【作者】 赵杰；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2008， 博士

【摘要】 第一部分:Toll样受体2和4信号通路介导的人角膜上皮细胞对烟曲霉菌的炎性反应目的研究Toll样受体(Toll-like receptors,TLR)2和TLR4信号通路介导的人永生化角膜上皮细胞(THCE)对烟曲霉菌(Aspergillus fumigatus,AF)孢子、上清、菌丝体抗原的炎性反应。方法采用自制的烟曲霉菌孢子、上清和菌丝体抗原,刺激培养的THCE细胞。以Real-time PCR检测THCE细胞TLR2、TLR4 mRNA的表达;以Western blot检测THCE细胞TLR2、TLR4的表达及pIkB的活化;以酶联免疫吸附试验(ELISA)方法检测THCE细胞培养上清中IL-1β、IL-6、TNF-α和IL-8的浓度;用TLR2和/或TLR4中和抗体特异性封闭TLR2和/或TLR4,或用尾叶香茶菜丙素(kamebakaurin,KA)特异性抑制核转录因子(nuclear factor,NF)-kB活性,观察调节TLRs信号转导通路对角膜上皮细胞IL-1β、IL-6、TNF-α和IL-8表达的影响。结果①TLRs信号通路活化实验:正常THCE细胞有TLR2及TLR4 mRNA及蛋白质的表达。烟曲霉菌抗原以时间依赖方式促进THCE中TLR2、TLR4的表达及细胞因子的分泌。烟曲霉菌孢子刺激THCE细胞30min后TLR2及TLR4的表达开始增多,分别为对照组的2和2.75倍(P＜0.05),刺激4h时达高峰,分别为对照组的23.5和27倍(P＜0.01)。上清抗原或菌丝抗原刺激THCE 2h后,TLR2、TLR4 mRNA表达较对照组升高,分别为对照组的35.5倍、33.5倍和9.75倍、18倍(P＜0.01),刺激6h时达高峰,分别为对照组的53倍、58.5倍和29.75倍、57.5倍(P＜0.01)。烟曲霉菌孢子抗原刺激THCE细胞2h后IL-1β、IL-6、TNF-α和IL-8的表达开始增多,分别为对照组的1.33倍、2.1倍、1.2倍、1.3倍(P＜0.05),刺激4h后达更多,分别为对照组的2.7倍、5.3倍、2.6倍、2.6倍(P＜0.01)。烟曲霉菌上清及菌丝刺激THCE细胞2h后IL-1β、IL-6、TNF-α和IL-8的表达开始增多,分别为对照组的1.4倍、2.2倍、1.3倍、1.3倍和1.3倍、2倍、1.2倍、1.2倍(P＜0.05),刺激后6h达更多,分别为对照组的3.7倍、6.6倍、3.2倍、3.2倍和3.2倍、6.3倍、2倍、2.8倍(P＜0.01)。烟曲霉菌孢子、上清或菌丝抗原刺激THCE细胞12h后,细胞TLR2及TLR4表达增高(P＜0.01),PIkB活化(P＜0.01),其培养上清内IL-1β、IL-6、TNF-α和IL-8浓度升高(P＜0.01),分别为未刺激组的3.6倍、7.3倍、4.1倍和4.5倍;4.2倍、7.4倍、4.1倍和4.5倍;3.3倍、7.3倍、3.9倍和4.4倍。②TLRs信号通路调节实验:在烟曲霉菌抗原刺激组,单纯封闭TLR2或TLR4可抑制pIkB的活性(P＜0.05),联合封闭TLR2和TLR4可明显抑制pIkB-α的活性(P＜0.01)。烟曲霉菌孢子抗原刺激THCE细胞12h后,其培养上清内IL-1β、IL-6、TNF-α和IL-8浓度升高,分别为未刺激组的3.6倍、7.3倍、4.1倍和4.5倍;烟曲霉菌上清抗原刺激THCE细胞12h后,其培养上清内IL-1β、IL-6、TNF-α和IL-8浓度升高,分别为未刺激组的4.2倍、7.4倍、4.1倍和4.5倍;烟曲霉菌菌丝体抗原刺激THCE细胞12h后,其培养上清内IL-1β、IL-6、TNF-α和IL-8浓度升高,分别为未刺激组的3.3倍、7.3倍、3.9倍和4.4倍。在烟曲霉菌孢子抗原刺激组,联合封闭TLR2和TLR4受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了70.3%、85.8%、54.4%、57.4%;单纯封闭TLR2受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组减少了63.1%、0.01%、29.3%、26.3%;单纯封闭TLR4受体,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了48.3%、84.2%、28.3%、18.5%。在烟曲霉菌上清抗原刺激组,联合封闭TLR2和TLR4受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了74.3%、86.0%、53.7%、56.6%;单纯封闭TLR2受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组减少了67.1%、0.01%、29.2%、25.4%;单纯封闭TLR4受体,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了47.1%、84.6%、28.2%和22.7%。在烟曲霉菌菌丝抗原刺激组,联合封闭TLR2和TLR4受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了67.6%、86%、53.7%、57.5%;单纯封闭TLR2受体后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组减少了58.3%、0.01%、26.8%、26.2%;单纯封闭TLR4受体,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了51.7%、84.4%、25.3%、23.1%。在烟曲霉菌孢子、上清、菌丝抗原刺激组,阻断NF-κB后,IL-1β、IL-6、TNF-α和IL-8的分泌较刺激组分别减少了71.4%、86.2%、71.9%、74.4%,75%、86.3%、71.1%、74.1%,68.3%、86.2%、71.1%、74.5%。结论TLR2和TLR4参与了THCE对烟曲霉菌孢子、上清抗原及菌丝抗原的识别;并通过TLRs-NF-κB通路诱导下游炎性细胞因子的产生,介导角膜上皮细胞的炎性反应。第二部分:TLR2和TLR4在大鼠烟曲霉菌性角膜炎中的作用研究目的建立大鼠烟曲菌角膜炎动物模型,了解真菌性角膜炎病变角膜的病理特点,进一步研究TLR2和TLR4信号通路在此病理过程中的表达及作用机制。方法以基质注射法建立Wistar大鼠烟曲霉菌(1×10~8 CFU/角膜)角膜炎动物模型。于菌液接种后1、3、5、7、11天处死大鼠,裂隙灯观察角膜大体形态并进行临床评分;角膜组织真菌培养行烟曲霉菌菌落计数及感染真菌鉴定;髓过氧化物酶(myeloperoxidase,MPO)活力测定中性粒细胞活性;病理学检查检测角膜及真菌形态;Real-time PCR检测TLR2、TLR4 mRNA的表达;免疫荧光组织化学及Western blot检测TLR2、TLR4的表达;ELISA检测IL-1β和IL-10的表达。结果在Wistar大鼠可成功建立烟曲霉菌性角膜炎的动物模型。裂隙灯检查可见菌液接种后第1天角膜水肿;第5天角膜炎症最严重,可见角膜溃疡;第7、11天炎症反应开始减轻,可见角膜新生血管。角膜组织真菌培养鉴定为烟曲霉菌感染,菌落计数在菌液接种后第1天最多,随后逐渐减少。髓过氧化物酶活力在菌液接种后第1天开始增强,第5天活力最强,第7天开始逐渐减弱。病理结果示感染角膜组织水肿、坏死,基质纤维排列紊乱,并可查见菌丝及多形核细胞浸润。Real-time PCR、免疫荧光组织化学及Western blot结果示角膜TLR2和TLR4在感染后第1天表达开始增多,第5天表达最多,随后逐渐降低。ELISA示正常角膜有极微量的IL-1β和IL-10表达;角膜中IL-1β的表达在接种后第1天开始增多(为对照组的16.7倍),持续升高到第5天达最多(为对照组的24倍),第7天开始减少;而角膜中IL-10的表达在接种后第1天开始增多(为对照组的6.9倍),呈持续性升高趋势,在第11天达最多(为对照组的31倍)。结论在大鼠角膜可成功的建立真菌性角膜炎的动物模型;角膜组织中功能性的TLR2和TLR4介导炎性细胞因子的表达,参与角膜对真菌的免疫反应及角膜炎的病理过程。第三部分:Toll样受体在真菌性角膜炎组织中的表达及其意义目的研究健康角膜组织及真菌性角膜炎角膜组织中Toll样受体(toll-likereceptors,TLRs)2和4及其介导的炎性细胞因子的表达及功能,为真菌性角膜炎发病机制研究提供理论依据。方法收集37例真菌性角膜炎标本及11例健康角膜标本,刮片及培养鉴定真菌的类型;病理学检查检测角膜及真菌形态;Real-time PCR检测TLR2、TLR4 mRNA的表达;Western blot及免疫荧光组织化学检测TLR2、TLR4蛋白质的表达;ELISA检测IL-1β和IL-10的表达。结果37例真菌性角膜炎患者中,平均病程为19.9天,10～12月份发病率高(54.1%);男女性别之比为1.47:1,平均年龄42岁,其中农民占35例(94.6%),21例(56.8%)患者可追溯到明确的眼部外伤史,如植物外伤史、铁丝划伤史、玻璃刺伤史等。37例标本均经临床检查与实验室诊断确认,角膜刮片镜检阳性率为91.9%,角膜标本真菌培养阳性率为62.2%,以镰刀菌、曲霉菌为主要致病菌。病理学检查示真菌性角膜炎病变角膜为广泛化脓性炎症,菌丝平行、斜行或垂直生长于胶原纤维之间。Real-time PCR检测结果示37例真菌性角膜炎标本中TLR2、TLR4 mRNA的表达均较11例健康角膜标本减少(P＜0.05)。Western blot及免疫荧光组织化学结果示健康人角膜组织表达TLR2和TLR4,真菌性角膜炎组织中TLR2、TLR4的表达水平较健康角膜标本降低。ELISA结果示真菌性角膜炎组织中IL-1β和IL-10的表达较健康角膜标本增加。结论镰刀菌和曲霉菌为真菌性角膜炎主要致病菌种。TLR2、TLR4及Th1/Th2型细胞因子在调节角膜真菌感染免疫和炎症程度方面可能发挥重要作用。更多还原

【Abstract】 Part one: TLR2 and TLR4 signaling in immune responses against Aspergillusfumigatus in human corneal epithelial cellsPurpose.Cornea epithelial cells play an early and crucial role in the initiation of ocular surface responses to pathogens.Participation of Toll-like receptor(TLR)2 and TLR4, which are major forms of fungi receptors,may be involved in the Aspergillus fumigatus induced immune responses.The objective of the present study was to examine whether Aspergillus fumigatus antigens induce NF-κB activation and production of proinflammation cytokines,and whether the expression of Toll-like receptor(TLR)2 and TLR4 was amplified by Aspergillus fumigatus antigens in cultured immortalized human corneal epithelial cells(THCEs),which may contribute to our knowledge of the mechanism by which the host comea can successfully defend against invasive fungi.Methods.THCEs were stimulated with inactive antigens from Aspergillus fumigatus.The expressions of TLR2 and TLR4 were determined by Real-Time Quantitative PCR, immunofluorescence,and western blot.The activation of pIκB was determined by western blot in the presence or absence of TLR2 or TLR4 antibody.The concentration of interleukin(IL)-1β,IL-6,TNF-αand IL-8 in the cell supernatant were also assessed by enzyme-linked immunosorbent assays(ELISA)in the presence or absence of TLRs antibodies or NF-κB inhibitor.Results.TLR2 and TLR4 were expressed in THCEs.Aspergillus fumigatus antigens induced the activation of corneal epithelial cells as characterized by TLR2 and TLR4 expression,activation of pIκB,and upregulation of IL-1β,IL-6,TNF-αand IL-8.The Aspergillus fumigatus antigens -induced NF-κB activation was blocked by TLR2 and TLR4 antibodies.In addition,the Aspergillus fumigatus antigens-induced production of IL-1β, IL-6,TNF-αand IL-8 in supernatants of corneal epithelial cells was also attenuated by NF-κB inhibitor.Conclusions.Aspergillus fumigatus antigens contribute to the inflammatory responses of corneal epithelium in a TLR2 and TLR4-NF-κB signaling pathway-dependent manner.Part two: Activation of Toll-Like Receptor 2 and 4 in a rat Model of Aspergillusfumigatus KeratitisPurpose.To establish,in the Wistar rat eye,a new model of Aspergillus fumigatus keratitis suitable for studies of pathogenesis and TLRs signaling pathways. Methods.Corneas of 280 rats were infected with spores of Aspergillus fumigatus. Aspergillus fumigatus spores were intrastromally injected into rat corneas(1×10~8 CFU per cornea).Corneas injected with sterile physiological saline served as controls.Rats underwent slit lamp examination(SLE)at 1,3,5,7,and 11 days post infection and were killed.Histopathologic analyses,quantitative fungal cultures,and myeloperoxidase(MPO) activity assays were performed at each time point.Corneal TLR2 and TLR4 levels were tested by real time quantitative PCR and western blot,IL-1βand IL-10 levels were measured by ELISA at each time point.Results.Aspergillus fumigatus keratitis developed in Wistar rat,as evidenced by high SLE scores and viable fungi per infected eye than in control rats at 1,3,5,7,and 11 days after infection,and the severity of which peaked on day 5 after infection.Histopathologic analysis and MPO assays of infected rat both revealed an influx of polymorphonuclear leukocytes(PMNs).Production of IL-1βand IL-10,which mediate neutrophil recruitment to the corneal stroma,was elevated in the corneal epithelium and stroma of infected corneas. Rats injected with Aspergillus fumigatus exhibited an increase in corneal mRNA and protein for TLR2 and TLR4 over controls.Conclusions.These studies demonstrate the establishment of Aspergillus fumigatus keratitis in the rat eye.These findings indicate that the cornea has functional TLR2 and TLR4,and activation of TLR2 and TLR4 through NF-κB may contribute to pathogenesis of keratomycosis.Part three: Expression of TLR2 and TLR4 in the Healthy and Fungi-infected CorneaPurpose.To investigate the toll-like receptor(TLR)2,TLR4 and cytokines levels in healthy corneas and corneas with fungal keratitis(FK).Methods.37 corneas with FK and 11 healthy corneas were examined for histopathology. The expression of TLR2 and TLR4 were evaluated by quantitative reverse transcription-polymerase chain reaction(QRT-PCR),western blot and immunofluorescence staining.The secretion of IL-1βand IL-10 in the corneas was determined by ELISA.Results.Of 37 human corneal buttons with FK obtained by keratoplasty,23(62.2%) showed positive culture identification and in which 17 showed Fusarium infection,5 showed Aspergillus infection,1 showed combined fungi infection.Corneal sections from FK group revealed a large proportion of polymorphonuclear leukocytes(PMNs).TLR2 and TLR4 mRNA was expressed in both healthy and FK corneas.The mRNA expression of TLR2 and TLR4 was downregulated,whereas the expression of IL-1βand IL-10 was upregulated in the FK corneas compared with the healthy corneas.The immunofluorescence staining showed that the expression of TLR2 and TLR4 was slightly stronger in the healthy corneas than that of FK corneas.The western blot also showed that the expression of TLR2 and TLR4 was slightly stronger in the healthy cornea than that of FK corneas.Conclusions.Fusarium species and Aspergillus species were the most commonly isolated pathogens of fungal keratitis.TLR2 and TLR4,as well as Th1/Th2 type cytokines may be implicated in the pathogenesis of fungi infection in the cornea,and may play key roles in fungal keratitis.更多还原