摘要
目的探讨miR-132-3p对内皮祖细胞增殖的影响及调控机制，为深静脉血栓的治疗提供新的理论依据。方法随机（随机数字法）挑选血栓患者22例，健康志愿者27例，采集静脉血，分离血浆和内皮祖细胞，实时定量PCR检测miR-132-3p在血浆及内皮祖细胞中的表达；实时定量PCR检测常氧和低氧条件下内皮祖细胞中miR-132-3p的表达；用电转法将miR-132-3p的模拟物（实验组）与特异性siRNA（对照组）分别转染入内皮祖细胞，MMT和CCK-8法检测miR-132-3p对内皮祖细胞增殖的影响；利用萤光素酶实验和免疫印迹实验分析miR-132-3p对内皮细胞中FOXO1表达的影响。结果血栓患者血浆中miR-132-3p平均水平为健康志愿者的（0．45±0．05）倍（P〈0．05）；低氧条件下内皮祖细胞中miR-132-3p表达水平为常氧状态下的（0．23±0．13）倍（P〈0．05）；实验组中miR-132-3p的表达水平是对照组的（15．72±2．06）倍，MMT法检测结果显示在低氧条件下，实验组细胞增殖是对照组的（7．79±1．37）倍（P〈0．01），CCK-8法检测结果表明实验组的细胞增殖是对照组的（6．46±0．38）倍（P〈0．01）；软件分析显示FOXO1为miR-132-3p的直接靶标，在低氧条件下miR-132-3p模拟物mimic转染的内皮祖细胞中荧光素酶活性是siRNA处理组的0．47倍，免疫印迹结果显示在低氧条件下miR-132-3p模拟物mimic转染的内皮祖细胞中FOXO1蛋白表达水平是siRNA处理组的0．18倍。结论与健康志愿者相比，血栓患者miR-132-3p表达明显降低，结果促进了FOXO1的表达，抑制了内皮祖细胞的增殖，这可能与静脉血栓的形成密切相关。
Objective To investigate the effect of miR-132-3p on the proliferation of endothelial progenitor cells and its regulatory mechanism in order to provide a new theoretical basis for the treatment of deep venous thrombosis. Methods Real-time quantitative PCR （qPCR） was used to detect the expression of miR-132-3p in the plasma and endothelial progenitor cells of 27 healthy volunteers and 22 thrombus patients, and in endothelial progenitor cells under normoxic and hypoxic conditions. The miR-132-3p analogue and the specific siRNA were transferred into endothelial progenitor cells by the electroporation method. The effect of miR-132-3p on the proliferation of endothelial progenitor cells was detected using MMT and Cell Counting Kit-8 （CCK-8） methods. The effects of miR-132-3p on the expression of FOXO1 in endothelial cells were analyzed using the luciferase assay and western blots.Results The expression of miR-132-3p in clinical patients with thrombosis was significantly decreased to 0.45 ± 0.05 times of that of the healthy volunteers （P〈0.05）. The expression of miR-132-3p in endothelial progenitor cells under hypoxia was down-regulated to （0.23 ± 0.13） times of that of under normoxia （P〈0.05）. The expression of miR-132-3p of experiment group under hypoxia was up-regulated to （15.72 ± 2.06） times of that of control group （P〈0.05）. MMT assay showed that the proliferation of cells in the experimental group under hypoxic condition was up-regulated to （7.79 ± 1.37） times of that in the control group （P〈0.01）. CCK-8 assay showed that cell proliferation in experimental group was up-regulated to （6.46 ± 0.38） times of that in the control group （P〈0.01）. Software analysis showed that FOXO1 was a direct target of miR-132-3p. Luciferase activity of miR-132-3p mimics transfected endothelial progenitor cells under hypoxic conditions were 0.47 times of that in siRNA treatment group. Western blot showed that the expression of FOXO1 protein in endothelial progenitor cells transfected with miR-132-3p mimics in hypoxia was 0.18 times of that in siRNA treatment group. Conclusions Compared with healthy volunteers, miR-132-3p expression in the blood of patients with thrombosis was significantly reduced that can promote transcription of the FOXO1 gene （and protein expression） and inhibit the proliferation of endothelial progenitor cells. It could be closely related to the formation of venous thrombosis.
作者
孙克玉
解梓琛
王继芹
刘梅
赵缜
宋振举
封启明
Sun Keyu;Xie Zichen;Wang Jiqin;Liu Mel;Zhao Zhen;Song Zhenju;Feng Qiming(Department of Emergency Medieine , Clinical Laboratory, Minghang Central Hospital, Shanghai, 201199, China;Department of Emergency Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032, China;Department of Emergency Medicine, Shanghai Sixth People＇s Hospital Affiliated to Shanghai Jiao Tong University, Shanghai 200023, China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2018年第6期652-656,共5页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金（81471840）
上海市重点专科建设项目（ZK2015B15）
上海市卫计委项目（201740127）
