内源性醛类代谢失调致认知损伤的机制研究

发布时间：2018-06-12 22:30

本文选题：内源性甲醛 + 内源性核糖　；参考：《重庆医科大学》2017年硕士论文

【摘要】：背景:内源性醛类(甲醛、核糖)在机体内维持一定的生理浓度且具有重要的生理功能。内源性甲醛作为甲基供体,参与体内叶酸循环、DNA甲基化、表观遗传学修饰、细胞分化、基因表达调控等多种细胞生物学过程。内源性核糖参与体内能量提供物质-三磷酸腺苷(ATP)的合成,同时也是DNA和RNA的重要组成部分。当体内内源性甲醛、核糖代谢失调,会造成机体认知功能损伤。已有实验结果显示,随着老龄化年龄的增加,体内甲醛逐渐升高。阿尔兹海默病病人体内甲醛与认知损伤呈正相关。甲醛能导致细胞凋亡、认知功能损伤。然而,关于低浓度甲醛长时间处理神经细胞是否会导致细胞功能损伤,形态改变尚未见文献报道。众所周知,II型糖尿病病人体内葡萄糖含量增加,但已有实验结果显示II型糖尿病病人体内的核糖含量同样显著增加,并且核糖与蛋白质发生非酶促糖基化反应的速度显著快于葡萄糖,产生的糖基化产物(AGEs)具有更强的细胞毒性。胰岛素可以改善学习、认知损伤,但胰岛素能否能挽回核糖产生的糖基化终末产物造成的神经细胞毒性、认知功能损伤尚不可知。目的:探讨低浓度甲醛长时间处理神经细胞是否会出现形态改变、功能损伤。继而研究阿尔兹海默病致病机制并对其早期预防、诊断、治疗提供理论依据。探讨胰岛素是否能挽回核糖代谢失调导致的细胞毒性、认知功能损伤及可能的作用机制。方法:运用连续传代的方法,建立甲醛导致神经系统的慢性损伤模型,全息激光成像系统,观察细胞体积、面积、厚度、粗糙度、椭圆度等形态变化以及贴壁情况。激光共聚焦显微镜观察甲醛处理原代神经元后其树突、轴突数目、长度、复杂度的变化。运用小鼠神经母瘤细胞(n2a)建立核糖细胞毒性损伤模型,cck-8检测细胞活力,结晶紫染色检测细胞数目,全息激光成像系统检测细胞形态变化,westernblotting检测不同处理组细胞内糖基化终末产物、胰岛素受体、胰岛素受体磷酸化水平以及grp78蛋白表达变化,细胞免疫荧光检测细胞内ages定位以及表达变化,tht染色检测细胞内纤维缠结变化。高效液相色谱检测细胞培养基内的核糖含量。免疫共沉淀检测细胞内胰岛素受体与糖基化终末产物(ages)是否有相互作用。采用腹腔注射核糖,建立核糖导致认知功能损伤动物模型。拉力检测小鼠前肢力量,水迷宫、y迷宫检测小鼠学习记忆能力,转棒实验检测小鼠平衡能力。westernblotting、免疫组化检测各组织内糖基化终末产物、胰岛素受体、磷酸化胰岛素受体、组织蛋白酶-d表达变化。结果:病理浓度甲醛长时间处理神经细胞,会导致细胞变厚、变圆,贴壁能力减弱。同时使神经元突起的一级结构减少,神经连接减少。核糖处理细胞,细胞活力随核糖处理的浓度增加显著的下降,细胞的的糖基化终末产物(ages)增加。胰岛素通过激活胰岛素受体,使胰岛素受体磷酸化增加,继而激活细胞内清除ages的途径,从而挽回核糖处理24h产生的细胞毒性,使细胞活力增加、细胞形态改变,细胞内AGEs表达量显著下降。相同浓度的葡萄糖处理细胞,细胞活力与对照组比较无明显变化,胞内AGEs也无变化。长时间注射,小鼠的体重增加,但各处理组小鼠增加的速度低于对照组。在第32天,核糖处理组、核糖加胰岛素处理小鼠体重显著低于对照组。腹腔注射会使小鼠的前肢拉力逐渐下降,核糖处理组小鼠下降的速度高于对照组和其他实验处理组,并在第20天时与对照组存在显著差异,但结束给药后,各处理组小鼠的前肢拉力恢复到最初状态,核糖处理组小鼠上升最慢。腹腔注射实际对小鼠存在一种损伤,影响小鼠的肌肉能力。各处理组血糖无明显变化。运用水迷宫、Y迷宫检测各处理组小鼠的学习记忆能力。水迷宫结果显示核糖处理小鼠,小鼠的学习能力增加,但随后的检测结果显示核糖处理组小鼠的空间记忆能力损伤。Y迷宫结果提示核糖处理组,小鼠的工作记忆与对照组相比有损伤,与核糖加胰岛素处理组的记忆能力比存在显著差异。运用转棒实验排除运动的因素的影响。转棒实验结果显示,核糖处理组小鼠的耐力、平衡能力与对照组没有变化,但与胰岛素组、核糖加胰岛素处理组存在显著差异。蛋白免疫印迹检测了各处理组血清中蛋白以及糖基化终末产物的量,实验结果显示,核糖处理组小鼠血清中蛋白质含量显著低于对照组,而血清中糖基化终末产物的表达量显著高于其他各处理组。
[Abstract]:Background: endogenous aldehydes (formaldehyde, ribose) maintain a certain physiological concentration in the body and have important physiological functions. Endogenous formaldehyde is used as a methyl donor and participates in various biological processes, such as folic acid cycle, DNA methylation, epigenetic modification, cell differentiation, gene expression regulation and so on. Endogenous ribose participates in the energy extraction in vivo. The synthesis of adenosine triphosphate (ATP) is also an important component of the DNA and RNA. When endogenous formaldehyde and ribose metabolism are disordered, the body's cognitive impairment can be caused. The experimental results show that formaldehyde in the body increases with the aging age. Related. Formaldehyde can cause apoptosis and cognitive impairment. However, no literature has been reported about whether the long time formaldehyde treatment of low concentration formaldehyde can cause cell function damage. It is known that the glucose content in type II diabetic patients increases, but the experimental results show the nucleus of II type diabetic patients. Sugar content also increased significantly, and the rate of non enzymatic glycosylation of ribose and protein was significantly faster than glucose, and the production of glycosylated products (AGEs) had stronger cytotoxicity. Insulin could improve learning and cognitive impairment, but could insulin be able to redeem the neurocytotoxicity caused by the glycosylation end products produced by the nuclear sugar. Objective: To explore the morphological changes and functional damage of neural cells with low concentration of formaldehyde for long time treatment, and to study the pathogenesis of Alzheimer's disease and to provide theoretical basis for its early prevention, diagnosis and treatment. Cognitive impairment and possible mechanism of action. Methods: a chronic injury model of the nervous system caused by formaldehyde was established by the method of continuous passage. The holographic laser imaging system was used to observe cell volume, area, thickness, roughness, ellipticity and other morphological changes as well as the condition of adherence. Laser confocal microscopy was used to observe formaldehyde treatment of primary neurons. The changes in the dendrites, the number of axons, the length and the complexity of the axons. Using the mouse neuroblastoma cells (N2a) to establish the toxic damage model of the ribose cells, the CCK-8 detection of cell vitality, the number of cells in the crystal violet staining, the morphological changes of the cells by the holographic laser imaging system, and the detection of the end products of the glycosylation in the cells of different treatment groups by westernblotting. Insulin receptor, insulin receptor phosphorylation level and GRP78 protein expression change, cell immunofluorescence detection of intracellular ages localization and expression changes, tht staining detection of intracellular fibrous tangles. High performance liquid chromatography for detection of ribose content in cell culture base. Immunoprecipitation detection of intracellular insulin receptor and glycosylation end Whether the end product (ages) has interaction. By intraperitoneal injection of ribose, the animal model of cognitive impairment caused by ribose was established. The force of the forelimb, water maze, and the maze of Y maze were detected to detect the learning and memory ability of mice. The balance ability of mice was detected by the rotation bar test, and the end products of glycosylation in the tissues were detected by immunohistochemistry. ISL receptor, phosphorylated insulin receptor, and cathepsin -d expression change. Results: pathological concentration of formaldehyde for long time treatment of nerve cells can cause cells to become thicker, round, and weaker. At the same time, the primary structure of the neurites is reduced and the nerve connections are reduced. Ribose is treated with ribose concentration. With the decrease, the cell's glycosylation end product (ages) increases. Insulin activates the insulin receptor to increase the phosphorylation of the insulin receptor, and then activates the intracellular removal of ages, thereby redeem the cytotoxicity of the ribose to treat 24h, increase the cell vitality, change the cell morphology, and decrease the expression of AGEs in the cell significantly. In the same concentration of glucose treated cells, there was no significant change in cell viability compared with the control group, and there was no change in the intracellular AGEs. Long time injection, the weight of mice increased, but the rate of increase in each treatment group was lower than that of the control group. In the thirty-second day, the ribose treatment group and the ribose plus insulin were significantly lower than the control group. Intraperitoneal injection would make it possible. In the ribose treatment group, the speed of the mice's forelimb tension gradually decreased, the rate of the mice in the ribose treatment group was higher than that of the control group and the other experimental group, and there was a significant difference between the control group and the control group at twentieth days. But after the end of the drug, the forelimb pulling force of the mice in each treatment group was restored to the original state, and the mice in the ribose treatment group rose the slowest. One kind of injury affected the muscle ability of mice. There was no significant change in blood sugar in each treatment group. The learning and memory ability of mice in each treatment group was detected by water maze and Y maze. The results of water maze showed that the learning ability of the mice was increased by the ribose treatment, but the subsequent test results showed that the spatial memory ability of the mice in the nuclear sugar treatment group was damaged by the.Y maze. The results showed that the working memory of the ribose treatment group was more damaged than the control group, and the memory ability of the ribose plus insulin treatment group was significantly different. The effect of the use of the rod experiment to exclude the movement factors. The result of the rotation bar experiment showed that the endurance of the ribose treatment group was not changed with the control group, but it was with the islets of the islet. The protein content in serum and the amount of glycosylated end products in the treatment groups were detected by Western blot. The experimental results showed that the protein content in the serum of the ribose treatment group was significantly lower than that of the control group, while the expression of the end products of glycosylation in the serum was significantly higher than that of the other treatment groups. Group.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

【参考文献】