自然杀伤细胞受体NKp80与NKG2F的生物学功能研究

发布时间：2018-04-13 13:02

本文选题：NK细胞 + 受体　；参考：《中国科学技术大学》2010年博士论文

【摘要】： 自然杀伤细胞(NK细胞)是固有免疫系统中非常重要的成员,能够有效的清除机体内的异常细胞,包括受到病原微生物感染的细胞和发生癌变的细胞。与T细胞不同,NK细胞可以直接识别异常信号,不需要抗原递呈细胞对抗原进行加工、递呈,能够快速对机体内的异常信号产生免疫应答,是机体的第一道免疫防线。 NK细胞能够有效的区分机体内的正常细胞和异常细胞,主要通过表达于NK细胞表面的NK细胞受体来完成自我与非我的识别过程。目前解释该现象公认的理论为'Missing Self"机制：机体正常细胞表面表达足量的HLA分子,它们能够结合位于NK细胞表面的抑制性受体(人白细胞抗原特异性抑制受体),抑制NK细胞的细胞毒活性；而在感染或癌变的细胞表面,HLA分子缺失或表达量大幅降低,不能有效的抑制NK细胞的杀伤功能,导致其被NK细胞清除。 NK细胞受体根据它们传导的信号不同,主要分为活化性受体和抑制性受体。它们对于NK细胞发挥各项生物学功能起着至关重要的作用。目前已经发现了大量NK细胞受体,其中部分受体的生物学功能研究得已比较透彻,已经发现了相应的配体,制备了单克隆抗体,解析了三维空间结构等。但是随着NK细胞受体研究不断向前进展,新的NK细胞受体不断被发现,它们的生物学功能尚不明晰。 NKp80和NKG2F是新近发现的两个NK细胞受体,目前关于它们的研究较少,生物学功能尚不清楚,我们的工作主要围绕这两个NK细胞受体展开,旨在更进一步了解这两个NK细胞受体的生物学功能。之前的两项研究发现NKp80能够向NK细胞内传递活化信号,增强NK细胞的细胞毒活性,提高NK细胞分泌炎性细胞因子的能力；同时还鉴定出了NKp80的配体——表达于单核细胞上的AICL分子。然而,目前NKp80和AICL的三维空间结构都未被解析,无法根据三维空间结构来推测它们相互结合的位点,难以了解它们相互作用的机理。为了探究NKp80与AICL相互作用的位点,我们首先在蛋白结构数据库(PDB)中找到了目前已经解析了三维空间结构、且同源性与AICL最高的蛋白分子CD69,然后根据CD69的三维空间结构,利用生物信息学工具3D-JIGSAW构建了AICL的模拟空间结构。随后在所有生物总蛋白库中对AICL氨基酸序列进行BLAST分析,找出同源性最高的7个蛋白,用ClustalW2软件分析比对这8个蛋白的氨基酸序列,在AICL氨基酸序列中确定出7个保守性区域。在AICL的模拟空间结构中展示这7个保守性区域发现,其中三个保守性区域位于AICL的模拟空间结构的外表面,按照这三个保守性序列合成了三条多肽。流式细胞术分析发现这三条多肽能够竞争性的抑制anti-NKp80单克隆抗体结合NK细胞表面的NKp80。细胞毒活性检测实验结果发现,其中的两条多肽能够部分阻断NKp80与AICL所介导的NK细胞的杀伤功能。结合生物信息学分析和功能实验的结果,我们认为两条多肽序列为潜在的分子相互作用位点。 NKG2F的功能目前尚不清楚,之前的研究显示NKG2F胞内段有一个类似于免疫受体酪氨酸抑制性基序(ITIM)的序列,但在细胞膜表面,它又能与DAP12结合,通过DAP12胞内的免疫受体酪氨酸活化性基序(ITAM)传递活化信号。NKG2F是活化性受体还是抑制性受体,尚存在争论。我们第一次利用重组表达技术,在大肠杆菌表达系统中表达出了人NKG2F重组蛋白,然后以重组人NKG2F为免疫原,免疫小鼠制备了anti-NKG2F多克隆抗体,以此为工具,通过荧光定量PCR和流式细胞术检测发现：细胞因子IL-2和IL-15活化NK细胞后,NKG2F在NK细胞表面表达量上调,从侧面印证了NKG2F为活化性受体。此外,流式细胞术细胞表面标记检测的结果还证实：同其他NKG2家族受体类似,NKG2F表达于外周血NK细胞的细胞膜上。
[Abstract]:Natural killer cells ( NK cells ) are very important members of the innate immune system , and can effectively remove abnormal cells in the organism , including cells infected by pathogenic microorganisms and cells that have canceration . Unlike T cells , NK cells can directly identify abnormal signals without the need of antigen - presenting cells for processing and presenting antigens , and can rapidly generate immune responses to abnormal signals in the organism , and are the first immune defense lines of the organism .

NK cells can effectively distinguish normal cells and abnormal cells in the organism , and accomplish self - and non - self - recognition processes mainly through NK cell receptors expressed on the surface of NK cells .
In the infected or cancerous cell surface , the deletion or expression of HLA molecules is greatly reduced , and the killing function of NK cells can not be effectively inhibited , and the NK cells can be removed by NK cells .

NK cell receptors are mainly divided into activating receptors and inhibitory receptors based on the signals they conduct . They play an important role in NK cells play a vital role in the biological function of NK cells .

NKp80 and NKG2F are newly discovered NK cell receptors . At present , there are few researches on these NK cell receptors . Our work mainly revolves around the two NK cell receptors , and aims at further understanding the biological functions of these two NK cell receptors . Two previous studies have found that NKp80 can transfer the activation signal to NK cells , enhance the cytotoxicity of NK cells , and improve the ability of NK cells to secrete inflammatory cytokines ;
At the same time , the expression of NKp80 and NKp80 on monocytes was identified . However , the three - dimensional spatial structure of NKp80 and AICH was not analyzed , and it was difficult to understand the mechanism of interaction . In order to investigate the interaction between NKp80 and AELL , we first found a three - dimensional spatial structure in the protein structure database ( PDB ) , and then constructed the simulated spatial structure of AEL5 using bioinformatics tool 3D - JIGSAW , according to the three - dimensional spatial structure of CD69 .

A BLAST analysis was performed in all the biological total protein libraries to identify the 7 proteins with the highest homology , and the amino acid sequence of the eight proteins was analyzed by the ClustalW2 software , and seven conserved regions were identified in the AICH amino acid sequence .

These seven conserved regions were found in the simulated spatial structure of AEL5 , among which three conserved regions were located on the outer surface of the simulated spatial structure of AICH , and three polypeptides were synthesized in accordance with these three conserved sequences .

Flow cytometry analysis found that these three polypeptides were capable of competitively inhibiting NKp80 binding to NK cell surface by anti - NKp80 monoclonal antibody . Two of these polypeptides were able to partially block the killing function of NKp80 and NKp80 - mediated NK cells . In combination with the results of bioinformatics analysis and functional experiments , we considered that the two polypeptide sequences were potential molecular interaction sites .

NKG2F is an activated receptor or an inhibitory receptor . NKG2F is an activated receptor or an inhibitory receptor . NKG2F is an activated receptor or an inhibitory receptor . NKG2F is an activated receptor or an inhibitory receptor .

【学位授予单位】：中国科学技术大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【参考文献】