兔角膜缘上皮细胞培养与移植的实验研究

Experimental study on the culture and transplantation of limbal epithelium cells of rabbit

目的观察以明胶为载体培养的角膜缘上皮细胞移植治疗兔角膜缘干细胞缺乏症的疗效。方法切除实验兔角膜缘组织并用NaOH处理制作角膜缘干细胞缺乏动物模型,切除的角膜缘上皮细胞在铺有明胶裁体的细胞培养板上培养5 d后(角膜上皮细胞移植术前24 h)用3H胸腺嘧啶核苷标记,于模型动物上行角膜缘上皮细胞移植术,术后观察角膜修复情况,并做病理学检查及同位素检测。结果 1.细胞培养:兔角膜缘上皮细胞可以在明胶载体上培养,增殖、分化为密集的角膜上皮细胞层。2.移植眼情况:行角膜缘上皮细胞移植术后,模型兔眼角膜上皮完整,新生血管减少;病理检查显示移植眼角膜缘及周边部上皮细胞为多层结构,角膜新生血管消失,基质内炎性细胞浸润减轻;角膜缘上皮细胞移植术后4周移植眼角膜仍可测到3H胸腺嘧啶核苷。结论角膜缘上皮细胞移植治疗角膜缘干细胞缺乏症可恢复角膜缘干细胞缺乏病变角膜上皮结构的完整性,减少角膜新生血管的形成,维持角膜缘干细胞的屏障功能,为角膜移值提供更好的条件。

】OBJECTIVE To study the treatment of autotransplantation on rabbit limbal stem cells lack by cultured limbal epithelium cells cultured on gelatin.METHODS Limbal epithelium cells had been cultured on gelatin in DMEM/HamF12 medium for one week,and cultured 1imbal epithelium cell had been marked with 3HTDR for one day,then limbal epithelium cells and gelatin were transplanted on the limbal and scleral of the rabbit model with lack of corneal stem cells by autotransplantation.The corneal changes were observed by a slitlamp every day,the corneaI pathological changes and 3HTDR content were examined.RESULTS Rabbit limbal epithelium cells continued to proliferate,differentiate and form multiple cell layers on gelatin.After autotransplantation with cultured epithelium cells and gelatin the rabbit epithelium showed corneal phenotype and progressive decrease of vascularity and stromal infiltration in the limbal and peripheral zone.Pathological examination verified that the limbal and peripheral corneal epithelium was composed of multilayer cells;the neu-vascularization was reduced and stromal inflammatory cells were decreased.The 1imbal content 3HTDR by isotope radioisotope was examined four weeks after operation.CONCLUSIONS Transplantation with cultured limbal epithelium cells could restore the composition of corneal epithelial cell,decrease neo-vascularization,maintain the function of limbal cellular barrier and provide better condition for later keratoplasty.