中华皮肤科杂志 ›› 2019, Vol. 52 ›› Issue (5): 310-313.doi: 10.3760/cma.j.issn.0412-4030.2019.05.003

• 论著 • 上一篇    下一篇

人甲母质细胞无血清原代培养方法的建立

陈朝丰    于波    吕超    刘晓云    胡小平   

  1. 北京大学深圳医院皮肤科,深圳  518036
  • 收稿日期:2018-08-17 修回日期:2019-03-03 出版日期:2019-05-15 发布日期:2019-04-30
  • 通讯作者: 胡小平 E-mail:xiaoping7752@sohu.com
  • 基金资助:
    深圳市卫计委课题(201606004);深圳市三名工程项目资助(SZSM201812059)

Establishment of a culture method for primary human nail matrix cells in serum-free media

Chen Chaofeng, Yu Bo, Lyu Chao, Liu Xiaoyun, Hu Xiaoping   

  1. Department of Dermatology, Peking University Shenzhen Hospital, Shenzhen 518036, China
  • Received:2018-08-17 Revised:2019-03-03 Online:2019-05-15 Published:2019-04-30
  • Contact: Hu Xiaoping E-mail:xiaoping7752@sohu.com
  • Supported by:
    Program of Shenzhen Health and Family Planning Commission (201606004); Fund of “San?ming” Project of Medicine in Shenzhen (SZSM201812059)

摘要: 【摘要】 目的 建立人甲母质细胞无血清原代培养方法。方法 甲母质组织来自9例2016年1 - 12月在北京大学深圳医院行截指(趾)术与甲床修整术的患者。采用无血清DEME/F?12培养基于37 ℃、5% CO2条件下培养组织块2 ~ 3 d,换无血清的角质形成细胞生长(CnT?07)培养基培养原代甲母质细胞,显微镜下观察培养过程中的细胞形态。以角蛋白5(K5)、角蛋白10(K10)为标记,采用免疫荧光鉴别培养获得的细胞,流式细胞仪分析细胞纯度。结果 培养2 ~ 3 d时,有细胞沿组织块爬出,在第10天左右,形成了大的细胞团块,部分细胞形态为上皮样细胞,呈铺路石样排列,部分呈扁平状,形态为纺锤形或星形。免疫荧光示,部分细胞可同时表达K5与K10,证明甲母质细胞的存在。K10阳性细胞约占37.6%。结论 采用组织块培养法结合无血清培养基可成功培养出人原代甲母质细胞,体外培养的人甲母质细胞可同时表达K5与K10。

关键词: 指(趾)甲; 原代细胞培养; 培养基, 无血清; 角蛋白5; 角蛋白10; 甲母质细胞

Abstract: 【Abstract】 Objective To establish a culture method for primary human nail matrix cells in serum-free media. Methods Nail matrix tissues were collected from 9 patients, who received nail or toe amputation and nail bed repair in Peking University Shenzhen Hospital between January 2016 and December 2016, and cultured in the serum-free DEME/F-12 media at a 37℃ incubator with an atmosphere of 5% CO2 in air for 2 - 3 days. Then, primary human nail matrix cells were cultured in keratinocyte serum-free media (CnT-07), and the morphology of human nail matrix cells was observed by microscopy during the culture process. Immunofluorescence cytochemistry with anti-keratin 5 (K5) and K10 was performed to identify the acquired cells, and flow cytometry to analyze the cell purity. Results After 2 or 3 days of the culture, some cells began to crawl out from the tissue. On day 10, large cell masses were formed, some cells were morphologically similar to epithelioid cells arranged in a paving stone-like pattern, and some were flat giving a spindle-shaped or star-shaped appearance. Immunofluorescence cytochemistry showed that some cells could express both K5 and K10, which proved the existence of nail matrix cells, and 37.6% of the cells expressed K10. Conclusion Human primary nail matrix cells could be successfully cultured by using the tissue culture method with serum-free culture media, and the nail matrix cells cultured in vitro can express both K5 and K10.

Key words: Nails, Primary cell culture, Culture media, serum?free, Keratin?5, Keratin?10, Nail matrix cell   

中图分类号: 

  • R75