自噬调控脂多糖诱导的巨噬细胞炎症因子水平

发布时间：2018-08-09 13:23

【摘要】：目的探讨细胞自噬调控对脂多糖(LPS)刺激巨噬细胞引发炎症反应的影响及其分子机制。方法分离小鼠骨髓间充质干细胞,经过诱导分化获得骨髓来源的巨噬细胞,通过LPS刺激巨噬细胞建立炎症反应细胞模型。利用绿色荧光蛋白-微管相关蛋白1轻链3(GFP-LC3)质粒转染观察自噬体的形成;利用3-甲基腺嘌呤(3-MA)和雷帕霉素(Rap)分别抑制和促进细胞自噬,研究自噬干预对LPS诱导巨噬细胞炎症反应的影响;实时定量PCR检测LC3B、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、IL-6和IL-12p40的mRNA水平,Western blot法检测LC3B、核因子κB(NF-κB)、NF-κB抑制蛋白α(IκBα)的蛋白水平。结果 LPS刺激巨噬细胞2、4、6 h,诱导巨噬细胞LC3B mRNA和蛋白高表达,巨噬细胞中自噬体增加。与单独LPS刺激相比,3-MA可抑制细胞自噬,显著增加炎症因子TNF-α、IL-1β、IL-6和IL-12p40 mRNA表达,而Rap处理减少自噬数量,降低TNF-α、IL-1β、IL-6和IL-12p40 mRNA水平;自噬抑制后,可通过降低IκBα和增加p-NF-k Bp65的表达而影响NF-κB信号通路,进而调节炎症细胞因子表达。结论自噬可调控LPS刺激巨噬细胞引起的炎症反应。
[Abstract]:Objective to investigate the effect of autophagy regulation on the inflammatory response induced by lipopolysaccharide (LPS) stimulated macrophages and its molecular mechanism. Methods Murine bone marrow mesenchymal stem cells were isolated and the macrophages derived from bone marrow were obtained by induced differentiation. The inflammatory response cell model was established by LPS stimulation of macrophages. The formation of autophagy was observed by transfection of green fluorescent protein-microtubule-associated protein-1 light chain 3 (GFP-LC3) plasmid, and 3-methyladenine (3-MA) and rapamycin (Rap) were used to inhibit and promote autophagy, respectively. To study the effect of autophagy intervention on the inflammatory response of macrophages induced by LPS, the levels of LC3B, TNF- 伪, IL-1 尾, IL-6 and IL-12p40 were detected by real-time PCR. The protein levels of LC3B, NF- 魏 B, NF- 魏 B inhibitory protein 伪 (I 魏 B 伪) were detected by Western blot. Results LPS stimulated macrophages for 6 h and induced the high expression of LC3B mRNA and protein, and the increase of autophagy in macrophages. Compared with the stimulation of LPS alone, 3-MA inhibited autophagy, significantly increased the expression of TNF- 伪, IL-1 尾, IL-6 and IL-12p40 mRNA, while Rap treatment decreased the number of autophagy and the levels of TNF- 伪, IL-1 尾, IL-6 and IL-12p40 mRNA. It can affect NF- 魏 B signaling pathway by decreasing I 魏 B 伪 and increasing the expression of p-NF-k Bp65, and then regulate the expression of inflammatory cytokines. Conclusion autophagy can regulate the inflammatory response of macrophages stimulated by LPS.
【作者单位】：山西医科大学第二临床医学院;首都医科大学附属北京佑安医院;
【基金】：国家十二五科技重大专项(2012ZX10002004-006,2012ZX10004904-003-001,2013ZX10002002-006-001) 国家自然科学基金(81270532) 北京市自然科学基金(7162085) 首都特色临床应用研究(Z121107001012167) 北京市卫生系统高层次卫生技术人才培养计划(2013-3-075) 北京市属医学科研院所公益发展改革试点项目(京医研2016-2)
【分类号】：R575.3

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