【作者】 何龙；

【导师】 曹雪涛；

【作者基本信息】 第二军医大学 ， 免疫学， 2001， 博士

【摘要】 革兰氏阴性细菌胞壁外膜中的脂多糖（LPS）以及细菌DNA和含非甲基化CpG基序的寡聚脱氧核糖核苷酸（CpG ODN）等都被机体当成危险信号，可激活单核巨噬细胞、树突状细胞等抗原提呈细胞合成和分泌多种细胞因子及其它炎症介质。本研究主要探讨了一种新型树突状细胞来源的G蛋白信号转导调节蛋白（DC-RGS）、核受体过氧化体增殖物激活受体（PPAR）及主要组织相容性复合物Ⅱ类分子（MHC-Ⅱ）在LPS和CpG ODN活化单核巨噬细胞等抗原提呈细胞产生细胞因子及一氧化氮（NO）过程中的作用。我们通过基因转染研究发现DC-RGS对LPS诱导人单核细胞THP-1产生细胞因子（TNF-α、IL-1β、IL-6和IL-12p70）具有微调作用，但不影响IL-8的分泌；CpG ODN也具有与LPS相似的脱敏现象而且它们可以互相诱导对方脱敏；脱敏时THP-1分泌IL-8不受抑制；用PPAR的配体和基因转染研究发现PPARα和PPARγ能以配体和转录活性非依赖性机制抑制巨噬细胞RAW264.7产生IL-12p40和NO，但不影响THP-1细胞产生IL-8；脱敏后PPARγ表达增高提示其可能反馈性参与LPS和CpG ODN脱敏过程；抗小鼠MHC-Ⅱ的单克隆抗体B21-2能诱导APCs产生NO，合用LPS、CpG ODN、超抗原SEA或热休克蛋白HSP60则具有协同效应，表明MHC-Ⅱ的信号转导过程对LPS、CpG ODN、超抗原SEA和HSP60等危险信号的效应以及信号转导过程具有调节作用。这些发现对炎症感染、动脉粥样硬化和一些自身免疫性疾病的发病机制的了解以及防治具有一定的意义。更多还原

【Abstract】 Part I: Identification of a Novel RGS Molecule and Its TuningEffects on Cytokine Production by Lipopolysaccharide TreatedTHP-1 MonocytesRegulator of G-protein signaling (RGS) proteins are GTPase activating proteins thatinhibit signaling in various cellular responses controlled by heterotrimeric G proteins. Here wereport a novel RGS molecule cloned from human dendritic cells (DCs), designated DC-RGS,and its role in regulating the responsiveness of monocytes to lipopolysaccharide (LPS) in vitro.We show that DC-RGS is widely distributed and mainly expressed in myeloid cells. Inhibitionof DC-RGS expression in THP-l cells by antisense technique differentially sensitizedLPS-induced production of tumor necrosis factor (TNF)-ct, interleukin (IL)-1 j3, IL-6, IL-8 and11-12. Moreover, inhibition of DC-RGS expression partially restored secretion of TNF-ct and11-6by LPS desensitized TFW-1 cells. Overexpression of DC-RGS in THP-1 cells did notsignificantly affect cytokine production compared with mock control. Thus, DC-RGS, a novelRGS cloned from DCs, plays a fine-timing role in LPS responses and participates in regulatingresponsiveness of monocytes to LPS. We proposed that RGS proteins together with G proteinsmight participate in innate immunity and subsequently affect the adaptive immune responses.Part II: Evidence for Activation-independent RepressionMechanism of PPARy in LPS and CpG ODN Responses: Relevanceto DesensitizationLipopolysaccharide (LPS) desensitization or endotoxin tolerance, a state of hypo-responsiveness to LPS induced by pretreatment of low dose of LPS, is characterized bydecreased proinflammatory factors production in response to secondary LPS challenge even in10high dose. Here we showedoligodeoxynucleotides containing unmethylated CpG motif (CpGODN), similar to LPS, could also resulted in desensitization as evidenced from reduced nitricoxide (NO) and cytokine IL-12p40 production by murine macrophage Raw264.7 cells. LPSand CpG ODN could cross-desensitize each other to induce NO and IL-12p40 production butCpG ODN was more potential. Interestingly, 1-8 production by THIP-l human monocytescouldn’t be desensitized by LPS or/and CpG ODN. We then tested the hypothesis thatperoxisome proliferator-activated receptor (PPAR) a and PPARy, ligand-dependent nuclearreceptors that have been implicated in negative-modulating macrophage cell activation, mightbe involved in desensitization state induction. We showed that pretreatment with ligands ofPPARcC (agonist Wy-14643) and PPARy (agonist 15-d-PGJ2 and antagonist BADGE), but notwith PPARy agonist pioglitazone, reduced LPS or CpG ODN-induced NO and IL-12p4Oproduction. Further, enhanced expression of PPARQ or PPARy (including a dominant-negativePPARy mutant) by transient transfection, mimics desensitization induction, attenuated LPS orCpG ODN-induced NO and ]IL-l2p4O production, but did not affect IL-S production. Westernblot analysis showed that LPS or CpG ODN treatment resulted in altered kinetics of PPAR7and NF-icB p50 subunit expression while no alteration of PPARcL expression in THP-1 cells.LPS or CpG ODN pretreatment significantly increased expression of PPART. Thus we suggestthat PPARy might participate in signaling and desensitization induced by LPS and CpG ODNin activation-independent manner and constitutively.Part Ill: Modulating Effects of B21-2, a Monoclonal AntibodyAgainst MHC class II molecules, on Danger Signals-Induced NOProduction by Antigen-Presenting CellsMajor histocompatibilty complex class II molecules (M1HC II) are efficiently expressedby antigen-presenting cells (APCs), including macrophages, dendritic cells (DCs) and B cells.Macrophages and DCs could be induced to produce nitric oxide (NO) by danger signals, suchas bacterial endotoxin lipopolysaccharide (LPS)更多还原