摘要
目的:建立不含佛波醇酯(TPA)和霍乱毒素(CT)培养液培养人类正常表皮黑素细胞的方法,避免TPA的致瘤危险和霍乱毒素的毒性。方法:临床环切包皮经中性酶-胰酶(D ispaseⅡ-Trypsin)两步消化后,获得表皮基底细胞悬液,于不含TPA和CT的KC-SFM角质形成细胞无血清培养基培养,用差速贴壁法及胰酶差速消化法去除角质形成细胞,用两段法去除成纤维细胞污染,观察黑素细胞的增殖情况和树突伸展状态,采用多巴(Dopa)染色和免疫组化对黑素细胞进行鉴定。结果:经过7天的培养,黑素细胞达到70%汇合,细胞呈双极、三极或多极的树突状。经1-2次传代,黑素细胞纯度达98%以上,Dopa染色和抗S-100单抗染色阳性。结论:用不含TPA和CT的KC-SFM角质形成细胞培养基可以获得大量高纯度黑色素细胞。
Objective：To establish a procdure of culturing melanocytes in the absence of phorbol esters （TPA） and cholera toxin （CT）. This method can avoid tumorigenesis of TPA and the toxicity of the CT. Methods：We used dispase 11 and trypsin to digest the skin tissue and obtain the melanocytes, and cultured the cells in medium which free of serum, phorbol esters and cholera toxin. The melanocytes were purified in the medium at first and then proliferated in the medium with serum. We observed the proliferation and cytodendrites of the melanocytes. Dopa and anti- 100 antibody staining were used to identify the cell type. Results： After seven days of culture, the melanocytes were 70% confluence. They were dendritic cells with dipolar, tripolar or multipolar. After one or two passages, The cells can reach a purification of 98%. The results of dopa staining and anti-S100 immunocytochemistry were positive. Conclusion ：This procdure can obtain the highly purified melanocytes which grew well in the keratinocyte serum free medium in the absence of phorbol esters and cholera toxin.
出处
《岭南皮肤性病科杂志》
2006年第5期336-339,共4页
Southern China Journal of Dermato-Venereology
基金
深圳市科技计划基金项目(基金编号:200404090)
