發(fā)布時間：2018-04-13 13:02

  本文選題：NK細胞 + 受體　； 參考：《中國科學技術(shù)大學》2010年博士論文

【摘要】： 自然殺傷細胞(NK細胞)是固有免疫系統(tǒng)中非常重要的成員,能夠有效的清除機體內(nèi)的異常細胞,包括受到病原微生物感染的細胞和發(fā)生癌變的細胞。與T細胞不同,NK細胞可以直接識別異常信號,不需要抗原遞呈細胞對抗原進行加工、遞呈,能夠快速對機體內(nèi)的異常信號產(chǎn)生免疫應(yīng)答,是機體的第一道免疫防線。 NK細胞能夠有效的區(qū)分機體內(nèi)的正常細胞和異常細胞,主要通過表達于NK細胞表面的NK細胞受體來完成自我與非我的識別過程。目前解釋該現(xiàn)象公認的理論為'Missing Self"機制：機體正常細胞表面表達足量的HLA分子,它們能夠結(jié)合位于NK細胞表面的抑制性受體(人白細胞抗原特異性抑制受體),抑制NK細胞的細胞毒活性；而在感染或癌變的細胞表面,HLA分子缺失或表達量大幅降低,不能有效的抑制NK細胞的殺傷功能,導致其被NK細胞清除。 NK細胞受體根據(jù)它們傳導的信號不同,主要分為活化性受體和抑制性受體。它們對于NK細胞發(fā)揮各項生物學功能起著至關(guān)重要的作用。目前已經(jīng)發(fā)現(xiàn)了大量NK細胞受體,其中部分受體的生物學功能研究得已比較透徹,已經(jīng)發(fā)現(xiàn)了相應(yīng)的配體,制備了單克隆抗體,解析了三維空間結(jié)構(gòu)等。但是隨著NK細胞受體研究不斷向前進展,新的NK細胞受體不斷被發(fā)現(xiàn),它們的生物學功能尚不明晰。 NKp80和NKG2F是新近發(fā)現(xiàn)的兩個NK細胞受體,目前關(guān)于它們的研究較少,生物學功能尚不清楚,我們的工作主要圍繞這兩個NK細胞受體展開,旨在更進一步了解這兩個NK細胞受體的生物學功能。之前的兩項研究發(fā)現(xiàn)NKp80能夠向NK細胞內(nèi)傳遞活化信號,增強NK細胞的細胞毒活性,提高NK細胞分泌炎性細胞因子的能力；同時還鑒定出了NKp80的配體——表達于單核細胞上的AICL分子。然而,目前NKp80和AICL的三維空間結(jié)構(gòu)都未被解析,無法根據(jù)三維空間結(jié)構(gòu)來推測它們相互結(jié)合的位點,難以了解它們相互作用的機理。為了探究NKp80與AICL相互作用的位點,我們首先在蛋白結(jié)構(gòu)數(shù)據(jù)庫(PDB)中找到了目前已經(jīng)解析了三維空間結(jié)構(gòu)、且同源性與AICL最高的蛋白分子CD69,然后根據(jù)CD69的三維空間結(jié)構(gòu),利用生物信息學工具3D-JIGSAW構(gòu)建了AICL的模擬空間結(jié)構(gòu)。 隨后在所有生物總蛋白庫中對AICL氨基酸序列進行BLAST分析,找出同源性最高的7個蛋白,用ClustalW2軟件分析比對這8個蛋白的氨基酸序列,在AICL氨基酸序列中確定出7個保守性區(qū)域。 在AICL的模擬空間結(jié)構(gòu)中展示這7個保守性區(qū)域發(fā)現(xiàn),其中三個保守性區(qū)域位于AICL的模擬空間結(jié)構(gòu)的外表面,按照這三個保守性序列合成了三條多肽。 流式細胞術(shù)分析發(fā)現(xiàn)這三條多肽能夠競爭性的抑制anti-NKp80單克隆抗體結(jié)合NK細胞表面的NKp80。細胞毒活性檢測實驗結(jié)果發(fā)現(xiàn),其中的兩條多肽能夠部分阻斷NKp80與AICL所介導的NK細胞的殺傷功能。結(jié)合生物信息學分析和功能實驗的結(jié)果,我們認為兩條多肽序列為潛在的分子相互作用位點。 NKG2F的功能目前尚不清楚,之前的研究顯示NKG2F胞內(nèi)段有一個類似于免疫受體酪氨酸抑制性基序(ITIM)的序列,但在細胞膜表面,它又能與DAP12結(jié)合,通過DAP12胞內(nèi)的免疫受體酪氨酸活化性基序(ITAM)傳遞活化信號。NKG2F是活化性受體還是抑制性受體,尚存在爭論。我們第一次利用重組表達技術(shù),在大腸桿菌表達系統(tǒng)中表達出了人NKG2F重組蛋白,然后以重組人NKG2F為免疫原,免疫小鼠制備了anti-NKG2F多克隆抗體,以此為工具,通過熒光定量PCR和流式細胞術(shù)檢測發(fā)現(xiàn)：細胞因子IL-2和IL-15活化NK細胞后,NKG2F在NK細胞表面表達量上調(diào),從側(cè)面印證了NKG2F為活化性受體。此外,流式細胞術(shù)細胞表面標記檢測的結(jié)果還證實：同其他NKG2家族受體類似,NKG2F表達于外周血NK細胞的細胞膜上。
[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 .

【學位授予單位】：中國科學技術(shù)大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前1條

1 ;The Role of Innate Immune Cells in the Response of Heat-Treated Mycobacterium tuberculosis (M.tb) Antigens Stimulating PBMCs[J];Cellular & Molecular Immunology;2004年06期

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本文編號：1744603