【作者】 徐晓飞；

【导师】 张学武；

【作者基本信息】 华南理工大学 ， 制糖工程， 2014， 博士

【摘要】 真菌多糖是一大类的重要大分子物质，从20世纪70年代开始发现香菇多糖Lentinan的抗肿瘤活性以来，越来越多的生物活性被发现，包括抗肿瘤、免疫调节、抗菌、抗病毒、降血糖、降血脂、抗氧化等。近年来许多研究发现，真菌多糖口服后也依然能发挥生物活性。然而，真菌多糖口服后是如何发挥作用的机制和特点了解非常有限，特别是多糖口服后与机体接触的肠道部位发生怎样的变化。香菇作为药食两用真菌，在食品、保健食品和药品领域都有广泛的应用。随着技术发展和研究深入，许多种香菇多糖已从香菇中得到分离和鉴定。在本课题的前期研究中，采用水提、醇沉从香菇子实体中得到一种香菇杂多糖（命名为L2），平均分子量（Mw）为26KDa。主要由葡萄糖(87.5%)、半乳糖(9.6%)和阿拉伯糖(2.8%)组成。香菇多糖L2在121℃及以下、pH4.0-8.0环境下，具有良好的免疫增强活性，其中TLR2（Toll-like receptor2）是L2的免疫细胞受体之一。在本课题中，利用NMR技术进一步表征L2的主链结构为β-D-(16)连接葡萄糖带少量-D-(16)连接阿拉伯糖，并在部分糖基上C-O3和C-O4上有分支。选用C57BL/6成年和老龄小鼠评估L2口服后免疫功能特性，L2使用40mg/kg day剂量连续灌胃小鼠四周，提高成年小鼠胸腺指数和脾脏指数（增加胸腺和脾脏重量），增加脾T淋巴细胞增殖能力和NK（Natural killer）细胞的杀伤活性，增加外周血清中IL-2(Interleukin2)、IFN-γ (Interferon γ)和IL-12的浓度；提高老龄小鼠的免疫功能，包括增加老龄小鼠的脾脏指数、脾淋巴细胞增殖能力、外周血中IL-2和IL-6的浓度。针对L2对小鼠肠道细菌组成影响，利用第二代高通量焦磷酸测序技术（测16S rRNA基因），检测成年及老年小鼠粪便菌群组成结构。结果表明L2显著改变成年小鼠肠道菌群结构，包括增加小鼠粪便菌群的丰富度，但降低粪便菌群的多样性。然而，L2提高老龄小鼠粪便菌群组成的丰富度、多样性和均匀性。表明香菇多糖对肠道菌群结构的多样性、均匀性具有双向调节作用，调节的方向与属主免疫功能状态相关。具体地，对成年小鼠而言，在门(phylum)分类水平上，变形菌门（Proteobacteria）比例显著性增加；在纲(class)分类水平上，-变形菌纲（Epsilonproteobacteria）比例显著增加；在科(family)分类水平上，螺杆菌科（Helicobacteraceae）比例显著增加，而S24-7比例显著降低。特别地，在种（species）水平上，Helicobacter suncus显著增加；而对老龄小鼠，在门（phylum）、纲（class）和科(family)分类水平上，香菇多糖L2能部分恢复小鼠老龄相关的肠道粪便菌群结构的变化，如扭转衰老相关的F/B值和Bacteroidia比例的增加。这些变化可能与L2对肠粘膜免疫系统的调节特性和肠道菌群的营养性增殖作用有关。L2对成年小鼠和老年小鼠的免疫调节和肠道菌群组成影响的结果表明了L2的免疫调节作用可能与肠道菌群中Helicobacter（如Helicobacter suncus）、Alistipes和Bacteroides acidifaciens中的一些细菌比例变化有关。在沿肠道的菌群空间影响方面，L2降低成年小鼠盲肠和结肠部位菌群的丰富度、多样性和均匀性，但对小肠部位菌群的丰富度、多样性和均匀性无明显影响。具体而言，在门(phylum)分类水平上，增加小肠部位的菌群门类的种类；在盲肠部位，变形菌门（Proteobacteria）的比例上升而蓝细菌门（Cyanobacteria）的比例下降；在结肠部位，拟杆菌门（Bacteroidetes, B）,变形菌门（Proteobacteria）的比例增加，而厚壁菌门（Firmicutes, F）的比例下降，呈现偏向拟杆菌为主导。结果提示免疫功能变化与肠道菌群的多样性、均匀性和丰富度的变化有关联。L2对肠道菌群的影响既可能包括通过影响免疫系统功能对肠道菌群的间接作用，也有香菇多糖L2被肠道菌群发酵利用的作用。为了考察L2对肠道上皮细胞的直接影响，利用Caco-2/RAW264.7cells模型，考察L2对肠上皮细胞免疫因子分泌的作用特点。研究显示，L2刺激肠上皮细胞Caco-2分泌的免疫因子模式与酵母多糖Zymosan A不同，提示不同结构的多糖口服后可能呈现不同特征的免疫调节作用。同时，L2对Caco-2细胞分泌的免疫因子模式受到肠道革兰氏阴性菌成分LPS和免疫细胞参与影响，提示香菇多糖L2、肠粘膜免疫系统和肠道菌群之间复杂的相互作用。利用RT-PCR(Real time-PCR)对成年小鼠沿肠道免疫基因相关基因表达检测表明，L2导致在小肠部位的基因表达以上调为主，而盲肠、结肠部位的基因表达则以下调为主。L2上调趋化因子、干扰素和白介素、肿瘤坏死因子、病原菌模式识别受体基因的转录；对TNF超家族成员则表现为下调作用；对细胞连结屏障功能和抗菌相关基因则既有上调也有下调；对TLRs先天免疫信号通路及细胞免疫调节相关基因则以下调为主。L2对沿肠道免疫相关基因的转录调节可能是通过直接对肠粘膜免疫系统的刺激作用和改变肠道菌群组成结构来实现。对差异表达基因利用R spider分析，分别构建出由15（小肠）、5（盲肠）、10（结肠）个基因组成的3个功能网络。在小肠部位功能主要涉及：免疫反应、调控细胞凋亡、异蛋白低聚化、血小板源生长因子受体信号通路、细胞内信号通路、正向调控DNA复制。在盲肠部位涉及功能是：B细胞激活、异蛋白低聚化。在结肠部位涉及功能是：发炎反应、细胞增殖、LPS调控的信号通路、T细胞激活和正向调控NF-kB转录因子活性。提示L2在肠道不同部位（小肠、盲肠、结肠）所影响及发挥的生理功能可能不一样，反映了肠道菌群与属主相互作用的复杂性和菌群—机体组织影响之间的特异性。最后，利用蛋白组学技术对成年小鼠小肠和结肠组织蛋白表达检测，L2对小肠组织蛋白的表达影响大于结肠组织蛋白。在小肠处鉴定出25个差异表达蛋白，在结肠处鉴定出22个差异表达蛋白。小肠、结肠处的差异表达蛋白仅有少量种类重叠，绝大部分均不同，提示L2的生物学作用广泛，进一步验证了L2口服后对肠道作用的组织部位特异性。大部分的差异表达蛋白生物学功能主要集中于细胞组成成分（cellular componentorganization）、发育过程调节(developmental process)、细胞内调节（cellular process）、免疫调节(immune system process)、代谢调节(metabolic process)、多细胞组织过程(multicellular organismal process)、刺激反应(response to stimulus)，并选取了12个蛋白进行Western blot验证，结果与2DE趋势符合。STRING蛋白互作网络分析，印证了L2对肠道组织蛋白的表达呈现组织部位特异性。表明L2对蛋白表达影响可能主要是通过肠道菌群调节和直接刺激作用而实现。更多还原

【Abstract】 Fungal polysaccharides are wildly recognized for their bioactivities in anti-tumor,immuno-modulating, anti-bacteria, anti-virus, anti-hyperglycemia, anti-oxidant, etc. In recentyears, many researches revealed that fungal polysaccharides still owed high bioactivities afteroral administration. However, how the polysaccharides exerted their effects were still poorlyunderstood. Especially, what specific changes occured in the gut where the polysaccharidesdirectly contacted with intestine musical barrier. Lentinula edodes is the most popular edibleand medical mushrooms in the world. With the development of purification and structuredetermination methods, many types of polysaccharides have been obtained from the fruitbody of Lentinula edodes. In our previous study, a new heteropolysaccharide named L2wasseparated from the fruit body of Lentinula edodes by boiling water, followed with traditionalpurification procedure. Based on chemical and physical analyses, L2were mainly consisted ofglucose (87.5%), galactose (9.6%), and arabinose (2.8%) with average molecular weight of26K Da. The polysaccharides L2showed significant and toll-like receptor (TLR)2-involvedimmuno-stimulating activities which exhibited high stability in wide ranges of pH from4.0to10.0and temperature below121℃treatment with RAW264.7cells model.In present studies, the main chain structure characteristics were investigated by NMR.Subsequently, the immuno-modulating properties and mechanism of L2after oraladministration in mice were further investigated. NMR analysis indicated that the mainbackbone chain of L2were β-D-(16)-linked glucose and small proportion of-D-(16)-linked arabinose connected with side chains at C-O3and C-O4inmonosaccharide unit. Oral administration of L2with dosage of40mg/kg day for four weeks,the increased of spleen and thymus indices, natural killer cell (NK cell) activity, andproliferation activity of T cell in splenocytes as well as the increased cytokines of IL-2,IFN-γ,and IL-12in serum were observed in adult C57BL/6mice. Interestingly, the results alsoshowed that L2restored the age-attenuated immune responses by improving the proliferationactivity of lymphocytes in splenocytes and cytokines concentration (IL-2and IL-6) in serum.The gut microbes play a critical role in modulation of immune function. The impacts ofL2on the gut microbial communities were examined by high-throughput pyrosequencingtechnology targeting to16S rRNA gene. In the fecal microbial communities of adult mice, L2induced the increase of observed richness while reduced the-diversity. Specifically, thesignificantly increased Proteobacteria were observed in L2-treated mice, which werecharacterized by the increase of Epsilonproteobacteria at class level. The increased Helicobacteraceae and reduced S24-7were present in fecal microbiota after L2treatment inadult mice. In particular, some OTUs (Operational taxonomic units, OTUs), corresponding toHelicobacter suncus, were found to be significantly increased in L2treated-mice. However,L2partly reversed the age-altered composition of gut microbiota such as the decline of-diversity and evenness and the disproportions of some bacteria groups at phylum, class, andfamily levels in aged mice, such as the skewing ratio of Firmicutes/Bacteroidetes, theincreased proportion of Bacteroidia in fecal microbiota. These changes in fecal microbiotastructure might be related to the immune-stimulating of L2to intestinal immune system andfertilizing properties to gut microbiota. Taken together, L2showed dual-direction regulationactivities to fecal microbiota which depended on the status of host immunity. Specifically,some bacteria in Helicobacter（such as Helicobacter suncus）、Alistipes、and Bacteroidesacidifaciens might played important roles in the L2-induced immune-modulating responses inC57BL/6mice.In adult mice, the microbial communities exhibit distinctly different space distributionalong the intestine. L2treatment reduced the observed richness,-diversity, and evenness ofgut microbiota, especially in the cecum and colon. No obvious changes in richness,-diversity, and evenness were observed except for the increase number of bacterial phylumin the small intestine. However, the relative abundance of Proteobacteria was increasedaccompanying of the decreased of Cyanobacteria in cecum. In colon, the microbialcommunities were shifted to Bacteroidetes-dominated characterized by the increase ofBacteroidetes and Proteobacteria and the decrease of Firmicutes. These results furthersuggested the associations between the immune function and the richness,-diversity, andevenness of gut microbiota. The impacts of L2on gut microbial communities might beattributed to the immune-stimulating to intestinal immune system and utilization by gutmicrobiota.Furthermore, the immuno-stimulating profiles of L2were evaluated in Caco-2cells andCaco-2/RAW264.7co-culture system. Subsequently, the customized RT-PCR array containing112genes were employed to investigate the effects of L2on immune-related gene expressionsin small intestine, cecum and colon. The results showed that the patterns of cytokinessecretion induced by L2were significantly different from that of Zymosan A in vitro,indicating different immuno-modulating profiles might be observed after different structure ofpolysaccharides consumption. Especially, the patterns of cytokines secretion by Caco-2cellswere changed in presence of Gram-negative component, LPS, and RAW264.7macrophagecells, indicating the exsting of complicated interaction among polysaccharides L2, intestinal immune system, and gut microbiota. Interestingly, L2differentially impacted the geneexpressions of small intestine, cecum and colon, in which55,26and25genes were markedlychanged, respectively. Most of differential genes were up-regulated in the small intestinewhile majority of differential genes were down-regulated in the cecum and colon. Specificly,L2up-regulated genes related to chemokines, interferon, interleukins, TNF, and patternrecognition receptors, while down-regulated genes related to TLRs-involved signalingpathway and cellular immunity. These results indicated that the gut microbiota might playedimportant roles in the L2-treated gene expression along the intestine. Especially,3coreregulation networks were identified for small intestine (15-gene sub-network), cecum (5-genesub-network) and colon (10-gene sub-network) with R Spider tool. In small intestine, thedifferential genes were involved in biological processes such as regulation of apoptosis,immune response, protein heterooligomerization, platelet derived growth factor receptorsignaling pathway, intracellular signaling pathway, positive regulation of DNA replication. Incecum, B cell activation and protein heterooligomerization were related to differential genesin sub-network. In colon, biological processes such as immune responses (inflammatoryresponse, T cell activation), LPS mediated signaling pathway, cell proliferation, positiveregulation of NF-kB transcription factor activity were involved in sub-network. These datademonstrated a detailed landscape of tissue-specific gene expressions and core regulationnetworks in response to L2.At last, proteomic analysis on the colon and small intestine tissues of mice fed with L2were applied compared with normal mice. Two-dimensional gel electrophoresis (2-DE)combined with MALDI-TOF-MS were employed to detect and identify the protein expressionprofiles.22proteins in colon tissue and25proteins in small intestine tissues were identifiedshowing significant changes in abundance induced by L2, respectively. Little of thedifferential expressed proteins were overlapped, indicating the tissue-specific effects of L2treatment. Among of the differential expressed proteins,12of them were choosed to validateby Western blot. These differentially expressed proteins were mainly involved inphysiological processes related to cellular component organization, developmental process,cellular process, immune system process, metabolic process, multicellular organismal process,response to stimulus. The results suggested the wide range of biological activities of L2.STRING network analysis demonstrated that21colon proteins were mapped to a7-proteinand a3-protein sub-networks,25small intestine proteins were mapped to a17-proteinnetwork, and all the45altered proteins were integrated into a unified network containing29proteins. This suggested the existence of a concert mechanism, although acting on colon and small intestine separately. These findings facilitated the understanding of regulatorymechanism in response to L2treatment in mice.更多还原