【作者】 何海艳；

【导师】 黄茂；

【作者基本信息】 南京医科大学 ， 呼吸内科学， 2008， 硕士

【摘要】 支气管哮喘是一种严重危害人类健康的常见病,多发病。目前认为,支气管哮喘是以气道可逆性阻塞、高反应性和气道炎症为特征的一组综合征,气道炎症是前两者的基础。在多种遗传和环境因素共同作用下,经过巨噬细胞（包括树突状细胞）的抗原呈递作用,Na（?）ve T辅助细胞（Th 0）被活化,分化为Th 2细胞,释放IL-4、IL-5、IL-13等多种Th 2细胞因子,促进了支气管哮喘的形成和发展。其中IL-13是一种重要的Th2细胞因子,它通过诱导哮喘气道炎症、增加气道高反应性、促进粘液分泌,并参与了哮喘气道重建,成为支气管哮喘发病机制中的重要介质。但迄今为止,对IL-13基因表达的分子调控机制仍知之甚少。T细胞是产生IL-13的主要来源。CD3/CD28双信号通路对T细胞的刺激能够模拟T细胞在体内受抗原活化产生Th2细胞因子的过程。转录因子在基因的表达和调控上具有核心作用,GATA3是Th2细胞特异性的转录因子,细胞中增高的GATA3能够同时促进Th2细胞中IL-4、IL-5和IL-13的基因表达。许多研究表明在IL-13基因启动子区域GATA位点对IL-13的基因表达起重要作用。活化T细胞核因子（nuclear factor of activated T cell,NFAT）是TCR介导的信号转导通路中关键的转录因子。我们的前期研究表明在CD3/CD28双信号通路刺激后NFAT1与IL-13启动子结合增强。在人IL-13基因启动子区域GATA3与NFAT1位点邻近,作为同时参与IL-13基因转录的GATA3和NFAT1间是否存在相互作用?如何相互作用?均值得进一步探讨。本课题通过研究T细胞经CD3/CD28活化后观察GATA3和NFAT1在IL-13启动子区域功能上的相互作用,探索IL-13基因转录的分子机制,并进一步研究糖皮质激素丙酸氟替卡松（Fluticasonepropionate,FP）和传统哮喘治疗药物三氧化二砷（Arsenic Troxide,AT）对该相互作用的影响。第一部分GATA3-NFAT1相互作用在人活化T细胞IL-13基因表达中的作用目的:研究T细胞在CD3/CD28双信号通路共刺激活化后,细胞内GATA3与NFAT1的结合情况及NFAT1抑制剂FK-506对GATA3与IL-13启动子结合的影响,探讨GATA3-NFAT1相互作用在T细胞IL-13基因表达中的作用。方法:采用CD3/CD28单克隆抗体（浓度分别为10μg/ml和5μg/ml）双信号通路对Hut-78细胞进行共刺激,分别在0min、30min、1h、2h时采用免疫共沉淀（IP）的方法观察GATA3与N-FAT1结合情况及FK-506对两者结合的影响。通过染色质免疫共沉淀（CHIP）的方法研究NFAT1被FK-506阻断后GATA3与IL-13启动子区域的结合情况。结果:Hut-78细胞在CD3/CD28共刺激活化后,细胞内NFAT1和GATA3结合增强,在刺激30min时两者即有结合,随着刺激时间的延长在1h、2h时两者结合有进一步增强的趋势,呈明显的时间依赖性。FK-506（1n M）组在30min、1h、2h时均能抑制NFAT1与GATA3的结合,以2h时抑制更为明显（2.25±0.1 VS 1.18±0.09）,P＜0.05。用FK-506对NFATl进行抑制后,GATA3与IL-13的启动子区域结合减弱。T细胞经CD3/CD28免疫共刺激活化30分钟时FK-506抑制GATA3与IL-13启动子结合的作用不明显,而在1h时能够抑制GATA3同IL-13启动子的结合（3.95±0.26 VS 2.82±0.44）,2h时这种抑制效应更为明显,其抑制效应约为50%（4.34±1.42 VS 1.73±0.09）,P＜0.05。结论:T细胞经过CD3/CD28共刺激活化后细胞内NFAT1和GATA3结合增强,FK-506能抑制两者的结合。FK-506能抑制GATA3与IL-13启动子区域结合。FK-506可能通过影响两者的结合,抑制了GATA3对IL-13基因转录的启动。第二部分:FP抑制活化T细胞IL-13基因表达的机制目的:研究T细胞在CD3/CD28共刺激活化后,FP对NFAT1和GATA3结合的影响及FP对GATA3与IL-13启动子结合的影响,探讨FP抑制活化T细胞中IL-13基因表达的可能机制。方法:采用CD3/CD28单克隆抗体（浓度分别为10μg/ml和5gg/ml）双信号通路对Hut-78细胞进行共刺激,分别在0min、30min、1h、2h时采用免疫共沉淀（IP）的方法观察GATA3与NFAT1结合情况及FP对两者结合的影响。通过染色质免疫共沉淀（CHIP）的方法研究FP对GATA3与IL-13启动子结合的影响。结果:与对照组相比,经CD3/CD28刺激组GATA3与NFAT1结合增强,且在30min、1h、2h各时间点逐渐增高,呈时间依赖性。FP(10^-8M)能够部分抑制GATA3与NFAT1的结合,在细胞活化30min（1.33±0.08VS 0.91±0.083）、1h（1.97±0.18 VS 1.56±0.13）、2h（2.65±0.18 VS 1.80±0.23）时FP均可抑制两者的结合（P＜0.05）。HUT-78细胞经CD3/CD28双信号通路共刺激活化后在30min、1h及2h各时间点GATA-3与IL-13启动子区域结合逐渐增强。FP在细胞活化1h能显著抑制GATA3与IL-13启动子区域结合（8.19±2.1 8 VS 2.69±1.33）,P＜0.05。结论:T细胞经过CD3/CD28活化后GATA3与NFAT1结合增强,并促进GATA3与IL-13启动子区域结合。FP可能部分通过影响GATA3和NFAT1的相互作用,抑制T细胞IL-13基因表达。第三部分:砷剂对活化T细胞IL-13基因表达的影响目的:研究T细胞在CD3/CD28双信号通路刺激后IL-13的基因表达以及三氧化二砷对该基因表达的影响,并探讨三氧化二砷（AT）抑制IL-13基因表达的可能机制。方法:采用CD3/CD28单克隆抗体（浓度分别为10μg/ml和5μg/ml）双信号通路对Hut-78细胞进行共刺激,在30min及19h时用RT-PCR方法观察IL-13mRNA表达及AT对IL-13mRNA表达的影响,并采用IP方法观察在细胞活化0min、30min、1h、2h时GATA3与NFAT1结合情况及AT对两者结合的影响。结果:CD3/CD28双信号通路共刺激后,Hut-78细胞的IL-13mRNA表达增加,在19h时砷剂（5μM）能够明显抑制该基因的表达（6.77±2.78 VS 2.24±0.57）,P＜0.05。Hut-78细胞在经过CD3/CD28免疫活化30分钟后,GATA3和NFATl两者即有结合且在2小时内两者结合呈逐渐增强趋势,AT在2h时能够部分抑制两者的结合(2.47±1.83VS 1.41±1.11,P＜0.05。结论:对Th2细胞IL-13基因转录表达的抑制可能是AT平喘抗炎的机制之一,该过程可能是通过部分抑制GATA3和NFAT1两者相互作用实现的。更多还原

【Abstract】 Asthma is a complex inflammatory disease of the lung characterized by airway hyperresponsiveness（AHR）,eosinophilic inflammation and mucus hypersecretion.The inflammatory process,which is the basis of AHR and mucus hypersecretion,is believed to be a result of inappropriate immune responses to common aeroallergens in genetically susceptible individuals. It has been hypothesized that CD4⁺T cells that produce a Th2 pattern of cytokines（IL-4,IL-5,IL-13）play a pivotal role in the pathogenesis of asthma.However,the therapy targeted at IL-4 and IL-5 proved little efficacy.IL-13 has been recognized as a critical regulator of the allergic response.Blockade of IL-13 markedly inhibits allergen-induced airway eosinophilia,hyperresponsiveness,mucus production and airway remodeling.However,the molecular mechanisms regulating IL-13 production are still largely unknown.GATA3 and NFAT1 both play an important role in the expression of IL-13 gene.To investigate the interaction between GATA3 and NFAT1 on IL-13 gene expression,human T cell lines（Hut-78 cells）were stimulated with anti-CD3 and anti-CD28 antibodies to mimic antigen-mediated co-stimulation in vivo. PARTⅠ:The synergistic interaction between NFAT1 and GATA3 On gene expression of IL-13 in human T cellsObjective:To investigate the synergistic interaction between NFAT1 and GATA3 on IL-13 gene expression in human T cells due to CD3/CD28 co-stimulation.Methods:Hut-78 cells were stimulated with anti-CD3/CD28 monoclonal antibodies at the concentration of 10μg/ml and 5μg/ml respectively.The interaction between NFAT1 and GATA3 in several times（0min,30min, 1h,2h）was detected by immunoprecipitation assays.Chromatin immunoprecipitation assays were used to investigate the binding of GATA3 to the promoter of IL-13.Results:Anti-CD3/CD28 co-stimulation induced the binding of GATA3 and NFAT1 in a time-dependant manner.FK-505 can inhibit the binding of GATA3 to NFAT1,and thus the binding of GATA3 to IL- 13 promoter.Conclusions:These results demonstrate the important role of the synergistic interaction between NFAT1 and GATA3 in the regulation of IL-13 gene expression in Hut-78 cells following CD3/CD28 co-stimulation. PARTⅡ:The role of FP on the gene expression of IL-13 in actived human T cellsObjective:To study the effect of FP on the GATA3-NFAT1 interaction and subsequent GATA3 binding to IL-13 promoter in human T cells due to CD3/CD28 co-stimulation and investigate the anti-inflammatory mechanism of FP.Methods:Hut-78 cells were stimulated with anti-CD3/CD28 monoclonal antibodies at the concentration of 10μg/ml and 5μg/ml respectively.The effect of FP on the GATA3-NFAT1 interaction in 0min,30min,1h and 2h were detected by immunoprecipitation assays.Chromatin immunoprecipitation assays were used to investigate the binding of GATA3 to the promoter of IL- 13.Results:Anti-CD3/CD28 co-stimulation induced the binding of GATA3 and NFAT1 in a time-dependant manner,which was partially inhibited by FP at the concentration of 10^-8M FP also inhibited the binding of GATA3 to the promoter of IL- 13.Conclusions:FP can inhibit IL-13 gene expression in actived human T cells,which may be associated with inhibiting of the formation of GATA3-NFAT1 compound and the binding of GATA3 to the promoter of IL-13. PARTⅢ:Arsenic trioxide inhibits the gene expression of IL-13 in activated T cellsObjective:To study the interleukin 13（IL-13）gene expression in T cells due to CD3/CD28 co-stimulation and the effect of arsenic trioxide（AT） on IL-13 gene expression.Methods:Hut-78 cells were stimulated with anti-CD3/CD28 monoclonal antibodies（10μg/ml and 5μg/ml,respectively）.IL-13 mRNA level in Hut-78 cells was measured in groups with or without AT treatment.The effect of AT on the GATA3-NFAT1 compound in 0min,30min,1h and 2h were detected by immunoprecipitation assays.Results:The level of IL-13 mRNA increased in Hut-78 cells due to CD3/CD28 co-stimulation.However AT inhibited the gene expression of IL-13.AT inhibited the binding of GATA3 and NFAT1 in actived human T cells in 2h.Conclusions:AT would inhibit the gene expression of IL-13 in activated T cells,which might be partially through inhibiting the formation of the GATA3-NFAT1 compound.更多还原