【作者】 钟文贤；

【导师】 谢立信；

【作者基本信息】 浙江大学 ， 眼科学， 2007， 博士

【摘要】 目的探索建立一种理想的模拟临床人角膜真菌感染的小鼠真菌性角膜炎模型并对其角膜组织损伤的临床表现和组织病理学改变进行全面观察；同时在此基础上利用分子生物学技术，对真菌性角膜炎炎症发生过程中作为核心环节的主要炎性因子的表达、作用及调控进行研究，以促进对真菌性角膜炎发病机制的研究和发展更为有效的真菌性角膜炎治疗手段。方法1、动物模型创建选取茄病镰刀菌、烟曲霉菌和白色念珠菌等三种主要角膜致病真菌标准菌株以及健康6～8周龄近交系BALB／c小鼠，采用“角膜表面镜片术辅助上皮刮除法”建立角膜真菌感染模型，接种菌液浓度为1×10~6CFU／ml，感染术后缝合小鼠睑裂，24 h后打开眼睑，每天于裂隙灯显微镜下观察角膜感染情况，数码相机照相记录，分别于感染后1、3、5、7和12 d采用半定量方法对感染角膜予以临床评分，并在感染后不同时间点取小鼠实验眼角膜刮片物进行病原学检测。2、组织病理学检测分别于感染后1、3、5、7和12 d摘取各实验组小鼠眼球，均置于10％福尔马林快速固定液中固定24 h，常规制作石蜡切片，并分别行苏木素-伊红(HE)染色和过碘酸Schiff(PAS)染色，光学显微镜下进行分析。观察项目包括炎症反应、真菌负荷和真菌菌丝的生长方式，其中对炎症反应和真菌负荷程度采用半定量方法分为轻、中、重三级并予以评分。对菌丝在角膜中的生长方式，以真菌菌丝走行方向与角膜板层成角大小作为判断标准，成角大于45度者被判定为垂直生长，小于45度者为水平生长。3、主要炎性因子表达的研究分别于感染后1、3、5、7和12 d取各感染组小鼠实验眼病变角膜，采用RT-PCR和ELISA技术对不同菌种感染角膜组织中的巨噬细胞炎症蛋白2(MIP-2)、细胞因子诱生性中性粒细胞趋化物(KC)、白介素1β(IL-1β)和白介素6(IL-6)等4种炎性因子的mRNA和蛋白质表达水平进行检测，RT-PCR结果采用Image J计算机图像分析软件进行半定量分析。4、炎性因子特异性多克隆抗体干预实验选取健康6～8周龄近交系BALB／c小鼠，分为3大组，每大组再分为3个小组，分别接种三种主要角膜致病真菌。第1大组为感染对照组，于术前1 h和术后24 h对小鼠实验眼分别给予20μl灭菌生理盐水结膜下注射，第2大组给予MIP-2多克隆抗体20μl(2ng／μl)、第3大组则给予IL-1β多克隆抗体20μl(2ng／μl)结膜下注射，采用“角膜表面镜片术辅助上皮刮除法”建立小鼠角膜真菌感染模型，接种菌液浓度为1×10~6CFU／ml。感染24 h拆除眼睑缝线后每天用裂隙灯显微镜观察动物角膜感染情况并予以临床评分，并分别于感染后1、3、5、7和12 d摘取各组小鼠眼球制成常规石蜡切片，HE和PAS染色观察病变组织炎症反应、真菌负荷和真菌菌丝的生长方式。此外，分别于感染后1、3、5、7和12 d摘取第2和第3大组各小组小鼠实验眼角膜，采用ELISA法对其MIP-2和KC的蛋白质表达水平进行检测。结果1、感染小鼠病变特征病原学检测所有小鼠均成功感染所接种真菌，三种真菌的感染病变发展过程具有一定差异，但高峰期均为感染后3～7 d，以5 d时最为严重和典型，7 d后病变逐步减轻。烟曲霉菌和白色念珠菌性病变持续时间约为10 d，茄病镰刀菌性约12 d。烟曲霉菌性病变最重，绝大多数于3～5 d时出现明显的角膜中央溃疡；白色念珠菌性病变较重，高峰期病变区糜烂呈豆腐渣样；茄病镰刀菌组则病变较平稳，大多出现典型的苔被状外观，但病程持续较长时间。各实验组在1、3、5和7 d时病变临床评分彼此差异有极显著统计学意义(P=0.000)。2、组织病理学改变HE染色观察角膜炎症反应发现在感染早期，角膜中炎症细胞浸润较少，随着病程进展，炎症细胞逐渐增多，角膜组织破坏加重，以第5 d时为著，而7 d时炎症细胞开始明显减少，角膜上皮逐渐修复。三组病变中以烟曲霉菌组炎症反应最重，白色念珠菌组次之，茄病镰刀菌组最轻。PAS染色各菌种感染后第1 d和第3 d标本均查见真菌菌丝，5 d时除白色念珠菌有2例标本查见菌丝外，其余标本均未查见菌丝，病变特点倾向于早期菌丝多，而随着病程进展菌丝逐渐减少。病原菌的负荷方面，白色念珠菌性病变真菌菌丝数量最多，密度最高。对炎症反应和真菌负荷程度分级的统计学分析表明，炎症反应程度与真菌负荷量变化呈负相关关系(r=-0.806，P=0.029)，而不同菌种导致的角膜炎症反应与其病变大体表现呈正相关关系(r=0.961，P=0.000)。在角膜中的生长方式上，茄病镰刀菌菌丝主要以水平生长为主，而烟曲霉菌和白色念珠菌主要为垂直生长。3、主要炎性因子的表达三种菌种感染的小鼠角膜病变组织以及损伤对照和正常小鼠角膜各时间点标本均有MIP-2、KC、IL-1β和IL-6的mRNA和蛋白质的表达，但不同实验组之间和相同实验组不同时间点之间各因子的表达量和表达高峰时间点有差异。MIP-2、KC和IL-1β的表达变化规律保持一致，三者表达量在各实验组中的排序为：烟曲霉菌组＞白色念珠菌组＞茄病镰刀菌组＞损伤对照组＞正常对照；而IL-6的排序为：茄病镰刀菌组＞白色念珠菌组＞烟曲霉菌组＞损伤对照组＞正常对照。MIP-2、KC和IL-1β的表达高峰时间在茄病镰刀菌组中为感染后5 d，烟曲霉菌组为感染后1 d，白色念珠菌组为感染后3 d。IL-6的两个较高水平表达时间点在茄病镰刀菌和烟曲霉菌组中均为感染后1 d和7 d，而在白色念珠菌组中为感染后1 d和5 d。4、MIP-2和IL-1β活性中和实验MIP-2多克隆抗体干预后，各菌种感染角膜病变临床评分较感染对照组均有不同程度降低，其中感染后各时间点的烟曲霉菌性病变显著减轻(P＜0.05)；白色念珠菌性病变早期(1～3 d)减轻明显(P＜0.05)，中后期(5～12 d)仅略减轻(P＞0.05)；茄病镰刀菌性病变早期轻度减轻，中、后期减轻不明显。IL-1β多克隆抗体干预后，三组小鼠角膜病变在早期有明显减轻(P＜0.05)，中后期病变减轻不明显。两种中和抗体应用后，各菌种感染角膜炎症反应程度与相应时间点临床病变变化一致，但真菌负荷和菌丝生长方式与感染对照组无差异。IL-1β多克隆抗体干预病变早期标本的MIP-2和KC的蛋白表达水平明显下降(P＜0.05)，中后期的表达则无明显差异(P＞0.05)。结论1、利用“角膜表面镜片术辅助上皮刮除法”可成功建立稳定、可靠的模拟临床人角膜病变的小鼠真菌性角膜炎模型，为进一步研究真菌性角膜炎的发病机制等奠定了坚实的基础。2、真菌性角膜炎病变过程可区分为早、中、晚期三个阶段，分别表现为病变的浸润扩展、化脓坏死和瘢痕愈复等主要特征，但不同菌种导致的病变严重程度各有差异，炎症反应是导致真菌性角膜炎病变损害的主要因素，这为真菌性角膜炎的基础和临床研究指出了一个新的方向。3、不同角膜致病真菌菌种在角膜中表现出有不同的生长方式，这一现象对临床上真菌性角膜炎患者采取手术治疗时手术方式和时机的选择具有一定的参考价值。4、小鼠真菌性角膜炎角膜病变组织中均有MIP-2、KC、IL-1B和IL-6等趋化因子和细胞因子的mRNA和蛋白质的表达。其中MIP-2是小鼠真菌性角膜炎发病中关键的趋化因子，IL-1β则是最重要的细胞因子并对MIP-2和KC的表达有调控作用，MIP-2和IL-1β的持续高水平表达在角膜病变损害中起着重要甚至是主要的作用，而IL-6的表达是小鼠真菌性角膜炎中机体重要的防御和保护性因素。5、采用特异性多克隆中和抗体对真菌性角膜炎发病过程中的主要趋化因子和细胞因子进行阻断，可有效地减轻真菌性角膜炎的病变损害，这一结果令人鼓舞，对于以主要炎性因子为靶点的真菌性角膜炎治疗方面的新药研发是一个有益的探索和重要的实验基础。更多还原

【Abstract】 ObjectiveTo establish an idealized mouse model of fungal keratitis that mimics clinical human corneal fungal infection and permits the evaluation of the clinical manifestation and histopathology of corneal damages. On the basis, meanwhile, applying technique of molecular biology to investigate the expression, regulation of predominant inflammatory cytokines that play a crucial role in the inflammation process of fungal keratitis, which is beneficial to promote the investigation of the pathogenesis and development of more effective therapeutic means of fungal keratitis.Methods1. Establishment of animal modelFusarium solani, Aspergillus fumigatus, and Candida albicans as the common corneal pathogenic species were respectively selected to induce fungal keratitis in inbred BALB/c mice aged 6～8 weeks old using "epikeratophakia with the aid of corneal epithelium erasion". The concentration of fungal suspension was 10~6 colony-forming units (CFU) per ml. Mouse palpebral fissure was sutured after the infection surgery and opened 24 hours later. All mice were observed daily for corneal involvement under slit-lamp biomicroscope and taken photographs using digital camera. At 1, 3, 5, 7 and 12d, infectious corneas were given semiquantitative clinical scoring and corneal scrapings were obtained from experimental eyes to perform etiologic examination.2. Histopathology examinationAt 1, 3, 5, 7, and 12d after infection, several mouse eyes in experimental groups were enucleated, immersed in 10% formalin for 24 hours, made into routine paraffinsections, then applied hematoxylin-eosin(HE) and periodic acid-Schiff (PAS) staining respectively. The stained sections were analyzed under light microscope, including inflammatory reaction, fungal burden and hyphal growth pattern. The degree of inflammation and fungal burden was categorized to mild, moderate, and severe grades and was given accordingly scoring. According to the angulation degrees of hyphal direction to the plane of stromal lamellae, we defined the hyphal growth patterns of fungal pathogens as vertical when hyphae grew at an angle >45 degrees, parallel when <45 degrees.3. Expression of predominant inflammatory cytokinesAt 1, 3, 5, 7, and 12 d after infection, four inflammatory cytokines in infectious corneas, including macrophage inflammatory protein (MIP)-2, cytokine-induced neutrophil chemoattractant (KC), interleukin (IL)-1β, IL-6, were detected respectively for expression of mRNA and protein levels, using RT-PCR and ELISA. Results of RT-PCR were made for semiquantitative analysis by Image J software for image analysis.4. Intervention experiment of polyclonal antibodies of inflammatory cytokines Inbred 6～8weeks old BALB/c mice were selected to 3 groups with 3 subgroups ineach and were inoculated 3 common corneal pathogenic species respectively. Group 1 was infection-control group, with subconjunctival injection of 20μl sterilized saline. Mice in group 2 and 3 were injected with 20μl(2ng/μl) MIP-2 polyclonal antibodies and 20 μl (2ng/μl) IL-1β polyclonal antibodies subconjunctivally respectively 1 h before and 24 h after surgery. Models of fungal keratitis were established in mice of 3 groups using "epikeratophakia with the aid of corneal epithelium erasion" with the concentration of 10~6CFU/ml in fungal suspension. 24 h after infection, eyelids were opened All mice were observed daily for corneal involvement under slit-lamp biomicroscope and given clinical scoring. At 1, 3, 5, 7 and 12 d, whole eyes were enucleated from several mice in each group and made into routine paraffin sections, observing tissue inflammatory reaction, fungal burden, and growth patterns of fungal hyphae by HE and PAS staining. Corneas of experimental eyes in group 2 and group 3were incised at different time points, MIP-2 and K.C protein levels in which were measured by ELISA assay. Results1. Features of infectious miceAll mice were successful to challenge the fungi inoculated by etiologic assay. The infectious process of 3 fungi had certain variances, except that the peak time of disease was at 3～7d after infection with involvement on 5 d most severe and typical and relieving gradually after 7 d. The involvement duration of Aspergillus fumigatus and Candida albicans was about 10 days, 12 days of Fusarium solani. The involvement of Aspergillus fumigatus was most severe with conspicuous central ulcer in most corneas in 3～5 d after infection, followed by Candida albican with tofukasu anabrosis in involvement region in the peak time. Fusarium solani had steady process, typical musci appearance and longer duration. At 1, 3, 5, and 7 d after infection, the difference of clinical scorings was statistical significance among each other (P=0.000).2. Histopathology changesThe inflammatory reaction in HE staining displayed infiltrated inflammatory cells at earlier period. With the process going on, inflammatory cells increased gradually and corneal tissues demolished more severely, which was most obvious in 5 d after infection. Then inflammatory cells diminished obviously and corneal tissues repaired after 7 d. Aspergillus fumigatus possessed most severe inflammatory reaction followed by Candida albicans and Fusarium solani was mildest. Fungal hyphae were exhibited in PAS staining in all 3 groups on 1 d and 3 d and only 2 cases in Candida albicans group were exhibited on 5 d after infection. The features tended to be that hyphae is multitude in earlier period and diminish gradually. As to fungus burden, fungal hyphae of Candida albicans possessed most in quantity and density. The statistical analysis of inflammation and fungus burden indicated inverse correlation (r= -0.806, P=0.029) between inflammation and fungus burden but positive correlation (r=0.961, P=0.000) between corneal inflammation and its manifestation induced by different strains. On the growth patterns, the hyphae of Fusarium solani lay parralel to the corneal lamellae; whereas thehyphea of Aspergillus fumigatus and Candida albicans were observed growing perpendicularly to the stromal lamellae.3. Expression of predominant inflammatory cytokinesMIP-2, KC, IL-1, and IL-6 were expressed at mRNA and protein levels in corneas of all mice including infectious group, injury control group and normal group with various expression amount and peak time point between the groups and different time points in the same group. The expression regularity of MIP-2, KC, and IL-1β was in coincidence and the expression amount sequence was: Aspergillus fumigatus groups > Candida albicans groups > Fusarium solani groups > injury control groups > normal control groups. IL-6 sequence was: Fusarium solani groups > Candida albicans groups > Aspergillus fumigatus groups > injury control groups > normal control groups. The expression peak time points of MIP-2, KC, and IL-1β were 5 d in Fusarium solani groups, 1 d in Aspergillus fumigatus groups and 3 d in Candida albicans groups after infection. The time points of high-levels expression of IL-6 were 1 d and 7 d in Fusarium solani groups and Aspergillus fumigatus groups but 1 d and 5 d in Candida albicans groups.4. Neutralization of MIP-2 and IL-1β activitiesAfter intervened by MIP-2 polyclonal antibodies, clinical scorings of all infectious groups degraded in certain extent with obvious extent of Aspergillus fumigatus groups (P<0.05), obvious extent (P<0.05) at earlier period (1～3 d) and mild (P>0.05) at midanaphase (5～12 d) in Candida albicans groups, and mild extent at earlier periods but not obvious at midanaphase in Fusarium solani groups. As to IL-1β polyclonal antibodies, the degrading extent was obvious at earlier periods (P<0.05) but not at midanaphase in all 3 groups. After neutralization antibodies were applied, the inflammatory reaction extent and the manifestation at corresponding time points were in coincidence of all groups with no difference of fundus burden and hyphae growth patterns. Protein levels of MIP-2 and KC in corneas intervened by IL-1β polyclonal antibodies descended obviously at earlier periods (P<0.05) but had no difference at midanaphase (P>0.05).Conclusions1. Stable and reliable fungal keratitis that mimics clinical keratopathy can be established by "epikeratophakia with the aid of corneal epithelium erasion", which is a substantial foundation for further reseach such as pathogenesis of fungal keratitis.2. The process of fungal keratitis is devided to 3 phases: early phases, intermediate phases, and later phases with infiltration and extending, suppuration and necrosis, scarring and recover as the key features of manifestation respectively. The severity of involvements by each strain is diverse and inflammatory reaction is predominant factors of corneal impairment, which indicates a new direction for basic and clinical reseach of fungal keratitis.3. The different corneal pathogenic species of fungi display various growth patterns in cornea, which have referred roles in the choice of modus and opportunity of operation for clinical patients with fungal keratitis.4. The chemokines and cytokines such as MIP-2, KC, IL-1β, and IL-6 are expressed in mice corneal tissues of fungal keratitis.. MIP-2 is crucial chemokine to fungal keratitis in mice and IL-1β is the predominant cytokine which regulate the expression of MIP-2 and KC. The persistent high-level expression of MIP-2 and KC is the important factors even being predominant factors in corneal impairment, but the expression of IL-6 is the defensive and protective factors in fungal keratitis of mouse.5. Specific polyclonal neutralizing antibodies can be adopted to inhibit predominant chemokines and cytokines and effectivly relieve the corneal damage by fungal keratitis. This result is encouraging, which is a beneficial explore and significant experiment basis for new drugs aimed with predominant inflammatory cytokines as targets for fungal keratitis.更多还原