缺氧对大鼠肺成纤维细胞甲酰肽受体1表达的影响  

Effects of hypoxia on expression of formyl peptide receptor 1 in rat lung fibroblasts in vitro

作　　者：李晓栩[1,2,3] 黄缄[1,2,3] 矫力 崔宇[1,2,3] 杨诚忠 魏铭 LI Xiaoxu;HUANG Jian;JIAO Li;CUI Yu;YANG Chengzhong;WEI Ming(Department of High Altitude Physiology and Pathology,Facalty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Key Laboratory of Extreme Environmental Medicine of Ministry of Education,Facalty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Key Laboratory of High Altitude Medicine of PLA,Facalty of High Altitude Military Medicine,Army Medical University(Third Military Medical University),Chongqing,400038;Department of Pathophysiology,Department of Stem Cell and Tissue Engineering,College of Basic Medical Sciences,Chongqing Medical University,Chongqing,400016,China)

机构地区：[1]陆军军医大学(第三军医大学)高原军事医学系:高原生理学与病理学教研室,重庆4400038 [2]陆军军医大学(第三军医大学)高原军事医学系:极端环境医学教育部重点实验室,重庆4400038 [3]陆军军医大学(第三军医大学)高原军事医学系:全军高原医学重点实验室,重庆4400038 [4]重庆医科大学基础医学院干细胞与组织工程研究室病理生理学教研室,重庆400016

出　　处：《第三军医大学学报》2020年第6期568-575,共8页Journal of Third Military Medical University

基　　金：军队医学科技青年培育计划(20QNPY002);军队后勤科研项目-1226工程(BWS17J031)。

摘　　要：目的研究缺氧对大鼠肺成纤维细胞甲酰肽受体1(formyl peptide receptor 1,FPR1)表达的影响。方法分离大鼠肺成纤维细胞,用48孔Boyden趋化小室观察大鼠成纤维细胞在不同浓度(0、10、100、1000 nmol/L组)的FPR激动剂(fMLF)作用下的趋化能力。将细胞置于低氧培养箱中培养(1%O2,5%CO2,94%N2,37℃)24 h,取细胞培养上清,结合FPR特异性拮抗剂tBoc处理,检测缺氧培养上清中FPR介导的趋化活性。将细胞分为常氧组[普通培养箱(5%CO2,95%空气,37℃)]和缺氧组[置低氧培养箱中培养(1%O2,5%CO2,94%N2,37℃)2、12、24 h],用免疫印迹、PCR检测大鼠肺成纤维细胞FPR1和FPR激活物膜联蛋白A1(annexin A1,AnxA1)的蛋白、mRNA水平。不同浓度fMLF作用下细胞中FPR1、AnxA1的蛋白水平。使用tBoc预处理细胞并缺氧24 h后,检测FPR1、AnxA1的蛋白水平。结果fMLF可明显引起大鼠肺成纤维细胞趋化100 nmol/L组引起趋化至膜下侧的细胞数增多最为显著(P<0.05)。缺氧后的细胞培养上清趋化活性显著高于常氧组(P<0.05),加入tBoc后其趋化活性显著降低(P<0.05),降低程度显著高于常氧组。缺氧12 h,肺成纤维细胞的FPR1蛋白水平显著增加(P<0.05)。缺氧12、24 h与缺氧0 h组相比较,AnxA1的蛋白水平显著增加(P<0.05)。与常氧组相比较肺成纤维细胞的FPR1、AnxA1的mRNA水平显著增加(P<0.05);随着fMLF浓度的升高,FPR1和AnxA1的蛋白表达水平逐渐增加,100、1000 nmol/L组与0 nmol/L组相比,具有显著性差异(P<0.05)。甲酰肽受体拮抗剂tBoc可以显著削弱此效应。结论原代培养的大鼠肺成纤维细胞表达FPR1,缺氧促进大鼠肺成纤维细胞FPR1及其激活物表达与分泌,FPR1激活可进一步增强受体及其激活物的表达与分泌。Objective To study the effect of hypoxia on the expression of formyl peptide receptor 1(FPR1)in rat lung fibroblasts.Methods We tested the chemotactic activity of 10,100,and 1000 nmol/L FPR agonist(fMLF)for lung fibroblasts isolated from adult female SD rats using Boyden chemotaxis chamber assay.The cells were cultured in a hypoxic incubator(1%O2,5%CO2,and 94%N2)at 37℃for 24 h,and the culture supernatant was collected and treated with the FPR-specific antagonist tBoc before assessing fibroblast chemotactic activity mediated by FPR.Rat lung fibroblasts cultured in a normoxic condition or in a hypoxic incubator(1%O2)for 2,12,and 24 h were tested for mRNA and protein levels of FPR1 and FPR agonist annexin A1(AnxA1)using real-time PCR and Western blotting.The protein levels of FPR1 and AnxA1 were also examined in cells treated with different concentrations of fMLF and in cells treated with tBoc prior to hypoxia for 24 h.Results fMLF induced obvious chemotactic migration of rat lung fibroblasts,and 100 nmol/L exhibited the strongest chemotactic activity for the fibroblasts(P<0.05).The supernatant of the cells with hypoxic exposure showed a significantly higher chemotactic activity than that of the cells cultured in a normoxic condition(P<0.05);the addition of tBoc significantly decreased in the chemotactic activity of the supernatants,and the reduction was more obvious with the hypoxic cell culture supernatant(P<0.05).Hypoxia for 12 h significantly increased FPR1 protein expression in the lung fibroblasts(P<0.05);hypoxia for 12 and 24 h significantly enhanced the protein expression of AnxA1 in the fibroblasts(P<0.05).The mRNA levels of FPR1 and AnxA1 increased significantly in the lung fibroblasts after hypoxia for 2,12,and 24 h(P<0.05);increasing the concentration of fMLF in the cell culture resulted in progressively increased protein levels of FPR1 and AnxA1,and their increments became significant after treatment with fMLF at 100 and 1000 nmol/L(P<0.05);this effect was significantly attenuated by the application o

关 键 词：成纤维细胞 缺氧 趋化 甲酰酞受体 

分 类 号：R322.35[医药卫生—人体解剖和组织胚胎学] R363.22[医药卫生—基础医学]