MULT1和MULT1S转基因小鼠的鉴定和初步分析及γδT细胞对类脂A识别机制的研究

发布时间：2018-08-19 16:18

【摘要】： NKG2D是一种激活性受体,由同源二聚体构成,可表达于NK细胞、NKT细胞、CD8+αβT细胞和γδT细胞等多种免疫细胞表面,在固有免疫和适应性免疫应答中都起着非常重要的作用。近年来,UL16结合蛋白(ULBPs)作为一类新发现的人NKG2D配体家族日益受到关注。MULT1及其的剪切变体MULT1S,是ULBPs在小鼠的同类物,是小鼠的NKG2D配体。MULT1在正常的细胞膜上不表达或表达水平很低。在病理条件下,如在应激细胞、细菌或病毒感染细胞和肿瘤细胞,MULT1表达可上调,并通过与小鼠NKG2D受体结合,介导抗感染和肿瘤免疫。 MULT1/MULT1S是跨膜蛋白,其胞内段较长,除了通过NKG2D受体介导抗感染和肿瘤免疫外,是否还具有其它功能,如是否与胸腺的发育有关,是否有反向的信号转导的功能以及抗原提呈细胞上是否会表达MULT1/MULT1S作为危险信号或作为协同刺激分子发挥作用,MULT1和MULT1S两者在功能上有何区别等问题,目前文献尚无报道,有必要进一步的研究。因此,我们建立了MULT1/MULT1S的转基因小鼠模型,旨在通过过表达MULT1/MULT1S,比较两种转基因小鼠MULT1/MULT1S在组织中的表达及其对免疫系统的影响,以了解它们的免疫学功能。本论文的第一部分主要是将构建的两个品系的转基因小鼠MULT1Tg (3'S和4'S)及3个品系的MULT1STg (7'S、10'S和41’S)与野生型C57BL／6小鼠交配,以产生Fl代小鼠,并对F1代小鼠进行基因型和表型的鉴定,为筛选符合进一步研究需要的转基因小鼠提供依据。通过提取F1代转基因小鼠的基因组DNA进行基因组PCR及Southern blotting,以鉴定转基因小鼠的基因型。结果获得鉴定阳性的小鼠3’S 25只、4'S 13只、7'S 16只、10'S 42只和41’S 9只。对小鼠尾部组织和尾血mRNA的实时定量RT-PCR检测结果表明,3’S和4'S品系的MULT1Tg小鼠以及10’S和41’S品系的MULT1STg小鼠的基因转录产物阳性,可用于进行下一步研究。应用流式细胞分析以及免疫组化对MULT1Tg小鼠多种组织细胞进行初步分析的结果显示,MULT1主要表达在胸腺和小肠上皮间淋巴细胞,在脾细胞上基本不表达,且在小肠上皮间淋巴细胞表面的表达与小鼠的年龄有关。这提示下一步研究应主要开展胸腺、肠道及皮肤组织的相关研究。本文的第二部分是有关人γδT细胞对类脂A的识别机制的研究(此部分与崔永春博士共同完成)。首先,应用流式细胞术、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)的条件下,类脂A诱导人外周血来源的γδT细胞的增殖情况。结果显示,当moDC存在时,LA能诱导人类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。这提示γδT细胞仅识别由moDC递呈的LA抗原。其次,抗CD1a、CD1b、CD1c、CD1d、TCRγδ、LA、MHCI和MHCⅡ抗体对LA诱导的γδT细胞增殖的阻断实验结果表明,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；提示γδT细胞对LA的识别,是由moDC细胞提呈,由moDC细胞上的CD1b和CD1c分子介导,并依赖于TCRγδ的过程。最后,对LA反应性γδT细胞的TCRγδCDR3区基因序列的测定结果,未见有特异性保守序列,但发现优势取用J1片段,尚需对其结合特性作进一步验证。上述发现丰富了对γδT细胞识别脂类抗原机制的认识。
[Abstract]:NKG2D is an active receptor, which is composed of homologous dimers and can be expressed on the surface of NK cells, NKT cells, CD8 + alpha beta T cells and gamma delta T cells. It plays an important role in innate and adaptive immune responses. In recent years, UL16 binding proteins (ULBPs) have become a new class of human NKG2D ligand family. MuLT1 and its shear variant MULT1S, the analogue of ULBPs in mice and the NKG2D ligand in mice, are not expressed or expressed at very low levels on normal cell membranes. Anti infection and tumor immunity.
MULT1/MULT1S is a transmembrane protein with a long intracellular segment. It has other functions besides anti-infection and tumor immunity mediated by NKG2D receptor, such as whether it is related to the development of thymus, whether it has the function of reverse signal transduction and whether MULT1/MULT1S is expressed as a dangerous signal or as a co-stimulus on antigen presenting cells. There is no report on the functional differences between MULT1 and MULT1S, so it is necessary to study them further. Therefore, we have established a transgenic mouse model of MULT1/MULT1S to compare the expression of MULT1/MULT1S in tissues and its immune system by overexpressing MULT1/MULT1S. The influence of the system is to understand their immunological functions.
In the first part of this paper, two murine strains MULT1Tg (3'S and 4'S) and three mutant strains MULTT1STg (7'S, 10'S and 41'S) were mated with wild type C57BL/6 mice to produce Fl mice. Genotype and phenotype of F1 mice were identified to screen transgenic mice for further study. The genomic DNA of F1 transgenic mice was extracted for genomic PCR and Southern blotting to identify the genotype of transgenic mice. Results The positive mice were identified as 3'S 25, 4'S 13, 7'S 16, 10'S 42 and 41'S 9. The results of real-time quantitative RT-PCR showed that the mRNA of tail tissue and tail blood of mice were positive. The results of flow cytometry and immunohistochemistry showed that MULT1 was mainly expressed in thymus and small intestinal epithelial lymph nodes. The expression of lymphocytes on the surface of intestinal epithelial lymphocytes is related to the age of mice, which suggests that the next step of research should mainly focus on thymus, intestine and skin tissue.
The second part of this paper is about the recognition mechanism of lipid A by human gamma delta T cells (this part was completed with Dr. Cui Yongchun). Firstly, flow cytometry, 3H-TdR incorporation assay and carboxyfluorescein diacetate succinimidyl ester (CFSE) staining were used to detect the lipid A. Lipid A could induce the proliferation of human peripheral blood-derived gamma delta T cells with or without monocyte-derived dendritic cells (moDC). The results showed that LA could induce the proliferation of human gamma delta T cells in the presence of moDC, but the proliferation of human gamma delta T cells could not be observed in the absence of moDC. These results suggest that gamma delta T cells recognize only LA antigens presented by moDC. Secondly, anti-CD1a, CD1b, CD1c, CD1d, TCR gamma delta, LA, MHCI and MHCII antibodies can block the proliferation of LA-induced gamma delta T cells. The results show that only anti-CD1b, CD1c, TCR gamma delta and LA antibodies can significantly block the proliferation of LA-induced gamma delta T cells. These results suggest that the recognition of LA by gamma delta T cells is mediated by CD1b and CD1c molecules on moDC cells and depends on the process of TCR gamma delta. Finally, the sequencing of TCR gamma delta CDR3 region gene in LA reactive gamma delta T cells showed no specific conservative sequence, but the J1 fragment was found to be the dominant candidate for LA binding. The above findings enrich the understanding of the mechanism of the recognition of lipid antigens by gamma delta T cells.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【相似文献】