摘要
背景CD4＋效应T细胞（Th1／Th2）的平衡模式以往常用于解释真菌感染性疾病的免疫机制。近年来发现，除Th1和Th2细胞亚群外，Th17细胞亚群也参与抗真菌感染的免疫应答，但其在真菌性角膜炎中的作用却鲜有研究。目的探讨白细胞介素-17（IL-17）及Th17特异性转录因子维甲酸相关核孤儿受体γt（RORγt）在真菌性角膜炎中的表达变化及其意义。方法将清洁级BALB／c小鼠96只按随机数字表法随机分为真菌性角膜炎模型组和损伤对照组，真菌性角膜炎模型组小鼠采用角膜表面镜术辅助上皮刮除并层间注入1×10^6CFU／ml真菌菌液5μl法建立小鼠茄病镰刀菌性角膜炎模型，损伤对照组在角膜表面镜术辅助上皮刮除后层间注入等体积（5111）的PBS液。采用质量分数10％KOH湿片法检查菌丝，部分用接种法进行真菌培养并鉴定菌种，另一部分进行细菌培养以排除细菌污染。于造模后第1、3、5、7天在裂隙灯显微镜下观察小鼠角膜炎症的变化特点，参照Wu和Hu的方法对角膜炎症进行评分。分别于造模后第1、3、5、7天处死小鼠并获取角膜组织行苏木精一伊红染色，观察角膜组织的病理改变。采用real—timePCR技术检测IL-17mRNA及其特异性转录因子RORγtmRNA的表达水平，并用ELISA法检测IL-17蛋白在角膜组织中的表达。结果造模后第1、3、5、7天角膜的炎症评分分别为（3．2±0．8）、（6．6±1．1）、（9．4±1．1）、（6．8±0．8）分，差异有统计学意义（F=89．786，P=0．010），其中第3天和第7天角膜炎症评分明显高于第1天，但低于第5天，差异均有统计学意义（P〈0．05）；角膜的组织病理学检测炎性细胞浸润情况与角膜炎症评分变化趋势一致。造模后第3、5、7天，IL-17mRNA在真菌性角膜炎模型组小鼠角膜的表达分别为4．12±0．73、20．72±1．81、14．16±1．88，高于损伤对照组的0．35±0．17、0．28±0．09、0．22±0．09，差异均有统计学意义（P〈0．01），且组内比较第5天时表达量高于第1、3、7天值，差异有统计学意义（P〈0．01），真菌性角膜炎模型组IL-17蛋白在造模后第1、3、5、7天的表达量均明显高于损伤对照组，差异均有统计学意义（P〈0．叭）。真菌性角膜炎模型组各时间点RORγt mRNA在角膜组织中的表达变化与IL-17mRNA的表达趋势一致（P〈0．01）。结论IL-17及其特异性转录因子RORγt在真菌性角膜炎局部组织中的表达量均上调，其表达量变化的趋势与其炎症反应程度相关，推测Th17在真菌性角膜炎的免疫反应中发挥重要作用。
Background In the past few decades, the balance of Thl/Th2 is often used to explain the immune mechanisms of fungal infection and fungal disease. More recently, a novel subset of CD4＋ effector Th cells has been found to participate in anti-fungal infection response. However, whether Thl7 is involved in the immune response in fungal keratitis is unclear up to now. Objective Present study was to investigate the expression change of Thl7 type cytokine and its specific transcription factor, retinoid-related orphan nuclear receptor gamma t （ RORγt） , in the cornea of Fusarium solani keratitis. Methods Ninety-six clean BALB/c mice were divided into Fusarium solani keratitis model group and control group by randomized digital table. Fusarium solani keratitis models were established by epikeratophakia-assisted corneal epithelial erasion and interlayerly injection of 5 μ; （1 × 10^6 CFU/ml） Fusarium solani solution in the right eyes, and the equal volume of PBS was injected in the same way in the control group. 10% KOH wet film was used to examine the fungal hyphea and funga strain was identified by inoculation. The corneas were examined under the slit lamp microscope 1 day, 3, 5, 7 days after modeling and the inflammatory response was scored based on the criteria of Wu and Hu. The histopathological examination of corneas was performed in the time points above. Real time fluorescence quantitative PCR was used to detect the expression levels of interleukin-17 （IL-17） mRNA and RORγt mRNA in the corneas. The expression of IL-17 protein in the corneas was detected by ELISA. The use and raise of the mice followed the Statement of Association for Research in Vision and Ophthalmology. Results The inflammatory scores were 3.2±0. 8,6. 6± 1.1,9.4 ± 1.1 and 6.8 ±0. 8 in 1 day, 3, 5, 7 days after modeling, showing a significant difference among them（ F= 89. 786, P = 0. 010）. The inflammatory scores were higher in the third and seventh day than that in the first day （ P〈0. 05 ）, but they were significantly lower than that in the fifth day （ P〈O. 05 ）. The infiltration of inflammatory cells showed a coincident tendency with the score. The expressing levels of IL-17 mRNA （2-△△Ct） in the corneas were 4. 12±0. 73,20. 72±1.81 and 14. 16±1.88 in 3, 5, 7 days after modeling, with statistically significant differences in comparison with those in the control group （P〈0. 01 ） , and the expression level was significantly higher in the fifth day than those in the first, third and seventh day in the model group（P〈0. 01 ）. The expression levels of IL-17 protein （ng/g） were significantly increased 1 day, 3, 5, 7 days in the model group compared with the control group （P〈0. 01 ）. A similar change was found in the expression of RORγt mRNA to that of IL-17 mRNA. Conclusions Expressions of IL-17 and its transcription factor RORγt upregulate in the fungal keratitis and has an association with inflammatory degree, which suggests that Th17 subset may play an important role in the immune responses of fungal keratitis.
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2013年第7期653-658,共6页
Chinese Journal Of Experimental Ophthalmology
基金
福建医科大学教授学术发展基金项目（js09009）
