【摘要】：腦血管病是威脅人類生命的主要疾病之一。目前很多研究證實動脈粥樣硬化易損斑塊的破裂和栓子的脫落是引起急性心血管事件及腦卒中的重要原因,頸動脈斑塊造成的血管狹窄甚至閉塞、斑塊的突然破裂導(dǎo)致急性的動脈阻塞都可以引起缺血性腦卒中。因此,頸動脈斑塊的早期識別、斑塊穩(wěn)定性的評價以及干預(yù)阻止其進展對于心腦血管病的防治意義重大。第一部分TLR4在動脈粥樣硬化斑塊形成和進展中的作用機制目的:觀察動脈粥樣硬化斑塊(AP)形成和發(fā)展中Toll樣受體4(TLR4)表達的變化,探討TLR4在AP形成和發(fā)展的過程中的作用機制。材料與方法:8周齡ApoE(-/-)小鼠20只,適應(yīng)性喂養(yǎng)1周后,隨機分為4組,每組5只,包括對照組、普通飲食組、10周模型組和20周模型組。對照組處死,普通飲食組給予普通飼料喂養(yǎng)10周后處死,10周模型組和20周模型組分別予高脂飲食喂養(yǎng)10周和20周后處死,取血檢測血清總膽固醇(TC)、甘油三酯(TG)、氧化型低密度脂蛋白(oxLDL),留取頸動脈和主動脈標(biāo)本,觀察斑塊情況,對標(biāo)本進行HE染色、油紅O染色、彈力纖維染色以及天狼猩紅染色。并通過免疫組化檢測頸動脈斑塊巨噬細胞(MOMA-2)、平滑肌肌動蛋白(α-actin)蛋白水平、TLR4、白介素1β(IL-1β)、白介素6(IL-6)、腫瘤壞死因子(TNF-α)的表達。結(jié)果:1.普通飼料組TC、TG、oxLDL水平較對照組升高(P0.05);10周模型組較普通飼料組血脂明顯升高(P0.05),AP形成。2.20周模型組較對照組和10周模型組血清TC、TG、oxLDL水平升高(P0.05),AP明顯增多,斑塊脂質(zhì)成分增加,彈力纖維中斷、減少,膠原成分減少,巨噬細胞增加,平滑肌細胞減少。3.20周模型組較對照組和10周模型組頸動脈斑塊TLR4、IL-1β、IL-6和TNF-α表達增加(P0.05)。結(jié)論:1.ApoE(-/-)小鼠高脂飲食可以成功建立AP動脈的動物模型,且高脂飲食的時間越長,AP越明顯。2.ApoE(-/-)小鼠通過高脂飲食建立動脈粥樣硬化動物模型后oxLDL水平增高,動脈及斑塊炎癥因子水平增高,巨噬細胞增多,血管平滑肌細胞減少,膠原合成減少,從而促進斑塊的進展,并降低斑塊的穩(wěn)定性。3.apoe(-/-)小鼠隨著動脈粥樣硬化的加劇,tlr4和炎性因子的表達增加,可能參與促進動脈粥樣硬化斑塊進展。第二部分tlr4誘導(dǎo)自噬與動脈粥樣硬化斑塊穩(wěn)定性的關(guān)系目的:觀察apoe(-/-)小鼠動脈粥樣硬化斑塊tlr4表達與自噬的關(guān)系,探討tlr4誘導(dǎo)自噬對動脈粥樣硬化斑塊(ap)穩(wěn)定性的影響。方法:8周齡apoe(-/-)小鼠24只,適應(yīng)性喂養(yǎng)1周后,給予高脂飲食10周后隨機分為3組,每組8只,包括lps組、對照組、tak組。lps組給予lps(1mg/kg)腹腔注射,每周2次;tak組給予tak-242(0.3mg/kg)腹腔注射,每周2次;對照組給予相應(yīng)的等容積的生理鹽水腹腔注射,每周2次。三組仍繼續(xù)高脂飲食,腹腔注射10周后取血檢測血清總膽固醇(tc)、甘油三酯(tg)、氧化型低密度脂蛋白(oxldl)。留取頸動脈和主動脈標(biāo)本,觀察斑塊情況,對標(biāo)本進行he染色、油紅o染色、彈力纖維染色以及天狼猩紅染色。并通過免疫組化檢測斑塊內(nèi)巨噬細胞(moma-2)、平滑肌肌動蛋白(α-actin)蛋白水平、toll樣受體4(tlr4)、il-1β、il-6、tnf-α的表達。westernblot檢測lc3Ⅱ/Ⅰ,beclin-1,p62水平。結(jié)果:1.lps組小鼠血清tc、tg、oxldl水平高于對照組和tak組小鼠(p0.05);tak組tg、oxldl水平低于對照組(p0.05)。2.lps組小鼠ap較對照組和tak組巨噬細胞含量(mamo-2免疫組化染色陽性率)增多(p0.05),tak組較對照組小鼠ap巨噬細胞含量減少(p0.05)。lps組較對照組和tak小鼠ap平滑肌細胞含量(α-actin免疫組化染色陽性率)減少(p0.05);tak組較對照組平滑肌含量增加(p0.05)。3.lps組小鼠ap與對照組和tak組比較,tlr4、il-1β、il-6、tnf-α的表達增加(p0.05);tak組較對照組tlr4、il-1β、il-6、tnf-α的表達減少(p0.05)。4.對照組、lps組和tak組小鼠動脈lc3Ⅱ/Ⅰ、p62和beclin-1蛋白表達量有差異(p0.05)。lps組較對照組和tak組lc3Ⅱ/Ⅰ表達增加(p0.001)。lps組較對照組和tak組beclin-1表達增加(p0.001);tak組較對照組beclin-1表達減少(p0.001)。lps組較對照組和tak組p62表達增加(p0.001);tak組較對照組p62表達增加(p0.01)。結(jié)論1.lps可以上調(diào)aptlr4的表達,tak-242下調(diào)tlr4的表達。2.lps上調(diào)tlr4表達后,促進炎癥因子的分泌,加重脂代謝紊亂,自噬水平明顯增高,但p62蛋白水平同時升高,可能lps導(dǎo)致過度自噬而出現(xiàn)自噬功能異常,反而加劇了ap進展和易損性增加。3.tak-242通過下調(diào)tlr4的表達,減少巨噬細胞,增加平滑肌細胞的含量,增加斑塊的穩(wěn)定性。第三部分阿托伐他汀及聯(lián)合普羅布考對tlr4誘導(dǎo)自噬的影響目的:評價阿托伐他汀及聯(lián)合普羅布考對tlr4誘導(dǎo)的自噬的影響,探討其抗動脈粥樣硬化的作用機制。方法:8周齡apoe(-/-)小鼠24只,適應(yīng)性喂養(yǎng)1周后,予以高脂飲食10周,再隨機分為3組,每組8只,包括對照組、他汀組、聯(lián)合組。對照組給予等容積超純水灌胃10周,每天1次;他汀組,高脂飲食的同時給予阿托伐他汀(10mg/kg/d)灌胃10周,每天1次;(3)聯(lián)合組,換喂食普羅布考飼料,同時給予阿托伐他汀(10mg/kg/d)灌胃10周,每天1次。取血檢測血清總膽固醇(tc)、甘油三酯(tg)、氧化型低密度脂蛋白(oxldl)。留取頸動脈和主動脈標(biāo)本,觀察斑塊情況,對標(biāo)本進行he染色、油紅o染色、彈力纖維染色和天狼猩紅染色。并通過免疫組化檢測斑塊內(nèi)巨噬細胞(moma-2)、平滑肌肌動蛋白(α-actin)蛋白水平、toll樣受體4(tlr4)、il-1β、il-6、tnf-α的表達。westernblot檢測lc3Ⅱ/Ⅰ,beclin-1,p62水平。結(jié)果:1.他汀組和聯(lián)合組tc和oxldl水平較對照組降低,聯(lián)合組較他汀組oxldl水平降低(p0.05)。2.他汀組和聯(lián)合組與對照組比較,頸動脈ap彈力纖維較完整,脂質(zhì)含量減少,膠原含量增加,tlr4、il-1β、il-6、tnf-α的表達減低、平滑肌細胞增加,易損指數(shù)降低(p0.05)。聯(lián)合組較他汀組小鼠頸動脈ap巨噬細胞含量減少,易損指數(shù)降低(p0.05)。3.對照組、他汀組和聯(lián)合組小鼠動脈lc3Ⅱ/Ⅰ、p62和beclin-1蛋白表達量有差異(P0.05)。他汀組和聯(lián)合組與較對照組動脈Beclin-1表達增加(P0.05);聯(lián)合組較對照組和他汀組LC3Ⅱ/Ⅰ表達增加,P62表達減少(P0.05)。結(jié)論:1.阿托伐他汀可下調(diào)TLR4表達,降低血脂,抑制氧化應(yīng)激和炎癥因子的分泌,增加斑塊的穩(wěn)定性。2.聯(lián)合應(yīng)用阿托伐他汀和普羅布考較單獨應(yīng)用阿托伐他汀具有更強的抑制氧化應(yīng)激的作用,減少巨噬細胞含量,適當(dāng)?shù)卦黾恿俗允伤?發(fā)揮更強的抗動脈粥樣硬化作用,更有效地增加了斑塊的穩(wěn)定性。
[Abstract]:Cerebrovascular disease is one of the major diseases that threaten human life. Many studies have confirmed that rupture of vulnerable atherosclerotic plaques and loss of embolus are important causes of acute cardiovascular events and stroke. Carotid plaques cause vascular stenosis or occlusion, and sudden rupture of plaques leads to acute arterial obstruction. Therefore, early identification of carotid plaques, evaluation of plaque stability and intervention to prevent their progression are of great significance for the prevention and treatment of cardiovascular and cerebrovascular diseases. Materials and Methods: Twenty eight-week-old ApoE (-/-) mice were randomly divided into four groups after one week of adaptive feeding. Five mice in each group were divided into control group, normal diet group, 10-week model group and 20-week model group. The rats in the 10-week model group and the 20-week model group were sacrificed after 10 and 20 weeks of high-fat diet respectively. The serum total cholesterol (TC), triglyceride (TG), oxidized low-density lipoprotein (oxLDL) and carotid artery and aorta specimens were taken to observe the plaque, HE staining, oil red O staining and elastic fiber staining were performed. The levels of macrophages (MOMA-2), smooth muscle actin (a-actin), TLR4, interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), tumor necrosis factor (TNF-a) in carotid plaque were detected by immunohistochemistry. Results: 1. The levels of TC, TG and oxLDL in the normal diet group were higher than those in the control group (P The levels of TC, TG and oxLDL in serum of model group at 2.20 weeks were higher than those of control group and model group at 10 weeks (P 0.05). The levels of AP, lipid components in plaque increased, elastic fibers interrupted, decreased, collagen components decreased, macrophages increased and smooth muscle cells decreased. The levels of TC, TG and oxLDL in model group at 3.20 weeks were higher than those of control group and model group at 10 weeks. The expression of TLR4, IL-1beta, IL-6 and TNF-alpha in carotid artery plaque increased in group A (P 0.05). Conclusion: 1. ApoE (-/-) mice could successfully establish AP artery model by high-fat diet, and the longer the high-fat diet, the more obvious the AP. 2. ApoE (-/-) mice established atherosclerotic animal model by high-fat diet, the oxLDL level increased, arterial and plaque inflammation. 3. Apoe (-/-) mice with increased atherosclerosis, increased expression of TLR4 and inflammatory factors, may participate in the promotion of atherosclerotic plaque progression. Part 2: Tlr4 induction Objective: to observe the relationship between the expression of TLR4 and the stability of atherosclerotic plaque in ApoE (-/-) mice, and to explore the effect of autophagy induced by TLR4 on the stability of atherosclerotic plaque (ap). LPS (1 mg / kg) was injected intraperitoneally twice a week in the LPS group; tak-242 (0.3 mg / kg) was injected intraperitoneally twice a week in the Tak group; and the control group was injected intraperitoneally with the corresponding normal saline of equal volume twice a week in the control group. The three groups continued to eat high-fat diet. After 10 weeks of intraperitoneal injection, the serum total cholesterol was detected. (tc), triglyceride (tg), oxidized low density lipoprotein (oxldl). carotid artery and aorta specimens were taken to observe the plaque condition. he staining, oil red O staining, elastic fiber staining and Sirius scarlet staining were performed. macrophages (moma-2), smooth muscle actin (a-actin) protein level and Toll-like were detected by immunohistochemistry. The expression of receptor 4 (tlr4), interleukin-1 beta, interleukin-6 and TNF-a were detected by Western blot. the levels of tc, TG and oxLDL in serum of mice in LPS group were higher than those in control group and Tak Group (p0.05). the levels of TG and oxLDL in Tak Group were lower than those in control group (p0.05). 2. the contents of macrophages in AP group were higher than those in control group and Tak Group (mamo-2 immunohistochemical staining positive). Sex rate increased (p0.05), Tak group compared with the control group AP macrophage content decreased (p0.05). LPS group compared with the control group and Tak mice AP smooth muscle cell content (a-actin immunohistochemical staining positive rate) decreased (p0.05); Tak group compared with the control group smooth muscle content increased (p0.05). 3. LPS group mice AP compared with the control group and Tak group, tlr4, IL-1 beta, il-6, TNF-a. The expression of tlr4, IL-1 beta, IL-6 and TNF-a in the Tak Group was lower than that in the control group (p0.05). 4. the expression of lc3ii / _, p62 and beclin-1 in the arteries of the LPS group and the Tak Group were different (p0.05). the expression of lc3ii / _in the LPS group was higher than that in the control group and the Tak Group (p0.001). the expression of beclin-1 in the LPS group was higher than that in the control group and the Tak Group (p0.001). The expression of beclin-1 in Group K was lower than that in control group (p0.001). the expression of p62 in LPS group was higher than that in control group and Tak Group (p0.001). the expression of p62 in Tak Group was higher than that in control group (p0.01). conclusion 1. LPS can up-regulate the expression of aptlr4, and tak-242 can down-regulate the expression of tlr4. 2. LPS can up-regulate the expression of tlr4, promote the secretion of inflammatory factors, aggravate the disorder of lipid metabolism, and significantly increase the level of autophagy. High levels of p62 protein may lead to excessive autophagy and autophagy dysfunction, but aggravate the progress and vulnerability of ap. 3. tak-242 down-regulates the expression of tlr4, reduces macrophages, increases the content of smooth muscle cells, and increases the stability of plaque. part three: atorvastatin and probucol combined with TLR4 AIM: To evaluate the effect of atorvastatin and probucol on autophagy induced by TLR4 and explore the mechanism of its anti-atherosclerosis effect. METHODS: Twenty-four apoE (-/-) mice aged 8 weeks were fed with high-fat diet for 10 weeks, and then randomly divided into three groups, eight in each group, including control group, statin group and combined group. The control group was given isovolumetric ultrapure water once a day for 10 weeks, while the statin group was given atorvastatin (10mg/kg/d) for 10 weeks, once a day; (3) the combined group was given probucol feed, and atorvastatin (10mg/kg/d) for 10 weeks, once a day. Carotid artery and aorta specimens were taken to observe the plaque. he staining, o il red O staining, elastic fiber staining and Sirius red staining were performed. the levels of macrophages (moma-2), smooth muscle actin (a-actin) protein, Toll-like receptor 4 (tlr4), IL-1 beta, il-6, TN in the plaque were detected by immunohistochemistry. The levels of TC and oxLDL in statin group and combined group were lower than those in control group, and the levels of oxLDL in combined group were lower than those in statin group (p0.05). 2. compared with the control group, the elastic fibers of carotid artery AP in statin group and combined group were more intact, the content of lipid decreased, the content of collagen increased, tlr4, IL-1 beta, and IL-1 beta. Compared with statin group, the content of AP macrophages in carotid artery decreased, and the vulnerability index decreased (p0.05). 3. the expression of LC3 II / i, p62 and beclin-1 in arteries of control group, statin group and combination group were different (p0.05). statin group and combination group were compared with control group. Compared with the control group and statin group, the expression of LC3 II/I increased and the expression of P62 decreased in the combined group (P 0.05). Conclusion: 1. Atorvastatin can down-regulate the expression of TLR4, reduce blood lipid, inhibit the secretion of oxidative stress and inflammatory factors, and increase the stability of plaque. 2. Atorvastatin combined with probucol is more effective than alone. Atorvastatin can inhibit oxidative stress, decrease macrophage content, increase autophagy level, exert stronger anti-atherosclerosis effect, and increase plaque stability more effectively.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R543.5

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