促红细胞生成素促进内皮祖细胞增殖依赖于PI3K/Akt信号通路  

Erythropoietin promotes endothelial progenitor cells proliferation depending on PI3k/Akt pathway

作　　者：吴海卫[1] 张雷[1] 胡若愚[2] 李好[3] 景华[1] 董国华[1] 许飚[1] 李德闽[1] 

机构地区：[1]南京大学临床学院解放军南京军区南京总医院心胸外科,江苏省南京市210002 [2]东南大学附属中大医院心胸外科,江苏省南京市210009 [3]上海市肺科医院胸外科,上海市200433

出　　处：《中国组织工程研究》2014年第45期7312-7319,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(30972969);南京军区医学科技创新课题(10MA100)~~

摘　　要：背景:促红细胞生成素能促进组织损伤部位血管生成,与其促进内皮祖细胞增殖、分化密切相关,但促红细胞生成素促进内皮祖细胞增殖、分化的机制尚不清楚。目的:观察促红细胞生成素对小鼠骨髓来源内皮祖细胞功能活性的影响,并初步阐明其信号机制。方法:密度梯度离心法分离获取小鼠骨髓内皮祖细胞,鉴定后传代培养,以PI3K特异性抑制剂LY294002作干预处理,实验细胞分为EGM-2组、促红细胞生成素处理组(培养液中促红细胞生成素浓度分别为1,5,10 U/m L)、促红细胞生成素+LY组(培养液中分别含有10 U/m L促红细胞生成素及10 mmol/L LY294002)、LY组(培养液中含10 mmol/L LY294002)、二甲基亚砜组(培养液中含1 m L/L二甲基亚砜),分别采用CCK8试剂盒、流式细胞法检测细胞增殖和凋亡,采用ELISA法检测细胞裂解液内皮型一氧化氮合酶、血管内皮生长因子含量,Western blot法测定细胞裂解液中Akt及p-Akt表达。结果与结论:促红细胞生成素能显著促进内皮祖细胞增殖,并随培养基中促红细胞生成素含量增加而呈现量效关系,而促红细胞生成素的促增殖作用可被LY294002完全抑制。促红细胞生成素处理组细胞凋亡率明显低于促红细胞生成素+LY组。LY组、促红细胞生成素+LY组细胞裂解液中内皮型一氧化氮合酶、血管内皮生长因子含量显著低于促红细胞生成素处理各组。各组Akt表达无明显差异,而促红细胞生成素+LY组p-Akt表达显著低于促红细胞生成素各组。上述结果提示,促红细胞生成素能显著促进体外培养的内皮祖细胞的增殖、降低内皮祖细胞的凋亡率,其作用依赖于PI3K/Akt信号通路。BACKGROUND：It has been proved that erythropoietin can promotes angiogenesis in injured tissue, which is closely related to the proliferation and differentiation of endothelial progenitor cel s. However, the involved mechanism remains unclear yet. OBJECTIVE：To investigate the effect of erythropoietin on the function and activity of bone marrow-derived endothelial progenitor cel s in mice, and to explore the signal pathway. METHODS：The endothelial progenitor cel s from the bone marrow of mice were separated by means of density gradient centrifugation and then cultured. The cel s were preconditioned by specific inhibitor of PI3K 〈br〉 （LY294002）, and were divided into the fol owing groups：EGM-2 group, three erythropoietin preconditioned groups （the concentrations of erythropoietin in medium were 1, 5, 10 U/mL respectively）, erythropoietin＋LY group （10 U/mL erythropoietin and 10 mmol/L LY294002 in medium）, LY group （10 mmol/L LY294002 in medium）, dimethyl sulfoxide group （1 mL/L dimethyl sulfoxide in medium）. The cel proliferation and apoptosis were evaluated by cel counting kit-8 and flow cytometry respectively. The contents of endothelial nitric oxide synthase and vascular endothelial growth factor in cel lysates were detected by the method of ELISA, and the expressions of Akt and p-Akt were by western blot assay. 〈br〉 RESULTS AND CONCLUSION：Erythropoietin could promote the proliferation of endothelial progenitor cel s in a dose-dependent manner, which was, however, completely inhibited by LY294002. The apoptosis rate in the erythropoietin preconditioned groups was significantly lower than that in the erythropoietin＋LY group. The contents of endothelial nitric oxide synthase and vascular endothelial growth factor in cel lysates of LY group and erythropoietin＋LY group were significantly lower than those in the erythropoietin groups. There was no difference in Akt expression found in each group, while the p-Akt expression in the erythropoietin＋LY group was significantl

关 键 词：干细胞 培养 内皮祖细胞 促红细胞生成素 磷脂酰肌醇3-激酶 信号通路 国家自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]