FXa对动脉粥样硬化斑块稳定性的影响及其机制的研究

发布时间：2018-01-19 22:43

本文关键词： 动脉粥样硬化斑块稳定性炎症活化的第十凝血因子蛋白酶激活受体-2 ERK1/2信号通路　出处：《东南大学》2016年博士论文　论文类型：学位论文

【摘要】：动脉粥样硬化是一种进展性的炎症性疾病,不稳定动脉粥样硬化斑块破裂并继发血栓形成可导致危及生命的急性心血管事件。FXa不仅在凝血级联中占据着重要位置,对凝血酶的形成起到关键作用,而且越来越多的证据显示FXa在炎症反应中也扮演着不可或缺的角色,因此本课题拟探讨FXa对动脉粥样硬化斑块稳定性的影响及相关机制。目的：1.探讨抑制FXa是否影响小鼠动脉粥样硬化斑块的病理进展和稳定性；2.探讨FXa影响动脉粥样硬化斑块稳定性的可能机制；3.探讨FXa对巨噬细胞生物学特性的影响及可能机制。方法：1.FXa抑制剂对ApoE-/-小鼠动脉粥样硬化斑块病理进展的影响：选取6周龄大小的雄性ApoE-/-小鼠作为实验小鼠,全程进行高脂饲养,建立动脉粥样硬化斑块模型,20周龄时进行为期4周的Fondaparinux (FXa抑制剂)腹腔注射,5mg/kg,1次／天。24周龄时麻醉称重,心脏取血,检测小鼠血浆中总胆固醇、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇的水平。显微镜下取小鼠头臂干,HE染色法测量动脉粥样硬化斑块的病变面积、病变厚度、管腔狭窄程度、纤维帽厚度和坏死核比例,Masson染色法检测斑块中胶原含量,Perls染色法检测斑块内出血情况,Van Kossa染色法检测斑块钙化程度,免疫组化法检测斑块中Factor X、α-SMA、Mac-2、VCAM-1、CD-31、 MMP-9、PAR-1、PAR-2的表达,明胶酶谱法检测斑块中MMP-9的酶活性,Western Bolt法检测斑块中PAR-1和PAR-2信号。显微镜下取小鼠胸主动脉,提取RNA,通过Real-time PCR法检测主动脉组织中炎症因子Egr-1、TNF-α、 IFN-γ、IL-6、IL-10、MCP-1的表达水平。2.FXa通过激活PAR-2发挥生物学特性：Western Blot法检测巨噬细胞中PAR-1和PAR-2的表达情况,分别用thrombin(PAR-1特异性激动剂)、trypsin(PAR-2特异性激动剂)刺激细胞30min,收集细胞,Western Blot法检测ERK1/2磷酸化水平(PAR-1和PAR-2激活的指标),评估巨噬细胞中PAR-1和PAR-2的功能活性。分别用不同浓度的FXa预处理细胞30min,收集细胞,Western Blot法检测ERK1/2磷酸化水平,评估FXa的最佳刺激浓度。分别用PBS、 hirudin(凝血酶抑制剂)、TAP(FXa抑制剂)预处理细胞30min,然后用FXa刺激细胞30min,收集细胞,Western Blot法检测ERK1/2磷酸化水平,评估FXa诱导细胞内信号传导的特异性。分别用PBS、ATAP2(PAR-1封闭抗体)孵化细胞30min,然后分别用thrombin、FXa刺激细胞30min,收集细胞,Western Blot法检测ERK1/2磷酸化水平,证实FXa诱导细胞内信号传导不需要依赖PAR-1的激活。分别用thrombin和trypsin对细胞进行脱敏处理,然后用FXa对脱敏细胞进行刺激,收集细胞,Western Blot法检测ERK1/2磷酸化水平,证实FXa通过激活PAR-2诱导细胞内信号传导。分别用PBS、 TAP、SAM 11(PAR-2封闭抗体)、U0126(ERK1/2抑制剂)预处理细胞,然后用FXa刺激细胞,收集细胞,提取RNA,通过Real-time PCR法检测细胞中炎症因子TNF-α、IFN-γ、IL-6、MCP-1的表达情况,评估FXa对巨噬细胞致炎水平的影响。结果：1. Fondaparinux对小鼠的体重、血脂水平没有影响,与对照组相比,Fondaparinux不影响斑块的面积、厚度及管腔狭窄程度,但是显著增加斑块的纤维帽厚度(56.78±6.18μm vs 42.35±2.62μm, P=0.049)和胶原含量(30.79±0.84%vs 23.52±1.04%,P= 0.006),降低斑块的坏死核比例(34.8±0.91%vs 41.32±1.44%,P=0.001)。2. Fondaparinux不影响斑块中Factor X的表达,显著降低斑块中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)的表达,Fondaparinux降低斑块中MMP-9的酶活性(0.74±0.04 vs 1.0±0.07,P=0.032),并减弱斑块中PAR-1和PAR-2的信号传导。3. Fondaparinux降低小鼠体内炎症因子Egr-1、TNF-α、IFN-γ、IL-6、IL-10和MCP-1的表达水平(P0.05)。4.PAR-1和PAR-2在巨噬细胞中有所表达,thrombin和trypsin能分别特异性激活巨噬细胞中的PAR-1和PAR-2。5.FXa能诱导巨噬细胞的信号传导,0.75U/ml时效应最强,TAP能阻断这种信号传导,而hirudin对FXa诱发的信号传导则没有影响。6. ATAP2能阻断thrombin诱导的信号传导,但是对FXa诱导的信号传导没有影响。与对照组相比,thrombin脱敏后的细胞用FXa刺激仍能诱发细胞的信号传导,而trypsin脱敏后的细胞用FXa刺激则不能诱发细胞的信号传导。7.FXa增加巨噬细胞中炎症因子TNF-α、IFN-γ、IL-6和MCP-1的表达(P0.05),而TAP、SAM11和U0126则能阻断FXa的这种效应。结论：1-抑制FXa不影响小鼠的体重、脂质水平和动脉粥样硬化斑块的病理进展,但是可以降低斑块的炎症水平、血管新生以及MMP-9的表达量和酶活性,增加斑块的纤维帽厚度和胶原含量,降低斑块的坏死核比例,从而增加斑块的稳定性,FXa对斑块稳定性的影响与PARs信号传导关系密切。2.FXa通过激活巨噬细胞中的PAR-2引发特异性的信号传导,从而增加巨噬细胞的促炎活性,FXa的这种效应与ERK1/2信号通路关系密切。
[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.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.5

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