重組人Delta-like1蛋白對外周血單個核細胞來源的DC分化和功能的作用
發(fā)布時間：2019-05-27 00:25
【摘要】： 白血病高復發(fā)率是困擾臨床血液工作者的一個難題,殘留的白血病細胞被認為是引起白血病復發(fā)及影響預后的根本原因。應用免疫治療有效的抑制和去除微小殘留病就成為現(xiàn)階段研究的重要方向。樹突狀細胞(DC)是目前所知的機體內(nèi)功能最強的專職抗原提呈細胞,是自體、異體混合淋巴細胞反應中重要的刺激細胞,因其具有膜樣或樹突狀突起而得名。未成熟樹突狀細胞的主要功能是抗原的識別、內(nèi)吞和細胞內(nèi)抗原的處理。成熟的DC可表達豐富的MHC、B7等免疫分子,能激活靜息T細胞,激發(fā)初始免疫反應從而直接指導效應T細胞的分化和反應,殺傷腫瘤細胞產(chǎn)生抗腫瘤免疫反應。近年來,DC的相關(guān)研究受到廣泛關(guān)注,特別是DC參與細胞毒性T細胞抗腫瘤免疫作用方面的研究取得了一些重要進展。不同形式的腫瘤抗原(蛋白抗原、抗原多肽、腫瘤細胞裂解物、凋亡細胞)及LPS致敏的DC體內(nèi)注射可誘導出特異性抗腫瘤免疫應答,動物實驗及臨床應用已證明DC免疫療法對白血病有一定療效。 Notch信號途徑是從果蠅到人類進化高度保守的三大信號通路之一。它主要通過膜表面受體介導的細胞與細胞間相互作用來調(diào)節(jié)前體細胞和干細胞的發(fā)育、分化以及命運決定。單個核細胞作為巨噬細胞,樹突狀細胞及成骨細胞的前體細胞高表達Notch1及Notch2。研究發(fā)現(xiàn),利用固化hDll1-ex(t即通過表達Delta1-ext-myc融合蛋白,利用myc抗體將其固定于培養(yǎng)皿中)和相應的細胞因子M-CSF的共同作用下,可促進人外周血中單核細胞的凋亡。與之相反,在聯(lián)合細胞因子GM-CSF共同作用下促進了單核細胞向不成熟的樹突狀細胞分化而抑制其向巨噬細胞分化。本實驗室前期已證明Notch信號缺失或者信號增強可以影響小鼠DC的發(fā)育分化和部分功能,但是沒有研究其對抗腫瘤免疫的影響,因此本實驗旨在探討通過利用可溶性的重組hDll-1蛋白或Notch信號阻斷劑GSI改變?nèi)送庵苎獑蝹�核細胞來源DC的Notch信號,觀察其對DC發(fā)育和功能的影響,以期能給白血病的臨床治療提供一個比較好的實驗基礎(chǔ)和理論依據(jù),從而對目前臨床上白血病的生物免疫治療能有所改善。我們主要從以下幾方面進行了研究: 1確定重組hDll-1在DC培養(yǎng)體系中有效作用濃度范圍。 2通過FACS檢測重組hDll-1或Notch信號阻斷劑GSI誘導培養(yǎng)外周血單個核細胞來源的DC的增殖及表面分子CXCR4,HLA-DR,CD83表達的變化。 3測定重組hDll-1蛋白或GSI誘導培養(yǎng)的DC刺激同種異體淋巴細胞增殖反應。 結(jié)果顯示:可溶性的重組hDll1可以激活Notch信號通路,低濃度活化的重組hDll1促進DC的成熟,而高濃度則相反;GSI阻斷Notch信號通路后可以抑制DC的成熟,但是Notch信號的激活或抑制對外周血單個核細胞來源的DC增殖沒有影響。LPS刺激DC后其表面分子HLA-DR,CXCR4和CD83的表達均上調(diào),且重組hDll1蛋白刺激后的DC表面上述分子的表達要高于GSI作用組。Notch信號通路激活后其刺激CD8+ T淋巴細胞增殖能力增強,而GSI則與之相反。結(jié)論:激活Notch信號通路可以促進外周血單個核細胞來源的DC的成熟,其刺激同種異體CD8~+ T淋巴細胞的增殖能力增強,這一特征在白血病的免疫治療中有重要意義。
[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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
本文編號：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.
【學位授予單位】：第四軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392
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