本文選題：北五味子多糖 + 293T�。� 參考：《廣州中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:五味子是一種傳統(tǒng)中藥,來源于木蘭科的植物五味子Schisandrachinensis(Turcz.)Baill.的干燥成熟果實,習稱為"北五味子",而木蘭科華中五味子Schisandra sphenantheraRehd.et Wils.的干燥成熟果實則稱為"南五味子"。五味子中含有不少的多糖成分,多糖又稱為碳水化合物,其通用的分子式為(CH20)n。五味子多糖大多具有抗氧化、抗衰老的作用,在體內可以保護人的腎臟、肝臟。目前已有文獻中對五味子多糖抗氧化和衰老方面的研究并不深入,尤其是對其作用機制的研究更是少之又少,因此探究五味子多糖產生的抗氧化作用及其作用機制具有不小的意義。NF-E2(Nuclear factor erythriod 2-related factor 2,Nrf2)目前已經被證明是與抗氧化作用最密切相關的靶標之一,當體內、外受到氧化應激等相關刺激后,Nrf2在細胞漿中與Keap1迅速地解離開來并被活化,接著Nrf2從核外向核內運動。本實驗以人胚腎細胞293T為模型,研究北五味子多糖對293T細胞的抗氧化、增殖作用的影響以及作用機制,為氧化應激、衰老的預防和治療提供新的思路和方向,也為北五味子多糖開發(fā)出相關的食品、藥品提供理論依據。方法:1.細胞的培養(yǎng)按照常規(guī)方法培養(yǎng)293T細胞株,培養(yǎng)液按如下比例配制:DMEM高糖培養(yǎng)基:FBS:青鏈霉素混合液=(45ml:5ml:500ul),于恒溫37℃,恒量含5%CO2的細胞培養(yǎng)箱內每天觀察細胞生長情況,傳代培養(yǎng)。2.北五味子粗多糖(SCP)的提取、分離、純化到藥店購買北五味子的藥材,經過石油醚脫脂后得到水提粗多糖,經過脫色、脫蛋白及過DEAE-52、sephadexG-100層析柱進行分離、純化得到一個純化的多糖組分,收集液凍干成粉末后在冷凍干燥的條件下保存。3.北五味子多糖的抗氧化作用根據SOD、CAT、MDA、GSH含量或活性檢測試劑盒的說明書操作4.北五味子多糖對Nrf2及其下游因子的調節(jié)作用采用Western blot檢測不同濃度的純化后多糖組分作用于293T細胞后細胞內Nrf2及其下游的NQO1、HO-1的蛋白表達水平變化,用熒光酶標儀檢測不同濃度的多糖組分給藥后對ARE-luc熒光素酶的影響。5.北五味子多糖對細胞核內外Nrf2活性及其穩(wěn)定性的影響采用Western blot檢測不同濃度的多糖組分給藥后293T細胞Nrf2在核內外蛋白表達水平的變化。用Western blot檢測多糖組分預處理細胞2h后給或不給蛋白合成抑制劑CHX對不同時間段細胞內Nrf2蛋白水平表達的影響。6.北五味子多糖對Nrf2與DNA結合的活性的影響多糖組分處理293T細胞后收集并裂解細胞,提起核蛋白,用EMSA法檢測Nrf2與DNA結合活性的變化。7.北五味子多糖對細胞增殖的影響以293T細胞為研究對象,10umol·L-1的叔丁基對苯二酚(tBHQ)作為激動劑用于陽性對照組,實驗各組用不同濃度多糖組分作用于細胞,分別培養(yǎng)24,48,72 h后,MTT法檢測細胞增殖情況。結果:1.水提得到的粗多糖經過脫色、脫蛋白后總多糖含量為40%。以水為洗脫劑,依次經過DEAE-52層析柱分離純化得到多糖組分SCP-1、過sephadexG-100層析柱分離純化后得到純化組分SCP-2,總多糖含量為86%。2.SCP-2能上調Nrf2及其下游抗氧化因子NQOl、HO-1蛋白水平的表達,增強ARE熒光素酶的活性。3.SCP-2能增強293T細胞中GSH、CAT的含量,減少MDA的含量,增加293T細胞中SOD的活力。4.SCP-2能降低核外的Keap1、Nrf2蛋白水平的表達,使Nrf2活化并向細胞核內轉移,同時SCP-2還可以增加Nrf2的蛋白穩(wěn)定性,延長其半衰期。5.SCP-2能增強Nrf2的DNA結合活性。6.SCP-2可以促進正常細胞的增殖,抑制癌細胞的增殖。結論:北五味子多糖(SCP)能通過激活Nrf2/ARE信號通路,上調Nrf2及其下游抗氧化因子比如二相代謝酶NQO1、H0-1的表達,增加GSH、CAT等內源性抗氧化酶的表達,還能減少細胞內MDA的含量,增強SOD的活力,對正常細胞有促進增殖的作用,對癌細胞則是抑制其增殖。這些作用產生的機制與北五味子多糖能誘導Nrf2從核外向核內轉移,增強Nrf2的蛋白穩(wěn)定性,延長其半衰期,并能增強Nrf2的DNA結合活性有關。由此可以考慮將五味子多糖可以開發(fā)為一種天然的抗氧化劑。
[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.

【學位授予單位】：廣州中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R284
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本文編號：1803903