【作者】 刘嵘；

【导师】 胡丽华；

【作者基本信息】 华中科技大学 ， 临床检验诊断学， 2010， 博士

【摘要】 变应性哮喘是一种常见的与免疫相关的复杂呼吸道炎症性疾病,其特征主要是气道反应性增高和针对环境过敏原的特异性IgE或总IgE升高,一般受遗传、环境、个体免疫状态等多重因素的影响而发病。据报道人类的Tim-3是Tims基因家族重要的成员之一,它就位于与哮喘呈高度连锁的染色体5q31-33区域。目前已有不少研究将Tim-3基因作为哮喘重要的易感候选基因,进行Tim-3基因多态性与哮喘的关联研究。在部分人群中开展研究的结果显示,Tim-3基因多态性与哮喘有一定的相关性。近来的研究表明人体在正常情况下,TH1细胞和TH2细胞这两类亚型受不同转录因子的调控,分泌不同的细胞因子,并相互协调和拮抗,维持内环境的稳态。TH1／TH2细胞及其分泌的细胞因子的失衡是哮喘发病的影响因素。哮喘发病时,出现了TH2细胞的极化偏移,TH2细胞及其分泌的细胞因子相对增加。Tim-3最初被发现在TH1细胞对抗原产生增殖性反应后表达于终末分化的TH1细胞上,并与Tim-3的配体galectin-9结合后发挥终止TH1介导的免疫反应的功能。Tim-3与其配体galectin-9的结合,引发了一种抑制信号,导致了TH1细胞的凋亡,负调了TH1型免疫反应。但是,Tim-3的表达并不局限于T细胞。近来的研究通过逆转录-聚合酶链式反应证实了Tim-3在多种人的正常和恶性上皮组织,小神经胶质细胞,单核细胞,树突状细胞,M2巨噬细胞,肥大细胞和多种T细胞上均有表达,提示Tim-3可能与人体的多个组织器官以及免疫系统的功能相关。那么Tim-3是否在呼吸系统有所表达,它是否通过呼吸系统和免疫系统而与哮喘相关联正是需要进一步研究的问题。本研究我们首先在人呼吸道相关细胞株上筛选了Tim-3的表达,并建立hTim-3质粒稳定转染16HBE的细胞株,应用屋尘螨和地塞米松处理上调hTim-3和未上调hTim-3的16HBE细胞株,检测各处理组NF-κB和炎症相关因子表达水平,分析hTim-3对16HBE细胞株NF-κB和炎症相关因子表达水平的影响。目的：筛选Tim-3表达阳性的人呼吸道相关细胞株,为进一步阐明Tim-3可能与人呼吸道疾病相关奠定基础；构建pEGFP-C2-hTim-3真核表达载体,可供后续进一步研究hTim-3的功能使用。将测序完全正确的重组质粒pEGFP-C2-hTim-3转染入人支气管上皮细胞株16HBE中,筛选出阳性细胞克隆,为后续实验提供一个平台。方法：用含10%胎牛血清的高糖DMEM培养基于37℃5%CO2饱和湿度培养箱中体外培养人呼吸道相关细胞株16HBE,A549,GLC-82,调整细胞到最佳状态,对数生长期时收集细胞,抽提总RNA,并逆转录为cDNA。参照人GenBank中Tim-3基因的全长序列,使用Primer5.0设计并合成其引物,采用PCR检测以上三种细胞Tim-3的mRNA表达。将健康人外周血Tim-3基因全长cDNA片段克隆入真核表达载体pEGFP-C2,经菌落PCR、双酶切及测序鉴定,构建包含目的基因Tim-3的重组质粒pEGFP-C2-hTim-3。利用脂质体介导pEGFP-C2-hTim-3质粒和pEGFP-C2质粒分别转染16HBE细胞,荧光显微镜观察16HBE细胞的瞬时转染效率,并采用含G418的培养基筛选以获得整合了pEGFP-C2-hTim-3和pEGFP-C2的阳性细胞克隆,用荧光显微镜观察pEGFP-C2-hTim-3和pEGFP-C2在16HBE细胞上的定位表达,用Real-time PCR技术和Western Blot技术检测转染pEGFP-C2-hTim-3和转染pEGFP-C2的16HBE细胞上hTim-3的表达情况。结果：1.PCR结果显示人呼吸道相关细胞株16HBE,A549,GLC-82均表达Tim-3mRNA。2.构建成具有筛选特征和能够稳定表达的pEGFP-C2-hTim-3质粒载体,测序结果与预期设计完全一致。3.瞬时转染后经荧光显微镜检查可见阳性表达pEGFP-C2-hTim-3绿色荧光蛋白的16HBE细胞不足8%,而阳性表达pEGFP-C2绿色荧光蛋白的16HBE细胞不足15%。采用G418筛选后获得阳性表达野生型hTim-3和外源性pEGFP-C2-hTim-3的16HBE细胞表达的hTim3水平比转染pEGFP-C2空质粒的16HBE细胞显著增高(P<0.01),经荧光显微镜观察前者定位在胞膜上,而后者则定位在胞质上。结论：人呼吸道相关细胞株16HBE,A549,GLC-82均表达Tim-3 mRNA,Tim-3可能与人类呼吸道疾病的发生和发展相关。成功构建了真核表达载体pEGFP-C2-hTim-3。成功筛选获得了高表达pEGFP-C2-hTim-3的16HBE阳性克隆细胞,可供后续研究使用。目的：研究T细胞免疫球蛋白及粘蛋白域蛋白3(简称TIM-3)对人气道上皮细胞株16HBE经屋尘螨或／和地塞米松处理后NF-κB及相关细胞因子表达的调控,并探讨相应机制。方法：实验分两组,分别为转染了pEGFP-C2和转染了pEGFP-C2-hTim-3的16HBE细胞株。两组细胞株分别用一定浓度的屋尘螨或／和地塞米松处理。提取细胞mRNA,并逆转录为cDNA,实时荧光定量PCR方法检测转染后的不同处理组细胞株Tim-3,NF-κB, IFN-γ, galectin-9, T-bet,IL-4和GATA-3等的]mRNA表达水平。提取细胞蛋白后,Western blot方法检测转染后的不同处理组细胞株NF-κB的蛋白表达水平。分析hTim-3对经屋尘螨或／和地塞米松处理的16HBE细胞株NF-κB及相关细胞因子表达的调控,并探讨相应的机制。结果：1.实时荧光定量PCR结果显示,当16HBE细胞用屋尘螨或／和地塞米松处理时,转染了PEGFP-C2-hTim3质粒的16HBE细胞株上Tim-3(p<0.01), NF-κB(p< 0.01), IFN-γ(p<0.01), T-bet(p<0.01), galectin-9(p<0.01), IL-4(p<0.01)的表达与相应处理的转染了pEGFP-C2质粒的16HBE细胞的以上指标表达有显著性差异,而GATA-3并没有明显的改变(p>0.05)。而且,在转染了pEGFP-C2-hTim3质粒的16HBE细胞株中,Tim-3的表达与IFN-γ, T-bet和galectin-9的表达呈正相关,而与NF-κB和IL-4的表达呈负相关；但是,在转染了pEGFP-C2质粒的16HBE细胞中,并未存在这种较强的相关性。而且,当用一定浓度的屋尘螨或／和地塞米松处理转染了pEGFP-C2-hTim3质粒的16HBE细胞时,Tim-3对NF-κB表达的影响可能通过上调IFN-γ, T-bet, galectin-9和下调IL-4的表达实现。2.Western Blot结果显示,当用屋尘螨或／和地塞米松处理16HBE细胞时,转染了pEGFP-C2-hTim3质粒的16HBE细胞中Tim-3的表达与NF-κB的蛋白表达呈负相关；但是,在转染pEGFP-C2的16HBE细胞中未出现这种明显的趋势。这表明转染pEGFP-C2-hTim3质粒的16HBE细胞中Tim-3的上调抑制了NF-κB的蛋白表达。结论：在16HBE细胞中,Tim-3通路的上调可能通过上调IFN-γ, T-bet, galectin-9和下调IL-4的表达而减弱了NF-κB的表达,并以此发挥抑炎的功效。目的：研究T细胞免疫球蛋白及粘蛋白域蛋白3(简称TIM-3)对人气道上皮细胞株16HBE经屋尘螨或／和地塞米松处理后炎症相关因子表达的调控,并探讨相应机制。方法：实验分两组,分别为转染了pEGFP-C2和转染了pEGFP-C2-hTim-3的16HBE细胞株。两组细胞株分别用一定浓度的屋尘螨或／和地塞米松处理。提取细胞mRNA,并逆转录为cDNA,实时荧光定量PCR方法检测转染后的不同处理组细胞株Tim-3, NF-κB, TNF-α, IL-8,RANTES,ICAM-1等的mRNA表达水平。分离上清后,ELISA方法检测转染后的不同处理组细胞株NF-κB,TNF-α,IL-8的蛋白表达水平。分析hTim-3对经屋尘螨或／和地塞米松处理的16HBE细胞株NF-κB及相关炎症因子表达的调控,并探讨相应的机制。结果：1.实时荧光定量PCR结果显示,当16HBE细胞用屋尘螨或／和地塞米松处理时,转染了pEGFP-C2-hTim3质粒的16HBE细胞株上Tim-3(p<0.01), NF-κB(p< 0.01), TNF-α(p<0.01), IL-8(p<0.01), RANTES(p<0.01), ICAM-1(p<0.01)的表达与相应处理的转染了pEGFP-C2质粒的16HBE细胞的以上指标表达有显著性差异；而且,在转染了pEGFP-C2-hTim3质粒的16HBE细胞株中,Tim-3的表达与NF-κB, TNF-α, IL-8, RANTES和ICAM-1的表达呈负相关；但是,在转染了pEGFP-C2质粒的16HBE细胞中,并未存在这种较强的相关性。当用一定浓度的屋尘螨或／和地塞米松处理转染了pEGFP-C2-hTim3质粒的16HBE细胞时,在mRNA水平上TNF-α水平的减低可能参与了NF-κB表达的下调,Tim-3对IL-8,RANTES和ICAM-1表达的影响可能通过下调NF-κB这种依赖性的方式实现。2.ELISA结果显示,当用屋尘螨或／和地塞米松处理16HBE细胞时,转染了pEGFP-C2-hTim3质粒的16HBE细胞中TNF-α,IL-8的表达与NF-κB的蛋白表达呈正相关；但是,在转染pEGFP-C2的16HBE细胞中未出现这种明显的趋势。这表明转染了pEGFP-C2-hTim3质粒的16HBE细胞中,在蛋白水平上TNF-α水平的减低可能参与了NF-κB表达的下调,Tim-3对IL-8表达的影响可能通过下调NF-κB种依赖性的方式而产生。结论：在16HBE细胞中,Tim-3通路的上调可能通过NF-κB依赖性的方式下调了IL-8,RANTES和ICAM-1的表达,TNF-α水平的减低也可能参与了NF-κB表达的下调,并以此发挥抑炎的功效。更多还原

【Abstract】 Allergic asthma is a common complex respiratory tract inflammatory disease related with immunity, and its characteristics are mainly airway hyperreaction and specific IgE or all IgE upregulation to environmental allergen, which is caused by multiple factors such as genetic, environmental and individual immune state. It was reported that chromosome 5q31-33 region was strongly linked to asthma. Human Tim-3 is one member of Tims gene family, which is located in the chromosome 5q31-33 region strongly linked to asthma. At present some researchers think Tim-3 as a good candidate susceptibility gene for asthma, and perform the association study about Tim-3 polymorphism with asthma. The results of study conducted in several populations showed that Tim-3 polymorphism was linked to asthma.Recent study has showed that the two subtype TH1 cells and TH2 cells are regulated by different transcription factors, secrete different cytokines to coordinate and maintain internal environment homeostasis. The imbalance of TH1/TH2 cells and their secretary cytokines is the important factor for the development of asthma. When asthma developes, TH2 cells appears polarization, TH2 cells and their secretary cytokines increase correspondingly. Tim-3 was first identified to be a transmembrane protein expressed on terminally differentiated TH1 cells when they were stimulated by antigen. If Tim-3 binds to the Tim-3 ligand, galectin-9, it play the role to terminate TH1-mediated immunity. Interaction of Tim-3 and its ligand galectin-9 triggers an inhibitory signal to lead to apoptosis of TH1 cells, and thus negatively regulating TH1-mediated immunity. However, expression of Tim-3 is not restricted to T cells. Previous study demonstrated that Tim-3 was expressed in a wide range of human normal and malignant epithelial tissues by RT-PCR, on microglia, monocytes, DCs, M2 macrophages, mast cells, cytotoxic CD8+ T cells, activated CD4+ T cells, Th17 and Treg, which suggests Tim-3 may be associated with several tissue and immune functions. Therefore, it is to be further investigated that whether Tim-3 is expressed in respiratory system and whether Tim-3 is linked with asthma by respiratory system and immune system. In this study, we detect Tim-3 expression in 16HBE cell, GLC-82 cell and A549 cell by using PCR. We have cloned human full-length Tim-3 from peripheral blood monocytes. And we transfect the eukaryotic expression plasmids which respectively expresses human pEGFP-C2-flTim-3 and pEGFP-C2 into human bronchial epithelial cells and screen positive clones by G418. Then we stimulate the transfected 16HBE cells with house dust mite or/and Dexamethasone at some concentration, and detect NF-κB and inflammation-related genes expression level in treated groups to assess the effects of hTim-3 on NF-κB and inflammation-related genes expression level.OBJECTIVE:To Screen the respiratory tract related cell lines for Tim-3 positive expression and make foundation for elucidating that Tim-3 may be associated with respiratory tract related disease. To construct an eukaryotic expression vector pEGFP-C2-hTim-3 for further study of the function of hTim-3. To transfect 16HBE cells by human pEGFP-C2-hTim-3 and pEGFP-C2 and screen the positive clones for further study.METHOD:The human bronchial epithelial cell lines 16HBE, A549 and GLC-82 were cultured in DMEM with high glucose medium supplemented with 10% fetal bovine serum, and were maintained at 37℃and 5% CO2 in saturated humidity air. Monocytes were separated from peripheral blood anticoagulated by EDTA-K2 of healthy adults with Ficoll lymphocyte separation medium. The RNA of the four above cells was extracted by TRIzol and reverse transcripted into cDNA. PCR for detecting mRNA of Tim-3 in respiratory tract related cell lines was performed. The full-length hTim-3 gene was amplified with monocytes cDNA from peripheral blood of healthy adults. The full-length hTim-3 gene cDNA was cloned into pEGFP-C2 plasmid. And the extracted plasmid was verified by PCR, restrictedly enzyme analysis and DNA sequencing. The right plasmid was construction of an eukaryotic expression vector pEGFP-C2-hTim-3. The recombination plasmid pEGFP-C2-hTim-3 and the plasmid pEGFP-C2 were transfected into 16HBE cells by Lipofectamine 2000, respectively. The transient transfection results was observed by the fluorescence microscopy. The transfected 16HBE cells were screened by 0.6 mg/mL G418 and were maintained by 0.4 mg/mL G418 after two weeks. The fluorescence protein expression in 16HBE transfected pEGFP-C2-hTim-3 and pEGFP-C2 was observed by the fluorescence microscopy. The protein expression of Tim-3 in 16HBE transfected pEGFP-C2 and pEGFP-C2-hTim-3 was detected by Western Blot.RESULT:1. The PCR results showed that hTim-3 was expressed in monocytes from human peripheral blood,16HBE, GLC-82 and A549 cells.2. After DNA sequencing, the sequence of the constructed pEGFP-C2-hTim-3 plasmid was in accordance with GeneBank accession nos. BC063431 and AF450242.3. After transient transfection, transfected pEGFP-C2-hTim3 plasmid 16HBE with the green fluorescence protein expression occupied less than 8%; transfected pEGFP-C2 plasmid 16HBE with the green fluorescence protein expression occupied less than 15%. After screening, The fluorescence microscopy observation results presented that the green fluorescence of 16HBE transfected pEGFP-C2-hTim3 plasmid was located on the membrane, suggesting that hTim3 was expressed on the membrane; the green fluorescence of 16HBE transfected pEGFP-C2 plasmid was mainly located on the cytoplasma. The expression of Tim-3 in 16HBE transfected pEGFP-C2-hTim3 plasmid was higher than that in 16HBE transfected pEGFP-C2 plasmid by Real-time PCR and Western Blot analysis (p< 0.01).CONCLUSION:HTim-3 was expressed in monocytes from human peripheral blood, 16HBE, GLC-82 and A549 cells, suggesting that Tim-3 may be associated with respiratory tract-related disease. An eukaryotic expression vector pEGFP-C2-hTim-3 was constructed successfully. The 16HBE cells stably transfected by human pEGFP-C2-hTim-3 were established successfully.OBJECTIVE:To investigate the effect of Tim-3 pathway on NF-κB expression in 16HBE cell treated with house dust mite or/and Dexamethasone, and further elucidate the mechanism of regulation.METHOD:16HBE transfected pEGFP-C2-hTim-3 and 16HBE transfected pEGFP-C2 were treated with house dust mite or/and Dexamethasone at some concentration, respectively. The mRNA of cells were extracted by TRIzol and reverse transcripted to cDNA. The mRNA expression of Tim-3, NF-κB, IFN-γ, galectin-9, T-bet, IL-4 and GATA-3 in different treated cells after transfection were detected by Real-time quantative PCR. The protein of cells were extracted for detecting NF-κB protein expression in different treated cells after transfection by Western Blot analysis. Then we investigated the effect of hTim-3 on NF-κB and some cytokines expression in 16HBE cells treated with house dust mite or/and Dexamethasone and discussed corresponding mechanism.RESULT:1. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3(p<0.01), NF-κB(p<0.01), IFN-γ(p<0.01), T-bet(p<0.01), galectin-9(p<0.01), IL-4(p<0.01) in 16HBE cells transfected pEGFP-C2-hTim3 plasmid were different obviously from that of 16HBE cell transfected pEGFP-C2 in corresponding groups, and GATA-3 did not change obviously(p>0.05). Furthermore, expression of Tim-3 was positive correlation with expression of IFN-γ, T-bet and galectin-9, correlated negatively with expression of NF-κB and IL-4 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid; however, there were not the strong correlations in 16HBE cell transfected pEGFP-C2. It suggested that effect of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid when treated with house dust mite or/and Dexamethasone on NF-κB probably was influenced by upregulating of IFN-γ, T-bet, galectin-9 expression, and downregulating IL-4.2. When 16HBE cells were treated with house dust mite or/and Dexamethasone, Tim-3 expression in 16HBE cells transfected pEGFP-C2-hTim3 plasmid was negative correlation with NF-κB protein expression, however, there was not the tendency in 16HBE cells transfected pEGFP-C2. It showed that Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 suppressed protein expression of NF-κB.CONCLUSION:Upregulation of Tim-3 pathway may attenuate NF-κB expression by upregulation expression of IFN-γ, T-bet, galectin-9, and downregulation level of IL-4 in 16HBE cells, thus inhibiting inflammation.OBJECTIVE:To investigate the effect of Tim-3 pathway on inflammation-related factors expression in 16HBE cell treated with house dust mite or/and Dexamethasone, and further elucidate the mechanism of regulation.METHOD:16HBE transfected pEGFP-C2-hTim-3 and 16HBE transfected pEGFP-C2 were treated with house dust mite or/and Dexamethasone at some concentration, respectively. The mRNA of cells were extracted by TRIzol and reverse transcripted to cDNA. The mRNA expression of Tim-3, NF-κB, TNF-α, IL-8, RANTES, ICAM-1 in different treated cells after transfection were detected by Real-time quantative PCR. The supernants of cells were separated for detecting NF-κB, TNF-α, IL-8 protein expression in different treated cells after transfection by ELISA. Then we investigated the effect of Tim-3 on TNF-α, NF-κB and some inflammation-related factors expression in 16HBE cells treated with house dust mite or/and Dexamethasone and discussed corresponding mechanism.RESULT:1. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3(p<0.01), NF-κB(p<0.01), TNF-α(p<0.01), IL-8(p<0.01), RANTES(p<0.01), ICAM-1 (p<0.01) in 16HBE cells transfected pEGFP-C2-hTim3 plasmid were different obviously from that of 16HBE cell transfected pEGFP-C2 in corresponding groups. Furthermore, expression of Tim-3 was negative correlation with expression of NF-κB, TNF-α, IL-8, RANTES and ICAM-1 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid; however, there were not the strong correlations in 16HBE cell transfected pEGFP-C2. Further, the effects of Tim-3 on IL-8, RANTES and ICAM-1 expression may be achieved in downregulating NF-κB-dependent manner, TNF-a may downregulate NF-κB expression.2. When 16HBE cells were treated with house dust mite or/and Dexamethasone, expression of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 plasmid was negative correlation with NF-κB, TNF-αand IL-8 protein expression, however, there was not the tendency in 16HBE cells transfected pEGFP-C2. It showed that upregulation of Tim-3 in 16HBE cells transfected pEGFP-C2-hTim3 suppressed protein expression of NF-κB, TNF-αand IL-8.CONCLUSION:Upregulation of Tim-3 pathway may attenuate the expression of IL-8, RANTES and ICAM-1 in NF-κB-dependent manner, TNF-a may downregulate NF-κB expression, thus inhibiting inflammation development.更多还原