本文選題：小麥麩皮多糖 + 免疫調(diào)節(jié)��； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：小麥?zhǔn)鞘澜缟献钤缭耘嗟霓r(nóng)作物之一屬禾本科單子葉植物綱植物。已有文獻(xiàn)報(bào)道小麥麩皮中含有的膳食纖維具有降血糖、降低血清膽固醇、預(yù)防便秘、調(diào)節(jié)血糖水平及免疫調(diào)節(jié)的生理功能,但關(guān)于小麥麩皮多糖在細(xì)胞分子水平的免疫調(diào)節(jié)作用及機(jī)制鮮有報(bào)道。本文以小麥麩皮(淮麥33)為實(shí)驗(yàn)材料,首先研究不同提取方法對(duì)小麥麩皮多糖提取率、提取后麩皮殘?jiān)⒂^形態(tài)、糖醛酸含量、硫酸根含量、蛋白質(zhì)含量、單糖組成及免疫調(diào)節(jié)活性的影響;其次,通過(guò)體外實(shí)驗(yàn)研究小麥麩皮多糖對(duì)巨噬細(xì)胞RAW264.7細(xì)胞毒性、分泌細(xì)胞因子NO、PGE2、TNF-α水平等細(xì)胞活性的變化,驗(yàn)證其免疫調(diào)節(jié)作用及從小麥麩皮多糖在RAW264.7細(xì)胞中引起iNOS、COX-2、TNF-α的mRNA表達(dá)量及其蛋白表達(dá)情況的變化來(lái)探究可能的活化分子機(jī)制,并通過(guò)建立免疫抑制小鼠模型,小麥麩皮多糖灌胃小鼠,用ELISA試劑盒法檢測(cè)IL-2及IFN-γ的分泌水平,測(cè)定小鼠脾臟和胸腺指數(shù),探討小麥麩皮多糖對(duì)免疫抑制小鼠的免疫調(diào)節(jié)作用。最后,利用DEAE-52纖維素柱層析及葡聚糖凝膠G-100柱層析對(duì)初步純化的小麥麩皮多糖進(jìn)一步純化分級(jí);同時(shí),通過(guò)高效液相色譜(HPLC)和氣相色譜分析(GC)測(cè)定純化后小麥麩皮多糖的分子量大小及其單糖的組成、物質(zhì)的量之比;利用紅外光譜對(duì)小麥麩皮多糖初步結(jié)構(gòu)進(jìn)行解析,研究小麥麩皮多糖結(jié)構(gòu)與其藥理活性之間的關(guān)系。主要研究結(jié)果如下:(1)不同提取方法提取的小麥麩皮多糖,堿提法獲得的粗多糖得率最高為31.73%,其含量為57.79%;糖醛酸和硫酸根的含量依次為:堿提法酶提法酸提法水提法;四種提取方法所得小麥麩皮多糖均由阿拉伯糖、木聚糖及葡萄糖組成;MTT法檢測(cè)四種方法所得小麥麩皮多糖對(duì)細(xì)胞均無(wú)毒性,其中水提法獲得的多糖能顯著促進(jìn)RAW264.7細(xì)胞分泌NO因子并能促進(jìn)細(xì)胞iNOS和COX-2蛋白的表達(dá)。(2)體外實(shí)驗(yàn)研究結(jié)果表明:小麥麩皮多糖對(duì)巨噬細(xì)胞RAW264.7無(wú)毒性作用,且小麥麩皮多糖可通過(guò)調(diào)節(jié)NF-κB、AP-1、p38MAPK信號(hào)轉(zhuǎn)導(dǎo)通路促進(jìn)巨噬細(xì)胞的免疫功能。此外,體內(nèi)實(shí)驗(yàn)結(jié)果顯示:小麥麩皮多糖能夠增強(qiáng)環(huán)磷酰胺所致免疫低下小鼠的胸腺和脾臟器官指數(shù),并上調(diào)細(xì)胞因子的分泌水平。(3)小麥麩皮水溶性粗多糖經(jīng)分離純化后得到3個(gè)組分CWBP-I、CWBP-2、CWBP-3;通過(guò)HPLC測(cè)得純化后小麥麩皮多糖的3個(gè)組分的平均分子量大小分別是182.73 kDa、90.104 kDa、40.721 kDa;GC測(cè)得小麥麩皮多糖3個(gè)組分均由阿拉伯糖和木糖組成,其摩爾質(zhì)量之比依次為1:0.68、1:1.45、1:1.14;紅外光譜分析結(jié)果顯示在1045 cm-1波長(zhǎng)處的吸收峰為阿拉伯木聚糖的特征吸收峰。(4)小麥麩皮純化多糖的3個(gè)組分均可提高RAW264.7細(xì)胞的免疫活性,且CWBP-2組分的免疫調(diào)節(jié)功能尤為顯著。
[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- 偽 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- 偽 in RAW264.7 cells from wheat bran polysaccharides, and to establish an immunosuppressive mouse model. The secretion levels of IL-2 and IFN- 緯 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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R151

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