5-氮杂-2’-脱氧胞苷对内皮祖细胞分化的影响及机制研究  

Effects and Mechanism of 5-aza-2'-deoxycytidine on the Differentiation of Endothelial Progenitor Cells

作　　者：刘加远 杨国源[2] 杨志军[1] 曲美洁 戴宜武[1] 徐如祥[1] 

机构地区：[1]安徽医科大学北京军区总医院临床学院,100070 [2]上海交通大学生物医学工程学院,200030 [3]上海交通大学医学院附属瑞金医院神经内科,200025

出　　处：《心脑血管病防治》2016年第2期86-90,共5页CARDIO-CEREBROVASCULAR DISEASE PREVENTION AND TREATMENT

基　　金：国家自然科学基金(编号:812713156)

摘　　要：目的探讨5-氮杂-2’-脱氧胞苷诱导内皮祖细胞分化为内皮细胞的作用及初步机制研究。方法收集脐带血,Ficoll-Paque淋巴细胞分离液和6%羟乙基淀粉沉降法分离获得单核细胞并培养,采用免疫组化及倒置相差显微镜鉴定细胞为内皮祖细胞(EPCs),EPCs培养7d后,分别加入1μmol/L(即d C1组),3μmol/L(即d C2组)5-Aza-d C的培养液,阴性组为采用0.0076%DMSO进行干预。流式细胞术检测5-Aza-d C处理后第0、3、7d后CD34+、CD31+的表达,RT-PCR分析VE-cadherin、Tie-2及v WF基因表达,western blotting检测v WF蛋白表达,MSP法检测BMP4基因Cp G岛甲基化表达情况。结果 1单核细胞经倒置相差显微镜及VE-cadherin、Tie-2、v WF、KDR、CD34及CD133等因子证实为EPCs;2流式细胞术结果显示,d C2组在经5-Aza-d C处理后的第3d、第7d CD34+(分别为30.2%、6.7%)降低,同阴性组(46.7%、40.8%)比较差异具有统计学意义(均P<0.05);d C2组在经5-Aza-d C处理后的第7d C CD31+(47.9%)升高,同阴性组(31.3%)比较差异具有统计学意义(P<0.05);3RT-PCR结果显示,d C2组在处理后第14d Tie-2、VE-cadherin基因均明显高于阴性组(均P<0.05);d C1、d C2组在处理后的第3d、第7d、第14d Tie-2、v WF、VE-cadherin基因均明显高于处理后的第0d(均P<0.05);4Western blotting检测显示,d C2组第0d、第3d、第7d v WF蛋白表达量均高于阴性组(均P<0.05);d C1、d C2组在第3d、第7d v WF蛋白表达量均高于所对应组别的第0d v WF蛋白表达量(均P<0.05);5甲基化处理后d C1组和d C2组BMP4呈低表达,而非甲基化时d C1组和d C2组BMP4呈强表达。结论 5-Aza-d C具有定向且快速诱导EPCs分化为内皮细胞的可能,这一作用机制可能与促进BMP4分化基因表达,进而刺激Tie-2、VE-cadherin及v WF的表达的作用有关。Objective To investigate the effects and mechanism of 5_aza_2’_deoxycytidine induce endothelial progenitor cells to dif-ferentiate into endothelial cells.Methods mononuclear cells were separated from umbilical cord blood ,using Ficoll_Paque lymphocyte separation medium and 6% hydroxyethyl starch sedimentation ,which were cultured and differentiate into endothelial progenitor cells （EPCs）.The EPCs were identified using immunohistochemistry and inverted phase contrast microscope.After 7 days ,1μmol/L （ie dC1 group） or 3μmol/L （ie dC2 group） of 5_Aza_dC was added.0.0076% DMSO was used as intervention in the negative group.CD34＋ ,CD31＋ expression influenced by 5_Aza_dC Treatment after 0 ,3 ,7 ,14d were evaluated by flow cytometry ,VE_cadherin , Tie_2 ,gene expression of vWF were analyzed by RT_PCR ,protein expression of vWF was tested by western blotting ,gene expression of CpG island methylation of BMP4 was detected by MSP. Results ① monocytes were proved to differentiate into EPCs by inverted phase contrast microscope ,VE_cadherin ,Tie_2 ,vWF ,KDR ,CD34 and CD133 and other factors ;② It was showed in flow cytome-try that CD34＋ （respectively ,30.2% ,6.7% ） in dC2 group by 5_Aza_dC at 3d ,7d was statistical significantly higher than negative group （46.7% ,40.8% ;all P〈0.05） ,CD31＋ （47.9% ） in dC2 group by 5_Aza_dC at 7d was statistical significantly higher than negative group （31.3% ;all P〈0.05） ③It was showed in RT_PCR that Tie_2 ,VE_cadherin gene in dC2 group at 14d were significantly higher than negative group （ t=8.12 ,12.28 ,all P〈0.05 ）;Tie_2 ,vWF ,VE_cadherin gene in dC1 ,dC2 group at 3d ,7d ,14d t were higher than at 0d （all P〈0.05） ;④In Western blotting anal-ysis ,it was showed that vWF protein expression in dC2 group at 0d , 3d ,7d were higher than negative group in those days （ P〈0.05） ,WF protein expression in dC1 ,dC2 group at 3d ,7d were higher than the protein expression at 0d in each two group （all P〈0.05）;⑤After methylated

关 键 词：5_氮杂_2’_脱氧胞苷 内皮祖细胞 分化 内皮细胞 

分 类 号：R54[医药卫生—心血管疾病]