角膜缘上皮细胞移植重建眼表的实验研究

Reconstruction of corneal surface with transplantation of corneal limbal epithelial cells

目的探讨角膜缘上皮细胞构建的角膜上皮植片重建眼表的可行性和有效性。方法采用碱烧伤法建立完全性角膜缘干细胞缺陷的SD大鼠动物模型,以羊膜为载体培养人角膜缘上皮细胞,移植重建角膜缘干细胞缺陷眼表,观察术后眼表状态的改变,RT-PCR和免疫组化检测术后10、20、30、45 d及60 d角膜组织CK3/12和P63的表达。结果体外培养的人角膜缘上皮细胞移植术后,完全性角膜缘干细胞缺陷眼表状态较术前和对照组都有不同程度改善;免疫组化和RT-PCR检测表明体外培养角膜缘上皮细胞移植术后角膜组织60 d内P63和CK3/12表达无明显改变。结论以去上皮羊膜为载体,体外培养人角膜缘上皮细胞构建的角膜上皮植片能够重建完全性角膜缘干细胞缺陷眼表。

Objective To evaluate the feasibility and effect of reconstruction of corneal surface with limbal stem cell deficiency （LSCD） in rats. Methods Eyes of rats were rendered total limbal stem cell deficieny by alkaki burn. Human limbal epithelial cells were cultured on human amniotic membrane （AM） and transplanted to reconstruct corneal surface with LSCD. The expressions of CK3/ 12 and P63 proteins and mRNA were assessed by immunohistochemical staining and RT-PCR at 10, 20, 30, 45 and 60 days of postoperation; the changes of ocular surface status were also observed. Results After reconstructed with human limbal epithelial cells ex vivo expanded on human amniotic membrane （AM）, corneal surface in rats with total limbal stem cell deficiency was improved, compared to preoperation and contrast group. The expressiond of cytokeratin CK3/12 and P63 were not significantly changed during 60 days of postoperation. Conclusion Transplantation of human corneal limbal epithelial cells cultivated on de-epithelization amniotic membrane can successfully reconstruct corneal surfaces with total LSCD.