内皮克隆形成细胞旁分泌对人脐静脉内皮细胞生物学功能的影响  

Paracrine effect of endothelial colony-forming cells on biological functions of human umbilical vein endothelial cells

作　　者：王布霖 郭晓瑞 李勤[2] 

机构地区：[1]南方医科大学,广东省广州市510515 [2]解放军广州军区广州总医院,广东省广州市510010 [3]广东省中山市第二人民医院,广东省中山市528447

出　　处：《中国组织工程研究》2017年第8期1221-1228,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81571910);广东省科技计划项目(2014A020212256);广州市科技计划项目(201607010394)~~

摘　　要：背景:研究表明,内皮克隆形成细胞移植对缺血损伤组织的血管新生具有促进作用。然而,这一作用是否与其旁分泌效应有关,仍有待进一步研究证实。目的:检测人脐血来源内皮克隆形成细胞条件培养基中细胞因子分泌谱,并观察内皮克隆形成细胞条件培养基对人脐静脉内皮细胞增殖、迁移及成管能力的影响。方法:从人脐血中分离、培养内皮克隆形成细胞,并进行细胞鉴定。采用抗体芯片对无血清内皮克隆形成细胞条件培养基中的细胞因子进行检测。采用无血清EBM-2培养基作为对照组,应用CCK-8、细胞划痕实验、Matrigel成管实验检测内皮克隆形成细胞条件培养基对人脐静脉内皮细胞增殖、迁移及成管能力的影响。结果与结论:(1)原代培养细胞形态:培养的细胞呈"铺路石"样生长,表达CD34,KDR及CD144,不表达CD45及CD133,Dil-acLDL摄取及FITC-UEA-I结合双阳性,在Matrigel基质胶中可形成管腔样结构,以上均符合内皮克隆形成细胞的特征;(2)抗体芯片检测:内皮克隆形成细胞条件培养基高表达30种促血管新生因子;(3)CCK-8检测:实验组人脐静脉内皮细胞在24,48,72 h增殖活性均高于对照组(P<0.01);(4)细胞划痕实验结果显示,实验组人脐静脉内皮细胞迁移率在12,24 h均高于对照组(P<0.01);(5)与对照组相比,实验组人脐静脉内皮细胞在Matrigel基质胶上可形成更多的管状结构(P<0.01)。(6)结果表明:内皮克隆形成细胞能通过旁分泌效应促进成熟内皮细胞的生物活性。BACKGROUND： Endothelial colony-forming cells （ECFCs） transplantation exerts beneficial impact on angiogenesis of impaired tissues caused by ischemia. However, whether this impact is related to the paracrine effect of ECFCs needs to be studied further.OBJECTIVE： To detect the cytokine secretion profile of human umbilical cord blood derived-ECFCs conditioned medium （ECFCs-CM） and explore the effect of ECFCs-CM on the proliferation, migration and tube formation ability of human umbilical vein endothelial cells （HUVECs）.METHODS： Human cord blood derived-ECFCs were isolated, cultured in vitro, and then identified based on the previous studies. The cytokines in serum free ECFCs-CM were detected using a cytokines antibody array. HUVECs were cultured with ECFCs-CM, serum free EBM-2 as control. The proliferation, migration and tube formation abilities of HUVECs were examined by cell counting kit-8, scratch test and Matrigel assay, respectively.RESULTS AND CONCLUSION： The cultured cells demonstrated typical characteristics of ECFCs, which showed cobblestone appearance and were positive for CD34, KDR and CD144, but not CD45 or CD133, uptook Dil-acLDL and bond FITC-UEA-I, and tube-like structures were formed on Matrigel. The cytokine antibody array showed that ECFCs-CM significantly upregulated the expressions of 30 kinds of angiogenic factors. Compared with the control group,the HUVECs cultured with ECFCs-CM showed significantly improved proliferation ability at 24, 48 and 72 hours （P 〈0.01）. The migration rate of HUVECs in the experimental group was significantly higher than that in the control group at 12 and 24 hours （P 〈 0.01）. There were more tubular structures in the experimental group than those in the control group （P 〈 0.05）. These results indicate that ECFCs can promote the bioactivity of mature endothelial cells through paracrine action.

关 键 词：干细胞 组织工程 细胞增殖 组织构建 内皮细胞 内皮祖细胞 内皮克隆形成细胞 细胞培养 旁分泌 条件培养基 血管新生 血管生成 慢性创面愈合 国家自然科学基金 

分 类 号：R318[医药卫生—生物医学工程]