蛋白酶体及免疫亚单位在腹主动脉瘤发病中的作用及机制研究

发布时间：2018-06-15 19:36

本文选题：蛋白酶体 + β5i　；参考：《北京协和医学院》2016年博士论文

【摘要】：研究背景：动脉粥样硬化性腹主动脉瘤(AAA)是老龄化社会的常见病,动脉瘤破裂后,死亡率高,是60岁以上人群死亡的主要原因之一。炎症反应是其主要发病机制之一,其中T淋巴细胞浸润、分化作用重要,但具体调控机制尚待明确。泛素-蛋白酶体系统(UPS)在调节炎症反应中发挥重要作用,可通过激活NF-κB信号通路促进组织炎症反应,但蛋白酶体是否通过激活NF-κB促进AAA形成尚无报导。免疫蛋白酶体对T细胞分化、炎性因子分泌等过程具有重要调节作用,以p5i亚单位作用为主,但p5i是否参与AAA形成,目前尚无报导。目的：明确蛋白酶体及其免疫亚单位β5i在AAA形成中的作用及调节机制。实验方法：收集AAA患者及正常人腹主动脉组织,同时通过对雄性ApoE-/-小鼠皮下埋植血管紧张素Ⅱ(Ang Ⅱ)缓释泵(1,000 ng/min/kg)复制AAA动物模型。试剂盒测定蛋白酶体糜蛋白酶活性。采用免疫染色、Western Blot方法分析β5i在腹主动脉组织中表达水平。通过低剂量硼替佐米(Bortezomib, BTZ) (50μg/kg, i.p,每3日1次)部分抑制蛋白酶体活性,实验分为4组：假手术组(Sham),假手术给药组(BTZ),Ang Ⅱ灌注组(Ang Ⅱ),血管紧张素Ⅱ灌注+给药组(Ang Ⅱ+BTZ)。进一步采用PR-957(又称ONX 0914,10mg/kg, s.c,隔日1次)抑制p5i活性,实验分为4组：假手术组(Sham),假手术给药组(PR-957), Ang Ⅱ灌注组(AngⅡ), Ang Ⅱ灌注+给药组(Ang Ⅱ+PR-957)。大体取材计算腹主动脉平均直径、发生率、动脉瘤分型。通过HE染色、Masson染色和Gomori's醛品红法染色观察各组小鼠炎症反应和血管重塑情况。通过免疫染色、流式细胞分析技术明确炎症细胞浸润情况(包括巨噬细胞和CD3+T细胞)；通过免疫染色、qPCR分析明确各组小鼠动脉壁内炎症细胞亚型分泌细胞因子变化；通过免疫荧光染色a-SMA,并与末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法(TUNEL)共染色确定主动脉壁内血管平滑细胞(VSMC)凋亡情况；通过Western Blot等检测组织中炎症反应及相关信号通路变化；通过明胶酶谱法检测主动脉组织内基质金属蛋白酶(MMPs)的活性变化。结果：1、低剂量BTZ可有效抑制Ang Ⅱ诱导的ApoE-/-小鼠AAA的发生发展；2、BTZ可减轻腹主动脉组织炎症反应,减少组织中CD3+T淋巴细胞的数目；3、BTZ可减少Ang Ⅱ诱导的NF-κB信号通路激活及下游ICAM-1的表达；4、人及ApoE-/-小鼠AAA组织中免疫亚单位β5i表达及活性均增加；5、PR-957可有效抑制ApoE-/-小鼠组织中β5i的表达,并降低Ang Ⅱ诱导ApoE-/-小鼠AAA的发生率及严重程度；6、PR-957可有效抑制Ang Ⅱ诱导的ApoE-/-小鼠腹主动脉炎症反应及血管重塑；7、PR-957可减少组织中CD3+T淋巴细胞的数目,并抑制其向辅助型T淋巴细胞(Helper T cell, Th)和细胞毒性T细胞(Cytotoxic T cell, Tc)细胞分化；8、p5i高表达可通过促进主动脉壁辅助型T淋巴细胞17 (Helper T cell 17, Th17)细胞分化,同时抑制调节型细胞(Regular T cell, Treg)细胞分化,促进AAA形成：9、PR-957可抑制组织VSMC凋亡,同时抑制组织MMPs活化。结论：本研究表明蛋白酶体激活通过活化NF-κB信号通路促进炎症细胞在血管壁的黏附聚集促进AAA形成。进一步研究表明免疫蛋白酶体亚单位β5i的活化促进AAA形成,主要通过调节组织内特定炎性细胞分化。通过本研究,初步明确了固有蛋白酶体及p5i参与AAA发生的病理生理机制,为药物治疗AAA提供新的参考靶点。
[Abstract]:Background: atherosclerotic abdominal aortic aneurysm (AAA) is a common disease in the aging society. After aneurysm rupture, the mortality is high, and it is one of the main causes of death in people over 60 years of age. The inflammatory reaction is one of the main mechanisms of the disease. The infiltration of T lymphocytes is important, but the specific regulation mechanism remains to be clear. Ubiquitin egg is still to be defined. The white enzyme body system (UPS) plays an important role in regulating the inflammatory response. It can stimulate the inflammatory response by activating the NF- kappa B signaling pathway, but it is not reported that the proteasome promotes the formation of AAA by activating NF- kappa B. The immune proteasome has an important regulatory effect on the differentiation of T cells and the secretion of inflammatory factors, and the action of p5i subunit is the role of the proteasome. Main, but there is no report on whether p5i participates in the formation of AAA. Objective: to clarify the role and regulation mechanism of proteasome and its immune subunit beta 5I in the formation of AAA. Experimental methods: collecting AAA patients and normal human abdominal aorta tissue, and by subcutaneous implantation of angiotensin II (Ang II) sustained-release pump (1000 ng/min) to male ApoE-/- mice (1000 ng/min). /kg) replicate the AAA animal model. The activity of proteasome chymotrypsin was measured by the kit. The expression of beta 5I in the abdominal aorta was analyzed by immuno staining and Western Blot method. The activity of proteasome was suppressed by low dose boron (Bortezomib, BTZ) (50 u g/kg, i.p, 1 times every 3 days), and the experiment was divided into 4 groups: sham operation group (Sham). The sham operation group (BTZ), Ang II perfusion group (Ang II), angiotensin II perfusion + administration group (Ang II +BTZ). Further using PR-957 (also known as ONX 0914,10mg/kg, S.C, 1 times a day) to inhibit p5i activity, the experiment was divided into 4 groups: sham operation group (Sham), sham operation group (PR-957), perfusion group (II), perfusion II + administration group PR-957). The average diameter of the abdominal aorta, the incidence of the abdominal aorta, the aneurysm classification. The inflammatory reaction and vascular remodeling in each group were observed by HE staining, Masson staining and Gomori's aldehyde fuchsin staining. The inflammatory cell infiltration (including macrophages and CD3+T cells) was determined by immunization and flow cytometry. Through immunofluorescence staining, the changes of cytokines secreted by inflammatory cells in the arterial wall of the mice were determined by qPCR analysis. The apoptosis of vascular flat slide cells (VSMC) in the aortic wall was determined by immunofluorescence staining a-SMA and TUNEL (terminal deoxynucleotidyl transferase mediated dUTP nick end labeling assay); and Western Bl was used. OT and other changes in the inflammatory response and related signal pathways in the tissue. The changes in the activity of matrix metalloproteinase (MMPs) in the aorta were detected by gelatin zymography. Results: 1, low dose BTZ could effectively inhibit the development of AAA in ApoE-/- mice induced by Ang II; 2, BTZ can reduce the inflammatory reaction in the abdominal aorta and reduce the C in the tissue. The number of D3+T lymphocyte, 3, BTZ can reduce the activation of NF- kappa B signaling pathway induced by Ang II and the expression of downstream ICAM-1; 4, the expression and activity of the immune subunit beta 5I in human and ApoE-/- mice AAA tissues are increased, and 5, PR-957 can effectively inhibit the expression of beta 5I in ApoE-/- mouse tissues, and reduce the incidence of induced mice. 6, PR-957 can effectively inhibit the inflammatory response and vascular remodeling in the abdominal aorta of ApoE-/- mice induced by Ang II, and 7, PR-957 can reduce the number of CD3+T lymphocytes in the tissues, and inhibit the differentiation to the auxiliary T lymphocyte (Helper T cell, Th) and the cytotoxic T cells (Cytotoxic). By promoting the differentiation of T lymphocyte 17 (Helper T cell 17, Th17) cells and inhibiting the differentiation of regulatory cells (Regular T cell, Treg) cells and promoting the formation of AAA: 9, PR-957 can inhibit apoptosis of tissue VSMC, and inhibit tissue activation. Conclusion: the activation of proteasome activation by activation of the activated kappa activation signal The pathway promotes the adhesion and aggregation of inflammatory cells in the vascular wall to promote the formation of AAA. Further studies have shown that the activation of the immuno proteasome subunit beta 5I promotes the formation of AAA, mainly by regulating the differentiation of specific inflammatory cells in the tissue. Through this study, the pathophysiological mechanism of the inherent proteasome and p5i and AAA is preliminarily identified for the treatment of drugs. The treatment of AAA provides a new reference target.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.1

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