發(fā)布時間：2018-06-06 23:05

  本文選題：動脈粥樣硬化易損斑塊 + 血管新生　； 參考：《重慶大學》2015年博士論文

【摘要】：目前的研究和臨床表明,位列全球死亡人數(shù)的前兩位的疾病分別為缺血性心臟病和腦血管疾病,高達死亡人數(shù)的21.9%。伴隨全國人民生活水平的提高,心腦血管疾病已經(jīng)躍居為我國的第一位殺手。冠狀動脈粥樣硬化發(fā)病最顯著的特征是冠狀動脈的不穩(wěn)定斑塊破裂,形成血栓堵塞冠狀動脈。因此,對預防冠狀動脈粥樣硬化最重要的手段是早期預判和治療不穩(wěn)定性易損斑塊。冠狀動脈粥樣硬化病理學研究表明,易損斑塊具有以下組織病理學特征:在血管狹窄處容易形成較大的斑塊;斑塊中有薄的纖維帽;炎癥細胞和大的脂核浸潤;斑塊內(nèi)有較多的新生血管。動脈粥樣硬化易損斑塊內(nèi)出現(xiàn)的病理性新生血管,能加速粥樣硬化病變的發(fā)展,從而誘發(fā)斑塊破裂脫落,形成血栓堵塞血管,促進其急性心血管事件的發(fā)生。研究報道指出,AS(Atherosclerosis)易損斑塊的形成和破裂的病理原因,一直沒能完全闡述明白,尤其是在高脂和變化的應力場環(huán)境下的趨勢更是無法解釋清楚。而有觀點指出,滋養(yǎng)血管增生以及在斑塊內(nèi)高密度富集,是其核心關(guān)鍵點。此時出現(xiàn)的滋養(yǎng)血管,其功能和結(jié)構(gòu)不完整,尤其外膜的不穩(wěn)定,極易發(fā)生側(cè)漏,從而誘發(fā)斑塊內(nèi)出血和破損出現(xiàn)。因而本文從斑塊內(nèi)血管新生入手,探索AS易損斑塊形成、發(fā)展的病因;引入高脂環(huán)境和高切應力因素,研究損傷血管在復雜環(huán)境下,斑塊內(nèi)血管新生與其病變過程之間的關(guān)系。VEGF(Vascular Endothelial Growth Factor)是特異性和作用最強的血管新生因子,參與了多種體內(nèi)的病理性血管新生,在腫瘤和斑塊血管新生中起著關(guān)鍵調(diào)控蛋白的作用。在腫瘤血管新生的研究中,已經(jīng)系統(tǒng)的探索了VEGF及下游通路蛋白在腫瘤病變中的作用機理。然而在AS易損斑塊的研究中,只是證實了VEGF參與了斑塊的病變和血管新生過程,但是對于VEGF影響斑塊血管新生的機理、下游調(diào)控蛋白在AS斑塊中的作用、以及與AS易損斑塊的高危因素高脂高切應力之間的關(guān)系研究,還無準確的定論�；诖�,本文通過對血管新生經(jīng)典通路VEGF-VEGFR2-FAK(Focal adhesion kinase)/paxillin在oxLDL(Oxidized low density liporprotein)和高切應力介導的血管新生中的作用研究,明確了VEGF及下游通路蛋白在AS易損斑塊形成中的作用機制,為臨床治療AS貢獻了新的基因靶點。本論文中,我們使用的動物模型為小鼠左頸總動脈套環(huán)模型。在此基礎(chǔ)上給小鼠飼喂高脂飼料(1%膽固醇和5%豬油),進而研究斑塊的組成、血管新生數(shù)量。通過血管新生抑制劑(TNP-470)對模型組注射治療,研究治療組斑塊的形成和血管新生情況。從動物模型上,證明血管新生能促進動脈粥樣硬化易損斑塊的形成。在正常組和模型組血管中分別對血管新生相關(guān)基因vegf、cd31(plateletendothelialcelladhesionmolecule-1)進行免疫染色,發(fā)現(xiàn)模型組中vegf和cd31表達都集中在斑塊的肩部和纖維帽區(qū)域(較正常組)。并通過合成vegfsirna、vegfr1sirna、vegfr2sirna、vegf重組蛋白對高脂高切應力影響下的內(nèi)皮細胞刺激,觀察下游通路蛋白fak/paxillin的活化(磷酸化),以及對內(nèi)皮細胞遷移和管腔形成的影響,來探索高脂高切應力介導下的斑塊血管新生的信號傳導途徑。主要研究內(nèi)容和結(jié)果如下:①構(gòu)建小鼠左頸動脈套環(huán)模型,右側(cè)頸動脈進行假手術(shù)對照。單一套環(huán)模型只能刺激內(nèi)膜增生,不誘導易損斑塊的形成。其中增生的體積在高低切應力區(qū)域基本一致。加入高脂因素,左頸動脈高切應力區(qū)域(狹窄近心端)出現(xiàn)大量不穩(wěn)定斑塊,低切應力區(qū)域(遠心端)無斑塊形成,僅有一定內(nèi)膜增生。而正常組血管,在高脂環(huán)境下,其血管壁有少量脂肪顆粒沉積和內(nèi)膜增生。通過he染色,顯示高切應力區(qū)域易損斑塊內(nèi)的新生血管主要為外膜不完整、內(nèi)皮細胞有凋亡趨勢、管腔內(nèi)有大量淋巴細胞浸潤的易泄漏微血管。而tnp-470治療組,血管內(nèi)膜增生減弱,易損斑塊中的血管新生有所減弱,斑塊面積減少。②通過對小鼠頸動脈進行血管新生相關(guān)蛋白vegf、cd31免疫染色。發(fā)現(xiàn)在正常組中,血管無vegf、cd31的陽性表達。而模型組高切應力組中,vegf、cd31在斑塊的肩部和纖維帽區(qū)域都存在陽性高表達。研究顯示,vegf參與了斑塊血管新生的病理過程,從而為我們選擇vegf作為關(guān)于血管新生的研究靶點提供了依據(jù)。③通過用oxldl和切應力分別刺激huvecs(humanumbilicalvascularendothelialcell),研究兩者對huvecs遷移和管腔形成的影響。發(fā)現(xiàn)正常組內(nèi)皮細胞無遷移和管腔形成。而oxldl組內(nèi)皮細胞遷移和管腔形成明顯,其中最佳濃度為20μg/ml。高切應力組內(nèi)皮細胞遷移和管腔形成明顯,最佳壓力為25dyn/cm2。研究顯示,oxldl和高切應力能促進內(nèi)皮細胞功能主導的血管新生。④通過合成vegfsirna、vegf重組蛋白對高脂高切應力影響下的內(nèi)皮細胞刺激,研究vegf在oxldl和高切應力介導的血管新生中的作用。實驗結(jié)果表明,vegf的過表達,能促進huvecs遷移和管腔形成;同時高脂高切應力都能促進huvecs中vegf及下游受體(vegfr1、vegfr2)的過表達;vegfsirna組內(nèi)皮細胞用oxldl和高切應力刺激,內(nèi)皮細胞的遷移和管腔形成明顯減少(較oxldl和高切應力組)。研究顯示,vegf是oxldl和高切應力調(diào)控血管新生的關(guān)鍵蛋白。⑤通過合成vegfr1sirna、vegfr2sirna、vegf重組蛋白、vegfsirna對高脂高切應力影響下的內(nèi)皮細胞刺激,研究vegf-vegfr2-fak/paxillin信號通路在ox LDL和高切應力介導的血管新生中的作用。實驗結(jié)果說明,VEGF能調(diào)控VEGFR2下游蛋白FAK/paxillin的磷酸化;oxLDL和高切應力通過VEGF調(diào)控VEGFR2-FAK/paxillin下游信號通路的表達;同時VEGFR2能調(diào)控下游蛋白FAK/paxillin的磷酸化;VEGFR2/VEGFR1是VEGF調(diào)控血管新生的關(guān)鍵因子。因此,在高脂高切應力調(diào)控的血管新生中,VEGF-VEGFR2-FAK/paxillin信號通路起著關(guān)鍵調(diào)控通路作用。綜上所述,通過本研究工作,我們闡明了血管新生在動脈粥樣易損斑塊中的作用,同時驗證了VEGF-VEGFR2-FAK/paxillin信號通路在高脂高切應力所調(diào)控的動脈粥樣硬化斑塊內(nèi)血管新生中的作用。
[Abstract]:Current research and clinical studies show that the top two diseases in the world are ischemic heart disease and cerebrovascular disease, and the 21.9%. of the death toll is accompanied by the improvement of the National People's living standard. Cardiovascular and cerebrovascular diseases have leaped into the first killer in our country. The most significant characteristic of coronary atherosclerosis is the characteristic of coronary atherosclerosis. The most important means of preventing coronary atherosclerosis is early prediction and treatment of unstable vulnerable plaques. The pathological study of coronary atherosclerotic plaque shows that vulnerable plaques have the following histologic features: easy to form in vascular stenosis. Large plaques; thin fibrous caps in plaques; inflammatory cells and large fat nuclei; there are more neovascularization in the plaque. Atherosclerotic Vulnerable Atherosclerotic neovascularization can accelerate the development of atherosclerotic lesions, resulting in plaque rupture and abscission, thrombosis and blockage of blood vessels, and the promotion of acute cardiovascular events. The report points out that the pathological causes of the formation and rupture of vulnerable plaques of AS (Atherosclerosis) have not been fully explained, especially in the high fat and changing stress field environment, and it is pointed out that the key point is to increase the growth of the nourishing blood vessels and to enrich the density in the plaque. In this case, the function and structure of the nourishing blood vessels are incomplete, especially the instability of the outer membrane and the occurrence of hemorrhage and breakage in the plaque. Therefore, this paper, starting with the angiogenesis in the plaque, explores the formation of AS vulnerable plaques, the cause of development, the introduction of high fat environment and high shear stress factors, and the complexity of the damage vessels in complex research. .VEGF (Vascular Endothelial Growth Factor) is a specific and strongest angiogenic factor in the environment, which is a specific and most powerful angiogenic factor. It participates in a variety of pathological angiogenesis in the body and plays the role of key regulatory proteins in tumor and plaque angiogenesis. The mechanism of VEGF and downstream pathway proteins in tumor lesions has been systematically explored. However, in the study of AS vulnerable plaques, it is only confirmed that VEGF is involved in plaque lesion and angiogenesis, but the mechanism that VEGF affects plaque angiogenesis, the role of the downstream modulation protein in the AS plaque, and the vulnerability to AS There is no accurate conclusion of the relationship between high risk of plaque and high fat and high shear stress. Based on this, this paper studies the role of VEGF-VEGFR2-FAK (Focal adhesion kinase) /paxillin in the angiogenesis in oxLDL (Oxidized low density liporprotein) and high shear stress, and makes clear the VEGF and downstream. The mechanism of pathway protein in the formation of AS vulnerable plaque contributes to the new gene target for clinical treatment of AS. In this paper, we use the animal model of left neck total artery ring model in mice. On this basis, we feed mice with high fat diet (1% cholesterol and 5% lard), and then study the composition of the plaque, the number of angiogenesis, and the blood flow through blood. The formation of plaque and angiogenesis in the treatment group were studied by injection of neonatal inhibitor (TNP-470) to the model group. From the animal model, it was proved that angiogenesis could promote the formation of vulnerable plaque in atherosclerosis. In the normal group and the model group, the neovascularization gene VEGF, CD31 (plateletendothelialcelladhesionmo), respectively, was used in the normal and model group. Lecule-1) immunohistochemical staining showed that the expression of VEGF and CD31 in the model group was concentrated in the shoulder and the fiber cap region of the plaque (the normal group). The activation of the downstream pathway protein fak/paxillin (phosphorylation) was observed through the synthesis of vegfsirna, vegfr1sirna, vegfr2sirna, and VEGF recombinant protein on the endothelial cells under the influence of high lipid high shear stress. The main research contents and results are as follows: (1) the model of the left carotid artery in mice is constructed and the right carotid artery is controlled by the false operation. The single ring model can only stimulate the hyperplasia of the intima, and it does not induce easily. The volume of the lesion was basically the same in the area of high and low shear stress. A large number of unstable plaques appeared in the high shear stress region (narrow near the heart end) in the left carotid artery, and the low shear stress region (Yuan Xinduan) had no plaque formation and only a certain intimal hyperplasia. He staining showed that the neovascularization in the vulnerable plaque of the high shear stress area was mainly the incomplete outer membrane, the endothelial cells had the tendency of apoptosis, and the intravascular infiltration of lymphocytes were easy to leak microvessels in the lumen. In the TNP-470 treatment group, the intravascular membrane hyperplasia was weakened, and the angiogenesis in vulnerable plaque was found. The plaque area decreased. (2) the vascular neovascularization related protein VEGF, CD31 immunostaining was performed on the carotid artery in mice. It was found that in the normal group, the blood vessels were not VEGF and CD31 positive. While in the high shear stress group, VEGF and CD31 were positive and high in the shoulder and fiber cap regions of the plaque. The study showed that VEGF was involved in the plaque blood. The pathological process of neovascularization provides a basis for our selection of VEGF as a target for angiogenesis. (3) the effect of both oxLDL and shear stress on HUVECs (humanumbilicalvascularendothelialcell), respectively, on the migration of HUVECs and the formation of the lumen in the lumen. The migration of endothelial cells and the formation of the lumen in the oxLDL group were obvious. The best concentration was the migration of endothelial cells and the formation of the lumen in the high shear stress group of 20 mu g/ml.. The best pressure was the 25dyn/cm2. study. The oxLDL and high shear stress could promote the angiogenesis of endothelial cells. (4) through the synthesis of vegfsirna, the recombinant protein of VEGF should be highly cut to high fat. The effect of VEGF on the angiogenesis in oxLDL and high shear stress was studied under the influence of force. The results showed that the overexpression of VEGF could promote the migration of HUVECs and the formation of the lumen, and the high fat and high shear stress could promote the overexpression of VEGF and the downstream receptor (VEGFR1, VEGFR2) in HUVECs; the vegfsirna group of endothelial cells used oxLDL. The migration of endothelial cells and the formation of endothelium were significantly reduced (compared with oxLDL and high shear stress groups). The study showed that VEGF was a key protein in the regulation of angiogenesis by oxLDL and high shear stress. 5. Through the synthesis of vegfr1sirna, vegfr2sirna, VEGF recombinant protein, and vegfsirna to the endothelial cell stimulation under the influence of high fat and high shear stress, study veg The role of f-vegfr2-fak/paxillin signaling pathway in ox LDL and high shear stress mediated angiogenesis. The experimental results show that VEGF regulates the phosphorylation of the downstream protein FAK/paxillin of VEGFR2; oxLDL and high shear stress regulate the expression of the downstream signal pathway of VEGFR2-FAK/paxillin through VEGF; and VEGFR2 can regulate the FAK/paxillin downstream protein. Phosphorylation; VEGFR2/VEGFR1 is a key factor in the regulation of angiogenesis in VEGF. Therefore, the VEGF-VEGFR2-FAK/paxillin signaling pathway plays a key regulatory pathway in the angiogenesis of high fat and high shear stress. In this study, we elucidated the role of new vascular neovascularization in atherosclerotic plaque. The role of VEGF-VEGFR2-FAK/paxillin signaling pathway in angiogenesis in atherosclerotic plaques regulated by high fat and high shear stress.
【學位授予單位】：重慶大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R543.5

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