【作者】 郭慧；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2010， 博士

【摘要】 第一部分：TLRs介导的角膜上皮细胞对烟曲霉菌的天然免疫反应目的：研究角膜上皮细胞Toll样受体(TLR)2和TLR4分别对配体刺激的免疫反应及其介导角膜上皮细胞对烟曲霉菌炎症反应的作用。方法：培养永生化人角膜上皮细胞,利用TLR2配体酵母聚糖、TLR4配体LPS及烟曲霉菌菌丝分别刺激角膜上皮细胞,于不同时间点(1h,3h,6h,12h)收取培养上清进行酶联免疫吸附试验(ELISA)检测细胞因子IL-1β和IL-6水平。分别设计靶向人TLR2、TLR4的siRNA序列,构建表达TLR2-或TLR4-siRNA的质粒,分别转染角膜上皮细胞,利用RT-PCR及Western blot检测TLR2、TLR4表达以评价抑制效果。利用烟曲霉菌菌丝并分别刺激转染siRNA的角膜上皮细胞及未转染对照细胞,于不同时间点(1h,3h,6h,12h)利用ELISA检测上清中细胞因子IL-1β和IL-6水平。结果：TLR2配体酵母聚糖、TLR4配体LPS及烟曲霉菌均可刺激永生化人角膜上皮细胞(THCE)分泌炎性细胞因子IL-1β和IL-6。酵母聚糖刺激角膜上皮细胞后6h IL-1β和IL-6水平开始升高,并呈时间依赖性。LPS刺激后12h可见细胞因子显著性升高,但表达水平不如酵母聚糖刺激明显。烟曲霉菌刺激后3h细胞因子水平即明显升高,随刺激时间延长进一步上调。TLR2-或TLR4-siRNA贡粒转染角膜上皮细胞后,TLR2、TLR4 mRNA及蛋白水平表达均受到显著抑制。烟曲霉菌菌丝刺激后对照组IL-1β和IL-6表达明显上调,而TLR2-或TLR4-siRNA处理组IL-1β和IL-6水平均较对照组显著降低。结论：TLR2配体和TLR4配体可诱导角膜上皮细胞的天然免疫反应；角膜上皮细胞通过TLR2和TLR4识别烟曲霉菌并介导炎性细胞因子表达。角膜上皮细胞表达的TLR2和TLR4在角膜抗真菌感染天然免疫中发挥重要作用。第二部分：TLR2介导茄病镰刀菌诱导的人角膜基质细胞抗炎细胞因子表达目的：研究TLR2在茄病镰刀菌诱导的人角膜基质细胞促炎细胞因子和抗炎细胞因子表达中的作用。方法：设计靶向人TLR2 siRNA序列,连接入p-silencer 2.0 U6载体构建能表达TLR2-siRNA的质粒。分别转染永生化人角膜基质细胞,利用免疫荧光、RT-PCR及Western blot检测TLR2的表达。制备茄病镰刀菌菌丝和孢子刺激液并分别刺激转染siRNA的角膜基质细胞及未转染对照细胞,刺激12h后利用实时荧光定量PCR检测细胞因子IL-1β和IL-10的表达。结果：TLR2-siRNA质粒转染人角膜基质细胞后,TLR2 mRNA及蛋白水平表达均明显被抑制,mRNA抑制效率为60%,蛋白抑制效率可达65%左右。对照siRNA转染组、空载体组及空白对照组TLR2表达无显著差异。单纯镰刀菌菌丝和孢子均可刺激人角膜基质细胞IL-10和IL-1β表达明显上调,与对照组有显著差异(P<0.01),其中菌丝刺激组细胞因子表达水平比孢子刺激组高。分别使用茄病镰刀菌菌丝及孢子刺激后,对照组IL-1β及IL-10的表达均明显上调,且菌丝诱导的表达上调比孢子更显著；RNA干扰组,IL-10表达较对照组明显降低,菌丝刺激组IL-10表达下调82%,孢子刺激组下调70%,与未干扰组表达水平比较均具有显著差异(P<O.01)；IL-1β的表达亦有降低,菌丝刺激组下调60%,孢子刺激组约下调54%,具有统计学差异,但不如IL-10下调显著。结论：TLR2是人角膜基质细胞识别茄病镰刀菌的重要识别受体,它可能通过介导IL-10的表达发挥一种抗炎性作用。这将为探索角膜真菌感染发病机理和免疫防御反应机制提供新的思路。第三部分：RNA干扰大鼠角膜TLR2表达对角膜真菌感染的影响目的：研究RNA干扰大鼠角膜TLR2对角膜烟曲霉菌感染的影响。方法：分别合成对照siRNA或TLR2siRNA,与转染试剂混合制备siRNA悬液,于感染前1天给予结膜下注射联合表面点眼。利用角膜接触镜法建立大鼠烟曲霉菌性角膜炎模型,进行裂隙灯检查、临床评分及组织病理学分析评价感染情况。利用免疫荧光技术检测大鼠角膜TLR2表达以评价TLR2 siRNA转染的效率。利用实时定量PCR技术检测炎性细胞因子TNFa, IL-1β, IL-4, IL-6, IL12p40和趋化因子MCP-1和MIP-2表达水平。利用髓过氧化物酶(MPO)法检测多型核白细胞(PMN)浸润。结果：TLR2 siRNA转染后,大鼠角膜TLR2表达明显减少,呈区域性、节段性分布。对照siRNA转染组角膜TLR2呈正常表达。对照组大鼠角膜给予烟曲霉菌接种后,角膜炎呈进行性发展,第1天可见角膜上皮呈毛玻璃样水肿,第3天可见明显的黄白色溃疡,边界不清,角膜缘充血明显；病变发展迅速,第4-5天为感染高峰期,溃疡面进一步扩大,表面凹凸不平,角膜中央区坏死组织脱落呈典型的龛状溃疡,可见后弹力层膨出、角膜葡萄肿甚至角膜穿孔。转染组感染后第1天角膜见轻度炎性浸润,第3天角膜浸润较前加重,但未见明显溃疡,第5天浸润开始减轻,面积逐渐局限、缩小,于2周左右完全愈合,仅存小片云翳及新生血管。对照组大鼠角膜炎性细胞因子TNFa, IL-1β, IL-4, IL-6, IL12p40及趋化因子MCP-1、MIP-2表达水平在第1天开始上调,于3-5天达到高峰,第7天开始下降并于2周后恢复至低水平。相比之下,TLR2 siRNA转染组炎性细胞因子TNFa, IL-1β, IL-6, IL12p40及趋化因子MCP-1、MIP-2水平明显降低,IL-4水平未见显著下调。TLR2 siRNA转染组PMN浸润较对照组明显减轻。结论：siRNA可通过结膜下注射联合点眼的方式成功转入角膜组织。TLR2是角膜识别烟曲霉菌感染的主要受体,在诱导炎性细胞因子产生和炎性浸润发挥关键作用。TLR2siRNA可通过下调炎性细胞因子及炎性细胞浸润达到抑制角膜过度炎症反应和减少角膜损伤的目的,为探索利用TLR2基因沉寂治疗角膜真菌感染的新方法提供了理论依据。更多还原

【Abstract】 PartⅠ:TLRs-mediated innate responses of corneal epithelial cells against Aspergillus fumigatus challengePurpose. To determine the responses of Toll-like receptor (TLR) 2 and TLR4 to their ligands in corneal epithelial cells and their roles in mediating inflammatory responses against Aspergillus fumigatus challenge.Methods. Telomerase-immortalized human corneal epithelial cells (THCE) were challenged by TLR2 ligand zymosan, TLR4 ligand LPS and Aspergillus fumigatus hyphae, respectively. Culture media collected at different time points (1h,3h,6h,12h) was subjected to cytokine ELISA to detect the levels of IL-1βand IL-6. THCEs treated with TLR2- or TLR4-siRNA plasmid to knock down TLR2 and TLR4 expression were challenged with Aspergillus fumigatus hyphae. Culture media were collected at different time points (1h,3h,6h,12h) for ELISA to detect IL-1βand IL-6 levels.Results. THCEs responded to the challenge of TLR2 or TLR4 ligand by expressing and secreting inflammatory cytokines IL-1βand IL-6. Zymosan-induced cytokines level began to increase at 6h in a time-dependent manner. LPS caused significant increase of IL-1βand IL-6 at 12h after challenge but exhibit lower levels than that of zymosan. And exposure of THCEs to Aspergillus fumigatus hyphae resulted in the upregulation of IL-1βand IL-6 since 3h after stimulation which continued to increase with prolonged incubation. Knockdown of TLR2 or TLR4 expression by specific siRNA caused a significant decrease in Aspergillus fumigatus-induced production of IL-1βand IL-6.Conclusions. THCEs can respond to TLR2 and TLR4 ligands challenge by secreting IL-1βand IL-6. They recognize Aspergillus fumigatus hyphae via TLR2 and TLR4 receptors and initiate innate immune responses against Aspergillus fumigatus challenge. Corneal epithelial cells play a role in innate defense against fungal infection through producing inflammatory cytokines.Part II:Toll-like receptor 2 mediates anti-inflammatory cytokine expression in human corneal fibroblasts in response to Fusarium soluPurpose To determine the role of Toll-like receptor (TLR) 2 in the expression of proinflammatory and anti-inflammatory cytokines in corneal fibroblasts challenged by fungi.Methods The sequence of siRNA targeting TLR2 was designed and cloned into the p-silencer 2.0 U6 vector to be a combined expression vector that expresses TLR2-siRNA. The cultured telomerase-immortalized human stroma fibroblasts (THSF) were transfected with the plasmid containing TLR2-siRNA and the expression of TLR2 was assessed by immunocytochemistry, RT-PCR, and Western blotting analyses. The transfected cells were stimulated by hyphae or conidia of Fusarium solu, respectively, and mRNA levels of IL-1βand IL-10 were measured by real time RT-PCR.Results THSF transfected with TLR2-siRNA exhibited a reduced level of TLR2 when compared with the control cells transfected with empty plasmid. TLR2-siRNA exhibited a more dramatic reduction in mRNA level of about 60% and TLR2 protein was decreased over controls approximately 65%. There is no difference between the group of control siRNA, empty vector and blank controls. The IL-lbeta and IL-10 levels increased dramatically in blank controls after stimulation of Fusarium solu and the levels induced by hyphae were much higher than by conidia. In contrast, THSF treated with TLR2-siRNA compared with control exhibited reduced levels of IL-1beta and IL-10 after fungi stimulation. After RNA interference, IL-10 level significantly decreased over controls approximately 82% in hyphae stimulation (P＜0.01) and 70% in conidia stimulation(P＜0.01). IL-lbeta level showed a reduction of 60%(P＜0.01) in hyphae stimulation and 54% (P＜0.01) in conidia stimulation, respectively. Fusarium solu-stimulated IL-10 production by THSF transfected with TLR2-siRNA was severely impaired while IL-iβproduction was partially inhibited.Conclusions TLR2 appears to be a major pattern recognition receptor to detect Fusarium solu in vitro and may play the anti-inflammatory role through the induction of IL-10. This may lead to a better understanding of the pathogenesis of fungal infections in cornea and may help in designing more efficient strategies in inhibiting the fungi-triggered inflammatory reaction.PartⅢ:Effect of RNA interfering of Toll-like receptor (TLR) 2 in rat cornea on Aspergillus fumigatus keratitisPurpose To investigate the effect of RNA interfering of Toll-like receptor (TLR) 2 in rat cornea on Aspergillus fumigatus keratitis.Methods The rat corneas were wounded and Aspergillus fumigatus strain was applied to the cornea surface. Slit lamp, clinical scoring and histopathological analysis were performed to monitor disease. Control or TLR2 siRNA was injected subconjunctively before infection and applied topically to cornea. TLR2 expression was measured by immunofluorescence staining to determine the feasibility and efficiency of TLR2 siRNA delivery. Production of inflammatory cytokines such as TNFa, IL-1β,IL-4, IL-6, IL12p40 and chemokines such as MCP-1 and MIP-2 were determined by real time quantitative PCR. PMN infiltration was assessed by myeloperoxidase (MPO) activity assay.Results Rat corneas treated with TLR2 siRNA showed a significant reduction of TLR2 expression which distributed in clusters through the corneal epithelium. Keratitis occurred and developed progressively after inoculation of Aspergillus fumigatus. Corneal ulcers were evident at day 3 p.i. with rough surface and obscure boundary. The ulceration expanded deeper and larger at day 4-5 p.i. accompanied by the destruction and degradation of cornea tissue. Descementocele, corneal staphyloma and perforation were observed in several cases. In contrast, TLR2 siRNA-treated corneas displayed mild infiltration at 1-3 days p.i. and only slight corneal opacity at day 5 p.i. with preserved corneal integrity. Compared to the control siRNA-treated corneas, mRNA levels of inflammatory cytokines TNFa, IL-1β, IL-6, IL12p40 and chemokines MCP-1, MIP-2 in the corneas of TLR2 siRNA-treated rats was dramatically reduced whereas IL-4 level was unchanged. While there was no measurable MPO activity in non-infected corneas (data not shown), MPO activity was detected in Aspergillus fumigatus-infected corneas at day 1 p.i. and upregulated at day 3 and 5 p.i. in the control corneas. This increase in PMN infiltration, however, was dramatically attenuated by TLR2 siRNA treatment, suggesting an enhanced resolution of inflammation.Conclusions TLR2 siRNA treatment reduces inflammation of Aspergillus fumigatus keratitis by the downregulation of inflammatory cytokines and cell infiltration, which may suggest a novel avenue to control infection and reduce damage caused by excessive inflammation.更多还原