体外研究人LOX-1受体功能的细胞模型建立  

A study model for LOX-1 receptor in vitro

作　　者：徐婷[1] 戴亚蕾[1] 尹岚[1] 杨红[1] 田菲[1] 陈春霞[1] 

机构地区：[1]同济大学医学院免疫学教研室,上海200092

出　　处：《同济大学学报（医学版）》2009年第1期14-19,共6页Journal of Tongji University(Medical Science)

基　　金：国家自然科学基金资助项目(30671969)

摘　　要：目的研究LOX-1受体在早期动脉粥样硬化中的作用,建立体外LOX-1高表达细胞模型,用于探讨LOX-1与Ox-LDL的相互作用及参与泡沫细胞形成的机制。方法根据人类cDNA文库设计引物,采用巢式PCR技术用人单核细胞转化的巨噬细胞中扩增出LOX-1完整编码区,克隆至pEGFP-C1重组真核表达载体,经测序证实后,用脂质体转染法转染至293T细胞,采用RT-PCR鉴定外源基因的表达,倒置荧光显微镜检测质粒转染效率和融合蛋白的细胞膜上定位情况,流式细胞技术检测EGFP-LOX-1融合蛋白表达情况以及其对Ox-LDL的结合能力。结果pEGFP-LOX-1重建质粒经测序证实克隆的LOX-1序列完整,插入方向正确,转染后实验组LOX-1 mRNA大量表达,融合蛋白正确定位在细胞膜表面,并显示结合配体Dil-Ox-LDL的功能提高到6.3倍。表明所克隆的LOX-1受体具有特异性结合Ox-LDL的功能,从而建立了高表达LOX-1受体的细胞。结论成功克隆人类LOX-1基因,使其在293T细胞中高度表达并获得了相应的功能,为进一步研究LOX-1提供有利工具。Objective To study the effect of LOX-1 on atherosclerosis at the early stage, by establishing a cell expression model for LOX-1 to investigate the interaction between LOX-1 and its ligand Ox-LDL and the mechanism involved in foam cell formation. Methods Human LOX-1 gene fragment was obtained from human macrophage by nest RT-PCR and subcloned into eukaryotic expression pEGFP-C1 plasmid. After sequencing, the recombinant plasmid was transfected into 293T cells by lipidosome. The expression of exogenous LOX-1 in 293T cells was detected by RT-PCR. The fusion protein localization on the cell surface was detected by inverted fluorescence microscope. The expression of the fusion protein and the ability of binding its ligand Ox-LDL were detected by flow cytometry. Results The cloned ORF of LOX-1 gene fragment was confirmed by DNA sequencing and the subcloned recombinant pEGFP-LOX-1 plasmid was in right position. After 48 h transfection, there abundant LOX-1 mRNA was expressed in the cells transfected with pEGFP-LOX-1 plasmid. The fusion protein was also highly expressed and localized on the surface of cell. The cells expressing exogenous LOX-1 protein had increased 6.3 fold binding ability with Dil-Ox-LDL.These results indicated that high expression LOX-1 cell could bind Ox-LDL which could be used for further studies. Conclusion Human LOX-1 gene was cloned successfully and expressed effectively with corresponding functions in 293T cells which provides powerful tool for LOX-1 function studying.

关 键 词：LOX-1 基因克隆 真核表达 动脉粥样硬化 

分 类 号：R363.2[医药卫生—病理学]