人腹膜间皮细胞的改良分离及其鉴定

背景:腹膜间皮细胞作为腹膜的重要组分,分泌多种细胞因子,在参与抗炎、免疫调节、腹膜纤维化等方面起着重要作用,如何获得优质、均一的腹膜间皮细胞成为解决这些问题的关键。目的:拟建立改良的人腹膜间皮细胞消化培养法。方法:0.1%胶原酶Ⅰ消化腹部大网膜组织,去除红细胞后用含体积分数为10%胎牛血清的1640培养基培养。在培养过程中以倒置显微镜观察细胞形态变化,CCK-8观察培养液对间皮细胞生长的促进作用,透射电子显微镜观察细胞超微结构,激光共聚焦免疫荧光鉴定细胞。结果与结论:分离培养的细胞为多角形,汇合时呈铺路石样排列,培养细胞的纯度达90%以上;生长良好,迅速,可传代至四五代。透射电子显微镜下见细胞表面大量的微绒毛,免疫荧光显示角蛋白、波形蛋白抗原阳性,白细胞CD45、第Ⅷ因子相关抗原阴性;所有被鉴定特征均符合间皮细胞的特点。结果提示胶原酶Ⅰ消化法是一种简单、高效、重复性高的分离腹膜间皮细胞的方法。

Isolation and identification of improved human peripheral peritoneum mesothelial cells

BACKGROUND:Peripheral peritoneum mesothelial cells are served as an important component of peritoneum which secrete many kinds of cytokines,and play an important role in anti-inflammatory,immunoloregulation,and peritoneal fibrosis.How to obtain high-quality and homogeneous peritoneal mesothelial cells has become the key to solving problems.OBJECTIVE:To develop an improved method for digestion culture of human peritoneal mesothelial cells(HPMCs).METHODS:HPMCs were digested by 0.1% collagenaseⅠ,after eliminating erythrocytes,the cells were cultured by 10% fetal bovine serum of 1640 medium.Inverted microscope was used to observe the morphological structures of cells;CCK-8 was used to test the promotion of medium on the growth of mesothelial cell.The ultrastructure of cells was observed by transmission electron microscope(TEM),and isolated cells were identified by laser confocal immunofluorescence microscope.RESULTS AND CONCLUSION:The isolated and cultured mesothelial cells were polygonal and confluenced gradually and grew well like the slab stone.The purity of cultured mesothelial cells was more than 90%;the cells grew well,quickly,and could be passaged to 4 and 5 generations.Microvilli of the cells were evident on the surface of cells under TEM.Immunofluorence analysis showed the cells were positively express cytokeratin and vimentin,but negative for leucocyte CD45 and factor Ⅷ fos-related antigen.All identified marks are accorded with the characteristics of mesothelial cells.The results indicated that collagenaseⅠdigestion method is simple,efficient,and reproducible separation method of HPMCs.