表皮黑素细胞的纯化  

Purification of epidermal melanocytesin culture

作　　者：李娣[1] 张汝芝[1] 

机构地区：[1]苏州大学附属第三医院,213003

出　　处：《天津医药》2017年第1期5-8,I0001,共5页Tianjin Medical Journal

基　　金：国家自然科学基金资助项目(81171516)

摘　　要：目的比较不同包被材料、不同培养条件对黑素细胞的纯化作用。方法将包皮组织、包皮的表皮组织及消化所得的细胞悬液,分别培养于层黏连蛋白或基质胶包被的培养板中,传代时进行差别胰酶处理、80%~90%融合时加入G418(0.05%)4 min或5-Brd U,通过黑素细胞的标记性抗体HMB-45荧光染色,观察黑素细胞纯度。结果原代培养中,细胞悬液比组织块获得细胞更多更快,单纯表皮比带真皮的组织块更早有细胞迁移并增殖;层黏连蛋白包被比基质胶包被更有利于黑素细胞生长;差别胰酶处理以及加入一定浓度的G418或5-Brd U均可纯化黑素细胞,但G418、5-Brd U浓度较大时对细胞损伤明显。结论采用层黏连蛋白包被板培养包皮来源的细胞悬液,传代时采用差别胰酶处理,传代后加小剂量的G418,有利于黑素细胞纯化,而不影响其生长。Objective To compare the influence of different coating materials and cultural conditions on thepurification and growth of human epidermal melanocytes. Methods The full-thick foreskin, epidermis and cell suspensionobtained from human foreskin were cultured in the plates, which were precoated with matrigel or laminin respectively. Whenhaving reached 80%-90% confluence, the cells were treated with 0.05% trypsin-EDTA for 4 minutes and resuspended inM254 medium, which were supplemented with G418 and 5-Brd U, respectively. The purity of melanocytes was observed byan immunofluorescence staining with melanocyte markers. Results During the primary culture, the cell suspensiongenerated more cells at faster speed compared with those of skin explants and epidermal specimen. Moreover, the epidermisreleased cells earlier and proliferated quickly over skin explants. The melanocytes in the plates coated with laminin otherthan with matrigel displayed faster and better growth. The unwanted keratinocytes and fibroblasts were removed by usingdifferentiation trypsinition combined with supplement of G418 or 5-Brd U. Conclusion Using a plate coated with lamininto culture cell suspension from human foreskin, and via a differentiation trypsinization combined with supplement of smalldoses of G418 to subculture the cells, is advantageous to the melanocyte purification, without affecting their growth.

关 键 词：黑素细胞 细胞培养技术 纯化 包被材料 

分 类 号：R751[医药卫生—皮肤病学与性病学]