食蟹猴局灶性脑缺血的组织及血浆蛋白质组学分析

发布时间：2018-05-16 07:45

本文选题：食蟹猴模型 + 局灶性脑缺血　；参考：《华南理工大学》2016年硕士论文

【摘要】：缺血性脑卒中是死亡率最高的第三大疾病。目前,关于脑卒中的大多数研究还在大鼠模型上进行;而啮齿类动物和人类的生理、病理差别较大,并且大部分组学研究都没有单独分析细胞核及线粒体蛋白质组。本文采用基于n LC-MS/MS的鸟枪法蛋白组学研究方法,对灵长类动物疾病模型——局灶性脑缺血食蟹猴的脑组织和血浆中的蛋白进行测定,并对涉及不同生物学进程的相关蛋白进行了鉴定和分析。研究内容主要如下:使用灵长类动物食蟹猴构建局灶性脑缺血疾病模型,于缺血4 h收集梗死区、半影区及正常区的脑组织以及缺血4 h和再灌注0.5 h的血浆。建立基于阳离子交换的蛋白分级方法和基于LC-MS/MS的蛋白鉴定方法。1)分析比较了梗死区及正常区组织细胞线粒体及细胞核蛋白质组。对线粒体中有关有氧呼吸、产能代谢及细胞调控的蛋白进行分类;对细胞核中有关炎症、细胞凋亡的蛋白进行了分类比较。2)分析梗死区、半影区、正常区脑组织细胞浆中的蛋白。通过对物质代谢相关蛋白的分类比较,寻找不同缺血状态下物质代谢的差异;通过对炎症、凋亡相关蛋白及转录调节因子的比较,寻找不同区域的差异。3)分析了缺血4 h及再灌注0.5 h的血浆样本,寻找其中的脑特异性蛋白,并与临床的发现进行比较。结果:1)在梗死区线粒体及细胞核中分别鉴定到19、21个蛋白,而在正常区中的线粒体和细胞核中各鉴定到21、16个蛋白;2)梗死区、半影区、正常区的胞浆中分别鉴定到178、133、282个蛋白,梗死区中有关糖代谢的蛋白鉴定数量下降,而脂质代谢的蛋白数量上升,梗死区中有关炎症、凋亡的蛋白数量最高,半影区中转录调节因子的鉴定数量最高;3)缺血4 h血浆样品中鉴定到4个脑特异蛋白(LanC-like蛋白2、NELL-蛋白激酶C结合蛋白、微管蛋白β-2A、β-突触核蛋白),再灌注0.5 h血浆样品中鉴定到6个脑特异蛋白(神经分泌蛋白VGF、G protein regulated inducer of neurite outgrowth3、Synembryn B蛋白、小脑退行相关蛋白1、髓鞘脂碱性蛋白、Tau微管蛋白激酶)。结论:通过使用灵长类动物模型,研究了缺血性脑卒中疾病的分子机制,初步发现了在不同生理、病理状态下,脑组织物质能量代谢、免疫炎症反应及细胞凋亡过程的差异。通过对血浆蛋白质组学的分析研究,发现了多个蛋白可以作为脑缺血疾病的生物标志物。本研究为后续实验建立了方法,但其结果尚需要进一步实验进行验证。
[Abstract]:Ischemic stroke is the third major disease with the highest mortality. At present, most of the studies on stroke are still on the rat model, while the physiological and pathological differences between rodents and human beings are very different, and most of the histopathological studies have not analyzed the nucleus and the grain protein group alone. This paper uses the n LC-MS/MS based shotgun. The protein in the brain tissue and plasma of the focal cerebral ischemic cynomolgus monkey was determined by the method of protein composition, and the proteins related to different biological processes were identified and analyzed. The main contents are as follows: using the primate animal cynomolgus monkey to build the focal cerebral ischemic disease model Type, the cerebral tissue in the infarct area, the penumbra and normal regions and the plasma of the ischemic 4 h and reperfusion 0.5 h were collected at 4 h of the ischemia. The protein classification method based on the cation exchange and the LC-MS/MS based protein identification method.1) were used to analyze the mitochondria and nuclear proteome of the tissue cells in the infarcted and normal regions. The proteins of aerobic respiration, productivity metabolism and cell regulation were classified; the proteins related to inflammation and apoptosis in the nucleus were classified and compared.2) to analyze the proteins in the cytoplasm of the cerebral tissue of the infarct, the penumbra and the normal areas. The plasma samples of 4 h and 0.5 h of reperfusion were analyzed by comparison of inflammation, apoptosis related proteins and transcriptional regulators, and the specific brain proteins were found and compared with the clinical findings. Results: 1) the 19,21 protein was identified in the infarct area and in the nucleus of the nucleus. 21,16 proteins were identified in the mitochondria and nuclei in the normal region; 2) 178133282 proteins were identified in the infarct area, the penumbra region and the normal region. The number of protein identification related to glucose metabolism in the infarct area decreased, and the number of protein in the lipid metabolism increased. The number of apoptotic proteins in the infarct area was the highest, and the penumbra region was in the middle of the infarct area. The number of transcriptional regulators was highest; 3) 4 brain specific proteins (LanC-like protein 2, NELL- protein kinase C binding protein, microtubulin beta -2A, beta synuclein), and 0.5 h plasma samples were identified in 4 h plasma samples, and 6 brain specific proteins (neurosecretory protein VGF, G protein regulated inducer of) were identified. Outgrowth3, Synembryn B protein, cerebellar degenerative protein 1, myelin alkaline protein, Tau microtubule protein kinase. Conclusion: by using the primate animal model, the molecular mechanism of ischemic stroke disease was studied. The material energy metabolism of brain tissue, immune inflammatory response and cell apoptosis in different physiological and pathological conditions were preliminarily found. The analysis of plasma proteomics shows that many proteins can be used as biomarkers for cerebral ischemic diseases. This study has established a method for subsequent experiments, but the results are still needed to be verified by further experiments.

【学位授予单位】：华南理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R743.3

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