本文選題：食蟹猴模型 + 局灶性腦缺血��； 參考：《華南理工大學》2016年碩士論文

【摘要】：缺血性腦卒中是死亡率最高的第三大疾病。目前,關于腦卒中的大多數研究還在大鼠模型上進行;而嚙齒類動物和人類的生理、病理差別較大,并且大部分組學研究都沒有單獨分析細胞核及線粒體蛋白質組。本文采用基于n LC-MS/MS的鳥槍法蛋白組學研究方法,對靈長類動物疾病模型——局灶性腦缺血食蟹猴的腦組織和血漿中的蛋白進行測定,并對涉及不同生物學進程的相關蛋白進行了鑒定和分析。研究內容主要如下:使用靈長類動物食蟹猴構建局灶性腦缺血疾病模型,于缺血4 h收集梗死區(qū)、半影區(qū)及正常區(qū)的腦組織以及缺血4 h和再灌注0.5 h的血漿。建立基于陽離子交換的蛋白分級方法和基于LC-MS/MS的蛋白鑒定方法。1)分析比較了梗死區(qū)及正常區(qū)組織細胞線粒體及細胞核蛋白質組。對線粒體中有關有氧呼吸、產能代謝及細胞調控的蛋白進行分類;對細胞核中有關炎癥、細胞凋亡的蛋白進行了分類比較。2)分析梗死區(qū)、半影區(qū)、正常區(qū)腦組織細胞漿中的蛋白。通過對物質代謝相關蛋白的分類比較,尋找不同缺血狀態(tài)下物質代謝的差異;通過對炎癥、凋亡相關蛋白及轉錄調節(jié)因子的比較,尋找不同區(qū)域的差異。3)分析了缺血4 h及再灌注0.5 h的血漿樣本,尋找其中的腦特異性蛋白,并與臨床的發(fā)現進行比較。結果:1)在梗死區(qū)線粒體及細胞核中分別鑒定到19、21個蛋白,而在正常區(qū)中的線粒體和細胞核中各鑒定到21、16個蛋白;2)梗死區(qū)、半影區(qū)、正常區(qū)的胞漿中分別鑒定到178、133、282個蛋白,梗死區(qū)中有關糖代謝的蛋白鑒定數量下降,而脂質代謝的蛋白數量上升,梗死區(qū)中有關炎癥、凋亡的蛋白數量最高,半影區(qū)中轉錄調節(jié)因子的鑒定數量最高;3)缺血4 h血漿樣品中鑒定到4個腦特異蛋白(LanC-like蛋白2、NELL-蛋白激酶C結合蛋白、微管蛋白β-2A、β-突觸核蛋白),再灌注0.5 h血漿樣品中鑒定到6個腦特異蛋白(神經分泌蛋白VGF、G protein regulated inducer of neurite outgrowth3、Synembryn B蛋白、小腦退行相關蛋白1、髓鞘脂堿性蛋白、Tau微管蛋白激酶)。結論:通過使用靈長類動物模型,研究了缺血性腦卒中疾病的分子機制,初步發(fā)現了在不同生理、病理狀態(tài)下,腦組織物質能量代謝、免疫炎癥反應及細胞凋亡過程的差異。通過對血漿蛋白質組學的分析研究,發(fā)現了多個蛋白可以作為腦缺血疾病的生物標志物。本研究為后續(xù)實驗建立了方法,但其結果尚需要進一步實驗進行驗證。
[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.

【學位授予單位】：華南理工大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R743.3

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