中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2012年
9期
779-784
,共6页
张红敏%刘素素%许中中%马聪慧%谢艳亭%吴细丕%王丽娅
張紅敏%劉素素%許中中%馬聰慧%謝豔亭%吳細丕%王麗婭
장홍민%류소소%허중중%마총혜%사염정%오세비%왕려아
角膜%真菌/感染%免疫/局部%中性粒细胞%T淋巴细胞
角膜%真菌/感染%免疫/跼部%中性粒細胞%T淋巴細胞
각막%진균/감염%면역/국부%중성립세포%T림파세포
Cornea%Fungi/infection%Immunity/topical%Neutrophil cell%T lymphocyte
背景 在中国,感染性角膜病变是引起角膜盲的主要原因之一,而其中真菌性角膜炎的发病率高、危害大,是角膜盲防治工作的重点.研究表明,真菌性角膜炎后局部的免疫状态在疾病的发生发展过程中起着至关重要的作用,但其免疫机制尚待进一步研究. 目的 研究真菌性角膜炎发生发展过程中局部免疫细胞的作用及其机制. 方法 共选用SPF级12周龄雄性C57BL/6J小鼠48只,按照随机数字表法将小鼠随机分为对照组和模型组,每组24只小鼠,然后根据检测的时间点将成模的小鼠随机亚分为临床评分组,6、9、12、24、72和120 h组,每组3只小鼠.模型组小鼠用角膜划痕和带有真菌的竹签涂抹法接种腐皮镰孢菌以建立真菌性角膜炎模型,对照组仅用无菌刀片划痕而不接种任何真菌.各组小鼠分别于上述时间点进行裂隙灯显微镜检查,采用改良标准对炎症程度进行临床评分.其余组按照时间点处死小鼠,采用免疫荧光标记法对炎症局部中性粒细胞和T淋巴细胞进行鉴定,计数炎性细胞数量.实验动物的使用遵循ARVO声明,研究过程经郑州大学实验动物伦理委员会和河南省眼科研究所生命科学实验管理与伦理审查委员会批准. 结果 造模成功后24 h模型组小鼠出现角膜混浊,72 h时病变最为严重,此后病变逐渐减轻,而对照组24 h后上皮愈合.造模后24 ～72 h,模型组眼部炎症反应评分逐渐达峰值,与造模后6h相比差异均有统计学意义(P＜0.01)；造模后6、9、12h模型组与对照组比较,角膜炎症反应评分均明显升高,差异均有统计学意义(P＜0.05).造模后9h和12h模型组小鼠中性粒细胞角膜浸润数量增加,与对照组相比差异均有统计学意义(P＜0.05),与24、72、120 h比较差异亦有统计学意义(P=0.000).模型组造模后12、24、72 h角膜中性粒细胞与6h比较差异均有统计学意义(P=0.004、0.000、0.001),但造模后9 h、120h与造模后6h比较差异均无统计学意义(P=0.772、0.323).真菌接种后72 h和120 h角膜T淋巴细胞数与造模后6h比较差异均有统计学意义(P=0.000、0.000).造模后模型组中性粒细胞数目与炎症临床评分呈明显正相关(r=0.593,P=0.000),T淋巴细胞浸润数目与炎症临床评分间无明显相关性(r=0.315,P=0.062). 结论 真菌性角膜炎的免疫反应过程中,病变早期主要是中性粒细胞发挥关键作用.
揹景 在中國,感染性角膜病變是引起角膜盲的主要原因之一,而其中真菌性角膜炎的髮病率高、危害大,是角膜盲防治工作的重點.研究錶明,真菌性角膜炎後跼部的免疫狀態在疾病的髮生髮展過程中起著至關重要的作用,但其免疫機製尚待進一步研究. 目的 研究真菌性角膜炎髮生髮展過程中跼部免疫細胞的作用及其機製. 方法 共選用SPF級12週齡雄性C57BL/6J小鼠48隻,按照隨機數字錶法將小鼠隨機分為對照組和模型組,每組24隻小鼠,然後根據檢測的時間點將成模的小鼠隨機亞分為臨床評分組,6、9、12、24、72和120 h組,每組3隻小鼠.模型組小鼠用角膜劃痕和帶有真菌的竹籤塗抹法接種腐皮鐮孢菌以建立真菌性角膜炎模型,對照組僅用無菌刀片劃痕而不接種任何真菌.各組小鼠分彆于上述時間點進行裂隙燈顯微鏡檢查,採用改良標準對炎癥程度進行臨床評分.其餘組按照時間點處死小鼠,採用免疫熒光標記法對炎癥跼部中性粒細胞和T淋巴細胞進行鑒定,計數炎性細胞數量.實驗動物的使用遵循ARVO聲明,研究過程經鄭州大學實驗動物倫理委員會和河南省眼科研究所生命科學實驗管理與倫理審查委員會批準. 結果 造模成功後24 h模型組小鼠齣現角膜混濁,72 h時病變最為嚴重,此後病變逐漸減輕,而對照組24 h後上皮愈閤.造模後24 ～72 h,模型組眼部炎癥反應評分逐漸達峰值,與造模後6h相比差異均有統計學意義(P＜0.01)；造模後6、9、12h模型組與對照組比較,角膜炎癥反應評分均明顯升高,差異均有統計學意義(P＜0.05).造模後9h和12h模型組小鼠中性粒細胞角膜浸潤數量增加,與對照組相比差異均有統計學意義(P＜0.05),與24、72、120 h比較差異亦有統計學意義(P=0.000).模型組造模後12、24、72 h角膜中性粒細胞與6h比較差異均有統計學意義(P=0.004、0.000、0.001),但造模後9 h、120h與造模後6h比較差異均無統計學意義(P=0.772、0.323).真菌接種後72 h和120 h角膜T淋巴細胞數與造模後6h比較差異均有統計學意義(P=0.000、0.000).造模後模型組中性粒細胞數目與炎癥臨床評分呈明顯正相關(r=0.593,P=0.000),T淋巴細胞浸潤數目與炎癥臨床評分間無明顯相關性(r=0.315,P=0.062). 結論 真菌性角膜炎的免疫反應過程中,病變早期主要是中性粒細胞髮揮關鍵作用.
배경 재중국,감염성각막병변시인기각막맹적주요원인지일,이기중진균성각막염적발병솔고、위해대,시각막맹방치공작적중점.연구표명,진균성각막염후국부적면역상태재질병적발생발전과정중기착지관중요적작용,단기면역궤제상대진일보연구. 목적 연구진균성각막염발생발전과정중국부면역세포적작용급기궤제. 방법 공선용SPF급12주령웅성C57BL/6J소서48지,안조수궤수자표법장소서수궤분위대조조화모형조,매조24지소서,연후근거검측적시간점장성모적소서수궤아분위림상평분조,6、9、12、24、72화120 h조,매조3지소서.모형조소서용각막화흔화대유진균적죽첨도말법접충부피렴포균이건립진균성각막염모형,대조조부용무균도편화흔이불접충임하진균.각조소서분별우상술시간점진행렬극등현미경검사,채용개량표준대염증정도진행림상평분.기여조안조시간점처사소서,채용면역형광표기법대염증국부중성립세포화T림파세포진행감정,계수염성세포수량.실험동물적사용준순ARVO성명,연구과정경정주대학실험동물윤리위원회화하남성안과연구소생명과학실험관리여윤리심사위원회비준. 결과 조모성공후24 h모형조소서출현각막혼탁,72 h시병변최위엄중,차후병변축점감경,이대조조24 h후상피유합.조모후24 ～72 h,모형조안부염증반응평분축점체봉치,여조모후6h상비차이균유통계학의의(P＜0.01)；조모후6、9、12h모형조여대조조비교,각막염증반응평분균명현승고,차이균유통계학의의(P＜0.05).조모후9h화12h모형조소서중성립세포각막침윤수량증가,여대조조상비차이균유통계학의의(P＜0.05),여24、72、120 h비교차이역유통계학의의(P=0.000).모형조조모후12、24、72 h각막중성립세포여6h비교차이균유통계학의의(P=0.004、0.000、0.001),단조모후9 h、120h여조모후6h비교차이균무통계학의의(P=0.772、0.323).진균접충후72 h화120 h각막T림파세포수여조모후6h비교차이균유통계학의의(P=0.000、0.000).조모후모형조중성립세포수목여염증림상평분정명현정상관(r=0.593,P=0.000),T림파세포침윤수목여염증림상평분간무명현상관성(r=0.315,P=0.062). 결론 진균성각막염적면역반응과정중,병변조기주요시중성립세포발휘관건작용.
Background Infective keratopathy is a key cause of corneal blindness in China,and fungal keratitis is proved to have a higher incidence and bigger threats in infective keratitis.Researches showed that topical immunology plays an important effect during the development of fungal keratitis,but its mechanism is still studying.Objective This experiment was to explore the critical immunocyte during the process of fungal keratitis.Methods Forty-eight SPF 12-week-old male C57BL/6J mice were included and randomized into the control group and model group.The fungal keratitis model closely mimicking human cornea infections was established in the mouse using scratch followed by incubation of fusarium solani on the cornea,and the mice in the control group scratched on the cornea only.Cornea was examined under the slit lamp at 0,6,9,12,24,72 and 120 hours after operation.The severity of keratomycosis was clinically scored based on the literature criteria.The inflammatory cells were identified using immnofluorescence label,and the number of the inflammatory cells was calculated and compared among different groups and time points.This study complied with the Statement of ARVO in the use of experimental animal.Both Experimental Animal Ethic Commission in Zhengzhou University and Life Science Management Commission approved this study proposal.Results After inoculation of fusarium solani,typical fungul keratitis signs were seen on the cornea.Severe corneal opacifieation occurred within 24 hours and peaked at 72 hours.However,only mild edema of cornea was exhibited and gradually recovered normal in the control group within 24 hours.The clinical score of inflammation was higher in the model group in various time points than that in the control group,and it was seen that 24-72 hours after operation,the score attached peak in the model group with a significant difference in comparison with the control group(P＜0.01).In 9,12,24,72 and 120 hours after operation,the number of neutrophil cells was significantly increased in the model group compared with control group (P＜0.05),and that in 12,24,72 hours after operation was significantly higher than the 6 hours(P=0.004,0.000,0.001).However,no significant differences were seen in the number of neutrophil cells between 9 or 120 hours and 6 hours after operation(P=0.772,0.323).The number of T lymphocytes in cornea was significantly increased in 72 and 120 hours in comparison with 6 hours in the model group(P=0.000,0.000),and from 72 to 120 hours after operation,the number of T lymphocytes was significantly higher than that of the contral group (P＜0.01).The neutrophil cell number was positive correlated with the inflammatory score in the early phase (r =0.593,P =0.000).T limphocyte emerged in late phase but no significant correlation with the clinical score (r＝0.315,P=0.062).Conclusions Neutrophil cells play a critical role in the development of fungal keratitis in early stage.
