兔骨髓间充质干细胞在兔角膜缘干细胞完全缺乏模型眼表的分化  

Differentiation of rabbit bone marrow mesenchymal stem cells on the ocular surface of total limbal stem cell deficient rabbit models

作　　者：兰梅 杨芳 唐俊明[2] 张蕾[2] 余锦强 LAN Mei;YANG Fang;TANG Jun - ruing;ZHANG Lei;YU Jin - qiang(Department of Oph- thalmology, People＇s Hospital, Hubei University of Medicine, Shiyan442000 , Hubei, Chin)

机构地区：[1]湖北医药学院附属人民医院眼科,湖北十堰442000 [2]十堰市人民医院临床医学研究所,湖北十堰442000

出　　处：《广东医学》2018年第11期1616-1624,共9页Guangdong Medical Journal

基　　金：国家自然科学基金资助项目(编号:81170095);十堰市科学技术研究与开发项目基金(编号:2010-014z)

摘　　要：目的探讨兔骨髓间充质干细胞(BMSC)移植到兔角膜缘干细胞完全缺乏模型后是否能分化成角膜上皮细胞。方法 3~5月龄清洁级日本大耳白兔24只,采用全周角膜缘板层切除术加中央角膜上皮刮除术建立兔右眼角膜缘干细胞完全缺乏模型,4周后观察眼表形态,行苏木素-伊红(HE)染色、过碘酸-雪夫(PAS)染色及DAPI染色鉴定模型成功与否,选取成功模型18只(36只眼),应用随机数字表法分为对照组(18只兔左眼)、模型组、羊膜组、羊膜加BMSC组,后3组每组6只(兔右眼)。取第三代(P3)兔BMSC,以去上皮羊膜为载体培养观察,待细胞铺满羊膜达90%以上时进行移植手术,于移植术后12周观察兔右眼表形态,行HE染色观察角膜结构,免疫荧光检测兔BMSC分化方向。结果建模后4周有21只兔右眼眼表形态评分达到7分及7分以上,行HE染色、PAS染色、DAPI染色,结果显示角膜缘及角膜上皮细胞层被清除,浅基质层大量炎症细胞浸润及新生血管形成,可见杯状细胞形成,说明模型构建成功。移植术后12周照相观察眼表形态,与自身兔左眼角膜相比,模型组兔右眼角膜混浊,有大量新生血管,羊膜组右眼角膜轻度混浊,有少量新生血管,羊膜加BMSC组兔右眼角膜透明,未见新生血管;HE染色显示羊膜加BMSC组角膜结构修复完整,免疫荧光检测到羊膜加BMSC组有角膜分化的特异性标记因子CK3+12表达,而羊膜组未见表达。结论兔BMSC移植到兔角膜缘干细胞完全缺乏模型后,在体内能够分化成角膜上皮细胞或角膜样上皮细胞,恢复眼表结构及功能。Objective To observe differentiation of the rabbit BMSC on the ocular surface in total limbal stem cell deficient rabbit models into CEC. Methods Twenty - four Japanese clean white rabbits （3 - 5 months） were taken as models. Right eye limbal stem cell deficient models were prepared with annulus limbal dissection and central corneal epithelium scraping. Ocular surfaces were observed 4 weeks later, and HE staining, PAS staining and DAPI staining were applied to verify establishment of model. Eighteen successful models （36 eyes） were randomly grouped. The control group consisted of the 18 normal left eyes, total limbal stem cell deficient model group consisted of 6 right eyes, amniotic mem- brane transplantation group consisted of 6 right eyes and the experimental group consisted another 6 right eyes. The 3rd generation BMSC, with denuded amniotic membrane as a carrier, were planted on model eyes while growing to cover more than 90% of amniotic membrane surface. Twelve weeks later, the pictures of ocular surfaces were obtained by digital cam- era where corneal structures were displayed by HE staining and differentiation of rabbit BMSC was tested by immunoflures- ence. Results After 4 weeks of establishing the models, ocular surface scores of 21 rabbits were no less than 7. HE stai- ning, PAS staining, and DAPI staining showed that limbal and corneal epithelial cell layer were removed, plenty of in- flammatory cells infiltrated and neovascularization in the sunerficial basal laver_ with the formation of goblet cells, indica-ting that models were successfully established. After 12 weeks of transplantation, compared with the normal left cornea, opacity of the model eyes （right cornea） with a large number of neovascularization was observed in model group; slightly turbid with minimal neovascularization was observed in amniotic membrane transplantation group; and transparent without any neovascularization was observed in experiment group. HE staining showed that corneal structure was complete in amni- otic m

关 键 词：兔骨髓间充质干细胞 兔角膜缘干细胞完全缺乏模型 角膜上皮细胞 移植 分化 

分 类 号：R772.2[医药卫生—眼科]