密度梯度离心法分离和纯化视网膜色素上皮细胞

目的 观察密度梯度离心法分离、纯化原代及传代视网膜色素上皮(retinal pigment epithelium,RPE)细胞的效果。 方法 将原代及传代后仍有成纤维细胞污染的人RPE细胞分别各分为实验组和对照组，实验组用聚蔗糖(ficoll) 泛影葡胺(ficoll hypaque, F／H )（d=1.077 g/ml）进行等密度梯度离心分离，对照组用常规离心分离纯化 ，0.4％台盼蓝拒染活细胞计数；抗人细胞角蛋白（cytokeratin，CK）间接免疫荧光染色, 流式细胞术分析；荧光显微镜观察细胞形态；观察细胞的生长状态和初步克隆形成率；共聚焦显微镜观察细胞纯化结果及其生长状态。 结果 实验组细胞存活率均高于对照组（P＜0.00 1），但是细胞数量减少；流式细胞术分析结果显示实验组CK阳性细胞率高于对照组（P＜0.001）；ficoll 离心组涂片细胞呈圆形，边界清晰，荧光染色均匀，活力好；实验组RPE细胞形态较均一，贴壁时间短，进入分裂期快，初步细胞克隆形成率高于对照组（P＜0.001）；爬片生长的RPE细胞CK阳性细胞率两组组间比较差异有显著性的意义（P均<0.001）。 结论 ficoll 密度梯度离心法分离的RPE细胞形态均一，生长一致性好，进入细胞倍增期时间短，细胞活力保持好；细胞纯化效果较好。 （中华眼底病杂志，2003，19：333-404）

Dissociation and purifying of the retinal pigment epithelial cells by gradient centrifugation

Objective To investigate the effect of dissociation of the human retinal pigment epithelium (RPE) cells by ficoll hypaque gradient centrifugation. Methods The primary human RPE cells and subcultured human RPE cells were dissociated with ficoll gradient centrifugation solution (d= 1.077 g/ml) and the same divided cells as the control were dissociated with routine normal culture medium centrifugation. The Trypan blue (0.4%) rejection staining was used, and the mouse anti-human monoclonal antibody and fluorescein isothiocyanate (FITC) labeled rabbit anti-mouse IgG were utilized for indirect immunoreactivity for the test of human cytokeratin (CK) in active RPE cells cytoplasm. Flow cytometry assay was used to analyzed the percentages of CK positive staining RPE cells. simultaneously, the cells configuration, growth condition, the rate of clone formation, and the purifying result were observed under the fluorescent and confocal microscope. Results The survival rate and positive rate of CK of RPE cells in experimental group were higher than those in the control ( P< 0.001), but the number of the cells was reduced. The cells in the experimental group were integrated round with smooth border, symmetrical staining, homogeneous configuration and higher rate of clone formation ( P< 0.001). Conclusions RPE cells disassociated with ficoll gradient centrifugation have the better dissociation effects.