本文選題：內(nèi)源性甲醛 + 內(nèi)源性核糖�。� 參考：《重慶醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:內(nèi)源性醛類(甲醛、核糖)在機體內(nèi)維持一定的生理濃度且具有重要的生理功能。內(nèi)源性甲醛作為甲基供體,參與體內(nèi)葉酸循環(huán)、DNA甲基化、表觀遺傳學(xué)修飾、細胞分化、基因表達調(diào)控等多種細胞生物學(xué)過程。內(nèi)源性核糖參與體內(nèi)能量提供物質(zhì)-三磷酸腺苷(ATP)的合成,同時也是DNA和RNA的重要組成部分。當(dāng)體內(nèi)內(nèi)源性甲醛、核糖代謝失調(diào),會造成機體認知功能損傷。已有實驗結(jié)果顯示,隨著老齡化年齡的增加,體內(nèi)甲醛逐漸升高。阿爾茲海默病病人體內(nèi)甲醛與認知損傷呈正相關(guān)。甲醛能導(dǎo)致細胞凋亡、認知功能損傷。然而,關(guān)于低濃度甲醛長時間處理神經(jīng)細胞是否會導(dǎo)致細胞功能損傷,形態(tài)改變尚未見文獻報道。眾所周知,II型糖尿病病人體內(nèi)葡萄糖含量增加,但已有實驗結(jié)果顯示II型糖尿病病人體內(nèi)的核糖含量同樣顯著增加,并且核糖與蛋白質(zhì)發(fā)生非酶促糖基化反應(yīng)的速度顯著快于葡萄糖,產(chǎn)生的糖基化產(chǎn)物(AGEs)具有更強的細胞毒性。胰島素可以改善學(xué)習(xí)、認知損傷,但胰島素能否能挽回核糖產(chǎn)生的糖基化終末產(chǎn)物造成的神經(jīng)細胞毒性、認知功能損傷尚不可知。目的:探討低濃度甲醛長時間處理神經(jīng)細胞是否會出現(xiàn)形態(tài)改變、功能損傷。繼而研究阿爾茲海默病致病機制并對其早期預(yù)防、診斷、治療提供理論依據(jù)。探討胰島素是否能挽回核糖代謝失調(diào)導(dǎo)致的細胞毒性、認知功能損傷及可能的作用機制。方法:運用連續(xù)傳代的方法,建立甲醛導(dǎo)致神經(jīng)系統(tǒng)的慢性損傷模型,全息激光成像系統(tǒng),觀察細胞體積、面積、厚度、粗糙度、橢圓度等形態(tài)變化以及貼壁情況。激光共聚焦顯微鏡觀察甲醛處理原代神經(jīng)元后其樹突、軸突數(shù)目、長度、復(fù)雜度的變化。運用小鼠神經(jīng)母瘤細胞(n2a)建立核糖細胞毒性損傷模型,cck-8檢測細胞活力,結(jié)晶紫染色檢測細胞數(shù)目,全息激光成像系統(tǒng)檢測細胞形態(tài)變化,westernblotting檢測不同處理組細胞內(nèi)糖基化終末產(chǎn)物、胰島素受體、胰島素受體磷酸化水平以及grp78蛋白表達變化,細胞免疫熒光檢測細胞內(nèi)ages定位以及表達變化,tht染色檢測細胞內(nèi)纖維纏結(jié)變化。高效液相色譜檢測細胞培養(yǎng)基內(nèi)的核糖含量。免疫共沉淀檢測細胞內(nèi)胰島素受體與糖基化終末產(chǎn)物(ages)是否有相互作用。采用腹腔注射核糖,建立核糖導(dǎo)致認知功能損傷動物模型。拉力檢測小鼠前肢力量,水迷宮、y迷宮檢測小鼠學(xué)習(xí)記憶能力,轉(zhuǎn)棒實驗檢測小鼠平衡能力。westernblotting、免疫組化檢測各組織內(nèi)糖基化終末產(chǎn)物、胰島素受體、磷酸化胰島素受體、組織蛋白酶-d表達變化。結(jié)果:病理濃度甲醛長時間處理神經(jīng)細胞,會導(dǎo)致細胞變厚、變圓,貼壁能力減弱。同時使神經(jīng)元突起的一級結(jié)構(gòu)減少,神經(jīng)連接減少。核糖處理細胞,細胞活力隨核糖處理的濃度增加顯著的下降,細胞的的糖基化終末產(chǎn)物(ages)增加。胰島素通過激活胰島素受體,使胰島素受體磷酸化增加,繼而激活細胞內(nèi)清除ages的途徑,從而挽回核糖處理24h產(chǎn)生的細胞毒性,使細胞活力增加、細胞形態(tài)改變,細胞內(nèi)AGEs表達量顯著下降。相同濃度的葡萄糖處理細胞,細胞活力與對照組比較無明顯變化,胞內(nèi)AGEs也無變化。長時間注射,小鼠的體重增加,但各處理組小鼠增加的速度低于對照組。在第32天,核糖處理組、核糖加胰島素處理小鼠體重顯著低于對照組。腹腔注射會使小鼠的前肢拉力逐漸下降,核糖處理組小鼠下降的速度高于對照組和其他實驗處理組,并在第20天時與對照組存在顯著差異,但結(jié)束給藥后,各處理組小鼠的前肢拉力恢復(fù)到最初狀態(tài),核糖處理組小鼠上升最慢。腹腔注射實際對小鼠存在一種損傷,影響小鼠的肌肉能力。各處理組血糖無明顯變化。運用水迷宮、Y迷宮檢測各處理組小鼠的學(xué)習(xí)記憶能力。水迷宮結(jié)果顯示核糖處理小鼠,小鼠的學(xué)習(xí)能力增加,但隨后的檢測結(jié)果顯示核糖處理組小鼠的空間記憶能力損傷。Y迷宮結(jié)果提示核糖處理組,小鼠的工作記憶與對照組相比有損傷,與核糖加胰島素處理組的記憶能力比存在顯著差異。運用轉(zhuǎn)棒實驗排除運動的因素的影響。轉(zhuǎn)棒實驗結(jié)果顯示,核糖處理組小鼠的耐力、平衡能力與對照組沒有變化,但與胰島素組、核糖加胰島素處理組存在顯著差異。蛋白免疫印跡檢測了各處理組血清中蛋白以及糖基化終末產(chǎn)物的量,實驗結(jié)果顯示,核糖處理組小鼠血清中蛋白質(zhì)含量顯著低于對照組,而血清中糖基化終末產(chǎn)物的表達量顯著高于其他各處理組。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16

【參考文獻】

相關(guān)期刊論文 前2條

1 苗君葉;盧靜;張子劍;童志前;赫榮喬;;甲醛導(dǎo)致細胞周期異常的濃度選擇性[J];生物化學(xué)與生物物理進展;2013年07期

2 ;Formaldehyde stress[J];Science China(Life Sciences);2010年12期

，

本文編號：2011309