羊膜和结膜基质诱导胚胎干细胞分化为上皮样细胞的初步研究  

The preliminary study on differentiate into epithelial-like cells of embryonic stem cells by amniotic membrane and conjunctival stroma

作　　者：黄丹平[1] 葛坚[1] 高前应[1] 卢蓉[1] 郑健樑[1] 

机构地区：[1]中山大学中山眼科中心,广州市510060

出　　处：《中华显微外科杂志》2007年第5期363-366,406,共5页Chinese Journal of Microsurgery

基　　金：广东省自然科学基金(031734)

摘　　要：目的观察小鼠胚胎干细胞在保存人羊膜及兔眼表结膜基质上分化的情况。方法36只新西兰兔随机分为3组,每组12只。A组:将表达绿色荧光蛋白(green fluorescent protein,GFP)基因的小鼠ES-GFP细胞用5-溴脱氧尿嘧啶(BrdU)标记后行实验兔结膜下移植;B组:将小鼠ES-GFP细胞接种到人羊膜上共培养4d,用BrdU标记后移植到实验兔结膜缺损区;C组:采用保存羊膜移植到实验兔兔结膜缺损区。分别于移植后1、2、3、4,6和8周,摘取各组实验眼行荧光显微镜、组织学和免疫组织化学检查,荧光检测ES-GFP细胞在组织中的荧光表达,组织学检查移植到结膜基质的ES-GFP细胞的存活、形态变化以及移植局部的反应等情况;免疫组织化学检测移植到结膜基质的带有BrdU标记的ES-GFP细胞CK3/CK12和CK13的表达。结果小鼠ES-GFP细胞接种到人羊膜后能在羊膜上贴附生长,与羊膜共培养4 d后部分ES-GFP细胞分化为多角形上皮样细胞,免疫组化显示β1整合素阳性。将负载有ES-GFP细胞的羊膜移植到兔结膜缺损区,术后荧光显微镜可在结膜上皮层检测到绿色荧光带,免疫组织化学检测结膜上皮层CK13表达阳性,CK3/CK12表达阴性,在重建的结膜上皮层可检测到BrdU核阳性细胞,未见异常增殖细胞。结论采用保存人羊膜负载小鼠ES细胞行兔结膜移植,小鼠ES细胞能在兔结膜基质存活并增殖。Objective To investigate the differentiation of murine embryonic stem cells （ESCs） by human amniotic membrane and conjunctival stroma of rabbit. Methods Thirty-six New Zealand albino rabbits were equally and randomly divided into 3 groups：group A ：The ESCs expressed with green flurescent protein （ES-GFP） and labeled with 5 -Bromo-2-deoxyuridine （BrdU） wer injected under the conjunctiva of the rabbits group B：The ES-GFP were cultured on amniotic membrane for 4 days and then labeled with BrdU, they were transplanted onto the scleral area of the rabbits, group C： the amniotic membrane was transplanted onto the scleral area of the rabbits. The eyeballs were taken off after 1,2,3,4,6 and 8 weeks. The growth and differentiation of ES-GFP were observed by histopathological and immunohistochemical examination. Results The ES-GFP cells grew well on amniotic membrane when they were bred on amniotic membrane, Some of the cells could differentiate into epithelial-like cells after co-culture with amniotic membrane for 4 days. The immunohistochemical staining results showed that these cells were positive in integin-β1. When the ES-GFP cells cultured on amniotic membrane transplanted on the scleral area of rabbits, Immunocytochemical examination reveals that the conjunctival epithelial were positive in cytokeratin 13 staining and negative in CK3/CKl2staining,and the cells which postive in BrdU in reconstructive conjunctival epithelial were found. There were no abnormal proliferative cells. Conclusion The murine ESCs may be cultured on human amniotic membrane in vitro. When they were cultured on amniotic membrane and transplanted onto the scleral area of rabbits they can survive, proliferate and without abnormal proliferative cells.

关 键 词：胚胎干细胞 羊膜 结膜上皮细胞 诱导分化 

分 类 号：R777[医药卫生—眼科] R318.0[医药卫生—临床医学]