中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2013年
2期
151-155
,共5页
柯红琴%刘磊%栗静%王虎杰%黎冬平
柯紅琴%劉磊%慄靜%王虎傑%黎鼕平
가홍금%류뢰%률정%왕호걸%려동평
角膜缘干细胞缺乏%动物模型%兔
角膜緣榦細胞缺乏%動物模型%兔
각막연간세포결핍%동물모형%토
Limbal stem cell deficiency%Animal model%Rabbit
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1 mm、角膜缘外2 mm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 ～ 150 μm.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-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.
揹景 角膜緣榦細胞缺乏(LSCD)可導緻各種眼錶功能異常,嚴重的LSCD會導緻角膜結膜化、慢性炎癥、持續的角膜上皮缺失、角膜混濁和伴髮的長期視力喪失. 目的 研究成功構建兔全LSCD模型的適宜方法和最佳時間,觀察造模後各時間點模型眼角膜的病理結構改變. 方法 選擇3～5月齡健康日本大耳白兔20隻,用手術刀切除左眼360°角膜緣內1 mm、角膜緣外2 mm的角膜緣上皮組織和剩餘的全部中央角膜上皮組織和淺層基質,切除組織厚度為100 ～ 150 μm.分彆在術後2～5週行裂隙燈檢查,以角膜混濁、角膜新生血管情況和上皮熒光素鈉染色情況為觀察指標,按照國際通用LSCD模型評分標準對模型眼進行評分.分彆于術後2、3、4、5週穫取模型眼的角膜組織標本,行囌木精-伊紅染色和過碘痠希伕染色觀察角膜組織結構和杯狀細胞的改變,用間接免疫熒光染色法檢測細胞角蛋白3(CK3)在角膜組織中的錶達.手術後不同時間點造模成功率的差異比較採用Fisher精確概率法.結果 術後第2、3、4、5週時的模型成功率分彆為12.5％、62.5％、81.3％和87.5％,術後第3週造模成功率明顯高于第2週,差異有統計學意義(P=0.009)；第3週與第4週相比差異無統計學意義(P=0.465),第3週與第5週相比差異有統計學意義(P=0.049),第4週與第5週相比差異無統計學意義(P=0.200).造模成功的平均時間為(3.21±0.80)週.造模成功的標準為模型眼角膜明顯混濁,角膜新生血管形成和熒光素鈉染色暘性.模型眼的組織病理學檢查示角膜基質水腫,較多炎性細胞浸潤和新生血管形成.過碘痠希伕染色髮現,僅在造模後第5週的角膜組織中髮現杯狀細胞.各時間點造模成功模型眼角膜組織中均未見到CK3暘性錶達. 結論 用手術切除角膜緣及全部中央角膜上皮的方法可以成功構建全LSCD動物模型,造模成功的時間約為手術後3.2週.
배경 각막연간세포결핍(LSCD)가도치각충안표공능이상,엄중적LSCD회도치각막결막화、만성염증、지속적각막상피결실、각막혼탁화반발적장기시력상실. 목적 연구성공구건토전LSCD모형적괄의방법화최가시간,관찰조모후각시간점모형안각막적병리결구개변. 방법 선택3～5월령건강일본대이백토20지,용수술도절제좌안360°각막연내1 mm、각막연외2 mm적각막연상피조직화잉여적전부중앙각막상피조직화천층기질,절제조직후도위100 ～ 150 μm.분별재술후2～5주행렬극등검사,이각막혼탁、각막신생혈관정황화상피형광소납염색정황위관찰지표,안조국제통용LSCD모형평분표준대모형안진행평분.분별우술후2、3、4、5주획취모형안적각막조직표본,행소목정-이홍염색화과전산희부염색관찰각막조직결구화배상세포적개변,용간접면역형광염색법검측세포각단백3(CK3)재각막조직중적표체.수술후불동시간점조모성공솔적차이비교채용Fisher정학개솔법.결과 술후제2、3、4、5주시적모형성공솔분별위12.5％、62.5％、81.3％화87.5％,술후제3주조모성공솔명현고우제2주,차이유통계학의의(P=0.009)；제3주여제4주상비차이무통계학의의(P=0.465),제3주여제5주상비차이유통계학의의(P=0.049),제4주여제5주상비차이무통계학의의(P=0.200).조모성공적평균시간위(3.21±0.80)주.조모성공적표준위모형안각막명현혼탁,각막신생혈관형성화형광소납염색양성.모형안적조직병이학검사시각막기질수종,교다염성세포침윤화신생혈관형성.과전산희부염색발현,부재조모후제5주적각막조직중발현배상세포.각시간점조모성공모형안각막조직중균미견도CK3양성표체. 결론 용수술절제각막연급전부중앙각막상피적방법가이성공구건전LSCD동물모형,조모성공적시간약위수술후3.2주.
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 conjunctivalization,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 μm 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 slitlamp 2,3,4,5 weeks after operation,and the inflammatory score was performed according to corneal opacity,corneal neovascularization and corneal flurescein 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 cytokeratin 3(CK3) in the cornea was detected using indirect immuno-fluorescence 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.
