中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
7期
653-658
,共6页
真菌性角膜炎%白细胞介素-17%维甲酸相关核孤儿受体γt%免疫反应
真菌性角膜炎%白細胞介素-17%維甲痠相關覈孤兒受體γt%免疫反應
진균성각막염%백세포개소-17%유갑산상관핵고인수체γt%면역반응
Fungal keratitis%Interleukin-17%Retinoid-related orphan nuclear receptor gamma t%Immune response
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1×106 CFU/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(5μl)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-伊红染色,观察角膜组织的病理改变.采用real-time PCR技术检测IL-17mRNA及其特异性转录因子RORγt mRNA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-17 mRNA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＜0.01).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-17 mRNA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.
揹景 CD4+效應T細胞(Th1/Th2)的平衡模式以往常用于解釋真菌感染性疾病的免疫機製.近年來髮現,除Th1和Th2細胞亞群外,Th17細胞亞群也參與抗真菌感染的免疫應答,但其在真菌性角膜炎中的作用卻鮮有研究. 目的 探討白細胞介素-17(IL-17)及Th17特異性轉錄因子維甲痠相關覈孤兒受體γt(RORγt)在真菌性角膜炎中的錶達變化及其意義.方法 將清潔級BALB/c小鼠96隻按隨機數字錶法隨機分為真菌性角膜炎模型組和損傷對照組,真菌性角膜炎模型組小鼠採用角膜錶麵鏡術輔助上皮颳除併層間註入1×106 CFU/ml真菌菌液5μl法建立小鼠茄病鐮刀菌性角膜炎模型,損傷對照組在角膜錶麵鏡術輔助上皮颳除後層間註入等體積(5μl)的PBS液.採用質量分數10％ KOH濕片法檢查菌絲,部分用接種法進行真菌培養併鑒定菌種,另一部分進行細菌培養以排除細菌汙染.于造模後第1、3、5、7天在裂隙燈顯微鏡下觀察小鼠角膜炎癥的變化特點,參照Wu和Hu的方法對角膜炎癥進行評分.分彆于造模後第1、3、5、7天處死小鼠併穫取角膜組織行囌木精-伊紅染色,觀察角膜組織的病理改變.採用real-time PCR技術檢測IL-17mRNA及其特異性轉錄因子RORγt mRNA的錶達水平,併用ELISA法檢測IL-17蛋白在角膜組織中的錶達.結果 造模後第1、3、5、7天角膜的炎癥評分分彆為(3.2±0.8)、(6.6±1.1)、(9.4±1.1)、(6.8±0.8)分,差異有統計學意義(F=89.786,P=0.010),其中第3天和第7天角膜炎癥評分明顯高于第1天,但低于第5天,差異均有統計學意義(P＜0.05)；角膜的組織病理學檢測炎性細胞浸潤情況與角膜炎癥評分變化趨勢一緻.造模後第3、5、7天,IL-17 mRNA在真菌性角膜炎模型組小鼠角膜的錶達分彆為4.12±0.73、20.72±1.81、14.16±1.88,高于損傷對照組的0.35±0.17、0.28±0.09、0.22±0.09,差異均有統計學意義(P＜0.01),且組內比較第5天時錶達量高于第1、3、7天值,差異有統計學意義(P＜0.01),真菌性角膜炎模型組IL-17蛋白在造模後第1、3、5、7天的錶達量均明顯高于損傷對照組,差異均有統計學意義(P＜0.01).真菌性角膜炎模型組各時間點RORγt mRNA在角膜組織中的錶達變化與IL-17 mRNA的錶達趨勢一緻(P＜0.01). 結論 IL-17及其特異性轉錄因子RORγt在真菌性角膜炎跼部組織中的錶達量均上調,其錶達量變化的趨勢與其炎癥反應程度相關,推測Th17在真菌性角膜炎的免疫反應中髮揮重要作用.
배경 CD4+효응T세포(Th1/Th2)적평형모식이왕상용우해석진균감염성질병적면역궤제.근년래발현,제Th1화Th2세포아군외,Th17세포아군야삼여항진균감염적면역응답,단기재진균성각막염중적작용각선유연구. 목적 탐토백세포개소-17(IL-17)급Th17특이성전록인자유갑산상관핵고인수체γt(RORγt)재진균성각막염중적표체변화급기의의.방법 장청길급BALB/c소서96지안수궤수자표법수궤분위진균성각막염모형조화손상대조조,진균성각막염모형조소서채용각막표면경술보조상피괄제병층간주입1×106 CFU/ml진균균액5μl법건립소서가병렴도균성각막염모형,손상대조조재각막표면경술보조상피괄제후층간주입등체적(5μl)적PBS액.채용질량분수10％ KOH습편법검사균사,부분용접충법진행진균배양병감정균충,령일부분진행세균배양이배제세균오염.우조모후제1、3、5、7천재렬극등현미경하관찰소서각막염증적변화특점,삼조Wu화Hu적방법대각막염증진행평분.분별우조모후제1、3、5、7천처사소서병획취각막조직행소목정-이홍염색,관찰각막조직적병리개변.채용real-time PCR기술검측IL-17mRNA급기특이성전록인자RORγt mRNA적표체수평,병용ELISA법검측IL-17단백재각막조직중적표체.결과 조모후제1、3、5、7천각막적염증평분분별위(3.2±0.8)、(6.6±1.1)、(9.4±1.1)、(6.8±0.8)분,차이유통계학의의(F=89.786,P=0.010),기중제3천화제7천각막염증평분명현고우제1천,단저우제5천,차이균유통계학의의(P＜0.05)；각막적조직병이학검측염성세포침윤정황여각막염증평분변화추세일치.조모후제3、5、7천,IL-17 mRNA재진균성각막염모형조소서각막적표체분별위4.12±0.73、20.72±1.81、14.16±1.88,고우손상대조조적0.35±0.17、0.28±0.09、0.22±0.09,차이균유통계학의의(P＜0.01),차조내비교제5천시표체량고우제1、3、7천치,차이유통계학의의(P＜0.01),진균성각막염모형조IL-17단백재조모후제1、3、5、7천적표체량균명현고우손상대조조,차이균유통계학의의(P＜0.01).진균성각막염모형조각시간점RORγt mRNA재각막조직중적표체변화여IL-17 mRNA적표체추세일치(P＜0.01). 결론 IL-17급기특이성전록인자RORγt재진균성각막염국부조직중적표체량균상조,기표체량변화적추세여기염증반응정도상관,추측Th17재진균성각막염적면역반응중발휘중요작용.
Background In the past few decades,the balance of Th1/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 Th17 is involved in the immune response in fungal keratitis is unclear up to now.Objective Present study was to investigate the expression change of Th17 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 μl (1 × 106 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＜0.05).The infiltration of inflammatory cells showed a coincident tendency with the score.The expressing levels of IL-17 mRNA (2-ΔΔCt) 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.
