黄精多糖的分离、鉴定及免疫调节功效研究

发布时间：2018-01-15 10:32

本文关键词：黄精多糖的分离、鉴定及免疫调节功效研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：黄精是一种药食同源的中药材,其中含有多种对人体有益的活性成分。黄精多糖作为黄精的主要成分,具有多种生物活性,因此具有很高的应用价值。多糖是一种广泛存在于动物和植物细胞中天然大分子物质,是构成生命的四大基本物质之一。研究表明,多糖具有抗肿瘤、调节血糖和血脂、抑菌和抗炎、调节免疫等作用,在功能食品、医疗保健方面具有良好的发展应用前景。近年来,中药多糖的免疫调节作用成为研究热点,取得了很大的进展。虽然对黄精及其多糖的研究有不少文献报道,但大多集中在黄精多糖的提取分离纯化,粗提物抑制肿瘤生长、调节血糖、改善记忆等功效的研究,而黄精多糖纯提取物的结构鉴定及其免疫调节等生物活性的研究相对较少,并且从体内和体外实验、细胞分子水平和基因水平阐明黄精多糖对细胞信号转导途径的调节及其交互作用,找出其关键环节,对于揭示生物学功能的发生机制具有重大意义。巨噬细胞作为一种免疫细胞,具有吞噬和介入炎症反应的作用,它广泛分布于机体不同组织中,在天然免疫中发挥着重要作用。近年来,大量体内外研究证明,许多天然多糖具有调节巨噬细胞功能的作用。黄精多糖具有多种重要的生物学活性,其中最重要的就是免疫调节作用,其可以作为免疫调节剂,通过免疫调节来提升机体的免疫防御能力。本课题有关黄精多糖的分离、鉴定及免疫调节功效的主要研究内容分为三部分:(1)黄精多糖的制备、纯化、分离以及抗氧化性能评价,(2)黄精多糖在巨噬细胞RAW264.7体外免疫调节以及信号通路研究,(3)环磷酰胺所致免疫低下小鼠的体内免疫调节作用及其机制。黄精多糖的制备和评价研究内容包括提取、纯化、分离以及抗氧化性能的考察。利用水提醇沉法将黄精多糖从黄精中提取出来,利用Sevage试剂脱蛋白、活性炭吸附法脱色,将所得到的黄精粗多糖进行纯化。采用DEAE-SepharoseFast Flow离子交换色谱对黄精多糖进一步分离纯化,得到四种组分,分别命名为PSP1、PSP2、PSP3、PSP4。采用红外光谱和核磁共振分析对分离得到的黄精多糖进行初步结构鉴定,通过Size exclusion chromatography测定了黄精多糖的分子量,通过HPLC柱前衍生化法测定了黄精多糖的单糖组成;体外抗氧化实验表明,黄精多糖具有较好的自由基清除能力和还原能力。黄精多糖体外免疫疗效的考察由巨噬细胞RAW264.7吞噬能力、一氧化氮和细胞因子调节以及信号通路研究组成。本实验选用的细胞模型为小鼠RAW264.7巨噬细胞系,利用中性红实验测定了巨噬细胞的吞噬能力,通过硝酸还原酶法测定细胞上清中的一氧化氮(NO)含量,细胞因子IL-6和TNF-α的含量以及它们对应的mRNA的表达分别通过酶联免疫吸附法和RT-PCR来测定。利用Western Blot 方法检测了 RAW264.7 细胞中 p38 MAPK、I1κB-α、NF-κB p65、iNOS 和 COX-2的蛋白表达,并用免疫荧光法检测了 NF-κBP65的核转移。结果表明黄精多糖可以促进巨噬细胞吞噬,增加NO、TNF-α和IL-6的分泌。在机制方面,黄精多糖促进了 IκB-α降解,增强了 p38 MAPK、NF-κB p65、iNOS 和 COX-2 的表达,并呈良好的剂量依赖关系,同时提升了 NF-κB的核转移过程。实验结果说明,黄精多糖具有明显的免疫增强活性,进一步表明其有助于促进细胞介导免疫反应。黄精多糖体内免疫调节作用研究包括:环磷酰胺所致免疫低下小鼠的腹腔巨噬细胞吞噬能力,脾脏T和B淋巴细胞增殖能力,血清中细胞因子的免疫应答。采用环磷酰胺建造非特异性免疫功能低下的实验动物模型,分别从免疫器官指数、免疫细胞(T淋巴细胞、B淋巴细胞、腹腔巨噬细胞)活性以及细胞因子(TNF-α、IL-2、IL-8、IL-10)的变化等方面考察了黄精多糖免疫调节作用。结果表明:黄精多糖不但能够提高免疫细胞的活性,同时还可以调节血清中TNF-α、IL-2、IL-8和IL-10等因子的水平,进而平衡Th1和Th2。换句话说,黄精多糖能够提高机体免疫力。综上所述,本课题首先提取纯化了黄精多糖并对其结构和理化性质进行了评价;巨噬细胞RAW264.7体外免疫活性实验结果表明,黄精多糖能够显著提高细胞吞噬能力,增强一氧化氮和细胞因子免疫调节活性,其免疫作用机制是通过调节P38 MAPK和NF-κB信号通路来实现的;环磷酰胺所致免疫低下小鼠体内实验显示,黄精多糖能够增强腹腔巨噬细胞吞噬能力以及脾脏T淋巴细胞和B淋巴细胞增殖能力,提升血清中细胞因子的免疫应答。本课题从体内体外两个方面探索了黄精多糖免疫调节活性和作用机制,为进一步开发利用黄精中药材提供了崭新的免疫治疗思路与途径。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285

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