促红细胞生成素诱导骨髓内皮祖细胞功能活化与JAK2依赖ERK信号途径的作用

Effects of JAK2-dependent ERK signal pathway on erythropoietin-induced functional activation of bone marrow-derived endothelial progenitor cells

背景:促红细胞生成素能够促进内皮细胞增殖、迁延,形成新生血管,因此促红细胞生成素可能诱导内皮祖细胞功能活化。除了经典的造血功能以外,是否内皮祖细胞也是促红细胞生成素促血管生成效应的一个靶点。目的:观察促红细胞生成素对大鼠骨髓内皮祖细胞功能活性的影响,并分析其信号途径。方法:用密度梯度离心法分离大鼠骨髓内皮祖细胞。首先将细胞分为无血清DMEM培养液对照组、重组人促红细胞生成素500,1000,2000U/L组、促红细胞生成素(2000U/L)与ERK抑制剂FR180204(50μmol/L)共同作用组,分别用MTT比色法、Transwell小室、Matrigel、Westernblot法检测内皮祖细胞的增殖活性、迁移、管腔形成能力及Bcl-2表达。然后将细胞分为无血清DMEM培养液对照组、重组人促红细胞生成素组2000U/L组,促红细胞生成素(2000U/L)与JAK2抑制剂SD1029(10μmol/L)共同作用组,用Westernblot法检测P-ERK的表达。结果与结论:与对照组相比,随着浓度的增加,促红细胞生成素可明显增强内皮祖细胞的增殖活性,增加迁移的细胞数及形成的管腔数,上调Bcl-2蛋白的表达,在一定范围内呈剂量依赖效应。当加入ERK抑制剂FR180204后,与促红细胞生成素2000U/L组相比,这些效应明显减弱。此外,促红细胞生成素上调p-ERK1/2的表达,当加入JAK2抑制剂SD1029后,p-ERK1/2的表达明显下调。提示促红细胞生成素能够促进内皮祖细胞功能活化,这种效应可能是通过依赖JAK2的ERK信号途径调节的。

BACKGROUND：Erythropoietin（EPO） can promote the proliferation and migration of endothelial cells to form new vessels,so EPO may play an important role in functional activation of endothelial progenitor cells（EPCs）.Except hematopoiesis,whether EPCs are a target of EPO remains poorly understood.OBJECTIVE：To investigate the effects of EPO on functional activation of EPCs and its signal pathway.METHODS：EPCs were separated from bone marrow of rats using density gradient centrifugation.First,cells were divided into control group（serum-free DMEM culture medium）,recombinant human erythropoietin（rhEPO） in different dose groups（500,1 000,2 000 U/L）,EPO（2 000 U/L） and the ERK inhibitor FR180204（50 μmol/L） interaction group.Proliferation,migration,tubes formation and Bcl-2 expression of EPCs were detected with MTT assay,Transwell chamber,Matrigel and Western blot assay respectively.Secondly,cells were divided into control group（serum-free DMEM culture medium）,rhEPO group（2 000 U/L）,rhEPO（2 000 U/L） and JAK2 inhibitor SD1029（10 μmol/L） interaction group.P-ERK expression of EPCs was detected by Western blot assay.RESULTS AND CONCLUSION：Compared with control group,EPO significantly increased EPCs proliferation,the number of migrating cells and tubes formation,up-regulated Bcl-2 protein expression in a dose-dependent manner in a certain rage.After added ERK inhibitor FR180204,these effects were significantly decreased compared with the rhEPO group（2 000 U/L）.In addition,compared with the control group,the p-ERK1/2 expression in the rhEPO group（2000 U/L） was significantly up-regulated,JAK2 inhibitor SD1029 made the result reduced markedly.These indicate that EPO can promote functional activity of EPCs,which might be modulated by JAK2-dependent ERK signal pathway.