膜突蛋白及其抗体对人肺微血管内皮细胞损伤机制的研究  

Human pulmonary microvascular endothelial cells injury could be mediated by the co-effect of moesin and anti-moesin antibody

作　　者：李梦涛[1] 尹雷[1] 王迁[1] 郑文洁[1] 何建国[2] 曾小峰[1] 

机构地区：[1]中国医学科学院北京协和医院风湿免疫科,100032 [2]中国医学科学院阜外心血管病医院肺血管病研究中心

出　　处：《中华风湿病学杂志》2010年第4期232-235,289,共5页Chinese Journal of Rheumatology

基　　金：“十一五”国家科技支撑计划（2006BAI01A07、2008BA159B02）;中华医学会临床医学科研专项资金（08010-270105）

摘　　要：目的通过抗膜突蛋白单克隆抗体（MmAb）介导人肺微血管内皮细胞（HPMEC）损伤模型，探讨膜突蛋白及其抗体在肺血管损伤和肺动脉高压（PAH）形成过程中的作用。方法以磷酸盐缓冲液（PBS）作为对照组，肿瘤坏死因子（TNF）-α、MmAb及TNF—α＋MmAb作为实验组分别作用于HPMEC，在不同时相应用免疫印记法检测细胞上清液中膜突蛋白表达量，流式细胞仪对细胞表面膜突蛋白抗原表达量和细胞凋亡进行检测，同时应用激光共聚焦显微镜和电子显微镜评判HPMEC形态和功能的变化，用独立样本t检验和单因素方差分析进行统计学处理。结果①TNF-α作用后HPMEC上清液中和细胞膜表面的膜突蛋白表达量均较对照组显著增加（P〈0．01）；②TNF—α＋MmAb组致HPMEC凋亡率显著高于TNF-α组（31．4％与14．1％，P〈0．01），亦显著高于MmAb组（31．4％与3．89％，P〈0．01）；③TNF—α＋MmAb共同作用在激光共聚焦显微镜下可见HPMEC骨架结构F-actin功能丧失、逐渐出现核浓聚和细胞凋亡，在电镜下可见HPMEC微绒毛明显减少，近乎消失。结论HPMEC有膜突蛋白表达，在TNF—α作用下细胞外可检测到膜突蛋白，MmAb可以对HPMEC造成损伤，主要表现为对数生长期的细胞出现骨架蛋白结构改变和早期凋亡。Objective To establish the anti-moesin antibody-mediated human pulmonary microvascular endothelial cell （HPMEC） injury model and investigate the possible mechanism of HPMEC injury. Methods With PBS as control, the experimental groups were divided into tumor necrosis factor （TNF）-α, anti-moesin monoclonal antibody （MmAb） and TNF—α＋MmAb combined groups. Incubating with HPMECs separately, moesin expression in the supernatant was detected by Western-blotting. Membrane expression of moesin and apoptosis of HPMECs were tested by flow cytometry. Morphologic and functional changes of HPMECs were also observed by laser cofocal and electronic microscopes. Independent samples t-test and oneway ANOVA were used for statistical analysis. Results （1） More expressions of moesin were detected in the TNF-α group compared to the controls （P〈0.01）, both in the supernatant and the endothelial surface. （2）More apo-ptosis in TNF—α＋MmAb group could be found compared to the TNF—α group （31.4% vs 14.1%, P〈0.01 ） and-also to the MmAb group （31.4% vs 3.89%, P〈0.01）. （3） Dysfunction of F-actin and HPMEC skeletal structure were observed in the TNF-α ＋MmAb group, which led to apoptosis subsequently. Microvilli of HPMECs were nearly disappeared under electronic microscopy. Conclusion HPMEC could express moesin, particularly when stimulated by inflammation. Co-effect of moesin and MmAb has been confirmed to cause injury of HPMECs, which is manifested as dysfunction of endothelial skeletal proteins and premature apoptosis.

关 键 词：抗体 单克隆 细胞凋亡 人肺微血管内皮细胞 膜突蛋白 

分 类 号：R593.2[医药卫生—内科学] R543.2[医药卫生—临床医学]