本文關(guān)鍵詞：他汀類藥物治療動脈粥樣硬化的免疫機(jī)制研究　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 他汀類藥物 AS NK細(xì)胞 NKT細(xì)胞 Tim-3

【摘要】：研究背景及目的:心腦血管疾病是目前全球范圍內(nèi)致死率和致殘率都非常高的疾病,其本質(zhì)上的病理變化是相關(guān)血管的動脈粥樣硬化(atherosclerosis,AS)。研究認(rèn)為AS是一種免疫性疾病,感染、免疫缺陷及自身免疫都參與了 AS的病理、生理過程。固有免疫和適應(yīng)性免疫的多種免疫細(xì)胞和免疫因子參與了 AS的發(fā)生和發(fā)展,自然殺傷細(xì)胞(NK細(xì)胞)和自然殺傷T細(xì)胞(NKT細(xì)胞)被發(fā)現(xiàn)存在于AS斑塊中,參與了 AS的形成。研究表明,AS中NK細(xì)胞數(shù)量下降,細(xì)胞毒性功能減弱。NK細(xì)胞可分為功能不同的兩個(gè)亞群:CD3-CD56bright NK細(xì)胞和CD3-CD56dim NK細(xì)胞。NKT細(xì)胞是兼有NK細(xì)胞和T細(xì)胞特征的一群細(xì)胞,表面同時(shí)表達(dá)NK細(xì)胞和T細(xì)胞上存在的部分分子結(jié)構(gòu)。NK細(xì)胞和NKT細(xì)胞活化后均可分泌干擾素-γ(IFN-γ),動物實(shí)驗(yàn)顯示IFN-y有促進(jìn)AS形成的作用。T細(xì)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家族的一員,被發(fā)現(xiàn)表達(dá)于T細(xì)胞、NK細(xì)胞、NKT細(xì)胞等多種細(xì)胞表面。在慢性乙型肝炎患者中,NKT細(xì)胞上Tim-3表達(dá)上調(diào);在多種疾病狀態(tài)下,NK細(xì)胞上Tim-3表達(dá)均升高,同時(shí)伴有NK細(xì)胞的細(xì)胞毒性及IFN-γ分泌減少。在動脈粥樣硬化患者外周血中,NK細(xì)胞上Tim-3分子也被發(fā)現(xiàn)表達(dá)升高。他汀類藥物是一種3-羥基-3-甲基戊二酸單酰輔酶A(HMG-CoA)還原酶抑制劑,臨床上廣泛用于AS的治療,對AS及相關(guān)疾病有良好的治療效果。研究提示,他汀類藥物發(fā)揮抗AS的作用并不完全依賴于其降血脂的功效,他汀類藥物的免疫調(diào)節(jié)作用在其中有著重要影響。他汀類藥物通過抑制HMG-CoA還原酶而影響下游多種成分的生成,進(jìn)而影響下游小分子參與的生物過程,發(fā)揮免疫調(diào)節(jié)作用。然而,他汀類藥物通過免疫調(diào)節(jié)作用治療AS的機(jī)制尚不完全清楚,本研究旨在通過研究他汀類藥物對AS外周血NK細(xì)胞和NKT細(xì)胞表面Tim-3分子和IFN-y分子的影響,初步探索他汀類藥物治療AS可能通過的免疫途徑,為臨床治療提供更多的實(shí)驗(yàn)和理論基礎(chǔ)。研究對象和方法:1.研究對象:研究對象為2016年1月-12月期間于山東省千佛山醫(yī)院就診的35歲-79歲的患者,經(jīng)影像學(xué)或者超聲檢查診斷患有AS。嚴(yán)格按照入選標(biāo)準(zhǔn)和排除標(biāo)準(zhǔn)篩選患者。本研究由山東大學(xué)千佛山醫(yī)院倫理委員會批準(zhǔn)。每一位入組的患者均被告知了本研究,并同意了相關(guān)事宜。有14例服用瑞舒伐他汀治療的患者(其中2位患者每天10 mg,其余患者每天20 mg,口服,連續(xù)服用3個(gè)月以上)和12例服用阿托伐他汀治療的患者(其中1位患者每天5 mg,其余患者每天10 mg,口服,連續(xù)服用3個(gè)月以上)被納入實(shí)驗(yàn),以及20例未服用他汀類藥物治療的患者被納入實(shí)驗(yàn)作為對照。抽取每位被納入實(shí)驗(yàn)的患者的空腹外周靜脈血。2.實(shí)驗(yàn)方法:將收集的外周血盡快使用淋巴細(xì)胞分離液處理,提取外周單個(gè)核細(xì)胞(PBMCs),制備細(xì)胞懸液。取PBMCs細(xì)胞懸液,加入FITC標(biāo)記的抗人CD3、PerCP標(biāo)記的抗人CD56及APC標(biāo)記的抗人Tim-3的抗體,于4℃避光環(huán)境下孵育30分鐘后上機(jī)檢測NK細(xì)胞(CD3-CD56+細(xì)胞),CD3-CD56bright NK細(xì)胞,CD3-CD56dim NK 細(xì)胞,CD3-CD56+Tim-3+ 細(xì)胞,CD3-CD56brightTim-3+ 細(xì)胞,CD3-CD56dimTim-3+ 細(xì)胞,NKT 細(xì)胞(CD3+CD56+ 細(xì)胞),CD3+CD56+Tim-3+ 細(xì)胞,CD3+Tim-3+細(xì)胞以及Tim-3分子在NK細(xì)胞及其亞群上的平均熒光素強(qiáng)度(mean fluorescence intensity,MFI)。另取PBMCs細(xì)胞懸液在含刺激阻斷劑的完全培養(yǎng)基中培養(yǎng)2小時(shí)之后,加入FITC標(biāo)記的抗人CD3、PerCP標(biāo)記的抗人CD56抗體于4℃避光環(huán)境下孵育30分鐘后,固定、破膜,加入PE標(biāo)記的抗人IFN-γ抗體,于4℃避光環(huán)境下孵育30分鐘后重懸上機(jī),流式檢測CD3-CD56+IFN-γ 細(xì)胞、CD3+CD56+IFN-γ+ 細(xì)胞和 CD3+IFN-γ+ 細(xì)胞。3.統(tǒng)計(jì)學(xué)處理:采用SPSS 20統(tǒng)計(jì)軟件分析數(shù)據(jù),計(jì)量資料中,符合正態(tài)分布及方差齊的數(shù)據(jù)采用單因素方差分析(one-way ANOVA),其余數(shù)據(jù)采用非參數(shù)檢驗(yàn)(Kruskal-Wallis one-wayANOVA)比較三組樣本的組間差異。分類變量資料采用χ2檢驗(yàn)(chi-square test)進(jìn)行分析。p0.05為差異有統(tǒng)計(jì)學(xué)意義。結(jié)果:1.共有46例患者被納入研究,在瑞舒伐他汀治療組、阿托伐他汀治療組和對照組中,男性患者所占比例無明顯差異(78%,67%,75%,p=0.78),年齡無統(tǒng)計(jì)學(xué)差異(63.3±2.4,67.1 ±2.3,64.0±1.9,p=0.48),糖尿病患者所占比例無統(tǒng)計(jì)學(xué)差異(21%,33%,20%,p=0.67)。2.他汀類藥物治療降低了 NK細(xì)胞及其細(xì)胞亞群中Tim-3+細(xì)胞所占的比例以及三群細(xì)胞上Tim-3的MFI(瑞舒伐他汀治療組vs對照組和阿托伐他汀治療組vs對照組的p值依次為:Tim-3+ NK細(xì)胞p=0.003,p=0.003;NK細(xì)胞上Tim-3的MFI p=0.019,p=0.011;CD3-CD56brightTim-3+ NK 細(xì)胞p=0.002,p=0.001;CD3-CD56bright NK細(xì)胞中 Tim-3 的 MFI,p=0.004,p=0.004;CD3-CD56dimTim-3+ NK細(xì)胞p=0.004,p=0.005;CD3-CD56dimNK細(xì)胞中Tim-3 的 MFI,p=0.021,p=0.013)。3.他汀類藥物治療降低了 NKT細(xì)胞和CD3+T細(xì)胞中Tim-3+細(xì)胞的比例(瑞舒伐他汀治療組vs對照組:p0.001,p0.001;阿托伐他汀治療組vs對照組:p0.001,p=0.009);增加了外周血單個(gè)核細(xì)胞中NKT細(xì)胞的比例(瑞舒伐他汀治療組vs對照組:p=0.092;阿托伐他汀治療組vs對照組:p=0.017)。4.各個(gè)細(xì)胞群中,干擾素-γ的產(chǎn)生無明顯差異。結(jié)論:他汀類藥物可降低AS中NK細(xì)胞及其細(xì)胞亞群表面、NKT細(xì)胞表面和CD3+T細(xì)胞表面Tim-3分子表達(dá),增加外周血中NKT細(xì)胞的比例,但不影響IFN-γ的產(chǎn)生,這可能在他汀類藥物的抗動脈粥樣硬化治療中發(fā)揮了一定作用,可能是他汀類藥物治療AS通過的免疫機(jī)制,但是具體的作用機(jī)制有待于進(jìn)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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R543.5

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