Klotho蛋白促进内皮祖细胞旁分泌和分化的作用及机制研究

Role and mechanism of Klotho protein in promoting paracrine secretion and differentiation of endothelial progenitor cells

目的探讨Klotho蛋白促进内皮祖细胞(EPC)旁分泌和分化的作用及机制。方法选取健康人外周血分离培养EPC,分为对照组、Klotho组、AKT阻断组及ERK阻断组(n=6)。采用流式细胞术检测其表型;ELISA方法检测血管内皮生长因子(VEGF)、基质细胞衍生因子(SDF1)和碱性成纤维生长因子(bFGF)水平;MTT检测内皮细胞增殖率;划痕实验检测内皮细胞的迁移;流式细胞术检测EPC向CD31阳性内皮细胞的分化。结果EPC培养到第7天后,CD34的阳性率为93.6%,VEGF受体2的阳性率为94.9%。与对照组比较,Klotho组、AKT阻断组及ERK阻断组内皮细胞增殖率、内皮细胞划痕区域闭合率、CD31阳性细胞比例更高(P<0.05,P<0.01);AKT阻断组VEGF、SDF1、bFGF表达明显低于Klotho组和ERK阻断组[(46.44±4.62)ng/ml vs(70.29±5.87)ng/ml和(65.21±4.69)ng/ml;(16.56±2.03)ng/ml vs(37.84±3.53)ng/ml和(34.29±3.72)ng/ml;(5.09±1.40)ng/ml vs(7.52±2.61)ng/ml和(7.36±2.18)ng/ml,P<0.05]。与Klotho组比较,AKT阻断组内皮细胞增殖率、划痕区域闭合率及CD31阳性细胞比例显著降低[(29.81±2.55)%vs(43.37±4.29)%,(29.41±3.46)%vs(56.56±6.42)%,(7.08±1.11)%vs(23.91±3.69)%,P<0.01]。结论Klotho蛋白能够通过AKT信号通路,促进EPC旁分泌和分化,进而促进血管生成。

Objective To studye the role and mechanism of Klotho protein in predicting the paracrine secretion and differentiation of EPC.Methods The EPC isolated from the peripheral blood of healthy people were divided into control group,Klotho group,AKT block group and ERK block group(n=6).The phenotype of EPC was identified by flow cytometry.The expressions of VEGF,SDF1 and bFGF were detected by ELISA.The proliferation of endothelial cells was detected with MTT.The migration of endothelial cells was assayed by scratch assay.The differentiation of EPC into CD31-positive endothelial cells was detected by flow cytometry.Results The positive rate of CD34 and VEGFR-2 was 93.6%and 94.9%respectively on day 7 after the EPC were cultured.The proliferation rate and closure rate of endothelial cells in scratch area and the rate of CD31-positive cells were significantly higher in Klotho group,AKT block group and ERK block group than in control group(P<0.05,P<0.01).The expression levels of VEGF,SDF1 and bFGF were significantly lower in AKT block group than in Klotho group and ERK block group(46.44±4.62 ng/ml vs 70.29±5.87 ng/ml,65.21±4.69 ng/ml;16.56±2.03 ng/ml vs 37.84±3.53 ng/ml,34.29±3.72 ng/ml;5.09±1.40 ng/ml vs 7.52±2.61 ng/ml,7.36±2.18 ng/ml,P<0.05).The proliferation rate of endothelial cells and closure rate of endothelial cells in scratch area were significantly lower in AKT block group than in Klotho group(29.81%±2.55%vs 43.37%±4.29%,29.41%±3.46%vs 56.56%±6.42%,P<0.01).The rate of CD31-positive cells was significantly lower in AKT block group than in control group(7.08%±1.11%vs 23.91%±3.69%,P<0.01).Conclusion Klotho protein can promote the secretion of paracrine and differentiation of EPC through the AKT signaling pathway,thus stimulating the vascularization.