【作者】 刘娜；

【导师】 赵忠熙；

【作者基本信息】 山东大学 ， 药学（专业学位）， 2017， 硕士

【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。更多还原

【Abstract】 Polygonatum sibiricum,as a kind of medicinal and edible Chinese herbal medicine,has high application value.Polygonatum sibiricum contains polysaccharides,saponins,anthraquinone compounds,alkaloids,cardiac glycosides,lignans,vitamins and other beneficial to the human body active ingredients.Among them,Polygonatum sibiricum polysaccharide as the main component of Polygonatum sibiricum has a variety of biological activity,and doesn’t have obvious side effects,making Polygonatum sibiricum to be a natural medicine and health food.Polysaccharide is a kind of natural macromolecules,which widely exists in animals and plants cells and it is one of the four basic substances of life.Studies have shown that polysaccharide has anti-tumor,regulate blood sugar and blood lipids,anti-bacterial,anti-inflammatory and immune regulation activities.Polysaccharide has good application prospect in the development of functional food and health care.In recent years,the immunomodulatory effect of Polysaccharide from traditional Chinese medicine(TCM)has been a hot topic.Although the research on Polygonatum sibiricum and its polysaccharides has been reported in many literatures,most of them have been concentrated on the extraction and purification of polysaccharides,the effects of crude extracts on tumor growth,blood glucose regulation and memory improvement.Structure identification and immunoregulation of Polygonatum sibiricum polysaccharides were relatively few,and the regulation of the cell signal transduction pathway and its interaction were elucidated by in vivo and in vitro experiments,cell molecular level and gene level,and the key link was found which has great significance to reveal the mechanism of their various biological functions.Macrophages are widely distributed in different tissues of the body.They are a kind of immune cells with phagocytosis and inflammatory reaction,and play an important role in innate immunity.In recent years,a large number of in vivo and in vitro studies have confirmed that many natural polysaccharides can regulate the function of macrophages.Polygonatum sibiricum polysaccharide has many important biological activities and the most important one is the immune activity.Polygonatum sibiricum polysaccharide can be used as immunomodulator to enhance immune defenses by immune regulation.The main contents of this study are as follows:(1)Preparation,purification,isolation and evaluation of antioxidant properties of Polygonatum sibiricum polysaccharide,(2)Immunization regulation and signal pathway studies of Polygonatum sibiricum polysaccharide in macrophages RAW264.7,(3)The effect and its mechanism of immune regulation in cyclophosphamide-induced immunocompromised mice.Studies on the preparation and evaluation of Polysaccharides from Polygonatum sibiricum include extraction,purification,isolation and antioxidant activity.The polysaccharides of Polygonatum sibiricum were extracted by water extraction and alcohol precipitation and were purified by Sevage reagent and activated carbon adsorption.DEAE-Sepharose Fast Flow ion exchange chromatography was used to separate and purify Polygonatum sibiricum polysaccharide,and the four components were PSP1,PSP2,PSP3,PSP4,respectively.The preliminary structure identification of Polygonatum polysaccharides was identified by IR and NMR,and the molecular weight was determined by Size exclusion chromatography.The monosaccharide composition of polysaccharides from Polygonatum sibiricum was identified by HPLC pre-column derivatization.The in vitro antioxidant experiments showed that the polysaccharides had good free radical scavenging ability and reducing ability.The study on the immune effect of Polygonatum sibiricum polysaccharide in vitro was studied by the phagocytic capacity of macrophages,the regulation of nitric oxide and cytokines,as well as the signal pathway.In this study,the RAW264.7 cell was selected as the cell model,and the phagocytic capacity of macrophages was determined by neutral red assay.Nitric oxide(NO)content was determined by nitrate reductase method.The levels of cytokines IL-6 and TNF-α and the expression of their corresponding mRNA was determined by ELISA and RT-PCR,respectively.The expression of p38 MAPK,IκB-α,NF-κB p65,iNOS and COX-2 in RAW264.7 cells was detected by Western Blot,and the nuclear transfer of NF-κB p65 was detected by immunofluorescence assay.The results showed that Polygonatum Polysaccharide could promote the proliferation and phagocytic activity of macrophages,enhance the immune activity of NO,TNF-α and IL-6.In the aspect of mechanism,Polygonatum sibiricum polysaccharide promoted the degradation of IκB-α,enhanced the expression of p38 MAPK,NF-κB,iNOS and COX-2 in a dose-dependent manner,and promoted the nuclear transfer process of NF-κB.The results showed that Polygonatum sibiricum polysaccharide had obvious immune enhancing activity,and further indicated that it could promote the cell mediated immune response.Polygonatum sibiricum polysaccharide in vivo immunomodulatory effects on the immunocompromised mice induced by cyclophosphamide included phagocytosis of mouse peritoneal macrophages,the proliferation of spleen T and B lymphocytes,and the immune response of cytokines in serum.Immune cells(T lymphocytes,B lymphocytes,peritoneal macrophages)and cytokines(TNF-a,IL-2,IL-8,IL-10)were used to investigate the immumoregulation of Polygonatum sibiricum polysaccharide.The results show that Polygonatum sibiricum polysaccharide can improve the index of immune organs,the ability of the phagocytosis of mouse peritoneal macrophages,enhance the proliferation ability of T and B lymphocytes in the spleen.The levels of TNF-a,IL-2 and IL-8 in serum decreased and the level of IL-10 increased significantly,controlling the imbalance of Th 1 and Th 2.The results indicated that Polygonatum sibiricum polysaccharide could activate the specific immune response,and had a positive effect on cellular immunity and humoral immunity.In summary,the polysaccharide was extracted and purified and its structure and physicochemical properties were evaluated.The activity of RAW264.7 cells in vitro showed that the polysaccharide of Polygonatum sibiricum could significantly improve phagocytosis,enhance nitric oxide and Cytokine immunoregulatory activity.The immune mechanism is the regulation of p38 MAPK and NF-κB signaling pathway.Immunocompromised mice induced by cyclophosphamide showed that Polygonatum sibiricum polysaccharide can enhance the phagocytic capacity of peritoneal macrophages and the spleen T and B lymphocyte proliferation ability,enhance the immune response of cytokines in serum.In this study,the immunoregulatory activity and mechanism of polysaccharides from Polygonatum sibiricum were explored in vitro and in vivo,which provided a new idea and approach for the further development and utilization of Polygonatum sibiricum.更多还原