本文選題：類脂 + 活化��； 參考：《中國協(xié)和醫(yī)科大學(xué)》2006年博士論文

【摘要】： γδT細(xì)胞在機(jī)體的免疫防御和免疫調(diào)節(jié)過程中發(fā)揮著重要作用,但目前對(duì)其抗原識(shí)別譜的認(rèn)識(shí)還很局限,尤其是γδT細(xì)胞對(duì)脂類抗原的識(shí)別還知之甚少。澄清γδT細(xì)胞與脂類抗原之間的相互作用機(jī)制,可為進(jìn)一步闡明γδT細(xì)胞在免疫學(xué)中的地位,全面描繪免疫應(yīng)答的本質(zhì)提供了重要線索和實(shí)驗(yàn)依據(jù),并也為免疫學(xué)應(yīng)用提供新的策略與手段。 本論文旨在研究人類γδT細(xì)胞對(duì)lipidA(LA)的識(shí)別機(jī)制,初步探討LA反應(yīng)性γδT細(xì)胞的TCRδ-CDR3長度及序列特點(diǎn),以及進(jìn)一步闡釋LA反應(yīng)性γδT細(xì)胞的免疫學(xué)功能。 (一)人類γδT細(xì)胞對(duì)LA識(shí)別機(jī)制的研究 LA是脂多糖(lipopolysaccharide,LPS)的重要組成部分和生物活性中心,具有較強(qiáng)的免疫原性。本研究集中討論了如下問題:人類γδT細(xì)胞對(duì)其識(shí)別的機(jī)制究竟如何?是否需要抗原提呈細(xì)胞的作用?如果需要,那么其分子機(jī)制是什么?與哪種抗原提呈分子相關(guān)?LA反應(yīng)性γδT細(xì)胞的TCR又有什么樣的特征可尋?針對(duì)以上問題,首先,我們分別應(yīng)用流式分析技術(shù)、~3H-TdR摻入實(shí)驗(yàn)和羥基熒光素二醋酸鹽琥珀酰亞胺脂(Carboxyfluorescein diacetate succinimidyl ester,CFSE)染色方法分別檢測了在加與不加單核細(xì)胞來源的樹突狀細(xì)胞(monocyte-derived dendritic cell,moDC)情況下,LA誘導(dǎo)人外周血來源的γδT細(xì)胞的增殖情況;其次,分別使用抗CD1a、CD1b、CD1c、CD1d、TCRγδ、LA、MHCⅠ和MHCⅡ抗體對(duì)LA誘導(dǎo)的γδT細(xì)胞增殖過程進(jìn)行阻斷;再次,對(duì)LA反應(yīng)性γδT細(xì)胞的TCRδCDR3區(qū)序列進(jìn)行測定,并使用基因掃描技術(shù)分析其長度特征;最后,分析了在抗CD1a、CD1b、CD1c、CD1d、TCRγδ、LA等抗體存在和不存在情況下,LA對(duì)γδT細(xì)胞刺激引發(fā)的包括IFN-γ、TNF-α、IL-2、IL-4、IL-5、IL-10在內(nèi)的細(xì)胞因子分泌情況。結(jié)果表明,人類γδT細(xì)胞與LA相互作用過程中,當(dāng)moDC存在時(shí),LA能誘導(dǎo)γδT細(xì)胞明顯增殖,而無moDC存在時(shí),基本上觀察不到γδT細(xì)胞有增殖現(xiàn)象;在各種抗體阻斷實(shí)驗(yàn)中,只有抗CD1b、CD1c、TCRγδ和LA的抗體能明顯阻斷LA誘導(dǎo)的γδT細(xì)胞增殖,而其它抗體沒有此阻斷效應(yīng);另外,我們進(jìn)一步驗(yàn)證了抗CD1b和抗CD1c抗體之間沒有交叉阻斷功能;基因掃描結(jié)果發(fā)現(xiàn)LA反應(yīng)性γδT細(xì)胞的TCRδCDR3區(qū)的片斷長度各不相同,但有長度約為75bp的優(yōu)勢片段,對(duì)LA反應(yīng)性γδT細(xì)胞的TCRδCDR3區(qū)的序列分析的結(jié)果表明沒有發(fā)現(xiàn)有特異性氨基酸序列,但發(fā)現(xiàn)其優(yōu)勢氨基酸序列的組成是TDRV2D-J1;LA反應(yīng)性γδT細(xì)胞主要分泌Th1類細(xì)胞因子,尤其是IFN-γ和TNF-α,并且與功能阻斷實(shí)驗(yàn)結(jié)果一致,抗CD1b、CD1c、TCRγδ和LA的抗體能完全阻斷γδT細(xì)胞分泌細(xì)胞因子。上述結(jié)果說明γδT細(xì)胞在識(shí)別LA的過程中,需要抗原提呈細(xì)胞的提呈作用,而且該過程是受CD1b和CD1c的限制,并依賴于TCRγδ。這一發(fā)現(xiàn)豐富γδT細(xì)胞識(shí)別脂類抗原的認(rèn)識(shí),為豐富γδT細(xì)胞識(shí)別抗原機(jī)制理論、全面描繪免疫應(yīng)答的本質(zhì)提供了依據(jù)。另外,我們的結(jié)果表明LA反應(yīng)性γδT細(xì)胞的TCRδCDR3區(qū)的序列沒有發(fā)現(xiàn)高度保守的,但無論其長度還是氨基酸序列組成都有優(yōu)勢序列存在,這為進(jìn)一步尋找LA高度特異性的人類γδT細(xì)胞克隆和高度親和力的TCRγδ序列提供了線索,并奠定了基礎(chǔ)。 (二)LA反應(yīng)性γδT細(xì)胞的功能研究 我們主要研究內(nèi)容如下:LA反應(yīng)性γδT細(xì)胞對(duì)E.coli的直接細(xì)胞毒功能研究;LA反應(yīng)性γδT細(xì)胞對(duì)自體巨噬細(xì)胞吞噬功能的調(diào)節(jié)作用研究;LA反應(yīng)性γδT細(xì)胞對(duì)B細(xì)胞產(chǎn)生抗LA抗體的調(diào)節(jié)功能研究。 首先,我們通過氧化還原染料-碘硝基四氮唑(p-iodonitrotetrazolium,INT)和聯(lián)甲苯四唑氯(5-cyano-2,3-ditolyl tetrazolium chlorid,CTC)還原生成相應(yīng)的產(chǎn)物四唑甲(INF和CTF)的量,在LA反應(yīng)性γδT細(xì)胞與E.coli共孵育4hr后,測定Ecoli的生物活性。結(jié)果發(fā)現(xiàn),E.coli的生物活性在與LA反應(yīng)性γδT細(xì)胞孵育后較孵育前有非常顯著的降低(P＜0.01);并且隨著效應(yīng)細(xì)胞-LA反應(yīng)性γδT細(xì)胞數(shù)量的增加及反應(yīng)時(shí)間的延長,Ecoli的生物活性越來越弱。以上結(jié)果提示LA反應(yīng)性γδT細(xì)胞對(duì)E.coli的確具有細(xì)胞毒作用,但是作用機(jī)制有待于進(jìn)一步澄清。 其次,通過構(gòu)建綠色熒光蛋白(green fluorescence protein,GFP)的原核表達(dá)載體pET-22b(+)-GFP,并用IPTG誘導(dǎo)該載體在大腸桿菌BL21(DE3)中進(jìn)行表達(dá),然后利用這種表達(dá)綠色熒光蛋白的E.coli(E.coli-GFP)來檢測巨噬細(xì)胞的吞噬功能。使用流式細(xì)胞儀技術(shù)分析與LA反應(yīng)性γδT細(xì)胞共孵育前后的吞噬了E.coli-GFP的巨噬細(xì)胞百分比的差異。結(jié)果提示,兩組中Ecoli-GFP~+巨噬細(xì)胞百分含量有顯著差異,LA反應(yīng)性γδT細(xì)胞能促進(jìn)自體巨噬細(xì)胞的吞噬功能。 最后,我們利用人源化Severe combined immune deficiency(SCID)小鼠檢測LA反應(yīng)性γδT細(xì)胞是否能夠調(diào)節(jié)B淋巴細(xì)胞產(chǎn)生抗LA抗體的功能。SCID小鼠腹腔接種人外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cell,PBMC)后2周,通過檢測其外周血中是否含有人類抗體來鑒定其免疫重建是否成功。然后,對(duì)免疫重建成功的小鼠用LA進(jìn)行免疫,其中部分實(shí)驗(yàn)組小鼠同時(shí)腹腔注射LA反應(yīng)性γδT細(xì)胞,隔周進(jìn)行LA加強(qiáng)免疫。然后分別在免疫后的第2、3、5、8、10周斷尾取血,用ELISA方法檢測血清中抗LA抗體含量。結(jié)果發(fā)現(xiàn),分別在第3和第5周,加LA反應(yīng)性γδT細(xì)胞組小鼠血清中抗LA抗體含量明顯高于未加LA反應(yīng)性γδT細(xì)胞組(P＜0.05)。結(jié)果提示,LA反應(yīng)性γδT細(xì)胞可能會(huì)增強(qiáng)B淋巴細(xì)胞產(chǎn)生抗體的功能。 上述結(jié)果不僅闡明了LA反應(yīng)性γδT細(xì)胞不僅對(duì)E.coli的活性有直接抑制作用,而且能夠調(diào)節(jié)巨噬細(xì)胞和B淋巴細(xì)胞對(duì)細(xì)菌的清除,這為γδT細(xì)胞橋接固有免疫和獲得性免疫的理論提供了實(shí)驗(yàn)支持。
[Abstract]:Gamma delta T cells play an important role in the immune defense and immunomodulatory process of the body, but the recognition of its antigen recognition spectrum is still limited, especially the recognition of lipid antigens by gamma delta T cells. Clarifying the mechanism of interaction between gamma delta T cells and lipid antigens can further clarify the immunology of gamma delta T cells. The status of the disease provides an important clue and experimental basis for comprehensively describing the nature of the immune response, and also provides new strategies and means for immunological application.
The purpose of this thesis is to study the identification mechanism of human gamma delta T cells to lipidA (LA), to explore the TCR Delta -CDR3 length and sequence characteristics of LA reactive gamma delta T cells, and to further explain the immunological function of LA reactive gamma delta T cells.
(1) study on the identification mechanism of human gamma delta T cells to LA
LA is an important component and bioactive center of lipopolysaccharide (LPS) and has a strong immunogenicity. This study focuses on the following questions: what is the mechanism of human delta T cell recognition and the need for antigen presenting cells? What is its molecular mechanism, if necessary, and which antigen to be extracted? What are the characteristics of the molecular correlation? LA reactive gamma delta T cells? For the above problems, first of all, we applied flow analysis, ~3H-TdR incorporation and hydroxyl fluorescein two acetate succinimide (Carboxyfluorescein diacetate succinimidyl ester, CFSE) staining methods to detect the addition and absence of the method respectively. In the case of monocyte-derived dendritic cell (moDC) with mononuclear cells, LA induces the proliferation of gamma delta T cells from human peripheral blood, followed by the use of anti CD1a, CD1b, CD1c, CD1d, TCR gamma, LA, LA, and antibodies against the proliferation of gamma delta T cells. The TCR Delta CDR3 region sequence was measured and its length characteristics were analyzed by gene scanning technique. Finally, the secretion of cytokines including IFN- gamma, TNF- a, CD1b, LA and LA were analyzed in the presence and absence of anti CD1a, CD1b, CD1c, CD1d, TCR gamma delta, LA and other antibodies. During the interaction of delta T cells with LA, when moDC exists, LA can induce the proliferation of gamma delta T cells. While no moDC exists, the proliferation of gamma delta T cells can not be observed. In various antibody blocking experiments, only the anti CD1b, CD1c, TCR gamma delta and LA antibodies can obviously obstruct the proliferation of the LA induced delta cells, while other antibodies have no resistance to this resistance. In addition, we further verified that there was no cross blocking function between anti CD1b and anti CD1c antibodies, and the results of gene scanning found that the fragment length of TCR Delta CDR3 region of LA reactive gamma delta T cells was different, but there was a dominant fragment length about 75bp, and the sequence analysis of TCR Delta CDR3 region of LA reactive gamma delta T fine cells showed that no The specific amino acid sequence was found, but it was found that the composition of the dominant amino acid sequence was TDRV2D-J1; LA reactive gamma delta T cells mainly secreted Th1 cell factors, especially IFN- gamma and TNF- alpha, and were consistent with the functional blocking experimental results. The antibodies against CD1b, CD1c, TCR gamma delta and LA could completely block the secreting cytokine of gamma delta T cells. It is indicated that gamma delta T cells need antigen presenting cells in the process of identifying LA, and that the process is restricted by CD1b and CD1c and depends on TCR gamma. This discovery enriches the recognition of the recognition of lipid antigens by gamma delta T cells, which provides a basis for enriching the mechanism theory of the recognition of the antigen of the gamma delta T cells and describing the essence of the immune response in an all-round way. In addition, our results show that the TCR Delta CDR3 region of the LA reactive gamma delta T cells has not been found highly conservative, but there is an advantageous sequence in both its length and amino acid sequence, which provides clues to the further search for the LA highly specific human gamma delta T cell clones and the highly compatible TCR gamma delta sequences. Foundation.
(two) study on the function of LA reactive gamma delta T cells
Our main contents are as follows: the study on the direct cytotoxic function of LA reactive gamma delta T cells to E.coli, the regulation of LA reactive gamma delta T cells on the phagocytosis of autologous macrophages, and the study of the regulation function of LA reactive gamma delta T cells on the production of anti LA antibodies in B cells.
First, the amount of the corresponding product four Zolo a (INF and CTF) was generated by the reduction of p-iodonitrotetrazolium, INT and 5-cyano-2,3-ditolyl tetrazolium chlorid (CTC), and the biological activity was determined after the LA reactivity gamma delta T cells were incubated with E.coli, and the results were found. The biological activity of I was significantly reduced (P < 0.01) before incubation with LA reactive gamma delta T cells, and the biological activity of Ecoli was becoming weaker with the increase in the number of -LA reactive -LA T cells in the effector cells and the prolongation of the reaction time. These results suggest that LA reactive gamma delta T cells do have cytotoxic effects on E.coli. But the mechanism of action needs to be further clarified.
Secondly, by constructing the prokaryotic expression vector pET-22b (+) -GFP of green fluorescence protein (GFP), and using IPTG to induce the expression of the carrier in Escherichia coli BL21 (DE3), and then use the E.coli (E.coli-GFP) expressing green fluorescent protein to detect the phagocytic function of macrophages. Flow cytometry is used. The difference between the percentage of macrophages phagocytic in E.coli-GFP was analyzed before and after reincubating with LA reactive gamma delta T cells. The results showed that the percentage of Ecoli-GFP~+ macrophages in the two groups was significantly different. The LA reactive gamma delta T cells could promote the phagocytosis of autologous macrophages.
Finally, we use human derived Severe combined immune deficiency (SCID) mice to detect the ability of LA reactive gamma delta T cells to regulate the function of B lymphocytes to produce anti LA antibodies in.SCID mice, 2 weeks after the peritoneal immunization of human peripheral blood mononuclear cells (peripheral blood), by detecting whether there is a human in the peripheral blood. The antibody was used to identify the success of the immune reconstruction. Then, the mice immunized with successful reconstruction were immunized with LA. Some of the mice in the experimental group were intraperitoneally injected with LA reactive gamma delta T cells and were immunized with LA in the other week. Then the blood was taken at the end of 2,3,5,8,10 after immunization, and the content of anti LA antibody in serum was detected by ELISA. The results showed that the anti LA antibody content in the serum of LA reactive gamma delta T cell group was significantly higher than that of the non LA reactive gamma delta T cell group (P < 0.05) in the third and fifth weeks respectively. The results suggest that LA reactive gamma delta T cells may enhance the function of the antibody production by B lymphocyte.
These results not only elucidate that LA reactive gamma delta T cells not only inhibit the activity of E.coli directly, but also regulate the clearance of macrophages and B lymphocytes to bacteria, which provides experimental support for the theory of bridging inherent immunity and acquired immunity of gamma delta T cells.
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【共引文獻(xiàn)】

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