依鲁替尼对ox-LDL诱导的巨噬细胞泡沫化和焦亡的调节作用及其与NLRP3炎症小体关系的研究

发布时间：2018-03-11 01:16

本文选题：依鲁替尼　切入点：NLRP3　出处：《大连医科大学》2016年博士论文　论文类型：学位论文

【摘要】：动脉粥样硬化早期,单核细胞在趋化因子的作用下被招募至病变处,与内皮细胞黏附,随后迁移至内皮下,转化为巨噬细胞。巨噬细胞摄入ox-LDL(oxidized low-density lipoprotein,氧化低密度脂蛋白)形成泡沫细胞,并大量死亡,形成动脉粥样硬化斑块的坏死核心,削弱纤维帽,增加斑块不稳定性。巨噬细胞的泡沫化和死亡是动脉粥样硬化发生、发展的关键环节。近年来NLRP3(nucleotide-binding oligomerization domain-like receptor protein3,核苷酸结合寡聚结构域样受体蛋白3)炎症小体在动脉粥样硬化中的作用越来越受到重视。研究显示NLRP3炎症小体参与动脉粥样硬化的形成,并与斑块的稳定性相关。NLRP3炎症小体可被ox-LDL激活,激活的炎症小体可剪切caspase-1前体(Cysteine-requiring Aspartate Protease-1,半胱氨酸天冬氨酸蛋白酶-1),促进成熟IL-1β(Interleukin-1β,白介素1β)的释放,诱导巨噬细胞焦亡,还可能参与细胞脂质沉积。依鲁替尼是一种口服的BTK(Bruton’s tyrosine kinase,布鲁顿酪氨酸激酶)抑制剂,临床上被用来治疗白血病。研究发现依鲁替尼可抑制明矾诱导的巨噬细胞NLRP3炎症小体激活,尚不知其对ox-LDL诱导的NLRP3炎症小体激活是否同样有抑制作用。我们据此提出假说:依鲁替尼可通过抑制NLRP3炎症小体的激活调节ox-LDL诱导的巨噬细胞泡沫化和焦亡。目的:观察依鲁替尼对ox-LDL诱导的THP-1起源巨噬细胞泡沫化和焦亡的影响,探讨其与NLRP3炎症小体的关系。方法:以PMA诱导的THP-1或THP-1-defNLRP3起源巨噬细胞为研究对象。给予ox-LDL,构建细胞泡沫化、焦亡、迁移和黏附模型,并分别用依鲁替尼和MCC950(NLRP3特异性抑制剂)进行处理。采用Western Blot、ELISA方法检测巨噬细胞NLRP3、pro-caspase-1、caspase-1、pro-IL-1、IL-1β水平,观察ox-LDL对巨噬细胞NLRP3炎症小体激活的诱导作用,以及依鲁替尼对ox-LDL诱导的巨噬细胞NLRP3炎症小体激活的影响。采用油红O染色、酶学法、免疫荧光、流式等方法观察依鲁替尼对巨噬细胞泡沫化、细胞内胆固醇含量、脂质摄入及胆固醇外流的影响,并采用Western Blot方法检测依鲁替尼对调节巨噬细胞胆固醇平衡的关键蛋白水平的影响,及其与NLRP3炎症小体的关系。分别用依鲁替尼、MCC950、VX765(caspase-1特异性抑制剂)等处理巨噬细胞,进行乳酸脱氢酶释放检测、Hoechst 33342和碘化丙啶双染及TUNEL染色,验证ox-LDL对巨噬细胞焦亡的诱导作用,观察依鲁替尼对ox-LDL诱导的巨噬细胞焦亡的影响,以及其与NLRP3炎症小体的关系。采用transwell小室、划痕实验及细胞黏附实验观察依鲁替尼对单核巨噬细胞迁移、黏附的影响及其与NLRP3炎症小体的关系。结果:(1)依鲁替尼可抑制ox-LDL诱导的THP-1起源的巨噬细胞NLRP3炎症小体的激活。(2)依鲁替尼、MCC950和NLRP3基因敲除均可以调节THP-1起源的巨噬细胞CD36、ABCA1和SR-BI蛋白表达水平,抑制细胞对ox-LDL的摄入,促进游离胆固醇外流,减轻细胞泡沫化程度。(3)Ox-LDL可促进THP-1起源巨噬细胞乳酸脱氢酶的释放,增加PI染色细胞比率及TUNEL染色阳性率;依鲁替尼、MCC950、VX765和NLRP3基因敲除均可抑制ox-LDL诱导的巨噬细胞乳酸脱氢酶的释放,降低PI染色细胞比率及TUNEL染色阳性率。(4)依鲁替尼和MCC950可不同程度的抑制THP-1细胞的迁移,但对THP-1细胞与内皮细胞的黏附无显著影响。结论:依鲁替尼可能通过抑制NLRP3炎症小体的激活抑制ox-LDL诱导的巨噬细胞泡沫化和焦亡。
[Abstract]:In the early stage of atherosclerosis, monocyte chemotactic factors are recruited to the lesion, adhesion and endothelial cells, then migrate to the endothelium, transformed into macrophages. Macrophages (oxidized low-density lipoprotein, ox-LDL uptake of oxidized low density lipoprotein) to form foam cells, and a large number of deaths, the formation of atherosclerotic plaque necrosis core. Weaken the fibrous cap, increased plaque instability. Macrophage foam and death is the occurrence of atherosclerosis, the key to development. In recent years, NLRP3 (nucleotide-binding oligomerization domain-like receptor protein3, nucleotide binding oligomerization domain protein like receptor 3) inflammasome in atherosclerosis is more and more attention. Research shows that the formation of NLRP3 inflammasome in atherosclerosis and plaque stability, and related.NLRP3 inflammasome Can be activated by ox-LDL, the activation of the inflammasome can shear the precursor of Caspase-1 (Cysteine-requiring Aspartate Protease-1, caspase -1), promote the mature IL-1 beta (Interleukin-1 beta, interleukin 1 beta) release of macrophages induced by focal dead, also may be involved in cellular lipid accumulation. Ibrutinib is an oral BTK (Bruton 's tyrosine kinase, Bruton tyrosine kinase inhibitor), were clinically used to treat leukemia. The study found that Yilu for alum can inhibit macrophages induced by NLRP3 inflammasome activation, is not known for its NLRP3 inflammasome activation of ox-LDL induced by whether it has the same inhibitory effect. We put forward the hypothesis: Yilu for Nicole through inhibition of NLRP3 inflammasome activation of ox-LDL induced macrophage foam and focal apoptosis. Objective: To observe the effect of ibrutinib on ox-LDL induced THP-1 derived macrophages Effects of cell foam and focal apoptosis, to explore the relationship between NLRP3 and inflammatory corpuscle. Methods: PMA induced THP-1 or THP-1-defNLRP3 macrophage origin as the research object. To construct ox-LDL, foam cell apoptosis, focal adhesion and migration model, and respectively with ibrutinib and MCC950 (NLRP3 specific inhibitor) were processing. Using Western Blot ELISA method for detection of macrophage NLRP3, pro-caspase-1, caspase-1, pro-IL-1, IL-1 levels, to observe the effect of ox-LDL on macrophage activation induced by NLRP3 inflammasome, and ibrutinib effect on ox-LDL induced macrophage NLRP3 inflammasome activation. The oil red O staining, immunofluorescence, enzyme method, observation Yilu imatinib on macrophage foam flow cytometry method, cellular cholesterol content, lipid intake and cholesterol efflux, and using the Western Blot method for detection of ibrutinib. Effect of key protein macrophage cholesterol homeostasis, and its relationship with NLRP3 inflammasome. With ibrutinib, MCC950, VX765 (Caspase-1 inhibitor) treatment of macrophages, lactate dehydrogenase release test, Hoechst 33342 and propidium iodide double staining and TUNEL staining, to verify the effect of ox-LDL on macrophage induced by focal dead the observation of ibrutinib effect on ox-LDL macrophages induced by focal death, and its relationship with NLRP3 inflammasome. By Transwell assay, scratch test and cell adhesion test observation of ibrutinib on mononuclear macrophage migration, adhesion and the relationship with NLRP3 inflammasome. Results: (1) Yilu for activation Nicole inhibited ox-LDL induced THP-1 macrophage origin NLRP3 inflammasome. (2) ibrutinib, MCC950 and NLRP3 gene knockout can regulate THP-1 derived macrophages Cell CD36, ABCA1 and SR-BI protein expression, inhibiting cell ox-LDL uptake, promote free cholesterol efflux, reduce the degree of foam cells. (3) Ox-LDL can promote THP-1 macrophage origin of lactate dehydrogenase release, increased PI staining and TUNEL staining positive cell ratio; ibrutinib, MCC950, VX765 and NLRP3 gene knockout in addition to macrophages can inhibit ox-LDL induced lactate dehydrogenase release, reduce PI staining and TUNEL staining. The ratio of the positive rate (4) ibrutinib and MCC950 can transfer different degrees of inhibition of THP-1 cells, but the adhesion of THP-1 cells and endothelial cells had no significant effect. Conclusion: ibrutinib can inhibit the inflammation of NLRP3 body activation inhibited ox-LDL induced macrophage foam and focal dead.

【学位授予单位】：大连医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543

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