本文關(guān)鍵詞： 腦缺血再灌注 能量代謝障礙 氧化應(yīng)激 內(nèi)質(zhì)網(wǎng)應(yīng)激 自噬 囊泡融合 p62 NSF　出處：《吉林大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：能量代謝障礙是腦缺血再灌注損傷的重要原因。腦缺血再灌注時(shí)線粒體氧化磷酸化障礙在導(dǎo)致ATP生成減少的同時(shí)，還產(chǎn)生大量的活性氧(reactive oxygenspecies，ROS)，引發(fā)氧化應(yīng)激。腦缺血再灌注時(shí)，除了ATP含量減少、自由基生成增加等引起細(xì)胞損傷之外，一些適應(yīng)性調(diào)控機(jī)制在腦缺血再灌注過(guò)程中的作用也引起了人們的關(guān)注。 內(nèi)質(zhì)網(wǎng)應(yīng)激與自噬是細(xì)胞對(duì)損傷刺激的適應(yīng)性反應(yīng)。內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬可以維持細(xì)胞內(nèi)環(huán)境的穩(wěn)態(tài)平衡，具有一定的保護(hù)性作用。內(nèi)質(zhì)網(wǎng)應(yīng)激時(shí)未折疊蛋白反應(yīng)可以激活自噬，自噬又可以通過(guò)降解錯(cuò)誤折疊或未折疊蛋白減輕內(nèi)質(zhì)網(wǎng)的負(fù)荷，抑制內(nèi)質(zhì)網(wǎng)應(yīng)激的過(guò)度激活，同時(shí)產(chǎn)生的降解產(chǎn)物也為機(jī)體細(xì)胞新蛋白質(zhì)的合成、細(xì)胞結(jié)構(gòu)的重建以及ATP的生成提供原料。但過(guò)度激活的內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬又能夠加重細(xì)胞損傷，甚至導(dǎo)致細(xì)胞死亡。最近的研究結(jié)果提示，內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬在腦缺血再灌注引起的能量代謝障礙和氧化應(yīng)激過(guò)程發(fā)揮重要的作用，但其作用機(jī)制尚不十分明了。 Keap1-Nrf2-ARE信號(hào)轉(zhuǎn)導(dǎo)途徑是機(jī)體氧化應(yīng)激條件下主要的防御機(jī)制。自噬相關(guān)蛋白p62作為一種多功能蛋白，不僅僅具有清除損傷的細(xì)胞器、降解異常聚集的蛋白等作用，而且，具有多種蛋白相互作用區(qū)的p62在細(xì)胞應(yīng)激、細(xì)胞生存等多種信號(hào)轉(zhuǎn)導(dǎo)途徑中發(fā)揮銜接分子的作用。已有研究表明在氧化應(yīng)激條件下，p62可與Nrf2之間形成一個(gè)正反饋環(huán)路，通過(guò)Keap1-Nrf2-ARE信號(hào)途徑，促進(jìn)抗氧化基因的表達(dá)。因此，探討多功能蛋白p62在腦缺血再灌注損傷過(guò)程中的作用，可能進(jìn)一步闡明細(xì)胞自噬與氧化應(yīng)激之間的調(diào)控機(jī)制。 自噬晚期自噬體和溶酶體的融合是一個(gè)典型的囊泡融合事件。NSF，作為一種ATP酶，通過(guò)利用自身水解ATP產(chǎn)生的能量，介導(dǎo)囊泡融合完成后SNARE復(fù)合物的解離，釋放SNARE進(jìn)入下一個(gè)囊泡融合進(jìn)程，是囊泡融合順利進(jìn)行必需的一個(gè)關(guān)鍵蛋白。體外研究表明，只有細(xì)胞質(zhì)內(nèi)可溶性的NSF在ATP存在條件下才能介導(dǎo)囊泡的融合，如果可溶性的NSF發(fā)生聚集或ATP缺失將會(huì)喪失該功能�？梢姡珹TP產(chǎn)生與囊泡融合關(guān)鍵蛋白NSF的功能存在著關(guān)聯(lián)，通過(guò)復(fù)制體外細(xì)胞實(shí)驗(yàn)性腦缺血再灌注模型，觀察能量代謝障礙時(shí)囊泡融合關(guān)鍵蛋白NSF的變化，進(jìn)而探討腦缺血損傷過(guò)程中能量代謝障礙與細(xì)胞自噬之間可能的調(diào)控機(jī)制。 目的： 本研究基于能量代謝障礙/氧化應(yīng)激在引起細(xì)胞損傷的同時(shí)，可能會(huì)激活細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬等適應(yīng)性反應(yīng)，探討內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬在細(xì)胞能量代謝障礙與氧化應(yīng)激中的作用，為闡明腦缺血再灌注損傷的機(jī)制提供新的線索。 方法： （1）體內(nèi)實(shí)驗(yàn)： 1）線栓法阻塞大腦中動(dòng)脈復(fù)制大鼠局灶性腦缺血再灌注模型。 2）TTC染色和HE染色判斷腦缺血再灌注后腦皮質(zhì)損傷情況。 3）免疫組織化學(xué)染色檢測(cè)Keap1和Nrf2的表達(dá)；免疫印跡檢測(cè)內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白、自噬相關(guān)蛋白和凋亡相關(guān)蛋白的表達(dá)、RT-PCR法檢測(cè)Keap1-Nrf2-ARE下游基因的表達(dá)。 （2）體外實(shí)驗(yàn)： 1）建立體外培養(yǎng)PC12細(xì)胞ATP缺失再恢復(fù)模型；MTT法檢測(cè)細(xì)胞生存率的變化；Western Blot檢測(cè)自噬和內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白的表達(dá)。 2）建立體外培養(yǎng)CHO細(xì)胞ATP缺失再恢復(fù)模型；LDH法檢測(cè)細(xì)胞的損傷改變；差速離心和線性甘油梯度離心獲取不同蛋白組分；Western Blot檢測(cè)不同組分NSF蛋白表達(dá)。 結(jié)果： （1）體內(nèi)實(shí)驗(yàn)： 1）神經(jīng)缺陷評(píng)分、TTC及HE染色結(jié)果表明，隨著缺血時(shí)間的延長(zhǎng)，大鼠腦缺血再灌注引發(fā)的大腦皮質(zhì)損傷逐漸加重。 2）缺血1H再灌注24h，，大腦皮質(zhì)泛素化蛋白表達(dá)增加，Grp78表達(dá)升高，Bcl-2的表達(dá)升高，Bax的表達(dá)下降；隨著缺血時(shí)間的延長(zhǎng)，至缺血3H再灌注24h，大腦皮質(zhì)泛素化蛋白表達(dá)明顯增加， CHOP/GADD153表達(dá)顯著升高，Bax表達(dá)升高，Bcl-2表達(dá)下降。 3）缺血1H再灌注24h及缺血1.5H再灌注24h時(shí)，自噬共軛蛋白Atg12-Atg5和LC3-PE表達(dá)增加；在缺血3H再灌注24h，自噬共軛蛋白Atg12-Atg5和LC3-PE表達(dá)顯著下降。 4）腦缺血1H再灌注24h時(shí)，Nrf2核定位增加，上調(diào)其下游抗氧化基因NQO1、GCLM和HO1mRNA的表達(dá)。隨著缺血時(shí)間的延長(zhǎng)，缺血3H再灌注24h時(shí)，Nrf2核定位減少，激活下游抗氧化基因NQO1和HO1的能力下降。 5）隨著缺血時(shí)間的延長(zhǎng)，p62mRNA表達(dá)水平先增加后降低，而p62蛋白水平僅僅在缺血3H再灌注24h顯著增加。 （2）體外實(shí)驗(yàn)： 1）無(wú)糖條件下輕度ATP缺失再恢復(fù)，PC12細(xì)胞生存率略有升高，此時(shí)LC3-II和p62蛋白變化不明顯；有糖條件下輕度ATP缺失再恢復(fù)，PC12細(xì)胞生存率相對(duì)于重度ATP缺失再恢復(fù)明顯升高，此時(shí)Grp78蛋白表達(dá)顯著升高，p62蛋白表達(dá)顯著下降；重度ATP缺失再恢復(fù)，PC12細(xì)胞生存率明顯下降，此時(shí)LC3-II和p62蛋白表達(dá)顯著升高；3-MA抑制自噬降低PC12細(xì)胞在ATP缺失條件下的生存率。 2）ATP缺失引起CHO細(xì)胞細(xì)胞質(zhì)內(nèi)可溶性NSF蛋白聚集和細(xì)胞損傷。NSF高表達(dá)可減輕ATP缺失再恢復(fù)誘導(dǎo)的CHO細(xì)胞損傷。 結(jié)論： 1.內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬途徑參與調(diào)控腦缺血再灌注損傷。短時(shí)間腦缺血后再灌注，內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬的激活起一定的保護(hù)性作用；長(zhǎng)時(shí)間腦缺血后再灌注，內(nèi)質(zhì)網(wǎng)應(yīng)激反應(yīng)過(guò)度激活，同時(shí)機(jī)體自噬的降解能力下降，加重腦損傷。 2.腦缺血再灌注時(shí)，自噬通過(guò)p62與Keap1-Nrf2-ARE信號(hào)途徑相關(guān)聯(lián)，共同作用減輕氧化應(yīng)激、抑制內(nèi)質(zhì)網(wǎng)應(yīng)激的過(guò)度激活。 3.體外實(shí)驗(yàn)抑制自噬能夠加重細(xì)胞損傷，提示內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬途徑在體外培養(yǎng)細(xì)胞能量代謝障礙過(guò)程中具有一定的細(xì)胞保護(hù)作用。 4.體外實(shí)驗(yàn)發(fā)現(xiàn)，能量代謝障礙導(dǎo)致細(xì)胞質(zhì)內(nèi)可溶性的自噬相關(guān)蛋白NSF聚集失活，喪失介導(dǎo)囊泡融合的功能，進(jìn)而可能抑制自噬降解途徑，引發(fā)細(xì)胞損傷。 綜上，我們認(rèn)為腦缺血導(dǎo)致的腦細(xì)胞損傷與能量代謝密切相關(guān)，而適度的內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬在能量代謝障礙引起的細(xì)胞損傷中具有一定的保護(hù)性作用。表明腦細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激-自噬的相關(guān)研究可能為腦缺血再灌注損傷的預(yù)防與治療提供了新的線索。
[Abstract]:Energy metabolism is an important cause of cerebral ischemia reperfusion injury. Cerebral ischemia reperfusion mitochondrial oxidative phosphorylation disorders resulting in decreased ATP generation at the same time, also produce large amounts of reactive oxygen species (reactive OXYGENSPECIES, ROS), caused by oxidative stress. Cerebral ischemia reperfusion, but reduced ATP content and increased free radical generation caused by cell damage, some adaptive mechanisms of reperfusion in the process of action also attracted the attention of people in cerebral ischemia.
Endoplasmic reticulum stress and autophagy is adaptive reaction to injury stimulation. Endoplasmic reticulum stress autophagy can maintain the homeostasis of intracellular environment, has a protective effect. The endoplasmic reticulum stress when unfolded protein response can activate autophagy, autophagy and misfolding by mistake or degradation of unfolded protein reducing Er load, inhibit excessive activation of endoplasmic reticulum stress, degradation products generated at the same time for the synthesis of new proteins in the body's cells, cell structure reconstruction and ATP production to provide raw materials. But the excessive activation of endoplasmic reticulum stress autophagy and can aggravate the injury of the cells, and even lead to cell death. Recent studies showed that energy metabolism and the process of oxidative stress in the endoplasmic reticulum stress autophagy induced by cerebral ischemia and reperfusion plays an important role, but its mechanism is not very clear.
The Keap1-Nrf2-ARE signal transduction pathway is the primary defense mechanism for oxidative stress. Under the condition of autophagy related protein p62 as a multifunctional protein, not only can eliminate the damage of organelles, protein degradation, abnormal aggregation and interaction with various proteins in the cell area of p62 stress, play a role in cell survival and molecular cohesion many signal transduction pathways. Studies have shown that under oxidative stress conditions, a positive feedback loop between p62 and Nrf2, through the Keap1-Nrf2-ARE signaling pathway, promote antioxidant gene expression. Therefore, to explore the process of multifunctional protein p62 in cerebral ischemia reperfusion injury in the role may further clarify the regulatory mechanism between cells autophagy and oxidative stress.
The late autophagosome and lysosome fusion of autophagy is a typical vesicle fusion event.NSF, as a ATP enzyme, produced by using its ATP hydrolysis energy mediated vesicle fusion dissociation of SNARE complex after the release of SNARE into a vesicle fusion process, vesicle fusion is carried out smoothly a key protein required. In vitro studies showed that only in the cytoplasm of soluble NSF in the presence of ATP conditions can be mediated by fusion of vesicles, if the soluble NSF aggregation and ATP deletion will lose the function. Obviously, ATP fusion protein NSF function key are associated with vesicles by replication in vitro experimental cerebral ischemia reperfusion model, to observe the changes of energy metabolism during vesicle fusion protein NSF, and to explore the regulation of cerebral ischemic injury between energy metabolism and autophagy may. Mechanism.
Objective:
This study is based on the energy metabolism disorder / oxidative stress on cell damage caused by at the same time, may activate adaptive response to endoplasmic reticulum stress - autophagy, to investigate the role of endoplasmic reticulum stress and autophagy in cell energy metabolism and oxidative stress, and provides new clues for elucidating the mechanism of cerebral ischemia reperfusion injury.
Method錛

本文編號(hào)：1516858