兔全角膜缘干细胞缺乏模型的构建和鉴定

Establishment of assessment of rabbit model of total limbai stem cell deficiency

背景角膜缘干细胞缺乏（LSCD）可导致各种眼表功能异常，严重的LSCD会导致角膜结膜化、慢性炎症、持续的角膜上皮缺失、角膜混浊和伴发的长期视力丧失。目的研究成功构建兔全LSCD模型的适宜方法和最佳时间，观察造模后各时间点模型眼角膜的病理结构改变。方法选择3～5月龄健康日本大耳白兔20只，用手术刀切除左眼360。角膜缘内1mm、角膜缘外2mm的角膜缘上皮组织和剩余的全部中央角膜上皮组织和浅层基质，切除组织厚度为100～150μm。分别在术后2～5周行裂隙灯检查，以角膜混浊、角膜新生血管情况和上皮荧光素钠染色情况为观察指标，按照国际通用LSCD模型评分标准对模型眼进行评分。分别于术后2、3、4、5周获取模型眼的角膜组织标本，行苏木精一伊红染色和过碘酸希夫染色观察角膜组织结构和杯状细胞的改变，用间接免疫荧光染色法检测细胞角蛋白3（CK3）在角膜组织中的表达。手术后不同时间点造模成功率的差异比较采用Fisher精确概率法。结果术后第2、3、4、5周时的模型成功率分别为12．5％、62．5％、81．3％和87．5％，术后第3周造模成功率明显高于第2周，差异有统计学意义（P=0．009）；第3周与第4周相比差异无统计学意义（P=0．465），第3周与第5周相比差异有统计学意义（P=0．049），第4周与第5周相比差异无统计学意义（P=0．200）。造模成功的平均时间为（3．21±0．80）周。造模成功的标准为模型眼角膜明显混浊，角膜新生血管形成和荧光素钠染色阳性。模型眼的组织病理学检查示角膜基质水肿，较多炎性细胞浸润和新生血管形成。过碘酸希夫染色发现，仅在造模后第5周的角膜组织中发现杯状细胞。各时间点造模成功模型眼角膜组织中均未见到CK3阳性表达。结论用手术切除角膜缘及全部中央角膜上皮的方法可以成功构建全LSCD动物模型，造模成功的时间约为手术后3．2周。

Background The deficiency of limbus stem cells will cause various kinds of the disorder of eye surface,in serious condition, it will lead to corneal eonjunetivalization, chronic inflammation, lasting deficiency of corneal epithelium and corneal opacity accompanying long-term visual loss. Objective The aim of this study was to investigate how to establish and evaluate the successful model of total limbal stem cell deficiency （ LSCD）. Methods Twenty Japanese white rabbits aged 3- 5 months were selected. The limbus corneal epithelium was removed from 1 mm inner to 2 mm outer of the limbus 100-150 p^m in deep, and the total central epithelium and shallow stroma were simultaneously removed in the left eyes using scalpel. The experimental eyes were examined by slit- lamp 2,3,4,5 weeks after operation, and the inflammatory score was performed according to corneal opacity, corneal neovascularization and corneal flureseein dye using international universal rating criteria of LSCD model. Cornea tissue was obtained in various time points mentioned above to examine the structural change and goblet cells. The expression of eytokeratin 3（CK3） in the cornea was detected using indirect immuno-fluoreseenee staining. Fisher Exact Probability method was used to compare the successful rate of models in different time points. Results The successful rate of models rate was 12.5% ,62.5% ,81.3% and 87.5% ,respectively 2,3,4,5 weeks after operation, showing a significant difference between the 3-week group and 2-week group （P = 0. 009 ）. However, no significant differences were found in the model successful rate between the 3-week group and the 4-week group,the 4-week group and the 5-week group （ P = 0. 465,0. 200 ）. There was a little difference between the 3-week group and the 5-week group （ P = 0. 049 ）. The mean time of achieving successful model was （ 3.21 ＋ 0.80 ） weeks. The result of hematoxylin ＆ eosin staining showed the edema of cornea stroma, a mass of inflammatory cells infiltration and new vessels. Goblet cells could be found by Periodic acid-Schiff staining 5 weeks after operation. Expression of CK3 was absent in the corneas of successful model eyes. Conclusions The LSCD model can be successfully established by cutting off the epithelium of limbus and central cornea, and the average time of model formation is about 3.2 weeks.