阿尔茨海默病转基因小鼠海马结构NIX、线粒体自噬的变化

发布时间：2018-06-02 13:20

本文选题：线粒体自噬 + NIX　；参考：《福建医科大学》2012年硕士论文

【摘要】：阿尔茨海默病（Alzheimer’sdisease,AD）是一种老年人易患的以进行性痴呆为主要特征的神经系统退行性疾病。主要临床表现为认知障碍、记忆功能进行性减退等，主要的病理改变包括在细胞外积聚的老年斑、细胞内的神经原纤维缠结（Neuronsfibertangles,NFT）以及海马神经元丢失。自噬是哺乳动物细胞通过降解细胞质中的长寿命蛋白和细胞器、清除损伤的胞质内容物等来维持细胞稳态的主要代谢通路，在保持细胞的健康过程中起着非常重要的作用，同时也是饥饿状态下的重要存活机制。自噬对防止某些疾病如神经退行性疾病有着积极的作用。NIX定位于线粒体外膜，NIX会引发线粒体的通透性增加，最终导致线粒体发生自噬被清除。LC3是一种磷蛋白质，LC3Ⅱ的量可以反映出是否发生自噬。线粒体的损坏会形成过量的ROS，解偶联状态下会消耗ATP，最终导致细胞功能紊乱、甚至死亡。因此，自噬对异常线粒体的清除是一种重要的细胞保护作用。线粒体自噬可以构成一个“质量检查站”来维持线粒体的生物能学、阻止年龄相关的紊乱和衰老，是细胞清除体内异常线粒体、维持其正常功能和基因组稳定及维持自身稳态的重要调节机制。若自噬不足，线粒体的动力受损会导致损伤细胞器的积累，从而导致衰老，尤其在负责线粒体清除和降解的途径受损时；自噬过度可能又会导致正常细胞的死亡，所以如何调控这个"度"也有待研究。通过调控细胞的自噬水平，充分利用自噬的有利方面消除不利方面，靶向治疗线粒体，有望控制神经退行性疾病如AD的发展，延缓衰老，提高生存质量。目的研究AD转基因小鼠海马结构NIX、LC3、线粒体自噬的变化，从而探讨线粒体自噬调控在AD中的可能作用，以期为AD提供一个新的治疗靶点。方法以Morris水迷宫检测野生型和突变型转基因小鼠学习记忆能力，刚果红、镀银染色观察比较海马结构的变化，免疫组化染色观察比较海马结构促凋亡蛋白NIX及LC3的变化，分别以每小格1.5cm×1.5cm、每小格0.5cm×0.5cm的网格对镀银染色、刚果红染色阳性物进行体视学计量分析，共聚焦激光扫描显微技术观察比较海马结构NIX-Mito荧光强度、NIX与线粒体共定位的数目及LC3-Mito荧光强度、线粒体自噬的数目的改变。结果（1）野生型和突变型转基因小鼠逃避潜伏期中位数分别为29.00s和38.00s，差异无统计学意义，P0.05；野生型和突变型小鼠搜索策略相比，突变型较野生型小鼠使用的搜索策略差，差异有统计学意义，P0.05。（2）野生型和突变型小鼠NIX累积光密度（integratedopticaldensity,IOD）中位数分别为103.83和128.85，差异有统计学意义，P0.05；野生型小鼠海马结构细胞排列整齐紧密、分布均匀，结构清晰，突变型小鼠海马结构细胞排列紊乱、稀疏，出现大量免疫反应阳性物呈棕黄色颗粒。（3）野生型和突变型小鼠海马结构LC3阳性物中位数分别为3.00和9.00，差异有统计学意义，P0.05；野生型小鼠海马结构在细胞胞质和突起可见阳性反应物呈棕黄色均质状，突变型小鼠海马结构在细胞胞质和突起可见阳性反应物呈棕黄色颗粒状，且胞外可见大量棕黄色阳性物聚集成团块状。（4）野生型和突变型小鼠海马结构NIX平均荧光强度分别为92.18±7.81和103.07±14.94，差异有统计学意义，P0.05；野生型和突变型小鼠海马结构Mito-TrackerGreen平均荧光强度分别为90.10±10.17和86.87±20.51，差异无统计学意义，P0.05；野生型和突变型小鼠海马结构NIX与线粒体共定位的数目分别为240.94±169.47和544.18±336.44，差异有统计学意义，P0.05；野生型小鼠海马结构细胞排列整齐紧密、分布均匀，结构清晰、外形规则，在细胞胞质和突起出现少量红绿荧光重合的黄色颗粒。突变型小鼠海马结构细胞排列紊乱、稀疏，在细胞胞质和突起出现大量红绿荧光重合的黄色颗粒。（5）野生型和突变型小鼠海马结构LC3平均荧光强度分别为91.69±14.69和130.89±28.90，差异有统计学意义，P0.05；野生型和突变型小鼠海马结构Mito-TrackerGreen平均荧光强度分别为70.29±29.72和65.05±29.06，差异无统计学意义，P0.05；野生型和突变型小鼠海马结构线粒体自噬的数目均值分别为229.67±79.54和312.26±101.55，差异有统计学意义，P0.05；野生型小鼠海马结构在细胞胞质和突起可见阳性反应物呈红色均质、少量颗粒状，突变型小鼠海马结构在细胞胞质和突起可见阳性反应物呈大量红色颗粒状，在胞质和突起出现大量红绿荧光重合的黄色颗粒-发生自噬的线粒体。结论（1）AD转基因小鼠出现行为学和典型病理改变，，为可靠模型。（2）AD转基因小鼠海马结构LC3总量增多，自噬增强。（3）AD转基因小鼠海马结构LC3与线粒体共定位数目增多，线粒体自噬增强。（4）NIX对线粒体自噬可能存在调节作用。
[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.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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