胰高血糖素样肽1对内皮祖细胞增殖分化能力的影响及其机制

Effect of glucagon like peptide-1 on proliferation and differentiation of endothelial progenitor cells and its mechanism

目的:探讨胰高血糖素样肽1(GLP-1)对体外培养的人外周血内皮祖细胞(EPCs)增殖分化能力的影响及可能的作用机制。方法:采用密度梯度离心法分离获得人外周血单个核细胞,培养7d后,收集贴壁细胞。贴壁细胞用不含FBS的M199培养液培养24h后,分成4组:对照组和GLP-1各浓度组(1,10,20nmol/L GLP-1共3组),分别用含0.2%BSA,1,10,20nmol/L GLP-1的M199培养液培养72h。倒置相差显微镜下观察EPCs生长情况,MTT法检测EPCs的增殖能力;RT-PCR技术检测EPCs中KDR,Flt-1,VE-cadherin,内皮型一氧化氮合酶(eNOS)mRNA的表达,ELISA法测定细胞上清液内皮细胞生长因子(VEGF)浓度,SP法检测细胞VEGF蛋白表达。同时用100ng/m LVEGF mAb对10nmol/LGLP-1培养的EPCs进行干预。结果:与对照组比较,GLP-1各浓度组EPCs增殖明显(P<0.05或P<0.01),EPCs中KDR,Flt-1,VE-cadherin,eNOS mRNA表达增强,细胞上清液中VEGF浓度显著升高、细胞VEGF蛋白表达增强(P<0.05或P<0.01)。VEGF mAb(100ng/mL)下调GLP-1培养的EPCs增殖能力,细胞KDR,Flt-1,VE-cadherin,eNOS mRNA表达下降。结论:GLP-1对体外培养的人外周血EPCs的增殖和分化能力具有一定的促进作用,上调VEGF自分泌是其可能的作用机制之一。

Objective To investigate the effect and mechanism of glucagon like peptide 1（GLP-1）on the proliferation and differentiation of endothelial progenitor cells（EPCs）derived from the peripheral blood. Methods Mononuclear cells were isolated from human peripheral blood by density gradient centrifugation. After 7 days of culture,attached cells were stimulated with different cultures of 0.2% BSA,and GLP-1（1,10,and 20 nmol/L）. Laser scanning confocal microscope was used to determine the EPCs from human peripheral blood.The activity of EPCs was observed under reverse microscope. MTT was used to determine the proliferation of EPCs. The expression of KDR,Flt-1,VE-cadherin,and eNOS mRNA was detected by RT-PCR.The concentration of serum VEGF was detected by ELISA. The expression of VEGF protein was detected by immunohistochemical SP method. The EPCs cultured in GLP-1 were intervened by VEGFmAb.Results EPCs was proliferated more in the GLP-1 group（1,10,and 20 nmol/L） than in the control group （P0.05 or P0.01）. The expression of KDR,FLT-1,VE-cadherin,eNOS mRNA and VEGF protein was higher than that in the control group（P0.05 or P0.01）. VEGFmAb（100 ng/mL）down-regulated the expression of KDR,Flt-1,VE-cadherin,and eNOS mRNA. Conclusion GLP-1 can promote the proliferation and differentiation of EPCs derived from the peripheral blood by up-regulating VEGF autocrine.