本文選題：皮層擴散性抑制預(yù)處理 + 缺血耐受�。� 參考：《吉林大學》2016年博士論文

【摘要】：背景:皮層擴散性抑制(Cortical spreading depression,CSD),基于其與梗死周圍去極化波(peri-infarct depolarization,PID)的相似性,已經(jīng)成為研究缺血半暗帶向梗死中心區(qū)演變機制的良好實驗?zāi)Ｐ�。很多研究都已證實,預(yù)先誘導(dǎo)CSD波可增加腦組織對抗缺血性損傷的耐受性,但是其潛在的病理生理機制仍不清楚。近年來,很多研究都已證明,自噬在腦缺血性損傷及腦對缺血耐受方面起重要作用。自噬在缺血/再灌注損傷中的作用成為腦保護研究領(lǐng)域的熱點,其中AMPK-m TOR-自噬通路研究較為廣泛。然而,其明確的作用在各種研究中結(jié)果不盡相同,且機制尚不明確,仍需要進一步探討。以往關(guān)于CSD預(yù)處理機制方面的研究多集中在能量代謝、一氧化氮、神經(jīng)生長因子、外生基因表達等方面,尚缺乏CSD預(yù)處理和自噬在缺血耐受方面相關(guān)性的研究。本實驗應(yīng)用大鼠大腦中動脈阻塞模型(Middle cerebral artery occlusion,MCAO),探討CSD預(yù)處理的病理生理機制,進一步明確自噬在缺血再灌注損傷中神經(jīng)保護作用機制及AMPK-m TOR-自噬通路在缺血耐受中的作用,為缺血性腦血管病的治療和預(yù)防提供新靶點。目的:通過研究CSD預(yù)處理,進一步探討缺血半暗帶向梗死中心區(qū)演變的機制。同時也首次將CSD預(yù)處理與自噬及其相關(guān)通路、自噬在缺血耐受中的作用聯(lián)系起來,并進一步明確自噬在缺血再灌注損傷中的作用及機制。方法:(1)皮層給1 mol/L氯化鉀溶液濕敷2小時誘導(dǎo)皮層擴散性抑制波(CSD),通過腦電圖監(jiān)測及電生理記錄直流場電位(direct current potential,DC)變化的方法證實CSD波的發(fā)生及其特征,建立CSD預(yù)處理的實驗?zāi)Ｐ?對照組用1 mol/L的氯化鈉溶液濕敷2小時。(2)CSD預(yù)處理與AMPK-m TOR-自噬相關(guān)通路研究。112只雄性Wistar大鼠隨機分組:鹽水對照組;預(yù)處理組(0、3、6、12、24小時)組;3-MA(i.c.v.)+CSD組;溶劑對照(i.c.v.)+CSD組;Compound C(i.p)+CSD組;溶劑對照(i.p)+CSD組。鹽水對照組皮層給予1 mol/L氯化鈉溶液濕敷2小時,12小時后取材;預(yù)處理組皮層給予1 mol/L氯化鉀溶液濕敷2小時,分別在0、3、6、12、24小時后取材;各給藥組在CSD預(yù)處理術(shù)前30 min分別給予自噬抑制劑3-methyladenine(3-MA,200 nmol,i.c.v.)和AMPK抑制劑Compound C(CC,20 mg/kg,i.p)。Western blotting檢測各組皮層自噬標記物LC3-II,Beclin-1,P62水平,AMPK-m TOR通路蛋白p-AMPK(Thr172)/AMPK,p-P70S6K(Thr389)、ULK1水平;免疫熒光檢測LC3-II陽性細胞數(shù)及分布,透射電鏡檢測自噬小體水平。(3)CSD預(yù)處理在缺血再灌注損傷中的神經(jīng)保護作用。84只雄性Wistar大鼠隨機分組:鹽水對照組,CSD預(yù)處理(0、3、6、12、24小時)組,3-MA組,溶劑對照組。鹽水對照組:鹽水預(yù)處理后12小時,行MCAO缺血2小時,再灌注12小時;CSD預(yù)處理組:分別在CSD預(yù)處理后0,3,6,12,24小時后分別行MCAO缺血2小時,再灌注12小時;3-MA組:CSD預(yù)處理前30分鐘給予自噬抑制劑3-MA(200 nmol,i.c.v.),預(yù)處理后12小時后行MCAO缺血2小時,再灌注12小時;溶劑對照組:CSD預(yù)處理前30分鐘給予相同體積的溶劑(i.c.v.),預(yù)處理后12小時后后行MCAO缺血2小時,再灌注12小時。應(yīng)用TTC染色法計算各組梗死體積,Longa法評估神經(jīng)功能缺損,并進行相關(guān)統(tǒng)計學分析。(4)自噬在缺血再灌注損傷中神經(jīng)保護作用的機制初步探討。30只雄性Wistar大鼠隨機分5組:假手術(shù)組(Sham),MCAO組,CSD+MCAO組,3-MA+CSD+MCAO組,溶劑+CSD+MCAO組。通過reverse transcriptase-PCR、Western blotting檢測MCAO缺血再灌注損傷后半暗帶區(qū)皮層凋亡相關(guān)蛋白Bcl-2、Caspase12,內(nèi)質(zhì)網(wǎng)應(yīng)激相關(guān)蛋白GRP78、XBP1,縫隙連接蛋白Connexin 36的m RNA水平及蛋白水平;TUNEL及免疫熒光雙標染色顯示凋亡細胞的比例及細胞類型。結(jié)果:(1)皮層給予1 mol/L氯化鉀溶液可引起誘導(dǎo)側(cè)皮層腦電圖波幅的持續(xù)性下降,由117.25±3.51μV下降到78.36±3.46μV(P0.01),而頻率和波形沒有明顯改變,腦電活動被抑制;電生理學改變?yōu)橹绷鲌鲭娢坏呢摲D(zhuǎn),證明CSD預(yù)處理模型建立成功,而對照組沒有相應(yīng)腦電圖及電生理改變。(2)CSD預(yù)處理對自噬及AMPK-m TOR通路的影響:Western blotting示,CSD可增加大鼠預(yù)處理側(cè)皮層LC3-II,Beclin-1水平及p-AMPK(Thr172)/AMPK比率,減少P62和p-P70S6K(Thr389)水平,這種改變在預(yù)處理后12小時達到高峰;應(yīng)用AMPK抑制劑Compound C(20 mg/kg)可下調(diào)皮層LC3-II,p-AMPK(Thr172)/AMPK比率,上調(diào)P62和p-P70S6K(Thr389)水平。免疫熒光顯示,LC3-II陽性細胞數(shù)在預(yù)處理組明顯高于對照組(P0.01),且主要分布于神經(jīng)元細胞中(87.2%),神經(jīng)膠質(zhì)細胞中較少(2.89%);應(yīng)用3-MA(P0.01)及CC(P0.05)后,LC3-II陽性細胞數(shù)減少。透射電鏡顯示預(yù)處理組皮層自噬小體的數(shù)量明顯高于對照組(P0.05)。(3)CSD預(yù)處理可顯著降低大鼠MCAO后的腦梗死體積,改善功能學評分,且這種保護作用在預(yù)處理后12小時達到高峰(P0.05);應(yīng)用自噬的抑制劑3-MA(200 nmol,i.c.v.)可減少CSD預(yù)處理對梗死體積及神經(jīng)功能評分的影響(P0.05)。(4)CSD預(yù)處理可減少缺血再灌注損傷的半暗帶區(qū)皮層內(nèi)凋亡相關(guān)標記物Caspase12蛋白質(zhì)水平和m RNA水平,增加抗凋亡標記物Bcl-2蛋白和m RNA水平,TUNEL染色顯示CSD預(yù)處理明顯減少半暗帶區(qū)神經(jīng)元細胞凋亡的比例(P0.01);CSD可降低內(nèi)質(zhì)網(wǎng)應(yīng)激標記物GRP78的蛋白質(zhì)水平和m RNA水平,減少XBP-1蛋白水平,增加其m RNA水平。應(yīng)用自噬抑制劑3-MA后可逆轉(zhuǎn)上述改變。CSD預(yù)處理后神經(jīng)元表面主要縫隙連接蛋白Connexin 36的蛋白質(zhì)和m RNA水平無明顯改變(P0.05)。結(jié)論:(1)皮層持續(xù)給予1 mol/L氯化鉀溶液2小時可引起預(yù)處理側(cè)皮層腦電活動的抑制,腦電圖波幅降低,誘導(dǎo)負性直流DC波的產(chǎn)生,成功建立CSD預(yù)處理模型;而給予1 mol/L氯化鈉溶液未引起上述改變。(2)CSD可通過AMPK-m TOR通路誘導(dǎo)自噬激活,且激活的自噬主要分布在神經(jīng)元細胞中,膠質(zhì)細胞中少見。(3)CSD預(yù)處理具有明確的神經(jīng)保護作用,減少梗死體積,改善神經(jīng)功能評分,且在預(yù)處理后12小時達高峰。(4)CSD預(yù)處理誘導(dǎo)的自噬可減少大鼠缺血再灌注損傷半暗帶區(qū)皮層的神經(jīng)元凋亡,抑制內(nèi)質(zhì)網(wǎng)應(yīng)激的過度激活,從而產(chǎn)生神經(jīng)保護作用,而對神經(jīng)元表面主要縫隙連接蛋白Connexin 36沒有影響。
[Abstract]:Background: Cortical spreading depression (CSD), based on its similarity to peri-infarct depolarization (PID) around infarct, has become a good experimental model to study the evolution mechanism of ischemic penumbra to infarct center. Many studies have confirmed that pre induced CSD wave can increase brain tissue antagonism. The tolerance of ischemic injury, but its potential pathophysiological mechanism is still unclear. In recent years, many studies have shown that autophagy plays an important role in ischemic brain damage and cerebral ischemia tolerance. The role of autophagy in ischemia / reperfusion injury has become a hot spot in the field of brain protection, in which the autophagy pathway of AMPK-m TOR- is studied However, its clear role is not the same in various studies, and the mechanism is still unclear. The previous studies on the mechanism of CSD preconditioning are mainly focused on energy metabolism, nitric oxide, nerve growth factor, exogenic gene expression, and so on, and lack of CSD preconditioning and autophagy in ischemic tolerance. In this study, the Middle cerebral artery occlusion (MCAO) model of rat middle cerebral artery (MCAO) was used to explore the pathophysiological mechanism of CSD preconditioning, and to further clarify the mechanism of the neuroprotective effect of autophagy in ischemic reperfusion injury and the role of the autophagy pathway of AMPK-m TOR- in ischemic tolerance. Objective: to provide new targets for the treatment and prevention of tubular diseases. Objective: to further explore the mechanism of the evolution of the ischemic penumbra to the center of the infarct through the study of CSD preconditioning. At the same time, the role of CSD preconditioning and autophagy and its related pathway, the role of autophagy in the ischemic tolerance, and the further study of autophagy in the ischemic reperfusion injury Methods: (1) (1) cortical diffusion inhibition wave (CSD) was induced by wet compress of 1 mol/L potassium chloride solution for 2 hours. The occurrence and characteristics of CSD wave were confirmed by electroencephalogram monitoring and electrophysiological recording DC field potential (direct current potential, DC), and the experimental model of CSD pretreatment was established, and 1 mol/L chlorination was used in the control group. Sodium solution wet compress for 2 hours. (2) CSD preconditioning and AMPK-m TOR- autophagy pathway related pathway study of.112 male Wistar rats randomly divided into saline control group, pretreated group (0,3,6,12,24 hour) group; 3-MA (i.c.v.) +CSD group; solvent control (i.c.v.) +CSD group; Compound restraint group; solvent control group. Saline control group was given 1 chlorine chloride. The sodium chloride solution was applied for 2 hours and 12 hours later. The pretreated group was treated with 1 mol/L potassium chloride solution for 2 hours and after 0,3,6,12,24 hours, respectively. The drug groups were given a autophagic inhibitor 3-methyladenine (3-MA, 200 nmol, i.c.v.) and Compound C of AMPK inhibitors before CSD pretreatment (CC, 20, 20, respectively). Ting detection of autophagic markers LC3-II, Beclin-1, P62 level, p-AMPK (Thr172) /AMPK, p-P70S6K (Thr389), ULK1 level, immunofluorescent detection of positive cell number and distribution, transmission electron microscopy to detect the level of autophagic corpuscle. (3) the neuroprotective effect of preconditioning in ischemia reperfusion injury R rats were randomly divided into two groups: saline control group, CSD preconditioning (0,3,6,12,24 hour) group, 3-MA group, solvent control group, saline control group: 12 hours after brine pretreatment, MCAO ischemia 2 hours, reperfusion 12 hours; CSD preconditioning group: MCAO ischemia for 2 hours after CSD preconditioning, and reperfusion for 12 hours respectively; 3-MA group: CSD prelocation The autophagic inhibitor 3-MA (200 nmol, i.c.v.) was given 30 minutes before treatment, and MCAO ischemia was performed for 2 hours after 12 hours of pretreatment and reperfusion for 12 hours. The solvent control group was given the same volume of solvent (i.c.v.) 30 minutes before CSD preconditioning. After 12 hours of pretreatment, MCAO ischemia was performed for 2 hours and reperfusion for 12 hours, and TTC staining was used to calculate each group of infarct bodies. Product, Longa method was used to evaluate neural function defect and to carry out statistical analysis. (4) the mechanism of neuroprotective effect of autophagy in ischemia-reperfusion injury was preliminarily discussed in.30 male Wistar rats randomly divided into 5 groups: sham operation group (Sham), MCAO group, CSD+MCAO group, 3-MA+CSD+MCAO group, and solvent +CSD+MCAO group. Reverse transcriptase-PCR, Western blo Tting detection of cortical apoptosis related protein Bcl-2, Caspase12, Caspase12, endoplasmic reticulum stress related protein GRP78, XBP1, m RNA level and protein level of gap connexin Connexin 36 after MCAO ischemia reperfusion injury; TUNEL and immunofluorescence double labeling staining showed the ratio of apoptotic cells and cell types. Results: (1) cortex was given 1 mol/L chlorination The potassium solution could cause a continuous decrease in the amplitude of the electroencephalogram of the induced lateral cortex, from 117.25 + 3.51 V to 78.36 + 3.46 mu V (P0.01), but the frequency and waveform did not change obviously, the EEG activity was suppressed, the electrophysiological change was negative reversal of the DC field potential, which proved that the model of CSD was established successfully, while the control group did not have the corresponding electroencephalogram and the control group. Electrophysiological changes. (2) the effect of CSD pretreatment on autophagy and AMPK-m TOR pathway: Western blotting shows that CSD can increase the LC3-II, Beclin-1 level and p-AMPK (Thr172) /AMPK ratio in the pretreated lateral cortex of rats, and reduce the level of P62 and Thr172. This change reaches the peak after 12 hours of preconditioning. The ratio of LC3-II, p-AMPK (Thr172) /AMPK, P62 and p-P70S6K (Thr389) was up-regulated in the naughty layer. Immunofluorescence showed that the number of LC3-II positive cells in the pretreated group was significantly higher than that of the control group (P0.01), and was mainly distributed in the neuron cells (87.2%) and in the glial cells (2.89%). The positive cells were used after 3-MA (P0.01) and CC. Transmission electron microscopy showed that the number of autophagic bodies in the pretreated group was significantly higher than that of the control group (P0.05). (3) CSD preconditioning significantly reduced the volume of cerebral infarction after MCAO and improved the functional score, and the protective effect reached its peak at 12 hours after preconditioning (P0.05), and the application of autophagy inhibitor 3-MA (200 nmol, i.c.v.) could reduce C The effect of SD preconditioning on infarct volume and nerve function score (P0.05). (4) CSD preconditioning can reduce the level of apoptosis related markers Caspase12 protein and m RNA in the semi dark zone of ischemia reperfusion injury, increase the Bcl-2 protein and m RNA levels of anti apoptotic markers, and TUNEL staining shows that CSD pretreatment significantly reduces the deity of the dark zone zone. The ratio of cell apoptosis (P0.01), CSD can reduce the protein level of the endoplasmic reticulum stress marker and the level of M RNA, reduce the level of XBP-1 protein, and increase the level of M RNA. The application of autophagy inhibitor 3-MA can reverse the above changes of the protein and M levels of the main gap junctional connexin 36 of the neuron surface after the pretreatment of.CSD. Significant changes (P0.05). Conclusion: (1) the continuous administration of 1 mol/L potassium chloride solution in the cortex for 2 hours can cause the inhibition of electroencephalogram activity in the pretreated lateral cortex, the decrease of the amplitude of electroencephalogram, the induction of negative direct current DC wave, and the successful establishment of the CSD preconditioning model, while 1 mol/L Sodium Chloride Solution does not cause the above changes. (2) CSD can be induced by AMPK-m TOR pathway. Autophagy is activated by autophagy, and the activation of autophagy is mainly distributed in neuron cells and in glial cells. (3) CSD preconditioning has a clear neuroprotective effect, reduces infarct volume, improves neurological function score, and reaches a peak at 12 hours after preconditioning. (4) CSD preconditioning induced autophagy can reduce the area of ischemia reperfusion injury in rats The neuronal apoptosis in the cortex inhibits the overactivation of endoplasmic reticulum stress and produces neuroprotective effects, but has no effect on the main gap connexin Connexin 36 on the surface of the neuron.

【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R743.3

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