【作者】 朱一蓓；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 内科血液学， 2004， 博士

【摘要】 树突状细胞(dendritic cells，DCs)是机体内最重要的、功能最强的一群专职性抗原提呈细胞(APC)，能有效摄取、加工及提呈抗原，刺激初始型T细胞(naive T cell)的增殖和活化，是启动、调控并维持免疫应答的中心环节。近年来，DCs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。DCs的分化成熟经历了一系列复杂而精细的过程，进一步探讨调节DCs分化、成熟和功能的生物因子、影响因素及其机制，将有助于以DCs为佐剂的生物治疗的开展。 本研究首先分析了人外周血单核细胞来源的DCs(MDDC)分化成熟过程中，共刺激分子、趋化因子受体的表达及其相互作用，比较CD40和TNF-α两条信号途径对MDDC分化成熟及功能的影响，并探讨IL-2及其受体在DCs活化和功能介导中的作用；继而以CD40和TNF-α信号激发的DCs联合细胞因子体外激发、扩增肿瘤特异性CTL，探讨共刺激信号对肿瘤特异性CTL生物学功能的影响及其机制，评价肿瘤特异性CTL体外趋化和杀伤肿瘤细胞的能力：最后研究肿瘤患者外周血单核细胞来源的DCs的体外诱导及其功能，分析IL-10对DCs分化和功能介导的影响及其机制，以此为恶性肿瘤的免疫治疗提供实验和理论依据。 第一部分 共刺激分子和趋化因子受体在树突状细胞中的表达及其相关生物学效应 目的：比较分析在TNF-α和CD40两种信号途径下，相关共刺激分子和趋化因子受体在人外周血单核细胞来源的DCs分化成熟过程中的表达，探讨共刺激信号和IL-2在肿瘤抗原特异性DCs生物学行为中的作用及其机制。 方法：采用透射电镜观察DCs超微结构：免疫荧光标记检测DCs分化发育过程中共刺激分子和趋化因子受体的表达：RT-PCR和Realtime-PCR检测PD-L1、PD-L2、4-1BB肿瘤特异性DCs体外诱导调节因素及其生物学功能研究中文提要和4一IBBL n1RNA的转录;ELISA检测IL一2、IL一12、IL一10的分泌水平;肠朋swell实验评价DCs对T细胞的趋化能力;Westem一blotting检测IL一2受体丫链的表达。结果:在CD4OmAb(5Cll)和TNF一a刺激成熟的人MDDC中，CD40、CD80、CD86、HLA一DR、GL50、PD一LI和PD一LZ等随着DCs成熟均逐渐上调表达，4一IBB和4一IBBL在抗原负载的 DCs上呈现共表达;CD40mAb激发的DCs中CD25和CD83的表达明显高于TNF一a组且高表达PD一LZ、中度表达PD一Ll(p<0.05);CD40mAb激发的DCs分泌IL一2和IL一12的量明显高于TNF一a组(P<0.05):CD40mAb诱导的Dcs适度表达CXCR4和CCR7，对T细胞的趋化能力明显高于TNF一a组护<0.05);DCs表达功能性几一2助链，IL一2能促进DCs增殖和分泌IFN一Y。结论:共刺激分子和趋化因子受体在人MDDC的分化、成熟和功能介导中起着重要的调节作用，CD40信号对人MDDC分化成熟起到至关重要的作用，DCs表达功能性IL一ZRY链，IL一2及其受体在DCs活化和功能介导中有重要意义。DCs通过4一IBB/4一IBBL共表达的形式可建立激发DCs和功能介导的新途径。更多还原

【Abstract】 Dendritic cells (DCs), the most potent professional antigen presenting cells (APC), are efficient in phagocytosing, processing and presenting antigens to naive T cells, stimulating the proliferation and activation of naive T cells, and play a central role in initiating, regulating and sustaining immune responses.DCs reside in unperturbed tissues in an immature form, where they are adapted for capturing and accumulating Ags. Indeed, DCs possess a wide spectrum of recognition systems for an efficient screening of the tissue environment. A variety of danger signals, including microorganisms, dying cells, or proinflammatory cytokines, induce the terminal differentiation, also known as maturation, of DCs. DCs undergo a complex and exquisite differentiation and maturation procedure. Mature DCs migrate to lymph nodes, acquire potent Ag presenting capacity, and stimulate T cell responses vigorously. Moreover, maturation of DCs is strengthened during interactions with T cells by signals such CD40 ligand (CD40L) provided by T cells themselves. Mature DCs express high levels of Ag presenting and costimulatory molecules, and release large amounts of IL-12, thereby stimulating preferentially Thl responses. Thus, DC maturation is a key checkpoint in the initiation of immunity and has important consequences also on the quality of the immune response.Recently, applications of DCs in tumor, transplantation, and anti-infection immunology have been paid more and more attention. Therefore, investigation of the associated influential factors and mechanisms contributed to the development of DCs based vaccination protocols. Our research were divided into three sections as the foliowings:In the first section our research focused on expressions and interactions of costimulatory molecules and chemokine receptors during the procedure of the human peripheral blood monocytes derived dendritic cells (MDDC) differentiation and maturation, comparison the different biological effects between CD40 and TNF-stimulated MDDC, and investigation the roles of IL-2 and IL-2R complex in MDDC’s activation and functions. The second section was about in vitro generation and activation of tumor specific CTL by CD40 or TNF-a stimulated MDDC combining a cocktail of cytokines. Costimulatory signals’ effects on tumor specific CTL and the associated mechanisms were also investigated. Moreover, chemoattraction and migration ability, cytotoxic activity against tumor cells were assayed in vitro. The third section focused on study of the repressive effects of IL- 10 on the differentiation, maturation and biological functions of MDDC in vitro. These studies all contributed to the development of immunotherapy on malignant tumor in clinic.Part I : Expression of costimulatory molecules and chemokine receptors in dendritic cells and the related biological effectsObjective: Our study aimed to compare the different biological effects between CD40and TNF-a stimulated MDDC, including expressions of costimulatory molecules and chemokine receptors during the procedure of the human peripheral blood MDDC differentiation and maturation; investigate the roles of costimulatory signals and IL-2 in activation of tumor specific DCs and the associated mechanisms.Methods: DCs ultrastructures were observed by transmission electron microscopy; Expressions of costimulatory molecules and chemokine receptors of MDDC were analyzed by FCM. PD-L1, PD-L2, 4- 1BB and 4-1BBL mRNA transcription were detected by RT-PCR and Real-time PCR. Concentrations of IL-2, IL-10 and IL-12 in supernatant ofDCs were determined by ELISA. The ability of DCs chemoattracting T lymphocytes was measured by transwell assay. Expression of IL-2Ry chain were detected by western-blotting.Results: During the process of maturation, both of CD40mAb (5C11) or TNF-stimulated MDDC up-regulated expressions of CD40, CD80, CD86, HLA-DR, GL50, PD-L1 and PD-L2 (P>0.05); 4-1 BB and 4-1BBL were co-expressed in Ag loaded MDDC; Expressions of CD25 and CD83 were higher in CD40 activated MDDC than TNF-a stimulated MDD更多还原