本文關(guān)鍵詞： 低血糖 腦損傷 iTRAQ 自噬 DJ-1　出處：《上海交通大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：背景低血糖是糖尿病患者強(qiáng)化血糖控制的常見并發(fā)癥。低血糖最容易導(dǎo)致腦損傷�！捌咸烟窃俟嘧⑿阅X損傷”概念的提出,使得人們對低血糖性腦損傷發(fā)病機(jī)制的認(rèn)識更加復(fù)雜,也引發(fā)了對傳統(tǒng)臨床低血糖救治的再思考。研究低血糖發(fā)作及恢復(fù)過程中與腦損傷有關(guān)的病理機(jī)制,有可能為臨床低血糖救治過程中提供腦保護(hù)治療的靶標(biāo)和思路。目的本研究旨在采用同位素相對標(biāo)記與絕對定量技術(shù)(Isobaric tag for relative and absolute quantitation,ITRAQ)連同多維液相色譜-串聯(lián)質(zhì)譜聯(lián)用技術(shù)(LC-MS/MS)尋找低血糖大鼠與正常大鼠腦組織內(nèi)的差異表達(dá)蛋白,對其進(jìn)行表達(dá)量的驗證及相關(guān)生物學(xué)功能探討,初步闡明低血糖性腦損傷發(fā)病的相關(guān)分子機(jī)制,為低血糖性腦損傷的治療提供理論依據(jù)。方法注射胰島素建立低血糖大鼠腦損傷模型,提取腦組織蛋白,酶解后用i TRAQ化學(xué)標(biāo)簽標(biāo)記,進(jìn)行多維液相色譜分離—串聯(lián)質(zhì)譜鑒定,篩選潛在的可能與低血糖性腦損傷相關(guān)的差異蛋白。選取差異表達(dá)明顯且生物學(xué)功能相關(guān)者在重新建立的動物模型上進(jìn)行m RNA和蛋白水平的表達(dá)量驗證。最后在模擬低血糖的體外無糖培養(yǎng)星形膠質(zhì)細(xì)胞模型上利用RNA干擾技術(shù)進(jìn)行相關(guān)生物學(xué)功能的初步研究。結(jié)果通過比較低血糖大鼠與假造模大鼠腦組織的蛋白表達(dá),以相對表達(dá)量≥1.5或≤0.67為標(biāo)準(zhǔn),共篩選到147種差異表達(dá)蛋白。采用實時熒光定量聚合酶鏈反應(yīng)和免疫印跡法在復(fù)制的動物模型上證實AQP4,NDRG1,DJ-1和Clusterin四個蛋白的表達(dá)量與在蛋白組學(xué)中發(fā)現(xiàn)的表達(dá)量變化趨勢一致。在模擬的體外星形膠質(zhì)細(xì)胞缺糖模型上進(jìn)一步驗證了缺糖上調(diào)AQP4和DJ-1兩個蛋白的表達(dá)量。沉默DJ-1蛋白后,星形膠質(zhì)細(xì)胞在無糖培養(yǎng)時死亡率明顯增加。將星形膠質(zhì)細(xì)胞進(jìn)行無糖培養(yǎng),觀察到AMPK通路被激活,自噬被啟動,但自噬通量受損。工具藥抑制自噬后無糖培養(yǎng)致星形膠質(zhì)細(xì)胞死亡明顯增加。與正常表達(dá)DJ-1的細(xì)胞比較,沉默DJ-1表達(dá)的星形膠質(zhì)細(xì)胞在無糖培養(yǎng)時AMPK通路受損,m TOR活性升高,自噬活性明顯降低。結(jié)論DJ-1蛋白在低血糖性腦損傷中發(fā)揮保護(hù)作用,減少缺糖導(dǎo)致的星形膠質(zhì)細(xì)胞損傷,其部分機(jī)制通過調(diào)節(jié)自噬來實現(xiàn)。
[Abstract]:Background hypoglycemia is a common complication of intensive blood glucose control in diabetic patients. Hypoglycemia is most likely to lead to brain injury. The concept of "glucose-reperfusion brain injury" has made people's understanding of the pathogenesis of hypoglycemic brain injury more complicated. It also triggered a reconsideration of the traditional clinical treatment of hypoglycemia. The pathological mechanism related to brain injury during hypoglycemia attack and recovery was studied. Objective the aim of this study was to use isotopic tag for relative and absolute quantitative technique to study Isobaric tag for relative and absolute quantification of Isobaric and Isobaric for relative and absolute, and multidimensional liquid chromatography-tandem substance. [WT5 "HZ] [WT5" HZ] [WT5 "BZ] [WT5" HZ]. LC-MS / MS was used to search for differentially expressed proteins in the brain of hypoglycemic rats and normal rats. The molecular mechanism of hypoglycemic brain injury was preliminarily elucidated by the verification of its expression and the discussion of its related biological functions. Methods the model of hypoglycemic brain injury in rats was established by insulin injection. Brain tissue proteins were extracted and labeled with I TRAQ chemical label after enzymatic hydrolysis, and identified by multi-dimensional liquid chromatography-tandem mass spectrometry. Potential differentially expressed proteins associated with hypoglycemic brain injury were screened. The expression levels of m RNA and protein were tested on the reestablished animal model with distinct differential expression and biological functional correlation. RNA interference technique was used to study the biological function of hypoglycemic astrocytes in vitro. Results the expression of protein in the brain of hypoglycemic rats was compared with that of hypoglycemic rats. If the relative expression is 鈮，

本文編號：1531606