本文選題：腦出血 + 自噬�。� 參考：《蘇州大學(xué)》2014年碩士論文

【摘要】：目的：自噬是一個(gè)涉及到細(xì)胞自身結(jié)構(gòu)通過溶酶體機(jī)制而被降解的過程，其中包括細(xì)胞器、衰老及錯(cuò)誤折疊或破損的蛋白質(zhì)等。在不同的狀況下，自噬可以引起細(xì)胞的死亡，也可以促進(jìn)細(xì)胞生存。研究表明，，自噬在腦出血（intracerebralhemorrhage, ICH）后被激活。造成腦出血后繼發(fā)的神經(jīng)功能缺損的主要原因包括腦出血后腦血腫周邊腦水腫的形成和繼發(fā)的神經(jīng)細(xì)胞死亡。本研究中，我們分別應(yīng)用了自噬的抑制劑3-methyladenine（3-MA）和誘導(dǎo)劑雷帕霉素（rapamycin，RAP）來調(diào)控自噬的水平，探索不同自噬水平對(duì)小鼠腦出血后腦損傷的作用，并通過對(duì)自噬和凋亡相關(guān)蛋白的監(jiān)測(cè)來探討自噬與凋亡的關(guān)系，以期為腦出血的治療提供新的方向。 方法：以ICR小鼠為實(shí)驗(yàn)動(dòng)物，采用在紋狀體部位注射膠原酶IV的方法建立ICH模型。在ICH前15min，小鼠側(cè)腦室給予自噬的抑制劑3-MA或激活劑雷帕霉素（RAP），并以等量的配制溶劑（DMSO與生理鹽水1:1混溶）注入側(cè)腦室作為對(duì)照組；于ICH后1d-7d，應(yīng)用行為學(xué)試驗(yàn)（Motor Test）評(píng)估ICH后各組試驗(yàn)動(dòng)物的運(yùn)動(dòng)功能；取ICH后24h和72h作為檢測(cè)時(shí)間點(diǎn)處死小鼠取全腦，用干-濕重法檢測(cè)腦出血側(cè)及對(duì)側(cè)紋狀體和腦皮質(zhì)以及小腦的含水量；取ICH后24h和72h作為檢測(cè)時(shí)間點(diǎn)，于處死前1h腹腔注射碘化丙啶（propidium iodide, PI）后取全腦，采用體視學(xué)顯微鏡觀察小鼠大腦出血中心區(qū)及周邊區(qū)PI陽性細(xì)胞數(shù)，研究不同的自噬激活水平對(duì)ICH引起的神經(jīng)細(xì)胞死亡的影響。為了進(jìn)一步探索不同自噬水平對(duì)ICH后腦損傷的作用機(jī)制，本研究中同樣取ICH后24h和72h作為檢測(cè)時(shí)間點(diǎn)處死小鼠取全腦，運(yùn)用免疫印跡法分別檢測(cè)ICH后24h和72h的自噬活性相關(guān)蛋白（Beclin-1，LC3-II，p62）和凋亡相關(guān)蛋白（Bcl-2，Bax，cleavedcaspase-3）的表達(dá)水平。 結(jié)果：（1）3-MA預(yù)處理后，Beclin-1及LC3-II的蛋白表達(dá)水平均明顯降低，p62蛋白水平增加；3-MA預(yù)處理組腦出血后的細(xì)胞死亡（PI陽性細(xì)胞數(shù)）顯著減少，腦水腫減輕，運(yùn)動(dòng)功能的損害也得到明顯恢復(fù)。相反，雷帕霉素預(yù)處理上調(diào)了Beclin-1和LC3-II的表達(dá)，降低了p62的蛋白水平，但增加了腦出血后的細(xì)胞死亡數(shù)量、加重了腦水腫及運(yùn)動(dòng)功能損害程度。（2）3-MA預(yù)處理顯著降低了ICH引起的Bax和cleaved caspase-3的蛋白表達(dá)的上調(diào)，但逆轉(zhuǎn)了ICH后Bcl-2的下調(diào)；相反，雷帕霉素預(yù)處理后，Bax和cleaved caspase-3的蛋白表達(dá)增加，同時(shí)進(jìn)一步降低了Bcl-2的表達(dá)。 結(jié)論：（1）ICH可引起自噬激活增強(qiáng)，3-MA預(yù)處理降低了ICH后的自噬表達(dá)水平，RAP預(yù)處理進(jìn)一步升高了ICH后的自噬表達(dá)水平；（2）抑制自噬減輕了ICH引起的腦損傷，而上調(diào)自噬加重了ICH后的腦損傷程度；（3）在小鼠ICH模型中，自噬可以通過激活細(xì)胞凋亡通路調(diào)節(jié)ICH引起的神經(jīng)細(xì)胞損傷。
[Abstract]:Objective: autophagy is a process involving the degradation of cell structures through lysosomal mechanisms, including organelles, senescence, and misfolded or damaged proteins. Autophagy can cause cell death and promote cell survival in different conditions. Studies have shown that autophagy is activated after intracerebral hemorrhage (ICH). The main causes of the secondary neurological deficit after ICH include the formation of cerebral edema around the intracerebral hematoma and the secondary nerve cell death. In this study, we used 3-methyladenine (3-MA), an inhibitor of autophagy, and rapamycin rap, an inducer, to regulate the level of autophagy and to explore the effects of different levels of autophagy on brain injury after intracerebral hemorrhage in mice. The relationship between autophagy and apoptosis was studied by monitoring autophagy and apoptosis-related proteins in order to provide a new direction for the treatment of intracerebral hemorrhage. Methods: ICH model was established by injecting collagenase IV into striatum of ICR mice. At 15 min before ICH, mice lateral ventricle were given autophagy inhibitor 3-MA or activator rapamycin (rap), and the same amount of preparation solvent (DMSO and normal saline 1:1 mixture) was injected into the lateral ventricle as control group. The motor test was used to evaluate the motor function of the experimental animals after ICH at 1 d ~ 7 d after ICH, and the whole brain was killed at 24 h and 72 h after ICH. The water content in the striatum, cortex and cerebellum of cerebral hemorrhage side and contralateral side were measured by dry wet weight method, 24 and 72 hours after ICH were taken as time points, and the whole brain was taken after intraperitoneal injection of (propidium iodide, Pi 1 hour before death. The number of Pi positive cells in central and peripheral areas of cerebral hemorrhage in mice was observed by stereological microscope to study the effect of different levels of autophagy activation on the neuronal death induced by ICH. In order to further explore the mechanism of different autophagy levels on brain injury after ICH, the mice were killed at 24 and 72 hours after ICH. The expression of autophagy active-associated protein (Beclin-1) LC3-IIP62 and apoptosis-related protein (Bcl-2AXA cleavedcaspase-3) at 24 and 72 hours after ICH were detected by Western blotting. Results: (1) the protein expression levels of Beclin-1 and LC3-II were significantly decreased after 3-MA preconditioning. The impairment of motor function was also significantly recovered. In contrast, rapamycin pretreatment up-regulated the expression of Beclin-1 and LC3-II, reduced the protein level of p62, but increased the number of cell death after intracerebral hemorrhage. (2) 3-MA pretreatment significantly decreased the up-regulated expression of Bax and cleaved caspase-3 protein, but reversed the down-regulation of Bcl-2, on the contrary, the protein expression of Bax and cleaved caspase-3 increased after rapamycin preconditioning. At the same time, the expression of Bcl-2 was further decreased. Conclusion: (1) ICH can induce autophagy enhancement and 3- MA pretreatment can decrease the level of autophagy expression after ICH. Rap pretreatment can further increase the level of autophagy expression after ICH, (2) inhibition of autophagy reduces the brain injury induced by ICH. In mouse ICH model autophagy can regulate the neuronal damage induced by ICH by activating apoptosis pathway.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743.34

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