本文選題：腦保護(hù) + 高壓氧預(yù)處理��； 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：背景：目前腦血管疾病已成為成年人死亡致殘的第二大原因，但其治療方法還很有限且療效不盡人意。我們前期實(shí)驗(yàn)已證實(shí)，高壓氧（hyperbaric oxygen，HBO）預(yù)處理可顯著縮小實(shí)驗(yàn)動(dòng)物缺血再灌注損傷后梗死容積，改善神經(jīng)功能表現(xiàn)[1,2]。在對(duì)HBO預(yù)處理的機(jī)制研究中我們證實(shí)，HBO預(yù)處理可通過(guò)上調(diào)腦缺血再灌注損傷后大鼠腦缺血半暗帶組織內(nèi)自噬水平，，減少細(xì)胞凋亡壞死，發(fā)揮腦保護(hù)作用[3]。但HBO預(yù)處理是如何促進(jìn)自噬的機(jī)制仍然不清楚，對(duì)HBO預(yù)處理腦保護(hù)機(jī)制的深入研究有助于探索新的臨床干預(yù)靶點(diǎn)。 胱抑素C（cystatin C）作為一種半胱氨酸蛋白酶抑制劑，可作用于溶酶體系統(tǒng)，調(diào)節(jié)多種蛋白水解酶的活性。在氧化應(yīng)激狀態(tài)下，Cystatin C可顯著地促進(jìn)細(xì)胞存活：強(qiáng)烈抑制組織蛋白酶活性，保護(hù)細(xì)胞內(nèi)膜結(jié)構(gòu)的穩(wěn)定性，并通過(guò)調(diào)節(jié)分子信號(hào)通路促進(jìn)自噬，延長(zhǎng)細(xì)胞壽命。有研究表明外源性補(bǔ)充Cystatin C可提高神經(jīng)細(xì)胞對(duì)氧糖剝奪損傷的耐受性[4,85]。我科通過(guò)同位素標(biāo)記相對(duì)與絕對(duì)定量（i-TRAQ）蛋白篩選技術(shù)發(fā)現(xiàn)HBO預(yù)處理后血清及腦組織中Cystatin C表達(dá)均明顯提高。但Cystatin C的表達(dá)增加是否與HBO預(yù)處理腦保護(hù)作用有關(guān)，尚不清楚。 基于上述研究我們推測(cè)，HBO預(yù)處理通過(guò)促進(jìn)少量氧自由基產(chǎn)生，上調(diào)CystatinC的表達(dá)，作用于自噬關(guān)鍵調(diào)節(jié)蛋白mTOR，促進(jìn)自噬信號(hào)通路，從而發(fā)揮腦保護(hù)效應(yīng)。本實(shí)驗(yàn)運(yùn)用SD大鼠大腦中動(dòng)脈阻塞（MCAO）及原代神經(jīng)元細(xì)胞氧糖剝奪（OGD）模型，利用siRNA干擾及外源性補(bǔ)充方法調(diào)節(jié)Cystatin C蛋白水平，對(duì)HBO預(yù)處理的腦保護(hù)機(jī)制進(jìn)行了深入探索。 實(shí)驗(yàn)一Cystatin C在HBO預(yù)處理誘導(dǎo)大鼠腦缺血耐受中的作用研究 目的：探討Cystatin C在HBO預(yù)處理腦保護(hù)中的作用及可能機(jī)制方法：1)觀察HBO預(yù)處理后腦缺血半暗帶區(qū)Cystatin C的表達(dá)。成年雄性SD大鼠，HBO預(yù)處理（2.5ATA，100%O2，1h/d×5d）后24h，行右側(cè)大腦中動(dòng)脈阻閉，缺血維持2h后給予再通。檢測(cè)缺血前、再灌注后2、4、6、12、24h共6個(gè)時(shí)間點(diǎn)CystatinC蛋白表達(dá)。2) Cystatin C對(duì)HBO預(yù)處理腦保護(hù)作用的影響。應(yīng)用側(cè)腦室注射技術(shù)，通過(guò)補(bǔ)充外源性Cystatin C蛋白及siRNA干擾方法上調(diào)及下調(diào)Cystatin C，檢測(cè)再灌注后24h后大鼠神經(jīng)功能及腦梗死容積。3)觀察HBO預(yù)處理后腦缺血半暗帶區(qū)mTOR磷酸化及自噬水平。檢測(cè)缺血前、再灌注后2、4、6、12、24h共6個(gè)時(shí)間點(diǎn)p-mTOR、mTOR及自噬相關(guān)蛋白LC3II/I、Beclin-1蛋白表達(dá)。免疫熒光雙標(biāo)定位。 結(jié)果：HBO預(yù)處理可有效減小大鼠腦缺血再灌注損傷后腦梗死容積，改善神經(jīng)功能狀況。再灌注后4h開始Cystatin C蛋白表達(dá)增加，至少可持續(xù)至24h；缺血再灌注損傷后mTOR磷酸化升高，HBO預(yù)處理可顯著下調(diào)mTOR磷酸化，自再灌注后4h開始至少可持續(xù)至24h；再灌注后6h開始自噬相關(guān)蛋白LC3II及Beclin-1表達(dá)增加，至少可持續(xù)至24h，mTOR磷酸化改變主要體現(xiàn)在神經(jīng)元細(xì)胞質(zhì)中，Beclin-1主要在腦缺血半暗帶區(qū)神經(jīng)元核中表達(dá)。Cystatin C-siRNA抑制HBO預(yù)處理誘導(dǎo)的CystatinC的增加，減弱HBO預(yù)處理的腦保護(hù)作用。外源性補(bǔ)充Cystatin C可模擬HBO預(yù)處理發(fā)揮腦保護(hù)作用。 結(jié)論：HBO預(yù)處理通過(guò)上調(diào)Cystatin C而誘導(dǎo)大鼠腦缺血耐受，且HBO預(yù)處理可抑制再灌注損傷后mTOR磷酸化，促進(jìn)自噬。 實(shí)驗(yàn)二Cystatin C在HBO預(yù)處理誘導(dǎo)原代神經(jīng)元氧糖剝奪耐受中的作用及機(jī)制研究 目的：觀察Cystatin C是否參與HBO預(yù)處理對(duì)原代神經(jīng)元的保護(hù)作用，并探討HBO預(yù)處理是否是通過(guò)Cystatin C調(diào)節(jié)mTOR及自噬而發(fā)揮作用。 方法：1) HBO預(yù)處理對(duì)原代神經(jīng)元氧糖剝奪損傷后Cystatin C表達(dá)的影響。孕16-18周SD大鼠，取胚胎鼠腦皮層進(jìn)行原代神經(jīng)元培養(yǎng)。第6天行HBO預(yù)處理（3.5ATA，90%O2，3h），24h后進(jìn)行氧糖剝奪（OGD），維持2h后給予復(fù)氧復(fù)糖。復(fù)氧后24h行MTT（檢測(cè)細(xì)胞存活率）、LDH漏出率（檢測(cè)細(xì)胞存活狀態(tài)）檢測(cè)及Western Blot（檢測(cè)Cystatin C蛋白的表達(dá)）。2) Cystatin C對(duì)HBO預(yù)處理腦保護(hù)作用的影響。向細(xì)胞培養(yǎng)液中外源性補(bǔ)充Cystatin C及siRNA干擾來(lái)上調(diào)及下調(diào)CystatinC，RT-PCR（檢測(cè)Cystatin C-mRNA，分別于干擾后1、2、3d進(jìn)行）及Western Blot(檢測(cè)Cystatin C蛋白，干擾后24h進(jìn)行)檢測(cè)不同時(shí)間點(diǎn)干預(yù)措施效率，復(fù)氧24h后檢測(cè)MTT及LDH漏出率。3) Cystatin C在HBO預(yù)處理調(diào)節(jié)原代神經(jīng)元細(xì)胞內(nèi)mTOR磷酸化及自噬水平中的作用。復(fù)氧24h檢測(cè)不同處理組細(xì)胞內(nèi)p-mTOR、mTOR及自噬相關(guān)蛋白LC3II/I、Beclin-1蛋白表達(dá)。 結(jié)果：免疫熒光染色顯示，皮層原代神經(jīng)元純度95%。HBO預(yù)處理可提高原代神經(jīng)元氧糖剝奪/復(fù)氧損傷24h后細(xì)胞存活率、降低細(xì)胞毒性，提高Cystatin C的表達(dá)。外源性補(bǔ)充Cystatin C可有效上調(diào)原代神經(jīng)元細(xì)胞內(nèi)Cystatin C水平，CystatinC-siRNA可顯著抑制Cystatin C表達(dá)，最佳轉(zhuǎn)染濃度為10μM。Cystatin C-siRNA抑制了HBO預(yù)處理誘導(dǎo)的Cystatin C的增加，減弱HBO預(yù)處理的神經(jīng)元保護(hù)作用；外源性補(bǔ)充Cystatin C可模擬HBO預(yù)處理的細(xì)胞保護(hù)作用。上調(diào)原代神經(jīng)元細(xì)胞內(nèi)Cystatin C后，抑制mTOR磷酸化、提高自噬水平（LC3II及Beclin-1表達(dá)增加）；下調(diào)HBO預(yù)處理后Cystatin C表達(dá)，mTOR磷酸化升高，自噬水平降低。 結(jié)論：Cystatin C參與HBO預(yù)處理的神經(jīng)細(xì)胞保護(hù)作用，且HBO預(yù)處理是通過(guò)上調(diào)Cystatin C、抑制mTOR磷酸化、促進(jìn)自噬而減輕體外培養(yǎng)大鼠皮層原代神經(jīng)元的氧糖剝奪/復(fù)氧損傷。 小結(jié)：在體實(shí)驗(yàn)與離體實(shí)驗(yàn)結(jié)果共同提示，Cystatin C在HBO預(yù)處理誘導(dǎo)的缺血耐受中發(fā)揮重要作用；HBO預(yù)處理通過(guò)上調(diào)Cystatin C蛋白表達(dá)并抑制其下游mTOR的磷酸化而促進(jìn)自噬，從而誘導(dǎo)缺血耐受產(chǎn)生。
[Abstract]:Background: at present, cerebrovascular disease has become the second major cause of death and disability in adults, but the treatment method is still limited and the curative effect is not satisfactory. Our previous experiments have proved that hyperbaric oxygen (HBO) preconditioning can significantly reduce the infarct volume after ischemia reperfusion injury in experimental animals and improve the neurological function of [1,2]. in the experimental animals. In the study of the mechanism of HBO preconditioning, we have confirmed that HBO preconditioning can increase the level of autophagy within the ischemic penumbra of the rat brain after the brain ischemic reperfusion injury, reduce apoptosis and necrosis, and play the role of [3]., but the mechanism of promoting autophagy by HBO preconditioning is still unclear. Further research on the brain protection mechanism of HBO preconditioning It is helpful to explore new targets for clinical intervention.
Cystatin C (cystatin C), as a cysteine protease inhibitor, acts in the lysosome system and regulates the activity of a variety of protein hydrolases. Under oxidative stress, Cystatin C can significantly promote cell survival: strongly inhibit cathepsin activity, protect the stability of the microstructure of the fine cell, and regulate the molecular signaling through the regulation of the activity of cystatin. The road promotes autophagy and prolongs cell life. Studies have shown that exogenous supplementation of Cystatin C can increase the tolerance of nerve cells to oxygen glucose deprivation injury. [4,85]. in our family by isotope labeling relative and absolute quantitative (i-TRAQ) protein screening technique, the expression of Cystatin C in serum and brain tissues is significantly increased after HBO pretreatment, but Cystatin C Whether the increase of expression is related to the protective effect of HBO on brain is unclear.
Based on the above study, we speculate that HBO preconditioning can increase the expression of CystatinC by promoting the production of a small amount of oxygen free radicals, acting on the key regulatory protein mTOR of autophagy, promoting autophagic signaling pathway, and thus exerting the effect of brain protection. In this experiment, the middle cerebral artery occlusion (MCAO) and the primary neuron cell oxygen glucose deprivation (OGD) model in SD rats were used in this experiment. SiRNA interference and exogenous supplementation were used to regulate the level of Cystatin C protein, and the mechanism of HBO pretreatment for brain protection was further explored.
Effect of Experiment 1 Cystatin C on ischemic tolerance induced by HBO pretreatment in rats
Objective: To explore the role and possible mechanism of Cystatin C in HBO preconditioning brain protection: 1) to observe the expression of Cystatin C in cerebral ischemic penumbra after HBO preconditioning. Adult male SD rats, HBO preconditioning (2.5ATA, 100%O2,1h/d x 5d) 24h, right middle cerebral artery occlusion and reperfusion after ischemia, and reperfusion before ischemia and reperfusion. The effect of Cystatin C on the protective effect of HBO pretreatment on the brain protection by.2 Cystatin C at 6 time points in a total of 6 time points. The application of lateral ventricle injection technique, up regulation and downregulation of Cystatin C by supplementing the exogenous Cystatin C protein and siRNA interference method, and detecting the neural function and the volume of cerebral infarction after reperfusion after reperfusion The level of mTOR phosphorylation and autophagy in the ischemic penumbra region of the posterior brain. Before and after reperfusion, 2,4,6,12,24h was detected at 6 time points: p-mTOR, mTOR and autophagy related protein LC3II/I, Beclin-1 protein expression, and immunofluorescence double labeling.
Results: HBO pretreatment could effectively reduce the volume of cerebral infarction after cerebral ischemia-reperfusion injury and improve the state of nerve function. After reperfusion, the expression of Cystatin C protein increased at least at 4h, at least to 24h; mTOR phosphorylation was increased after ischemia reperfusion injury, and mTOR phosphorylation could be reduced significantly by HBO preconditioning, and 4H began to begin at least after reperfusion. After reperfusion, the expression of autophagy related protein LC3II and Beclin-1 increased at least after reperfusion, at least to 24h, and the change of mTOR phosphorylation was mainly reflected in the cytoplasm of neurons. Beclin-1 mainly expressed the CystatinC increase of.Cystatin C-siRNA inhibitory HBO preconditioning induced in the neuronal nuclei of the ischemic penumbra region, and weakened HBO preconditioning. Exogenous Cystatin C can simulate the protective effect of HBO on brain protection.
Conclusion: HBO pretreatment can induce cerebral ischemic tolerance in rats by up regulating Cystatin C, and HBO pretreatment can inhibit mTOR phosphorylation and promote autophagy after reperfusion injury.
Experiment two the role and mechanism of Cystatin C in HBO preconditioning induced oxygen glucose deprivation tolerance in primary neurons
Objective: To observe whether Cystatin C participates in the protective effect of HBO preconditioning on primary neurons and the effect of HBO preconditioning on the regulation of mTOR and autophagy through Cystatin C.
Methods: 1) the effect of HBO preconditioning on the expression of Cystatin C after primary neuron oxygen deprivation injury. 16-18 weeks pregnant SD rats were taken to take the embryonic rat cerebral cortex for primary neuron culture. HBO preconditioning (3.5ATA, 90%O2,3h) was performed on sixth days, oxygen glucose deprivation (OGD) after 24h, and 2h after 2h, and 24h line MTT (detection of cell survival) after reoxygenation. LDH leakage rate (detection of cell survival state) detection and Western Blot (detection of the expression of Cystatin C protein).2) the effect of Cystatin C on the brain protection of HBO preconditioning. Estern Blot (detection of Cystatin C protein, 24h after interference) detection of the efficiency of intervention measures at different time points, the detection of MTT and LDH leakage after reoxygenation 24h, Cystatin C is the role of Cystatin C in the regulation of phosphorylation and autophagy in the primary neurons of the HBO preconditioning. White LC3II/I, Beclin-1 protein expression.
Results: immunofluorescence staining showed that the preconditioning of primary cortical neuron purity 95%.HBO could increase the survival rate of 24h cells after oxygen deprivation / reoxygenation injury in primary neurons, reduce cytotoxicity and increase the expression of Cystatin C. Exogenous supplementation of Cystatin C can effectively increase the level of Cystatin C in the cells of primary neurons, and CystatinC-siRNA can be significantly increased. Inhibition of Cystatin C expression, the optimal transfection concentration of 10 u M.Cystatin C-siRNA inhibited the increase of Cystatin C induced by HBO pretreatment and weakened the neuronal protection of HBO preconditioning. Exogenous Cystatin C could simulate the cell protection of HBO pretreatment. Autophagy level (increased expression of LC3II and Beclin-1), down regulated Cystatin C expression after pretreatment of HBO, increased mTOR phosphorylation and decreased autophagy level.
Conclusion: Cystatin C participates in the neuroprotective effect of HBO preconditioning, and HBO preconditioning is by up regulation of Cystatin C, inhibiting the phosphorylation of mTOR, promoting autophagy and alleviating the oxygen deprivation / reoxygenation injury in the primary neurons of the rat cortex in vitro.
Summary: both in vivo and in vitro results suggest that Cystatin C plays an important role in the ischemic tolerance induced by HBO preconditioning; HBO preconditioning promotes autophagy by up regulating the expression of Cystatin C protein and inhibiting the phosphorylation of the downstream mTOR, thus inducing ischemic tolerance.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

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2 趙德強(qiáng);潘速躍;陳建輝;楊文俊;;腦梗死與胱抑素C的關(guān)系[J];南方醫(yī)科大學(xué)學(xué)報(bào);2009年04期

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本文編號(hào)：1796426