【作者】 许云云；

【导师】 王泳； 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2009， 硕士

【摘要】 胸腺是T细胞分化、发育、成熟的主要器官。从骨髓迁入的淋巴样祖细胞在与独特的胸腺微环境基质细胞的相互作用下,经过复杂的分化发育过程,最终成为功能性CD4+T细胞及CD8+T细胞。由于在体内很难进行单一细胞群体或某种特殊分子对胸腺发育影响的研究,使胸腺发育的研究一直难以取得突破性进展,体外培养体系—胚胎胸腺器官培养(fetal thymus organ culture, FTOC)的建立和发展,为胸腺细胞发育、成熟及其相关调节机制的探讨提供了有效的研究手段。树突状细胞(dendritic cells, DCs)是机体中最主要的和最有效的抗原提呈细胞,对启动T细胞的免疫应答起着至关重要的作用。胸腺作为中枢免疫器官,存在着胸腺树突状细胞(thymic dendritic cells, TDCs)。近年来,不断有TDCs的相关文献报道,TDCs在调控阴性选择和诱导自身免疫耐受方面发挥着重要的作用。FMS样酪氨酸激酶3配体(Fms-like tyrosine kinase 3 ligand, Flt3L)是一种重要的可促进多能干细胞分化的细胞因子。体内和体外实验表明, Flt3L在诱导树突状细胞分化、发育方面发挥着重要的作用。本研究在成功建立FTOC实验体系的基础上,利用该实验体系研究了Flt3L对胸腺T细胞及胸腺树突状细胞的分化发育的影响。本论文分为两部分。第一部分主要阐述了FTOC实验体系的建立,并在此基础上研究细胞因子白细胞介素-7(Interleukin-7, IL-7)和Flt3L对胸腺T细胞和胸腺树突状细胞分化发育的影响。首先是FTOC常规实验体系的建立,选取15.5～16.5dpc(day postcoitus)的孕鼠作为实验对象,于实体显微镜下摘取胚胎胸腺,在组织器官培养皿中进行FTOC常规培养。实验分为Flt3L组、IL-7组和对照组,12d后收集各实验组经FTOC培养所获得的胸腺细胞,通过细胞计数检测细胞数目的变化,用流式细胞仪检测细胞表面分子CD4、CD8、CD11c、B220、Ia等的表达,通过光学显微镜观察细胞形态。获得以下实验结果:(1)细胞计数显示,添加外源性IL-7组的胸腺细胞数目明显减少,而Flt3L组和对照组的胸腺细胞数目无明显统计学的差异。(2)流式细胞仪检测显示:IL-7组胸腺CD4-CD8-双阴性细胞及CD8+单阳性细胞比例有所增加,而CD4+CD8+双阳性细胞比例显著下降,CD4+单阳性细胞比例没有明显变化;Flt3L组胸腺T细胞大部分为CD4+CD8+双阳性细胞及CD4+?CD8+单阳性细胞;Flt3L组的胸腺T细胞亚群分布与对照组无明显统计学的差异。(3)形态学观察显示:Flt3L组和IL-7组的胸腺细胞中均可见一些聚集的有突起的具有树突状细胞形态特征的细胞存在,而对照组绝大部分为圆形的胸腺细胞。由此可见IL-7和Flt3L在胸腺T细胞及胸腺树突状细胞的分化发育中发挥重要的调节作用。第二部分研究利用常规FTOC实验体系并联合悬滴培养方法,将小鼠骨髓来源的c-kit+造血干/祖细胞植入胸腺,从而探讨Flt3L对小鼠骨髓源造血干/祖细胞向胸腺树突状细胞分化、发育过程的影响。摘取15.5～16.5dpc胎鼠的胸腺,用药液2-脱氧鸟苷(2-deoxyguanosine, 2-dGuo)处理胸腺以去除胸腺中的造血细胞,随后将骨髓来源的c-kit+造血干/祖细胞通过悬滴培养方法植入处理过的胸腺,最后将胸腺置于组织器官培养皿中,将含有和未含有细胞因子Flt3L的培养基分别滴加在胸腺小叶上,进行FTOC培养,实验分为对照组和Flt3L组。12d后收集不同条件下FTOC培养获得的胸腺细胞,通过流式细胞仪对胸腺细胞的表型进行分析;将在不同条件下FTOC培养的获得胸腺细胞分别进行MACS分选,从而获得胸腺树突状细胞两亚群—髓系树突状细胞(B220-CD11c+, conventional dendritic cells, cDC)和浆细胞样树突状细胞(B220+CD11c+, plasmacytoid dendritic cells, pDC),再通过Giemsa染色法对其进行形态学分析,通过RT-PCR方法检测其Toll样受体的表达,通过ELISA方法检测其特异性细胞因子的分泌情况,再将经FTOC培养获得的胸腺cDC和胸腺pDC分别与异源的T细胞进行混合淋巴细胞反应,通过MTT法检测T细胞的增殖。获得以下实验结果:(1)流式细胞仪检测显示:与对照组相比较,Flt3L组树突状细胞和B细胞、NK细胞数量均有不同程度的增加。(2)Giemsa染色显示:经过CpG2216刺激成熟后,胸腺cDC和胸腺pDC均表现出成熟树突状细胞的形态特征。(3)RT-PCR分析显示:CpG2216刺激后,胸腺cDC低表达或不表达TLR7和TLR9,而胸腺pDC高表达TLR7和TLR9。(4)ELISA分析显示:CpG2216刺激后,小鼠的胸腺cDC中IFN-α和IL-12p70的分泌量均低于胸腺pDC。(5)MTT检测表明,无CpG2006刺激时,胸腺cDC和胸腺pDC刺激T细胞增殖的能力均比较弱;但添加CpG2006刺激后,胸腺cDC和胸腺pDC趋向于成熟,刺激T细胞增殖的能力均有所增强。由此可见,来自Flt3L组的胸腺细胞中的部分细胞不仅具有类似树突状细胞的形态和表达树突状细胞表面特异分子,而且也具有激发T细胞的作用。以上结果表明Flt3L能促进胸腺树突状细胞的生成。综上所述,本研究成功建立了FTOC的实验体系,研究了Flt3L和IL-7对胸腺T细胞及胸腺树突状细胞分化发育的影响。并且在此基础上,进一步研究了Flt3L对小鼠骨髓源c-kit+造血干/祖细胞向胸腺树突状细胞分化发育过程的影响。通过该实验体系,可以在体外扩增胸腺cDC和胸腺pDC,这将为体外研究胸腺树突状细胞的分化发育及其相关分子机理提供了有效的实验手段。更多还原

【Abstract】 Thymus is an important organ of T-cell differentiation, development and maturation. Marrow-derived T lymphoid precursor cells can develop into funtional CD4 postive T cells or CD8 positive T cells only through the complicated interactions with thymic stroml cells in the microenvironment in the thymus. There was no breakthrough progress on the researches of development of thymus so far, as there were no good ways to monitor the effection of signal cell population or specific molecules on thymus development. The establishment and development of fetal thymus organ culture technology now offer new and effective means of the further study on the development, maturation and related regulation mechanism of the thymocytes.Dendritic cells, the most important and effective member of antigen presenting cells and are essential for the T cells immunoresponse as a kind of accessory immune cells. As the central immune organ, thymus contains plenty of thymic dendritic cells (TDCs), which can enhance the proliferation of thymic T lymphocytes, regulate the negative selection and induce central tolerance through presenting various antigens to play an important role in regulating cellular and humoral immunity. In this study, the effects of Fms-like tyrosine kinase 3 ligand (Flt3L) on the development of thymic T cells and TDCs were investigated basis on the establishment of the FTOC experimental system.This study combined two parts. In the first part, we focused on the establishment of FTOC experimental system, and then conducted the analysis of the effect of Interleukin-7 (IL-7) and Flt3L on the development of thymic T cells and thymic dendritic cells. Thymic lobes were removed from 15-day-old or 16-day-old mouse fetueses respectively and were cultured under the fetal thymus organ cultures (FTOC) system in vitro. Being cultured, thymic lobes were treated with IL-7 or Flt3L and the group within which no cytokines was added was adopted as the control, then collected the cultured cell respectively, to detect the expression of CD4, CD8, CD11c, B220, Ia and CD11b by flow cytometry, and to observe the morphology under the ligtmicroscope. The results showed that:(1)The result of cell counting indicated that the total amount of the thymocytes was decreased obviously after been cultured with IL-7, while the total amount of thymocytes in the Flt3L and control group was the same. (2) The result of FACS indicated that in IL-7 group the proportion of CD4+CD8+thymocytes decreased, the proportion of CD4-CD8- and CD4-CD8+ thymocytes increased, and no much difference of the amount of CD4+CD8- thymocytes was found, while in Flt3L group CD4+CD8+ thymocytes and CD4-CD8+/CD4+CD8- thymocytes occupied the main part and its T Lymphocyte Subsets distribution was as same as the control group. (3) A group of cells with the characteristic morphology of the dendritic cells was observed under the lightmicroscope in IL-7 group and Flt3L group. So IL-7 and Flt3L played important role in the differentiation and development of thymic T lymphocytes and dendritic cells in thymus.In the second part, bone marrow hematopoietic stem/progenitor cells (HSCs/HPCs) were seeded into 2-deoxyguanosine (2-dGuo)-treated thymus by the way of hanging drop culture, and the effect of Flt3L on the differentiation of thymic dendritic cells were thoroughly investigated by using FTOC.The thymic lobes of fetal mice were treated with 2-dGuo, and then the treated thymic lobes were seeded with c-kit+ HSCs/HPCs by hanging drop culture in the Teraski plate, followed by FTOC in the presence or absence of Flt3L. After 12 days of culture, the cells of the thymic lobes were collected for analysis. The expression of CD4, CD8, CD11c, B220, Ia and CD11b were detected by flow cytometry, the thymic conventional dendritic cells (cDC) and plasmacytoid dendritic cells (pDC) were sorted by MACS. The morphology of the isolated cells was observed by Giemsa staining. In addition, the expressions of Toll-like receptors were screened by RT-PCR. Furthermore, the expressions of IFN-αand IL-12 were assayed by ELISA. The isolated cDCs and pDCs were used as the stimulators for allogeneic T cells with or without CpG stimultion, whose proliferation were then detected by MTT method. The results show as fllows: (1) The results of FCM indidated that the numbers of B lymphocytes, natural killer cells and dendritic cells were increased in the presence of Flt3L compared with control group. (2) The results of Giemsa indicated that both thymic cDC and pDC had the typic morphology of dendritic cells. (3) The results of RT-PCR showed that thymic cDCs expressed little TLR7 and TLR9, whereas thymic pDCs highly expressed TLR7 and TLR9. (4) The results of ELISA indicated that thymic pDC secreted more amount of IFN-αand IL-12 than thymic cDC did. (5) MTT assay showed that with the stimulation of CpG2006, both thymic pDCs and cDCs had the capacity of stimulating allogeneic T cell proliferation. There results indicate that Flt3L can promote the differentiation and generation of thymic dendritic cells from c-kit+ HSCs/HPCs by using FTOC.In summary, the FTOC experimental system was established successfully, on the base of which the effects of IL-7 and Flt3L on the development of thymic T cells and TDCs were extensively investigated. Moreover, the effect of Flt3L on the differentiation of TDCs from murine c-kit+ HSCs/HPCs was studied by using FTOC. Both of the generation of thymic cDC and pDC could be promoted in the presence of Flt3L. All these results may lay a foundation for the clarification of the cellular and molecular mechanisms on the development and differentiation of T cells and TDCs in vitro.更多还原