胸腺基质淋巴细胞生成素和白细胞介素-4在变应性结膜炎鼠模型中的促炎作用

背景 研究表明胸腺基质淋巴细胞生成素(TSLP)为类白细胞介素(IL)-17的炎性细胞因子,在多种变应性疾病的发生和发展中发挥关键作用,但是尚未见到关于TSLP是否参与中国常见变应原诱导的变应性结膜炎的报道. 目的 研究TSLP和IL-4在由中国常见的黄花蒿诱导的变应性结膜炎小鼠模型眼表组织和颈部淋巴结中的表达变化,探讨TSLP和IL-4在变应性结膜炎发病中的作用. 方法 采用随机数字表法将6～8周龄雌性BALB/c小鼠20只随机分为正常对照组和模型组.将400μl黄花蒿花粉提取液溶解于400μl变应原(抗原)溶媒中制备成抗原溶液,将50μl抗原溶液注射于模型组小鼠足底皮垫下初次致敏,于注射后第10 ～12天用黄花蒿花粉提取液局部点右眼再次致敏,每日点眼1次;正常对照组小鼠未做任何处理.于造模后第13天采用颈椎脱臼法处死小鼠并摘取眼球,在眼科显微镜下各组分别取16只小鼠32只眼角膜上皮、结膜和颈部淋巴结组织,采用实时定量PCR法检测各种靶组织中TSLP mRNA及IL-4 mRNA的相对表达量,另每组各取4只小鼠8只眼眼睑及眼球制备石蜡切片,采用免疫组织化学法检测角膜、结膜及结膜下组织中TSLP和IL-4蛋白的表达. 结果 模型组小鼠第2次致敏后0.5h可见小鼠眼睑水肿、结膜充血水肿、溢泪、搔抓眼睑等症状,症状持续6 ～24 h,造模成功率为80％.实时定量PCR结果显示,模型组与正常对照组小鼠角膜上皮均未发现IL-4表达.模型组小鼠角膜上皮、结膜组织和颈部淋巴结组织中TSLP mRNA表达量分别是正常对照组的(1.63±0.20)、(2.71±0.48)和(1.48±0.05)倍,差异均有统计学意义(=4.44、14.16、5.01,均P＜0.05);模型组小鼠结膜和颈部淋巴结中IL-4 mRNA表达量分别是正常对照组的(2.94±0.39)倍和(1.74±0.09)倍,差异均有统计学意义(t=9.92、14.54,均P＜0.05).免疫组织化学法检测显示,正常对照组小鼠角膜上皮和结膜上皮细胞中TSLP蛋白表达强度微弱,模型组小鼠TSLP蛋白表达增强.模型组小鼠结膜下组织中可见IL-4呈强阳性表达,正常对照组小鼠结膜下组织中未发现IL-4表达.结论 TSLP主要表达于变应性结膜炎小鼠的角膜、结膜和颈部淋巴结组织,提示其参与变应性结膜炎角膜和结膜的炎症反应;IL-4主要表达于变应性结膜炎小鼠结膜下组织中,提示其主要参与结膜的炎症反应,TSLP和IL-4共同参与变应性结膜炎的发生和发展过程,这2种炎症因子为变应性结膜炎的临床治疗提供了新的靶点.

Inflammatory-promoting roles of thymic stromal lymphopoietin and interleukin-4 in allergic conjunctivitis mice

Background Researches showed that thymic stromal lymphopoietin (TSLP) is an interleukin-17-like inflammatory factor and plays important roles in the pathogenesis and development of allergic diseases.However,the study whether TSLP plays roles in allergic conjunctivitis is rare.Objective This study was to investigate the expression change of TSLP and IL-4 in ocular surface tissue and cervical lymph node in the mice with allergic conjunctivitis induced by artemisia annua,a common plant in China,and to explore the role of TSLP and IL-4 in the pathogenesis and development of allergic conjunctivitis.Methods Twenty female BALB/c mice aged 6-8 weeks were randomized into normal control group and model group.Antigen solution was prepared using 400 μl extracting solution of artemisia annua pollen with 400 μl antigen solvent.The mouse models of allergic conjunctivitis were established by injection of 50 μl antigen solution in footpad followed by ocular topical administration of extracting solution once a day from day 10 to 12 after injection,and no any intervention was given in the normal control group.The mice were sacrificed and eyeballs were obtained 13 days after modeling,and corneal epithelium,conjunctiva and cervical lymph nodes were harvested for the detection of TSLP mRNA and IL-4 mRNA by real-time PCR.Immunochemistry was employed to assay the expression of TSLP and IL-4 proteins in corneal,conjunctival and subconjunctival tissues.Results Ocular inflammatory signs appeared 0.5 hours after ocular topical administration of extracting solution,including eyelid edema,conjunctival congestion,tears,scratching eyelids,etc.The symptoms lasted for 6-24 hours,with the model successful rate 80％.Real-time PCR indicated that the expression of IL-4 was absent in corneal epithelium in both model group and normal control group.The relative expression levels of TSLP mRNA in the corneal epithelium,conjunctiva and cervical lymph nodes in the model group were more (1.63±0.20)times,(2.71±0.48) times and (1.48 ±0.05) times than the normal control group,showing significant differences between the two groups (t =4.44,14.16,5.01,all at P＜0.05).Compared with the normal control group,the relative expression levels of IL-4 mRNA in the model group increased (2.94±0.39) times and (1.74±0.09) times,with significant differences between the two groups (t =9.92,14.54,both at P＜0.05).Immunochemistry assay showed that the expression of TSLP protein in the corneal and conjunctival epithelial cells were enhanced in the model group compared with the normal control group.In addition,an intensive expression of IL-4 protein was seen in subconjunctival tissue in the model group,while the expression of IL-4 was absent in the normal control group.Conclusions TSLP is mainly expressed in the cornea,conjunctiva and cervical lymph nodes of the mice with allergic conjunctivitis,suggesting that TSLP promotes the inflammatory process of cornea and conjunctiva;IL-4 is mainly expressed in conjunctiva,showing IL-4 participates in conjunctival inflammatory process.TSLP and IL-4 play synergistic roles in promoting the inflammatory process of ocular surface in the mice with allergic conjunctivitis,which may be new therapeutic targets.