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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。
OBJECTIVE: To investigate the effects of oxidized low-density lipoprotein (ox-LDL) on the expression of LOX-1 (LOX-1) and angiotensinⅡ (AngⅡ) using bimolecular fluorescence complementation Type receptor (AT1) interaction, suggesting that it may be involved in atherosclerosis and other cardiovascular diseases, one of the important mechanisms. METHODS: COS7 cells were co-transfected with LOX-1 and AT1 receptor plasmids, and then stimulated with ox-LDL (50ug / ml) to detect the expression of p-ERK. According to the sequences of BiFC vector and AT1, LOX-1 gene primers, AT1 and LOX-1 genes were cloned into BiFC specific fluorescent vector to obtain recombinant BiFC plasmids: phmKGN-MN-AT1, phmKGC-MN- AT and phmKGC- HEK293 cells were co-transfected with MC-LOX-1, phmKGN-MC-LOX-1 and stimulated with ox-LDL (50ug / ml) to study the interaction between AT1 receptor and LOX- BiFC recombinant plasmids: phmKGN-MN-β2AR and phmKGC-MN-β2AR, respectively, of the other family members were transfected into HEK293 cells with BiFC plasmid corresponding to LOX-1, After stimulated with ox-LDL (50ug / ml), the fluorescence of cells was observed. Results The expression of p-ERK was induced by ox-LDL stimulation of COS7 cells transfected with AT1 and LOX-1 receptor plasmids. BiFC recombinant plasmids of AT1, LOX-1 and β2AR were successfully constructed. The AT1 and LOX- BiFC plasmid co-transfected HEK293 cells, ox-LDL stimulation, green fluorescence can be observed. The BiFC plasmid transfected with β2AR and LOX-1 did not detect any fluorescence after ox-LDL stimulation. Conclusion The preliminary validation ox-LDL can induce LOX-1 and AT1 receptor specific interaction.