【作者】 郭亮；

【导师】 顾建新；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 第一部分DC-SIGN与Tollip的相互作用研究DC-SIGN（（?）endritic（?）ell-（?）pecific（?）CAM-3（?）rabbing（?）onintegrin）是C型凝集素受体超家族的成员,它主要在树突状细胞（Dendritic cell,DC）表面表达。DC-SIGN是Ⅱ型跨膜受体,分为胞浆尾部、跨膜区以及胞外段三部分。DC-SIGN能够通过其胞外段的碳水化合物识别结构域（carbohydrate recognition domain,CRD）以钙离子依赖的方式去识别一些病原体表面或组织表面特异性的糖结构,比如HIV-1、结核杆菌表面的高甘露糖结构,结肠癌细胞表面的Lewis^x/y。DC-SIGN可以介导DC多方面的功能,比如参与DC的迁移,介导DC与T细胞的粘附和对T细胞的激活,捕获病原体和抗原呈递等。但是,DC-SIGN分子也存在不利于人体的一面。目前的研究发现,DC-SIGN可以促进一些病原体或肿瘤逃避机体的免疫监视。比如,DC-SIGN捕获HIV-1以后,可将其内化至细胞中的一个非溶酶体区域,保护HIV-1免受降解,并通过反式作用的方式促进HIV-1对CD4+T细胞的感染;在识别结核杆菌细胞壁成分以后,DC-SIGN所产生的信号可以抑制DC的成熟,干扰Toll样受体信号,促进免疫抑制因子IL-10的表达;DC-SIGN与CEA（carcinoembryonic antigen）表面的Lewis^x/y抗原结合以后,可以诱导2型T辅助细胞反应,有利于肿瘤免疫逃避。DC-SIGN与病原体或肿瘤免疫逃避的关系和机制的研究,将为人们治疗相关疾病提供新的思路。目前,对于DC-SIGN的研究主要集中在其胞外段对病原体的识别方面,而DC-SIGN胞浆段的相关研究还很少。为此,本实验室以DC-SIGN的胞浆段为诱饵,在人的胸腺cDNA文库中进行酵母双杂交并筛选到Tollip（Toll interactingprotein）这一基因。Tollip是IL-1/Toll样受体信号通路中的信号分子,对于调节炎症反应发挥一定的作用。Tollip在蛋白质运输方面也发挥作用,比如介导内化后的IL-1受体向溶酶体的转运。通过GST-pull down,免疫共沉淀,荧光共定位等方法,我们证实了Tollip和DC-SIGN胞浆段相互作用的存在,并且Tollip的C2结构域,即57-164位氨基酸残基对于该相互作用是必需的。我们在实验中发现,当DC-SIGN内化进入细胞浆以后与Tollip存在着点状共定位。并且,用小干扰RNA技术将Tollip的表达抑制以后,内化的DC-SIGN无法有效地转运至溶酶体。此外,用DC-SIGN特异性的抗体激活DC-SIGN信号通路以后,Tollip可以被酪氨酸磷酸化。我们的研究结果表明Tollip对于DC-SIGN内化后向溶酶体的转运发挥重要作用,同时也可能作为DC-SIGN信号通路中的潜在效应分子。对于DC-SIGN与Tollip相互作用的研究的深入,将为进一步阐明DC-SIGN介导病原体或肿瘤免疫逃避的分子机制提供新的线索。第二部分灵芝孢子粉多糖成分GSG的生物学活性探讨灵芝是（Ganoderma lucidum）担子菌纲多孔菌科灵芝属真菌赤芝和紫芝的总称,有扶正固本等功效,被《本经》列为上品。灵芝孢子（Spores of Ganodermalucidum）是由灵芝发育后期释放出来的担孢子。近年来,随着孢子粉采集和破壁技术的提高,对灵芝孢子粉有了进一步的认识。药理试验表明,灵芝孢子粉比灵芝子实体具有更强更全面的作用,它是灵芝的精华部分,具有抑制肿瘤细胞生长,调节、提高人体免疫力,降低胆固醇,提高肌体耐缺氧能力等功能。灵芝孢子化学成分有蛋白质、氨基酸类、多糖类和维生素类等多种成分。目前研究认为,灵芝多糖类化合物是灵芝中主要的活性成分之一,国内外已有报道指出从孢子粉中可提取出具免疫调节活性的多糖。中国科学院上海有机所通过水提、乙醇分级沉淀的方法,并通过采用DEAE-cellulose和SephadexG-50色谱柱分离纯化,从灵芝破壁孢子粉中得到单一级的多糖组分GSG（（?）.lucidum（?）pores（?）lucan）。经过红外、NMR以及GC-MS的分析,该多糖被鉴定为是以β（1→3）Glcp构成主链,且Glc残基在6—O处有分枝,侧链由β（1→6）Glcp、β（1→4）Glcp组成,由α（1→）Glcp构成侧链末端。通过HPGPC（High performance gel permeation chromatography）分析,GSG的分子量为5000～11000Da。因此GSG是可溶性低分子量的β葡聚糖。我们在体外研究中发现,GSG能够刺激小鼠腹腔巨噬细胞的活化,引起活性氧（reactive oxygenspecies,ROS）的产生以及细胞因子TNF-α和IL-6的分泌。并且,GSG刺激细胞因子的分泌是依赖于MAPK（mitogen-activated protein kinase）信号通路的,包括ERK、p38和JNK信号。通过表面胞质团能量共振以及流式细胞仪检测技术,我们证实GSG能够被C型凝集素受体Dectin-1所识别。进一步研究发现,GSG能够部分通过Dectin-1行使其免疫激活的效应。在体内,GSG具有抗肿瘤活性以及增强ConA所诱发的小鼠脾脏细胞增殖反应的功能。我们的实验证实了从灵芝孢子中提取的β葡聚糖GSG在体内和体外均具有免疫激活效应,并且GSG分子量低、水溶性好,跟一些大分子量的葡聚糖比如香菇多糖相比,更有利于人体吸收,降低抗原性。因此GSG具有很大的开发潜力。Dectin-1在介导GSG的免疫激活效应方面发挥重要作用,这一研究结果将为人们探寻灵芝抗肿瘤和免疫调节的机制提供新的思路和线索。更多还原

【Abstract】 PartⅠStudy on the interaction between DC-SIGN and TollipDC-SIGN（Dendritic cell-specific ICAM-3 grabbing nonintegrin） is a typeⅡmembrane protein mainly expressed on DCs（dendritic cells）.Based on its structure, DC-SIGN belongs to the C-type lectin superfamily and it contains a short, cytoplasmic N-terminal tail,transmembrane region and an extracellular carbohydrate recognition domain（CRD）.Via its extracellular CRD,DC-SIGN could recognize various kinds of pathogens or tissue protein containing high-mannose moieties or nonsialylated Lewis^x/y,such as HIV-1,Mycobacterium tuberculosis and carcinoembryonic antigen（CEA） on colorectal tumor cells.DC-SIGN plays an important role in mediating DCs adhesion,migration,and primary T cell activation. However,many studies have showed that DC-SIGN may contribute to the pathogenesis and the immune escape of some pathogens or tumors.For instance,upon binding HIV,DC-SIGN internalizes it rapidly and enhances its infection in trans of the target cells.ManLAM,a component of the Mycobacterium tuberculosis,targets DC-SIGN to induce IL-10 and inhibit the maturation of DCs.The interactions between DC-SIGN and colorectal tumor-associated Lewis glycans on CEA impair the function and differentiation of DC and may induce generalized failure of a host to mount an effective antitumor response.Although the ligands of DC-SIGN have been well defined,few studies have been done on its cytoplasmic tail.In the present study,we identified Tollip（Toll interacting protein） as a binding partner of the cytoplasmic tail of DC-SIGN using yeast two-hybrid screening,in human thymus cDNA library.Tollip is a integral part of the IL-1RI/TLR signaling cascade.It is also required for the sorting of the IL-1RI at late endosomes suggesting its involvement in protein sorting.GST pull-down assays showed that Tollip interacted with the cytoplasmic tail of DC-SIGN in vitro. Furthermore,the amino acid residues 57-164 of Tollip,which is part of the C2 domain was crucial for this interaction.The interaction was further confirmed in vivo by co-immunoprecipitation assay and confocal microscopic analysis.After internalization,DC-SIGN co-localized with Tollip on punctate structures throughout the cytoplasm.Knockdown of Tollip by siRNA impaired the lysosomal trafficking of DC-SIGN.Additionally,Tollip could be tyrosine phosphorylated upon DC-SIGN ligation by its specific antibody.Our results suggested that Tollip plays an important role in DC-SIGN sorting and might be an effector molecule in DC-SIGN signaling cascade.These results may be helpful in elucidating the immune escape by DC-SIGN and deserves further investigation. PartⅡStudy on the biological activities of the polysaccharide from the spores of Ganoderma lucidumGanoderma lucidum（G.lucidum）,a species of basidiomycetes,has been widely used as a tonic in promoting longevity and health in China and other Asian countries. G.lucidum has been studied extensively in recent years because of its intrinsic immunomodulatory and anti-tumor properties.Though the fruit bodies of G.lucidum have been widely utilized,the spores of G.lucidum were realized and utilized only in the 20th century.The spores of G.lucidum also contain a large amount of bioactive substances like the fruit bodies of G.lucidum.The bioactivity of the spores may be higher than that of the fruit bodies of G.lucidum.However,the bioactive components in the spores of G.lucidum have been rarely studied due to the difficulties in collecting and sporoderm-breaking of the spores.In recent years,with the successful cultivation of G.lucidum indoors on a large scale and a breakthrough in sporoderm-breaking technology,much attention has been paid to chemical components of G.lucidum spores and their versatile biological activities.Recently,a number of triterpenes from the spores were isolated and characterized.However,only a few glucans from the spores were isolated and their functions remain to be investigated.GSG（G.lucidum spores glucan）,a water-soluble polysaccharide from the spores of G.lucidum was extracted with the water extract and sequential alcohol precipitating method.The molecular weight of GSG ranges from 5000 Da to 11000 Da as determined by HPGPC（High performance gel permeation chromatography）.IR, NMR and GC-MS confirmed the structure of GSG:it contains a backbone chain ofβ-1-3-linked D-glucopyranosyl residues and two 1-6-1inkedβ-D-glucose side chains that are further elaborated with additionalα（1,6） orα（1,4）-linked glucose regions.In vitro,GSG was an effective inducer of ROS production and MAPKs-dependent TNF-αand IL-6 secretion in murine resident peritoneal macrophages.Dectin-1 could recognize GSG and partially mediate its immunostimulatory activities.Additionally, in vivo administration of GSG potentiated the ConA-induced proliferative response of splenocytes and induced anti-tumor activity against Lewis lung cancer in mice.Our study demonstrated the biological activities of GSG both in vitro and in vivo. Due to the low molecular weight,great water-solubility,high purity and immunoenhancing effects,it is hopeful that GSG might be utilized as an intravenous injection for anti-tumor therapy and immunomodulation.The ability of Dectin-1 to mediate the activities of GSG would help to provide insights into the immunomodulatory mechanism by GSG.更多还原