本文選題：Aliskiren + 動脈粥樣硬化��； 參考：《青島大學》2016年博士論文

【摘要】：研究背景動脈粥樣硬化(AS)是缺血性心腦血管疾病主要的病理生理基礎。臨床研究發(fā)現(xiàn)不穩(wěn)定性AS斑塊內(nèi)出血或斑塊發(fā)生破裂是引起缺血性血管事件急性發(fā)作的主要原因。AS斑塊內(nèi)有病理性新生血管,這些血管的滲透性高、脆性大,易發(fā)生破裂,從而導致斑塊內(nèi)出血,引起急性心腦血管事件。研究證實炎癥與斑塊內(nèi)新生血管的形成關系密切,炎癥因子刺激斑塊內(nèi)部的血管新生。TLR4/NF-κB信號傳導通路可通過刺激炎癥細胞分泌炎癥因子來介導炎癥反應。有研究顯示TLR4/NF-κB途徑可能會誘導MMPs表達,從而參與AS的病理過程。研究表明AS內(nèi)的多種MMPs含量升高。AS的動脈壁中MMP-2、MMP-9水平明顯增加,且增加程度與病變嚴重程度正相關。Aliskiren是新發(fā)現(xiàn)的腎素抑制劑,可明顯減少AS病變。研究證實RAAS激活后可以通過TLRs依賴的信號途徑誘導Apo E-/-小鼠AS斑塊的血管炎癥,但其作用機制與途徑尚不明確。目的:觀察Aliskiren對動脈粥樣硬化斑塊內(nèi)新生血管形成和斑塊穩(wěn)定性的影響,探討Aliskiren是否通過抑制TLR4/NF-κB信號通路介導的炎癥反應及MMP-2、MMP-9的生成來影響新生血管的形成,增強斑塊的穩(wěn)定性。方法:取30只健康雄性、8周齡的載脂蛋白E基因敲除(Apo E-/-)小鼠,將其隨機分為3組:模型組10只、Aliskiren低劑量組10只、Aliskiren高劑量組10只,另取C57BL/6小鼠10只作為對照組。Aliskiren低劑量組、高劑量組分別給予Aliskiren25mg/kg/d,50mg/kg/d灌胃;C57BL/6小鼠及模型組給予生理鹽水灌胃1m L/100g/d,灌胃時間為12周。制作主動脈根部切片,免疫組化染色觀察分析斑塊內(nèi)新生血管的密度;HE染色分析斑塊形態(tài),計算血管斑塊面積、管腔面積;油紅O染色觀察斑內(nèi)脂質成分含量;Masson染色觀察斑塊中膠原含量;ELISA檢測血清中炎癥因子TNF-α、IL-6、IL-1β、MCP-1水平;RT-PCR法檢測主動脈組織中MMP-2、MMP-9、TLR4 m RNA水平變化;Western Blot法檢測主動脈組織中MMP-2、MMP-9、TLR4及NF-κB蛋白水平變化。結果:1.血脂測定:模型組小鼠血清TC、TG、LDL-C濃度較對照組明顯升高(P0.05);Aliskiren低劑量組和高劑量組小鼠血清TC、TG、LDL-C濃度與模型組無明顯變化(P0.05)。2.HE染色:模型組小鼠主動脈AS斑塊形成顯著,管腔狹窄較重,動脈內(nèi)膜不連續(xù)、不光滑,內(nèi)皮細胞部分缺失。與模型組相比,Aliskiren低劑量組和高劑量組斑塊面積、斑塊面積/管腔面積比值明顯降低(P0.05);與Aliskiren低劑量組相比,Aliskiren高劑量組降低更顯著(P0.05)。3.油紅O染色:模型組小鼠主動脈AS斑塊內(nèi)紅染的沉積的脂質量大。Aliskiren低劑量組和高劑量組斑塊內(nèi)脂質含量較模型組明顯減少(P0.05);與Aliskiren低劑量組相比,Aliskiren高劑量組降低更顯著(P0.05)。4.Masson染色:模型組小鼠主動脈斑塊內(nèi)藍染膠原量少。與模型組相比,Aliskiren低劑量組和高劑量組斑塊內(nèi)膠原含量增加明顯(P0.05);與Aliskiren低劑量組相比,Aliskiren高劑量組增加更顯著(P0.05)。5.斑塊內(nèi)新生血管的密度:模型組可見大量的棕褐色的顆粒,CD34陽性表達多。Aliskiren低劑量組和高劑量組新生血管較模型組顯著減少(P0.05);與Aliskiren低劑量組相比,Aliskiren高劑量組新生血管減少更顯著(P0.05)。6.TNF-α、IL-6、IL-1β、MCP-1水平:與對照組相比,模型組TNF-α、IL-6、IL-1β、MCP-1水平顯著升高(P0.05);與模型組相比,Aliskiren低劑量組和高劑量組模型組TNF-α、IL-6、IL-1β、MCP-1水平明顯降低(P0.05),Aliskiren高劑量組降低更明顯(P0.05)。7.主動脈組織中MMP-2、MMP-9、TLR4 m RNA表達水平:與對照組相比,模型組MMP-2、MMP-9、TLR4 m RNA表達明顯升高(P0.05);與模型組相比,Aliskiren低劑量組和高劑量組模型組MMP-2、MMP-9、TLR4 m RNA表達明顯降低(P0.05),Aliskiren高劑量組降低更明顯(P0.05)。8.主動脈組織中MMP-2、MMP-9、TLR4及NF-κB蛋白表達水平:與對照組相比,模型組MMP-2、MMP-9、TLR4及NF-κB蛋白表達顯著升高(P0.05);與模型組相比,Aliskiren低劑量組和高劑量組MMP-2、MMP-9、TLR4及NF-κB蛋白表表達明顯降低(P0.05),Aliskiren高劑量組降低更明顯(P0.05)。結論:1.Aliskiren能夠減少AS斑塊內(nèi)新生血管的形成,增強斑塊的穩(wěn)定性。2.Aliskiren可能是通過抑制TLR4/NF-κB信號通路介導的炎癥反應及MMP-2、MMP-9的生成來影響新生血管的形成,增強斑塊穩(wěn)定性。
[Abstract]:Background atherosclerosis (AS) is the main pathophysiological basis of ischemic cardio cerebrovascular disease. Clinical studies have found that bleeding or plaque rupture in unstable AS plaques is the main cause of acute ischemic vascular events in.AS, and there are pathological new blood tubes in plaque. These vessels have high permeability, large brittleness and easy onset. The rupture, which leads to hemorrhage in plaque, causes acute cardiovascular and cerebrovascular events. Studies have confirmed that inflammation is closely related to the formation of neovascularization in the plaque. Inflammatory factors stimulate the angiogenesis.TLR4/NF- kappa B signal transduction pathway within the plaque within the plaque to mediate inflammatory reactions by stimulating inflammatory cells to secrete inflammatory factors. Research shows that TLR4/NF- The kappa B pathway may induce the expression of MMPs and thus participate in the pathological process of AS. The study shows that a variety of MMPs levels in the AS increase the MMP-2, MMP-9 level in the arterial wall of.AS, and the degree of increase is positively related to the severity of the lesion, which is a newly discovered renin inhibitor, which can significantly reduce the AS lesion. TLRs dependent signaling pathway induces vascular inflammation in the Apo E-/- mouse AS plaque, but its mechanisms and pathways are not yet clear. Objective: To observe the effect of Aliskiren on the formation of neovascularization and plaque stability in atherosclerotic plaques and to explore whether Aliskiren is mediated by the TLR4/ NF- kappa B signaling pathway and MMP-2, MMP -9 formation to influence the formation of new blood vessels and enhance plaque stability. Methods: 30 healthy males and 8 weeks old apolipoprotein E gene knockout (Apo E-/-) mice were randomly divided into 3 groups, 10 in the model group, 10 in the Aliskiren low dose group and 10 in the Aliskiren high dose group, and 10 in the C57BL/6 mice were used as the low dose of the control group as the low dose of the control group.Aliskiren. Group, high dose group were given Aliskiren25mg/kg/d, 50mg/kg/d gavage respectively; C57BL/6 mice and model group were given 1m L/100g/d with saline for 12 weeks. The density of the neovascularization in the aortic root was made by immunohistochemistry. The plaque morphology was analyzed by HE staining, and the area of plaque and the area of the lumen were calculated. The content of lipid components in the plaque was observed by oil red O staining, the content of collagen in the plaque was observed by Masson staining, and the levels of inflammatory factors TNF- a, IL-6, IL-1 beta and MCP-1 in the serum were detected by ELISA, and the levels of MMP-2, MMP-9, TLR4 m in aorta were detected by RT-PCR. Results: 1. blood lipid measurement: the serum levels of TC, TG and LDL-C in the model group were significantly higher than those in the control group (P0.05). The serum TC, TG, LDL-C concentration in the low dose group and the high dose group of the Aliskiren group had no significant changes (P0.05).2.HE staining (P0.05).2.HE staining: the aortic AS plaque in the model group was significant, the stenosis of the lumen was heavier, the intima of the artery was not continuous and unsmooth. Compared with the model group, the patch area of the Aliskiren low dose group and the high dose group decreased significantly (P0.05). Compared with the low dose group of Aliskiren, the Aliskiren high dose group decreased more significantly (P0.05).3. oil red O staining: the lipid quality of the red dye in the aorta AS plaque of the model mice. The lipid content in the plaque in the large.Aliskiren low dose group and the high dose group was significantly lower than that in the model group (P0.05). Compared with the low dose group of Aliskiren, the Aliskiren high dose group decreased more significantly (P0.05).4.Masson staining: the amount of blue stained collagen in the aortic plaque in the model group was less. Compared with the model group, the low dose group and the high dose group were within the plaque group. The increase of collagen content was significant (P0.05); compared with the low dose group of Aliskiren, the density of the neovascularization in the Aliskiren high dose group increased significantly (P0.05) in the.5. plaque: the model group showed a large number of brown brown particles, the CD34 positive expression of the multi.Aliskiren low dose group and the high dose group decreased significantly compared with the model group (P0.05), and the low level of Aliskiren (P0.05). Compared with the control group, the level of.6.TNF- alpha, IL-6, IL-1 beta, MCP-1 was significantly higher in the Aliskiren high dose group than in the control group. Compared with the control group, the level of TNF- alpha, IL-6, IL-1 beta and MCP-1 increased significantly (P0.05). Compared with the model group, the low dose group and the high dose group were significantly lower than the model group. Iskiren high dose group decreased the expression level of MMP-2, MMP-9, TLR4 m RNA in the aorta of (P0.05).7.: compared with the control group, the expression of MMP-2, MMP-9, TLR4 m was significantly higher than that in the control group. The expression level of MMP-2, MMP-9, TLR4 and NF- kappa B in the aorta of (P0.05).8. was significantly reduced in the dose group. Compared with the control group, the expression of MMP-2, MMP-9, TLR4 and NF- kappa B protein in the model group was significantly higher than that in the model group. The decrease of skiren in high dose group is more obvious (P0.05). Conclusion: 1.Aliskiren can reduce the formation of neovascularization in AS plaque and enhance the stability of plaque by inhibiting the inflammatory response mediated by TLR4/NF- kappa B signaling pathway and MMP-2, MMP-9 formation to influence the formation of neovascularization and enhance plaque stability.
【學位授予單位】：青島大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R543.5

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