Fucoidan对β淀粉样蛋白和D-gal联合诱导PC12细胞和小鼠学习记忆能力损伤的保护作用

发布时间：2018-05-22 17:31

本文选题：褐藻多糖硫酸酯(Fucoidan) + 阿尔茨海默症　；参考：《大连医科大学》2016年硕士论文

【摘要】：目的:阿尔茨海默病(Alzheimer’s disease,AD)是一种进行性神经退行性疾病,临床表现为记忆和其他认知功能的丧失。现代医学研究表明,AD发病主要与β-淀粉样肽25~35(Aβ25~35)在脑组织异常沉积、脑内神经血管单元功能异常导致Aβ25~35清除障碍、胆碱能神经元受损、氧化应激、神经炎症反应或基因突变等因素有关。Fucoidan是富含岩藻糖和硫酸酯基的水溶性杂聚糖,目前发现仅存在于褐藻和某些棘皮动物,如海参、海胆体内,而褐藻来源的Fucoidan具有独特的分支结构,并且不同种属具有不同比例的半乳糖、木糖、甘露糖和糖醛酸等。Fucoidan具有抗衰老、抗凝血、抗血栓、免疫调节、抗肿瘤等活性,是重要的海洋药源物质。本实验研究Fucoidan对Aβ25~35和D-gal联合应用致PC-12细胞损伤的保护作用,检测与衰老相关的生化指标和与细胞凋亡相关的蛋白的变化;采用D-gal用药诱导建立衰老小鼠模型,研究Fucoidan对衰老小鼠学习记忆的改善作用和机制。方法:1.采用乙醇分级沉淀法从粗品Fucoidan中纯化获得Fucoidan。2.采用苯酚-硫酸法、硫酸-咔唑法、自动指示旋光仪、核磁共振仪分别测定Fucoidan的总糖含量、糖醛酸含量、旋光度和官能团。3.凝聚态Aβ25~35和D-gal的制备,采用酶学检测、Western blot、流式细胞术得到最佳药物浓度为25μM10m M。4.MTT检测Fucoidan细胞毒性作用。5.光学显微镜和Hoechst33258染色观察观察细胞的外部形态和凋亡细胞核形态。6.流式细胞术检测Fucoidan对细胞的影响。7.采用Western blot方法检测细胞中caspase3、caspase3/17p、caspase8、caspa7e9、Cyt C和Livin、XIAP在蛋白水平以及酶学方法检测细胞中SOD活力和GSH的含量。8.建立D-gal衰老模型,动物行为学实验,酶学检测组织中SOD和ACh E的活力,GSH和Ch AT含量,尼氏染色检测尼氏小体数量。结果:1.Fucoidan的得率为38.91%。2.Fucoidan总糖含量、硫酸基团含量、糖醛酸含量、分子量和旋光度分别为44.7%、18.5%、12.5%、26.61×104Da和0.99o(20℃)3.随着药物浓度的升高,细胞的存活率下降,与Control组相比,AβD-gal(15μM10m M、20μM10m M、25μM10m M、30μM10m M)在处理36h、48h、72h后细胞的存活率下降。酶学检测、Western blot、流式细胞术得到药物浓度为10m M25μM,处理时间为48h诱导效果最佳。4.Fucoidan处理细胞24h后,各组细胞和用药组细胞相比,细胞的存活率没有显著性差异,说明Fucoidan没有明显的细胞毒性作用。5.Fucoidan保护细胞24h后,Fucoidan各组与对照组相比,细胞神经丝的长度有所恢复,细胞密度增大。PC12细胞逐步呈现弥漫均匀的低荧光强度,出现了正常细胞的特征,说明Fucoidan能够有效的减少细胞凋亡,使凋亡细胞显著减少。流式细胞实验结果显示,细胞的存活率明显下降。6.Fucoidan处理细胞24h后,Fucoidan各组与对照组相比,细胞早期凋亡和晚期凋亡的数量比明显下降。7.Fucoidan保护细胞24h后,200μg/m L,400μg/m L组的caspase3、caspase8、caspase9、Cyt C的表达量明显下降,Livin、XIAP的表达量明显上升。Fucoidan各组细胞的SOD的活力和GSH的含量显著提高。8.水迷宫结果显示,用药组Fucoidan100mg/kg和200mg/kg组与模型组相比,小鼠找到平台的潜伏期明显缩短。脑组织中GSH,Ach的含量,Ch AT的活性上升,ACh E活性下降。血清中SOD活力和GSH含量增加,Fucoidan能够提高抗氧化防御酶的活性。结论:Fucoidan对衰老的小鼠的学习记忆能力有改善作用,通过水迷宫、酶学检测相关衰老的指标,Fucoidan是通过调节神经因子的表达来改善小鼠的学习和记忆能力。细胞实验证明,Fucoidan能够抑制与细胞凋亡相关的蛋白表达,促进凋亡抑制因子的表达从而对细胞有保护作用。
[Abstract]:Objective: Alzheimer 's disease (AD) is an progressive neurodegenerative disease. The clinical manifestation is the loss of memory and other cognitive functions. Modern medical research shows that the pathogenesis of AD is mainly with the abnormal deposition of beta amyloid peptide 25~35 (A beta 25~35) in the brain tissue, and the abnormal function of the neurovascular unit in the brain leads to the clearance of A beta 25~35. Hindrence, the damage of cholinergic neurons, oxidative stress, neuroinflammatory reactions, or gene mutations, and other factors related to.Fucoidan are water-soluble heterosans rich in fucose and sulfuric acid esters, which are found only in brown algae and some acanthoderms, such as sea cucumbers, sea urchins, and Fucoidan derived from brown algae, with unique branch structures and different species. .Fucoidan, with different proportions of galactose, xylose, mannose and uronic acid, is an important marine source of antiaging, anticoagulant, antithrombotic, immunoregulation and anti-tumor activity. This experiment studied the protective effect of Fucoidan on A beta 25~35 and D-gal combined application of PC-12 cell damage and detection of biochemical markers associated with aging. And the changes in the protein related to apoptosis; using D-gal to induce the aging mice model, to study the effect and mechanism of Fucoidan on the learning and memory of aging mice. Method: 1. the purification of Fucoidan.2. by ethanol precipitation method from the crude Fucoidan was obtained by the phenol sulfuric acid method, the sulfuric acid carbazole method and the automatic optical polarimeter. The total sugar content of Fucoidan, the content of glucuronic acid, the rotation luminosity and the.3. condensed state A beta 25~35 and D-gal were prepared by NMR. The optimum drug concentration was 25 u M10m M.4.MTT detection Fucoidan cytotoxicity by the.5. optical microscope and the Hoechst33258 staining observation by the method of enzymatic detection, Western blot, and flow cytometry. The external morphology and apoptotic cell nucleus morphology.6. flow cytometry detection of the effect of Fucoidan on cells..7. used Western blot to detect Caspase3, caspase3/17p, caspase8, caspa7e9, Cyt C and Livin. The activity of SOD and ACh E, the content of GSH and Ch AT in the tissues and the number of Nissl corpuscles were detected by Nissl staining. Results: the yield of 1.Fucoidan was 38.91%.2.Fucoidan total sugar, the content of sulphuric acid group, the content of uronic acid, the molecular weight and the rotation luminosity were 44.7%, 18.5%, 12.5%, 26.61 * 104Da and 0.99o (20) 3. with the drug. The survival rate of cells decreased. Compared with the Control group, A beta D-gal (15 mu M10m M, 20 mu M10m M, 25 mu M10m M, 30 mu M10m M) decreased the survival rate of the cells after processing 36h. After the cells were compared with the drug group, the survival rate of the cells was not significantly different, indicating that Fucoidan had no obvious cytotoxic effect of.5.Fucoidan to protect the cell 24h, and the length of the cell nerve filament was recovered and the density of.PC12 cells gradually showed a diffuse and homogeneous low fluorescence intensity, compared with the control group. The characteristics of normal cells showed that Fucoidan could effectively reduce apoptosis and reduce apoptotic cells significantly. Flow cytometry results showed that after the survival rate of.6.Fucoidan cells decreased significantly, the number of apoptotic and late apoptotic cells decreased significantly compared with the control group, compared with the control group, the number of Fucoidan cells decreased significantly by.7.Fucoida. After n 24h, the expression of Caspase3, caspase8, caspase9, Cyt C decreased in 200 mu g/m L and 400 g/m L group. The incubation period of the platform was significantly shortened. The content of GSH, Ach, the activity of Ch AT increased, the activity of ACh E decreased. The SOD activity and GSH content in the serum increased, and Fucoidan could improve the activity of antioxidant defense enzymes. Conclusion: Fucoidan can improve the learning and memory ability of aging mice, and detect the related senescence by water maze. Fucoidan can improve the learning and memory ability of mice by regulating the expression of neural factors. Cell experiments have shown that Fucoidan can inhibit the expression of protein related to apoptosis and promote the expression of apoptosis inhibiting factors and thus protect the cells.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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