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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 observe the differentiation of mouse embryonic stem cells on the human amniotic membrane and conjunctival matrix of rabbits. Methods Thirty-six New Zealand rabbits were randomly divided into three groups of 12 rats each. Group A: Mouse ES-GFP cells expressing green fluorescent protein (GFP) gene were labeled with 5-bromodeoxyuridine (BrdU) and then subcutaneously transplanted into rabbits. Group B: Mouse ES- GFP cells were inoculated into human amnion for 4 days, then labeled with BrdU and then transplanted to the defect area of experimental rabbit conjunctiva. Group C: transplanted amniotic membrane was used to transplanted the rabbit conjunctiva defect. At 1, 2, 3, 4, 6 and 8 weeks after transplantation, the experimental eyes of each group were harvested for fluorescence microscopy, histology and immunohistochemistry. The fluorescence of ES-GFP cells was detected by fluorescence. The survival and morphological changes of ES-GFP cells transplanted to the conjunctival stroma and the local reactions of transplantation were examined. The expression of CK3 / CK12 and CK13 in BrdU-labeled ES-GFP cells transplanted to the conjunctival stroma was detected by immunohistochemistry. Results After ES cells were inoculated into human amnion, mouse ES-GFP cells could attach to the amnion, and some ES-GFP cells differentiated into polygonal epithelial-like cells after co-cultured with amniotic membrane for 4 days. Immunohistochemistry showed that β1 integrin was positive. The amniotic membrane loaded with ES-GFP cells was transplanted into the rabbit conjunctiva defect area. Green fluorescence band was detected in the conjunctival epithelium by fluorescence microscope after operation. Immunohistochemistry showed positive expression of CK13 in the conjunctival epithelium and negative expression of CK3 / CK12 in the reconstructed Of the conjunctival epithelium can detect BrdU nuclear positive cells, no abnormal proliferation of cells. CONCLUSION: Conjugated human amnion-loaded mouse ES cells can survive and proliferate in rabbit conjunctival stroma.