《ApoA-I调节LPS处理泡沫细胞锌指蛋白36去磷酸化的抗炎作用及其机制研究-病理学与病理生理学》由会员分享，可在线阅读，更多相关《ApoA-I调节LPS处理泡沫细胞锌指蛋白36去磷酸化的抗炎作用及其机制研究-病理学与病理生理学（54页珍藏版）》请在金锄头文库上搜索。
1、中 文 摘 要目的: 观察载脂蛋白A-I (apolipoproteinA-I, apoA-I) 对脂多糖 (Lipopolysaccharides, LPS) 诱导THP-1巨噬细胞源性泡沫细胞锌指蛋白36 (Tristetraprolin, TTP) 去磷酸化和炎症因子表达的影响，探讨TTP 翻译后修饰在apoA-I抑制巨噬细胞炎症因子表达中的作用及其机制。方法: 体外培养 THP-1 细胞株，用 100 mol/L 的佛波酯 (phorbol 12-myristate 13-acetate, PMA) 刺激细胞24h，使其诱导分化为巨噬细胞，并以 50 g/ml 的氧化型低密度脂蛋白 (
2、oxidized low density lipoprotein, oxLDL) 孵育细胞使其转化为 THP-1 巨噬细胞源性泡沫细胞。以 apoA-I 和/或 LPS 处理细胞，采用 ELISA检测白细胞介素-1 (interleukin-1, IL-1)、肿瘤坏死因子 (tumor necrosis factor , TNF-)、IL-6 和 IL-8 的表达；采用 Western-Blot 观察 apoA-I 对 LPS 诱导 THP-1 巨噬细胞源性泡沫细胞锌指蛋白36 (tristetraprolin, TTP) 和 磷酸化 TTP (tristetraprolin phosphor
3、ylation, phospho-TTP)、钙调蛋白(calmodulin, CaM)、钙调神经磷酸酶 (Calcineurin, CaN) 表达的影响。放线菌素D (actinomycin D, Act D) 终止转录后，实时定量 PCR 检测 IL-1 等多种炎症因子 mRNA 的表达。分别使用 CaM 抑制剂(W7)、CaN 抑制剂(FK506)、TTP 小 RNA干扰(siRNA) 处理细胞，以ELISA、实时定量 PCR 等方法分别检测巨噬细胞炎症因子蛋白质和 mRNA 的表达；发色底物法检测 CaM 和 CaN 的活性。结果：相对于 LPS 单独处理组，apoA-I 预处理 THP
4、-1 巨噬细胞源性泡沫细胞2h后 显著降低 LPS 诱导的炎症因子 IL-1、TNF-、IL-6 和 IL-8 表达水平；Act D 终止转录后，apoA-I 促进 IL-1 mRNA 的降解；apoA-I 促进 LPS 刺激THP-1 巨噬细胞源性泡沫细胞 TTP 去磷酸化，对 CaN 和 CaM 表达没有影响，但增强 CaM 和 CaN 活性。TTP siRNA下调 TTP 的表达，并明显抑制 apoA-I促进 THP-1 巨噬细胞源性泡沫细胞 IL-1 mRNA 降解的效应。W7 和 FK506分别抑制 CaM 和 CaN 活性，并明显抑制 apoA-I 对 phospho-TTP 表达
5、的下调作用，也抑制了apoA-I 促进 IL-1 mRNA 降解的效应。结论：在 THP-1 巨噬细胞源性泡沫细胞中，apoA-I 通过激活细胞内 CaM/CaN 信号转导途径，促进 TTP 去磷酸化，增强 TTP 功能，从而促进炎症因子 mRNA 的降解。关键词：锌指蛋白36；载脂蛋白A-I；泡沫细胞；炎症；动脉粥样硬化52ApoA-I inhitits LPS-induced inflammatory cytokines expression in macrophage-derived foam cells via regulating tristetraprolin dephosphor
6、ylationABSTRACTObjective: To observe the effect of apolipoprotein A1 (apoA-I) on the expression of inflammatory cytokines and explore the role of TTP-translational modification in the anti-inflammatory effect of apoA-I in macrophage-derived foam cells.Methods: Human THP-1 monocytes were treated in v
7、itro with phorbol 12-myristate 13-acetate (PMA) (100mol/L) for 24h, and then the medium was replaced by serum-free medium containing oxLDL (50 g/ml) to become fully differentiated macrophage foam cells before their use in experiments. Cells were cultured and treated with apoA-I and (or) LPS. Protein
8、 was extracted and subjected to ELISA analysis for interleukin-1 (IL-1), tumor necrosis factor- (TNF-), IL-6 and IL-8. Total protein extracts from cells were subjected to Western-Blot analyses for tristetraprolin (TTP), phospho-TTP, calmodulin (CaM), Calcineurin (CaN). The Act D was added to stop th
9、e transcription, RNA were extracted and subjected to RT-PCR analysis for inflammatory cytokines mRNA analysis. Cells were treated with CaM antagonist W7, CaN antagonist FK506 and siRNA of TTP. Protein and RNA were extracted and subjected to ELISA or RT-PCR analysis for inflammatory cytokines. Protei
10、n was extracted and subjected to chromogenic substrate analysis for CaM and CaN activity.Results: Human THP-1 macrophage-derived foam cells were pretreated by apoA-I for 2h, LPS-induced up-regulation of IL-1, TNF-, IL-6 and IL-8 were significantly suppressed relative to LPS alone group. The results
11、of RT-PCR indicated that apoA-I reduced the mRNA levels of IL-1 after treatment of act D. ApoA-I treatment significantly decreased the levels of tristetraprolin phosphorylation, had no effect on expression, but enhanced the activity of CaM and CaN in LPS-stimulated THP-1 macrophage-derived foam cell
12、s. Treatment with siRNA for TTP significantly down-regulated apoA-I-induced IL-1 mRNA decay in LPS-treated THP-1 cells. W7, a CaM inhibitor, and FK506 for CaN inhibitor significantly inhibited activity of CaM and CaN, which further abolished the inhibition effect of apoA-I on phospho-TTP expression
13、in LPS-treated THP-1 cells as well as IL-1 mRNA decay.Conclusions: ApoA-I enhanced the interaction of CaN and TTP through a CaM/CaN signaling pathway-dependent manner in LPS-stimulated human THP-1 macrophage-derived foam cells, and apoA-I increased some inflammatory cytokines mRNA decay via promotin
14、g the function and dephosphorylation of TTP, which can bind and target ARE-containing mRNAs for rapid degradation.Postgraduate: Jin-feng Li (Pathology & Pathophysiology) Directed by Professors: Yu-lin Tu & Kai YinKey words: Zinc finger protein 36, Apolipoprotein A1, Foam cells, Inflammation, atheros
15、clerosis引 言动脉粥样硬化(atherosclerosis, As) 是一种慢性免疫炎症性病变，同时也是心血管系统最常见的疾病，严重威胁人类健康，针对其预防和治疗是当前医学研究领域的重要课题。As的病理变化存在变质、渗出和增生等炎症的基本特征，其中血管壁炎症反应在As从发病到出现临床事件过程中都发挥重要作用。单核细胞进入血管壁是炎症反应激活的特征，已被认为是 As 发生发展的关键因素1。TTP 是一种具有双锌指结构域的RNA结合蛋白，主要在巨噬细胞表达，通过与多种炎症因子mRNA的AU重复序列结合，降低mRNA 的稳定性，促进其降解2。TTP主要功能为靶向调节体内多种与As相关的炎症因
16、子（其mRNA 的3UTR均存在与TTP结合的AREs），其中包括：TNF-、IL-1、IL-10、IFN-等3-5。然而，TTP磷酸化失活后就不具备降解炎症因子mRNA的效应6-8。炎症状态下TTP的磷酸化失活与p38MAPK激活有关：促炎因素LPS通过TLR4/MyD88途径激活p38MAPK，p38MAPK激活后进一步激活其下游MAPK激酶-2(MK2)，MK2磷酸化TTP的52和178丝氨酸位点，而磷酸化的TTP与适配蛋白14-3-3结合，并与去腺苷化酶分离，失去了对炎症因子mRNA降解的能力9, 10。研究发现，活化CaM/CaN信号途径后，CaN能够迅速去磷酸化p38MAPK并使其失活11。最近，CaN的同源性蛋白磷酸酯酶-2A(protein phosphatase-2A, PP-2A)也被发现可以直
