ANGPTL4促进脑梗死后血管生成及神经再生和减轻脑出血后水肿及神经缺损的研究

发布时间：2018-03-05 13:15

  本文选题：血管生成素样蛋白4　切入点：缺血性脑卒中　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：本研究旨在探讨ANGPTL4能否通过促进血管生成及神经再生增强脑血管功能储备及梗死后神经修复能力、相关机制。方法：在本研究中,C57BL/6小鼠被随机分为两组：生理盐水干预组(对照组)和ANGPTL4干预组(实验组)。手术造模之前5min,分别接受经尾静脉注射人重组ANGPTL4 (rhANGPTL4,40 ug/kg body weight)或生理盐水。将所有小鼠远端大脑中动脉电凝阻塞构建急性缺血性脑卒中模型。梗死后1天,利用MRI检测造模是否成功,使行为学评估免受造模失败的干扰：利用TTC染色评估两组间梗死体积差异。梗死后7天,对BrdU/vWF和BrdU/DCX (bromodeoxyuridine/doublecortin)进行免疫荧光标记,前者评估梗死周边区及侧脑室室管膜下区(subventricular zone, SVZ)血管生成,后者评估SVZ区、侧脑室外侧壁及海马齿状回颗粒下层区(subgranular zone,SGZ)神经再生；对Iba-1/BrdU进行免疫荧光标记,评估梗死周边区小胶质细胞增殖及活化程度。利用免疫组织化学方法分别半定量检测梗死后1天、3天MPO(myeloperoxidase)表达水平,评估梗死周边区中性粒细胞数目；梗死后7天梗死周边区神经元数目。利用Western blot检测梗死后1天T-AKT, p-AKT, VEGF、MPO、Fas及FasL表达水平。结果：(1)脑梗死后1天,实验组梗死体积显著低于对照组;(2)脑梗死后7天,实验组梗死周边区及SVZ区BrdU+细胞数及BrdU+/vWF+细胞数显著高于对照组;(3)脑梗死后7天,实验组SVZ区及SGZ区BrdU+细胞数及BrdU+/DCX+细胞数显著高于对照组;免疫组化半定量检测亦显示实验组神经元数目显著高于对照组,表明ANGPTL4保护神经元免遭丢失；(4)脑梗死后1天及3天免疫组化半定量检测MPO表达,实验组显著低于对照组,表明ANGPTL4抑制急性期炎症反应；(5)脑梗死后7天,实验组小胶质细胞增殖及活化程度显著低于对照组,说明ANGPTL4抑制了小胶质细胞的活化；(6)实验组phospho-AKT表达水平及phospho-AKT/T-AKT比值显著高于对照组,两组之间Total-AKT及VEGF无显著性差异；(7)实验组MPO、Fas及FasL表达水平显著低于对照组。结论：ANGPTL4可通过增强AKT激酶磷酸化促进缺血性脑卒中后血管生成和神经再生。同时减少梗死周边区神经元数量,抑制缺血半暗带急性期及慢性期炎症反应,其可能是通过抑制FasL及Fas表达和其下游通路产生上述作用的。目的：脑出血后脑水肿与不良预后高度相关。本研究目的是探索ANGPTL4对脑出血后脑水肿及神经功能缺损的治疗作用、相关机制。方法：共选取168只12周左右体重25-28克的清洁级C57BL/6J小鼠,经细菌胶原酶诱导脑纹状体出血。手术前5min随机接受尾静脉注射生理盐水或重组人血管生成素样蛋白4(recombinant human angiopoietin-like protein 4, rhANGPTL4;40微克/千克体重)。在术前、脑出血后1天、3天、7天、14天、21天及28天,对照组及实验组分别接受转筒实验(rotarod test)、改良神经功能缺损评分(mNSS).转角实验(corner turn test)等行为学测验。通过干湿重法及HE染色分别检查脑水肿程度及血肿体积。应用免疫荧光标记量化紧密连接及粘附连接的完整性,应用透射电子显微镜观察血脑屏障超微结构。应用蛋白质印迹法检测VE-cadherin、claudin-5、Src及phospho-Src相对含量。结果：(1)系列行为学测验表明,脑出血后1天神经功能缺损最为严重,随后4周逐渐恢复且实验组显著优于对照组：(2)实验组小鼠脑出血后1天及3天时同侧脑组织含水量显著低于对照组；脑出血后3天时血肿体积亦显著低于对照组;(3)免疫荧光结果显示,实验组VE-cadherin、claudin-5阳性区域及其相对比例均显著高于对照组；(4)透射电镜结果表明出血后24h及72h实验组血脑屏障超微结构状态显著优于对照组；(5)实验组Src、phospho-Src表达水平及phospho-Src/Src值显著低于对照组,VE-cadherin、claudin-5显著高于对照组。结论：本研究发现ANGPTL4能够阻止脑出血所致血脑屏障的破坏,促进神经功能恢复。实验组p-Src表达下降同血管通透性下降及抑制水肿形成相并行,说明ANGPTL4能通过抑制Src激酶活性减轻脑水肿及改善神经功能缺损。
[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SGZ鍖築rdU+缁嗚優鏁板強BrdU+/DCX+缁嗚優鏁版樉钁楅珮浜庡�圭収缁�;鍏嶇柅缁勫寲鍗婂畾閲忔��娴嬩害鏄剧ず瀹為獙缁勭�炵粡鍏冩暟鐩�鏄捐憲楂樹簬瀵圭収缁，

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