胃癌中CD40信号调节髓源性抑制细胞（MDSC）生物学功能的研究

发布时间：2018-03-09 04:13

  本文选题：胃癌　切入点：CD40　出处：《苏州大学》2015年博士论文　论文类型：学位论文

【摘要】：胃癌是我国最常见的恶性肿瘤之一,占消化道肿瘤的50%~60%,病死率居各类恶性肿瘤之首,5年生存率只有30%。近年来发病率明显上升,而且越来越年轻化,但因早期症状不明显,缺乏特异性的初筛指标,多数患者在就诊时已经处于进展期,传统根治手术及放化疗对进展期胃癌的疗效不理想。因此,迫切需要新的治疗方法。免疫治疗是目前的研究热点,启动和建立有效免疫应答正成为肿瘤治疗的新希望。目前众多研究发现一些协同刺激分子在多种肿瘤中异常表达。CD40/CD40L是介导特异性免疫应答的一对极其重要的共刺激分子。CD40表达于淋巴系统的恶性细胞,包括几乎所有的B细胞恶性肿瘤,以及许多实体瘤,例如膀胱癌,肾癌,胰腺癌,胃癌,乳腺癌,大肠癌,肺癌等。临床研究表明CD40的表达与肿瘤的进展和转移有密切关联。肿瘤细胞协同刺激分子的表达改变,包括正性协同刺激分子表达的下降或者突变,以及负性协同刺激分子的异常高表达,导致T细胞的凋亡及功能抑制,参与了肿瘤免疫逃逸。而肿瘤免疫逃逸通常与髓源性抑制细胞(myeloid derived suppressor cell,MDSC)的聚集和调节性T细胞(Regulatory T cell,Treg)的增多相关。Treg在维持免疫耐受和破坏抗肿瘤反应中扮演重要角色。MDSC通过诱导活化的T细胞产生凋亡、释放抑制性细胞因子、诱导Treg的产生以及与NK细胞和Treg细胞等形成复杂的网络而产生负向调控功能。其抑制作用主要与精氨酸酶1(arginase-1,ARG1)和诱导型一氧化氮合酶(inducible nitrieoxidesyntnase,i Nos)有关。已有研究表明MDSC可通过抑制T细胞免疫反应以及扩增Treg发挥其免疫抑制功能的。本研究以CD40为切入点,探讨其对MDSC生物学功能的调控作用及相关机制,为认识MDSC在免疫逃逸中的作用提供有价值的线索。第一部分:小鼠胃癌模型中MDSC上CD40的表达及其与肿瘤进展的关系目的:检测CD40分别在荷瘤小鼠脾脏来源和肿瘤浸润的MDSC上的表达水平,分析其表达在胃癌进展过程中可能的作用。方法:利用小鼠胃癌细胞系MFC皮下注射C57BL/6j构建小鼠胃癌模型。随着肿瘤进展,每隔5天脱颈处死3只模型鼠,剥离肿瘤和脾脏,通过流式细胞术检测肿瘤不同进展阶段脾脏及肿瘤浸润组织中MDSC比例及其表面CD40表达情况,统计学分析其表达的意义。结果:与正常小鼠脾脏MDSC相比,荷瘤小鼠脾脏来源的MDSC表面CD40的表达明显上升(P=0.0024);脾脏MDSC以及肿瘤组织浸润MDSC比例随着肿瘤生长逐渐增高;MDSC上CD40表达水平随肿瘤进展逐渐降低后稳定至一定水平。此外,我们尚发现Wilde Type(WT)小鼠大约在5~6天成瘤,而CD40-/-小鼠约在7天后成瘤,肿瘤生长速度和大小明显小于WT小鼠。随着肿瘤进展,WT小鼠肿瘤浸润性MDSC的累积水平明显高于CD40-/-小鼠。上述结果提示,在小鼠胃癌模型中,肿瘤相关MDSC表面共刺激分子CD40高表达,且与肿瘤进展相关。第二部分:探讨CD40对MDSC生物学功能的调控作用目的:以CD40分子为切入点,探讨其在调节MDSC凋亡、对T细胞增殖抑制以及诱导Treg方面的调控作用及相关机制。方法:自脾脏分离纯化的Gr-1+CD11b+细胞调整细胞浓度铺于24孔板。每孔加入2 l GM-CSF后,分别用IL-4、IL-6、IL-10、TNF-α、GM-CSF、IFN-γ、LPS、PEG2和MFC刺激24 h、48h、72h后,收集细胞,用流式细胞术分别检测CD40在MDSC上的表达。取经磁珠分选获得的小鼠脾脏MDSC悬液,铺入96孔板中,对照组加入PBS,实验组加入激发型CD40抗体,5%CO2孵箱培养24 h后,分别离心收集各孔细胞,流式细胞术分析MDSC细胞凋亡率。将分选获得的小鼠脾脏MDSC与CFSE染色T细胞按照1:3的比例,用anti-CD3和anti-CD28进行刺激,72小时后检测CFSE荧光衰减,分析T细胞增殖情况。将WT小鼠和CD40KO小鼠的脾脏细胞分离出来后,流式细胞术检测IL-4,IL-17,IFN-γ,Foxp3的表达。结果:LPS体外刺激MDSC后能够上调表面分子CD40的表达,且在48小时后,CD40呈进一步显著性上调。胃癌微环境下,CD40能够显著抑制MDSC的凋亡;而且CD40促进MDSC介导的抑制T细胞增殖功能;CD40参与了MDSC介导的Treg诱导-分化,促进Foxp3表达,增强MDSC免疫抑制功能,在胃癌免疫逃逸中发挥关键作用。第三部分:Genechip分析CD40-/-与CD40low胃癌荷瘤小鼠脾脏细胞差异基因表达目的:通过基因芯片技术分析CD40-/-与CD40low胃癌荷瘤小鼠脾脏细胞基因表达概况,寻找差异表达基因,分析CD40对MDSC生物功能作用的可能分子机制。方法:参照第一部分构建胃癌荷瘤小鼠模型,当肿瘤直径达1cm后,颈椎脱臼法处死WT荷瘤小鼠与CD40-/-荷瘤小鼠,手术取出完整的脾脏。经150目钢网研磨,裂红后,加入1ml Trizol备用。Genechip委托北京博奥生物有限公司进行检测。结果:通过基因芯片技术对CD40-/-和WT小鼠脾脏细胞进行全基因组检测,寻找差异表达基因,分析CD40对MDSC生物功能作用的可能分子机制。结果显示两组细胞基因表达谱有差异。筛选两组间差异倍数(经log2转换)2倍以上的基因为差异表达基因,筛选出2890个差异表达基因,其中表达上调593个,表达下调1419个。Pathway分析结果显示差异基因主要涉及信号通路、细胞代谢、免疫应答、炎症反应、细胞凋亡等。结论 本研究表明CD40是肿瘤相关MDSC的重要标志,能够调控MDSC介导的免疫抑制功能,从而影响肿瘤的发生和发展。这对分析肿瘤免疫耐受的产生机制并探索有效的免疫治疗方法具有重要意义。为胃癌的有效干预治疗提供了新的思路。
[Abstract]:Gastric cancer is one of the most common malignant tumors in China, accounting for 50%~60% and gastrointestinal tumors, the mortality rate among all malignant tumors, 5 year survival rate is only 30%. in recent years the incidence rate increased significantly, and more and more young, but because the early symptoms are not obvious, the lack of specific screening index, most patients have in the advanced stage at the time of treatment, the curative effect of traditional radical surgery and chemotherapy for advanced gastric cancer is not ideal. Therefore, an urgent need for new treatments. Immunotherapy is the research hotspot at present, start and establish an effective immune response is becoming the new hope for the treatment of tumor. At present, many studies have found that some costimulatory molecules in a variety of the abnormal expression of.CD40/CD40L in tumor specific immune response is mediated by a pair of important costimulatory molecules.CD40 expression in the lymphatic system malignant cells, including almost all of the B cell malignant tumor, to And many solid tumors, such as bladder cancer, kidney cancer, pancreatic cancer, gastric cancer, breast cancer, colorectal cancer, lung cancer. Clinical studies have shown that closely related to progression and metastasis of tumors. The expression of CD40 and expression of costimulatory molecules in tumor cells, including positive expression of costimulatory molecules decreased or mutation, abnormal high the expression of negative costimulatory molecules, induce apoptosis and function of T cells involved in tumor suppression, immune escape and tumor immune escape cells usually associated with myeloid derived suppressor (myeloid derived suppressor cell, MDSC) and aggregation of regulatory T cells (Regulatory T cell, Treg) increased in immune related.Treg damage tolerance and plays an important role in.MDSC induced apoptosis through activation of T cells in antitumor response, inhibit the release of cytokines, induce the expression of Treg and NK cells and Treg cells form a complex The network has a negative regulatory function. Its inhibitory effect and arginase 1 (arginase-1, ARG1) and inducible nitric oxide synthase (inducible nitrieoxidesyntnase, I Nos). Studies have shown that MDSC can inhibit T cell immune response and amplification of Treg exerts its immunosuppressive function. In this study, CD40 as the starting point, to explore its role in the regulation of MDSC biological function and mechanism, provide valuable clues for understanding the role of MDSC in immune escape. The first part: the objective relationship between the expression of MDSC CD40 on the mouse model of gastric carcinoma and advances in tumor: the detection of CD40 in spleen derived tumor bearing mice and tumor infiltration of MDSC on the expression level of its expression may be in progress in the process of gastric cancer. Methods: the construction of mouse gastric cancer mouse model of gastric cancer cell line MFC by subcutaneous injection of C57BL/6j with tumor. In every 5 days, 3 rats were sacrificed, tumor and spleen, spleen and tumor MDSC expression by flow cytometry in different stages of tumor invasive tissue proportion and the surface of CD40, the expression of the meaning of statistical analysis. Results: compared with normal mice spleen MDSC surface expression of MDSC CD40 in spleen the source of tumor bearing mice was significantly increased (P=0.0024); spleen MDSC and tumor infiltrating MDSC with tumor growth ratio gradually increased; the MDSC expression of CD40 with tumor progression decreased gradually after stable to a certain level. In addition, we have found that Wilde Type (WT) mice at about 5~6 and CD40-/- about tumor Tiancheng, in mice 7 days after tumor formation, tumor growth rate and size was less than WT mice. With the progression of cancer, the cumulative levels of tumor infiltrating MDSC WT mice was significantly higher than that of CD40-/- mice. The results suggest that in the mouse stomach In the model of cancer, tumor associated MDSC costimulatory molecules CD40 expression and associated with tumor progression. The second part: To investigate the role of CD40 in regulation of the biological functions of MDSC Objective: to CD40 molecules as the starting point, to investigate the regulation of MDSC apoptosis and related mechanism of regulation of T cell proliferation inhibition and induced by Treg since the separation of spleen. Methods: adjust the cell concentration of purified Gr-1+CD11b+ cells plated on 24 well plates. Each hole with 2 L GM-CSF, respectively IL-4, IL-6, IL-10, TNF- alpha, GM-CSF, LPS, PEG2 and IFN- gamma, MFC stimulated for 24 h, 72h, 48h, cells were collected and detected the expression CD40 on MDSC by flow cytometry. From multisort obtained mice spleen MDSC suspension, spread in 96 well plates, the control group joined PBS agonist CD40 antibody was added in experimental group, 5%CO2 incubator after 24 h culture were collected by centrifugation of the hole cells, flow cytometry analysis of MDSC cells 鑳炲噵浜＄巼.灏嗗垎閫夎幏寰楃殑灏忛紶鑴捐剰MDSC涓嶤FSE鏌撹壊T缁嗚優鎸夌収1:3鐨勬瘮渚，

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