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目的:靶向血凝素样氧化型低密度脂蛋白受体-1(LOX-1)基因的发卡样siRNA(shRNA)表达载体的构建及其对巨噬细胞源性泡沫细胞形成的影响。方法:(1)采用DNA重组技术,将LOX-1shRNA双链与线性化pGenesil-1质粒表达载体连接,构建LOX-1特异性小发夹RNA表达载体pLOX-1-shRNA1及pLOX-1-shRNA2,用脂质体法转染小鼠单核巨噬细胞(RAW264.7),半定量逆转录聚合酶链反应法检测LOX-1mRNA的表达,Western blot法检测LOX-1蛋白的表达,选择沉默效果最佳的作为下一步的干预序列。(2)ox-LDL诱导巨噬细胞建立泡沫细胞模型,pLOX-1-shRNA进行干预,干预组使用脂质体法进行细胞转染,转染24小时后,再加入ox-LDL作用24小时,用油红O染色法及细胞内游离胆固醇及总胆固醇测定法观察对泡沫细胞形成的影响,倒置荧光显微镜观察RAW264.7细胞对Dil-ox-LDL的摄取率。结果:测序鉴定发现插入的发卡样序列正确,成功合成了发卡样LOX-1基因RNA干扰表达载体,pLOX-1-shRNA1及pLOX-1-shRNA2转染RAW264.7细胞后,其LOX-1基因和蛋白表达显著下调,并且以pLOX-1-shRNA2沉默效果最佳。结论:成功构建了能有效抑制LOX-1基因及蛋白表达的干扰表达载体,同时可抑制巨噬细胞源性泡沫细胞形成及对Dil-ox-LDL的摄取,为进一步利用RNA干扰技术防治动脉粥样硬化提供理论基础。
OBJECTIVE: To construct a hairpin-like siRNA (shRNA) expression vector targeting the hemagglutinin-like oxidized low density lipoprotein receptor-1 (LOX-1) gene and its effect on the formation of macrophage-derived foam cells. Methods: (1) LOX-1 specific small hairpin RNA expression vector pLOX-1-shRNA1 and pLOX-1-shRNA2 were constructed by DNA recombination technology by ligating LOX-1 shRNA double strands with the linearized pGenesil-1 plasmid expression vector .Mouse mononuclear macrophages (RAW264.7) were transfected by lipofectamine 2000. LOX-1 mRNA expression was detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). The expression of LOX-1 protein was detected by Western blot. The best-performing sequence for the next intervention. (2) Foam cell model was induced by ox-LDL in macrophages and pLOX-1-shRNA was intervened. The cells in the intervention group were transfected by liposome method. After 24 hours of transfection, ox-LDL was added for 24 hours, Oil red O staining and intracellular free cholesterol and total cholesterol assay was used to observe the effect of foam cells formation. The uptake of Dil-ox-LDL by RAW264.7 cells was observed by inverted fluorescence microscope. Results: Sequencing and sequencing showed that the insertion of hairpin-like sequence was correct, and the LOX-1 gene RNAi expression vector was successfully synthesized. After transfecting RAW264.7 cells with pLOX-1-shRNA1 and pLOX-1-shRNA2, And protein expression was significantly down-regulated, and pLOX-1-shRNA2 was the best. CONCLUSION: The interfering expression vector that can effectively inhibit LOX-1 gene and protein expression was successfully constructed, and the formation of macrophage-derived foam cells and the uptake of Dil-ox-LDL were also inhibited. In order to further utilize RNA interference in the prevention and treatment of atherosclerosis Sample hardening provides the theoretical basis.