植物雌激素α-玉米赤霉醇对内皮祖细胞的促增殖效应

Phytoestrogen α-Zearalanol Promotes Proliferation of Endothelial Progenitor Cells in Vitro

目的:观察植物雌激素α-玉米赤霉醇(α-ZAL)对内皮祖细胞(EPCs)增殖能力的影响并探讨其机制。方法:密度梯度离心法获取大鼠外周血单个核细胞,加用内皮细胞培养基EGM-2培养,免疫荧光染色和流式细胞术等进行EPCs鉴定,培养2～3代后用于实验。分别用10-5mol/LH2O2和磷脂酰肌醇-3-激酶(PI3K)及下游靶蛋白Akt(PI3K/Akt)抑制剂LY294002处理细胞,并加用10-6和10-7mol/Lα-ZAL进行干预。检测EPCs上清液中乳酸脱氢酶(LDH)活性以反映细胞损伤情况,用MTT法、EPCs克隆数及细胞总数计数反映EPCs增殖情况,Westernbloting法检测Akt及磷酸化Akt(phosphor-Akt)蛋白表达。结果:经鉴定培养细胞为EPCs;10-5mol/LH2O2及PI3K/Akt抑制剂LY294002可明显降低EPCs存活率(P<0.05),减少细胞克隆,增加LDH活性(P<0.01);而加入10-6和10-7mol/Lα-ZAL干预后能减轻以上作用(P<0.05),同时10-6mol/Lα-ZAL能减少LY294002抑制EPCsAkt的表达和磷酸化。结论:α-ZAL能促进EPCs的体外增殖能力,其机制可能与激活PI3K/Akt信号通路有关。

Objective：To investigate the effect of phytoestrogen α-zearalanol（α-ZAL） on proliferation of rat endothelial progenitor cells（EPCs） and mechanism involved.Method：Mononuclear cells were isolated from rat peripheral blood in vitro by ficoll density gradient centrifugation and then the cells were seeded on rat tail collagenⅠcoated culture plates,and resuspended in EBM-2 medium.EPCs were identified by immunofluorescence staining and flow cytometry analysis.After 2~3 passages,EPCs were used for following experiments：stimulated by 10-5 mol/L H2O2 or PI3K/Akt inhibitor LY294002,and pretreated with 10-6 or 10-7 mol/L α-ZAL.The damage of EPCs was detected by LDH activity;the proliferation of EPCs was detected by MTT and counting of EPCs colony numbers and total cell number;the total Akt and phosphorylation of Akt were determined by Western blot.Results：After identification,cells cultured were EPCs;10-5 mol/L H2O2 and PI3K/Akt inhibitor LY294002 decreased EPCs survival rate（P0.05）,LDH activity and cloning formation ability（P0.01）;10-6 and 10-7 mol/L α-ZAL pretreatment alleviated those effect（P0.05）.10-6 mol/L α-ZAL could decrease the inhibition effect of LY294002 on attenuating the expression and phosphorylation of Akt.Conclusion：α-Zearalanol could promote the proliferation of EPCs in vitro and the PI3K/Akt pathway might be involved in this effect.