【作者】 戴俊；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 免疫学， 2004， 硕士

【摘要】 树突状细胞(dendritic ceils，DC)是机体内最重要的一群专职性抗原递呈细胞(antigen presenting cells，APC)，也是机体免疫应答的有力启动者。近年来，随着DC的功能和生物学特性被不断认识，DC在肿瘤免疫、移植免疫和抗感染免疫等方面日益受到人们的重视。 DC的前体细胞来源于骨髓造血干细胞，前体细胞伴随血流到达外周组织，分化为未成熟DC。在肿瘤免疫应答中，未成熟DC摄取肿瘤抗原物质，加工、处理成小分子的多肽片段，再把这些片段与主要组织相容性复合物(major histocompatibility complex，MHC)结合，表达在细胞表面。逐渐成熟的DC离开肿瘤抗原部位，进入淋巴组织，定居于淋巴结的T细胞区。在迁移的过程中，DC发生一系列的变化，逐渐失去摄取抗原的能力，上调表达共刺激分子和组成性趋化因子受体，分泌多种趋化因子及细胞因子。成熟DC在淋巴结与初始T细胞相互作用，表面的抗原肽-MHC分子复合物被T细胞表面受体(T cell antigen receptor，TCR)识别产生抗原特异性信号，同时DC表达的共刺激分子与T细胞表面的相应分子结合，为T细胞提供第二信号从而促进T细胞活化增值。此外，DC还分泌一些细胞因子，如IL-1、IL-6、IL-12等，使T细胞进一步活化、增殖发育为具有肿瘤特异性杀伤性的细胞毒性T淋巴细胞(cytotoxic T lymphocytes，CTL)。CTL通过分泌具有抗肿瘤作用的效应分子穿孔素和颗粒酶发挥作用，穿孔素单体在靶细胞膜上形成不同孔径的跨膜通道，使颗粒酶进入靶细胞，引起靶细胞凋亡，从而产生针对相应靶细胞的细胞免疫应答。 DC在体内迁徙活化，激发T细胞活化为CTL，发动抗肿瘤免疫应答是一系列复杂而精细的过程，涉及到多个因素的调控，包括协同刺激信号的刺激、趋化因子及其受体在DC不同周期的转变等。作为其中最强有效的激发信号之一，CD40信号激发能促进DC成熟，明显上调细胞表面其他共刺激分子的表达，为T细胞的活化提供所必需的第二信号，促进Dc产生几一2、IL一12和TNF一a、IFN邓等多种细胞因子，从而启动各类初始T细胞发生免疫应答。而CD40信号激发对于DC的迁徙能力，和由DC激发的活化CTL的迁徙及其抗肿瘤能力的影响尚未有系统确切的研究，如何理解上述各因素之间的相互作用和内在联系，如何将不同成熟阶段DC的迁徙特性，以及DC激发的活化CTL细胞的迁徙和抗肿瘤能力加以合理利用，对于设计更加有效的DC和CTL抗肿瘤疫苗应用于临床有着重要的意义。1.激发性CD40单抗SCn对树突状细胞迁徙能力影响的研究:本研究利用抗原负载后，联合CD40激发性单抗5Cll或TNF一a激发，促进外周血CD14+单核细胞来源的DC成熟活化;Real一T而e PCR和FACS检测在不同方法刺激和成熟阶段的DC趋化因子分泌、趋化因子受体的表达谱;Transwell微孔迁徙实验检测DC的迁徙能力。实验结果显示:(1)未成熟DC低表达共刺激分子和组成性趋化因子受体;抗原负载后DC上调表达共刺激分子和趋化因子受体的表达;联合SCll诱导成熟具有增强DC分泌组成性趋化因子SDF一la、ELC和IP一10的能力;高水平的趋化因子可以通过自分泌作用，适度下调成熟DC自身高表达的组成性趋化因子受体CXCR4、CCR7。 (2)未成熟DC对各趋化因子和肿瘤上清的迁徙能力强于各组成熟DC;抗原负载后24h的DC表现出对生理性低浓度趋化因子的迁徙特性，对肿瘤上清的迁徙能力明显下降;联合SCll诱导的成熟DC具有对高浓度趋化因子的迁徙倾向性，对肿瘤上清的迁徙能力强于其他组成熟DC，各组成熟DC的迁徙均具有一定的肿瘤特异性。以上结果提示:不同促成熟因素诱导和不同成熟阶段的DC具有各自独特的迁徙特性，综合应用DC的迁徙特性在肿瘤治疗中具有潜在的应用价值。2.激发性CD40单抗SCn对树突状细胞激发的活化CTL迁徙能力的影响:本研究采用FACS检测T细胞在活化过程中趋化因子受体的表达:Transwell微孔迁徙实验检测活化CTL向DC上清，肿瘤上清及趋化因子的迁徙能力。结果显示:(l)各组DC均能促使初始T细胞在活化发育成为CTL期间上调趋化因子受体CXCR3的表达，其中以CD40激发性单抗SCll诱导的成熟DC激发的活化CTL尤其明显，同时此组成熟DC通过分泌高浓度的相应配体IP一10，有效的趋化活化CTL向DC上清迁徙。(2)SCll诱导的成熟DC激发的活化CTL具有很强的向肿瘤上清的迁徙特子性，其迁徙具有肿瘤特异性，此组活化CTL穿越内皮细胞的能力亦强于其他各组活化CTL。以上结果提示:CD40激发性单抗SCll诱导的成熟DC可以有效的激活T细胞，促进T细胞上调表达CXCR3，增强CTL向趋化因子和肿瘤上清的迁徙和穿越血管内皮细胞的能力。3:激发性CD40单抗5Cll对DC激发的活化CTL体外抗肿瘤能力的影响:本研究采用ELISA试剂盒法检侧细胞培养体系上清中几一2、IFN一Y的分泌，溶血实验和DNTB十BLT显色法检侧活化CTL胞浆穿孔素和颗粒酶的释放，光镜观察和“‘Cr释放法检测各组活化C几的体外抗肿瘤能力。结果显示:(1) CD40激发性单抗SCll诱导的成熟DC具有很强的刺激T细胞增值活化的能力。(2) SCll诱导的成熟DC促进T细胞分泌IL一2和IFN斗，从而增强Dc激发活化的CTL抗肿瘤效应分子穿孔素和颗粒酶的释放。(3) SCll诱导的成熟DC?更多还原

【Abstract】 Dendritic cells (DC) , the most effective antigen presenting cell in vivo that we’ve ever known, can ingest and process antigens then onset specific immune response in vivo. When DC biological functions are realized gradually by people these years, DC are received more and more recognition in the tumor immunity, transplantation immunity, anti-infection immunity and so on.The precursors of DC origin from the marrow hemopoietic stem cells. When the precursors arrive at the periphery concomitance with the blood, they differentiate into immature DC. In the tumor immunity, immature DC ingest the tumor antigens, process them into small molecule polypeptides, then combine these polypeptides with MHC and express them on the cell membrane. The gradually mature DC leave the tumor site, migrate to the lymph nodes and settle down in the T cell area, In the migration process, DC lose their antigen ingesting ability, up-regulate the expression of costimulatory molecules and constitutive chemokine receptors, excrete many chemokines and cytokines. Mature DC promote T cells’ activation and proliferation in lymph nodes. The antigen polypeptides on DC membrane are recognized by TCR to afford the antigen special signal, at the same time costimulatory molecules combining with the corresponding molecules provide the second activation signal for T cells. Furthermore DC secrete some cytokines such as IL-12, IL-1, IL-6 to promote T cells to develop into CTL, then the activated CTL exert the mightiness anti-tumor immunity through excreting the effector molecules such as perfurin and granzyme. The perfurin engender channels on target tumorcell membrane. So that the granzyme can entry the target cells to induce the apoptosis of tumor cells.DC maturation and migration relate to many factors such as antigen stimulation. costimulatory signal stimulation, and the switch of the expression of chemokines and their receptors. As one of the most effective stimulus, CD40 signal can up-regulate the expression of other costimulatory molecules on DC; promote cytokines secretion such as IL-8, IL-12, IFN-r, so as to start the T cell immune response. The DC maturation is also based on DC migration in vivo. The migration process is under precise regulation of many chemokines and their receptors. There hasn’t been systemic and authentic investigation about the effect of CD40 signal on the DC and T cells’ migration and potent anti-tumor ability till now. Understanding of the interaction and internal relationship among these factors plays a significant role in designing more effective DC and CTL vaccine for clinical use.1. The effect of CD40 agonistic monoclonal antibody 5C11 on DC migration ability: DC loaded with apoptosis tumor antigens were cultured with CD40 agonistic monoclonal antibody 5C11 or TNF-a as stimulus to promote DC maturation; FACS was used to investigate the expression of molecules on DC membrane such as chemokine receptors; The mRNA expression of chemokines was detected by Real-Time PCR; Transwell polycarbonate filter was used to investigate the migration ability of DC. The results show that: (1): Immature DC expressed low-level costimulatory molecules > constitutive chemokine receptors; After loaded with antigens, DC up-regulate the expression of these molecules; Mature DC induced by 5C11 could excrete high concentration of chemokines such as SDF-1a, ELC which maybe moderately down-regulate the highly expressed corresponding receptors CXCR4, CCR7 by autocrine. (2): Compared with mature DC, immature DC had stronger migration ability in the migration experiment towards different chemokines and tumor supernatant; 24h after loaded antigen, DC inclined to migrate towards physiological low concentration of chemokines; Mature DC induced by 5C11 were apt to migrate towards high concentration of chemokines, and had stronger migrationability than other groups of mature DC towards the tumor supernatant. These results suggested that DC under dissimilar maturation stimulus and different maturation stage had different migration speciality.更多还原