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目的探讨氧化型低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)的致动脉粥样硬化作用与Toll样受体4(Toll-like receptor 4,TLR4)表达的相关性。方法构建针对TLR4的小干扰KNA(small interference RNA,siRNA)表达载体,转染人单核细胞株U937,于转染前后用不同浓度的ox-LDL刺激,用流式细胞术和实时荧光定量PCR法检测TLR4蛋白和mRNA表达变化,同时用ELISA测定NF-κB活性改变以及细胞上清液中动脉粥样硬化相关细胞因子MCP-1和IL-8的浓度变化。结果ox-LDL刺激可导致单核细胞TLR4表达上调,同时NF-κB p65活性增强,MCP-1和IL-8在细胞上清液中的浓度显著高于对照组(P<0.05),并且呈剂量依赖性;利用siRNA表达载体使TLR4表达抑制后,相同剂量的ox-LDL刺激,NF-κB p65相对活性以及MCP-1和IL-8的分泌显著低于TLR4未抑制组(P<0.05)。结论ox-LDL的致动脉粥样硬化作用至少部分是由TLR4介导的;调节TLR4的表达,可以减弱ox-LDL刺激引起的包括NF-κB活化以及MCP-1和IL-8分泌等致动脉粥样硬化效应。
Objective To investigate the relationship between atherosclerosis and expression of Toll-like receptor 4 (TLR4) in oxidized low density lipoprotein (ox-LDL). Methods Small interference RNA (siRNA) expression vector targeting TLR4 was constructed and transfected into human monocytic cell line U937. The cells were stimulated with different concentrations of ox-LDL before and after transfection. Flow cytometry and real-time fluorescence quantitative PCR Method was used to detect the changes of TLR4 protein and mRNA expression. At the same time, the changes of NF-κB activity and the concentrations of atherosclerosis-related cytokines MCP-1 and IL-8 in the supernatant were measured by ELISA. Results The stimulation of ox-LDL led to the up-regulation of TLR4 expression in monocytes and the increase of NF-κB p65 activity. The concentration of MCP-1 and IL-8 in the supernatant was significantly higher than that in the control group (P <0.05) Dose-dependent manner. The same dose of ox-LDL stimulation, relative activity of NF-κB p65 and secretion of MCP-1 and IL-8 were significantly lower than those of TLR4 untreated group (P <0.05) . CONCLUSIONS: The atherogenic effect of ox-LDL is mediated at least in part by TLR4. Regulating the expression of TLR4 can attenuate ox-LDL-induced arterioles including NF-κB activation and MCP-1 and IL-8 secretion Atherosclerosis effect.