發(fā)布時(shí)間：2018-01-15 10:32

  本文關(guān)鍵詞：黃精多糖的分離、鑒定及免疫調(diào)節(jié)功效研究　出處：《山東大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 黃精多糖 提取分離 巨噬細(xì)胞 免疫調(diào)節(jié)

【摘要】：黃精是一種藥食同源的中藥材,其中含有多種對(duì)人體有益的活性成分。黃精多糖作為黃精的主要成分,具有多種生物活性,因此具有很高的應(yīng)用價(jià)值。多糖是一種廣泛存在于動(dòng)物和植物細(xì)胞中天然大分子物質(zhì),是構(gòu)成生命的四大基本物質(zhì)之一。研究表明,多糖具有抗腫瘤、調(diào)節(jié)血糖和血脂、抑菌和抗炎、調(diào)節(jié)免疫等作用,在功能食品、醫(yī)療保健方面具有良好的發(fā)展應(yīng)用前景。近年來(lái),中藥多糖的免疫調(diào)節(jié)作用成為研究熱點(diǎn),取得了很大的進(jìn)展。雖然對(duì)黃精及其多糖的研究有不少文獻(xiàn)報(bào)道,但大多集中在黃精多糖的提取分離純化,粗提物抑制腫瘤生長(zhǎng)、調(diào)節(jié)血糖、改善記憶等功效的研究,而黃精多糖純提取物的結(jié)構(gòu)鑒定及其免疫調(diào)節(jié)等生物活性的研究相對(duì)較少,并且從體內(nèi)和體外實(shí)驗(yàn)、細(xì)胞分子水平和基因水平闡明黃精多糖對(duì)細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)途徑的調(diào)節(jié)及其交互作用,找出其關(guān)鍵環(huán)節(jié),對(duì)于揭示生物學(xué)功能的發(fā)生機(jī)制具有重大意義。巨噬細(xì)胞作為一種免疫細(xì)胞,具有吞噬和介入炎癥反應(yīng)的作用,它廣泛分布于機(jī)體不同組織中,在天然免疫中發(fā)揮著重要作用。近年來(lái),大量體內(nèi)外研究證明,許多天然多糖具有調(diào)節(jié)巨噬細(xì)胞功能的作用。黃精多糖具有多種重要的生物學(xué)活性,其中最重要的就是免疫調(diào)節(jié)作用,其可以作為免疫調(diào)節(jié)劑,通過(guò)免疫調(diào)節(jié)來(lái)提升機(jī)體的免疫防御能力。本課題有關(guān)黃精多糖的分離、鑒定及免疫調(diào)節(jié)功效的主要研究?jī)?nèi)容分為三部分:(1)黃精多糖的制備、純化、分離以及抗氧化性能評(píng)價(jià),(2)黃精多糖在巨噬細(xì)胞RAW264.7體外免疫調(diào)節(jié)以及信號(hào)通路研究,(3)環(huán)磷酰胺所致免疫低下小鼠的體內(nèi)免疫調(diào)節(jié)作用及其機(jī)制。黃精多糖的制備和評(píng)價(jià)研究?jī)?nèi)容包括提取、純化、分離以及抗氧化性能的考察。利用水提醇沉法將黃精多糖從黃精中提取出來(lái),利用Sevage試劑脫蛋白、活性炭吸附法脫色,將所得到的黃精粗多糖進(jìn)行純化。采用DEAE-SepharoseFast Flow離子交換色譜對(duì)黃精多糖進(jìn)一步分離純化,得到四種組分,分別命名為PSP1、PSP2、PSP3、PSP4。采用紅外光譜和核磁共振分析對(duì)分離得到的黃精多糖進(jìn)行初步結(jié)構(gòu)鑒定,通過(guò)Size exclusion chromatography測(cè)定了黃精多糖的分子量,通過(guò)HPLC柱前衍生化法測(cè)定了黃精多糖的單糖組成;體外抗氧化實(shí)驗(yàn)表明,黃精多糖具有較好的自由基清除能力和還原能力。黃精多糖體外免疫療效的考察由巨噬細(xì)胞RAW264.7吞噬能力、一氧化氮和細(xì)胞因子調(diào)節(jié)以及信號(hào)通路研究組成。本實(shí)驗(yàn)選用的細(xì)胞模型為小鼠RAW264.7巨噬細(xì)胞系,利用中性紅實(shí)驗(yàn)測(cè)定了巨噬細(xì)胞的吞噬能力,通過(guò)硝酸還原酶法測(cè)定細(xì)胞上清中的一氧化氮(NO)含量,細(xì)胞因子IL-6和TNF-α的含量以及它們對(duì)應(yīng)的mRNA的表達(dá)分別通過(guò)酶聯(lián)免疫吸附法和RT-PCR來(lái)測(cè)定。利用Western Blot 方法檢測(cè)了 RAW264.7 細(xì)胞中 p38 MAPK、I1κB-α、NF-κB p65、iNOS 和 COX-2的蛋白表達(dá),并用免疫熒光法檢測(cè)了 NF-κBP65的核轉(zhuǎn)移。結(jié)果表明黃精多糖可以促進(jìn)巨噬細(xì)胞吞噬,增加NO、TNF-α和IL-6的分泌。在機(jī)制方面,黃精多糖促進(jìn)了 IκB-α降解,增強(qiáng)了 p38 MAPK、NF-κB p65、iNOS 和 COX-2 的表達(dá),并呈良好的劑量依賴(lài)關(guān)系,同時(shí)提升了 NF-κB的核轉(zhuǎn)移過(guò)程。實(shí)驗(yàn)結(jié)果說(shuō)明,黃精多糖具有明顯的免疫增強(qiáng)活性,進(jìn)一步表明其有助于促進(jìn)細(xì)胞介導(dǎo)免疫反應(yīng)。黃精多糖體內(nèi)免疫調(diào)節(jié)作用研究包括:環(huán)磷酰胺所致免疫低下小鼠的腹腔巨噬細(xì)胞吞噬能力,脾臟T和B淋巴細(xì)胞增殖能力,血清中細(xì)胞因子的免疫應(yīng)答。采用環(huán)磷酰胺建造非特異性免疫功能低下的實(shí)驗(yàn)動(dòng)物模型,分別從免疫器官指數(shù)、免疫細(xì)胞(T淋巴細(xì)胞、B淋巴細(xì)胞、腹腔巨噬細(xì)胞)活性以及細(xì)胞因子(TNF-α、IL-2、IL-8、IL-10)的變化等方面考察了黃精多糖免疫調(diào)節(jié)作用。結(jié)果表明:黃精多糖不但能夠提高免疫細(xì)胞的活性,同時(shí)還可以調(diào)節(jié)血清中TNF-α、IL-2、IL-8和IL-10等因子的水平,進(jìn)而平衡Th1和Th2。換句話說(shuō),黃精多糖能夠提高機(jī)體免疫力。綜上所述,本課題首先提取純化了黃精多糖并對(duì)其結(jié)構(gòu)和理化性質(zhì)進(jìn)行了評(píng)價(jià);巨噬細(xì)胞RAW264.7體外免疫活性實(shí)驗(yàn)結(jié)果表明,黃精多糖能夠顯著提高細(xì)胞吞噬能力,增強(qiáng)一氧化氮和細(xì)胞因子免疫調(diào)節(jié)活性,其免疫作用機(jī)制是通過(guò)調(diào)節(jié)P38 MAPK和NF-κB信號(hào)通路來(lái)實(shí)現(xiàn)的;環(huán)磷酰胺所致免疫低下小鼠體內(nèi)實(shí)驗(yàn)顯示,黃精多糖能夠增強(qiáng)腹腔巨噬細(xì)胞吞噬能力以及脾臟T淋巴細(xì)胞和B淋巴細(xì)胞增殖能力,提升血清中細(xì)胞因子的免疫應(yīng)答。本課題從體內(nèi)體外兩個(gè)方面探索了黃精多糖免疫調(diào)節(jié)活性和作用機(jī)制,為進(jìn)一步開(kāi)發(fā)利用黃精中藥材提供了嶄新的免疫治療思路與途徑。
[Abstract]:Huang Jing is a Chinese herbal medicinal and edible active ingredients, which contains a variety of beneficial. Polygonatum Polysaccharide as the main component of polygonatum, has a variety of biological activity, so it has very high application value. The polysaccharide is a kind of widely exists in animal and plant cells in natural macromolecules. Is one of the four basic elements of life. The study shows that the polysaccharide with anti-tumor, regulate blood lipid, antibacterial and anti-inflammatory, regulate immune function in functional food, has good prospects for the development and application of medical care. In recent years, Chinese medicine immune regulation of polysaccharide has become a research hotspot, made great progress. Although the study of Polygonatum Polysaccharide and there are many reports, but mostly concentrated in the extraction and separation and purification of Polygonatum Polysaccharide, crude extracts inhibit the growth of tumor, regulate blood sugar, improve memory and other effects, and Huang Jing Study on structural identification and immune regulation of polysaccharide extracts from pure biological activity is relatively small, and from in vivo and in vitro experiments, molecular level and gene level to elucidate the regulation and the interaction of Polygonatum Polysaccharide on cell signal transduction pathways, and find out the key link, is of great significance to reveal the mechanism of the biological function of macrophages. As a kind of immune cells with phagocytosis and interventional inflammatory reaction, which are widely distributed in various tissues, plays an important role in innate immunity. In recent years, a large number of studies have proved that many kinds of natural polysaccharides with regulation of macrophage function. Polygonatum Polysaccharide has many important biological activities. Which is the most important immunomodulatory effects, which can be used as immunomodulator, through immune regulation to enhance the body's immune defense ability. This lesson The separation problem of Polygonatum Polysaccharide, the main research content identification and immune regulation function is divided into three parts: (1) Polygonatum Polysaccharide preparation, purification, separation and antioxidant performance evaluation, (2) Polygonatum Polysaccharide in regulating macrophage RAW264.7 in vitro and immune signal pathway, (3). Cyclophosphamide induced immunosuppressed mice immune regulation and its mechanism of Polygonatum Polysaccharide. The preparation and evaluation of research content including the extraction, purification, separation and antioxidant properties. Effects of extraction and alcohol precipitation method to Polygonatum Polysaccharide Extracted from Huang Jingzhong by water, removal of protein by Sevage reagent, activated carbon adsorption decolorization, will the crude polysaccharide was purified by DEAE-SepharoseFast Flow ion exchange chromatography of Polygonatum Polysaccharide were further purified by four components, which were named PSP1, PSP2, PSP3, PSP4. using infrared spectroscopy and nuclear magnetic resonance Analysis of Polygonatum isolated preliminary identification of polysaccharide, the molecular weight of chromatography was determined by Size exclusion of Polygonatum Polysaccharide, monosaccharide composition by HPLC derivatization method for the determination of Polygonatum Polysaccharide; that in vitro antioxidant experiment and Polygonatum Polysaccharide has good scavenging ability and reducing power effects of Polygonatum Polysaccharide in vitro immune efficacy of RAW264.7 by macrophage phagocytosis, nitric oxide and cytokine regulation and signaling pathway. The cell model used in the experiment of mouse RAW264.7 macrophages, macrophage phagocytosis was determined by neutral red test, determination of nitric oxide in the supernatant by the method of nitrate reductase (NO) the content, content of cytokines IL-6 and TNF- alpha expression and their corresponding mRNA respectively by enzyme-linked immunosorbent assay and RT-PCR were determined by Weste. RN Blot was detected in RAW264.7 cells p38 MAPK I1 kappa B- alpha kappa B, NF- p65, iNOS and the expression of COX-2 protein was detected by immunofluorescence and NF- kappa BP65 nuclear translocation. The results showed that Polygonatum Polysaccharide can promote macrophage phagocytosis, increase NO secretion of TNF- and IL-6. Machine system, Polygonatum Polysaccharide promotes I kappa B- alpha degradation, enhanced p38 MAPK, NF- p65 expression of iNOS kappa B, and COX-2, and showed a good dose-dependent relationship, while improving the nuclear transfer process of NF- kappa B. The experimental results show that Polygonatum Polysaccharide can increase the activity of the obvious immune, further that help to promote cell mediated immune response. Polygonatum polysaccharide immune regulation effects include: phagocytosis of peritoneal macrophages induced by cyclophosphamide immunosuppressed mice, spleen T and B lymphocyte proliferation, immune response cytokines in the serum of the ring. The experimental animal model of amidophosphoric built non specific immunity, respectively from the index of immune organs, immune cells (T lymphocytes, B lymphocytes, macrophages) activity and cytokine (TNF- alpha, IL-2, IL-8, IL-10) changes of Polygonatum Polysaccharide immune regulation effect. The results showed that: yellow the purified polysaccharides can not only improve the activity of immune cells, but also can adjust the TNF- alpha, serum IL-2, IL-8 and IL-10 levels of factor, and the balance between Th1 and Th2. in other words, Polygonatum Polysaccharide can enhance the immunity. In summary, this paper firstly extracted Polygonatum Polysaccharide was purified and its structure and principle chemical properties were evaluated; the experimental results of RAW264.7 macrophage immune activity in vitro showed that Polygonatum Polysaccharide can significantly improve cell phagocytosis, enhance nitric oxide and cytokine immune activity, its immune function The mechanism is realized by adjusting the P38 MAPK and NF- B signaling pathway; display immunosuppression mice induced by cyclophosphamide in vivo, Huang Jingduo sugar can enhance phagocytosis and spleen T lymphocyte and B lymphocyte proliferation ability of peritoneal macrophages, enhance the immune response of cytokines in serum. This topic from two aspects to explore the in vitro and in vivo of Polygonatum Polysaccharide immunomodulating activity and mechanism of action, provides a new way of thinking and immunotherapy for further development and utilization of polygonatumsibiricum from medicinal materials.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R284;R285

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