本文關(guān)鍵詞： 動(dòng)脈粥樣硬化 斑塊穩(wěn)定性 炎癥 活化的第十凝血因子 蛋白酶激活受體-2 ERK1/2信號(hào)通路　出處：《東南大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：動(dòng)脈粥樣硬化是一種進(jìn)展性的炎癥性疾病,不穩(wěn)定動(dòng)脈粥樣硬化斑塊破裂并繼發(fā)血栓形成可導(dǎo)致危及生命的急性心血管事件。FXa不僅在凝血級(jí)聯(lián)中占據(jù)著重要位置,對(duì)凝血酶的形成起到關(guān)鍵作用,而且越來(lái)越多的證據(jù)顯示FXa在炎癥反應(yīng)中也扮演著不可或缺的角色,因此本課題擬探討FXa對(duì)動(dòng)脈粥樣硬化斑塊穩(wěn)定性的影響及相關(guān)機(jī)制。目的：1.探討抑制FXa是否影響小鼠動(dòng)脈粥樣硬化斑塊的病理進(jìn)展和穩(wěn)定性；2.探討FXa影響動(dòng)脈粥樣硬化斑塊穩(wěn)定性的可能機(jī)制；3.探討FXa對(duì)巨噬細(xì)胞生物學(xué)特性的影響及可能機(jī)制。方法：1.FXa抑制劑對(duì)ApoE-/-小鼠動(dòng)脈粥樣硬化斑塊病理進(jìn)展的影響：選取6周齡大小的雄性ApoE-/-小鼠作為實(shí)驗(yàn)小鼠,全程進(jìn)行高脂飼養(yǎng),建立動(dòng)脈粥樣硬化斑塊模型,20周齡時(shí)進(jìn)行為期4周的Fondaparinux (FXa抑制劑)腹腔注射,5mg/kg,1次／天。24周齡時(shí)麻醉稱(chēng)重,心臟取血,檢測(cè)小鼠血漿中總膽固醇、甘油三酯、低密度脂蛋白膽固醇和高密度脂蛋白膽固醇的水平。顯微鏡下取小鼠頭臂干,HE染色法測(cè)量動(dòng)脈粥樣硬化斑塊的病變面積、病變厚度、管腔狹窄程度、纖維帽厚度和壞死核比例,Masson染色法檢測(cè)斑塊中膠原含量,Perls染色法檢測(cè)斑塊內(nèi)出血情況,Van Kossa染色法檢測(cè)斑塊鈣化程度,免疫組化法檢測(cè)斑塊中Factor X、α-SMA、Mac-2、VCAM-1、CD-31、 MMP-9、PAR-1、PAR-2的表達(dá),明膠酶譜法檢測(cè)斑塊中MMP-9的酶活性,Western Bolt法檢測(cè)斑塊中PAR-1和PAR-2信號(hào)。顯微鏡下取小鼠胸主動(dòng)脈,提取RNA,通過(guò)Real-time PCR法檢測(cè)主動(dòng)脈組織中炎癥因子Egr-1、TNF-α、 IFN-γ、IL-6、IL-10、MCP-1的表達(dá)水平。2.FXa通過(guò)激活PAR-2發(fā)揮生物學(xué)特性：Western Blot法檢測(cè)巨噬細(xì)胞中PAR-1和PAR-2的表達(dá)情況,分別用thrombin(PAR-1特異性激動(dòng)劑)、trypsin(PAR-2特異性激動(dòng)劑)刺激細(xì)胞30min,收集細(xì)胞,Western Blot法檢測(cè)ERK1/2磷酸化水平(PAR-1和PAR-2激活的指標(biāo)),評(píng)估巨噬細(xì)胞中PAR-1和PAR-2的功能活性。分別用不同濃度的FXa預(yù)處理細(xì)胞30min,收集細(xì)胞,Western Blot法檢測(cè)ERK1/2磷酸化水平,評(píng)估FXa的最佳刺激濃度。分別用PBS、 hirudin(凝血酶抑制劑)、TAP(FXa抑制劑)預(yù)處理細(xì)胞30min,然后用FXa刺激細(xì)胞30min,收集細(xì)胞,Western Blot法檢測(cè)ERK1/2磷酸化水平,評(píng)估FXa誘導(dǎo)細(xì)胞內(nèi)信號(hào)傳導(dǎo)的特異性。分別用PBS、ATAP2(PAR-1封閉抗體)孵化細(xì)胞30min,然后分別用thrombin、FXa刺激細(xì)胞30min,收集細(xì)胞,Western Blot法檢測(cè)ERK1/2磷酸化水平,證實(shí)FXa誘導(dǎo)細(xì)胞內(nèi)信號(hào)傳導(dǎo)不需要依賴(lài)PAR-1的激活。分別用thrombin和trypsin對(duì)細(xì)胞進(jìn)行脫敏處理,然后用FXa對(duì)脫敏細(xì)胞進(jìn)行刺激,收集細(xì)胞,Western Blot法檢測(cè)ERK1/2磷酸化水平,證實(shí)FXa通過(guò)激活PAR-2誘導(dǎo)細(xì)胞內(nèi)信號(hào)傳導(dǎo)。分別用PBS、 TAP、SAM 11(PAR-2封閉抗體)、U0126(ERK1/2抑制劑)預(yù)處理細(xì)胞,然后用FXa刺激細(xì)胞,收集細(xì)胞,提取RNA,通過(guò)Real-time PCR法檢測(cè)細(xì)胞中炎癥因子TNF-α、IFN-γ、IL-6、MCP-1的表達(dá)情況,評(píng)估FXa對(duì)巨噬細(xì)胞致炎水平的影響。結(jié)果：1. Fondaparinux對(duì)小鼠的體重、血脂水平?jīng)]有影響,與對(duì)照組相比,Fondaparinux不影響斑塊的面積、厚度及管腔狹窄程度,但是顯著增加斑塊的纖維帽厚度(56.78±6.18μm vs 42.35±2.62μm, P=0.049)和膠原含量(30.79±0.84%vs 23.52±1.04%,P= 0.006),降低斑塊的壞死核比例(34.8±0.91%vs 41.32±1.44%,P=0.001)。2. Fondaparinux不影響斑塊中Factor X的表達(dá),顯著降低斑塊中α-SMA (5.67±0.54 vs 8.10±0.52, P=0.002)、Mac-2(3.89±0.41 vs 5.24±0.35, P=0.017)、 VCAM-1 (6.22±0.60 vs 8.67±0.93, P=0.041)、CD-31 (4.33±0.53 vs 6.33±0.60,P=0.024)、MMP-9 (4.22±0.33 vs 7.45±0.44, P=0.000)、PAR-1(5.19±0.43 vs 7.78±0.56,P=0.001)和PAR-2 (3.13±0.30 vs 4.77±0.40, P=0.003)的表達(dá),Fondaparinux降低斑塊中MMP-9的酶活性(0.74±0.04 vs 1.0±0.07,P=0.032),并減弱斑塊中PAR-1和PAR-2的信號(hào)傳導(dǎo)。3. Fondaparinux降低小鼠體內(nèi)炎癥因子Egr-1、TNF-α、IFN-γ、IL-6、IL-10和MCP-1的表達(dá)水平(P0.05)。4.PAR-1和PAR-2在巨噬細(xì)胞中有所表達(dá),thrombin和trypsin能分別特異性激活巨噬細(xì)胞中的PAR-1和PAR-2。5.FXa能誘導(dǎo)巨噬細(xì)胞的信號(hào)傳導(dǎo),0.75U/ml時(shí)效應(yīng)最強(qiáng),TAP能阻斷這種信號(hào)傳導(dǎo),而hirudin對(duì)FXa誘發(fā)的信號(hào)傳導(dǎo)則沒(méi)有影響。6. ATAP2能阻斷thrombin誘導(dǎo)的信號(hào)傳導(dǎo),但是對(duì)FXa誘導(dǎo)的信號(hào)傳導(dǎo)沒(méi)有影響。與對(duì)照組相比,thrombin脫敏后的細(xì)胞用FXa刺激仍能誘發(fā)細(xì)胞的信號(hào)傳導(dǎo),而trypsin脫敏后的細(xì)胞用FXa刺激則不能誘發(fā)細(xì)胞的信號(hào)傳導(dǎo)。7.FXa增加巨噬細(xì)胞中炎癥因子TNF-α、IFN-γ、IL-6和MCP-1的表達(dá)(P0.05),而TAP、SAM11和U0126則能阻斷FXa的這種效應(yīng)。結(jié)論：1-抑制FXa不影響小鼠的體重、脂質(zhì)水平和動(dòng)脈粥樣硬化斑塊的病理進(jìn)展,但是可以降低斑塊的炎癥水平、血管新生以及MMP-9的表達(dá)量和酶活性,增加斑塊的纖維帽厚度和膠原含量,降低斑塊的壞死核比例,從而增加斑塊的穩(wěn)定性,FXa對(duì)斑塊穩(wěn)定性的影響與PARs信號(hào)傳導(dǎo)關(guān)系密切。2.FXa通過(guò)激活巨噬細(xì)胞中的PAR-2引發(fā)特異性的信號(hào)傳導(dǎo),從而增加巨噬細(xì)胞的促炎活性,FXa的這種效應(yīng)與ERK1/2信號(hào)通路關(guān)系密切。
[Abstract]:Atherosclerosis is an inflammatory disease progression, unstable atherosclerotic plaque rupture and can cause acute life-threatening cardiovascular events.FXa not only in the blood coagulation cascade plays an important role in secondary thrombosis, a key role in the formation of thrombin, and more and more evidence that FXa plays an important role in inflammatory reaction therefore, this paper intends to explore the effects of FXa on the stability of atherosclerotic plaque and its related mechanism. Objective: 1. to investigate whether FXa inhibition effect of atherosclerotic plaque in mice with pathological progress and stability; 2. to explore the possible mechanism of FXa on atherosclerotic plaque stability; 3. to explore the effects of FXa on the biological characteristics of macrophages and its possible mechanism. Methods: the effects of 1.FXa the progress of atherosclerotic plaque inhibitor on ApoE-/- pathology: The size of the 6 week old male ApoE-/- mice were used as experimental mice, the whole high fat feeding, the establishment of atherosclerotic plaque model, for a period of 4 weeks of Fondaparinux at 20 weeks of age (FXa inhibitor) intraperitoneal injection, 5mg/kg, 1 times / day of.24 week old anesthesia weighing, heart blood, serum total cholesterol, plasma of mice triglyceride, low density lipoprotein cholesterol and high density lipoprotein cholesterol levels in mice. Anonyma taken under a microscope, the lesion area, HE staining method to measure carotid atherosclerotic plaque thickness, luminal stenosis, fibrous cap thickness and necrotic core ratio, collagen content detection plaque Masson staining, Perls staining to detect plaque internal bleeding, Van Kossa staining method was used to detect the degree of plaque calcification, immunohistochemical method to detect plaque Factor X, alpha -SMA, Mac-2, VCAM-1, CD-31, MMP-9, PAR-1, PAR-2 expression, zymography The detection of enzyme activity in plaque MMP-9, PAR-1 PAR-2 and Western Bolt method in signal detection of plaque under the microscope. The mice thoracic aorta, RNA extraction, detection by inflammatory factor Real-time in aortic tissue by PCR Egr-1, TNF- alpha, IFN- gamma, IL-6, IL-10, play the biological characteristics of MCP-1.2.FXa expression by activating PAR-2 expression PAR-1 and PAR-2 Western Blot method in the detection of macrophages, respectively thrombin (PAR-1 specific agonist), trypsin (PAR-2 specific agonist) stimulation of 30min cells, cells were collected to detect the phosphorylation level of ERK1/2 Western Blot (PAR-1 and PAR-2 activation index), functional assessment of PAR-1 and PAR-2 in macrophages then using 30min cells, different concentrations of FXa pretreated cells were collected to detect the phosphorylation level of ERK1/2 Western Blot, the best concentration of assessment of FXa. Respectively PBS, hirudin (thrombin inhibition Preparation), TAP (FXa inhibitor) pretreatment of 30min cells, then the cells were stimulated with FXa 30min, a collection of cells, detecting the phosphorylation level of ERK1/2 Western Blot method, evaluation of FXa to induce specific intracellular signal transduction. Respectively PBS, ATAP2 (PAR-1 blocking antibody) incubation cell 30min, then use thrombin, FXa stimulation 30min cells, cells were collected to detect the phosphorylation level of ERK1/2 Western Blot, confirmed that FXa induced signal transduction in the cell does not require the activation of PAR-1 dependent respectively. The cells were treated with thrombin and trypsin desensitization, and then use FXa to desensitized cells was stimulated, cells were collected to detect the levels of ERK1/2 phosphorylation of Western induced by FXa Blot method. Intracellular signal transduction through the activation of PAR-2. Respectively PBS, TAP, SAM 11, U0126 (PAR-2 blocking antibody) (ERK1/2 inhibitor) pretreatment of cells, then FXa stimulated cells, cells were collected and RNA was extracted by Real- Inflammatory factor TNF- alpha, time PCR cell detection in IFN- gamma, IL-6, MCP-1 expression, impact assessment of FXa on macrophage inflammatory level. Results: 1. Fondaparinux had no effect on the weight of mice, blood lipid level, compared with the control group, Fondaparinux does not affect the plaque area, thickness and degree of stenosis, but significant increase in the thickness of fibrous cap plaques (56.78 + 6.18 m 42.35 + vs 2.62 m, P=0.049) and collagen content (30.79 + 23.52 + 1.04% 0.84%vs, 0.006 P=), reduced plaque necrosis nuclear ratio (34.8 + 0.91%vs 41.32 + 1.44% P, =0.001).2. Fondaparinux did not affect the expression of Factor in X plaques a significant reduction in plaque, alpha -SMA (5.67 + 0.54 vs 8.10 + 0.52, P=0.002), Mac-2 (3.89 + 0.41 vs 5.24 + 0.35, P=0.017), VCAM-1 (6.22 + 0.60 vs 8.67 + 0.93, P=0.041), CD-31 (4.33 + 0.53 vs 6.33 + 0.60, P=0.024), MMP-9 (4.22 + 0.33 vs 7.45 + 0.44, P=0.000), PAR-1 (5.19 + 0.43 vs 7.78 + 0.56, P=0.001) and PAR-2 (3.13 + 0.30 vs 4.77 + 0.40, P=0.003) the expression of Fondaparinux decreased enzyme activity in MMP-9 plaque (0.74 + 0.04 vs 1 + 0.07, P=0.032, PAR-1 and PAR-2) and attenuated plaque in signal.3. Fondaparinux conduction reduce inflammatory factors in mice Egr-1, TNF- alpha, IFN- gamma, IL-6, expression of IL-10 and MCP-1 (P0.05).4.PAR-1 and PAR-2 expression in macrophages, thrombin signaling and trypsin can respectively specific activation of macrophages in the PAR-1 and PAR-2.5.FXa can induce macrophages, 0.75U/ml had the strongest effect, TAP blocking this signaling, and hirudin on FXa induced signal transduction had no effect on.6. ATAP2 can block the signal transduction induced by thrombin, but had no effect on the signal transduction induced by FXa. Compared with the control group, thrombin cells after desensitization Can cell signal transduction induced by FXa stimulation, and trypsin desensitization cells after stimulation with FXa, signal transduction of.7.FXa cells induced by TNF- increased not inflammatory cytokines in macrophages IFN- alpha, gamma, expression of IL-6 and MCP-1 (P0.05), TAP, SAM11 and U0126 can block the effect of FXa. Conclusion: 1- inhibited FXa does not affect the weight of mice, the pathological progress of lipid level and atherosclerosis, but can reduce the level of plaque inflammation, angiogenesis and the expression of MMP-9 and enzyme activity, increased plaque fibrous cap thickness and collagen content, reduced plaque necrosis nuclear ratio, thereby increasing the effect of FXa on the stability of plaque the stability of plaque and PARs signaling is closely related to.2.FXa signal transduction by macrophage activation in PAR-2 induced specific, thereby increasing the proinflammatory activity of macrophages, the effect of FXa and ERK1 The /2 signal pathway is closely related.

【學(xué)位授予單位】：東南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R543.5

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