北五味子多糖的分离、纯化及其抗氧化作用的机制研究

发布时间：2018-04-26 01:32

  本文选题：北五味子多糖 + 293T　； 参考：《广州中医药大学》2017年硕士论文

【摘要】：目的:五味子是一种传统中药,来源于木兰科的植物五味子Schisandrachinensis(Turcz.)Baill.的干燥成熟果实,习称为"北五味子",而木兰科华中五味子Schisandra sphenantheraRehd.et Wils.的干燥成熟果实则称为"南五味子"。五味子中含有不少的多糖成分,多糖又称为碳水化合物,其通用的分子式为(CH20)n。五味子多糖大多具有抗氧化、抗衰老的作用,在体内可以保护人的肾脏、肝脏。目前已有文献中对五味子多糖抗氧化和衰老方面的研究并不深入,尤其是对其作用机制的研究更是少之又少,因此探究五味子多糖产生的抗氧化作用及其作用机制具有不小的意义。NF-E2(Nuclear factor erythriod 2-related factor 2,Nrf2)目前已经被证明是与抗氧化作用最密切相关的靶标之一,当体内、外受到氧化应激等相关刺激后,Nrf2在细胞浆中与Keap1迅速地解离开来并被活化,接着Nrf2从核外向核内运动。本实验以人胚肾细胞293T为模型,研究北五味子多糖对293T细胞的抗氧化、增殖作用的影响以及作用机制,为氧化应激、衰老的预防和治疗提供新的思路和方向,也为北五味子多糖开发出相关的食品、药品提供理论依据。方法:1.细胞的培养按照常规方法培养293T细胞株,培养液按如下比例配制:DMEM高糖培养基:FBS:青链霉素混合液=(45ml:5ml:500ul),于恒温37℃,恒量含5%CO2的细胞培养箱内每天观察细胞生长情况,传代培养。2.北五味子粗多糖(SCP)的提取、分离、纯化到药店购买北五味子的药材,经过石油醚脱脂后得到水提粗多糖,经过脱色、脱蛋白及过DEAE-52、sephadexG-100层析柱进行分离、纯化得到一个纯化的多糖组分,收集液冻干成粉末后在冷冻干燥的条件下保存。3.北五味子多糖的抗氧化作用根据SOD、CAT、MDA、GSH含量或活性检测试剂盒的说明书操作4.北五味子多糖对Nrf2及其下游因子的调节作用采用Western blot检测不同浓度的纯化后多糖组分作用于293T细胞后细胞内Nrf2及其下游的NQO1、HO-1的蛋白表达水平变化,用荧光酶标仪检测不同浓度的多糖组分给药后对ARE-luc荧光素酶的影响。5.北五味子多糖对细胞核内外Nrf2活性及其稳定性的影响采用Western blot检测不同浓度的多糖组分给药后293T细胞Nrf2在核内外蛋白表达水平的变化。用Western blot检测多糖组分预处理细胞2h后给或不给蛋白合成抑制剂CHX对不同时间段细胞内Nrf2蛋白水平表达的影响。6.北五味子多糖对Nrf2与DNA结合的活性的影响多糖组分处理293T细胞后收集并裂解细胞,提起核蛋白,用EMSA法检测Nrf2与DNA结合活性的变化。7.北五味子多糖对细胞增殖的影响以293T细胞为研究对象,10umol·L-1的叔丁基对苯二酚(tBHQ)作为激动剂用于阳性对照组,实验各组用不同浓度多糖组分作用于细胞,分别培养24,48,72 h后,MTT法检测细胞增殖情况。结果:1.水提得到的粗多糖经过脱色、脱蛋白后总多糖含量为40%。以水为洗脱剂,依次经过DEAE-52层析柱分离纯化得到多糖组分SCP-1、过sephadexG-100层析柱分离纯化后得到纯化组分SCP-2,总多糖含量为86%。2.SCP-2能上调Nrf2及其下游抗氧化因子NQOl、HO-1蛋白水平的表达,增强ARE荧光素酶的活性。3.SCP-2能增强293T细胞中GSH、CAT的含量,减少MDA的含量,增加293T细胞中SOD的活力。4.SCP-2能降低核外的Keap1、Nrf2蛋白水平的表达,使Nrf2活化并向细胞核内转移,同时SCP-2还可以增加Nrf2的蛋白稳定性,延长其半衰期。5.SCP-2能增强Nrf2的DNA结合活性。6.SCP-2可以促进正常细胞的增殖,抑制癌细胞的增殖。结论:北五味子多糖(SCP)能通过激活Nrf2/ARE信号通路,上调Nrf2及其下游抗氧化因子比如二相代谢酶NQO1、H0-1的表达,增加GSH、CAT等内源性抗氧化酶的表达,还能减少细胞内MDA的含量,增强SOD的活力,对正常细胞有促进增殖的作用,对癌细胞则是抑制其增殖。这些作用产生的机制与北五味子多糖能诱导Nrf2从核外向核内转移,增强Nrf2的蛋白稳定性,延长其半衰期,并能增强Nrf2的DNA结合活性有关。由此可以考虑将五味子多糖可以开发为一种天然的抗氧化剂。
[Abstract]:Objective: Schisandra chinensis is a traditional Chinese medicine derived from the dry and mature fruit of Schisandra Schisandrachinensis (Turcz.) Baill., a plant from Magnoliaceae, which is called "North Schisandra", while the dried fruit of Schisandra sphenantheraRehd.et Wils. in Magnolia Schisandrae is called "Schisandra chinensis". Polysaccharide is also called carbohydrate, and its general molecular formula (CH20) n. Fructus Schisandrae polysaccharide mostly has antioxidant and anti-aging effect, and it can protect human kidney and liver in the body. The research on antioxidant and senescence of Schisandra polysaccharide is not thorough in the literature, especially the research on its mechanism is less. Therefore, it is very important to explore the antioxidant effect and mechanism of Schisandra polysaccharide,.NF-E2 (Nuclear factor erythriod 2-related factor 2, Nrf2), which has been proved to be one of the most closely related targets of antioxidant activity, and Nrf2 is in the cytoplasm and K in the cytoplasm after being stimulated by oxidative stress and other related stimuli. EAP1 was quickly removed and activated, then Nrf2 was activated in the nucleus and extrovert. In this experiment, the human embryonic kidney cell 293T was used as a model to study the effect of the polysaccharide of Schisandra chinensis on the antioxidant and proliferation of 293T cells, and to provide new ideas and directions for oxidative stress, the prevention and treatment of senescence, and the opening of the polysaccharide of Schisandra chinensis. The related food, the medicine provided the theoretical basis. Methods: 1. cells were cultured in accordance with the conventional methods to cultivate 293T cell lines. The culture medium was prepared according to the following proportion: DMEM high sugar medium: FBS: green streptomycin mixture = (45ml:5ml:500ul), at constant temperature at 37 C, and in a cell culture box containing 5%CO2, to observe cell growth every day and culture.2. The extraction, separation and purification of the crude polysaccharide (SCP) of Fructus Schisandrae (North Schisandra chinensis) was purified to the medicinal herb of Schisandra chinensis. After degreasing petroleum ether, the crude polysaccharide was obtained. After decolorization, deproteinization and DEAE-52, sephadexG-100 chromatography column was separated and purified to get a purified polysaccharide component. The collected liquid was frozen dry under the conditions of freeze-drying. Preservation of the antioxidant effect of.3. North Schisandra polysaccharide on the basis of SOD, CAT, MDA, GSH content or activity detection kit instruction operation 4. the regulation of Fructus Schisandrae polysaccharide on Nrf2 and its downstream factors using Western blot to detect different concentrations of purified polysaccharide components after 293T cells in cell Nrf2 and downstream NQO1, HO-1 eggs Changes in the level of white expression, the effect of different concentrations of Polysaccharide on ARE-luc luciferase was detected by a fluorescent enzyme scale. The effect of.5. North Schisandra polysaccharide on Nrf2 activity and stability in and out of the nucleus by Western blot was used to detect the changes in the expression level of 293T fine cell Nrf2 in the nucleus and inside the nucleus after the different concentration of polysaccharide group. Using Western blot to detect the effect of CHX on the expression of Nrf2 protein level in cells of different time periods after preprocessing of 2h in the polysaccharide component, the effect of.6. North Schisandra polysaccharide on the activity of Nrf2 and DNA binding, the polysaccharide components were collected and lysed by the polysaccharide component and lysed the cell, and the nucleoprotein was raised, and Nrf2 and D were detected by EMSA method. NA binding activity changes.7. North Schisandra polysaccharide on cell proliferation of cell proliferation, 293T cells as the research object, 10umol L-1 tert butyl hydroquinone (tBHQ) as an agonist in the positive control group, the experimental groups using different concentration of polysaccharide components in cells, respectively, after the culture of 24,48,72 h, MTT method to detect cell proliferation. Results: 1. water The crude polysaccharide was decolorized and the total polysaccharide content was 40%. with water as the eluant. The polysaccharide component SCP-1 was purified by DEAE-52 chromatography column. The purified component was purified after the separation and purification by sephadexG-100 chromatography column. The total polysaccharide content was 86%.2.SCP-2 can up Nrf2 and its downstream antioxidant factor NQOl, HO-1 protein. The expression of ARE luciferase can enhance the content of GSH, CAT, reduce the content of MDA, increase the activity of SOD in 293T cells and reduce the expression of Keap1 and Nrf2 protein in the 293T cells, and make Nrf2 activate and transfer to the nucleus. Meanwhile, it can also increase the stability of the protein and prolong its protein stability. The half-life.5.SCP-2 can enhance the DNA binding activity of Nrf2 and promote the proliferation of normal cells and inhibit the proliferation of cancer cells. Conclusion: SCP can up regulate the Nrf2 and its downstream antioxidant factors such as the two phase metabolic enzyme NQO1, the expression of H0-1, and increase the endogenous antioxidant enzymes such as GSH, CAT and so on. Expression can also reduce the content of MDA in cells, enhance the vitality of SOD, promote the proliferation of normal cells and inhibit the proliferation of cancer cells. The mechanism of these effects and the polysaccharide of Schisandra chinensis can induce the transfer of Nrf2 from nucleus to nucleus, enhance the protein stability of Nrf2, prolong its half-life, and enhance the DNA binding activity of Nrf2. Therefore, we can consider the polysaccharide from Schisandra chinensis can be developed as a natural antioxidant.

【学位授予单位】：广州中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284
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本文编号：1803903