本文關(guān)鍵詞： 砷污染 灰樹花 含硒糖蛋白 分離純化 免疫毒性 營(yíng)養(yǎng)干預(yù)作用　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目前,砷污染已成為極其嚴(yán)重的全球環(huán)境及公共衛(wèi)生問題之一。砷可致機(jī)體多系統(tǒng)毒性,如免疫、神經(jīng)、發(fā)育和肝臟毒性等。針對(duì)慢性砷中毒,營(yíng)養(yǎng)干預(yù)是一種有效的預(yù)防方法。研究表明,硒可拮抗砷中毒,具有一定的免疫調(diào)節(jié)活性,其中有機(jī)硒具有易吸收、高活性和低毒性的特點(diǎn)�；覙浠ㄊ且环N藥食兩用真菌,課題組前期研究發(fā)現(xiàn),其子實(shí)體具有較好的富硒能力,含硒蛋白是富硒灰樹花子實(shí)體中硒的主要賦存形式,占有機(jī)硒的57.52%。目前,對(duì)于富硒灰樹花含硒糖蛋白的分離純化、結(jié)構(gòu)表征及其對(duì)砷致免疫毒性的干預(yù)作用研究未見報(bào)道。本文優(yōu)化了富硒灰樹花水溶性含硒糖蛋白的提取工藝制得粗含硒糖蛋白,采用柱純化法,獲得純化組分并對(duì)其結(jié)構(gòu)進(jìn)行初步表征;采用NaAs O2致小鼠巨噬細(xì)胞RAW264.7免疫毒性模型,研究純化組分對(duì)砷致免疫毒性RAW264.7細(xì)胞的干預(yù)作用及分子機(jī)制;該研究對(duì)人類健康具有重要意義。主要研究?jī)?nèi)容如下:(1)富硒灰樹花水溶性含硒糖蛋白的提取工藝研究。采用RSM法優(yōu)化了富硒灰樹花水溶性粗含硒糖蛋白的提取工藝,最佳提取工藝條件為:提取溫度42.3℃、提取時(shí)間7.8h、料液比1:23.9(g/mL),該條件下得率為4.36%。(2)富硒灰樹花水溶性含硒糖蛋白的純化和初步表征。采用DEAE-52和Sephacryl S-400對(duì)Se-GPr和GPr進(jìn)行純化,分別獲得4個(gè)純化組分,硒含量分別為20.25、10.63、94.01和855.27μg/g,顯著高于相應(yīng)的未富硒純化組分;紅外分析結(jié)果發(fā)現(xiàn),與相應(yīng)GPr及其各純化組分相比,Se-GPr及其各純化組分表現(xiàn)為基本相同的特征吸收峰,Se-GPr22與Se-GPr44在1220~1020 cm-1處的C-H伸縮振動(dòng)和606~537 cm-1的C=O面外彎曲振動(dòng)明顯減弱,可能與硒對(duì)硫的取代有關(guān);氨基酸組成分析發(fā)現(xiàn),Se-GPr各純化組分氨基酸種類相對(duì)齊全,其中Se-GPr11和Se-GPr44基本符合理想蛋白質(zhì)條件;Se-GPr各純化組分的單糖組成為:Se-GPr11由阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖組成,Se-GPr22由阿拉伯糖、甘露糖、葡萄糖和半乳糖組成,Se-GPr33和Se-GPr44均由甘露糖、葡萄糖和半乳糖組成;電泳分析結(jié)果表明,各純化含硒糖蛋白均為單一組分,Se-GPr11的分子量為14.32kDa,Se-GPr22由20.57和31.12 kDa的兩個(gè)亞基組成,Se-GPr33由15.08、20.57和32.78kDa的三個(gè)亞基組成,Se-GPr44由16.73、32.78和42.46 kDa的三個(gè)亞基組成。(3)富硒灰樹花水溶性含硒糖蛋白對(duì)砷致免疫毒性的干預(yù)作用及分子機(jī)制。研究發(fā)現(xiàn),Se-GPr11和Se-GPr44對(duì)砷致免疫毒性有拮抗作用,事前干預(yù)24 h為實(shí)驗(yàn)最佳干預(yù)方式;Se-GPr11和Se-GPr44可通過促進(jìn)SOD活性,抑制MDA水平和ROS的產(chǎn)生量,拮抗砷免疫毒性作用,同時(shí),Se-GPr11和Se-GPr44可通過作用于MAPK通路,下調(diào)p-ERK、p-JNK、p-P38蛋白的表達(dá),促進(jìn)IL-2和INF-γ的分泌,對(duì)砷致RAW264.7細(xì)胞免疫毒性產(chǎn)生干預(yù)作用。
[Abstract]:At present, arsenic pollution has become one of the most serious global environmental and public health problems. Arsenic can cause multiple systemic toxicity, such as immunity, nerve, development and liver toxicity. Nutritional intervention is an effective method of prevention. Studies have shown that selenium can antagonize arsenic poisoning and has certain immunomodulatory activity, among which organic selenium has the characteristics of easy absorption, high activity and low toxicity. The previous study found that its fruiting body has good selenium enrichment ability, and the selenium protein is the main form of selenium in the fruiting body of selenium enriched Grifola frondosa, which occupies 57.52% of the selenium content. At present, the isolation and purification of selenium rich glycoprotein of Grifola frondosa are studied. The structure characterization and its intervention on arsenic induced immunotoxicity were not reported. In this paper, the extraction process of water-soluble selenium-containing glycoprotein from Selenium-enriched Grifola frondosa was optimized, and the crude selenium-containing glycoprotein was prepared by column purification method. The purified components were obtained and their structures were preliminarily characterized. The effects of purified components on arsenic induced immunotoxic RAW264.7 cells and its molecular mechanism were studied by using NaAs O2 induced mouse macrophage RAW264.7 immunotoxicity model. This study is of great significance to human health. The main contents of this study are as follows: 1) the extraction process of water-soluble selenium-containing glycoprotein from Se-enriched Grifola frondosa was studied. The extraction process of water-soluble crude selenium-containing glycoprotein from Se-enriched Grifola frondosa was optimized by RSM method. The optimum extraction conditions were as follows: extraction temperature 42.3 鈩，

本文編號(hào)：1503044