人腋窝毛乳头细胞的分离与培养

In vitro isolation and cultivation of human axillary dermal papilla cells

目的探讨高效快速分离培养人腋窝毛乳头细胞的方法。方法收集2015年10月至2016年5月陆军军医大学第一附属医院皮肤科腋臭术后含毛皮肤标本，分别用改良二步酶消化法、一步酶消化法和显微解剖法分离腋窝毛乳头，比较3种方法操作过程差异以及毛乳头分离效率、贴壁率、细胞迁出时间、总操作时间、实际操作时间的差异，并鉴定培养的腋窝毛乳头细胞。结果与一步酶消化法和显微解剖法相比，改良二步酶消化法操作更简单，分离效率可达30％以上，1周后毛乳头贴壁率高达96％，细胞迁出时间缩短3～4d，总耗时长于一步酶消化法和显微解剖法，但实际操作时间短于一步酶消化法和显微解剖法，且污染率低于显微解剖法。培养的腋窝毛乳头细胞早期可呈凝集性生长，6代以后的细胞呈非凝集性生长。免疫荧光结果示腋窝毛乳头细胞层黏连蛋白和Ⅳ型胶原阳性。结论改良二步酶消化法是一种简单、高效、快速分离人腋窝毛乳头细胞的方法，少量标本即可培养出腋窝毛乳头细胞。

Objective To investigate an efficient rapid method for the isolation and cultivation of human axillary dermal papilla ceils. Methods Skin specimens with hair follicles were obtained from the axillary area of patients who received bromhidrosis surgery in the Department of Dermatology of the First Affiliated Hospital to Army Medical University from October 2015 to May 2016. The axillary dermal papilla cells were isolated by two-step enzyme digestion method, one-step digestion method and micro-dissection method separately. Then, axillary dermal papilla ceils were cultured and identified. Differences in the operative procedure, separation efficiency and adhesion efficiency of dermal papilla cells, cell emigration duration, total operation duration and actual operation duration were compared among the above 3 methods. Results Compared with the one-step digestion method and micro-dissection method, the two-step enzyme digestion method showed simpler operative procedure, more than 30% separation rate and 96% adhesion rate of dermal papilla cells after 1 week. Moreover, the cell emigration duration was shortened by 3 - 4 days by the two-step enzyme digestion method. The two-step enzyme digestion method also showed longer total operation duration, but shorter actual operation duration compared with the one-step digestion method and micro-dissection method, as well as lower contamination rate compared with the micro-dissection method. Cultured axillary dermal papilla cells grew in an aggregative pattern in the early stage, but grew in a non- aggregative pattern after 6 passages. Immunofluorescence assay showed positive staining for laminin and collagen Ⅳ in axillary dermal papilla cells. Conclusion The modified two-step enzyme digestion method is a kind of simple, efficient and rapid method for the isolation of human axillary dermal papilla cells, and axillary dermal papilla cells can be harvested through this method by using a few specimens.