兔口腔黏膜上皮细胞羊膜种植及生物学特性研究

Implanting and biological character of rabbit oral epithlial cells on amniotic membrane

目的研究羊膜作为载体种植兔口腔黏膜上皮细胞的可行性及生物学特征,探索眼表重建良好和来源丰富的移植材料和方法。方法将兔口腔黏膜上皮细胞种植于去上皮羊膜基底膜进行体外培养扩增。倒置显微镜观察细胞在羊膜上的动态生长过程,将培养获得的复合上皮片作常规病理切片HE染色和透射电镜观察。采用AE5单克隆抗体鉴定培养组织中有无角蛋白3表达。结果兔口腔黏膜上皮细胞在去上皮羊膜基底膜面能黏附生长并增殖,体外培养3周左右即可在羊膜上汇合成片并分层。形成的复合组织由羊膜基底膜和较扁平的2～3层上皮细胞组成,基底层细胞与羊膜之间有半桥粒连接,细胞之间可见桥粒连接,细胞表达角蛋白3。结论兔口腔黏膜上皮细胞在去上皮羊膜上体外培养扩增能获得类似于角膜上皮的复层组织,为解决眼表重建供体来源不足的现状带来了新的希望。

[ Objective ] To investigate the cultivation and biological characteristics of rabbit oral epithelial cells planted on amniotic membrane （AM） as a substrate and try to find out satisfactory methods and rich material for ocular surface reconstruction. [Methods] Rabbit oral epithelial cells（EC） were implanted on denuded AM. The resulted sheet was examined by inverted microscope, HE staining pathological section and electron microscopy. The sheet was also immanohistochemically labeled with AE5 monoconal antibody of cytokeratin 3. [Results] Rabbit oral ECs could stick and confluence on denuded AM. After 3 weeks in culture, two to three layers of stratification oral epithelial cells had been established with the presence of CK3. EM revealed that numerous desmosomal junctions were among the multiple layers of oral ECs which attached to the basement membrane of AM with hemidesmosomes. [ Conclusion ] Stratification epithelium similar to normal rabbit corneal epithelium can be obtained by implanting rabbit oral ECs on denuded AM. That brings a probe to ocular surface reconstruction.