羊膜载体培养标记兔角膜内皮细胞移植的研究

目的探讨以羊膜基底膜为载体培养兔角膜内皮细胞移植的可能性,为培养内皮细胞移植治疗角膜内皮失代偿疾病提供依据与方法。方法体外培养扩增兔角膜内皮细胞,采用亲脂性碳青染料(CM-DiI)对细胞进行标记,种植于去除上皮细胞的羊膜基底膜上,体外培养形成单层角膜内皮层,并进行形态学、组织学、超微结构及细胞标记情况的观察;将羊膜为载体培养的内皮层对切除后板层的兔角膜进行移植,同时以无培养内皮细胞的羊膜移植和单纯角膜后板层切除为对照,术后观察角膜透明度与厚度,对其进行组织学与细胞标记情况的检测。结果 5～7d 角膜内皮细胞即在羊膜基底膜上融合成单层,细胞为扁平多角形,排列紧密,密度可达(3202.84±347.77)个/mm~2,荧光显微镜下可见内皮细胞被 CM-DiI 标记后显现红色荧光;培养内皮层移植后角膜维持相对的透明与薄度,而无内皮细胞羊膜移植和单纯后板层切除两组对照角膜严重水肿、混浊,厚度明显超过实验组角膜;培养内皮移植后角膜形成新的内皮层,通过标记的细胞发现移植后细胞仍为培养移植的内皮细胞而非周边细胞的移行。结论羊膜基底膜是角膜内皮细胞生长和移植的良好载体,体外培养角膜内皮细胞移植有望代替供体角膜移植,具有广阔的应用前景。(中华眼科杂志,2006,42:925-929)

Amniotic membrane as a carrier for cultivated and labeled corneal endothelial cell transplantation

OBJECTIVE: To investigate the feasibility of using amniotic membrane (AM) as a carrier for cultured corneal endothelial cells transplantation. METHODS: Rabbit corneal endothelial cells were cultivated, passaged, and labeled with the fluorescent tracker CM-DiI, then transplanted onto denuded AM. The cell density, morphology, histology and ultrastructure of the cultured cells on AM were examined by light microscope, fluorescent microscopy and scanning electron microscope. To determine whether these composite endothelial sheets were functional in vivo, the sheets were transplanted onto rabbit corneas whose deep lamellar with Descemet's membrane and endothelial cells had been completely removed. The deep layer excised rabbit corneas without any transplantation or with transplantation of denuded AM were treated as controls. After transplantation, the corneal appearance and corneal thickness were examined. The grafts were examined for cell density, morphology and histology 4 weeks after the surgery. RESULTS: A monolayer of endothelial cells were confluent on the AM 5 - 7 days after seeding. The density of the endothelial cells was (3202.84 +/- 347.77)/mm(2). Morphologically, the sheets consisted of a fairly continuous layer of flat polygonal endothelial cells that appeared uniform in size with tight junctions. The labeled cells were red in color under fluorescent microscopy. After transplantation, the corneas that received cultured endothelial cells had little edema and retained their thinness and transparency, while the corneas of controls showed prominent edema and opacity, indicating that these cells were transplanted cells rather than the cells migrated from peripheral cornea. Four weeks after surgery, the labeled endothelial cells were still found in the cornea with red fluorescence. CONCLUSIONS: AM maintains cultured endothelial cells' morphology and function both in vitro and in vivo. AM could serve as a carrier for corneal endothelial cells transplantation. These results provide a promising method for the treatment of diseases caused by corneal endothelial disorders.