本文選題：線粒體自噬 + NIX��； 參考：《福建醫(yī)科大學(xué)》2012年碩士論文

【摘要】：阿爾茨海默�。ˋlzheimer’sdisease,AD）是一種老年人易患的以進行性癡呆為主要特征的神經(jīng)系統(tǒng)退行性疾病。主要臨床表現(xiàn)為認知障礙、記憶功能進行性減退等，主要的病理改變包括在細胞外積聚的老年斑、細胞內(nèi)的神經(jīng)原纖維纏結(jié)（Neuronsfibertangles,NFT）以及海馬神經(jīng)元丟失。自噬是哺乳動物細胞通過降解細胞質(zhì)中的長壽命蛋白和細胞器、清除損傷的胞質(zhì)內(nèi)容物等來維持細胞穩(wěn)態(tài)的主要代謝通路，在保持細胞的健康過程中起著非常重要的作用，同時也是饑餓狀態(tài)下的重要存活機制。自噬對防止某些疾病如神經(jīng)退行性疾病有著積極的作用。NIX定位于線粒體外膜，NIX會引發(fā)線粒體的通透性增加，最終導(dǎo)致線粒體發(fā)生自噬被清除。LC3是一種磷蛋白質(zhì)，LC3Ⅱ的量可以反映出是否發(fā)生自噬。線粒體的損壞會形成過量的ROS，解偶聯(lián)狀態(tài)下會消耗ATP，最終導(dǎo)致細胞功能紊亂、甚至死亡。因此，自噬對異常線粒體的清除是一種重要的細胞保護作用。線粒體自噬可以構(gòu)成一個“質(zhì)量檢查站”來維持線粒體的生物能學(xué)、阻止年齡相關(guān)的紊亂和衰老，是細胞清除體內(nèi)異常線粒體、維持其正常功能和基因組穩(wěn)定及維持自身穩(wěn)態(tài)的重要調(diào)節(jié)機制。若自噬不足，線粒體的動力受損會導(dǎo)致?lián)p傷細胞器的積累，從而導(dǎo)致衰老，尤其在負責(zé)線粒體清除和降解的途徑受損時；自噬過度可能又會導(dǎo)致正常細胞的死亡，所以如何調(diào)控這個"度"也有待研究。通過調(diào)控細胞的自噬水平，充分利用自噬的有利方面消除不利方面，靶向治療線粒體，有望控制神經(jīng)退行性疾病如AD的發(fā)展，延緩衰老，提高生存質(zhì)量。 目的研究AD轉(zhuǎn)基因小鼠海馬結(jié)構(gòu)NIX、LC3、線粒體自噬的變化，從而探討線粒體自噬調(diào)控在AD中的可能作用，以期為AD提供一個新的治療靶點。 方法以Morris水迷宮檢測野生型和突變型轉(zhuǎn)基因小鼠學(xué)習(xí)記憶能力，剛果紅、鍍銀染色觀察比較海馬結(jié)構(gòu)的變化，免疫組化染色觀察比較海馬結(jié)構(gòu)促凋亡蛋白NIX及LC3的變化，分別以每小格1.5cm×1.5cm、每小格0.5cm×0.5cm的網(wǎng)格對鍍銀染色、剛果紅染色陽性物進行體視學(xué)計量分析，共聚焦激光掃描顯微技術(shù)觀察比較海馬結(jié)構(gòu)NIX-Mito熒光強度、NIX與線粒體共定位的數(shù)目及LC3-Mito熒光強度、線粒體自噬的數(shù)目的改變。 結(jié)果（1）野生型和突變型轉(zhuǎn)基因小鼠逃避潛伏期中位數(shù)分別為29.00s和38.00s，差異無統(tǒng)計學(xué)意義，P0.05；野生型和突變型小鼠搜索策略相比，突變型較野生型小鼠使用的搜索策略差，差異有統(tǒng)計學(xué)意義，P0.05。（2）野生型和突變型小鼠NIX累積光密度（integratedopticaldensity,IOD）中位數(shù)分別為103.83和128.85，差異有統(tǒng)計學(xué)意義，P0.05；野生型小鼠海馬結(jié)構(gòu)細胞排列整齊緊密、分布均勻，結(jié)構(gòu)清晰，突變型小鼠海馬結(jié)構(gòu)細胞排列紊亂、稀疏，出現(xiàn)大量免疫反應(yīng)陽性物呈棕黃色顆粒。（3）野生型和突變型小鼠海馬結(jié)構(gòu)LC3陽性物中位數(shù)分別為3.00和9.00，差異有統(tǒng)計學(xué)意義，P0.05；野生型小鼠海馬結(jié)構(gòu)在細胞胞質(zhì)和突起可見陽性反應(yīng)物呈棕黃色均質(zhì)狀，突變型小鼠海馬結(jié)構(gòu)在細胞胞質(zhì)和突起可見陽性反應(yīng)物呈棕黃色顆粒狀，且胞外可見大量棕黃色陽性物聚集成團塊狀。（4）野生型和突變型小鼠海馬結(jié)構(gòu)NIX平均熒光強度分別為92.18±7.81和103.07±14.94，差異有統(tǒng)計學(xué)意義，P0.05；野生型和突變型小鼠海馬結(jié)構(gòu)Mito-TrackerGreen平均熒光強度分別為90.10±10.17和86.87±20.51，差異無統(tǒng)計學(xué)意義，P0.05；野生型和突變型小鼠海馬結(jié)構(gòu)NIX與線粒體共定位的數(shù)目分別為240.94±169.47和544.18±336.44，差異有統(tǒng)計學(xué)意義，P0.05；野生型小鼠海馬結(jié)構(gòu)細胞排列整齊緊密、分布均勻，結(jié)構(gòu)清晰、外形規(guī)則，在細胞胞質(zhì)和突起出現(xiàn)少量紅綠熒光重合的黃色顆粒。突變型小鼠海馬結(jié)構(gòu)細胞排列紊亂、稀疏，在細胞胞質(zhì)和突起出現(xiàn)大量紅綠熒光重合的黃色顆粒。（5）野生型和突變型小鼠海馬結(jié)構(gòu)LC3平均熒光強度分別為91.69±14.69和130.89±28.90，差異有統(tǒng)計學(xué)意義，P0.05；野生型和突變型小鼠海馬結(jié)構(gòu)Mito-TrackerGreen平均熒光強度分別為70.29±29.72和65.05±29.06，差異無統(tǒng)計學(xué)意義，P0.05；野生型和突變型小鼠海馬結(jié)構(gòu)線粒體自噬的數(shù)目均值分別為229.67±79.54和312.26±101.55，差異有統(tǒng)計學(xué)意義，P0.05；野生型小鼠海馬結(jié)構(gòu)在細胞胞質(zhì)和突起可見陽性反應(yīng)物呈紅色均質(zhì)、少量顆粒狀，突變型小鼠海馬結(jié)構(gòu)在細胞胞質(zhì)和突起可見陽性反應(yīng)物呈大量紅色顆粒狀，在胞質(zhì)和突起出現(xiàn)大量紅綠熒光重合的黃色顆粒-發(fā)生自噬的線粒體。 結(jié)論（1）AD轉(zhuǎn)基因小鼠出現(xiàn)行為學(xué)和典型病理改變，，為可靠模型。（2）AD轉(zhuǎn)基因小鼠海馬結(jié)構(gòu)LC3總量增多，自噬增強。（3）AD轉(zhuǎn)基因小鼠海馬結(jié)構(gòu)LC3與線粒體共定位數(shù)目增多，線粒體自噬增強。（4）NIX對線粒體自噬可能存在調(diào)節(jié)作用。
[Abstract]:Alzheimer 'sdisease (AD) is a kind of nervous system degenerative disease characterized by progressive dementia. The main clinical manifestations are cognitive impairment, memory function progressive degeneration, and the main pathological changes include the senile plaques that accumulate outside the cell, and the neurofibrillary tangles in the cells (Neuronsf Ibertangles, NFT) and the loss of hippocampal neurons. Autophagy is an important role in maintaining cell homeostasis by mammalian cells to maintain the main metabolic pathways of cell homeostasis by degrading long life proteins and organelles in the cytoplasm to maintain the main metabolic pathways of cell homeostasis, and is also important in the state of starvation. The survival mechanism. Autophagy has a positive effect on preventing certain diseases, such as neurodegenerative disease, that.NIX is located in the outer membrane of the mitochondria, and NIX causes the increase of mitochondrial permeability, which eventually leads to mitochondrial autophagy to be cleared of.LC3 as a kind of phosphorus protein, and the amount of LC3 II can reflect the occurrence of autophagy. Mitochondrial damage will be formed. The amount of ROS, which will consume ATP in the uncoupling state, eventually leads to cell dysfunction and even death. Therefore, autophagy is an important cytoprotective effect on abnormal mitochondria. Mitochondria autophagy can form a "quality checkpoint" to maintain mitochondrial bioenergy, prevent age related disorders and senescence, and be cell clear. In addition to abnormal mitochondria in the body, an important regulatory mechanism for maintaining normal function and stability of the genome and maintaining homeostasis. If autophagy is insufficient, the impairment of mitochondria can cause damage to the accumulation of organelles, resulting in senescence, especially when the path of mitochondrial clearance and degradation is damaged; autophagy may lead to normal fines. Cell death, so how to regulate this "degree" is still to be studied. By regulating the level of autophagy, making full use of the favorable aspects of autophagy, eliminating adverse aspects and targeting the mitochondria, it is expected to control the development of neurodegenerative diseases such as AD, postpone senility and improve the quality of life.
Objective to study the changes of NIX, LC3 and mitochondrial autophagy in the hippocampal structure of AD transgenic mice, and to explore the possible role of mitochondrial autophagy regulation in AD, in order to provide a new therapeutic target for AD.
Methods the learning and memory ability of wild type and mutant transgenic mice was detected by Morris water maze. The changes of hippocampal structure were compared in Congo red and silver plating. The changes of hippocampal structure promoting apoptotic protein NIX and LC3 were observed by immunohistochemical staining, and each small lattice of 1.5cm x 1.5cm and 0.5cm x 0.5cm in each small lattice were stained with silver plating. Stereological quantitative analysis of fruit red staining and confocal laser scanning microscopy were used to compare the NIX-Mito fluorescence intensity of the hippocampal structure, the number of CO localization of NIX and mitochondria, the intensity of LC3-Mito fluorescence, and the change of the number of autophagy in mitochondria.
Results (1) the median of escape incubation period of wild type and mutant transgenic mice were 29.00s and 38.00s respectively, the difference was not statistically significant, P0.05. Compared with wild type and mutant mice, the search strategy of mutant type mice was less than that of wild type mice, and the difference was statistically significant. P0.05. (2) wild type and mutant mouse NIX accumulated light. The median of density (integratedopticaldensity, IOD) were 103.83 and 128.85 respectively. The difference was statistically significant, P0.05. The hippocampal cells in the wild type mice were arranged neatly, evenly distributed and clear in structure. The hippocampal cells in the mutant mice were arranged in disorder and sparse, and a large number of immunoreactive positive compounds appeared brown yellow granules. (3) wild type. The median of LC3 positive compounds in the hippocampal structure of the mutant mice were 3 and 9, respectively. The difference was statistically significant, P0.05. The positive reaction of the hippocampal structure in the cytoplasm and protuberance of the wild type mice was brown and yellow, and the mutant mouse hippocampal structure was brown and yellow granules in the cytoplasm and process of the cell cytoplasm and process. A large number of brown and yellow positive compounds were found to be aggregated. (4) the average fluorescence intensity of the hippocampal NIX in the wild and mutant mice was 92.18 + 7.81 and 103.07 + 14.94 respectively, and the difference was statistically significant, P0.05. The average fluorescence intensity of the hippocampal Mito-TrackerGreen in the wild and mutant mice was 90.10 + 10.17 and 86.87 + 20.51, respectively. There was no statistical significance, P0.05. The total number of NIX and mitochondria in the hippocampus of wild type and mutant mice was 240.94 + 169.47 and 544.18 + 336.44 respectively. The difference was statistically significant, P0.05. The hippocampal cells in the wild type mice were arranged in neat, uniform distribution, clear structure, regular shape, and appeared in cytoplasm and protuberance. A small amount of red and green fluorescence overlapped yellow granules. The mutant mouse hippocampal structure cells were arranged in disorder and sparse, and a large number of red and green fluorescence overlapped yellow granules appeared in the cytoplasm and protuberance. (5) the average fluorescence intensity of the hippocampal LC3 in the wild and mutant mice was 91.69 + 14.69 and 130.89 + 28.90 respectively, the difference was statistically significant, P0.05; The average fluorescence intensity of Mito-TrackerGreen in the hippocampal structure of wild and mutant mice was 70.29 + 29.72 and 65.05 + 29.06 respectively. The difference was not statistically significant, P0.05. The mean number of autophagy in the hippocampus of wild and mutant mice was 229.67 + 79.54 and 312.26 + 101.55 respectively. The difference was statistically significant, P0.05, and wild type. In the cytoplasm and protuberance of the rat hippocampal structure, the positive reactant in cytoplasm and protuberance showed a red homogenization and a small amount of granular. A large number of red granules were found in the cytoplasm and protuberance of the mutant mice. In the cytoplasm and the protuberances, a large number of red and green fluorescence overlapped yellow granules were found in the cytoplasm and protuberances, which occurred autophagic mitochondria.
Conclusion (1) the behavior and typical pathological changes of AD transgenic mice were found to be a reliable model. (2) the total amount of LC3 in the hippocampus of AD transgenic mice increased and autophagy enhanced. (3) the number of LC3 and mitochondria in the hippocampus of AD transgenic mice increased and mitochondrial autophagy enhanced. (4) NIX could regulate the autophagy of mitochondria.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.16

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