Background Herpetic stromal keratitis (HSK) is an immunopathologic eye disorder mediated by CD4+ T lymphocytes.Interleukin-9 (IL-9) is a cytokine linked to the process of many immune inflammatory diseases,but whether IL-9 is involved in the immunopathology of HSK remains unclear.Objective This study was to investigate the expression of IL-9 in murine cornea during the development of HSK and its relationship with the degree of HSK.Methods One hundred and eighty clean BALB/c mice were divided into the normal control group (20 mice) and experimental group (160 mice).HSK models were established by scratching on the surface of the cornea followed by inoculation of 1 × 106 plague forming unit(PFU) herpes simplex virus type 1 (HSV-1) KOS strain.Change of ocular surface was examined under the slit lamp biomicroscopy before and I day,3,5,7,8,10,14,21 days after inoculation of HSV-1.The corneas of mice were collected on the time points mentioned above.The relative expression level of IL-9 mRNA in the cornea of mice was detected by real-time PCR.Immunocytochemical localization of IL-9 protein in murine cornea was viewed by a confocal microscope after preparation of the corneal cryostat sections.All experimental manipulations were undertaken in accordance with the institutional guidelines for the care and use of laboratory animals.Results Punctiform,dendriform or geographic defect in corneal epithelium were seen 3 days and healing 6 days after inoculation of HSV-1.However,edema and opacity of the corneal stroma appeared 7 days and peaked 14 days following the inoculation.Real-time PCR assay showed that very little of IL-9 mRNA (A260/A280) was expressed in the cornea in the mice of the control group.But in 1 day,3,5,7,8,10 and 14 days,the relative level of IL-9 mRNA was 6.37±0.45,5.66±0.53,3.93±0.35,3.62±0.34,3.23±0.18,2.57±0.14,2.19±0.20,with a significant difference among the various time points (F=92.764,P=0.000),and IL-9 mRNA in 1 day,3,5,7,8,10 and 14 days after inoculation was significantly higher than before inoculation (P＜0.05);while no markedly difference was found in IL-9 mRNA expression between the 21-day group after inoculation and the normal control group (P =0.340).IL-9 protein was expressed in the epithelial layer,stromal layer and endothelial cell layer of the corneas,with a stronger green fluorescence in the HSK mice,but no expression of IL-9 protein was seen in the control mice.Conclusions HSK model can be successfully created by corneal scratching and inoculation of HSV-1 KOS strain in BALB/c mouse.IL-9 is involved in the pathogenesis and development of HSK mediated by immunoresponse.%背景 单纯疱疹病毒性角膜基质炎(HSK)是一种由CD4+T淋巴细胞介导的免疫病理性疾病.国外研究发现,白细胞介素-9(IL-9)在多种免疫性疾病的发病过程中发挥重要作用,但在HSK中是否起作用目前少有报道. 目的 研究小鼠HSK发生和发展过程中IL-9在角膜组织中的表达,探讨IL-9的表达与HSK进展程度之间的关系.方法 4～6周龄清洁级健康BALB/c小鼠180只分为对照组20只和实验组160只.对照组仅进行“#”字形划痕,不接种病毒；实验组小鼠角膜用注射针划痕后接种5μl含有1×106空斑单位(PFU)/ml的Ⅰ型单纯疱疹病毒(HSV-1) KOS毒株建立HSK小鼠模型.分别于病毒接种前(0 d),接种后1、3、5、7、8、10、14、21 d在裂隙灯显微镜下观察小鼠角膜的变化；于接种后14 d制备角膜组织切片,苏木精-伊红染色观察角膜的组织病理学改变；采用荧光实时定量PCR(real-time PCR)法检测IL-9 mRNA在小鼠角膜组织中的表达；采用免疫荧光组织化学方法检测IL-9蛋白在小鼠角膜组织中的表达. 结果 裂隙灯显微镜下观察结果显示,小鼠角膜接种HSV-1后第3天,实验组小鼠出现点状、树枝状、地图状角膜上皮缺损,接种后6d内上皮缺损痊愈,但于接种后第7天起出现角膜基质灰白色混浊,14d基质混浊加重并达高峰,之后逐渐减轻.Real-time PCR结果显示,未接种病毒(0 d)的正常小鼠角膜组织中仅有极微量的IL-9 mRNA表达,实验组小鼠接种病毒后1、3、5、7、8、10、14 d IL-9 mRNA在角膜组织中的相对表达量分别为6.37 ±0.45、5.66±0.53、3.93±0.35、3.62 ±0.34、3.23±0.18、2.57±0.14、2.19 ±0.20,总体表达差异有统计学意义(F=92.764,P=0.000),其中接种后各时间点IL-9 mRNA相对表达量与0d比较明显增高,差异均有统计学意义(P＜0.05),接种病毒后21 d,IL-9 mRNA相对表达量与0d比较差异无统计学意义(P=0.340).免疫荧光组织化学法检测显示,IL-9蛋白在实验组小鼠角膜的上皮层、基质层及内皮细胞层呈强阳性表达,而对照组小鼠角膜组织中未见IL-9蛋白的表达.结论 采用角膜划痕和HSV-1 KOS毒株接种法可成功建立BALB/c小鼠HSK模型,IL-9参与了HSK的免疫炎症反应.
展开▼
