培养的兔羊膜上皮细胞构建角膜表层移植片的研究

Reconstruction of transplant graft of corneal surface layers by using in vitro cultured AECs of rabbit

目的应用培养的兔羊膜上皮细胞(AECs)体外构建复层上皮细胞-角膜基质移植材料,探讨利用AECs重建角膜表层的可行性。方法取妊娠晚期新西兰大白兔(27～28孕周)的羊膜,制成AECs单细胞悬液,用含血清和表皮生长因子(EGF)的DMEM/F12培养液培养、传代,利用免疫组织化学单克隆抗体AE1/AE3、AE5检测培养的AECs中细胞角蛋白ck3/12的表达;将体外培养的2～3代兔AECs种植在新鲜兔角膜基质上,利用气-液界面培养法使之复层化,体外构建复层上皮细胞-角膜基质移植材料,进行光学显微镜和扫描电镜观察,并进行免疫组织化学测定。结果体外培养的兔AECs呈现单克隆抗体AE1/AE3、AE5表达阳性,AECs在新鲜兔角膜基质上能形成形态类似于正常角膜上皮细胞的3～5层复层结构,且复层化后的上皮细胞单克隆抗体AE5表达阳性。结论应用培养的AECs能成功构建类似角膜表层的复层上皮细胞-角膜基质移植材料,AECs可能成为重建角膜表层的一种新的细胞来源。

Objective The purposes of this study were to reconstruct the transplanting materials including stratified epithelial cells and corneal stroma by using cultured amniotie epithelial cells （AECs） of rabbit and explore the feasibility of reestablishing corneal surface layers using AECs. Methods AECs were obtained from the late trimester of pregnancy of New Zealand white rabbits （27-28E）. Cells were cultured and passaged in DMEM/F12 with 10% fetal bovine serum and 20ng/mL epidermic growth factor （EGF）. The AECs were detected to evaluate the expression of cytokeratin （CK3/12） by immunohistoehemistry with monoclonal antibody AE1/AE3 and AE5. The 2nd or 3rd generation of AECs was planted on fresh corneal stroma without epithalium. The AECs were stratified and the corneal-like transplanted materials including stratified epithelial cells and corneal stroma were re-established using air-lifting cultivation method. The stratified transplanted materials were observed under the light microscope and scanning electron microscope and detected to evaluate the expression of CK3/12 with monoclonal antibody AE1/AE3 or AE5. Results Primarily cultured AECs exihibited good growth statue in 36 hours and formed confluce and membrane in 48 hours. AE1/AE3 and AE5I were positively expressed in cultured AECs,which was shown by the brown staining in cytoplasm. The morphology of AECs in fresh corneal stroma was similar to that of corneal epithelial cells and the AECs were successfully stratified on fresh corneal stroma and formed the corneal epithelium-like construction including 3-5 stratifications by airlifting cuhivation method. The stratified epithelial cells showed the positive reaction for AE1/AE3. Conclusion The transplanting materials can be re-established successfully by the usage of cultured AECs. AECs could be potential seed cells for the re-establishment of corneal surface layers.