CD137信号对CIK细胞增殖和功能调节的研究

发布时间：2018-06-28 05:40

本文选题：CD137mAb + CD3~+CD56~+细胞　；参考：《南京医科大学》2008年硕士论文

【摘要】： 共刺激分子CD137是近年新发现的TNFR超家族的新成员,其主要表达在活化的T细胞、自然杀伤细胞(natural killer,NK cells)和树突状细胞(dendritic cells,DCs)表面。研究发现CD137与其配体结合后,介导的共刺激信号可促进T细胞和NK细胞增殖、诱导细胞因子的分泌以及减少活化诱导的细胞死亡(activation-induced cell death,AICD),增加细胞存活能力。细胞因子诱导的杀伤细胞(cytokineinduced killer cells,CIK细胞)是一种新型的具有广谱杀瘤活性的非主要组织相容性复合物(non-major histocompatibility complex,MHC)限制性的免疫效应细胞。但目前CIK细胞的扩增效率和治疗实体瘤的疗效仍有待进一步提高。CIK细胞为一群异质细胞群,包括CD3~+CD56~+细胞和CD3~+CD8~+细胞,其中CD3~+CD56~+细胞是CIK细胞发挥直接和间接抗肿瘤作用最主要的效应细胞。因此通过提高CIK细胞中CD3~+CD56~+细胞的数量和抗肿瘤的活性可有效提高CIK细胞过继免疫治疗的疗效。然而目前CD137信号对CD3~+CD56~+细胞亚群的功能调节国内外未见报道。在本研究中,通过CD137mAb激活CD137共刺激信号通路,可以显著提高CIK细胞的增殖能力和杀伤活性。在此基础上,探讨CD137信号对CIK细胞功能的调节机制。本实验结果为提高CIK细胞治疗实体瘤的疗效提供新的理论依据。目的:探讨CD137信号对健康人和肺癌患者外周血来源的CIK细胞的扩增和功能调节作用。方法:分离健康人和肺癌患者外周血单个核细胞(peripheral bloodmononuclear cells,PBMCs),实验组在常规CIK培养体系中加入cD137单克隆抗体(monoclonal antibody,mAb),即CD137-CIK组;对照组在常规CIK培养体系中加入鼠IgG1同型对照,即IgG1-CIK组。健康人CIK细胞:(1)苔盼蓝拒染法细胞计数分析细胞增殖;(2)LDH酶释放法检测CIK细胞对A549细胞杀伤活性;(3)AnnexinV-FITC/PI试剂盒检测CIK细胞凋亡率;(4)流式细胞术检测CIK细胞表型分布及CD3~+CD56~+细胞内细胞因子及其表面NK细胞受体(NK cell receptor,NKR)的表达;(5)CIK细胞和CD4~+Th0细胞共培养后,流式细胞仪分析CD4~+T细胞内IFN-γ和IL-4的表达水平。肺癌患者CIK细胞:(1)流式细胞术分析CIK细胞表型;(2)LDH酶释放法检测CIK细胞对A549细胞杀伤活性。结果:健康人:(1)CD137-CIK组细胞体外扩增效率显著高于IgG1-CIK组,CD137-CIK组细胞浓度最高达(9.87±0.57)×10~6/ml;IgG1-CIK组浓度最高为(7.02±0.68)×10~6/ml(p＜0.05);(2)培养至28天,CD137-CIK组和IgG1-CIK组细胞中CD3~+CD56~+细胞比例分别达到(39.86±4.69)%和(29.14±5.12)%(p＜0.05);(3)培养至14天,CD137-CIK组细胞凋亡坏死率明显低于IgG1-CIK组;(4)CD137-CIK组细胞体外杀伤肺癌细胞株A549活性高于对照组(20:1,P＞0.05;10:1和5:1时,P＜0.05);(5)培养至14天,CD137mAb上调CD3~+CD56~+细胞内IL-2,IFN-γ,TNF-α表达,同时下调IL-4,TGF-β_1,IL-10表达;(6)与IgG1-CIK组相比,CD137-CIK组中CD3~+CD56~+细胞表面NKG2D的表达较IgG1-CIK组明显增强,而NKG2A的表达则减弱;(7)结果显示,CD137-CIK组细胞可显著提高CD4~+T细胞IFN-γ表达水平,同时降低IL-4的产生。肺癌患者:(1)第28天,CD137-CIK组和IgG1-CIK组中CD3~+CD56~+细胞比例都显著提高,分别达到(30.1±7.3)%和(21.7±5.7)%(p＜0.01);(2)CD137-CIK组细胞体外杀伤肺癌细胞株A549活性明显高于IgG1-CIK组(10:1和5:1,P＜0.01;20:1,P＜0.05)。结论:CD137共刺激信号可有效的提高健康人和肺癌患者外周血中CIK细胞的扩增效率并增强CIK细胞体外非MHC限制性杀伤肿瘤细胞的能力;同时CD137信号通过提高CD3~+CD56~+细胞分泌Th1类细胞因子的能力,促使CIK细胞诱导CD4~+Th0细胞向Th1方向分化,从而实现辅助激活体内细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)免疫反应,发挥MHC限制性的抗肿瘤作用。上述研究结果为提高CIK细胞在实体瘤中的治疗效果提供更多的可行性依据。
[Abstract]:Co stimulator CD137 is a new member of the newly discovered TNFR superfamily in recent years. It is mainly expressed in activated T cells, natural killer cells (natural killer, NK cells) and dendritic cells (dendritic cells, DCs). The study found that the co stimulatory signal mediated by CD137 and its ligand can promote the proliferation of T cells and cells and induce fine induction. Cytokine secretion and reduced activation induced cell death (activation-induced cell death, AICD), increasing cell viability. Cytokine induced killer cells (cytokineinduced killer cells, CIK cells) are a new type of non major histocompatibility complex (non-major histocompatibility) with broad-spectrum antitumor activity (non-major histocompatibility) Complex, MHC) restrictive immune effector cells. However, the amplification efficiency of CIK cells and the efficacy of the treatment of solid tumors still need to be further enhanced by.CIK cells as a group of heterogeneous cells, including CD3~+CD56~+ and CD3~+CD8~+ cells, in which CD3~+CD56~+ cells are the most important effects of CIK cells in direct and indirect antitumor effects. So by increasing the number of CD3~+CD56~+ cells in CIK cells and antitumor activity, the effect of the adoptive immunotherapy of CIK cells can be effectively improved. However, the function regulation of the CD137 signal on the CD3~+CD56~+ cell subgroup has not been reported at home and abroad. In this study, the activation of the CD137 co stimulatory signal pathway by CD137mAb can be significantly improved. On the basis of the proliferation and killing activity of high CIK cells, the regulation mechanism of CD137 signal on the function of CIK cells is discussed. The results of this experiment provide a new theoretical basis for improving the curative effect of CIK cells in the treatment of solid tumors.
Objective: To investigate the amplification and function regulation of CD137 signal in peripheral blood CIK cells from healthy and lung cancer patients.
Methods: the peripheral blood mononuclear cells (peripheral bloodmononuclear cells, PBMCs) were isolated from the healthy and lung cancer patients. The experimental group added the cD137 monoclonal antibody (monoclonal antibody, mAb) to the CD137-CIK group in the routine CIK culture system, and the control group added the rat IgG1 same type control in the routine CIK culture system, that is, the group.
Healthy human CIK cells: (1) trypan blue staining method cell count analysis and analysis of cell proliferation; (2) LDH enzyme release assay to detect the killing activity of CIK cells to A549 cells; (3) AnnexinV-FITC/PI kit to detect the apoptosis rate of CIK cells; (4) flow cytometry detection of CIK cell phenotype distribution and CD3~+CD56~+ cell cytokines and the surface NK cell receptor (NK cell R) Eceptor (NKR) expression; (5) CIK cells and CD4~+Th0 cells co cultured, and the expression levels of IFN- and IL-4 in CD4~+T cells were analyzed by flow cytometry.
CIK cells in lung cancer patients: (1) flow cytometry was used to analyze the phenotype of CIK cells; (2) LDH enzyme release assay was used to detect the killing activity of CIK cells against A549 cells.
Results: (1) the efficiency of cell amplification in CD137-CIK group was significantly higher than that in group IgG1-CIK, and the highest cell concentration in group CD137-CIK was (9.87 + 0.57) x 10~6/ml, and the highest concentration in IgG1-CIK group was (7.02 + 0.68) x 10~6/ml (P < 0.05); (2) culture to 28 days, the ratio of CD3~+CD56~+ cells in CD137-CIK and IgG1-CIK groups reached (39.86 + 4.69)% and (39.86 + 4.69)%, respectively. 29.14 + 5.12)% (P < 0.05); (3) the apoptosis and necrosis rate in group CD137-CIK was significantly lower than that in group IgG1-CIK; (4) the activity of A549 in group CD137-CIK was higher than that of the control group (20:1, P > 0.05; 10:1 and 5:1, P < 0.05); (5) cultured to 14 days, CD137mAb increased the expression of CD3~+CD56~+ cells. IL-4, TGF- beta _1, IL-10 expression; (6) compared with the IgG1-CIK group, the expression of NKG2D on the surface of CD3~+CD56~+ cells in the CD137-CIK group was significantly enhanced and the expression of NKG2A decreased. (7) the results showed that the CD137-CIK group cells could significantly increase the expression level of CD4~+T cells and reduce the production of the CD3~+CD56~+ cells.
Patients with lung cancer: (1) on the twenty-eighth day, the proportion of CD3~+CD56~+ cells in group CD137-CIK and group IgG1-CIK increased significantly (30.1 + 7.3)% and (21.7 + 5.7)% respectively (P < 0.01). (2) the cytotoxic activity of lung cancer cell line in CD137-CIK group was significantly higher than that of IgG1-CIK group (10:1 and 5:1, P < 0.01; 20:1, P < 0.05).
Conclusion: CD137 co stimulation signal can effectively improve the amplification efficiency of CIK cells in the peripheral blood of healthy and lung cancer patients and enhance the ability of non MHC restrictive killer cells in CIK cells in vitro, and CD137 signal can induce CD4~+Th0 cells to induce CD4~+Th0 cells to Th1 direction by enhancing the ability of CD3~+CD56~+ cells to secrete Th1 cell factors. Differentiation, so as to assist in activating the immune response of cytotoxic T lymphocyte (cytotoxic T lymphocyte, CTL) in vivo, and exerting MHC restrictive antitumor effect, the above results provide more feasible basis for improving the therapeutic effect of CIK cells in solid tumor.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R734.2

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