实验性自身免疫性葡萄膜炎眼球中自然杀伤细胞的致炎作用及其机制

Inflammation-causing effects and mechanism of natural killer cells in experimental autoimmune uveitis rats

背景实验性自身免疫性葡萄膜炎（EAU）是葡萄膜炎常见的动物模型，自然杀伤（NK）细胞是一种强烈的致炎细胞，但其在EAU中的作用及其机制仍有待研究。目的探讨EAU模型大鼠不同发病阶段视网膜组织中NK细胞的定位和分布及其机制。方法将36只SPF级Lewis大鼠应用随机数字表法随机分成对照组和造模后第6、9、12、16、21天组，每组各6只。造模后第6、9、12、16、21天组大鼠采用光感受器间维生素A类结合蛋白（IRBP）联合5mg／ml结核杆菌和完全福氏佐剂（CFA）乳化液双后足垫皮下注射，然后腹腔内注射400ng百日咳毒素免疫大鼠建立EAU大鼠模型。对照组大鼠双后足垫皮下注射生理盐水与等容量CFA乳化液，然后腹腔内注射400ng百日咳毒素。造模后每天用裂隙灯显微镜观察大鼠眼前段炎症反应过程，根据Caspi方法进行眼部炎症症状评分。分别于造模后第6、9、12、16、21天摘取各组大鼠眼球，采用苏木精一伊红染色法检测各组大鼠视网膜炎症反应和组织结构形态学改变；采用免疫荧光双标法检测大鼠视网膜中NK细胞的分布及浸润情况。另取25只Lewis大鼠应用随机数字表法随机分为造模后第0、3、6、9和12天组，每组5只大鼠，电动匀浆机破裂组织并匀浆为眼内液，采用实时荧光定量PCR法检测大鼠眼内液中NK细胞趋化因子CXCL10 mRNA和CXCL12 mRNA的表达。结果对照组大鼠眼前节未发现炎症反应。造模后第6天组大鼠虹膜血管扩张，随着造模后时间延长虹膜血管扩张明显，前房逐渐出现渗出或积脓，造模后第12天炎症反应达峰。视网膜组织病理学检查显示，对照组大鼠视网膜组织结构排列整齐，各EAU模型组大鼠造模后随时间延长均出现不同程度的视网膜结构排列紊乱，外核层细胞分离，层间组织松散，视细胞水肿且有炎性细胞浸润，以造模后第12天组最为严重。免疫荧光双标记显示对照组仅见蓝色标记的细胞核，视网膜各层细胞排列规则；造模后第6天组开始可见大鼠视网膜内层大量NK细胞浸润，呈红色荧光，随时间延长逐渐增加，造模后第9天组NK细胞浸润达峰。造模后第9天组大鼠视网膜中CXCL10 mRNA相对表达量为34．298±16．689，明显高于造模后第3、6、12天组的1．390±0．660、3．359±2．581和4．711±1．387，差异均有统计学意义（均P〈0．01）；各组大鼠视网膜中CXCL12 mRNA相对表达量的总体比较差异无统计学意义（F=2．851，P〉0．05）。结论EAU大鼠发病早期视网膜中NK细胞浸润，其严重程度和视网膜中CXCL10的表达动态与EAU炎症发展过程相吻合，提示NK细胞在EAU的早期炎症过程中发挥重要作用，CXCL10是NK细胞的主要趋化因子。

Background Experimental autoimmune uveitis （EAU） is a common animal model of uveitis. Natural killer （NK） cells have been confirmed to be a type of strong inflammation-causing cells, but its role in EAU is still studing. Objective This study was designed to explore the role and mechanism of NK cells in the pathogenesis of EAU. Methods Thirty-six SPF Lewis rats were randomly divided into expeimental control group and EAU 6-,9-, 12-, 16-, and 21-day groups （6 rats for each group）. Rats in EAU group received subcutaneous injection interphotoreeeptor retinoid binding protein （IRBP） combining 5 mg/ml tubercle bacillus with complete Freund＇s adjuvant （CFA） emulsion in foot pads, and then 400 ng pertussis toxin was intraperitoneally injeeted to extablish EAU models in the EAU 6-,9-, 12-, 16-, and 21-day group, and normal saline solution combined with CFA and 4-00 ng pertussis toxin was used in the same way in the experimental control group. The inflammatory response was observed by slit lamp daily after modeling and scored based on Caspi criteria. The eyeballs were extracted in 6,9,12, 16 and 21 days after modeling for retinal histopathological examination. Immunofluorescent double-staining was employed to detect and locate the expression of NK cells in the retina. In addition,25 model rats were divided into EAU 0-,3-, 6-, 9- and 12-day groups, with 5 rats for each group, and eyeballs were extracted to prepare tissue homogenate. The expression of CXCL10 mRNA, and CXCL12 mRNA NK cell chemokines, in the tissue homogenate was assayed by real-time quantitative PCR. The use and care of the rats followed Regulations for the Administration of Affair Concerning Experimental Animal by State Science and Technology Commission. Results No inflammatory sign in ocular anterior segment of the rats was seen in the experimental control group. The expansion of rat iris vessels was found in the EAU 6-day group, and exudes and hypopyon of the anterior chamber occurred in the EAU 9-day group and the inflammation peaked in the EAU 12-day gorup. The rat retinal structure was normal in the experimental control group, and the arrangement disorder of retinal structure, the cell separation in outer nuclear layer and damage of photoreceptors were found under the optical microscope in different degree in various EAU groups, with the most serious change in the EAU 12-day group. Immunofluorescent double staining showed normally arranged nucleus in the experimental control group,and a lot of NK infiltration was seen in the EAU 6-day group and peaked in the EAU 9- day group. The expression level of CXCL10 mRNA in the EAU 9-day group was 34. 298 ± 16. 689, which was significantly higher than that in the EAU 3-,6- and 12-day group, respectively （1. 390±0. 660,3. 359±2. 581,4. 711 ± 1. 387） （all at P〈0. 01 ）. No significant differences were found in the relative expression of CXCL12 mRNA among different EAU groups （F= 2. 851 ,P〉0. 05）. Conclusions Retinal NK cell infiltration occurs in the early stage of EAU,and the severity of NK cell infiltration is consistent with the inflammatory process and CXCL10 expression, suggesting NK ceils play an important role in the early stage of EAU,and CXCL10 is an important chemokine of NK cells in EAU rats.