摘要
目的从人头皮毛囊外根鞘分离纯化培养无色素黑素细胞。方法采用两步酶法0.50%分离酶和0.50%胶原酶Ⅴ获得游离的毛囊,高浓度0.50%胰酶30min消化游离的毛囊外根鞘细胞,并以优化的培养基进行细胞培养。用HMB-45和NKI/beteb单克隆抗体免疫组化染色、透射电镜对培养物进行鉴定。结果培养物中初始贴壁细胞大多数为无色素黑素细胞,仅有少量的角质形成细胞,无成纤维细胞污染。经二次传代差别胰酶处理很容易去除角质形成细胞。取培养3周的细胞经免疫组化和透射电镜鉴定证实为无色素黑素细胞。传3代后细胞得到完全纯化。结论两步酶法结合高浓度的胰蛋白酶处理细胞可彻底清除成纤维细胞,获得无色素黑素细胞的纯培养。
Objective: To isolate and culture hair outer root sheath amelanotic melanocyte derived from human scalp. Methods: Hair follicles in the remaining dermis were isolated by two-step enzyme treatment. Hair follicle cell suspensions were prepared by 0.50% trypsin treatment for about 30 min, and cultured in a optimized culture medium. Cell type was confirmed by staining with monoclonal antibodies to HMB-45 and NKI/beteb antigens and observed with transmission electron microscope. Results: Anchoring cells were mostly amelanotic melanocyte in early stage, only a small quantity of keratinocyte and none of fibroblast. The keratinocytes were removed by differential trypsinization. After 3 weeks culture, the cells were all amelanotic melanocytes confirmed by immunohistochemistry stain and observation with transmission electron microscope. Conclusions: The available of culture of amelanotic melanocyte was obtained successfully by two-step enzyme and high dose trypsin treatment.
出处
《临床皮肤科杂志》
CAS
CSCD
北大核心
2004年第2期76-79,共4页
Journal of Clinical Dermatology
基金
国家自然科学基金资助项目30170861
关键词
无色素黑素细胞
细胞培养
成纤维细胞
毛囊外根鞘
amelanotic melanocyte
cell culture
fibroblast
hair follicle outer root sheath
