本文關(guān)鍵詞：氧化應(yīng)激中calpain對GSK-3β的片段化在其活性調(diào)節(jié)中的作用及機(jī)制　出處：《華中科技大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

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【摘要】：研究背景：糖原合成酶激酶-3β（GSK-3β）是一種多功能的絲蘇氨酸蛋白激酶，其功能失調(diào)可導(dǎo)致多種疾病例如精神疾病、代謝系統(tǒng)疾病以及神經(jīng)系統(tǒng)退行性疾病的發(fā)生發(fā)展，而在此過程中，氧化應(yīng)激也發(fā)揮重要作用。多條細(xì)胞信號通路參與調(diào)節(jié)GSK-3β的活性，酪氨酸216位點的磷酸化可以增加GSK-3β的活性，而其絲氨酸9位點的磷酸化則降低GSK-3β的活性，另外，胰島素途徑、wnt信號通路以及與GSK-3結(jié)合蛋白之間相互作用等都可調(diào)控GSK-3β的活性。我們先前的實驗結(jié)果已報道過在氧化應(yīng)激的情況下，GSK-3β活性增高，但是氧化應(yīng)激是如何參與調(diào)節(jié)GSK-3β激酶活性的機(jī)制不明。已有研究報道，calpain對GSK-3βN端的片段化可導(dǎo)致GSK-3β的抑制結(jié)構(gòu)域喪失，并將GSK-3β切割為兩個具有激酶活性的片段，即calpain的激活可截斷GSK-3β并使其活性升高。 目的：探討在HEK293/Tau441細(xì)胞系中，過氧化氫（H2O2）如何影響GSK-3β的活性及其潛在機(jī)制，進(jìn)一步研究氧化應(yīng)激中calpain對GSK-3β的片段化在其活性調(diào)節(jié)中的作用及機(jī)制。 方法：細(xì)胞培養(yǎng)，藥物處理后，用CCK-8試劑盒測定細(xì)胞活力，同時測定SOD和MDA含量，評估氧化應(yīng)激模型是否成功建立，檢測GSK-3β活性，并運用westernblot技術(shù)檢測GSK-3β不同位點磷酸化程度以及可能參與磷酸化的相關(guān)激酶活性形式的表達(dá)；用I×71共聚焦顯微鏡檢測藥物處理后不同時間點熒光探針Fura-2-AM標(biāo)記的HEK293/Tau441細(xì)胞內(nèi)鈣離子濃度變化，并運用蛋白免疫印跡分析GSK-3β的片段化程度；隨后又用上述實驗中所使用的過氧化氫濃度以及calpain的特異性抑制劑Ⅳ同時處理HEK293/Tau441細(xì)胞，探究calpain介導(dǎo)的GSK-3β特異性切割。 結(jié)果：（1）在過氧化氫處理的HEK293/Tau441細(xì)胞中，GSK-3β的活性與空白對照組相比增加了兩倍，然而GSK-3β的絲氨酸9位點的磷酸化程度也明顯升高，即非激活形式的GSK-3β水平升高，同時GSK-3β的酪氨酸216位點的磷酸化程度未出現(xiàn)明顯改變； （2）用過氧化氫處理HEK293/Tau441細(xì)胞1分鐘后，細(xì)胞內(nèi)鈣離子濃度開始上升，3分鐘細(xì)胞內(nèi)鈣離子濃度達(dá)到高峰，而后免疫印跡分析結(jié)果顯示GSK-3β被切割為兩個片段； （3）用過氧化氫和calpain特異性抑制劑Ⅳ同時處理HEK293/Tau441細(xì)胞，發(fā)現(xiàn)GSK-3β的片段化效應(yīng)明顯受到抑制。 結(jié)論：過氧化氫通過增加細(xì)胞鈣離子內(nèi)流激活calpain，引起GSK-3β的片段化，進(jìn)而對抗由過氧化氫磷酸化絲氨酸9位點誘導(dǎo)的GSK-3β酶活性的下降，，從而上調(diào)GSK-3β的活性。
[Abstract]:Background: glycogen synthase kinase -3 beta (GSK-3 beta) is a multifunctional serine / threonine protein kinase, its dysfunction can lead to various diseases such as mental illness, the occurrence and development of metabolic diseases and neurodegenerative diseases, and in this process, oxidative stress also plays an important role in several cellular signaling pathways. Participate in the regulation of GSK-3 beta activity, tyrosine 216 phosphorylation can increase GSK-3 beta activity, while its serine 9 phosphorylation decreased GSK-3 beta activity, in addition, the insulin pathway, Wnt pathway and GSK-3 binding protein interaction can regulate GSK-3 beta activity. Our previous experiments the results have been reported in situations of oxidative stress, increased GSK-3 activity, but the oxidative stress is how to participate in the mechanisms regulating GSK-3 beta kinase activity is unknown. It has been reported that the calpain of GSK Fragmentation of -3 beta N ends can lead to loss of domain inhibition of GSK-3 beta and cut GSK-3 beta into two fragments with kinase activity, that is, activation of calpain can truncate GSK-3 beta and increase its activity.
Objective: To investigate the effect of hydrogen peroxide (H2O2) on the activity and potential mechanism of GSK-3 beta in HEK293/Tau441 cell line, and further study the role and mechanism of calpain in the regulation of GSK-3 beta activity in oxidative stress.
Methods: cell culture, drug treatment, cell viability was determined by CCK-8 kit, the simultaneous determination of SOD and MDA content, the evaluation of oxidative stress models, detection of GSK-3 beta activity, and using Westernblot technology to detect GSK-3 beta sites phosphorylation and expression of related kinase activity forms may be involved in the phosphorylation of the use; I * 71 confocal microscopy to detect drug treatment at different time points after Fura-2-AM labeled HEK293/Tau441 changes in intracellular calcium ion concentration, the degree of fragmentation and use the Western blot analysis of GSK-3 beta; then by using the experiment of hydrogen peroxide concentration and specific inhibitor of calpain IV and HEK293/Tau441 cells, explore calpain GSK-3 mediated by beta specific cutting.
Results: (1) in H2O2 treated HEK293/Tau441 cells, GSK-3 beta activity increased two times compared with the control group, but GSK-3 beta serine 9 phosphorylation level was significantly increased, namely increased expression and activation of GSK-3 beta level, while GSK-3 beta tyrosine 216 phosphorylation there is no obvious change degree;
(2) after 1 minutes of treatment with hydrogen peroxide, the intracellular calcium concentration began to increase, and the intracellular calcium concentration reached its peak in 3 minutes. Western blotting analysis showed that GSK-3 HEK293/Tau441 was cut into two fragments.
(3) HEK293/Tau441 cells were treated with hydrogen peroxide and calpain specific inhibitor IV, and the fragmentation effect of GSK-3 beta was obviously inhibited.
Conclusion: hydrogen peroxide increases GSK-3 beta fragmentation by increasing intracellular calcium influx and activating calpain, thereby antagonized the decrease of GSK-3 beta activity induced by hydrogen peroxide phosphorylation serine 9 site, thereby increasing the activity of GSK-3 beta.

【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R363

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

1 陳太忠;張艷;熊_";徐發(fā)良;龔建平;余正;;脂多糖預(yù)處理對GSK-3功能活性的影響[J];第三軍醫(yī)大學(xué)學(xué)報;2012年13期

2 范鳴s

本文編號：1396640