不同羊膜为载体的人角膜缘上皮细胞原代培养研究

Primary culture of human limbal stem cells on different human amniotic membrane

目的观察完整羊膜与去上皮羊膜两种不同载体上所培养的人角膜缘上皮细胞的生长特点及P63的表达情况。方法采用组织块培养法体外扩增培养人角膜缘上皮细胞,将来自同一供体的2块角膜缘组织随机种植于完整上皮羊膜(A组)和去上皮羊膜(B组)上培养。倒置显微镜下观察培养细胞体外生长的形态及特点,并运用免疫细胞化学方法观察所培养的细胞P63抗体的表达情况。结果 A、B两组细胞(每组10例)分别培养成功8例。两组培养成功的8例细胞均能在体外移行、扩增、连接成片。B组移行扩增的速度较A组快。两组细胞生长至14 d时做免疫细胞化学染色,共16孔细胞中均可见P63阳性细胞,其中A组细胞P63阳性率高于B组细胞(P<0.05)。结论角膜缘上皮细胞在完整羊膜和去上皮羊膜上均能体外扩增,去上皮羊膜为载体的角膜缘上皮细胞增殖分化快,完整羊膜为载体的细胞P63阳性率高于去上皮羊膜为载体的细胞。

Objective To observe the growth characteristics and the expression of P63 of human limbal epithelial cells,which were cultured on the intact or denuded amniotic membrane.Methods Two explants of human limbus which obtained from the same donor were randomly placed and cultured by the explant culture method in vitro on the intact（A group） and denuded（B group） amniotic membrane.To observe the morphology and growth characteristics of the cultured cells in vitro under inverted microscope and to compare the expression of P63 antibody by immunocytochemical method.Results The two groups of cells（10 cases each group） were cultured successfully respectively in 8 cases which could to migration,proliferation and connecting in pieces in vitro.The speed of migration and proliferation of B group was faster than A group.The two groups of cells were taken immunocytochemical staining when growth to the14 th day.P63 positive cells were observed in all the 16 cultured samples and the positive staining rate of A group was higher than that in B group（P〈0.05）.Conclusions Limbal epithelial cells can be cultured successfully on two kinds of amniotic membrane.The proliferation and differentiation of the cells which cultured on the denuded amniotic membrane were faster,and positive immunocytochemical staining of P63 in the intact amniotic membrane was higher than which in the denuded amniotic membrane at 14 th day.