间充质干细胞（MSCs）通过诱导骨髓来源的髓系细胞的抑制功能促进肿瘤进展

发布时间：2018-06-02 19:53

本文选题：间充质干细胞 + CD11b~+Gr1~+　；参考：《北京协和医学院》2014年硕士论文

【摘要】：背景：间充质干细胞(mesenchymal stem cells,MSCs)是中胚层来源的具有高度自我更新能力和多向分化能力的多能干细胞,广泛存在于全身多种组织中,可在体外培养扩增,并能在特定条件下分化为神经细胞、成骨细胞、软骨细胞、心肌细胞、脂肪细胞等,是细胞替代治疗和组织工程首选的种子细胞,具有广阔的临床应用前景。此外,间充质干细胞的染色体核型和端粒酶活性均稳定,不表达组织相容性复合物(MHC)等刺激分子,因此不易被宿主T细胞识别,可逃脱宿主免疫系统排斥,具有低免疫性。MSCs还可通过旁分泌作用抑制B淋巴细胞和T淋巴细胞的增殖,诱导T淋巴细胞的凋亡而起到免疫抑制作用。微环境中的多种因素儿可影响MSCs的免疫调节能力,TNF-a可诱导MSCs高表达iNOS,通过NO依赖的方式直接抑制免疫应答；也可促进COX-2和PGE2表达上调,通过PGE2依赖的方式直接抑制免疫应答。另外,MSCs可通过分泌大量的细胞因子吸引更多的单核细胞、巨噬细胞以及中性粒细胞,从而影响肿瘤的生长。但MSCs对骨髓中CD11b+Grl+细胞有何影响,这种影响又是如何作用于肿瘤过程的,还有待进一步探究。目的：探讨间充质干细胞(MSCs)诱导骨髓来源的CD11b+Grl+细胞免疫功能对肿瘤的作用。方法：(1)将小鼠骨髓中分选的CD11b+Gr1+细胞与MSCs共培养72h后,计数。应用流式细胞技术测定活细胞百分比与细胞免疫表型。(2)通过T细胞抑制性实验检测与MSCs共培养后CD11b+Grl+细胞：将与TNF-α刺激过的MSCs或MSCs共培养后CD11b+Grl+细胞加入抗-CD3抗体刺激的T淋巴细胞体系中,共培养72h。流式细胞检测T淋巴细胞的增殖和活化。(3)建立4T1肿瘤模型,体内检测与TNF-α刺激过的MSCs或MSCs共培养后CD11b+Grl+细胞对肿瘤的影响作用。结果：MSC能够促进骨髓中CD11b+Grl+细胞的存活和生成,同时可诱导正常小鼠骨髓中CD11b+Grl+细胞对T细胞的抑制作用。动物实验结果显示,MSCs处理后的骨髓CD11b+Grl+细胞可以促进肿瘤的生长,加速小鼠的死亡结论：MSCs可以通过促进骨髓来源的CD11b+Grl+细胞的抑制作用加速肿瘤的生长进程。背景：中性粒细胞大量存在于肿瘤微环境中,但是它对肿瘤进程的影响还未知。中性粒细胞特定因素的作用下,会发挥一定的促炎功能。此外,中性粒细胞可以从不具备免疫抑制功能的表型转换到具有免疫抑制功能的表型：有研究发现粒细胞-巨噬细胞集落刺激因子(GM-CSF)和TGF-β都可以将小鼠骨髓来源的中性粒细胞转换为具有免疫抑制功能的表型。但是,在癌症发展的过程中,中性粒细胞是如何被转换为免疫抑制表型,从而促进肿瘤发展的,我们还知之甚少。间充质干细胞(Mesenchymal stem cell, MSCs)是骨髓中一类具有再生能力的基质细胞,并作为骨髓造血干细胞的支持细胞支持造血,同时MSCs也具有免疫调节活性,参与体内各种免疫调节。近期研究表明,MSCs会分泌大量的细胞因子,招募CD11b+Ly6C+Ly6G单核细胞、F4/80+巨噬细胞和CD11b+Ly6CdimLy6G+中性粒细胞到肿瘤细胞周围加速肿瘤的生长。另外有研究发现,MSCs能够使单核-巨噬细胞转化为具有免疫抑制功能的表型,从而促进肿瘤进展。MSCs可以促进中性粒细胞的存活,但是MSCs对中性粒细胞功能的确切作用还有待进一步阐明。目的：本研究旨在观察MSCs对CD11b+Ly6G+中性粒细胞的作用,揭示MSCs诱导CD11b+Ly6G+中性粒细胞的免疫抑制功能,促进肿瘤进程。方法：(1)将正常小鼠骨髓中或荷瘤小鼠脾脏中分选的CD11b+Ly6G+中性粒细胞与MSCs共培养72h后,应用流式细胞技术测定活细胞百分比与细胞免疫表型。(2)通过T细胞抑制性实验检测与MSCs共培养后正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞：将与TNF-α刺激过的MSCs或MSCs共培养后CD11b+Ly6G+中性粒细胞加入抗-CD3抗体刺激的T淋巴细胞体系中,共培养72h。流式细胞检测T淋巴细胞的增殖和活化。(3)建立4T1肿瘤模型,体内检测与TNF-(I刺激过的MSCs或MSCs共培养后正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞对肿瘤的影响作用。(4)测量与TNF-α刺激过的MSCs或MSCs共培养后小鼠骨髓CD11b+Ly6G+中性粒细胞中精氨酸酶含量的变化。(5)通过基因芯片分析与TNF-α刺激过的MSCs共培养后正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞中各分子mRNA水平的变化,并通过RT-PCR验证。(6)通过阻断与TNF-α刺激过的MSCs共培养后正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞中精氨酸酶和NO,明确MSCs诱导的CD11b+y6G+中性粒细胞对T细胞抑制的机制。结果：(1)在体外培养过程中,MSCs可促进正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞的存活。(2)MSCs诱导正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞对T细胞的抑制功能。(3)MSCs诱导的正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞在体内促进肿瘤的生长。(4)MSCs诱导正常小鼠骨髓中CD11b+Ly6G+中性粒细胞精氨酸酶的升高。(5)通过基因芯片的分析,发现MSCs可诱导正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞中大量促进肿瘤生长的分子上调。(6)iNOS是MSCs诱导的正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞抑制T细胞增殖和活化的重要因子。结论：MSCs可以通过诱导正常小鼠骨髓中或荷瘤小鼠脾脏中CD11b+Ly6G+中性粒细胞的免疫抑制作用促进肿瘤进展
[Abstract]:Background: mesenchymal stem cells (MSCs) is a multipotent stem cell derived from mesoderm with high self-renewal ability and multidirectional differentiation. It exists widely in various tissues of the whole body. It can be cultured in vitro and can be differentiated into nerve cells, osteoblasts, chondrocytes, cardiomyocytes and lipids under specific conditions. Adipose cells, which are the preferred seed cells in cell replacement therapy and tissue engineering, have broad prospects for clinical application. In addition, the karyotype and telomerase activity of mesenchymal stem cells are stable, no expression of histocompatibility complex (MHC) and other stimulators are expressed. Therefore, the host T cells are not easily identified and can escape the rejection of the host immune system. Low immune.MSCs can also inhibit the proliferation of B lymphocyte and T lymphocyte by paracrine effect, induce apoptosis of T lymphocyte and induce immunosuppressive effect. A variety of factors in microenvironment can affect the immune regulation of MSCs, TNF-a can induce MSCs high expression iNOS, and directly inhibit the immune response by the way of NO dependence. It also promotes the up-regulated expression of COX-2 and PGE2 and directly inhibits the immune response through PGE2 dependence. In addition, MSCs can affect the growth of the tumor by producing more monocytes, macrophages and neutrophils by secreting a large number of cytokines. But how does MSCs affect the CD11b+Grl+ cells in the bone marrow, and how this effect is done The use of the tumor process remains to be further explored.
Objective: To investigate the effect of mesenchymal stem cells (MSCs) on the immune function of bone marrow derived CD11b+Grl+ cells.
Methods: (1) the CD11b+Gr1+ cells in mouse bone marrow were co cultured with MSCs for 72h, and the percentage of living cells and cell immunophenotype were measured by flow cytometry. (2) CD11b+Grl+ cells were co cultured with MSCs by T cell inhibition test, and CD11b+Grl+ cells were co cultured with MSCs or MSCs stimulated by TNF- alpha, and CD11b+Grl+ cells were added. In the T lymphocyte system stimulated by anti -CD3 antibody, the proliferation and activation of T lymphocytes were detected by co culture 72h. flow cytometry. (3) the 4T1 tumor model was established, and the effect of CD11b+Grl+ cells on the tumor after TNF- alpha stimulated MSCs or MSCs co culture was detected in vivo.
Results: MSC can promote the survival and formation of CD11b+Grl+ cells in the bone marrow and induce the inhibition of CD11b+Grl+ cells in the bone marrow of normal mice. The results of animal experiments show that the bone marrow CD11b+Grl+ cells treated by MSCs can promote the growth of the tumor and accelerate the death of the mice.
Conclusion: MSCs can accelerate the growth process of tumor by promoting the inhibition of CD11b+Grl+ cells from bone marrow.
Background: neutrophils exist in a large number of tumor microenvironments, but the effect of neutrophils on the progression of the tumor is unknown. Under the action of specific neutrophils, a certain proinflammatory function can be played. Furthermore, neutrophils can be transformed from phenotypes that never have immunosuppressive function to a phenotype with immunosuppressive function: a study has been found. Granulocyte macrophage colony stimulating factor (GM-CSF) and TGF- beta can convert neutrophils from bone marrow from mice to immunosuppressive phenotypes. However, in the process of cancer development, we know little about how neutrophils are converted to immunosuppressive phenotypes and thus promote tumor development.
Mesenchymal stem cell (MSCs) is a kind of regenerative matrix cells in bone marrow, and supports hematopoiesis as the support cells of bone marrow hematopoietic stem cells. Meanwhile, MSCs also has immunoregulation activity and participates in various immunoregulation in the body. In the near future, MSCs secretes a large number of cytokines and recruits CD11b+Ly6C+Ly. 6G mononuclear cells, F4/80+ macrophages and CD11b+Ly6CdimLy6G+ neutrophils accelerate the tumor growth around the tumor cells. Other studies have found that MSCs can convert mononuclear macrophages into immunosuppressive phenotypes, thus promoting tumor progression by.MSCs to promote neutrophils survival, but MSCs against neutrophils The exact role of cell function remains to be elucidated.
Objective: the purpose of this study was to observe the effect of MSCs on CD11b+Ly6G+ neutrophils, and to reveal the immunosuppressive function of MSCs induced neutrophils and promote the progression of tumor.
Methods: (1) after co culture of CD11b+Ly6G+ neutrophils in the spleen of normal mouse bone marrow or tumor bearing mice and MSCs co culture 72h, the percentage of living cells and cell immunophenotype were measured by flow cytometry. (2) CD11b+Ly6G in the spleen of normal mice or the spleen of tumor bearing mice was detected by T cell inhibition test and MSCs co culture. + neutrophils: CD11b+Ly6G+ neutrophils were co cultured with MSCs or MSCs stimulated by TNF- alpha, and CD11b+Ly6G+ neutrophils were added to the T lymphocyte system with anti -CD3 antibody stimulation. The proliferation and activation of T lymphocytes were detected by co culture 72h. flow cytometry. (3) a 4T1 tumor model was established, and TNF- (I stimulated MSCs or co cultured normal mice) The effect of CD11b+Ly6G+ neutrophils in the spleen of bone marrow or tumor bearing mice. (4) the changes in arginase content in bone marrow CD11b+Ly6G+ neutrophils of mice were measured with TNF- alpha stimulated MSCs or MSCs. (5) the bone marrow or charge of normal mice was cultured by gene chip analysis and TNF- alpha stimulated MSCs Co culture. The changes in the mRNA level of each molecule in the CD11b+Ly6G+ neutrophils in the spleen of the tumor mice and verified by RT-PCR. (6) the CD11b+Ly6G+ neutrophil and NO in the spleen of normal mice or the spleen of the tumor bearing mice were inhibited by MSCs co culture with TNF- alpha stimulated MSCs. The CD11b+y6G+ neutrophils induced by MSCs were clearly suppressed to T cells. The mechanism of the system.
Results: (1) in vitro culture, MSCs can promote the survival of CD11b+Ly6G+ neutrophils in the spleen of normal mice or tumor bearing mice. (2) MSCs induced inhibition of T cells by CD11b+Ly6G+ neutrophils in the spleen of normal mice or in the spleen of tumor bearing mice. (3) the spleen of normal mice or the spleen of tumor bearing mice induced by MSCs CD11b+Ly6G+ neutrophils promote tumor growth in the body. (4) MSCs induces the increase of CD11b+Ly6G+ neutrophil arginase in the bone marrow of normal mice. (5) through the analysis of gene chip, it is found that MSCs can induce a large number of molecules that promote tumor growth in the spleen of normal mice or in the spleen of tumor bearing mice, CD11b+ Ly6G+ neutrophils. (6) iNOS is an important factor for MSCs induced CD11b+Ly6G+ neutrophils to inhibit proliferation and activation of T cells in normal mouse bone marrow or tumor bearing mice.
Conclusion: MSCs can promote tumor progression by inducing the immunosuppressive effect of CD11b+Ly6G+ neutrophils in normal mouse bone marrow or spleen bearing mice.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R329.2

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