可溶性B、T淋巴细胞衰减因子及其配体在单纯疱疹性角膜基质炎小鼠体内的异常表达  

Aberrant expression of soluble B and T lymphocyte attenuator and its ligand in murine herpetic stromal keratitis

作　　者：夏丽坤[1] 李琰[1] 张胜男[1] 曹哲瑶[1] 陆岩[1] 杨宏伟[1] 

机构地区：[1]中国医科大学附属盛京医院眼科,辽宁省沈阳市110004

出　　处：《眼科新进展》2011年第12期1101-1106,共6页Recent Advances in Ophthalmology

基　　金：国家自然科学基金面上项目资助(编号:30772394);辽宁省自然科学基金项目资助(编号:20072091);辽宁省科技攻关计划项目资助(编号:2007225018);沈阳市科学技术计划项目(编号:1091171-1-02);沈阳市高技术产业发展项目计划(编号:2011-14)~~

摘　　要：目的研究B、T淋巴细胞衰减因子(B and T lymphocyte attenuator,BTLA)及其配体疱疹病毒侵入介体(herpes virus entry mediator,HVEM)在单纯疱疹性角膜基质炎(herpetic stromal keratitis,HSK)小鼠角膜组织中及外周血CD4+T细胞上的表达水平,探讨共抑制信号BTLA-HVEM是否参与了CD4+T细胞介导的HSK免疫病理反应。方法将106PFU的单纯疱疹病毒I型(herpes simplex virus type 1,HSV-1)KOS毒株接种于BALB/c鼠的角膜上建立HSK动物模型,分别于角膜接种病毒前(0d),接种病毒后的第3、7、10、14、21天,用毛细管取小鼠的左眼眼眶静脉窦血1mL,分离淋巴细胞,行荧光抗体染色,用流式细胞仪检测CD3+CD4+BTLA+T细胞和CD3+CD4+HVEM+T细胞阳性率;在裂隙灯显微镜下观察小鼠角膜变化;免疫组织化学方法检测BTLA蛋白及HVEM蛋白在角膜组织中的表达。结果 BALB/c鼠的角膜接种HSV-1后的1～5d,角膜擦拭液中均检测出HSV-1复制,表明小鼠感染了单纯疱疹病毒。裂隙灯显微镜观察显示:角膜接种HSV-1后第3天,所有小鼠均患了急性上皮性角膜炎,并于感染后1周内痊愈,自病毒接种后第8天起,小鼠出现角膜基质炎的改变,表现为角膜基质呈灰白色混浊,角膜基质混浊于病毒接种后的第10天达到高峰,持续至第14天后逐渐减轻。流式细胞仪检测显示,小鼠外周血淋巴细胞中CD3+CD4+BTLA+T细胞和CD3+CD4+HVEM+T细胞的阳性率,在角膜接种病毒前(0d)分别为(3.15±0.60)%和(9.84±1.06)%,在角膜接种病毒后第10天(HSK疾病程度最严重时)分别增加到(20.47±3.15)%和(45.18±3.90)%(与0d相比差异均有显著统计学意义,均为P<0.01)。免疫组织化学方法检查HSK小鼠角膜组织中BTLA和HVEM的蛋白表达结果一致:HSK临床表现最严重时,即病毒接种后第10天时,BTLA蛋白和HVEM蛋白在角膜组织中表达最强,主要表达于角膜基质层内浸润的炎性细胞上,角膜上皮层和内皮层也有表达。结论在HSK小鼠模型中,BTLA及其配�Objective To investigate the role of negative costimulatory signals lymphocyte attenuator（BTLA） and herpes virus entry mediator（HVEM） in murine herpetic stromal keratitis（HSK）mediated by CD4＋ T cells by testing the expression of BTLA and HVEM in corneal tissues and CD4＋ T cells of peripheral blood.Methods Corneas of BALB/c mice were infected with 106 plague forming unit（PFU） of herpes simplex virus type 1（HSV-1） KOS strain for establishing HSK animal models.About 1 mL orbital venous sinus blood was collected from left eyes of rats before corneal inoculation and on the 3rd,7th,10th,14th,21st day after corneal inoculation with HSV-1.Lymphocytes were segregated and taken fluorescent antibody staining.By flow cytometry,the expression of CD4＋ BTLA＋ T cells and CD3＋ CD4＋ HVEM in murine peripheral blood was evaluated.The clinical evaluation of infected eyes was taken under the slit lamp microscope,and the characteristic expression of BTLA and HVEM surface protein on corneas was evaluated by immunohistochemistry.Results Replication of HSV-1 was found in corneal clean and polish liquid at 1 to 5 days after corneal inoculation with HSV-1,which indicated that rats had infected herpes virus.Slit lamp microscope had shown that all rats had infected with acute epithelial keratitis on the 3rd day after corneal inoculation with HSV-1,and cured at 1 week after infection;There were changes of corneal interstitial keratitis on the 8th day after inoculation with HSV-1,showing gray opacity of corneal stroma,went to peak at the 10th day and gradually decreased at the 14th day after corneal inoculation.Flow cytometry detection had shown that positive rates of CD3＋ CD4＋ BTLA＋ T cells and CD3＋ CD4＋ HVEM＋ T cells were（3.15±0.60）% and（9.84±1.06）% at the day after corneal inoculation,reached（20.47±3.15）% and（45.18±3.90）% on the 10th day after corneal inoculation,and at this time the HSK was the most severe.There were statistical differences between the day and on the 10th day a

关 键 词：疱疹性角膜基质炎 B淋巴细胞 T淋巴细胞 细胞衰减因子 疱疹病毒侵入介体 

分 类 号：R772[医药卫生—眼科] R392.11[医药卫生—临床医学]