改良角膜表面镜片术法建立兔曲霉菌性角膜炎动物模型

背景真菌性角膜感染动物模型是研究真菌性角膜炎发病机制的工具,目前的制作方法主要有划痕法、基质注射法和角膜表面镜片术法,但均有其不足之处。目的探讨一种简便、易操作的改良兔曲霉菌性角膜炎动物模型制作方法。方法成年新西兰白兔18只,采用烟曲霉菌孢子附贴滤纸片的改良角膜表面镜片术法制作真菌性角膜炎动物模型。将浸有10^8孢子／ml（10^8孢子／ml组,6只）或10^6孢子／ml（106孢子／ml组,6只）真菌混悬液的滤纸贴敷于去上皮的角膜基质并用角膜接触镜覆盖,将浸有生理盐水的滤纸贴敷于另6只兔眼角膜作为对照组。分别于造模后3、7、14d裂隙灯下观察眼前节症状,参照Dong的标准进行症状评分。制备角膜刮片并用质量分数10％KOH和荧光白染色在荧光显微镜下检测真菌菌丝,角膜组织切片分别行苏木精-伊红和过碘酸希夫染色,光学显微镜下观察角膜形态学改变和菌丝生长情况。对感染组织进行真菌培养以验证模型的质量。结果10^8孢子／ml组和10^6孢子／ml组真菌性角膜炎模型成功者分别为6眼和4眼,裂隙灯检查表明造模3d后10^8孢子／ml组眼前节症状明显重于10^6孢子／ml组,且随着时间的延长,炎性损伤逐渐转向增生期。造模后3d和7d,2组感染的兔眼症状评分明显高于对照组,差异均有统计学意义（P〈0．01）,10^8孢子／ml组兔眼的症状评分均明显高于10^6孢子／ml组,差异有统计学意义（P〈0．01）,造模后14d,10^8孢子／ml组兔眼的症状评分明显高于对照组,差异有统计学意义（P〈0．05）。造模后3d和7d,2组兔眼角膜刮片中均可见真菌菌丝。角膜组织病理学检查显示,造模3d和7d后10^8孢子／ml组可见炎性细胞浸润和角膜基质细胞坏死,并可见真菌菌丝,造模后14d可见新生血管长入。10^6孢子／ml组炎症轻于10^8孢子／ml组。真菌培养结果表明,造模后3d和7d时10^8孢子／ml组均见菌丝生长,而10^6孢子／ml组仅在造模3d时可见菌丝生长。结论改良角膜表面镜片术法可成功制备兔曲霉菌性角膜炎动物模型,是一种简便、易于操作的真菌性角膜炎动物模型制作方法。     

羊膜移植对实验性HSK中基质金属蛋白酶表达的影响

目的研究羊膜移植（AMT）对单纯疱疹性角膜炎（HSK）中基质金属蛋白酶（MMP-2，-9）表达的影响。方法40只BALB／c小鼠角膜感染Ⅰ型单纯疱疹病毒（HSV-1），实验组角膜行AMT。术后第0、2、7、14d取出角膜。常规病理切片、免疫组化染色和计算机图像分析检测角膜中MMP-2及-9的表达及平均光度值的变化。结果对照组20只鼠眼中17只发生HSK；AMT组仅有9只眼发生，差异有显著统计学意义（P〈0．01）。AMT组角膜上皮、基质病变程度及新生血管发生率明显低于对照组（P〈0．05）。免疫组化及图像分析显示对照组角膜细胞和浸润炎性细胞中表达的MMP-2及-9在第2d增加，14d时达高峰。AMT组各时间点MMP-2及-9表达低于对照组，差异有显著统计学意义（P〈0．05）。结论羊膜移植可能通过抑制角膜细胞及浸润的炎症细胞产生MMPs，从而抑制HSK的发生和发展。     

加替沙星治疗非结核性分枝杆菌性角膜炎的实验研究

目的 探讨加替沙星对兔非结核分枝杆菌（NTM）性角膜炎的治疗效果。方法 利用龟分枝杆菌建立兔NTM角膜炎模型，随机分为6组，分别使用加替沙星、左氧氟沙星、环丙沙星、阿米卡星及加替沙星联合阿米卡星进行治疗，平衡盐溶液（BSS）作为对照；观察治疗前后角膜浸润面积及临床表现的变化，并对治疗1周后的角膜病灶进行细菌定量分析。结果 加替沙星可抑制角膜组织内NTM的生长，使NTM角膜炎角膜浸润面积明显减小，与阿米卡星联合使用可增强其抗菌作用。结论 加替沙星是治疗NTM角膜炎的有效药物之一。     

核转录因子抑制剂对大鼠角膜新生血管的抑制作用

目的 探讨二硫氨基甲酸肽吡咯烷（PDTC）对角膜新生血管的抑制作用及其机制。方法 48只SD大鼠,碱烧伤法制作角膜新生血管模型,按随机数字表法随机均分成A、B、C及D组,每组12只鼠（12只眼）,A、B、C组伤后分别滴用0．5、1及2mg／ml的PDTC眼液,对照组（D组）滴生理盐水;均4次／d,共28d。每日裂隙灯显微镜下观察角膜新生血管的生长情况、测量新生血管面积、记录角膜炎性反应及角膜混浊度,并在4和28d每组各处死6只鼠,取全角膜作病理学分析和核转录因子NF-κB的活化分析（Western Blot法）。结果 B、C组的角膜新生血管面积均显著少于D组（F值分别为964．51、766．37,P值均〈0．01）,B、C组的角膜炎性反应程度与混浊度均低于D组（F值分别为72．41、54．33,P值均〈0．01）,差异均有统计学意义;A组与D组角膜新生血管面积的比较（F=0．588,P=0．486）、角膜炎性反应程度与混浊度的比较（F=1．278,P=0．122）,差异均无统计学意义。B、C组NF-κB活化程度显著低于D组,而A、D组间NF-κB的表达无差异。结论 PDTC能有效阻止NF-κB的活化,抑制角膜新生血管的形成。     

角膜交联对真菌性角膜溃疡的治疗作用

背景 真菌性角膜溃疡是一种严重威胁视力的炎症性疾病,严重者需行角膜移植术或眼球摘除术.角膜交联术（CXL）是近年来治疗一些角膜疾病的有效方法,但其用于真菌性角膜溃疡的疗效方面少有研究.目的 观察CXL对真菌性角膜溃疡的治疗作用和效果.方法 选取8周龄健康新西兰白兔15只,其中5只作为正常对照组,另取10只兔刮除右侧角膜上皮行角膜划痕,涂抹镰刀菌液,然后行异种脱细胞角膜片覆盖,制备真菌性角膜溃疡动物模型.按照随机数字表法将模型兔随机分为非治疗组和CXL治疗组,每日行裂隙灯显微镜检查,测量角膜病灶的直径,并观察角膜水肿和炎性细胞浸润情况.于CXL治疗后第3、7、14、21、28天分别对非治疗组和CXL治疗组兔眼行眼前节照相和激光扫描共焦显微镜检查.治疗后4周,收集15只实验兔角膜组织,于扫描电子显微镜下检测正常对照组、非治疗组和CXL治疗组角膜胶原纤维的超微结构.结果 实验兔右眼造模后3d即可见角膜病灶区灰白色溃疡灶,激光扫描共焦显微镜下浅基质层局部见豆荚样菌丝.造模后l周角膜溃疡灶加深,范围扩大,激光扫描共焦显微镜下浅基质层见大量真菌孢子和短棒样菌丝,可见角膜内皮细胞层有炎性细胞及前房内渗出.CXL治疗后3、7、14、21 d,CXL治疗组兔角膜上皮缺失范围均小于非治疗组,差异均有统计学意义（P＜0.05）.治疗后28 d,正常对照组、非治疗组和CXL治疗组兔角膜胶原纤维束平均直径分别为（24.6±1.8）、（24.9±1.9）和（43.0±7.4）nm,3个组间差异有统计学意义（F=27.05,P=0.00）,其中CXL治疗组胶原纤维直径较非治疗组和正常对照组增粗,差异均有统计学意义（t =-5.30、5.40,P＜0.05）,胶原纤维间见较多成纤维细胞;而非治疗组兔角膜胶原纤维直径与正常对照组间差异无统计学意义（t=0.25,P＞0.05）,正常对照组胶原纤维间少见成纤?     

NS398对白介素1α诱导兔角膜基质细胞环氧化酶2表达的抑制

目的探讨氮-2,环己氧-4,硝基苯-甲基磺胺（NS398）对白介素1α（IL-1α）诱导的兔角膜基质细胞环氧化酶2（COX-2）表达的影响。方法体外培养兔角膜基质细胞,实验组分别以含0、25、50、100、200μmol／LNS398的培养液孵育2h后加入IL-1α诱导COX-2表达,24h后实时荧光定量聚合酶链反应（Real-Time PCR）检测兔角膜基质细胞中COX-2基因表达的差异,四甲基偶氮唑盐（MTT）比色法检测不同浓度NS398对细胞生长的影响,并与对照组进行比较。结果Real—Time PCR结果显示IL—1α诱导后24h各组COX-2 mRNA表达量差异有统计学意义（F=988．45,P〈0．01）;对照组与各实验组以及各实验组之间COX-2 mRNA表达量进行多重比较可见,0μmol／LNS398浓度组与对照组之间差异无统计学意义（q=1．3322,P〉0．05）,其他NS398浓度组与对照组相比差异均有统计学意义（q=34．7896,48．3298,65．8010,70．8131,P〈0．01）,随培养液中NS398浓度的增加,IL-1α刺激后兔角膜基质细胞中COX-2 mRNA表达量不断下降（q=36．1218,49．6620,67．1332,72．1453,13．5402,31．0114,36．0235,17．4712,22．4833,5．0121,P〈0．01）;MTT法检测结果显示,100μmol／L、200μmol／L的NS398对兔角膜基质细胞生长均有明显的抑制作用（q=12．7693,20．9087,P〈0．01）。结论NS398能够有效抑制IL-1α诱导的兔角膜基质细胞COX-2的表达,但超过一定浓度会对细胞产生明显毒性作用。     

兔棘阿米巴角膜炎的组织病理学研究

目的 观察兔棘阿米巴角膜炎角膜组织感染的病理变化及其特点,探讨棘阿米巴角膜炎的发病机制.方法 新西兰白兔20只,其中16只角膜基质内注射纯化培养的棘阿米巴原虫悬液建立兔棘阿米巴角膜炎模型,观察兔角膜组织的病理学及免疫学的相应变化以及与组织降解和修复相关的细胞因子基质金属蛋白酶13(MMP13)及碱性成纤维细胞生长因子2(bFGF2)的表达;4只为对照.结果 兔棘阿米巴角膜炎感染,初期以中性粒细胞浸润为主,21d后以巨噬细胞为主,并伴有成纤维细胞的增生,7d和14d时 CD4+CD8+细胞比值异常;感染初期,组织内开始出现MMP13阳性表达,从14d开始MMP13合成减少,FGF2的表达逐渐增强,28d时达峰值,其后减弱.结论 兔棘阿米巴角膜炎早期的自然病程以感染性炎症为主,后期主要特征为角膜组织修复.MMP13和FGF2的表达变化与临床表现有一定相关.     

小牛血清去蛋白眼用凝胶对兔角膜碱烧伤的治疗作用

背景角膜化学烧伤,特别是碱烧伤对眼组织造成了严重危害,目前临床上多采用抗炎、抑制免疫反应、角膜移植术等方法进行治疗,但针对角膜碱烧伤微环境下的组织修复研究也十分必要。目的观察角膜修复剂小牛血清去蛋白眼用凝胶对兔角膜碱烧伤的治疗作用。方法采用浸有0．5mol／LNaOH溶液的滤纸贴附于角膜中央的方法制备兔眼角膜碱烧伤模型共24只兔24只眼,采用随机数字表法将模型眼随机分为生理盐水对照组、小牛血清去蛋白眼用凝胶组、空白凝胶基质组、碱性成纤维细胞生长因子（bFGF）阳性对照组4个组,分别采用生理盐水、小牛血清去蛋白眼用凝胶、凝胶空白基质和重组牛bFGF眼用凝胶点眼,每日4次。连续2周。分别于给药前及给药后3、5、7、10、14d于裂隙灯下观察各组眼的角膜溃疡和炎症反应,参照Ando等的评分标准对角膜炎症进行评分,进行角膜上皮荧光素钠染色,记录眼角膜溃疡面积的大小,并参照Trousdale评分标准对角膜溃疡进行评分。于造模后14d用过量麻醉法处死动物并摘除实验眼眼球,对角膜组织行常规组织病理学检查。结果造模后兔眼角膜混浊、水肿、灰白,造模成功率为100％。不同药物点眼后7d,裂隙灯下检查发现生理盐水对照组角膜呈瓷白色混浊,可见前房积脓;小牛血清去蛋白眼用凝胶组角膜溃疡愈合;空白凝胶基质组角膜上皮完整,但眼内结构窥不清;而bFGF阳性对照组角膜溃疡愈合,但可见新生血管。点眼后3、5、7、10、14d,小牛血清去蛋白眼用凝胶组及bFGF阳性对照组眼前节炎症评分均明显低于生理盐水对照组,差异均有统计学意义（P〈O．01）;小牛血清去蛋白眼用凝胶组眼前节炎症评分虽然稍低于bFGF阳性对照组,但二者的差异无统计学意义（P〉0．05）;与空白凝胶基质组相比,小牛血清去蛋白眼用凝胶组各时间点兔眼炎症评分值明显下降,差异均有统计学意义（P〈0．05）。小牛血清去蛋白眼用凝胶组点眼后,角膜溃疡评分均明显下降,与生理盐水对照组相比,角膜溃疡评分的差异有统计学意义（P〈0．01）,与bFGF阳性对照组比较,角膜溃疡评分有所下降,但差异无统计学意义（P〉0．05）,与空白凝胶基质组比较,角膜溃疡评分明显下降,差异有统计学意义（P〈0．05）。结论小牛血清去蛋白眼用凝胶可减轻家龟角膜碱焙伤的眼前节炎症反应,促进角膜上皮愈合,其疗效优于bFGF。     

水浸润分离法在SMILE手术角膜基质透镜分离中的应用

目的观察水浸润分离法在SMILE术中的安全性和有效性。方法前瞻性临床对照研究。将拟采用SMILE手术完成近视及近视散光患者72例（144眼）纳入研究,每例患者随机选取一眼常规分离角膜基质透镜,对侧眼水浸润分离角膜基质透镜。观察术中切口上皮损伤情况,光镜下观察透镜边缘的整齐程度。采用独立样本t检验比较2组术后视力、屈光状态、对比敏感度。结果术中发生角膜切口边缘上皮损伤水浸润分离组与常规分离组分别为2眼（3％）与14眼（19％）,差异有统计学意义（x2=6．41,P〈0．01）。采用水浸润法分离的角膜基质透镜边缘光滑整齐,常规分离的透镜边缘相对粗糙。水浸润法分离组与常规分离组术后第1天视力为4．98±0．06与4．89±0．53,等效球镜度为（＋0．20±0．42）D与（＋0．30±0．37）D,差异无统计学意义（t=-1．53,P〉0．05）,残余散光度为（0．14±0．40）D与（0．41±0．57）D,差异有统计学意义（t=-3．29,P〈0．05）。术后第1个月视力分别为4．99±0．06与4．97±0．06,等效球镜度分别为（＋0．11±0．40）D与（＋0．13±0．41）D,差异无统计学意义（t=-1．88,P〉0．05）,残余散光度为（0．05±0．46）D与（0．36±0．66）D,差异有统计学意义（t=-3．41,P〈0．05）。2组对比敏感度差异无统计学意义。结论与常规SMII正术中微透镜分离法相比,水浸润分离法可有效地减少术中角膜上皮的损伤,减小分离阻力,取出的角膜基质透镜表面更加平滑,边缘更加整齐,并明显减少了术后残余散光。     

机械法准分子激光上皮瓣下角膜磨镶术与去瓣Epi-LASIK术后兔角膜基质细胞凋亡的研究

目的观察机械法准分子激光上皮瓣下角膜磨镶术（Epi-LASIK）与去瓣Epi-LASIK术后不同时间点角膜基质细胞的凋亡,探讨2种手术方式对角膜创伤修复过程的影响。方法 42只新西兰大白兔随机编号,其中40只兔各取一侧眼行Epi-LASIK,对侧眼行去瓣Epi-LASIK,剩余2只兔4只眼作为正常对照组。分别于术后4h,1、3、7d共4个时间点取出角膜组织,透射电镜下观察各组兔角膜组织的超微结构改变,采用TUNEL法检测2组兔手术后不同时间点角膜基质细胞的凋亡情况,应用免疫组织化学法检测2组兔手术后不同时间点白细胞介素-1β（IL-1β）在角膜基质细胞中的表达。结果 Epi-LASIK组术后1d及3d角膜基质细胞凋亡细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.42,P〈0.05;t=4.04,P〈0.05）;Epi-LASI组术后1、3、7d角膜基质中IL-1β表达的阳性细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.13,P〈0.05;t=3.71,P〈0.05;t=3.06,P〈0.05）。Epi-LASIK组术后透射电镜下可见兔角膜基质细胞核固缩,染色质边集并可见凋亡小体。免疫组织化学染色显示Epi-LASIK组术后角膜基质中IL-1β的表达强于去瓣Epi-LASIK组。结论去瓣Epi-LASIK对角膜基质细胞的损害较Epi-LASIK轻,提示去瓣Epi-LASIK术后早期角膜创伤愈合反应的程度较轻,有利于角膜组织的快速修复。     

Improvement of HSV-1 necrotizing keratitis with amniotic membrane transplantation

PURPOSE: Stromal herpes simplex virus keratitis (HSK) is an immune-mediated disease. Previous studies have indicated that T cells, neutrophils, and macrophages contribute to the tissue damage in HSK. It has been shown that human amniotic membrane promotes epithelial wound healing and has diverse anti-inflammatory effects. In this study, the effect of amniotic membrane transplantation (AMT) on corneal wound healing and on inflammation in mice with necrotizing HSK was examined. METHODS: BALB/c mice were corneally infected with 10(5) plaque-forming units (PFU) of HSV-1 (KOS strain). In 16 mice that exhibited severe ulcerating HSK, the cornea was covered with a preserved human amniotic membrane as a patch. Corneas in 16 infected mice remained uncovered and served as a control. On days 2 and 7 after surgery, the amniotic membrane was removed (eight mice in each group), the HSV-1-infected cornea was evaluated clinically, and the eye was enucleated. Tissue sections were analyzed histologically for epithelialization and cellular infiltration and immunohistochemically with anti-CD3 mAb to T cells, anti-CD11b mAb to both macrophages and neutrophils, or anti-F4/80 mAb to macrophages. RESULTS: Profound regression of corneal inflammation and rapid closure of epithelial defects were observed clinically within 2 days in the amniotic membrane-covered eyes, whereas HSV-1 keratitis and ulceration progressed in all mice in the control group (P < 0.001). Histologically, corneal edema and inflammatory infiltration, and immunohistochemically the number of CD3(+), CD11b(+), and F4/80(+) cells in the cornea were markedly decreased at 2 and 7 days after amniotic membrane application, compared with the uncovered control corneas (P < 0.001). CONCLUSIONS: AMT promotes rapid epithelialization and reduces stromal inflammation and ulceration in HSV-1 keratitis. AMT in mice with HSV necrotizing stromal keratitis appears to be a useful model for investigating the effect and the action mechanism of human amniotic membrane.     

基质环形浸润性棘阿米巴角膜炎的治疗与分期初探

目的探讨有角膜环形基质浸润病变的棘阿米巴角膜炎的临床分期、治疗及预后。方法对17例（17眼）有角膜环形基质浸润病变的棘阿米巴角膜炎或合并真菌感染病例作了回顾性研究，根据角膜环形基质浸润的病程、环宽、浸润深浅、色泽分为早期和晚期，然后分别给予相应的药物和手术治疗。结果早期者8眼，药物治愈5眼，手术成功2眼，手术失败1眼；晚期者9眼，药物治愈2眼，手术成功5眼，手术失败2眼。结论早期诊断、正确分期及应用有效的药物治疗和选择适当的术式是成功治疗有环形基质浸润病变的棘阿米巴角膜炎的关键。     

激光原位角膜磨镶术后非结核分枝杆菌性角膜炎的研究现状

随着LASIK手术的普遍开展，术后并发感染性角膜炎的病例报道不断增多。非结核分枝杆菌(NTM)是导致术后感染性角膜炎的主要病原微生物之一，其中以龟分枝杆菌及偶发分枝杆菌最常见。LASIK术后NTM角膜炎的起病缓慢，早期无特异性症状，随病情的发展出现眼痛及视力下降。典型的临床体征为角膜基质多灶性点片状浸润，严重者可出现瓣下脓肿、角膜瓣坏死。对于LASIK术后2—3周角膜出现浸润病变或角膜瓣下出现结晶样角膜病变，应高度怀疑NTM角膜炎，其病因学诊断须依靠实验室检查。LASIK术后NTM角膜炎的治疗方法包括药物治疗、角膜瓣下的冲洗及手术治疗。治疗的原则为：药物治疗与手术治疗相结合，局部治疗与全身治疗相结合。目前最常用的抗生素为阿米卡星、克拉霉素及氟喹诺酮类抗生素。     

准分子激光原位角膜磨镶术后角膜细菌性感染的实验研究

目的:建立日本大耳白兔LASIK术后感染模型,了解LASIK术后细菌性角膜炎的病理过程,为LASIK术后细菌性角膜炎诊断、治疗提供依据。方法:选取健康成年日本大耳白兔20只,右眼行LASIK术后接种金黄色葡萄球菌,在术后12h;1,3,5,10d各时期行肉眼、数字化裂隙灯和共焦显微镜观察,10d后处死实验动物取角膜行病理切片。结果:18眼成功建立了LASIK术后细菌性角膜炎模型。其早期表现为角膜瓣浅基质层点状或小斑片状炎性浸润,随着时间的推移慢慢融合,并以层间为起点同时向前（角膜瓣）和向后（深基质层）发展,晚期造成角膜瓣移位、角膜瓣溃疡、穿孔伴前房积脓。结论:建立LASIK术后细菌性角膜炎模型可行。LASIK术后细菌性角膜炎会导致严重的病理损害。     

Influence of dimethylfumarate on experimental HSV-1 necrotizing keratitis

Background This study was performed to investigate the influence of fumaric acid esters on the course of herpes stromal keratitis (HSK).Methods The corneas of BALB/c mice were inoculated with 105 plaque-forming units of herpes simplex virus 1 (HSV-1, KOS strain). Groups of mice were treated intraperitoneally with phosphate buffered saline (PBS) (control mice), or with dimethylfumarate (DMF) at 15 mg/kg of body weight dissolved in PBS daily for 28 days pre-infection and for 14 days post-infection. The course of HSV-1 keratitis was studied clinically. Corneal sections were examined for inflammatory cell infiltration. The numbers of CD3, GR-1, CD11b and F4/80-expressing cells infiltrating the corneas were analyzed by immunohistochemistry.Results On day 14 after HSV infection, 72% of the mice in the control group had severe HSK. The development of HSK was reduced by DMF treatment in the DMF group (22%) (P=0.004). The total number of inflammatory cells and infiltration of polymorphonuclear-neutrophils (PMNs) were reduced in the corneas of DMF-treated mice. Compared to the PBS-treated mice, numbers of CD3, CD11b, GR-1 and F4/80-positive cells were reduced in the DMF group of mice.Conclusions The course of experimental herpes stromal keratitis can be improved with systemic fumaric acid ester treatment. The improvement of keratitis correlates with a reduced corneal infiltration of T cells and mononuclear cells.The authors have no financial interest in any of the reagents used in this study     

Corneal epithelial cells and stromal keratocytes efficently produce CC chemokine-ligand 20 (CCL20) and attract cells expressing its receptor CCR6 in mouse herpetic stromal keratitis

PURPOSE: CC chemokine-ligand 20 (CCL20) is known to be selectively expressed by surface-lining mucosal epithelial cells and skin epidermal keratinocytes and to attract cells such as immature dendritic cells and effector T cells via CCR6. This study evaluated the ability of corneal epithelial cells and stromal keratocytes to produce CCL20 in vitro and in vivo. METHODS: Human corneal epithelial cells (HCE) and corneal keratocytes (HCK) were treated without or with various cytokines and expression of CCL20 mRNA and secreion of its protein were evaluated by RT-PCR and ELISA. Induction of CCL20 mRNA in HCE and HCK was also examined upon in vitro infection with HSV-1. Using a mouse model of herpetic stromal keratitis (HSK), induction of CCL20 expression and accumulation of cells expressing CCR6 were evaluated by RT-PCR and immunohistochemistry. RESULTS: Not only corneal epithelial cells but also stromal keratocytes efficiently expressed CCL20 mRNA and protein upon stimulation with IL-1beta and TNF-alpha. In vitro infection with HSV-1 also induced CCL20 mRNA in both types of cells. In a mouse herpetic stromal keratitis model, prominent accumulation of CCL20 and CCR6 mRNA was revealed in HSV-1-infected corneas. Furthermore, immunohistochemistry demonstrated production of CCL20 by corneal epithelial cells as well as stromal keratocytes and stromal infiltration of DEC205+ dendritic cells, CD4+ T cells and CD8+ T cells. Double staining revealed that CCR6-expressing cells were mostly MHC class II+ dendritic cells. CONCLUSIONS: Not only epithelial cells but also stromal keratocytes are efficient producers of CCL20 in the cornea and recruit CCR6-expressing cells such as dendritic cells into inflamed cornea.     

细胞毒性T淋巴细胞相关抗原-4融合蛋白对鼠单纯疱疹性角膜基质炎抑制作用的研究

研究细胞毒性T淋巴细胞相关抗原-4融合蛋白(cytotoxic Tlymphocyte-associated antigen-4 immunoglobulin,CTIA-4Ig)对鼠单纯疱疹性角膜基质炎(herpetic stromal kerafifis,HSK)的抑制作用。方法用单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)接种于BALB／c鼠角膜上建立HSK动物模型,采用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,抑制T淋巴细胞增殖、分化为效应细胞;观察HSK的发病率、临床特征、角膜组织学改变、角膜病毒滴度、迟发型超敏反应及抗原刺激脾细胞分泌细胞因子的情况。结果CTIA-4Ig可减少小鼠外周血中CD4^ T淋巴细胞(81．6％)及CD8^ T淋巴细胞(67．9％),阻止鼠发生HSK、减轻角膜混浊程度及角膜内炎性细胞的浸润、损伤鼠的迟发型超敏反应能力,抑制鼠脾细胞分泌辅助性T淋巴细胞1型(T-helper 1,Th1)细胞因子;但不影响角膜病毒滴度及小鼠死亡率。结论用CTIA-4Ig阻断B7：CD28／CTIA-4协同刺激途径,能够抑制T淋巴细胞增殖并抑制CIM^ Th1细胞的功能,阻止HSK发病,减轻HSK的严重程度。     

Participation of T lymphocyte in corneal edema in the early stage of herpetic stromal keratitis

The participation of T lymphocytes in the immunopathogenesis of corneal opacity in herpetic stromal keratitis was investigated. In BALB/c mice infected intracorneally with herpes simplex virus type 1, corneal opacity was manifested 10 days after infection, while in athymic mice corneas remained almost clear. Histologically, all opaque corneas revealed stromal edema accompanied by the diffuse presence of polymorphonuclear cells and lymphocytes. When complement (C')-treated immune spleen cells were adoptively transferred into athymic mice 6 or 72 h after corneal infection, stromal keratitis with mild opacity was observed 10 days after transfer. The athymic mice given anti-Thy 1.2+C'-treated immune spleen cells failed to develop corneal opacity. The difference, as revealed by light and electron microscopy, was the presence or absence of lymphocytic infiltration and edema in the posterior third layers of the stroma and endothelial lesions. The endothelium was infiltrated by lymphocytes or macrophages and showed various stages of destruction. The main cause of corneal opacity in the early stage of herpetic stromal keratitis is thought to be stromal edema due to an adverse effect on the endothelium by immune T lymphocytes.     

白细胞介素10对小鼠单纯疱疹性角膜基质炎作用的实验研究

研究白细胞介素10(IL-10)在小鼠单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)中的作用。方法雌性BALB／c小鼠80只分为2组,每组40只;将单纯疱疹病毒1型接种于小鼠角膜上建立HSK动物模型;分别于鼠角膜接种病毒前的6h、当日及接种后2、4d,向实验组鼠的角膜内注射重组IL-10 20ng,腹腔内注射鼠重组IL-10 500 ng;对照组同步注射生理盐水;观察IL-10对小鼠HSK的发病率、临床特征、角膜病毒滴度、角膜细胞因子含量、角膜病理改变及迟发型超敏反应(DTH)的影响。结果IL-10降低了HSK发病率,减轻了HSK角膜混浊程度、角膜新生血管化程度及角膜内炎性细胞浸润,降低了角膜内IL-2和IL-6的含量,抑制了鼠的DTH反应。IL-10不影响病毒在角膜内的复制和清除。结论IL-10可抑制鼠的DTH反应和HSK的发病进展。     

Contribution of virus and immune factors to herpes simplex virus type I-induced corneal pathology

In vivo T-lymphocyte subpopulation depletion techniques were used to identify the roles of L3T4+ (CD4) and Lyt-2+ (CD8) T-lymphocytes in the pathogenesis of corneal stromal disease induced by two different strains of Herpes simplex virus type 1 (HSV-1). Histologic examination of infected corneas revealed significant differences in the composition of the inflammatory corneal infiltrates induced by the RE and KOS strains of HSV-1. The RE strain induced a predominantly polymorphonuclear leukocyte (PMN) infiltrate, which began approximately 1 week after infection and progressed through day 21. Depletion of CD4 cells before corneal infection with RE HSV-1 greatly reduced the incidence and severity of corneal disease; depletion of CD8 cells had no effect. The strain KOS HSV-1 induced an early PMN infiltrate that became predominantly mononuclear by day 21. Depletion of CD4 cells did not change the incidence or severity of KOS HSV-1-induced corneal stromal disease. The corneal lesions of these mice contained numerous CD8 cells. Depletion of CD8 cells before KOS HSV-1 infection of the cornea moderately reduced the incidence of stromal disease. However, in CD8-depleted mice with the disease, PMNs were the most prevalent infiltrating cells, and the disease appeared identical to that seen in RE HSV-1 infected corneas. Simultaneous depletion of CD4 and CD8 cells before KOS HSV-1 infection eliminated stromal disease. However, when T-cell depletion was discontinued in these mice, stromal disease developed in concert with the appearance of T-cells in the lymphoid organs and corneas. Thus, T-lymphocytes are a necessary component of HSV-1 corneal stromal disease. These results further suggest that RE HSV-1 preferentially activates CD4 cells in the cornea, and KOS HSV-1 preferentially activates CD8 cells in the cornea.