中文摘要：

目的研究内皮细胞高表达脂多糖相关因子1（EOLA1）与金属硫蛋白2A（MT2A）的相互作用关系,明确其相互作用位点及上下游关系。方法构建EGFP-EOLA1融合表达载体,转染HUVEC细胞,应用免疫荧光共定位成像技术观察EOLA1和MT2A在HUVEC中的共定位情况;应用截短突变和酵母双杂交确定EOLA1和MT2A的相互作用位置;应用RNAi技术建立EOLA1/MT2A上调和下调的HUVEC细胞模型,观测模型细胞EOLA1/MT2A的表达情况。结果 EOLA1和MT2A在HUVEC中存在共定位现象,共定位主要发生在核膜周围;酵母双杂交和免疫共沉淀实验结果显示,EOLA1的截短突变体1~77氨基酸（aa）片段和MT2A无结合活性,1~115aa片段及1~158aa片段和MT2A有结合活性。EOLA1上调/下调表达时MT2A同时相应上调/下调表达,MT2A的上调和下调表达对EOLA1表达无明显影响。结论 EOLA1和MT2A在HUVEC细胞内存在相互作用,相互作用位点位于EOLA1ORF的100~113功能域,EOLA1可以调控MT2A在HUVEC中的表达。

英文摘要：

Objective To elucidate the relationship of endothelial-overexpressed lipopolysaccharide-associated factor I（EOLA1 ）and metallothionein 2A （MT2A）. Methods The reconstructed plasmid pEGFP-N2-EOLA1 was transfeeted into HUVEC cells,then the cells were cultured with G418（400μg/mL）in M199 medium. The cells were stained with specific antibody and observed by confocal microscopy to confirm the colocalization of EOLA1 and MT2A. The interaction site of EOLA1 and MT2A was identified by Yeast Two=Hybrid analysis. Knockdown of EOLA1 and MT2A was accomplished by using RNAi,and over-expression of EOLA1 and MT2A was accomplished by transfection of reconstructed plasmid,the over-expression of EOLA1 and MT2A in HUVEC was detected by RTPCR. Results EOLA1 co-localized with MT2A mostly in nuclear membrane around and slightly in cytoplasm as shown by conSocal microscopy. The results of expression of EOLA1 and MT2A showed that EOLA1 could regulate the expression of MT2A in HUVEC cells. Conelusion EOLA1 interacts with MT2A at the site of 100-113 function domain,and EOLA1 could regulate the expression of MT2A in HUVEC cells.