本文選題：褐藻多糖硫酸酯(Fucoidan) + 阿爾茨海默癥�。� 參考：《大連醫(yī)科大學》2016年碩士論文

【摘要】：目的:阿爾茨海默病(Alzheimer’s disease,AD)是一種進行性神經退行性疾病,臨床表現(xiàn)為記憶和其他認知功能的喪失�，F(xiàn)代醫(yī)學研究表明,AD發(fā)病主要與β-淀粉樣肽25~35(Aβ25~35)在腦組織異常沉積、腦內神經血管單元功能異常導致Aβ25~35清除障礙、膽堿能神經元受損、氧化應激、神經炎癥反應或基因突變等因素有關。Fucoidan是富含巖藻糖和硫酸酯基的水溶性雜聚糖,目前發(fā)現(xiàn)僅存在于褐藻和某些棘皮動物,如海參、海膽體內,而褐藻來源的Fucoidan具有獨特的分支結構,并且不同種屬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、免疫調節(jié)、抗腫瘤等活性,是重要的海洋藥源物質。本實驗研究Fucoidan對Aβ25~35和D-gal聯(lián)合應用致PC-12細胞損傷的保護作用,檢測與衰老相關的生化指標和與細胞凋亡相關的蛋白的變化;采用D-gal用藥誘導建立衰老小鼠模型,研究Fucoidan對衰老小鼠學習記憶的改善作用和機制。方法:1.采用乙醇分級沉淀法從粗品Fucoidan中純化獲得Fucoidan。2.采用苯酚-硫酸法、硫酸-咔唑法、自動指示旋光儀、核磁共振儀分別測定Fucoidan的總糖含量、糖醛酸含量、旋光度和官能團。3.凝聚態(tài)Aβ25~35和D-gal的制備,采用酶學檢測、Western blot、流式細胞術得到最佳藥物濃度為25μM10m M。4.MTT檢測Fucoidan細胞毒性作用。5.光學顯微鏡和Hoechst33258染色觀察觀察細胞的外部形態(tài)和凋亡細胞核形態(tài)。6.流式細胞術檢測Fucoidan對細胞的影響。7.采用Western blot方法檢測細胞中caspase3、caspase3/17p、caspase8、caspa7e9、Cyt C和Livin、XIAP在蛋白水平以及酶學方法檢測細胞中SOD活力和GSH的含量。8.建立D-gal衰老模型,動物行為學實驗,酶學檢測組織中SOD和ACh E的活力,GSH和Ch AT含量,尼氏染色檢測尼氏小體數(shù)量。結果:1.Fucoidan的得率為38.91%。2.Fucoidan總糖含量、硫酸基團含量、糖醛酸含量、分子量和旋光度分別為44.7%、18.5%、12.5%、26.61×104Da和0.99o(20℃)3.隨著藥物濃度的升高,細胞的存活率下降,與Control組相比,AβD-gal(15μM10m M、20μM10m M、25μM10m M、30μM10m M)在處理36h、48h、72h后細胞的存活率下降。酶學檢測、Western blot、流式細胞術得到藥物濃度為10m M25μM,處理時間為48h誘導效果最佳。4.Fucoidan處理細胞24h后,各組細胞和用藥組細胞相比,細胞的存活率沒有顯著性差異,說明Fucoidan沒有明顯的細胞毒性作用。5.Fucoidan保護細胞24h后,Fucoidan各組與對照組相比,細胞神經絲的長度有所恢復,細胞密度增大。PC12細胞逐步呈現(xiàn)彌漫均勻的低熒光強度,出現(xiàn)了正常細胞的特征,說明Fucoidan能夠有效的減少細胞凋亡,使凋亡細胞顯著減少。流式細胞實驗結果顯示,細胞的存活率明顯下降。6.Fucoidan處理細胞24h后,Fucoidan各組與對照組相比,細胞早期凋亡和晚期凋亡的數(shù)量比明顯下降。7.Fucoidan保護細胞24h后,200μg/m L,400μg/m L組的caspase3、caspase8、caspase9、Cyt C的表達量明顯下降,Livin、XIAP的表達量明顯上升。Fucoidan各組細胞的SOD的活力和GSH的含量顯著提高。8.水迷宮結果顯示,用藥組Fucoidan100mg/kg和200mg/kg組與模型組相比,小鼠找到平臺的潛伏期明顯縮短。腦組織中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能夠提高抗氧化防御酶的活性。結論:Fucoidan對衰老的小鼠的學習記憶能力有改善作用,通過水迷宮、酶學檢測相關衰老的指標,Fucoidan是通過調節(jié)神經因子的表達來改善小鼠的學習和記憶能力。細胞實驗證明,Fucoidan能夠抑制與細胞凋亡相關的蛋白表達,促進凋亡抑制因子的表達從而對細胞有保護作用。
[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.
【學位授予單位】：大連醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R749.16

【相似文獻】

相關期刊論文 前1條

1 王玉琳;金澤;孫忠人;;針刺對D-gal致衰老模型大鼠腦、肝組織SOD、MDA影響的實驗研究[J];中醫(yī)藥信息;2011年04期

相關碩士學位論文 前1條

1 魏恒云;Fucoidan對β淀粉樣蛋白和D-gal聯(lián)合誘導PC12細胞和小鼠學習記憶能力損傷的保護作用[D];大連醫(yī)科大學;2016年

，

本文編號：1922991