【作者】 王浩；

【导师】 梁晓红；

【作者基本信息】 山东大学 ， 细胞生物学， 2025， 硕士

【摘要】 自然杀伤细胞(Natural killer cells,NK)是一种先天淋巴细胞,不仅能够直接杀伤靶细胞,而且可以分泌细胞因子(IFN-γ、TNF-α等),在免疫防御、免疫自稳、免疫监视中发挥重要作用,是机体抗肿瘤免疫的主要力量。然而,在肿瘤微环境中大量免疫抑制因子和抑制性免疫细胞亚群的作用下,NK细胞数量减少、效应功能障碍,介导肿瘤细胞免疫逃逸。因此,基于NK细胞的免疫治疗有望成为肿瘤治疗的重要候选策略之一。论文通过从药食同源化合物库中筛选能够增强NK细胞功能的小分子,利用体内外实验确定了小分子化合物——氨茶碱,能够促进NK细胞的存活和抗肿瘤功能,为肿瘤联合治疗提供了新思路。研究目的1.筛选能够增强NK细胞功能的小分子化合物。2.确定小分子化合物——氨茶碱对NK细胞生存和功能的作用。3.探究氨茶碱通过调控NK细胞抑制肿瘤发展进程的作用。研究方法和结果1.增强NK细胞杀伤能力的小分子化合物的筛选课题首先利用人NK细胞系NK-92杀伤靶细胞K562-luc作为实验体系,从包含486个小分子化合物的药食同源化合物库中筛选能够增强NK-92杀伤能力的小分子。通过两轮筛选,确定临床药物氨茶碱能够显著增强NK细胞杀伤K562细胞的能力。2.氨茶碱能够增强稳态和肿瘤微环境下NK细胞的存活能力和效应功能为进一步验证氨茶碱的功能,课题首先检测了其对稳态下NK细胞存活、增殖和功能的影响,流式细胞术结果显示,氨茶碱处理抑制NK-92细胞的凋亡,但不影响其增殖能力;同时能够显著增强NK-92细胞杀伤K562-luc、A549-luc细胞的能力,上调其细胞因子IFN-γ、TNF-α的表达水平。此外,课题利用transwell共培养的实验体系模拟肿瘤微环境,同时给予氨茶碱处理,明确氨茶碱对肿瘤微环境下NK细胞功能的影响。流式细胞术结果显示,氨茶碱能够增强与HepG2细胞共培养的NK-92的杀伤能力,上调其细胞因子IFN-γ、TNF-α的表达水平。相类似地,分别将人外周血NK细胞(h-NK细胞)、小鼠脾脏NK细胞(m-NK细胞)与肿瘤细胞共培养,并给予氨茶碱处理,结果显示氨茶碱处理亦能够增强肿瘤微环境下原代NK细胞的效应功能。3.氨茶碱以NK细胞依赖的方式抑制肿瘤的生长和转移NK细胞是抗肿瘤免疫的先锋力量,因此课题进一步探讨了氨茶碱对肿瘤生长和转移的影响,并明确NK细胞在其中的作用。首先,课题利用C57BL/6小鼠构建了小鼠黑色素瘤细胞系B16F10建立的皮下荷瘤模型和肺转移模型。结果显示,与对照组相比,氨茶碱治疗显著抑制B16F10皮下瘤的生长,以及肺转移能力;氨茶碱显著增加小鼠皮下肿瘤或转移肺组织中浸润NK细胞的数目,上调CD107a、IFN-γ的表达,但对脾脏NK细胞的数目和效应功能无明显影响。相一致地,氨茶碱处理亦能显著抑制小鼠肺癌细胞系LLC建立的肺转移瘤的生长,上调肺组织浸润NK细胞的数目和效应功能。更为重要的是,利用特异性抗体清除NK细胞后,能够消除氨茶碱治疗对肿瘤肺转移生长的抑制作用,以及其对肿瘤浸润CD4+T细胞、CD8+T细胞的数目和效应功能的影响。上述研究结果提示,氨茶碱能够显著抑制肿瘤的生长和转移,且该抗肿瘤效应主要依赖于NK细胞。研究结论和意义本课题通过筛选药食同源化合物库发现了氨茶碱对NK细胞杀伤能力的调控作用,明确了氨茶碱不仅能够抑制NK细胞凋亡,而且增强其杀伤功能和细胞因子表达,进而抑制肿瘤的生长和转移。本课题的研究揭示了临床药物氨茶碱在调控NK细胞生存和功能的新作用,为肿瘤联合治疗提供了新思路。更多还原

【Abstract】 Natural killer cells(NK)are one kind of innate lymphocytes that not only directly kill target cells,but also secrete cytokines(such as IFN-γ and TNF-α),playing an important role in immune defense,immune homeostasis,and immune surveillance.Thus,NK cells are the main force of the anti-tumor immunity.However,under the action of immunosuppressive factors and inhibitory immune cell subsets in the tumor microenvironment,the number of NK cells is reduced and the effector functions are impaired,leading to the immune escape of tumor cells.Therefore,NK cell-based immunotherapy is expected to be one of the candidate strategies for tumor therapy.In this project,small molecules capable of enhancing NK cell function were screened from the library of drug and food homologous compounds.Through in vitro and in vivo experiments,the small-molecule compound aminophylline was demonstrated to enhance the homeostasis of NK cells and promote their anti-tumor function.These findings disclose the novel role of approved clinical drugs in immune regulation,and provide a new idea for tumor combination therapy.Objectives:1.To screen small-molecule compounds of enhancing the function of NK cells.2.To determine the effects of the identified small-molecule compound—aminophylline—on the survival and effector function of NK cells.3.To investigate the role of aminophylline in suppressing tumor progression through the regulation of NK cells.Methods and Results:1.Drug screening for enhancing the cytotoxic activity of NK cellsIn this study,we initially employed an experimental system using the human NK cell line NK-92 against target cells(K562-luc)and screened small molecule compounds that enhanced the killing ability of NK-92 from the library of homologous compounds of medicine and food containing 486 small-molecule compounds.Through two rounds of screening,the clinical drug aminophylline was identified to enhance the ability of NK cells against K562 cells.2.Aminophylline enhances the survival and effector function of NK cells under both homeostatic and tumor microenvironment conditionsTo further validate the role of aminophylline,this study first investigated its effects on the survival,proliferation,and effector function of NK cells under homeostatic conditions.Flow cytometry analysis revealed that aminophylline significantly inhibited apoptosis in NK-92 cells without affecting their proliferative capacity.Concurrently,aminophylline markedly enhanced the cytotoxic activity of NK-92 cells against K562 and A549-luc cells,while upregulating the expression of cytokines IFN-y and TNF-α.Furthermore,a transwell system was employed to simulate the tumor microenvironment,and aminophylline was administered to effect on the function of NK cells within the tumor microenvironment.Flow cytometry results demonstrated that aminophylline enhanced both the cytotoxic capacity and the expression of IFN-y and TNF-α in NK-92 cells co-cultured with HepG2 cells.Similarly,when human peripheral blood NK cells(h-NK cells)and murine splenic NK cells(m-NK cells)were co-cultured with tumor cells in the presence of aminophylline,the results demonstrated that aminophylline also enhanced the effector function of primary NK cells within the tumor microenvironment.3.Aminophylline suppresses tumor growth and metastasis in a NK cell-dependent mannerAs NK cells serve as pivotal effector cells in antitumor immunity,this study further investigated the impact of aminophylline on tumor growth and metastasis,with a focus on elucidating the role of NK cells in this process.Firstly,the B16F10 subcutaneous tumor model and lung metastasis model were established in C57BL/6 mice.The results demonstrated that aminophylline treatment significantly inhibited the growth of B16F10 subcutaneous tumor and metastatic potential to the lungs compared with the control group.Furthermore,aminophylline markedly increased the infiltration of NK cells in subcutaneous tumor and metastatic lung tissues,and enhanced the expression of CD107a and IFN-y.However,it had no significant effect on the number or effector function of splenic NK cells.Consistently,aminophylline treatment also significantly suppressed the growth of LLC lung metastases,and enhanced both the infiltration and effector function of NK cells in the lungs.Most importantly,depletion of NK cells using a specific neutralizing antibody abolished the inhibitory effect of aminophylline on tumor metastasis and negated its influence on the infiltration and effector function of tumor-infiltrating CD4+T cells and CD8+T cells.These findings indicate that aminophylline significantly inhibits tumor growth and metastasis,and the antitumor effect of aminophylline primarily depends on NK cells.Conclusions and Significance:This study identifies the regulatory effects of aminophylline on the cytotoxic ability of NK cells by screening the small-molecule compounds from the library of drug and food homologous compounds.It is demonstrated that aminophylline not only inhibits NK cell apoptosis but also enhances their cytokine secretion and cytotoxic function,thereby suppressing tumor growth and metastasis.The findings reveal a novel role of the clinical drug aminophylline in regulating survival and function of NK cells,providing new insights for combination cancer therapy.更多还原