兔口腔黏膜上皮干细胞分化为角膜上皮样细胞的体外研究

目的以羊膜做载体将兔口腔黏膜上皮干细胞诱导为角膜上皮样细胞,探讨口腔黏膜上皮细胞作为种子细胞体外培养构建组织工程角膜的技术方法,为眼表重建提供材料。方法用Ⅳ型胶原黏附法分离纯化口腔黏膜上皮干细胞,对体外培养的口腔黏膜干细胞进行免疫表型鉴定。将筛选后获得的口腔黏膜上皮干细胞种植在去上皮羊膜表面体外培养,待细胞融合成单层后置入插入式培养皿中进行气液界面培养,促进细胞分化形成复层。培养数日后进行苏木素-伊红(HE)染色和免疫组织化学、光镜及透射电镜检测,观察羊膜-复层上皮组织结构,免疫荧光检测干细胞特异性标志物P63以及角膜上皮细胞标志物角蛋白3(K3)并与角膜上皮组织比较。结果体外诱导培养数天后,羊膜载体的口腔黏膜上皮细胞形成复层,在组织形态及生物学特性上与角膜上皮相似。并且组织细胞P63、K3表达明显,角膜特异性生物学标志物免疫组织化学染色呈阳性。结论在本实验的培养条件下,获得的口腔黏膜上皮干细胞可在体外培养扩增,呈克隆性生长,具有很强的增殖能力。口腔黏膜上皮干细胞体外培养可构建类角膜上皮。

In vitro study on the differentiation of rabbit oral epithelium stem cell into corneal epithelioid cell

Objective To investigate the technology of using oral mucosal epithelial stem cells as seed cell to fabricate tissue engineering mueosa, which studied on the corneal epithelioid cell induced by rabbit oral epithelium stem cell using amniotic membrane as vector. Methods Purification by adhesion method of IV type collagen and im- munohistochemistry to oral epithelium stem cells were performed. These purified cells were cultured on amniotie mem- brane, then were placed on a culture insert plate for air-lifting culture after formation of monolayer, in order to promote cell differentiation into multiple laters. Several days later, HE staining, immunohistoehemistry, light and transmission electron microscopy detection were performed, as well as the observation to the amniotie-muhiple layer epithelial orga- nization structure and comparison between special marker of neural stem cells （P63）, certain marker of corneal epithe- lial cells （K3） and corneal epithelial tissue by immunofluoreseence. Results Multilayered oral mueosal sheets were similar with cornea epithelia in organization and biological characteristics after induction culture for a few days. Ex- pression of P63 and K3 were evident and immunofluorescence of special marker showed positive. Conclusion Oral mueosal epithelial stem cells obtained like this study can culture in vitro with strong clonal proliferation and can be transformed into resemble corneal epithelial cells.