他汀类药物治疗动脉粥样硬化的免疫机制研究

发布时间：2018-01-02 11:27

本文关键词：他汀类药物治疗动脉粥样硬化的免疫机制研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

更多相关文章： 他汀类药物 AS NK细胞 NKT细胞 Tim-3

【摘要】：研究背景及目的:心脑血管疾病是目前全球范围内致死率和致残率都非常高的疾病,其本质上的病理变化是相关血管的动脉粥样硬化(atherosclerosis,AS)。研究认为AS是一种免疫性疾病,感染、免疫缺陷及自身免疫都参与了 AS的病理、生理过程。固有免疫和适应性免疫的多种免疫细胞和免疫因子参与了 AS的发生和发展,自然杀伤细胞(NK细胞)和自然杀伤T细胞(NKT细胞)被发现存在于AS斑块中,参与了 AS的形成。研究表明,AS中NK细胞数量下降,细胞毒性功能减弱。NK细胞可分为功能不同的两个亚群:CD3-CD56bright NK细胞和CD3-CD56dim NK细胞。NKT细胞是兼有NK细胞和T细胞特征的一群细胞,表面同时表达NK细胞和T细胞上存在的部分分子结构。NK细胞和NKT细胞活化后均可分泌干扰素-γ(IFN-γ),动物实验显示IFN-y有促进AS形成的作用。T细胞免疫球蛋白黏蛋白 3(T cell immunoglobulin-and mucin-domain-containing molecule-3,Tim-3)是一型跨膜蛋白Tim家族的一员,被发现表达于T细胞、NK细胞、NKT细胞等多种细胞表面。在慢性乙型肝炎患者中,NKT细胞上Tim-3表达上调;在多种疾病状态下,NK细胞上Tim-3表达均升高,同时伴有NK细胞的细胞毒性及IFN-γ分泌减少。在动脉粥样硬化患者外周血中,NK细胞上Tim-3分子也被发现表达升高。他汀类药物是一种3-羟基-3-甲基戊二酸单酰辅酶A(HMG-CoA)还原酶抑制剂,临床上广泛用于AS的治疗,对AS及相关疾病有良好的治疗效果。研究提示,他汀类药物发挥抗AS的作用并不完全依赖于其降血脂的功效,他汀类药物的免疫调节作用在其中有着重要影响。他汀类药物通过抑制HMG-CoA还原酶而影响下游多种成分的生成,进而影响下游小分子参与的生物过程,发挥免疫调节作用。然而,他汀类药物通过免疫调节作用治疗AS的机制尚不完全清楚,本研究旨在通过研究他汀类药物对AS外周血NK细胞和NKT细胞表面Tim-3分子和IFN-y分子的影响,初步探索他汀类药物治疗AS可能通过的免疫途径,为临床治疗提供更多的实验和理论基础。研究对象和方法:1.研究对象:研究对象为2016年1月-12月期间于山东省千佛山医院就诊的35岁-79岁的患者,经影像学或者超声检查诊断患有AS。严格按照入选标准和排除标准筛选患者。本研究由山东大学千佛山医院伦理委员会批准。每一位入组的患者均被告知了本研究,并同意了相关事宜。有14例服用瑞舒伐他汀治疗的患者(其中2位患者每天10 mg,其余患者每天20 mg,口服,连续服用3个月以上)和12例服用阿托伐他汀治疗的患者(其中1位患者每天5 mg,其余患者每天10 mg,口服,连续服用3个月以上)被纳入实验,以及20例未服用他汀类药物治疗的患者被纳入实验作为对照。抽取每位被纳入实验的患者的空腹外周静脉血。2.实验方法:将收集的外周血尽快使用淋巴细胞分离液处理,提取外周单个核细胞(PBMCs),制备细胞悬液。取PBMCs细胞悬液,加入FITC标记的抗人CD3、PerCP标记的抗人CD56及APC标记的抗人Tim-3的抗体,于4℃避光环境下孵育30分钟后上机检测NK细胞(CD3-CD56+细胞),CD3-CD56bright NK细胞,CD3-CD56dim NK 细胞,CD3-CD56+Tim-3+ 细胞,CD3-CD56brightTim-3+ 细胞,CD3-CD56dimTim-3+ 细胞,NKT 细胞(CD3+CD56+ 细胞),CD3+CD56+Tim-3+ 细胞,CD3+Tim-3+细胞以及Tim-3分子在NK细胞及其亚群上的平均荧光素强度(mean fluorescence intensity,MFI)。另取PBMCs细胞悬液在含刺激阻断剂的完全培养基中培养2小时之后,加入FITC标记的抗人CD3、PerCP标记的抗人CD56抗体于4℃避光环境下孵育30分钟后,固定、破膜,加入PE标记的抗人IFN-γ抗体,于4℃避光环境下孵育30分钟后重悬上机,流式检测CD3-CD56+IFN-γ 细胞、CD3+CD56+IFN-γ+ 细胞和 CD3+IFN-γ+ 细胞。3.统计学处理:采用SPSS 20统计软件分析数据,计量资料中,符合正态分布及方差齐的数据采用单因素方差分析(one-way ANOVA),其余数据采用非参数检验(Kruskal-Wallis one-wayANOVA)比较三组样本的组间差异。分类变量资料采用χ2检验(chi-square test)进行分析。p0.05为差异有统计学意义。结果:1.共有46例患者被纳入研究,在瑞舒伐他汀治疗组、阿托伐他汀治疗组和对照组中,男性患者所占比例无明显差异(78%,67%,75%,p=0.78),年龄无统计学差异(63.3±2.4,67.1 ±2.3,64.0±1.9,p=0.48),糖尿病患者所占比例无统计学差异(21%,33%,20%,p=0.67)。2.他汀类药物治疗降低了 NK细胞及其细胞亚群中Tim-3+细胞所占的比例以及三群细胞上Tim-3的MFI(瑞舒伐他汀治疗组vs对照组和阿托伐他汀治疗组vs对照组的p值依次为:Tim-3+ NK细胞p=0.003,p=0.003;NK细胞上Tim-3的MFI p=0.019,p=0.011;CD3-CD56brightTim-3+ NK 细胞p=0.002,p=0.001;CD3-CD56bright NK细胞中 Tim-3 的 MFI,p=0.004,p=0.004;CD3-CD56dimTim-3+ NK细胞p=0.004,p=0.005;CD3-CD56dimNK细胞中Tim-3 的 MFI,p=0.021,p=0.013)。3.他汀类药物治疗降低了 NKT细胞和CD3+T细胞中Tim-3+细胞的比例(瑞舒伐他汀治疗组vs对照组:p0.001,p0.001;阿托伐他汀治疗组vs对照组:p0.001,p=0.009);增加了外周血单个核细胞中NKT细胞的比例(瑞舒伐他汀治疗组vs对照组:p=0.092;阿托伐他汀治疗组vs对照组:p=0.017)。4.各个细胞群中,干扰素-γ的产生无明显差异。结论:他汀类药物可降低AS中NK细胞及其细胞亚群表面、NKT细胞表面和CD3+T细胞表面Tim-3分子表达,增加外周血中NKT细胞的比例,但不影响IFN-γ的产生,这可能在他汀类药物的抗动脉粥样硬化治疗中发挥了一定作用,可能是他汀类药物治疗AS通过的免疫机制,但是具体的作用机制有待于进一步深入研究。
[Abstract]:Background and purpose: cerebrovascular disease is present worldwide morbidity and mortality are very high disease, pathological changes of its essence is related to vascular atherosclerosis (atherosclerosis, AS). The study suggests that AS is an autoimmune disease, infection, immune deficiency and autoimmunity are involved in the AS the pathological and physiological process. A variety of immune cells and immune factors in innate immunity and adaptive immunity are involved in the occurrence and development of AS, natural killer cells (NK cells) and natural killer T cells (NKT cells) were found in the AS patch, is involved in the formation of AS. The results show that the number of NK cells in AS decreased cytotoxic function of.NK cells can be divided into the different functions of the two subsets: CD3-CD56bright NK cells and NK cells, CD3-CD56dim.NKT cells are a group of cells with NK cells and T cells of the surface at the same time, the expression of NK fine Interferon gamma secretion cells and T cells can exist on the part of the molecular structure of.NK cells and activated NKT cells (IFN-), animal experiments show that IFN-y can promote.T cell immunoglobulin AS formation of mucin 3 (T cell immunoglobulin-and mucin-domain-containing molecule-3, Tim-3) is a member of a type Tim transmembrane protein family the expression was found in T cells, NK cells, NKT cells and other cells surface. In patients with chronic hepatitis B, NKT cells Tim-3 expression; in a variety of disease states, NK cells on the expression of Tim-3 was increased, and the cell toxicity and IFN- gamma NK cells with reduced secretion in patients with atherosclerosis. In the peripheral blood of Tim-3 molecules on NK cells were also found. Increased expression of statins is a kind of 3- hydroxy -3- methyl glutaryl coenzyme A reductase inhibitors (HMG-CoA), widely used in clinical treatment of AS, Has a good therapeutic effect on AS and related diseases. Studies suggest that statins play the role of anti AS is not entirely dependent on the lipid-lowering efficacy of immunomodulatory effects of statins have important influence in the formation. Statins can inhibit HMG-CoA reductase and affect downstream of many ingredients, then influence the biological processes involved in downstream molecules, play a role in immune regulation. However, statins through immune regulation mechanism in the treatment of AS is not clear, the purpose of this research is to study on the influence of statins on peripheral blood AS NK cells and NKT cell surface Tim-3 and IFN-y molecules, explore the immune pathway of statins AS may be through drug treatment, to provide experimental and theoretical basis for more clinical treatment. Subjects and methods: 1. research object is January 2016 -12 months in the mountains Visit the East Hospital in Qianfo Hill province with 35 -79 years of age, by imaging or ultrasound with AS. in strict accordance with the inclusion criteria and exclusion criteria for screening patients. This study was approved by the ethics committee of Shandong University Qianfo Hill hospital. Every patients were informed of the study, and agreed with related matters. 14 patients taking rosuvastatin treatment (including 2 patients of 10 mg per day, the rest of the patients every 20 mg, oral, continuous use for more than 3 months) and 12 cases were treated with atorvastatin (including 1 patients every 5 mg, the remaining patients with 10 mg per day, orally, for taking 3 A month or more) were included in the experiment, and 20 patients not taking statins were included in the study as a control group. The patients included in the experiment were collected from each fasting peripheral venous blood.2. experimental methods: peripheral blood collection using the shower as soon as possible Pakistan cell separation, extraction of peripheral mononuclear cells (PBMCs), cell suspension was prepared. PBMCs cell suspension with FITC conjugated anti human CD3 antibody, PerCP labeled anti human CD56 and APC labeled anti human Tim-3, 4 DEG C to avoid light environment after 30 min of incubation the detection of NK cells (CD3-CD56+ cells), NK cells CD3-CD56bright, NK cells, CD3-CD56dim CD3-CD56+Tim-3+ cells, CD3-CD56brightTim-3+ cells, CD3-CD56dimTim-3+ cells, NKT cells (CD3+CD56+ cells), CD3+CD56+Tim-3+ cells, CD3+Tim-3+ cells and Tim-3 molecules in NK cells and its subsets of the average intensity of fluorescein (mean fluorescence intensity, MFI). Another PBMCs cells in the suspension containing stimulation blocker full medium after 2 hours, adding FITC labeled anti human CD3 and anti human CD56 antibody labeled with PerCP at 4 DEG C and dark environment after 30 min of incubation, fixed, Rupture of membranes, anti human IFN- antibodies with PE labeled, 4 DEG C to avoid light environment after 30 min incubation suspending machine, flow cytometry CD3-CD56+IFN- gamma cells, CD3+CD56+IFN- + cells and CD3+IFN- + cells.3. statistical analysis: data analysis using SPSS 20 statistical software, measurement data, accord with normal the distribution and homogeneity of variance data using single factor analysis of variance (one-way ANOVA), the rest of the data using non parametric test (Kruskal-Wallis one-wayANOVA) the difference between the three groups of sample groups. Categorical data using 2 test (chi-square test) of.P0.05 was considered significant. Results: 1. a total of 46 patients were included in the study, atorvastatin treatment group in rosuvastatin, atorvastatin treatment group and control group, the proportion of male patients with no significant difference (78%, 67%, 75%, p=0.78), there was no significant difference in age (63.3 + 2.4,67.1 + 2.3,64.0 + 1.9, P =0.48), the proportion of patients with diabetes had no statistical difference (21%, 33%, 20%, p=0.67).2. statin therapy reduces the proportion of Tim-3+ cell subsets in NK cells and the percentage of cells and three cells Tim-3 MFI (rosuvastatin treatment group, vs control group and atorvastatin treatment group vs control group P values were: Tim-3+ p=0.003 p=0.003; NK cells, NK cells of Tim-3 MFI p=0.019, p=0.011 CD3-CD56brightTim-3+ NK p=0.002; p=0.001; Tim-3 cells, NK cells in CD3-CD56bright, MFI, p=0.004, p=0.004; CD3-CD56dimTim-3+ p=0.004 p=0.005; Tim-3 NK cells, CD3-CD56dimNK cells in MFI, p=0.021, p=0.013).3. statin treatment reduced Tim-3+ NKT cell and CD3+T cell ratio (rosuvastatin treatment group vs control group: p0.001, p0.001; atorvastatin group vs control group: p0.001, p=0.009); the increase NKT cells in peripheral blood mononuclear cells (the proportion of rosuvastatin treatment group vs and control group p=0.092; atorvastatin group vs control group: p=0.017.4.) each cell group, no significant difference between the interferon gamma production. Conclusion: statins can reduce AS in NK cells and cell subsets the surface of cell surface expression of NKT and CD3+T cell surface molecules of Tim-3, increase of NKT cells in peripheral blood, but does not affect the IFN- gamma production, which may play an important role in anti atherosclerosis, statins, may be the immune mechanism of statin treatment by AS, but the specific mechanism of action further study is needed.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.5

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