【作者】 孟真；

【导师】 邵建忠；

【作者基本信息】 浙江大学 ， 细胞生物学， 2011， 博士

【摘要】 论文从分子和细胞水平围绕河豚清道夫受体(Tn-SR)的分子克隆和鉴定,以及它在LPS(lipopolysaccharide)引起的免疫反应中的功能和相关机制进行了研究,旨在阐明鱼类清道夫受体的基本生物学特征。利用生物信息学方法,本研究首次预测并克隆了哺乳动物清道夫受体A类分子SCARA5在鱼类中的同源物-河豚清道夫受体。河豚清道夫受体是典型的II.型跨膜受体,包括短的N端胞内区,跨膜区和长的C端胞外区。具备coiled-coil。胶原样和SRCR结构域,这些胞外区的结构特征与哺乳动物A类清道夫分子高度保守。位于胞内区的TRAF2结合位点和跨膜区的受体内化相关基序,与受体在免疫反应中的功能有密切关系。河豚清道夫受体主要以三聚体的形式存在,并且分布在白细胞表面。河豚清道夫受体组成型地分布在鱼类各个组织中,且当LPS刺激后,在脾脏、肌肉、脑和肾脏中表达量显著增加。河豚清道夫受体在LPS引I起的免疫反应中,具有双重功能。河豚清道夫受体胞外区能结合LPS。河豚清道夫受体参与河豚白细胞对LPS的识别和吞噬／结合,并且河豚清道夫受体内化至细胞内可能参与LPS的降解,减少LPS对细胞的毒作用和由此引起的减轻炎症反应。河豚清道夫受体不直接传递炎症反应信号从而激活NF-κB信号途径,但是对LPS引起的炎症反应起负调节作用。在斑马鱼胚胎模型上,不同浓度LPS刺激引起的NF-κB活性增强可被不同表达水平的河豚清道夫受体所抑制,导致炎症信号下降,并远远低于正常水平。体外互作实验证明河豚清道夫受体能够与肿瘤坏死因子受体相关因子2(Tumor necrosis factor receptor-associated factor2, TRAF2)直接结合。河豚清道夫受体通过结合TRAF2,竞争性地抑制肿瘤坏死因子-肿瘤坏死因子受体途径中以NF-κB活化为主要特征的炎症信号传递,从而减轻LPS对机体的影响。河豚清道夫受体的这一双重功能,为解释鱼类对LPS的高度耐受提供了证据,也为进一步认识和理解清道夫受体作为模式识别分子在先天免疫中的作用提供了新的视角。更多还原

【Abstract】 This article focus on the molecular cloning and identification of scavenger receptor in Tetraodon nigroviridis, Tn-SR and its function in immune response induced by LPS to clarify the biological characterization of scavenger receptor in fish.A novel Tn-SR was predicted and identified as an ortholog of mammalian SCARA5by bioinformatics methods, which has a typical structure of type II membrane receptor containing a short N-terminal cytoplasmic domain, a transmembrane domain and a long C-terminal extracelluar domain. In the extracelluar part of Tn-SR, the coiled-coil structure, collagenous domain and a scavenger receptor cysteine-rich domain (SRCR) are conserved organization of SR class A members in mammals. TRAF2-binding consensus motif at residue10-13and linear YXX? motif in transmembrane domain were found, which implied Tn-SR involving in the process of immune reaction. Expression in HEK293cells confirmed that Tn-SR assembles as a homotrimer and is expressed at the plasma membrane of leukocytes from Tetraodon nigroviridis.The gene expression profile showed that Tn-SR is detected in all tissues including liver, intestine, spleen, kidney, skin, gill, muscle and brain. After stimulated by LPS, expression levels of Tn-SR on spleen, muscle, brain and kidney were significantly up-regulated compared with other tissues.Tn-SR played a dual role in immune response stimulated by LPS. The extracelluar part of Tn-SR was ability to bind LPS detected by ELISA. Tn-SR involved in the LPS recognition and binding/phagocytosis of leukocytes from Tetraodon nigroviridis. Tn-SR was internalized to cytoplasma to ingest or clear LPS to reduce the toxic influence of LPS and inflammation by LPS. Although Tn-SR was not able to directly transduce the singling pathway induced by LPS, it involved in the negative regulation of NF-κB activation. In model of zebrafish embryos, fold induction of NF-κB were decreased remarkably in groups with different expression levels of Tn-SR induced by LPS at different concentrations. Tn-SR was certified to interacting with Tn-TRAF2by CO-IP under normal and LPS conditions.Tn-SR bind with TRAF2to inhibit NF-κB activation in tumor necrosis factor-tumor necrosis factor receptor signaling pathway to weaken the inflammation by LPS.These results suggested that scavenger receptor plays a key role in the resistant to toxic effect of LPS in fish and provided new supports to understanding the scavenger receptor as a pattern recognition receptor in innate immunity.更多还原