类脂A活化人类γδT细胞的机制与效应研究

发布时间：2018-05-25 08:35

本文选题：类脂 + 活化　；参考：《中国协和医科大学》2006年博士论文

【摘要】： γδT细胞在机体的免疫防御和免疫调节过程中发挥着重要作用,但目前对其抗原识别谱的认识还很局限,尤其是γδT细胞对脂类抗原的识别还知之甚少。澄清γδT细胞与脂类抗原之间的相互作用机制,可为进一步阐明γδT细胞在免疫学中的地位,全面描绘免疫应答的本质提供了重要线索和实验依据,并也为免疫学应用提供新的策略与手段。本论文旨在研究人类γδT细胞对lipidA(LA)的识别机制,初步探讨LA反应性γδT细胞的TCRδ-CDR3长度及序列特点,以及进一步阐释LA反应性γδT细胞的免疫学功能。 (一)人类γδT细胞对LA识别机制的研究 LA是脂多糖(lipopolysaccharide,LPS)的重要组成部分和生物活性中心,具有较强的免疫原性。本研究集中讨论了如下问题:人类γδT细胞对其识别的机制究竟如何?是否需要抗原提呈细胞的作用?如果需要,那么其分子机制是什么?与哪种抗原提呈分子相关?LA反应性γδT细胞的TCR又有什么样的特征可寻?针对以上问题,首先,我们分别应用流式分析技术、~3H-TdR掺入实验和羟基荧光素二醋酸盐琥珀酰亚胺脂(Carboxyfluorescein diacetate succinimidyl ester,CFSE)染色方法分别检测了在加与不加单核细胞来源的树突状细胞(monocyte-derived dendritic cell,moDC)情况下,LA诱导人外周血来源的γδT细胞的增殖情况;其次,分别使用抗CD1a、CD1b、CD1c、CD1d、TCRγδ、LA、MHCⅠ和MHCⅡ抗体对LA诱导的γδT细胞增殖过程进行阻断;再次,对LA反应性γδT细胞的TCRδCDR3区序列进行测定,并使用基因扫描技术分析其长度特征;最后,分析了在抗CD1a、CD1b、CD1c、CD1d、TCRγδ、LA等抗体存在和不存在情况下,LA对γδT细胞刺激引发的包括IFN-γ、TNF-α、IL-2、IL-4、IL-5、IL-10在内的细胞因子分泌情况。结果表明,人类γδT细胞与LA相互作用过程中,当moDC存在时,LA能诱导γδT细胞明显增殖,而无moDC存在时,基本上观察不到γδT细胞有增殖现象;在各种抗体阻断实验中,只有抗CD1b、CD1c、TCRγδ和LA的抗体能明显阻断LA诱导的γδT细胞增殖,而其它抗体没有此阻断效应;另外,我们进一步验证了抗CD1b和抗CD1c抗体之间没有交叉阻断功能;基因扫描结果发现LA反应性γδT细胞的TCRδCDR3区的片断长度各不相同,但有长度约为75bp的优势片段,对LA反应性γδT细胞的TCRδCDR3区的序列分析的结果表明没有发现有特异性氨基酸序列,但发现其优势氨基酸序列的组成是TDRV2D-J1;LA反应性γδT细胞主要分泌Th1类细胞因子,尤其是IFN-γ和TNF-α,并且与功能阻断实验结果一致,抗CD1b、CD1c、TCRγδ和LA的抗体能完全阻断γδT细胞分泌细胞因子。上述结果说明γδT细胞在识别LA的过程中,需要抗原提呈细胞的提呈作用,而且该过程是受CD1b和CD1c的限制,并依赖于TCRγδ。这一发现丰富γδT细胞识别脂类抗原的认识,为丰富γδT细胞识别抗原机制理论、全面描绘免疫应答的本质提供了依据。另外,我们的结果表明LA反应性γδT细胞的TCRδCDR3区的序列没有发现高度保守的,但无论其长度还是氨基酸序列组成都有优势序列存在,这为进一步寻找LA高度特异性的人类γδT细胞克隆和高度亲和力的TCRγδ序列提供了线索,并奠定了基础。 (二)LA反应性γδT细胞的功能研究我们主要研究内容如下:LA反应性γδT细胞对E.coli的直接细胞毒功能研究;LA反应性γδT细胞对自体巨噬细胞吞噬功能的调节作用研究;LA反应性γδT细胞对B细胞产生抗LA抗体的调节功能研究。首先,我们通过氧化还原染料-碘硝基四氮唑(p-iodonitrotetrazolium,INT)和联甲苯四唑氯(5-cyano-2,3-ditolyl tetrazolium chlorid,CTC)还原生成相应的产物四唑甲(INF和CTF)的量,在LA反应性γδT细胞与E.coli共孵育4hr后,测定Ecoli的生物活性。结果发现,E.coli的生物活性在与LA反应性γδT细胞孵育后较孵育前有非常显著的降低(P＜0.01);并且随着效应细胞-LA反应性γδT细胞数量的增加及反应时间的延长,Ecoli的生物活性越来越弱。以上结果提示LA反应性γδT细胞对E.coli的确具有细胞毒作用,但是作用机制有待于进一步澄清。其次,通过构建绿色荧光蛋白(green fluorescence protein,GFP)的原核表达载体pET-22b(+)-GFP,并用IPTG诱导该载体在大肠杆菌BL21(DE3)中进行表达,然后利用这种表达绿色荧光蛋白的E.coli(E.coli-GFP)来检测巨噬细胞的吞噬功能。使用流式细胞仪技术分析与LA反应性γδT细胞共孵育前后的吞噬了E.coli-GFP的巨噬细胞百分比的差异。结果提示,两组中Ecoli-GFP~+巨噬细胞百分含量有显著差异,LA反应性γδT细胞能促进自体巨噬细胞的吞噬功能。最后,我们利用人源化Severe combined immune deficiency(SCID)小鼠检测LA反应性γδT细胞是否能够调节B淋巴细胞产生抗LA抗体的功能。SCID小鼠腹腔接种人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)后2周,通过检测其外周血中是否含有人类抗体来鉴定其免疫重建是否成功。然后,对免疫重建成功的小鼠用LA进行免疫,其中部分实验组小鼠同时腹腔注射LA反应性γδT细胞,隔周进行LA加强免疫。然后分别在免疫后的第2、3、5、8、10周断尾取血,用ELISA方法检测血清中抗LA抗体含量。结果发现,分别在第3和第5周,加LA反应性γδT细胞组小鼠血清中抗LA抗体含量明显高于未加LA反应性γδT细胞组(P＜0.05)。结果提示,LA反应性γδT细胞可能会增强B淋巴细胞产生抗体的功能。上述结果不仅阐明了LA反应性γδT细胞不仅对E.coli的活性有直接抑制作用,而且能够调节巨噬细胞和B淋巴细胞对细菌的清除,这为γδT细胞桥接固有免疫和获得性免疫的理论提供了实验支持。
[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.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

【共引文献】