摘要
目的 研究移植内皮祖细胞（endothelial progenitor cell,EPC）对新生大鼠高氧肺损伤的影响及其机制. 方法 从4周龄Sprague-Dawyley大鼠骨髓中培养获取EPC并鉴定.另取新生Sprague-Dawley仔鼠60只,生后室温下适应性饲养24 h后,随机分为空气组、高氧组、移植组和Nω-硝基-L-精氨酸甲酯（Nω-nitro-L-argininemethylester,L-NAME）干预组（简称干预组）,每组15只.空气和高氧组分别在空气和85％高氧中饲养28d.移植组在85％高氧中暴露28d,其中在第21天尾静脉注射EPC 1×10 5个.干预组在移植组的基础上,自第21天开始连续腹腔注射L-NAME至第28天,每日剂量为20 mg/kg.第28天处死所有仔鼠,留取血标本,采用流式细胞技术检测CD34＋细胞,酶联免疫吸附方法检测血清血管内皮细胞生长因子（vescular endothelial growth factor,VEGF）.同时留取肺组织标本,观察辐射状肺泡计数和肺微血管计数、免疫荧光方法观察移植EPC在肺内的定植情况,实时荧光定量聚合酶链反应和蛋白质印迹技术检测肺组织中VEGF、VEGF受体（vescular endothelial growth factor receptor,VEGFR）2和内皮源性一氧化氮合酶（endothelial nitric oxide synthase,eNOS）的表达,并用硝酸还原酶法检测肺组织中一氧化氮的表达.采用单因素方差分析和Bonferroni方法进行统计学分析. 结果 （1）培养所得细胞具有典型的EPC形态改变;结合异硫氢基荧光素标记荆豆凝集素-1并摄取Dil荧光标记的乙酰化低密度脂蛋白的双阳性细胞约占总细胞数的85％;培养所得细胞中CD34＋细胞含量为68.2％～72.4％.（2）空气组、高氧组、移植组和干预组仔鼠外周血CD34＋细胞数量分别为（1.91±0.34）％、（1.06±0.10）％、（1.47±0.06）％和（0.77±0.11）％（F=32.710,P=0.000）,高氧组低于空气组,移植组高于高氧组,干预组又低于移植组（P值均＜ 0.05）.4组仔鼠血清VEGF水平分别为（7.90±2.72）、（6.38±0.72）、（1
Objective To study the effect of transplanted endothelial progenitor cell （EPC） on hyperoxia-induced lung injury in neonatal rats.Methods Rat bone marrow mononuclear cells were cultured in endothelial cell growth medium to obtain EPCs,which were identified by morphology,phagocytosis and CD34＋ analyses.Sixty neonatal Sprague-Dawley rats were allowed to acclimate in room air for 24 h after birth,and were then divided into four groups （15 per group）,including the air group,the hyperoxia group,the EPCs transplantation group and the N ω-nitro-L-arginine methyl ester （L-NAME） intervention group.Neoborn rats in the Air and Hyperoxia groups were fed in the room air or hyperoxia （85% oxygen） for 28 days.For rats in transplantation group were exposed continuously to hyperoxia for 28 days,and got an EPC （1 × 105 cells） injection on the 21st day.Rats in Intervention group were exposed continuously to hyperoxia for 28 days,got an EPC （1 × 105 cells） injection on the 21st day,and a daily injection of L-NAME from day 21 to day 28,with a daily dose of 20 mg/kg.Levels of circulating CD34＋ cells and serum VEGF expression were detected.Specimens from lung tissues were analyzed by immunohistochemistry or immunofluorescence.The expression of vascular endothelial growth factor （VEGF）,VEGF receptor 2 （VEGFR2） and eNOS were detected by realtime polymerase chain reaction and Western-blotting.NO production were detected by nitrate reductase assay.One way ANOVA and Bonferroni test were used for statistical analysis.Results （1） The cultured cells had a typical cobblestone appearance; double positive cell binding of fluorescein Ulex Europaeus agglutinin-1 and uptake of Dil-labeled acetylated low density lipoprotein accounted for approximately 85% of the total number of cells.CD34＋ cells accounted for 68.2%-72.4% of total cultured cells.（2） Circulating CD34＋ cells in the air group,hyperoxia group,EPC transplantation group and L NAME intervention group were （1.91 ± 0.34）%,（1.06 ± 0.10�
出处
《中华围产医学杂志》
CAS
CSCD
2015年第5期366-374,共9页
Chinese Journal of Perinatal Medicine
基金
国家自然科学基金（81070517、81270727）
关键词
内皮祖细胞
干细胞移植
支气管肺发育不良
一氧化氮合酶
大鼠
Endothelial progenitor cells
Stem cell transplantation
Bronchopulmonary dysplasia
Vascular endothelial growth factor A
Nitric Oxide Synthase
Rats
