管新生功能降低的原因之一。
Objective To investigate the effect of exogenous glycated fibroblast growth factor-2 (gFGF-2) on the number and in vitro biological activity of rat bone marrow endothelial progenitor cells (EPCs). Methods gFGF-2 was prepared by co-culture of 10μg / ml FGF-2 and 250mmol / L D-fructose at 37 ℃ for 48h. The protein fingerprinting analyzer was used to detect the glycosylation status. Bone marrow mononuclear cells were obtained by density gradient centrifugation. After 4 days of culture, the rats were divided into three groups: FGF-2 (150μg / ml) and gFGF-2 (150μg / ml) EPCs were identified by immunohistochemistry and immunofluorescence staining for detection of CD34, CD133, VEGFR-2, ac-LDL and UEA-1. Adherent cells were cultured with trypsin digestion for 7 days. The cells were cultured with FGF-2 and gFGF-2 respectively at 37 ℃ for 72 hours. The ability of the cells was detected by using angiogenesis kit. EPCs were collected and cultured for 7 days. Adherent cells were used for determination of EPCs adhesion capacity. EPCs migration ability was measured with a modified Boyden chamber. EPCs proliferation was assayed by MTT assay. GM-CSF, EPO and IL- 8 content. Results FGF-2 was successfully glycated into gFGF-2 by protein fingerprinting analysis. The EPCs in gFGF-2 group showed significantly lower adhesion (P <0.05), migration ability (P <0.01), weaker proliferation ability (P <0.05) and decreased angiogenesis ability P <0.01). The levels of EPO secreted by EPCs (P <0.05), GM-CSF (P <0.01) and IL-8 (P <0.01) decreased compared with the control group (P <0.01). Conclusions The number of gFGF-2 induced EPCs decreased and the biological activity decreased in vitro, and the ability of angiogenesis decreased. It is concluded that gFGF-2 may be one of the reasons for the impaired function of angiogenesis in diabetic patients.