【作者】 黄海华；

【导师】 孙汭；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2010， 博士

【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。更多还原

【Abstract】 Natural killer (NK) cells play important roles in innate immunity. They are able to distinguish malignant cells or pathogen-infected cells from normal cells and kill these abnormal cells without prior sensitization, as a result, natural killer cells react quickly to dangerous signals and act as the first line of defense against pathogens and transformed cells.NK cells are able to distinguish the abnormal cells from normal cells effectively, which is mediated by the NK cell receptors expressed on the NK cell surface.Currently accepted theory to explain this phenomenon as the "Missing Self" mechanism:NK cells received both activating signals and inhibitory signals through their surface receptors. Normal cells expressed sufficient HLA molecules, which was able to bind to the inhibitory receptors on the membrane of NK cell (human leukocyte antigen-specific inhibitory receptors) and transduce inhibitory signals, resulting in the inhibition of NK cell cytotoxicity. As a result, NK cells won’t attack normal cells. However, expression of HLA molecules of pathogen-infected cells or transformed cells was greatly down-regulated. As a result, NK cells couldn’t receive sufficient inhibitory signals, and NK cells were staying at activating state with strong cytotoxicity, resulting in elimination of these target cells.NK cell receptors were divided into two groups as activating receptors and inhibitory receptors depending on the signal they transduced. NK cell receptors play crucial roles in the biological functions of NK cells. A lot of NK cell receptors have been identified, some of which has been deeply investigated. The ligands and the 3-D structure information of these receptors has been identified, moreover, the specific monoclonal antibodies against these receptors has also been produced.However, few studies have been conducted for some newly identified NK cell receptors, whose biological functions were not clear yet.NKp80 and NKG2F were newly identified, whose biological functions were not clear. This work focused on the two NK cell receptors, aiming at a further progress of the biological functions.NKp80 has been reported to transduce activating signals, leading to NK cell activation and increase of NK cell cytotoxicity. AICL has been identified as the ligand of NKp80. But the interaction sites of NKp80-AICL were not clear. In this study,3-D model of AICL was constructed with the online server 3D-JIGSAW based on the crystal structure of CD69 which shared the highest sequence homology among released protein structures in PDB.7 proteins with highest sequence homology to AICL were listed by BLAST. Multiple sequence alignment of the amino acid sequence of these proteins was conducted by the tool Clustal W2, and 7 conserved sequences of AICL were found. Among these conserved sequences, three sequences were on the surface of the 3-D model of AICL which was constructed based on the crystal structure of CD69. Flow cytometric analysis demonstrated that these peptides were able to compete with anti-NKp80 mAb on NKp80 binding activity in a dose-dependent manner. Moreover, two peptides reduced NKp80-AICL mediated cytotoxicity of both fresh PBMCs and purified NK cells in 51Cr release cytotoxicity assay. Considering both the bioinformatic analysis and experimental results, we concluded that the P1 and P2 sequences on AICL might be the potential sites of NKp80-AICL interaction.Previous studies have demonstrated that NKG2F contained an immunoreceptor tyrosine-based inhibition motif (ITIM) like sequence in cytoplasmic domain, however, NKG2F also associated with DAP12 which contained an immunoreceptor tyrosine-based activation motif (ITAM). It was controversial whether NKG2F is an activating receptor or an inhibitory receptor.In this study, we recombinantly expressed the human NKG2F protein with the E. coli for the first time. Anti-NKG2F polyclonal antibody was produced by immunizing BALB/c mice with the recombinant NKG2F.With the anti-NKG2F polyclonal antibody, real-time PCR and flow cytometry were performed. The results demonstrated that IL-2 and IL-15 stimulation up-regulated the expression of NKG2F expression on the surface of peripheral blood NK cells. As NK cell were activated by IL-2 and IL-15 stimulation, we considered that NKG2F as an activating receptor. Furthermore, the flow cytometry results demonstrated that NKG2F was expressed on the surface of peripheral blood NK cells for the first time, which was consistent with other members of NKG2 family.更多还原