兔角膜缘干细胞的体外培养、鉴定及形态学特点

Culture,identification and morphological features of rabbit limbal stem cells in vitro

目的体外培养兔角膜缘干细胞(limbal stem cells,LSCs),观察其生物学特性。方法用含20%胎牛血清DMEM:F12(1:1)培养基对兔LSCs进行体外培养,倒置显微镜及HE染色观察培养细胞体外生长的形态,免疫细胞化学AE5和P63染色鉴定并进行图像分析,透射电镜观察超微结构特点。结果培养2～3d细胞从组织块游出,6～8d形成良好的单层,细胞多呈圆形或卵圆形。电镜观察,培养7dLSCs呈卵圆形,表面有少量微绒毛突起;胞质内细胞器少,核大,核仁明显,核浆比例大,部分细胞可见双核现象。免疫组化观察,培养第7天,大量细胞AE5免疫反应染色呈阴性,阳性细胞数量较少,大量细胞P63免疫反应染色呈阳性;培养第14天,AE5阳性细胞数明显增多,免疫反应染色增强,阳性细胞的平均灰度值显著降低(7d:184.30±9.85,14d:133.36±25.00,P<0.01)。P63免疫反应阳性细胞数第14天与第7天相比明显减少,而阳性细胞的平均灰度值显著增高(7d:108.13±17.96,14d:177.59±7.86,P<0.01)。结论应用组织培养方法获得的原始细胞具有与体内正常LSCs相一致的形态特征;培养第7天为LSCs生长的峰值期。

Objective To establish a method for culturing rabbit limbal stem cells （LSCs） in vitro and observe their biological characteristics.Methods The pieces of rabbit limbal tissue were cultured in the medium DMEM：F12 （1：1） containing 20% fetal bovine serum.The morphological feature of the cultured cells was observed by inverted phase contract microscopy and HE staining.LSCs were identified by immunocytochemical staining with AE5 and P63 antibodies.The ultrastructure of the cultured cells were studied by transmission electron microscopy.Results The cells were emigrated from pieces of limbal tissue after cultured for 2 to 3 d and then formed a favorable monolayer in 6 to 8 d,with most of the cells were round or oval in shape.Under transmission electron microscope,the cultured cells were oval with few microvilli on the surface and few organelles in the cytoplasm;their nuclei were large and the proportion of nucleus to cytoplasm volume was larger,the nucleolus was obvious and 2 nucleolei were found in some cells.Immunocytochemical technique showed that a few of the cultured cells were positive to AE5 staining and most of them were positive to P63 in cultured for 7 d,and in 14 d,the number of AE5-positive cells were obviously increased and their average gray scale were significant decreased from 184.30±9.85 in 7 d to 133.36±25.00 in 14 d （P0.01）.However,the P63-positive cells were obviously diminished in 14 d and their average gray scale were significant increased from 108.13±17.96 in 7 d to 177.59±7.86 in 14 d （P0.01）.Conclusion Our primary cultured rabbit LSCs have consistent biological characteristics with the normal LSCs in vivo.The peak growth period of LSCs is on the 7th day.