“去上皮”羊膜对体外培养结膜上皮细胞的作用

The effects of acellular human amniotic membrane on the culture of human conjunctival epithelial cells

目的 :探索羊膜基底膜对结膜上皮细胞形态功能的影响。方法 :把“去上皮”羊膜按照基底膜或实质面向上分为两组 ,接种结膜上皮细胞 ,同样条件下培养。取培养 14天的两组标本进行组织学 (HE和PAS染色 ) ,透射电镜 ,以及免疫组化检查 (包括抗角蛋白和抗波形蛋白染色 )。结果 :细胞 72小时内贴附在羊膜上。基底膜面接种的上皮细胞顶端见微绒毛 ,单层排列 ,细胞间有丰富桥粒 ,基底膜完整 ,可见半桥粒 ,抗角蛋白染色阳性 ,抗波形蛋白染色阴性 ;实质面接种的上皮细胞胞浆内有大量脂滴 ,表面未见微绒毛 ,细胞松散堆积 ,与羊膜连接不紧密 ,未见桥粒和半桥粒 ,抗角蛋白染色弱阳性 ,抗波形蛋白染色强阳性。结论 :羊膜基底膜是结膜上皮细胞体外培养的良好载体 ,有利于上皮细胞生长、粘附、分化 ,并能减缓上皮细胞老化。

Objective:To study the influence of amniotic membrane on the morphology and function of cultured human conjunctival epithelial cells. Methods:Isolated human conjunctival epithelial cells were cultured on the basement membrane or stroma surface of an acellular human amniotic membrane.After 14 days,the cultured epithelial cells were examined with staining of HE,PAS,transmission electronic microscopy,and immunohistochemical staining using monoclonal antibodies (anti Pan-Keratin and anti Vimentin). Results:The cultured cells attached to the amniotic membrane within 72 hours.Conjunctival epithelial cell cultured on basement membrane formed a better-organized monolayer and had more mitochondria endoplasmic reticulia under transmission election microscope than that on the stromal surface.There were a large amount of the desmosomes and hemidesmosomes in the cells cultured on basement membrane,while no one was observed in the cells cultured on stromal surface.The cells cultured on basement membrane were stained positively for keratin,but the cells cultured on stromal were stained positively for Vimentin. Conclusions:The basement membrane of amniotic membrane is the proper substrate for the growth of human conjunctival epithelial cells.It facilitates epithelial proliferation,prevents from the epithelial aging,and reinforces adhesion of the epithelial cells.It can guide the epithelial cells differentiation along the right direction.