本文選題：海藻糖 + 蛋白質聚集��； 參考：《吉林大學》2017年博士論文

【摘要】：海藻糖抑制短暫性缺血誘導的蛋白質聚集的機制研究目的:探討海藻糖對缺血性腦損害引起的蛋白質聚集的影響,并探索其機制。方法:建立大鼠的短暫性全腦缺血模型以及SH-SY5Y的氧糖剝奪模型。應用LDH釋放測定和臺盼藍測定、細胞內(nèi)活性氧測定、Morris水迷宮行為學評估、蘇木精及伊紅染色和組織學檢查、免疫組織化學分析、蛋白酶體活性測定、蛋白印跡分析和印跡密度分析探索海藻糖對缺血性損害引起的蛋白質聚集的影響及其機制。結果:(1)蘇木精-伊紅染色觀察發(fā)現(xiàn):與假手術組相比,缺血組中海馬CA1區(qū)神經(jīng)元在缺血后再灌注72小時后,其形態(tài)學表現(xiàn)為細胞質粉染、固定核呈多邊形。用3.0%濃度的海藻糖提前48小時預處理可顯著抑制短暫性腦缺血引起的海馬CA1區(qū)神經(jīng)元的形態(tài)學改變。缺血前使用3.0%濃度的海藻糖預處理后,缺血/再灌注后72小時時存活神經(jīng)元的百分比從5.52±2.31%增加到62.56±8.17%。水迷宮實驗中典型的游泳軌跡表明海藻糖預處理可以使缺血大鼠能以更為合適的方法搜索水中隱藏平臺,縮短尋找到水中隱藏平臺的潛伏期、減少在QII內(nèi)的游泳軌跡;與假手術組相比,缺血組大鼠在QII中停留時間更短暫,但是應用海藻糖預處理顯著延長了大鼠在目標象限中的停留時間。(2)蛋白印跡分析顯示,在3.0%海藻糖+缺血組,再灌注24小時時泛素標記蛋白質聚集體的增加明顯減弱。免疫組織化學染色顯示:在再灌注24小時時,經(jīng)3.0%濃度的海藻糖預處理的海馬ca1區(qū)神經(jīng)元中泛素標記的蛋白質聚集體明顯受到抑制。與缺血組相比,經(jīng)3.0%濃度的海藻糖預處理后在缺血/再灌注12小時、24小時和48小時時,海馬ca1區(qū)神經(jīng)元內(nèi)蛋白酶體活性明顯得到逆轉,其活性分別從47.56%±4.89%上升至71.88%±7.12%,53.25%±4.21%上升至73.35%±6.96%和46.88%±3.79%上升至75.28%±7.86%。(3)乳酸脫氫酶釋放測定顯示:在復氧6小時時,提前48小時應用海藻糖預處理(0.5或5.0mmol/l)可以顯著抑制氧糖剝奪誘導的sh-sy5y細胞死亡。蛋白印跡分析顯示:在復氧2小時時,經(jīng)5.0mmol/l濃度的海藻糖預處理后的sh-sy5y樣本,其逆轉了氧糖剝奪誘導的lc3ii、beclin-1水平的增加及自噬p62選擇性底物的減少。我們發(fā)現(xiàn)使用自噬抑制劑3ma以2mmol/l濃度預處理1小時后,它不僅抑制了氧糖剝奪誘導的lc3ii,beclin-1和p62蛋白水平的變化,而且在復氧6小時時減少了細胞的死亡。(4)5.0mmol/l濃度的海藻糖預處理可以顯著抑制mg-132誘導的蛋白酶體活性下降、乳酸脫氫酶釋放增加和泛素標記蛋白質聚集物的形成。(5)熒光顯微鏡下的圖像顯示用0.5mmol/l或5.0mmol/l的海藻糖提前48h預處理可以顯著抑制氧糖剝奪24小時并復氧2小時時人sh-sy5y細胞中綠色熒光的增加。熒光密度的統(tǒng)計分析證明0.5mmol/l和5.0mmol/l濃度的海藻糖均可以有效的抑制由氧糖剝奪引起的細胞內(nèi)氧化應激的增加。(6)蛋白印跡分析和印跡密度分析顯示,在復氧2小時時,經(jīng)5.0mmol/L濃度的海藻糖預處理的人SH-Y5Y細胞樣本中,氧糖剝奪誘導的PERK,磷酸化PERK,磷酸化eIF2α,IRE-1和GRP78的表達上調明顯受抑。這意味著海藻糖預處理抑制了人SH-SY5Y細胞樣本中氧糖剝奪誘導的內(nèi)質網(wǎng)應激。結論:在本研究中,我們證明海藻糖通過抑制氧化應激和內(nèi)質網(wǎng)應激來保護細胞內(nèi)蛋白酶體活性,從而實現(xiàn)其對缺血性損傷引起的蛋白質聚集的抑制作用。蛋白酶體在調節(jié)缺血性損傷引起的蛋白質聚集中起到關鍵性作用;海藻糖是一種有效的神經(jīng)細胞保護劑,可以抑制缺血性神經(jīng)元死亡。
[Abstract]:Study on the mechanism of trehalose inhibiting protein aggregation induced by transient ischemia: To explore the effect of trehalose on protein aggregation induced by ischemic brain damage and explore its mechanism. Methods: establishing rat transient global cerebral ischemia model and SH-SY5Y oxygen deprivation model. LDH release assay and trypan blue assay, intracellular Active oxygen assay, Morris water maze behavior assessment, hematoxylin and eosin staining and histological examination, immunohistochemical analysis, proteasome activity assay, Western blot analysis and imprinting density analysis to explore the effect and mechanism of trehalose on protein aggregation induced by ischemic damage. Results: (1) observation of hematoxylin eosin staining Compared with the sham operation group, the hippocampal CA1 area neurons in the ischemic group were stained with cytoplasm and polygonal after 72 hours of reperfusion after ischemia, and the preconditioning with 3% concentration of Trehalose before 48 hours preconditioning could significantly inhibit the morphological changes of the hippocampal neurons in the hippocampal CA1 region. Before ischemia, 3 were used. After the concentration of trehalose preconditioning, the percentage of surviving neurons at 72 hours after ischemia / reperfusion increased from 5.52 + 2.31% to 62.56 + 8.17%. in the water maze experiment, which showed that trehalose preconditioning could make ischemic rats search the hidden platform in water more appropriately and shorten the hidden platform in the water. Incubation period, reducing the swimming track in QII; compared with the sham group, the retention time of the rats in the ischemic group was shorter in the QII, but the application of trehalose preconditioning significantly prolonged the residence time of the rats in the target quadrant. (2) Western blot analysis showed that in the 3% trehalose + ischemia group and 24 hours of reperfusion, the ubiquitin labeled protein aggregates The increase in the immuno histochemical staining showed that the ubiquitin labeled protein aggregates in the hippocampal CA1 region neurons pretreated by 3% concentration of trehalose were significantly inhibited at 24 hours of reperfusion. Compared with the ischemic group, 3% concentration of trehalose was pretreated at 12 hours, 24 hours and 48 hours after ischemia / reperfusion. The activity of proteasome in the hippocampal CA1 region was obviously reversed, and its activity increased from 47.56% + 4.89% to 71.88% + 7.12% respectively. 53.25% + 4.21% increased to 73.35% + 6.96% and 46.88% + 3.79% to 75.28% + 7.86%. (3). L) could significantly inhibit the SH-SY5Y cell death induced by oxygen glucose deprivation. Western blot analysis showed that the SH-SY5Y sample pretreated by 5.0mmol/l concentration of trehalose at 2 hours reversed the lc3ii of oxygen deprivation induced, the increase of beclin-1 level and the decrease of autophagic p62 selective substrate. We found the use of autophagy inhibitor 3M. When a was pretreated with 2mmol/l concentration for 1 hours, it not only inhibited the changes in lc3ii, beclin-1 and p62 protein levels induced by oxygen glucose deprivation, but also reduced the cell death at the time of reoxygenation 6. (4) trehalose preconditioning in the concentration of 5.0mmol/l could significantly inhibit the decline of the activity of the white enzyme body induced by MG-132, the release of lactate dehydrogenase and the general release of lactate dehydrogenase. The formation of protein tagged protein aggregates. (5) images under the fluorescence microscope show that 0.5mmol/l or 5.0mmol/l trehalose preconditioning can significantly inhibit the increase of green fluorescence in human SH-SY5Y cells when oxygen glucose deprivation is 24 hours and reoxygenation for 2 hours. The statistical analysis of fluorescence density shows the trehalose concentration of 0.5mmol/l and 5.0mmol/l. The increase in intracellular oxidative stress caused by oxygen glucose deprivation can be effectively suppressed. (6) Western blot analysis and imprinting density analysis showed that the expression of oxygen glucose deprivation induced PERK, phosphorylated PERK, phosphorylated eIF2 a, IRE-1 and GRP78 was up-regulated in the samples of human SH-Y5Y cells pretreated by 5.0mmol/L concentration of trehalose at 2 hours of reoxygenation. This means that trehalose preconditioning inhibits endoplasmic reticulum stress induced by oxygen glucose deprivation in human SH-SY5Y cell samples. Conclusion: in this study, we have demonstrated that trehalose protects the activity of proteasome by inhibiting oxidative stress and endoplasmic reticulum stress to achieve protein aggregation caused by ischemic damage. The proteasome plays a key role in regulating the aggregation of proteins caused by ischemic injury; trehalose is an effective neuroprotective agent that inhibits the death of ischemic neurons.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R743.31

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