發(fā)布時(shí)間：2018-03-05 13:15

  本文選題：血管生成素樣蛋白4　切入點(diǎn)：缺血性腦卒中　出處：《華中科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的：本研究旨在探討ANGPTL4能否通過(guò)促進(jìn)血管生成及神經(jīng)再生增強(qiáng)腦血管功能儲(chǔ)備及梗死后神經(jīng)修復(fù)能力、相關(guān)機(jī)制。方法：在本研究中,C57BL/6小鼠被隨機(jī)分為兩組：生理鹽水干預(yù)組(對(duì)照組)和ANGPTL4干預(yù)組(實(shí)驗(yàn)組)。手術(shù)造模之前5min,分別接受經(jīng)尾靜脈注射人重組ANGPTL4 (rhANGPTL4,40 ug/kg body weight)或生理鹽水。將所有小鼠遠(yuǎn)端大腦中動(dòng)脈電凝阻塞構(gòu)建急性缺血性腦卒中模型。梗死后1天,利用MRI檢測(cè)造模是否成功,使行為學(xué)評(píng)估免受造模失敗的干擾：利用TTC染色評(píng)估兩組間梗死體積差異。梗死后7天,對(duì)BrdU/vWF和BrdU/DCX (bromodeoxyuridine/doublecortin)進(jìn)行免疫熒光標(biāo)記,前者評(píng)估梗死周邊區(qū)及側(cè)腦室室管膜下區(qū)(subventricular zone, SVZ)血管生成,后者評(píng)估SVZ區(qū)、側(cè)腦室外側(cè)壁及海馬齒狀回顆粒下層區(qū)(subgranular zone,SGZ)神經(jīng)再生；對(duì)Iba-1/BrdU進(jìn)行免疫熒光標(biāo)記,評(píng)估梗死周邊區(qū)小膠質(zhì)細(xì)胞增殖及活化程度。利用免疫組織化學(xué)方法分別半定量檢測(cè)梗死后1天、3天MPO(myeloperoxidase)表達(dá)水平,評(píng)估梗死周邊區(qū)中性粒細(xì)胞數(shù)目；梗死后7天梗死周邊區(qū)神經(jīng)元數(shù)目。利用Western blot檢測(cè)梗死后1天T-AKT, p-AKT, VEGF、MPO、Fas及FasL表達(dá)水平。結(jié)果：(1)腦梗死后1天,實(shí)驗(yàn)組梗死體積顯著低于對(duì)照組;(2)腦梗死后7天,實(shí)驗(yàn)組梗死周邊區(qū)及SVZ區(qū)BrdU+細(xì)胞數(shù)及BrdU+/vWF+細(xì)胞數(shù)顯著高于對(duì)照組;(3)腦梗死后7天,實(shí)驗(yàn)組SVZ區(qū)及SGZ區(qū)BrdU+細(xì)胞數(shù)及BrdU+/DCX+細(xì)胞數(shù)顯著高于對(duì)照組;免疫組化半定量檢測(cè)亦顯示實(shí)驗(yàn)組神經(jīng)元數(shù)目顯著高于對(duì)照組,表明ANGPTL4保護(hù)神經(jīng)元免遭丟失；(4)腦梗死后1天及3天免疫組化半定量檢測(cè)MPO表達(dá),實(shí)驗(yàn)組顯著低于對(duì)照組,表明ANGPTL4抑制急性期炎癥反應(yīng)；(5)腦梗死后7天,實(shí)驗(yàn)組小膠質(zhì)細(xì)胞增殖及活化程度顯著低于對(duì)照組,說(shuō)明ANGPTL4抑制了小膠質(zhì)細(xì)胞的活化；(6)實(shí)驗(yàn)組phospho-AKT表達(dá)水平及phospho-AKT/T-AKT比值顯著高于對(duì)照組,兩組之間Total-AKT及VEGF無(wú)顯著性差異；(7)實(shí)驗(yàn)組MPO、Fas及FasL表達(dá)水平顯著低于對(duì)照組。結(jié)論：ANGPTL4可通過(guò)增強(qiáng)AKT激酶磷酸化促進(jìn)缺血性腦卒中后血管生成和神經(jīng)再生。同時(shí)減少梗死周邊區(qū)神經(jīng)元數(shù)量,抑制缺血半暗帶急性期及慢性期炎癥反應(yīng),其可能是通過(guò)抑制FasL及Fas表達(dá)和其下游通路產(chǎn)生上述作用的。目的：腦出血后腦水腫與不良預(yù)后高度相關(guān)。本研究目的是探索ANGPTL4對(duì)腦出血后腦水腫及神經(jīng)功能缺損的治療作用、相關(guān)機(jī)制。方法：共選取168只12周左右體重25-28克的清潔級(jí)C57BL/6J小鼠,經(jīng)細(xì)菌膠原酶誘導(dǎo)腦紋狀體出血。手術(shù)前5min隨機(jī)接受尾靜脈注射生理鹽水或重組人血管生成素樣蛋白4(recombinant human angiopoietin-like protein 4, rhANGPTL4;40微克/千克體重)。在術(shù)前、腦出血后1天、3天、7天、14天、21天及28天,對(duì)照組及實(shí)驗(yàn)組分別接受轉(zhuǎn)筒實(shí)驗(yàn)(rotarod test)、改良神經(jīng)功能缺損評(píng)分(mNSS).轉(zhuǎn)角實(shí)驗(yàn)(corner turn test)等行為學(xué)測(cè)驗(yàn)。通過(guò)干濕重法及HE染色分別檢查腦水腫程度及血腫體積。應(yīng)用免疫熒光標(biāo)記量化緊密連接及粘附連接的完整性,應(yīng)用透射電子顯微鏡觀察血腦屏障超微結(jié)構(gòu)。應(yīng)用蛋白質(zhì)印跡法檢測(cè)VE-cadherin、claudin-5、Src及phospho-Src相對(duì)含量。結(jié)果：(1)系列行為學(xué)測(cè)驗(yàn)表明,腦出血后1天神經(jīng)功能缺損最為嚴(yán)重,隨后4周逐漸恢復(fù)且實(shí)驗(yàn)組顯著優(yōu)于對(duì)照組：(2)實(shí)驗(yàn)組小鼠腦出血后1天及3天時(shí)同側(cè)腦組織含水量顯著低于對(duì)照組；腦出血后3天時(shí)血腫體積亦顯著低于對(duì)照組;(3)免疫熒光結(jié)果顯示,實(shí)驗(yàn)組VE-cadherin、claudin-5陽(yáng)性區(qū)域及其相對(duì)比例均顯著高于對(duì)照組；(4)透射電鏡結(jié)果表明出血后24h及72h實(shí)驗(yàn)組血腦屏障超微結(jié)構(gòu)狀態(tài)顯著優(yōu)于對(duì)照組；(5)實(shí)驗(yàn)組Src、phospho-Src表達(dá)水平及phospho-Src/Src值顯著低于對(duì)照組,VE-cadherin、claudin-5顯著高于對(duì)照組。結(jié)論：本研究發(fā)現(xiàn)ANGPTL4能夠阻止腦出血所致血腦屏障的破壞,促進(jìn)神經(jīng)功能恢復(fù)。實(shí)驗(yàn)組p-Src表達(dá)下降同血管通透性下降及抑制水腫形成相并行,說(shuō)明ANGPTL4能通過(guò)抑制Src激酶活性減輕腦水腫及改善神經(jīng)功能缺損。
[Abstract]:Objective: This study aimed to investigate whether ANGPTL4 can promote angiogenesis and nerve regeneration by enhancing cerebral vascular function reserve and infarction after nerve repair ability, related mechanism. Methods: in this study, C57BL/6 mice were randomly divided into two groups: saline group (control group) and ANGPTL4 group (experimental group) operation. Before modeling, 5min, were treated by intravenous injection of recombinant human ANGPTL4 (rhANGPTL4,40 ug/kg body weight) or saline. The electrocoagulation all mouse distal middle cerebral artery occlusion model of acute ischemic stroke. 1 days after infarction, the success of modeling by MRI detection, the evaluation of the behavior from interference failure model: TTC staining was used to assess differences in infarct volume between the two groups. 7 days after infarction, BrdU/vWF and BrdU/DCX (bromodeoxyuridine/doublecortin) by immunofluorescence, the assessment of infarct area Under the membrane area and lateral ventricle tube (subventricular zone, SVZ) angiogenesis, which evaluates the SVZ area, SVZ and SGZ region (subgranular zone, SGZ) nerve regeneration; immunofluorescence labeling for Iba-1/BrdU, assessment of infarct area of microglia proliferation and activation by immunohistochemistry. Chemical methods are semi quantitative detection of infarction after 1 days, 3 days MPO (myeloperoxidase) expression level, evaluation of the infarcted area number of neutrophils; Infarction 7 days after the number of neurons in peri infarct area. Using Western blot detected 1 day after MI, T-AKT, p-AKT, VEGF, MPO, the expression level of Fas and FasL. Results: (1) 1 days after cerebral infarction, infarct volume in experimental group was significantly lower than the control group; (2) 7 days after cerebral infarction in experimental group, the infarcted area and SVZ area of BrdU+ cells and BrdU+/vWF+ cells was significantly higher than the control group; (3) 7 days after cerebral infarction. 楠岀粍SVZ鍖哄強(qiáng)SGZ鍖築rdU+緇嗚優(yōu)鏁板強(qiáng)BrdU+/DCX+緇嗚優(yōu)鏁版樉钁楅珮?shù)簬瀵圭収缁?鍏嶇柅緇勫寲鍗婂畾閲忔嫻嬩害鏄劇ず瀹為獙緇勭緇忓厓鏁扮洰鏄捐憲楂樹(shù)簬瀵圭収緇，

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