运用BiFC技术检测LOX-1与AT1受体的相互作用

Investigation into the association of LOX-1 with AT1 receptor using BiFC.

目的运用双分子荧光互补(bimolecular fluorescence complementation,BiFC)技术探讨氧化低密度脂蛋白(ox-LDL)介导血凝素样氧化低密度脂蛋白受体-1(LOX-1)和血管紧张素Ⅱ1型受体(AT1)之间的相互作用,推测其可能是参加动脉粥样硬化等心血管疾病过程的重要机制之一。方法将LOX-1与AT1受体质粒共转染COS7细胞,给予ox-LDL(50ug/ml)刺激后,检测细胞中p-ERK表达水平。根据BiFC载体和AT1、LOX-1基因序列设计引物,将AT1、LOX-1基因克隆到BiFC特异性的荧光载体上,得到重组BiFC质粒:phmKGN-MN-AT1,phmKGC-MN-AT和phmKGC-MC-LOX-1,phmKGN-MC-LOX-1,配对共转染HEK293细胞,用ox-LDL(50ug/ml)刺激,研究AT1受体和LOX-1的相互作用;构建G蛋白偶联受体家族中另一蛋白肾上腺素受体β2(β2-adrenergi creceptor,β2AR)的BiFC重组质粒:phmKGN-MN-β2AR和phmKGC-MN-β2AR,分别与LOX-1对应的BiFC质粒转染HEK293细胞,ox-LDL(50ug/ml)刺激后,观察细胞荧光。结果用ox-LDL刺激转染AT1与LOX-1受体质粒的COS7细胞,可以引起p-ERK表达升高;成功构建AT1、LOX-1、β2AR的BiFC重组质粒;将AT1和LOX-1的BiFC质粒共转染HEK293细胞,用ox-LDL刺激后,可以观察到绿色荧光。而转染β2AR和LOX-1的BiFC质粒,用ox-LDL刺激后未检测到荧光。结论初步验证ox-LDL可以诱导LOX-1与AT1受体特异性相互作用。

Ob ject ive: To study the as sociation of lectin-like oxidized low density lipoprotein receptor-1(LOX-1) with angiotens in Ⅱ type 1 receptor (AT1) induced by oxidized low densi ty l ipoprotein(ox-LDL) using a bimolecular ��uorescence complementation(BiFC) technique. Meth ods: LOX-1 and AT1 plasmids were trans fected into COS7 cells. After treatment with ox-LDL(50ug/ml), cells were collected and proteins were extracted from them to be measured the expression level of p-ERK. The gene encodingAT1 or LOX-1 was cloned and then subcloned into bimolecular ��uorescence complemented vectors .BiFC recombinant plasmids were obtained including phmKGN-MN-AT1, phmKGC-MN-AT as well as phmKGC-MCLOX- 1 and phmKGN-MC-LOX-1. The interaction between AT1 and LOX-1 was studied in HEK293 cells co-transfected with phmKGN-MN- AT1/ phmKGC-MC-LOX-1 or phmKGC-MN-AT/phmKGN-MC-LOX-1 after stimulation with ox-LDL(50ug /ml). Other BiFC recombinant plasmids of β2-adrenergic receptor (β2AR), phmKGN-MN-β2AR and phmKGC-MN-β2AR, were also constructed and co-transfected with the corresponding BiFC recombinant plasmid of LOX-1, respect ively. Fluorescence was observed after treatment wi th ox-LDL(50ug/ml). Results ：The expression of p-ERKincreased signi��cantly in COS7 cel ls transfected with AT1 and LOX-1 plasmids after t reatment wi th ox-LDL compared to control group. Plasmids of AT1, LOX-1, β2AR for bimolecular ��uores cence complementation as say were successfully constructed. Green ��uo rescence was detected in HEK293 cel ls co-transfected with BiFC plasmids of AT1 and LOX-1 after stimulation with ox-LDL, while no green ��uorescence was detected in HEK293 cells t ransfected wi th BiFC plasmids of β2AR and LOX-1. Conclusions: ox-LDL may induce the interaction between LOX-1 and AT1 receptor.