小麦麸皮多糖对巨噬细胞免疫调节作用的研究

发布时间：2018-04-23 04:26

本文选题：小麦麸皮多糖 + 免疫调节　；参考：《扬州大学》2017年硕士论文

【摘要】：小麦是世界上最早栽培的农作物之一属禾本科单子叶植物纲植物。已有文献报道小麦麸皮中含有的膳食纤维具有降血糖、降低血清胆固醇、预防便秘、调节血糖水平及免疫调节的生理功能,但关于小麦麸皮多糖在细胞分子水平的免疫调节作用及机制鲜有报道。本文以小麦麸皮(淮麦33)为实验材料,首先研究不同提取方法对小麦麸皮多糖提取率、提取后麸皮残渣微观形态、糖醛酸含量、硫酸根含量、蛋白质含量、单糖组成及免疫调节活性的影响;其次,通过体外实验研究小麦麸皮多糖对巨噬细胞RAW264.7细胞毒性、分泌细胞因子NO、PGE2、TNF-α水平等细胞活性的变化,验证其免疫调节作用及从小麦麸皮多糖在RAW264.7细胞中引起iNOS、COX-2、TNF-α的mRNA表达量及其蛋白表达情况的变化来探究可能的活化分子机制,并通过建立免疫抑制小鼠模型,小麦麸皮多糖灌胃小鼠,用ELISA试剂盒法检测IL-2及IFN-γ的分泌水平,测定小鼠脾脏和胸腺指数,探讨小麦麸皮多糖对免疫抑制小鼠的免疫调节作用。最后,利用DEAE-52纤维素柱层析及葡聚糖凝胶G-100柱层析对初步纯化的小麦麸皮多糖进一步纯化分级;同时,通过高效液相色谱(HPLC)和气相色谱分析(GC)测定纯化后小麦麸皮多糖的分子量大小及其单糖的组成、物质的量之比;利用红外光谱对小麦麸皮多糖初步结构进行解析,研究小麦麸皮多糖结构与其药理活性之间的关系。主要研究结果如下:(1)不同提取方法提取的小麦麸皮多糖,碱提法获得的粗多糖得率最高为31.73%,其含量为57.79%;糖醛酸和硫酸根的含量依次为:碱提法酶提法酸提法水提法;四种提取方法所得小麦麸皮多糖均由阿拉伯糖、木聚糖及葡萄糖组成;MTT法检测四种方法所得小麦麸皮多糖对细胞均无毒性,其中水提法获得的多糖能显著促进RAW264.7细胞分泌NO因子并能促进细胞iNOS和COX-2蛋白的表达。(2)体外实验研究结果表明:小麦麸皮多糖对巨噬细胞RAW264.7无毒性作用,且小麦麸皮多糖可通过调节NF-κB、AP-1、p38MAPK信号转导通路促进巨噬细胞的免疫功能。此外,体内实验结果显示:小麦麸皮多糖能够增强环磷酰胺所致免疫低下小鼠的胸腺和脾脏器官指数,并上调细胞因子的分泌水平。(3)小麦麸皮水溶性粗多糖经分离纯化后得到3个组分CWBP-I、CWBP-2、CWBP-3;通过HPLC测得纯化后小麦麸皮多糖的3个组分的平均分子量大小分别是182.73 kDa、90.104 kDa、40.721 kDa;GC测得小麦麸皮多糖3个组分均由阿拉伯糖和木糖组成,其摩尔质量之比依次为1:0.68、1:1.45、1:1.14;红外光谱分析结果显示在1045 cm-1波长处的吸收峰为阿拉伯木聚糖的特征吸收峰。(4)小麦麸皮纯化多糖的3个组分均可提高RAW264.7细胞的免疫活性,且CWBP-2组分的免疫调节功能尤为显著。
[Abstract]:Wheat is one of the earliest cultivated crops in the world. It has been reported that the dietary fiber contained in wheat bran has the physiological function of lowering blood sugar, lowering serum cholesterol, preventing constipation, regulating blood sugar level and immune regulation. However, there are few reports on the immunomodulation and mechanism of wheat bran polysaccharides at cellular and molecular level. In this paper, wheat bran (Huaimai 33) was used as experimental material. The extraction rate of polysaccharides from wheat bran by different extraction methods, the micromorphology of wheat bran residue, the content of uronic acid, the content of sulfate radical and the content of protein were studied. Effects of monosaccharide composition and immunomodulatory activity. Secondly, the effects of wheat bran polysaccharides on macrophage RAW264.7 cell toxicity and the level of the secreting cytokine NOPGE2TNF- 伪 were studied in vitro. To verify its immunomodulatory effect and to explore the possible mechanism of activation by inducing the changes of mRNA expression and protein expression of iNOSS-COX-2TNF- 伪 in RAW264.7 cells from wheat bran polysaccharides, and to establish an immunosuppressive mouse model. The secretion levels of IL-2 and IFN- 纬 were detected by ELISA kit, spleen and thymus index of mice were measured, and the immunomodulatory effects of wheat bran polysaccharides on immunosuppressive mice were studied. Finally, the primary purified wheat bran polysaccharides were further purified and classified by DEAE-52 cellulose column chromatography and dextran gel G-100 column chromatography. The molecular weight of purified wheat bran polysaccharides, the composition of monosaccharide and the ratio of monosaccharide to substance were determined by high performance liquid chromatography (HPLC) and gas chromatography (GC), and the preliminary structure of wheat bran polysaccharides was analyzed by infrared spectroscopy. The relationship between the structure of wheat bran polysaccharide and its pharmacological activity was studied. The main results are as follows: (1) the yield of crude polysaccharides extracted from wheat bran by different extraction methods was 31.73, the content of glyuronic acid and sulfate was 57.79, the content of uronic acid and sulfate was in turn: alkali extraction enzyme extraction acid extraction water extraction; Wheat bran polysaccharides obtained by four extraction methods were all composed of arabinose, xylan and glucose. MTT method was used to detect the cytotoxicity of wheat bran polysaccharides obtained by four methods. The polysaccharides obtained by water extraction can significantly promote the secretion of no factor by RAW264.7 cells and the expression of iNOS and COX-2 protein in vitro. The results showed that wheat bran polysaccharides had no toxic effect on RAW264.7 of macrophages. Wheat bran polysaccharides can promote the immune function of macrophages by regulating NF- 魏 B- AP-1 and p38 MAPK signal transduction pathway. In addition, the results of in vivo experiments showed that wheat bran polysaccharides could enhance the thymus and spleen organ index of immunocompromised mice induced by cyclophosphamide. After purification, three components, CWBP-IK, CWBP-2, CWBP-3, were obtained, and the average molecular weight of the three components of purified wheat bran polysaccharides were 182.73 kDa 90.104 kDa 40.721 kDa GC, respectively. The results showed that the three components of wheat bran polysaccharides were composed of arabinose and xylose. The molar ratio of the purified polysaccharides was 1: 0.68 / 1: 1: 1.14. The IR spectra showed that the absorption peak at the wavelength of 1045 cm-1 was the characteristic absorption peak of arabinoxylum. 4) the three components of purified polysaccharides from wheat bran could improve the immune activity of RAW264.7 cells. The immunomodulatory function of CWBP-2 components was particularly significant.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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