LPS诱导心肌成纤维细胞HMGB1的释放与Ⅰ和Ⅲ型胶原蛋白分泌的相关性研究  

Release of HMGB1 by LPS-treated cardiac fibroblasts and its contribution to the production of collagen type Ⅰ and Ⅲ

作　　者：尹晶平[1] 苏兆亮[1] 王映梅[2] 汪汀[1] 田莎莎[1] 徐鑫鑫[1] 邢丽丹[1] 张盼[1] 马珂[1] 许化溪[1] 

机构地区：[1]江苏大学免疫学研究所,江苏镇江212013 [2]第四军医大学西京医院病理科,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2012年第8期785-788,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81001319);中国博士后基金(2012M-511705);江苏省博士后基金(1102129C);江苏大学高级人才启动基金(11JDG128)

摘　　要：目的:探讨小鼠心肌成纤维细胞(CFs)在LPS刺激下高迁移率族蛋白(HMGB1)释放与Ⅰ、Ⅲ胶原蛋白分泌的相关性。方法:从7～14 d BALB/c小鼠心脏中分离培养心肌成纤维细胞,分别用LPS(500 ng/mL)刺激0,6,12,24,36,48 h后收取细胞及其上清。采用RT-PCR检测HMGB1以及Ⅰ、Ⅲ型胶原mRNA表达水平,通过Western blot检测细胞及其培养上清中HMGB1以及Ⅰ、Ⅲ型胶原含量;采用免疫荧光分析HMGB1在细胞内的定位。结果:LPS刺激CFs 6 h时HMGB1以及Ⅰ、Ⅲ型胶原水平无明显变化,12、24、36、48 h后HMGB1、Ⅰ、Ⅲ型胶原mRNA表达量均增加。Western blot检测发现,LPS刺激CFs 24 h后细胞上清可检测到HMGB1蛋白,且细胞中HMGB1的表达呈现先增加后降低的现象。免疫荧光观察到HMGB1的释放伴随着从细胞核到细胞质的转位过程。Ⅰ、Ⅲ型胶原蛋白的Western blot结果均呈现增加趋势。结论:LPS刺激CFs后HMGB1由细胞核转移到细胞质并可主动分泌到胞外,其作用呈现一定的时间依赖性。与心肌纤维化密切相关的Ⅰ、Ⅲ型胶原在LPS刺激后表达量明显增加,提示内源性HMGB1的定位及分泌可能与脓毒症引起的心功能紊乱后期的心肌纤维化可能有一定的关系。AIM： To investigate whether cardiac fibroblasts（CFs） treated by LPS can actively secrete high-mobility group box protein 1（HMGB1） and to analyze the correlation between HMGB1 releasing and the accumulation of collagen type Ⅰ,Ⅲ.METHODS： CFs were isolated from the heart of 7-14-day-old BALB/c mice and cultured in DMEM with 10% fetal bovine serum（FBS）.We collected the CFs and cell supernatants after treated by LPS for 0,6,12,24,36,48 h,respectively.The mRNA and protein expression levels of HMGB1,collagen 1a1（col1a1） and collagen 3a1（col3a1） in CFs after LPS stimulation were detected by RT-PCR and Western blotting,respectively.The intracellular localization of HMGB1 in treated CFs was investigated by immunofluorescence.RESULTS： After 0-6 h of LPS stimulation,the mRNA levels of HMGB1,col1a1,col3a1 had no significant changes;but increased obviously at 12,24,36,48 h.HMGB1 was found in the cell supernatant by Western blotting after 24 h LPS stimulation,and its expression decreased following the first rise in CFs.Meanwhile,immunofluorescence showed HMGB1 translocation from nucleus to cytoplasm.The levels of col1a1 and col3a1 were up-regulated in CFs after stimulation.CONCLUSION： LPS can induce HMGB1 translocation from nucleus to cytoplasm and across cellular membrane to the outside of CFs at a time-dependent manner.Col1a1 and Col3a1,which are closely associated with myocardial fibrosis,were obviously up-regulated by LPS stimulation,which indicates that actively released HMGB1 might contribute to myocardial fibrosis following the endotoxin induced-sepsis.

关 键 词：内毒素 高迁移率族蛋白1 心肌纤维化 心肌成纤维细胞 

分 类 号：R392.12[医药卫生—免疫学]