【作者】 刘书逊；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2003， 博士

【摘要】 补体(Complement)作为天然免疫(Innate immunity)中的一类重要和保守的体系，为机体提供了快速和高效地清除入侵微生物的途径。补体系统除了补体的直接杀伤机制外，在补体活化过程中释放的多种小片断分子具有广泛的生物学效应，包括趋化中性粒细胞和淋巴细胞、调理吞噬、参与调节细胞和体液免疫应答等等。因此，补体是连接机体天然免疫和获得性免疫(Adaptive immunity)的“桥梁”。然而，对机体来说，补体也具有潜在的威胁，其经过不适宜的活化后，可以导致机体多种组织的损伤。正常机体在进化过程中，形成了一套精细的保护自身免受补体攻击的调控机制。目前发现，机体多种细胞以及生殖系统的体液中表达和分泌丰富的补体调控蛋白，包括作用于补体活化早期阶段的CD55、CD46、CD35和作用于补体活化终末阶段的CD59，它们分别通过抑制补体活化过程中关键的C3、C5转化酶和抑制形成膜攻击单位，抵抗补体对自身组织细胞的攻击。 作为机体免疫系统中功能最强的专职性抗原提呈细胞(Antigen-presenting cells,APC)，树突状细胞(Dendritic cells,DC)能高效地摄取、加工处理和提呈抗原，具有较强的迁移能力，并能显著地激活初始型T细胞以启动T细胞免疫应答反应，此外，DC与B细胞以及NK细胞等也存在着相互作用，可见，DC在连接天然免疫和获得性免疫之间起着非常重要的作用。DC是一类异质性的细胞群体，不同的亚群，其免疫学功能特点不同，在免疫应答与免疫耐受、抗肿瘤、抗感染免疫中发挥不同的作用。考虑到补体和DC均能够连接天然免疫和获得性免疫的共同特性，而目前尚未见补体和DC之间相互作用、特别是补体对DC免疫学功能的直接调控作用的研究报道，因此，本研究探讨了补体与DC之间的相互关系，重点研究了补体对于不同亚群DC的杀伤作用、趋化作用以及对于DC抗原提呈功能的影响，具体包括：1)处于不同分化发育阶段的不同亚群的DC表达补体相关分子包括补体受体和补体调控蛋白的水平，以期了解DC对于补体及其活性片断作用的敏感性及相互作用的物质基础；2)活化补体博士论文补体分子对树突状细胞免疫学功能的调控作用及机制研究及其释放的活性片断(主要是Csa和Clq)对处于不同分化发育阶段的不同亚群DC的杀伤作用、趋化效应以及对DC激活T淋巴细胞的影响。本研究的完成有助于在免疫应答的调控机制方面获得新的认识，并对补体和DC在连接天然免疫和获得性免疫之间的作用及相互关系能有更全面的理解，为进一步从新的角度和思路深入研究DC在抗原特异性免疫应答与免疫耐受中的作用及其机制奠定基础。第一部分人树突状细胞表达补体受体及其相关分子的分析 因为DC是个异质性细胞群体，因此，我们分析了补体受体及其相关分子在不同分化发育阶段、不同亚群的树突状细胞上的表达。我们通过免疫磁珠分离了两种人DC前体，即髓系来源的单核细胞(MonocyteS，Mo)和淋巴系来源的浆细胞样DC(Plasmaeytoid dendritic eells，pDC)，对这两个不同DC亚群进行体外诱导培养，使其处于不同的分化发育阶段，然后检测了其表达补体受体一CD35( CRI)、CD21(CRZ)、CDI lb(CR3)、CDlle(CR4)，补体调控蛋白一CD46、CD55、以及部分补体片断分子受体一C3aR、CsaR、ClqRp的水平。曰补体受体及其相关分子在MoDC上的表达 不同分化阶段的单核细胞衍生性DC(Monocytes一derived DC，MoDC)的诱导:将新鲜分离的单核细胞Mo，在含有GM一CSF和工L一4培养体系中诱导5一7d，即分化为未成熟MoDC;对培养至sd的未成熟MoDC，用TNFa刺激Zd，即分化为成熟MoDC;此时再用LPS刺激24h，即为活化的MoDC。 结果发现，单核细胞Mo表达中等水平的CD35(CRz)、CD46、CD55和ClqRp;表达高水平的CDI lb(CR3)、CDI le(CR4)、CsaR;表达低水平的C3aR;不表达CD21 (CRZ)。与单核细胞Mo相比，未成熟的MoDC表达CD35、CDI lb、CDI le、CD46、CD55、CsaR和ClqRp的水平显著下调;其它分子的表达水平差异不显著。与未成熟MoDC相比，成熟MoDC表达CDI lb、CDI le、CD46和CD55的水平显著回升:其它分子的表达水平差异不显著;但与新鲜分离的单核细胞M。比较，成熟MoDC表达CD46、CD55、CsaR和ClqRp的表达水平依然显著降低。LPS刺激活化的MoDC，与成熟MoDC相比，补体相关分子的水平没有显著差异;与未成熟MoDC相比，其CD 1 lb、CDI Ic、CD46、CD55表达水平显著回升，其它补体相关分子的表达水平差异不显著;与新鲜分离的单核细胞Mo比较，LPS刺激活化的MoDC表达CD35、 博士论文补体分子对树突状细胞免疫学功能的调控作用及机制研究CD55、CsaR、ClqRp的水平显著低下，而其它分子表达水平没有显著差异。由此 可见，单核细胞M。在向DC自然分化成熟的过程中，表达补体受体(包括CsaR和ClqRp)和补体调控蛋白的水平下调，但是当TNFa或/和LPS作用于DC使之成熟和活化后，其中某些分子(包括CDllb、CDllC、CD55和CD46)的水平显著上调(但CD55的表达仍然显著低于Mo的CD55表达水平)。 这些结果提示，一方面，MoDC表达补体受体，提示补体可以作用于MoDC，补体介导的细胞毒效应可能更多还原

【Abstract】 The complement system, a key and conserved system of innate immunity, provides a rapid and efficient means of deleting invading microorganisms. In addition to direct killing, activation of the complement system results in the generation of numerous split products, which bind to complement receptors on various cells of the immune system, thereby modulating inflammation and mounting an immune response. Thus, the complement system act as a link between innate and acquired immunity. However, complement activation presents a potential threat to host cells, as inappropriate activation may lead to tissue damage in many diseases. Healthy individuals have developed several finely-tuned regulatory mechanisms to protect "self from attack by complement during evolution. An array of regulatory proteins have been found, which inhibit the formation of central enzymes involved in early stages of the complement activation pathway. These include membrane cofactor protein (MCP CD46), decay- accelerating factor protein (DAF CD55), complement receptor 1 (CR1, CD35), as well as CD59, which inhibits formation of the membrane attack complex during later stages. These regulatory factors are widely expressed and abundant on many cells, and in fluids of reproductive system.Dendritic cells are characterized by their ability to efficiently present antigen, and are uniquely equipped to stimulate naive T cell responses. Immature dendritic cells acquire antigens from throughout the peripheral body, then migrate to T-lymphocyte-dependent areas of lymph nodes. Thus, dendritic cells act as bridges, operating at the interface of innate and acquired immunity. Dendritic cells exist in heterogenous groups, which include several subsets with different roles in the regulation of immune responses and maintenance of immune homeostasis. In view of the common property of dendritic cells and complement, namely their ability to link innate and acquired immunity, and with no reports, to date, addressing the direct effects of complement on the biological functions of dendritic cells, we decided to study the interaction between the complement system and dendritic cells. We focusedon the following aspects; 1) we first assayed the expression of complement receptors and complement-associated molecules on distinct subsets of dendritic cells during their development in order to understand the physical basis of the sensitivity of dendritic cells to complement and its split products; we next studied the effects of complement activation on the survival of dendritic cells during their development; and finally examined the effects of the whole complement system, focusing on the ability of one of the split products of complement activation, C5a, and its first subcomponent-C1q, to influence chemotaxis of dendritic cells, as well as allo-T cell stimulatory activity of DC. We hope that our study will provide us with more comprehensive knowledge of the mechanisms of immune regulation and the roles that DC and complement play in innate and acquired immunity, as well as to lay a foundation for further exploration of the roles DC play in antigen-specific immune responses and immune tolerance from a new perspective.Part I Expression of complement receptors and complement-associated molecules on dendritic cells derived from distinct-origin at different stages of developmentTwo subsets of dendritic cells were generated from precursor cells isolated by means of magnetic cell separation system. Dendritic cells of myeloid origin were derived from monocytes, while dendritic cells of lymphoid origin, which have a plasmacytoid morphology, were derived from plasmacytoid dendritic cells. We assayed the expression of complement-associated molecules by the two subsets of dendritic cells during their development. These complement-associated molecules are: CD35(CR1), CD21(CR2), CDllb(CR3), CDllc(CR4), CD46, CD55, C3aR, CD88(C5aR), and C1qRp.The expression of complement receptors and complement-associated molecules on MoDC at different stages of developmentFreshly isolat更多还原