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目的通过转染小干扰RNA(siRNA)沉默RAW264.7细胞源性泡沫细胞神经轴突生长抑制因子B受体(Ng BR)表达,研究Ng BR对泡沫细胞胆固醇逆转运(RCT)的影响,探索从RCT途径抗动脉粥样硬化(As)的新方法,为冠心病的临床防治提供新思路。方法利用氧化型低密度脂蛋白(ox-LDL)诱导RAW264.7细胞形成泡沫细胞,油红O染色进行鉴定。将泡沫细胞分为四组:空白对照组、siRNA阴性对照组、Ng BR-siRNA1转染组(si Ng BR-1组)、Ng BR-siRNA2转染组(si Ng BR-2组)。利用siRNA沉默泡沫细胞Ng BR基因表达,并利用real-time PCR和Western blot对其进行干扰效率鉴定。随后采用real-time PCR检测各组细胞肝X受体α(LXRα)、三磷酸腺苷结合盒转运体A1(ABCA1)及三磷酸腺苷结合盒转运体G1(ABCG1)mRNA表达,Western blot检测各组细胞相应蛋白含量,液闪计数仪检测胆固醇流出率。结果 ox-LDL成功诱导泡沫细胞形成;si Ng BR-1组和si Ng BR-2组Ng BR mRNA及其蛋白明显下调(P<0.05);si Ng BR-1组和si Ng BR-2组LXRα、ABCA1和ABCG1的mRNA及其蛋白表达显著降低(P<0.05),胆固醇流出显著减少(P<0.05)。结论 Ng BR可以增加巨噬细胞源性泡沫细胞RCT的调控基因LXRα及其下游基因ABCA1、ABCG1的表达,从而减弱或者避免As的发生和发展,为冠心病的临床防治提供理论依据。
OBJECTIVE: To investigate the effect of Ng BR on the reversal of cholesterol transport (RCT) in foam cells by transfecting small interfering RNA (siRNA) into RAW264.7-derived foam cells, The new method of anti-atherosclerosis (As) from the RCT pathway provides a new idea for the clinical prevention and treatment of coronary heart disease. Methods Oxidized low density lipoprotein (ox-LDL) was used to induce the formation of foam cells in RAW264.7 cells. Oil red O staining was used to identify the cells. The foam cells were divided into four groups: blank control group, siRNA negative control group, Ng BR-siRNA1 transfection group (si Ng BR-1 group) and Ng BR-siRNA2 transfection group (si Ng BR-2 group). The expression of Ng BR gene in foam cells was silenced by siRNA, and the interference efficiency was identified by real-time PCR and Western blot. Then the expression of LXRα, ABCA1 and ABCG1 mRNA in each group were detected by real-time PCR. The relative protein content , Liquid scintillation counter cholesterol outflow rate. Results Ox-LDL induced the formation of foam cells successfully. Ng BR mRNA and protein were significantly down-regulated in si Ng BR-1 group and si Ng BR-2 group (P <0.05); si Ng BR-1 group and si Ng BR-2 group The mRNA and protein expressions of LXRα, ABCA1 and ABCG1 were significantly decreased (P <0.05) and the cholesterol efflux was significantly decreased (P <0.05). Conclusion Ng BR can increase the expression of LXRα and its downstream genes ABCA1 and ABCG1 in RCTs of macrophage-derived foam cells, thus weakening or avoiding the occurrence and development of As, providing a theoretical basis for the clinical prevention and treatment of coronary heart disease.