本文選題：NK細(xì)胞 + 受體��； 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2010年博士論文

【摘要】： 自然殺傷細(xì)胞(NK細(xì)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(jī)體內(nèi)的異常細(xì)胞,包括受到病原微生物感染的細(xì)胞和發(fā)生癌變的細(xì)胞。與T細(xì)胞不同,NK細(xì)胞可以直接識(shí)別異常信號(hào),不需要抗原遞呈細(xì)胞對(duì)抗原進(jìn)行加工、遞呈,能夠快速對(duì)機(jī)體內(nèi)的異常信號(hào)產(chǎn)生免疫應(yīng)答,是機(jī)體的第一道免疫防線。 NK細(xì)胞能夠有效的區(qū)分機(jī)體內(nèi)的正常細(xì)胞和異常細(xì)胞,主要通過表達(dá)于NK細(xì)胞表面的NK細(xì)胞受體來完成自我與非我的識(shí)別過程。目前解釋該現(xiàn)象公認(rèn)的理論為'Missing Self"機(jī)制：機(jī)體正常細(xì)胞表面表達(dá)足量的HLA分子,它們能夠結(jié)合位于NK細(xì)胞表面的抑制性受體(人白細(xì)胞抗原特異性抑制受體),抑制NK細(xì)胞的細(xì)胞毒活性；而在感染或癌變的細(xì)胞表面,HLA分子缺失或表達(dá)量大幅降低,不能有效的抑制NK細(xì)胞的殺傷功能,導(dǎo)致其被NK細(xì)胞清除。 NK細(xì)胞受體根據(jù)它們傳導(dǎo)的信號(hào)不同,主要分為活化性受體和抑制性受體。它們對(duì)于NK細(xì)胞發(fā)揮各項(xiàng)生物學(xué)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(xì)胞受體,其中部分受體的生物學(xué)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(xì)胞受體研究不斷向前進(jìn)展,新的NK細(xì)胞受體不斷被發(fā)現(xiàn),它們的生物學(xué)功能尚不明晰。 NKp80和NKG2F是新近發(fā)現(xiàn)的兩個(gè)NK細(xì)胞受體,目前關(guān)于它們的研究較少,生物學(xué)功能尚不清楚,我們的工作主要圍繞這兩個(gè)NK細(xì)胞受體展開,旨在更進(jìn)一步了解這兩個(gè)NK細(xì)胞受體的生物學(xué)功能。之前的兩項(xiàng)研究發(fā)現(xiàn)NKp80能夠向NK細(xì)胞內(nèi)傳遞活化信號(hào),增強(qiáng)NK細(xì)胞的細(xì)胞毒活性,提高NK細(xì)胞分泌炎性細(xì)胞因子的能力；同時(shí)還鑒定出了NKp80的配體——表達(dá)于單核細(xì)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(cè)它們相互結(jié)合的位點(diǎn),難以了解它們相互作用的機(jī)理。為了探究NKp80與AICL相互作用的位點(diǎn),我們首先在蛋白結(jié)構(gòu)數(shù)據(jù)庫(kù)(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(xué)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(kù)中對(duì)AICL氨基酸序列進(jìn)行BLAST分析,找出同源性最高的7個(gè)蛋白,用ClustalW2軟件分析比對(duì)這8個(gè)蛋白的氨基酸序列,在AICL氨基酸序列中確定出7個(gè)保守性區(qū)域。 在AICL的模擬空間結(jié)構(gòu)中展示這7個(gè)保守性區(qū)域發(fā)現(xiàn),其中三個(gè)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(gè)保守性序列合成了三條多肽。 流式細(xì)胞術(shù)分析發(fā)現(xiàn)這三條多肽能夠競(jìng)爭(zhēng)性的抑制anti-NKp80單克隆抗體結(jié)合NK細(xì)胞表面的NKp80。細(xì)胞毒活性檢測(cè)實(shí)驗(yàn)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(dǎo)的NK細(xì)胞的殺傷功能。結(jié)合生物信息學(xué)分析和功能實(shí)驗(yàn)的結(jié)果,我們認(rèn)為兩條多肽序列為潛在的分子相互作用位點(diǎn)。 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(gè)類似于免疫受體酪氨酸抑制性基序(ITIM)的序列,但在細(xì)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(hào)。NKG2F是活化性受體還是抑制性受體,尚存在爭(zhēng)論。我們第一次利用重組表達(dá)技術(shù),在大腸桿菌表達(dá)系統(tǒng)中表達(dá)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(xì)胞術(shù)檢測(cè)發(fā)現(xiàn)：細(xì)胞因子IL-2和IL-15活化NK細(xì)胞后,NKG2F在NK細(xì)胞表面表達(dá)量上調(diào),從側(cè)面印證了NKG2F為活化性受體。此外,流式細(xì)胞術(shù)細(xì)胞表面標(biāo)記檢測(cè)的結(jié)果還證實(shí)：同其他NKG2家族受體類似,NKG2F表達(dá)于外周血NK細(xì)胞的細(xì)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(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392

【參考文獻(xiàn)】

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(hào)：1744603