重组人Delta-like1蛋白对外周血单个核细胞来源的DC分化和功能的作用
发布时间：2019-05-27 00:25
【摘要】： 白血病高复发率是困扰临床血液工作者的一个难题,残留的白血病细胞被认为是引起白血病复发及影响预后的根本原因。应用免疫治疗有效的抑制和去除微小残留病就成为现阶段研究的重要方向。树突状细胞(DC)是目前所知的机体内功能最强的专职抗原提呈细胞,是自体、异体混合淋巴细胞反应中重要的刺激细胞,因其具有膜样或树突状突起而得名。未成熟树突状细胞的主要功能是抗原的识别、内吞和细胞内抗原的处理。成熟的DC可表达丰富的MHC、B7等免疫分子,能激活静息T细胞,激发初始免疫反应从而直接指导效应T细胞的分化和反应,杀伤肿瘤细胞产生抗肿瘤免疫反应。近年来,DC的相关研究受到广泛关注,特别是DC参与细胞毒性T细胞抗肿瘤免疫作用方面的研究取得了一些重要进展。不同形式的肿瘤抗原(蛋白抗原、抗原多肽、肿瘤细胞裂解物、凋亡细胞)及LPS致敏的DC体内注射可诱导出特异性抗肿瘤免疫应答,动物实验及临床应用已证明DC免疫疗法对白血病有一定疗效。 Notch信号途径是从果蝇到人类进化高度保守的三大信号通路之一。它主要通过膜表面受体介导的细胞与细胞间相互作用来调节前体细胞和干细胞的发育、分化以及命运决定。单个核细胞作为巨噬细胞,树突状细胞及成骨细胞的前体细胞高表达Notch1及Notch2。研究发现,利用固化hDll1-ex(t即通过表达Delta1-ext-myc融合蛋白,利用myc抗体将其固定于培养皿中)和相应的细胞因子M-CSF的共同作用下,可促进人外周血中单核细胞的凋亡。与之相反,在联合细胞因子GM-CSF共同作用下促进了单核细胞向不成熟的树突状细胞分化而抑制其向巨噬细胞分化。本实验室前期已证明Notch信号缺失或者信号增强可以影响小鼠DC的发育分化和部分功能,但是没有研究其对抗肿瘤免疫的影响,因此本实验旨在探讨通过利用可溶性的重组hDll-1蛋白或Notch信号阻断剂GSI改变人外周血单个核细胞来源DC的Notch信号,观察其对DC发育和功能的影响,以期能给白血病的临床治疗提供一个比较好的实验基础和理论依据,从而对目前临床上白血病的生物免疫治疗能有所改善。我们主要从以下几方面进行了研究: 1确定重组hDll-1在DC培养体系中有效作用浓度范围。 2通过FACS检测重组hDll-1或Notch信号阻断剂GSI诱导培养外周血单个核细胞来源的DC的增殖及表面分子CXCR4,HLA-DR,CD83表达的变化。 3测定重组hDll-1蛋白或GSI诱导培养的DC刺激同种异体淋巴细胞增殖反应。 结果显示:可溶性的重组hDll1可以激活Notch信号通路,低浓度活化的重组hDll1促进DC的成熟,而高浓度则相反;GSI阻断Notch信号通路后可以抑制DC的成熟,但是Notch信号的激活或抑制对外周血单个核细胞来源的DC增殖没有影响。LPS刺激DC后其表面分子HLA-DR,CXCR4和CD83的表达均上调,且重组hDll1蛋白刺激后的DC表面上述分子的表达要高于GSI作用组。Notch信号通路激活后其刺激CD8+ T淋巴细胞增殖能力增强,而GSI则与之相反。结论:激活Notch信号通路可以促进外周血单个核细胞来源的DC的成熟,其刺激同种异体CD8~+ T淋巴细胞的增殖能力增强,这一特征在白血病的免疫治疗中有重要意义。
[Abstract]:The high recurrence rate of the leukemia is a difficult problem for the clinical blood workers, and the residual leukemia cells are considered to be the root cause of the recurrence and the prognosis of the leukemia. The effective inhibition of immunotherapeutic and the removal of microresidual disease have become the important direction of the present research. Dendritic cells (DC) are the most potent full-time antigen-presenting cells in the body, which are the most important stimulating cells in the reaction of autologous and allogenic mixed lymphocytes, which are named after they have membrane-like or dendritic protrusions. The primary function of immature dendritic cells is the identification of antigens, endocytosis and the treatment of intracellular antigens. The mature DC can express rich MHC, B7 and other immune molecules, can activate the resting T cell, stimulate the initial immune response, direct the differentiation and reaction of the effector T cell, and kill the tumor cell to produce the anti-tumor immune response. In recent years, the related research of DC has been widely concerned, and in particular, the research of DC in cytotoxic T cell anti-tumor immune function has made some important progress. Different forms of tumor antigen (protein antigen, antigen polypeptide, tumor cell lysate, apoptosis cell) and LPS-sensitized DC in vivo injection can induce specific anti-tumor immune response, and animal experiments and clinical application have shown that the DC immune therapy has a certain curative effect on the leukemia. The Notch signaling pathway is the three signals that are highly conserved from Drosophila to human evolution One of the ways. It mainly regulates the development, differentiation, and life of precursor cells and stem cells by cell-to-cell interactions mediated by membrane surface receptors. Transport decision. Individual nuclear cells express Notch1 and Not as a high expression of the precursor cells of macrophages, dendritic cells, and osteoblasts Ch2. The study found that the human peripheral blood mononuclear cells can be promoted by the co-action of curing hDll1-ex (t, i.e., by expressing the Delta1-ext-myc fusion protein, using the myc antibody to fix it in the culture dish) and the corresponding cytokine M-CSF. In contrast to this, the differentiation of monocytes to immature dendritic cells is promoted by the co-action of the combined cytokine, GM-CSF, to inhibit its fine phagocytosis Cell differentiation. The absence or signal enhancement of the Notch signal in the early stage of the lab can affect the development and differentiation and partial function of the mouse DC, but it does not study it against tumor immunity The purpose of this experiment was to investigate the effect of the soluble recombinant hDll-1 protein or the Notch signal blocking agent GSI on the DC of human peripheral blood mononuclear cells, and to observe its effect on the development and function of DC. The effect of the present invention is to provide a better experimental basis and a theoretical basis for the clinical treatment of leukemia, so that the biological immunotherapy for the present clinical leukemia can be The improvement. We mainly proceed from the following aspects The results were as follows:1. The recombinant hDll-1 was determined to be in the DC culture system. GSI-induced proliferation and surface-molecule CXCR4, HLA-D in peripheral blood mononuclear cells by FACS detection of recombinant hDll-1 or Notch signaling blocker GSI Changes in expression of R, CD83.3. Determination of recombinant hDll-1 protein or GSI-induced culture D The results showed that the soluble recombinant hDll1 can activate the Notch signaling pathway, and the low-concentration activated recombinant hDll1 promotes the maturation of the DC, while the high concentration is the opposite; the GSI block The Notch signal path can inhibit DC maturation, but the Notch signal is activated or suppressed The expression of HLA-DR, CXCR4 and CD83 was up-regulated after LPS-stimulated DC and D-Dll1 protein was stimulated by the recombinant hDll1 protein. The expression of the above-mentioned molecules on the surface of the C-surface is higher than that of the GSI group. Conclusion: The activation of the Notch signaling pathway can promote the maturation of a single source of peripheral blood mononuclear cells, which can stimulate the proliferation of the allogeneic CD8 + T lymphocytes.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
本文编号：2485759
[Abstract]:The high recurrence rate of the leukemia is a difficult problem for the clinical blood workers, and the residual leukemia cells are considered to be the root cause of the recurrence and the prognosis of the leukemia. The effective inhibition of immunotherapeutic and the removal of microresidual disease have become the important direction of the present research. Dendritic cells (DC) are the most potent full-time antigen-presenting cells in the body, which are the most important stimulating cells in the reaction of autologous and allogenic mixed lymphocytes, which are named after they have membrane-like or dendritic protrusions. The primary function of immature dendritic cells is the identification of antigens, endocytosis and the treatment of intracellular antigens. The mature DC can express rich MHC, B7 and other immune molecules, can activate the resting T cell, stimulate the initial immune response, direct the differentiation and reaction of the effector T cell, and kill the tumor cell to produce the anti-tumor immune response. In recent years, the related research of DC has been widely concerned, and in particular, the research of DC in cytotoxic T cell anti-tumor immune function has made some important progress. Different forms of tumor antigen (protein antigen, antigen polypeptide, tumor cell lysate, apoptosis cell) and LPS-sensitized DC in vivo injection can induce specific anti-tumor immune response, and animal experiments and clinical application have shown that the DC immune therapy has a certain curative effect on the leukemia. The Notch signaling pathway is the three signals that are highly conserved from Drosophila to human evolution One of the ways. It mainly regulates the development, differentiation, and life of precursor cells and stem cells by cell-to-cell interactions mediated by membrane surface receptors. Transport decision. Individual nuclear cells express Notch1 and Not as a high expression of the precursor cells of macrophages, dendritic cells, and osteoblasts Ch2. The study found that the human peripheral blood mononuclear cells can be promoted by the co-action of curing hDll1-ex (t, i.e., by expressing the Delta1-ext-myc fusion protein, using the myc antibody to fix it in the culture dish) and the corresponding cytokine M-CSF. In contrast to this, the differentiation of monocytes to immature dendritic cells is promoted by the co-action of the combined cytokine, GM-CSF, to inhibit its fine phagocytosis Cell differentiation. The absence or signal enhancement of the Notch signal in the early stage of the lab can affect the development and differentiation and partial function of the mouse DC, but it does not study it against tumor immunity The purpose of this experiment was to investigate the effect of the soluble recombinant hDll-1 protein or the Notch signal blocking agent GSI on the DC of human peripheral blood mononuclear cells, and to observe its effect on the development and function of DC. The effect of the present invention is to provide a better experimental basis and a theoretical basis for the clinical treatment of leukemia, so that the biological immunotherapy for the present clinical leukemia can be The improvement. We mainly proceed from the following aspects The results were as follows:1. The recombinant hDll-1 was determined to be in the DC culture system. GSI-induced proliferation and surface-molecule CXCR4, HLA-D in peripheral blood mononuclear cells by FACS detection of recombinant hDll-1 or Notch signaling blocker GSI Changes in expression of R, CD83.3. Determination of recombinant hDll-1 protein or GSI-induced culture D The results showed that the soluble recombinant hDll1 can activate the Notch signaling pathway, and the low-concentration activated recombinant hDll1 promotes the maturation of the DC, while the high concentration is the opposite; the GSI block The Notch signal path can inhibit DC maturation, but the Notch signal is activated or suppressed The expression of HLA-DR, CXCR4 and CD83 was up-regulated after LPS-stimulated DC and D-Dll1 protein was stimulated by the recombinant hDll1 protein. The expression of the above-mentioned molecules on the surface of the C-surface is higher than that of the GSI group. Conclusion: The activation of the Notch signaling pathway can promote the maturation of a single source of peripheral blood mononuclear cells, which can stimulate the proliferation of the allogeneic CD8 + T lymphocytes.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392
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