【作者】 潘虹；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2012， 博士

【摘要】 研究背景棘阿米巴角膜炎(Acanthamoeba keratitis, AK)是棘阿米巴原虫感染角膜引起的一种严重的致盲性眼病。近年来,随着角膜接触镜的广泛使用,其发病率迅速升高。AK的发病与角膜创伤、接触污水源、配戴角膜接触镜明显相关,其中配戴角膜接触镜是其最主要的危险因素。研究报道,每百万个角膜接触镜配戴者中,AK的罹患者美国为1.36人,荷兰为3.06人,英国为17.53-21.14人；在我国报道的第一例AK是由角膜接触镜引起,并且与接触镜相关性AK,约占AK患者的30.8%。由于棘阿米巴包囊具有很强的环境适应性、抗药性并能长期缓慢在角膜生长,导致该病药物治疗效果差、术后易复发,使AK成为临床上治疗非常棘手、具有较高致盲率的角膜感染。因此,认识角膜感染发病机制和揭示宿主抗棘阿米巴感染的免疫防御机制具有至关重要的意义。角膜上皮细胞是角膜抵抗病原体感染的第一道屏障,也是角膜天然免疫防御系统的主要组成细胞,有赖于其模式识别受体(pattern recognition receptors, PRRs)对特定病原体的快速识别,通过产生丰富的炎性因子、炎性介质及抗菌肽等激活防御系统,清除病原。Toll样受体(Toll like receptors,TLRs)是一种重要的PRR,它通过识别许多病原微生物共有的特殊结构-病原相关分子模式(pathogen associated molecular patterns, PAMPs)激活细胞内的信号级联反应,诱导炎性细胞因子的生成和释放,趋化炎性细胞浸润,启动炎症和免疫反应,从而在防御细菌、真菌、病毒等病原体入侵中发挥重要作用。Toll样受体4(TLR4)是人们最早发现的TLR,其主要表达于免疫细胞如淋巴细胞、单核巨噬细胞等,也可以表达宿主-环境接触处的上皮细胞如呼吸道上皮细胞和肠上皮细胞等。TLR4识别相应配体后,可通过MyD88依赖途径或MyD88非依赖途径,激活下游信号通路,最后活化核转录因子(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB)、干扰素调控因子-3(interferon-regulated factor-3, IRF-3)等转录因子,诱导宿主细胞表达炎性细胞因子,诱导炎症反应和启动先天免疫。我们的前期研究发现,在人角膜上皮细胞中TLR4为阳性表达；应用棘阿米巴原虫刺激人角膜细胞检测TLR1-10表达的变化,发现TLR4显著升高,说明TLR4可能参与棘阿米巴角膜炎的炎症反应。众所周知,氧是人体新陈代谢和生命活动的关键物质,同时组织氧分压的变化也是重要的环境信号,参与机体的许多生理和病理过程如炎症、感染、缺氧等。角膜代谢所需要的氧80%来自外界空气,接触镜附着于角膜表面,使角膜处于低氧环境,从而影响角膜上皮细胞的生物特性,如上皮细胞水肿、凋亡、坏死、脱落等,破坏上皮屏障功能,降低上皮细胞的天然防御功能。研究发现低氧可促进绿脓杆菌对角膜上皮细胞的粘附、减少上皮细胞增殖、加速细胞凋亡,增加角膜上皮细胞对绿脓杆菌的易感性。近年研究发现低氧能够调控TLR4基因的表达,但因细胞类型和低氧培养条件的不同,低氧对TLR4的调控作用有所差异,然而低氧对人角膜上皮细胞TLR4的调控研究,目前尚未见相关报道。既然TLR4参与棘阿米巴角膜炎的炎症过程,因此有必要对低氧是否调控人角膜上皮细胞TLR4及其信号通路、低氧是否通过调控TLR4而影响角膜上皮细胞对棘阿米巴的易感性进行深入研究,这将为有效地预防角膜棘阿米巴感染提供新的思路。本研究分为两个部分：一、应用棘阿米巴刺激人角膜上皮细胞,检测TLR4基因与炎性因子白介素6(interleukin-6, IL-6)和IL-8分泌的变化,并应用RNA干扰(RNA interference, RNAi)技术沉默TLR4基因的表达,检测抑制TLR4后对炎性因子表达的影响,探讨TLR4在人角膜上皮细胞对棘阿米巴刺激的天然免疫反应中的作用。二、通过检测低氧对棘阿米巴刺激角膜细胞诱导的TLR4基因,下游信号通路蛋白髓样分化因子88(myeloid differentiation primary response gene88, MyD88)、NF-κB和细胞外调节蛋白激酶1/2(extracellular signal regulated protein kinase1/2, ERK1/2),以及炎性因子IL-8和干扰素-β(interferon-β, IFN-β)表达的影响,探讨低氧是否通过调控人角膜上皮细胞TLR4及其信号通路,从而影响角膜棘阿米巴感染的炎症反应,分析低氧对棘阿米巴角膜炎易感性的影响,揭示角膜接触镜配戴易患棘阿米巴角膜炎的原因,这将为棘阿米巴角膜炎的合理预防和治疗策略提供理论依据。第一部分Toll样受体4介导人角膜上皮细胞对棘阿米巴的天然免疫反应目的：研究Toll样受体4在人角膜上皮细胞对棘阿米巴炎性反应中的作用。方法：1.培养永生化的人角膜上皮细胞(telomerase-immortalized human epithelial cells, THCEs)和棘阿米巴虫株。先用不同浓度(1×103/ml、1×104/ml、1×105/ml、1×106/ml)的棘阿米巴刺激THCEs30min,孵育12h后收集细胞,Real Time-PCR检测TLR4mRNA的表达,Western blot检测TLR4蛋白表达。用1×106/ml浓度棘阿米巴刺激THCEs,于刺激的不同时间点(1h、3h、6h、12h)收集的细胞分别采用实时荧光定量聚合酶链反应(real-time quantitative polymerase chain reaction, Real-time PCR)和Western blot检测TLR4mRNA和蛋白表达；收集的培养上清采用酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测炎性细胞因子IL-6和IL-8水平。2.设计、合成特异性针对TLR4基因的小干扰RNA (small interference RNA, siRNA)序列：正义链为5’-GGCAAUUCUUUCCAGGAAATT-3’;反义链为5’-UUUCCUGGAAAGAAUUGCCAG-3’。阴性对照选择与人类基因没有任何同源的干扰序列,正义链为5’-UUCUCCGAACGUGUCACGUTT-3’;反义链为5’-ACGUGACACGUUCGGAGAATT-3’。采用脂质体(Lipofectamine)2000介导TLR4-siRNA及阴性对照siRNA(NC-siRNA)转染THCEs48h后,利用Real-time PCR和Western blot检测TLR4的：mRNA和蛋白表达以评价抑制效果。利用棘阿米巴(1×106/m1)分别刺激转染TLR4-siRNA及NC-siRNA的THCEs30min,孵育12h后利用ELISA检测上清中细胞因子IL-6和IL-8的表达。结果：1.随着棘阿米巴刺激浓度的增加,人角膜上皮细胞TLR4的mRNA和蛋白表达逐渐升高；在1×106/ml的棘阿米巴刺激浓度下,人角膜上皮细胞TLR4mRNA和蛋白表达在刺激后6h开始升高,并以时间依赖方式呈现上调。棘阿米巴刺激人角膜上皮细胞6h后,IL-6和IL-8的分泌明显升高,随着刺激时间的延长进一步上调。2. TLR4-siRNA转染人角膜上皮细胞后,TLR4mRNA及蛋白水平表达均受到显著抑制。棘阿米巴刺激后TLR4-siRNA处理组IL-6和IL-8的分泌水平较NC-siRNA组显著降低。结论：TLR4是人角膜上皮细胞识别棘阿米巴的重要受体并介导炎性细胞因子IL-6和IL-8的的表达,TLR4-siRNA可通过下调炎性因子的分泌达到抑制棘阿米巴角膜炎炎症反应的目的,为探索治疗角膜棘阿米巴感染的新方法提供了理论依据。第二部分低氧抑制Toll样受体4介导的人角膜上皮细胞对棘阿米巴的天然免疫反应目的：研究低氧对TLR4介导的人角膜上皮细胞对棘阿米巴炎性反应的调控作用。方法：1.培养永生化人角膜上皮细胞(THCEs)和棘阿米巴虫株。THCEs分别置于常氧(21%02)和低氧(1%02)条件下37℃培养24h,设置空白对照组和棘阿米巴刺激组(1×106/m1)。采用Annexin V-PE与7-AAD双染法检测细胞的生存活性。2.在常氧条件下,用棘阿米巴原虫(1×106/m1)刺激已预先在低氧孵箱培养24h的THCEs30min后,再放入低氧孵箱孵育6h。收取的细胞利用Real-time PCR检测TLR4\MyD88\IL-8和IFN-p mRNA的表达；利用Western blot检测TLR4、NF-κB抑制蛋白α (IκBα)、磷酸化NF-κB抑制蛋白(p-IκBα)、磷酸化细胞外调节蛋白激酶1/2(p-ERK1/2)的蛋白表达。收取的上清,采用酶联免疫吸附试验(ELISA)检测炎性细胞因子IL-8和IFN-p的分泌。3.在低氧条件下孵育THCEs,于不同的时间点(0h、6h、12h、24h、48h)收集细胞,分别采用Real-time PCR和Western blot检测TLR4mRNA和蛋白表达。4.将THCEs在低氧环境中孵育24h后,用TLR4的公认配体脂多糖(lipopolysaccharide, LPS)(1μg/ml)刺激6h。收取的细胞利用Real-time PCR检测MyD88、IL-6、和IL-8mRNA的表达；利用Western blot检测IκBα和p-IκBα的蛋白表达。结果：1.流式细胞技术分析细胞生存活性,显示低氧和棘阿米巴刺激对THCEs的生存活性均无显著影响。2. Real-time PCR结果显示,低氧可以下调棘阿米巴刺激THCEs引起的TLR4和通路分子MyD88的mRNA表达；Western blot结果显示,低氧可以降低棘阿米巴刺激THCEs引起的TLR4、p-IκBα和p-ERK1/2的蛋白表达,明显升高IκBα的表达；ELISA结果显示,低氧可以抑制棘阿米巴诱导的炎性细胞因子IL-8和IFN-β的分泌。3. Real-time PCR和Western blot的结果显示,低氧分别孵育THCEs24h和48h小时后,TLR4的mRNA和蛋白水平均明显减低。4.LPS刺激试验结果显示,低氧可以降低MyD88mRNA的表达,抑制IκBα的磷酸化,下调炎性细胞因子IL-6和IL-8的分泌。结论：低氧通过抑制TLR4的表达及其信号通路的活化,从而抑制人角膜上皮细胞对棘阿米巴刺激引起的炎性反应。低氧可能通过减弱人角膜上皮细胞抵抗棘阿米巴入侵的能力,从而增加细胞的易感性。这些发现可能解释了配戴角膜接触镜是棘阿米巴角膜炎最主要的危险因素的原因。更多还原

【Abstract】 BackgroundAcanthamoeba keratitis (AK) is a vision-threatening disease caused by a free-living, pathogenic Acanthamoeba. In the past few decades, with the wide-spread use of contact lenses, the incidence of AK has steadily increased. The United States Centers for Disease Control and Prevention found that several important risk factors are associated with AK, such as corneal injuries and contact with contaminated water, of which contact lens wear is the predominant risk factor. A study revealed that the incidence of AK is1.36per million contact lens weares in the United States,3.06in the Netherlands and17.53-21.14in England. In China, it has been reported the first case of AK was caused by contact lens wear in1992. Then another report showed that about30.8%of AK patients were associated with contact lenses. Acanthamoeba cysts have strong environmental adaptability resistance and long-term slow growth in the cornea, leading to poor results of drug treatment of the disease, postoperative recurrence. AK has become a corneal infection which is difficult to treat and with high rate of blind. Therefore, a better understanding of the pathogenesis and mechanisms responsible for the host defense against Acanthamoeba infection is of crucial importance.Corneal epithelia cells serve as the first-line defensive barrier of cornea and participate in the innate immune response to invasive pathogens through its pattern recognition receptors (PRRs). This rapid recognition triggers the production of inflammatory cytokines, chemokines, and the antimicrobial peptide, ultimately resulting in the clearance of invading pathogens. Toll-like receptors (TLRs) are important PRR, which recognize distinct pathogen-associated molecular patterns (PAMPs) that are shared by many pathogenic microorganisms. Recognition of these patterns by TLRs initiates cell signal cascade, induces the synthesis and secretion of inflammatory cytokines, triggers neutrophils infiltration, activates the inflammatory and immune responses, which plays an important role in the defense of bacteria, fungi, viruses and other pathogens invasion.Toll-like receptor4(TLR4) is the first discovered TLR, which mainly express on immune cells, such as lymphocytes, monocytes and macrophages, and also express on the epithelial cells at the host-environmental surface, such as airway epithelial and intestinal epithelial cells. On ligand binding, TLR4activates different downstream signaling through myeloid differentiation primary response gene88(MyD88)-dependent or MyD88-independent pathways, which leads to the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factor-3(IRF-3), induces the secretion of inflammatory cytokines and thus triggers the inflammation and innate immunity. In our previous study, we showed that human corneal epithelial cells expressed TLR4. When exposed to Acanthamoeba challenge, among TLR1-10, the expression of TLR4was significantly increased, indicating that TLR4may be involved in the inflammatory response of AK.As we know, oxygen is the key substance of metabolism and life activities, and the changes of oxygen pressure in tissue are also important environment signals involved in many physiological and pathological processes, such as inflammation, infection, hypoxia and so on. About80%of the oxygen which is needed to corneal metabolism is from air. Attached by contact lenses, the cornea is subjected to a hypoxic condition, thus affecting the biological properties of the corneal epithelial cells, such as edema, apoptosis, necrosis, shedding and so on, destructing the epithelial barrier function and reducing the natural defensive capacity. A study revealed that hypoxia can promote the adhesion of Pseudomonas aerugionsa on corneal epithelial cells, reduce the proliferation of epithelial cells, accelerate cells apoptosis and increase the susceptibility of corneal epithelial cells against P. aeruginosa. A study comparing high-vs low-Dk contact lenses showed increased incidence of P. aeruginosa keratitis (0:30%). It is suggested that incidence of the most severe adverse events is actually increased with low-Dk lenses when compared with high-Dk lenses. Recent reports showed that hypoxia can regulate the expression of TLR4. It was found that hypoxia also diminished TLR4expression in human umbilical vein endothelial cells for48-72h exposure. While, it has been demonstrated that hypoxia increased TLR4expression in microglia cells after8h exposure. These studies indicate that different cell types and hypoxic exposure durations lead to different cellular responses of TLR4expression. There is no report about the effect of hypoxia on human corneal epithelial cells. Since TLR4is involved in the inflammatory process of AK, it is necessary to investigate the effect of hypoxia on TLR4and its signaling and whether hypoxia affect the susceptibility of corneal epithelial cells to Acanthamoeba through regulating TLR4, which may help in designing more efficient strategies in prevention of AK.The research is divided into two parts:the one is through the use of RNA interference (RNAi) to silence the TLR4gene expression to investigate the effects of TLR4inhibition on inflammatory responses of human corneal epithelial cells against Acanthamoeba. The other is to determine whether hypoxia affects the corneal susceptibility to Acanthamoeba through regulating TLR4and its signaling, and to explain why contact lens use is one of the prominent risk factors for AK. This may help to develop strategies aimed at establishing rational prevention and useful treatment.Part I:Toll-Like Receptor4Mediates Innate Responses of Human Corneal Epithelial Cells against AcanthamoebaPurpose:To further determine the potential role of Toll-like receptor4(TLR4) in the inflammatory responses of human corneal epithelial cells against Acanthamoeba.Methods:1. Telomerase-immortalized human corneal epithelial cells (THCEs) were challenged by different concentrations (1×103/ml,1×104/ml,1×105/ml,1×106/ml) of Acanthamoeba with30min and after12h incubation, cells were collected. Real-time PCR was used to assess the mRNA level of TLR4, and Western blot was used to examine the protein level of TLR4. Then THCEs were stimulated with Acanthamoeba at the concentration of1×106/ml. Cells and culture media collected at different time points (1h,3h,6h,12h) were subjected to Real-time PCR, Western blot and enzyme-linked immunosorbent assay (ELISA) to detect the expression of TLR4, interleukin-6(IL-6) and interleukin-8(IL-8).2. The sequence of siRNA targeting TLR4was designed and synthesized. The sequences used for targeting TLR4are5’-GGCAAUUCUUUCCAGGAAATT-3’(for sense) and5’-UUUCCUGGAAAGAAUUGCCAG-3’(for antisense). The sequences of negative control siRNA (NC-siRNA) are5’-UUCUCCGAACGUGUCACGUTT-3’(for sense) and5’-ACGUGACACGUUCGGAGAATT-3’(for antisense). THCEs were transfected with siRNA using LipofectamineTM2000as instructed by the manufacturer. Forty-eight hours after transfection, the expression of TLR4was assessed by Real-time PCR and Western blot. After the transfected cells were challenged with Acanthamoeba, the production of IL-6and IL-8were measured by ELISA.Results:1. We observed that the mRNA and protein levels of TLR4were significantly upregulated after stimulation and this regulation effect was further enhanced with increasing severity of Acanthamoeba exposure. After Acanthamoeba challenge at the concentration of1×106/ml, the mRNA and protein levels of TLR4were upregulated in a time-dependent manner, with a significantly increase from6h after stimulation. And exposure of THCEs to Acanthamoeba resulted in the upregualtion of IL-6and IL-8from6h after stimulation, which continued to increase with prolonged incubation. 2. We found that TLR4mRNA and protein levels were evidently reduced after TLR4siRNA treatment in THCEs, compared with NC-siRNA. The secretion of IL-6and IL-8was obviously increased in response to Acanthamoeba challenge, while the secretion was reduced after pretreatment with TLR4-siRNA compared with NC-siRNA.Conclusions:Our results indicated that Acanthamoeba-induced inflammatory response was mediated by TLR4in human corneal epithelial cells. TLR4siRNA treatment reduced inflammatory response of AK by the downregulation of inflammatory cytokines, which may provide a novel gene therapeutic strategy for the treatment of AK. Part Ⅱ:Hypoxia Inhibits Acanthamoeba-Induced Inflammatory Responses of Human Corneal Epithelial Cells via Toll-Like Receptor4SignalingPurpose:To investigate the role of hypoxia in the expression of toll-like receptor4signaling and in the regulation of inflammatory response of human corneal epithelial cells against Acanthamoeba.Methods:1. THCEs challenged with Acanthamoeba (1×106/ml) were incubated at37℃for24h under normoxic (21%O2) or hypoxic (1%O2) conditions. Cell viability was analyzed by flurescence activating cell sorter (FACS) using the Annexin V-PE/7-AAD staining.2. After exposure to normoxia or hypoxia for24h, THCEs were challenged with Acanthamoeba resuspension for30min under normoxic condition followed by incubation with culture medium for an additional6h under normoxia or hypoxia, depending on the exposure condition. Real-time PCR was used to assess the mRNA of TLR4, MyD88, IL-8and IFN-p. Western blot was used to examine the protein level of TLR4, p-IκBα, p-ERK1/2and IκBα ELISA was used to detect the secretion of the inflammatory cytokines IL-8and IFN-(3. 3. THCEs were incubated under normoxia and hypoxia for6h,12h,24h and48h. The mRNA and protein levels of TLR4were measured by Real-time PCR and Western blot analysis.4. After exposure to hypoxia for24h, THCEs were challenged with TLR4ligand LPS (1μg/ml) for6h. Real-time PCR was used to assess the mRNA level of MyD88, IL-6and IL-8. Western blot was used to examine the protein level of p-IμBα and IκBα. ELISA was used to detect the secretion of the inflammatory cytokines IL-6and IL-8.Results:1. The data showed that there was no significant change in the percentage of cells undergoing apoptosis and necrosis when the cells were exposed to hypoxia, Acanthamoeba challenge or both compared with the untreated control, indicating that the viability of THCEs was maintained under hypoxia and Acanthamoeba challenge.2. Real-time PCR results showed that the mRNA levels of Acanthamoeba-induced TLR4, MyD88, IL-8and IFN-β in THCEs under hypoxia was remarkably decreased. Western blot analysis results demonstrated that the protein level of TLR4, p-IκBa and p-ERK1/2were significantly diminished, while the IκBα protein expression was increased. The secretions of IL-8and IFN-β under hypoxia were also reduced.3. The results of Real-time PCR and Western blot analysis showed that the expression of TLR4significantly decreased at24h and48h after hypoxic exposure, respectively. Moreover, the decrease of TLR4mRNA level occurred in a time-dependent manner.4. Hypoxia also inhibited LPS-induced IL-6and IL-8secretion, MyD88expression and NF-κB activation.Conclusions:Our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4signaling, indicating that hypoxia might impair the host cell’s ability to eliminate the Acanthamoeba invasion and could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.更多还原