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|本期目录/Table of Contents|

脂联素对高糖刺激下内皮祖细胞的作用及其可能的作用机制

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2013年第1期
页码:
10-016
栏目:
基础研究
出版日期:
2013-02-25

文章信息/Info

Title:
Effect of adiponectin on highglucosetreated endothelial progenitor cells and its possible mechanisms
作者:
冷 冰; 张 鹏; 任雨笙; 梁 春; 曹智勇; 吴宗贵
(第二军医大学附属长征医院心内科，上海 200003)
Author(s):
LENG Bing;  ZHANG Peng;  REN Yusheng;  LIANG Chun;  CAO Zhiyong;  WU Zonggui
(Department of Cardiology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China)
关键词:
人内皮祖细胞; 脂联素; 葡萄糖
Keywords:
human endothelial progenitor cells;  adiponectin;  glucose
分类号:
R329.2
DOI:
-
文献标识码:
A
摘要:
目的:研究脂联素(adiponectin,APN)对高糖刺激下内皮祖细胞(Endothelial Progenitor Cells,EPCs)的作用，并探讨其可能的机制。方法： 密度梯度离心法分离人外周血单个核细胞，经含血管内皮生长因子、碱性成纤维细胞生长因子和100 ml/L胎牛血清的M199培养基培养7 d，贴壁细胞进行形态学、流式细胞仪测细胞分子标志物(CD34、CD133和KDR)和激光共聚焦倒置显微镜下观察培养细胞摄取acLDL和结合UEAI经何种方法鉴定为EPCs。细胞同步化后，将其随机分为7组：正常糖浓度对照组(55 mmol/L)、高渗对照组（55 mmol/L葡萄糖+245 mmol/L甘露醇及其浓度）、高糖(30mmol/L)组及高糖APN干预组(30 mmol/L葡萄糖合并APN，125、25、5、10 μg/ml)。干预48 h后，分别采用MTT比色法、Transwell小室检测EPCs的增殖、迁移；以Annexin VFITC凋亡检测试剂盒处理细胞，流式细胞仪检测EPCs的凋亡；用荧光探针(DCFHDA)检测法进行细胞活性氧(reactive oxygen species,ROS)检测。结果： ①经密度梯度离心法分离出的外周血单个核细胞培养7 d后，细胞集落增加明显，梭形细胞增多并呈交叉性生长；用激光共聚焦倒置显微镜观察，细胞摄取DiIacLDL呈红色荧光，摄取FITCUEAI呈绿色荧光，摄取DiIacLDL并结合FITCUEAI的细胞呈黄色荧光，流式细胞仪分析结果提示：细胞表达KDR(9232%)、CD133(107%)、CD34(233%)，证实培养的细胞是正在分化的EPCs。②随着糖浓度的增高，EPCs的增殖能力下降，凋亡、ROS水平增加(P<001)；当糖浓度为30 mmol/L与50 mmol/L时，两者相比对EPCs的影响无统计学差异。③与正常糖浓度对照组相比，高渗对照组EPCs的增殖、迁移、凋亡和ROS水平无统计学差异。在30 mmol/L葡萄糖条件下，EPCs的数量和迁移功能较正常对照组明显下降，细胞凋亡增多，不同浓度APN干预后能明显提高高糖损伤后EPCs的功能(P<005，P<001)，并随着浓度的增加，EPCs的增殖与迁移能力增高，凋亡、ROS水平下降(P<001)。结论： ①与对照组相比，高糖干预可导致EPCs增殖能力下降，细胞凋亡增多；②高糖干预后加入APN，EPCs的增殖、迁移能力恢复，细胞凋亡减少，并在一定范围内，其作用随浓度的增加而增强；③高糖可以引起EPCs功能受损，其作用机制可能与ROS水平升高有关；而APN可以通过降低高糖引起的ROS水平，保护EPCs功能；渗透压对EPCs无影响。
Abstract:
AIM:To investigate the effect of adiponectin (APN) on hyperglycemiainduced oxidative damage of endothelial progenitor cells (EPCs). METHODS: Blood mononuclear cells were isolated from human peripheral blood by Ficoll density gradient centrifugation. After 7 days in M199 medium containing rhVEGF, rhbFGF and 10% fetal bovine serum, the attached cells were identified as EPCs by cell morphology. Cell molecular markers (CD34 CD133, and KDR) were measured by flow cytometry and the capabilities of cultured cells in absorbing acLDL and combining with UEAI were observed by confocal laserinverted microscope. Cells were synchronized and then divided into seven groups: control group (55 mmol/L), high glucose group (30 mmol/L), four adiponectintreated intervention groups, respectively, with different concentrations of adiponectin (125, 25, 5 or 10 μg/ml) and hypertonic control group. After 7 days of culture, adherent cells were collected and incubated with adiponectin for 48 h. Proliferation, migration, apoptosis rate and reactive oxygen species were tested by MTT, transwell chamber, flow cytometry and fluorescent probe (DCFH). RESULTS: In peripheral blood mononuclear cells isolated from peripheral blood by Ficoll density gradient centrifugation following the 7day treatment, an obvious increase in cell groups and growth of spindle cells were observed. The expressions of CD34, CD133 and KDR were positive and the expression rates were 233%, 107% and 9232%, respectively. By confocal microscopy, cell uptake of DiIAcLDL was combined with FITCUEAI. Doublestaining positive rate of cells proved that the cultured cells were EPCs. With the increase of the sugar concentrations, EPC proliferation decreased, whereas apoptosis and ROS levels increased (P<001). No statistical difference was observed in the effects on EPCs between glucose concentrations of 50 and 30 mmol/L. With the increase of APN concentration, EPC proliferation increased, whereas apoptosis and ROS levels dropped (P<001). No statistical difference was observed in the effects on EPCs between APN concentrations of 10 and 5 μg/ml. The osmotic pressure did not affect the results. CONCLUSION: Compared with the control group, the injured EPCs induced by high glucose showed lower proliferation and migration and a higher rate of apoptosis. EPCs incubated with adiponectin have higher capacities than those incubated by high glucose. High sugar increases ROS levels of EPCs and impairs the function of EPCs, whereas APN lowers high sugarinduced ROS levels and protects EPC function. The osmotic pressure has no effect on EPC function.

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备注/Memo

备注/Memo:
收稿日期：2012-02-24.基金项目：上海市科学技术委员会科研计划项目资助 (10411963900) 通讯作者：任雨笙，主任医师，主要从事冠心病临床及基础研究Email:renyusheng@gmail.com 作者简介：冷冰，硕士生Email:sunhaibosunhaibo@163.com
更新日期/Last Update: 2013-03-20