种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究  

Transplantation of porcine corneal matrix seeded with human bone marrow mesenchymal stem cells in a model of corneal injury

作　　者：施文建[1,2,3] 侯光辉[1,2] 吴静[4] 徐锦堂[4] 

机构地区：[1]暨南大学附属第三医院 [2]珠海市人民医院眼科,珠海519000 [3]福建医科大学附属第二医院眼科,泉州362000 [4]暨南大学医学院眼科学研究室,广州510630

出　　处：《眼科研究》2010年第4期300-305,共6页Chinese Ophthalmic Research

基　　金：珠海市科技计划项目资助(PB20051014)

摘　　要：目的研究种植人骨髓间充质干细胞(MSCs)的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。Background Tissue engineering cornea transplantation is a promising treatment way for keratopathy.ObjectiveThe present study attempts to investigate the possibility of porcine corneal matrix seeded with human bone marrow mesenchymal stem cells（MSCs） for the reparation of injury rabbit corneas.MethodsMSCs were isolated from the removed human bone during the sacrectomy and purified by whole bone marrow adherence method and passaged in DMEM/F12 containing fetal bovine serum in vitro.The immunophenotype of cultured MSCs was evaluated by flow cytometry.The differentiated adipocytes and osteoblasts from the MSCs by directional induce was identified using AgNO3 staining and Oil red O staining respectively.The generations 3 of MSCs were cultured on porcine corneal matrix without corneal epithelium for 4 days and then transplanted onto 12 left eyes of 12 New Zealand white rabbits,and porcine corneal matrix without corneal epithelium was transplanted onto the left eyes of other 12 New Zealand white rabbits as control group.The growth of MSCs and porcine corneal matrix was evaluated by histopathological examination after 2,4,8 weeks.The expression of cytokeratin 12（CK12）,a marker for corneal epithelial cells,on MSCs was identified by immunohistochemistry and immunofluorescence staining.Written informed consent was obtained from each patient prior to the initial of treating procedure.The experiment followed the Regulation for Administration of Affair Concerning Experimental Animals issued by State Science and Technology Commission.ResultsCultured MSCs showed the positive response for CD29（95.97%）,CD44（96.49%）,CD90（92.79%）,CD105（94.66%）,CD34（0.59%）,CD45（0.36%）.Differentiated osteoblasts presented the black staining for AgNO3 and adipocytes displayed the orange red color for Oil red O.MSCs grew well on the porcine corneal matrix.The corneal grafts survived well without rejection till the fourth week after transplantation in experimental eyes and the rejection of corneal graft occurred in control

关 键 词：骨髓间充质干细胞 猪角膜基质 组织工程角膜 角膜移植 

分 类 号：R779.65[医药卫生—眼科] Q81[医药卫生—临床医学]