内皮祖细胞移植对脓毒症大鼠的治疗作用  

Role of endothelial progenitor cell transplantation in rats with sepsis

作　　者：徐喜媛[1] 杨敬平[1] 那仁格日勒[1] 乌日娜[1] 田红军[1] 宋慧芳[1] 王慧[1] 

机构地区：[1]内蒙古医科大学第三附属医院呼吸与危重症医学科,内蒙古包头014010

出　　处：《中华危重病急救医学》2015年第6期514-519,共6页Chinese Critical Care Medicine

基　　金：内蒙古自治区自然科学基金（2012MS1163）

摘　　要：目的：探讨内皮祖细胞（EPCs）移植在细菌内毒素脂多糖（LPS）所致脓毒症中的治疗作用。方法选择同遗传背景的清洁级雄性SD大鼠60只，按随机数字表法将大鼠分为对照组、模型组和EPCs移植组3组，每组20只。尾静脉注射LPS 5 mg/kg诱导脓毒症模型，对照组注射等量生理盐水。分离、培养、鉴定大鼠EPCs，用绿色荧光蛋白腺病毒转染标记EPCs。EPCs移植组于LPS注射后1 h经尾静脉注入荧光标记的EPCs悬液，活体成像仪及冰冻切片检测组织中荧光标记的EPCs表达。移植后7 d解剖大鼠经腹主动脉采血，酶联免疫吸附试验（ELISA）检测外周血白细胞介素（IL-6、IL-10）水平；取肺、肝及肾组织，检测各组织湿/干质量（W/D）比值，苏木素-伊红（HE）染色观察各组织病理学改变，实时反转录-聚合酶链反应（RT-PCR）检测各组织Toll样受体4（TLR4）mRNA表达。结果流式细胞仪检测培养第5代时EPCs细胞CD133、CD34的双标记阳性率为99.0%；将标记绿色荧光蛋白的EPCs移植给大鼠后，荧光显示EPCs主要集中在大鼠的胸腹部，血管附近荧光较强。EPCs移植可显著减轻脓毒症大鼠肺、肝及肾组织炎细胞浸润、细胞损伤。与对照组比较，模型组外周血IL-6、IL-10水平明显升高，各器官组织W/D比值及TLR4 mRNA表达也明显升高。EPCs移植可显著降低模型大鼠外周血IL-6、IL-10水平〔IL-6（μg/L）：2.127±0.118比2.664±0.438，IL-10（ng/L）：24.5±3.9比31.5±3.8，均P＜0.01〕，降低肺、肝及肾组织W/D比值（肺组织：4.68±0.24比5.48±0.15，肝组织：3.33±0.11比3.94±0.09，肾组织：4.08±0.20比4.84±0.21，均P＜0.01），下调肺、肝及肾组织TLR4 mRNA表达（×10^3，肺组织：782±131比1136±126，肝组织：39.1±14.0比69.2±8.7，肾组织：52.2±15.2比83.5±17.1，均P＜0.01）。结论 EPCs静脉移植后能成功到达脓毒症大鼠的肺、肝及肾组织，下调促炎�Objective To investigate the role of endothelial progenitor cells （ EPCs ） transplantation in rats with sepsis induced by endotoxin （ lipopolysaccharides, LPS ）. Methods Sixty clean grade Sprague-Dawley （ SD ） rats with genetic background were divided into three groups according to random number table method：control group, model group, and EPCs transplantation group, with 20 rats in each group. The sepsis model was reproduced by intravenous delivery of LPS 5 mg/kg. Rats in control group were injected with the same amount of normal saline. EPCs were isolated, and cultured and identified were fluorescently labeled with the green fluorescent protein （ GFP ） adenoviral transfection method. The EPC transplantation group was injected with LPS, then a fluorescently labeled EPCs suspension was injected via the tail vein 1 hour later. The expression of fluorescent markers of EPCs was detected with both small animal in vivo imaging instrument and frozen section. Seven days after transplantation, abdominal aorta blood was collected to determine interleukins （ IL-6 and IL-10 ） in peripheral blood with enzyme linked immunosorbent assay （ ELISA ）, and the lung, liver, and kidney tissues were harvested, the wet/dry ratio of the lung （ W/D ） was calculated, and hematoxylin and eosin （ HE ） staining was performed to observe, the change in histopathology. Toll-like receptor 4 （ TLR4 ） mRNA expression in lung, liver, and kidney tissues was determined with real-time reverse transcription-polymerase chain reaction （ RT-PCR ）. Results The positive rate of EPCs cells with double marking of CD133 and CD34 was 99.0% at the 5th generation of subculture by using flow cytometry. After the transplantation of EPCs labeled with the green fluorescent protein, the appearance of fluorescence indicated that EPCs were mainly localized in the chest, and a stronger fluorescence was observed near the blood vessels. EPCs transplantation could significantly reduce the inflammatory cell infiltration and cell dama

关 键 词：内皮祖细胞 干细胞移植 脓毒症 炎症反应 TOLL样受体4 大鼠 TOLL-LIKE receptor 4 

分 类 号：R459.7[医药卫生—急诊医学]