ICAP1α及突变体T38A、I138A在内皮细胞ECV304整合素β1内化中的作用  

The effect of ICAP1α and its mutants T38A,I138A on internalization of integrin β1 on endothelial cells ECV304

作　　者：程忠良[1] 秦瑾[1] 刘正湘[1] 林敬阳[1] 刘毓[1] 左后娟[1] 汪道文[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院心血管内科,武汉430030

出　　处：《微循环学杂志》2009年第3期11-15,F0003,共6页Chinese Journal of Microcirculation

基　　金：国家自然科学基金资助项目(批准号:30670856)

摘　　要：目的:探讨ICAP1α及突变体T38A、I138A在内皮细胞ECV304内化整合素β1中的作用和机制。方法:构建pAAV-ICAP1α及其突变体pAAV-T38A和pAAV-I138A真核表达载体,转染ECV304。ECV304分为ICAP1α组、T38A组、I138A组、绿色荧光蛋白(GFP)组和未转染组,用NHS-SS-Biotin标记整合素β1,并利用生物素-亲和素系统以及Westernblot检测整合素β1内化情况。结果:各组细胞转染的质粒均稳定表达;Westernblot检测ICAP1α组、T38A组、I138A组、GFP组、未转染组内化的整合素β1相对光密度值分别为1.07±0.04、0.68±0.06、0.70±0.02、0.87±0.06、0.88±0.04;与GFP组和未转染组比较,ICAP1α组整合素β1内化明显增多(P<0.05);与GFP组、未转染组和ICAP1α组比较,T38A组和I138A组整合素β1内化明显减少(P<0.05),GFP组与未转染组无差别(P>0.05)。结论:转染ICAP1α后内皮细胞ECV304表面整合素β1内化增加,而转染T38A和I138A后内皮细胞ECV304表面整合素β1内化减少;ICAP1α可能通过第38位的苏氨酸残基(38Tyr)和第138位的异亮氨酸残基(138Ile)调控整合素β1的内化。Objective:To investigate the effect of ICAP1α and its mutants T38A,I138A oninternalization of integrin β1 on endothelial cell ECV304 and its mechanism.Method:The plasmids pAAV-ICAP1α,pAAV-T38A,pAAV-I138 were constructed and transfected into ECV304.ECV304 were assigned to ICAP1α group,T38A group,I138A group,GFP group and untransfected group.After transfection,integrin β1 on ECV304 was labeled by NHS-SS-Biotin.By the character of Biotin-Avidin,the internalization of integrin β1 was detected by western-blot.Results:The protein of the transfected cells in each group was expressed stably;The relative optical density of internalized integrin β1 in ICAP1α group,T38A group,I138A group,GFP group and untransfected group were 1.07±0.04,0.68±0.06,0.70±0.02,0.87±0.06,0.88±0.04,respectively;The internalization of integrin β1 in group ICAP1α was higher than that in GFP group and the untransfefted group(P<0.05);The internalization of integrin β1 in T38A group and I138A group was lower than that in ICAP1α group,the GFP group and the untransfefted group(P<0.05).Conclusion:ICAP1α can increase the internalization of integrin β1 on ECV304,while T38A and I38A decrease the internalization of integrin β1;ICAP1α regulates the internalization of integrin β1 on ECV304 by the key sites:38Tyr and 138Ile.

关 键 词：整合素Β1 ECV304 内皮细胞 突变体 细胞黏附因子 内化 小分子蛋白质 信号传递机制 

分 类 号：R739.41[医药卫生—肿瘤] R394[医药卫生—临床医学]