富硒灰树花水溶性含硒糖蛋白的分离纯化、初步表征及其对砷致免疫毒性的干预作用研究

发布时间：2018-02-11 12:20

  本文关键词： 砷污染 灰树花 含硒糖蛋白 分离纯化 免疫毒性 营养干预作用　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：目前,砷污染已成为极其严重的全球环境及公共卫生问题之一。砷可致机体多系统毒性,如免疫、神经、发育和肝脏毒性等。针对慢性砷中毒,营养干预是一种有效的预防方法。研究表明,硒可拮抗砷中毒,具有一定的免疫调节活性,其中有机硒具有易吸收、高活性和低毒性的特点。灰树花是一种药食两用真菌,课题组前期研究发现,其子实体具有较好的富硒能力,含硒蛋白是富硒灰树花子实体中硒的主要赋存形式,占有机硒的57.52%。目前,对于富硒灰树花含硒糖蛋白的分离纯化、结构表征及其对砷致免疫毒性的干预作用研究未见报道。本文优化了富硒灰树花水溶性含硒糖蛋白的提取工艺制得粗含硒糖蛋白,采用柱纯化法,获得纯化组分并对其结构进行初步表征;采用NaAs O2致小鼠巨噬细胞RAW264.7免疫毒性模型,研究纯化组分对砷致免疫毒性RAW264.7细胞的干预作用及分子机制;该研究对人类健康具有重要意义。主要研究内容如下:(1)富硒灰树花水溶性含硒糖蛋白的提取工艺研究。采用RSM法优化了富硒灰树花水溶性粗含硒糖蛋白的提取工艺,最佳提取工艺条件为:提取温度42.3℃、提取时间7.8h、料液比1:23.9(g/mL),该条件下得率为4.36%。(2)富硒灰树花水溶性含硒糖蛋白的纯化和初步表征。采用DEAE-52和Sephacryl S-400对Se-GPr和GPr进行纯化,分别获得4个纯化组分,硒含量分别为20.25、10.63、94.01和855.27μg/g,显著高于相应的未富硒纯化组分;红外分析结果发现,与相应GPr及其各纯化组分相比,Se-GPr及其各纯化组分表现为基本相同的特征吸收峰,Se-GPr22与Se-GPr44在1220~1020 cm-1处的C-H伸缩振动和606~537 cm-1的C=O面外弯曲振动明显减弱,可能与硒对硫的取代有关;氨基酸组成分析发现,Se-GPr各纯化组分氨基酸种类相对齐全,其中Se-GPr11和Se-GPr44基本符合理想蛋白质条件;Se-GPr各纯化组分的单糖组成为:Se-GPr11由阿拉伯糖、木糖、甘露糖、葡萄糖和半乳糖组成,Se-GPr22由阿拉伯糖、甘露糖、葡萄糖和半乳糖组成,Se-GPr33和Se-GPr44均由甘露糖、葡萄糖和半乳糖组成;电泳分析结果表明,各纯化含硒糖蛋白均为单一组分,Se-GPr11的分子量为14.32kDa,Se-GPr22由20.57和31.12 kDa的两个亚基组成,Se-GPr33由15.08、20.57和32.78kDa的三个亚基组成,Se-GPr44由16.73、32.78和42.46 kDa的三个亚基组成。(3)富硒灰树花水溶性含硒糖蛋白对砷致免疫毒性的干预作用及分子机制。研究发现,Se-GPr11和Se-GPr44对砷致免疫毒性有拮抗作用,事前干预24 h为实验最佳干预方式;Se-GPr11和Se-GPr44可通过促进SOD活性,抑制MDA水平和ROS的产生量,拮抗砷免疫毒性作用,同时,Se-GPr11和Se-GPr44可通过作用于MAPK通路,下调p-ERK、p-JNK、p-P38蛋白的表达,促进IL-2和INF-γ的分泌,对砷致RAW264.7细胞免疫毒性产生干预作用。
[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，

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