中华皮肤科杂志 ›› 2020, Vol. 53 ›› Issue (9): 725-728.doi: 10.35541/cjd.20190616

• 技术与方法 • 上一篇    下一篇

小标本二步酶消化法分离人头皮毛乳头细胞

胡天星    余南岚    杨海潮    朱琳    杨希川   

  1. 陆军军医大学第一附属医院皮肤科，重庆  400038
  • 收稿日期:2019-06-03 修回日期:2019-12-24 发布日期:2020-08-31
  • 通讯作者: 杨希川 E-mail:doctoryxc@msn.com
  • 作者简介:毕业要求6月之前，5月最好
  • 基金资助:
    国家自然科学基金（81573070）

In vitro isolation and cultivation of human scalp dermal papilla cells by two?step enzyme digestion of small specimens

Hu Tianxing, Yu Nanlan, Yang Haichao, Zhu Lin, Yang Xichuan    

  1. Department of Dermatology, The First Hospital Affiliated to Army Medical University, Chongqing 400038, China
  • Received:2019-06-03 Revised:2019-12-24 Published:2020-08-31
  • Contact: Yang Xichuan E-mail:doctoryxc@msn.com
  • Supported by:
    National Natural Science Foundation of China （81573070）

PDF

4

PDF (Mobile)

0

摘要： 【摘要】 目的 探索小标本高效快速分离培养头皮毛乳头细胞的方法。方法 2018年9月至2019年1月于陆军军医大学第一附属医院皮肤科收集3例头部色素痣、6例皮脂腺痣患者切除术后废弃的含毛皮肤标本，大小为0.5 cm × 0.5 cm ~ 0.5 cm × 1 cm。剪下含有毛囊的皮下脂肪层，用眼科镊分选出其中的毛囊，0.6%分离酶Ⅱ消化30 min，然后采用0.2%胶原酶Ⅳ 37 ℃消化30 ~ 60 min，离心获得毛乳头。对分离的毛乳头进行形态学观察，同时培养、传代、鉴定毛乳头细胞。结果 小标本二步酶消化法分离的头皮毛乳头镜下呈倒梨形，形态完整，部分可见残留真皮鞘，而一步酶消化法未分离出毛乳头。小标本二步酶消化法毛乳头分离率为60.8% ± 2.1%，72 h毛乳头细胞贴壁率为86.6% ± 3.9%，细胞迁出时间0.5 ~ 3.0 d，实验总操作时间2.0 ~ 3.0 h，实际操作时间1.0 ~ 1.5 h。小标本二步酶消化法分离的头皮毛乳头细胞早期可呈凝集性生长，从第9代细胞开始呈非凝集性生长。结论 小标本二步酶消化法是一种简单、高效分离人头皮毛乳头细胞的方法。

关键词: 毛囊, 细胞分离, 细胞培养技术, 头皮, 毛乳头细胞

Abstract: 【Abstract】 Objective To develop an efficient and rapid method for the isolation and cultivation of human scalp dermal papilla cells from small specimens. Methods Hair-bearing skin specimens measuring 0.5 cm × 0.5 cm - 0.5 cm × 1 cm in size were obtained from the scalp of 3 patients with pigmented nevus and 6 with sebaceous nevus during surgery in Department of Dermatology, the First Hospital Affiliated to Army Medical University from September 2018 to January 2019. The subcutaneous fat layer containing hair follicles was cut out of the specimens, and hair follicles were sorted with ophthalmic forceps, which were subsequently digested with 0.6% dispase Ⅱ for 30 minutes, then with 0.2% collagenase Ⅳ at 37 ℃ for 30 - 60 minutes, and were centrifuged to obtain hair papillae. Morphological observation was performed on the isolated hair papillae, and dermal papilla cells were cultured, passaged and identified. Results Under the microscope, the hair papillae isolated by two-step enzyme digestion of small scalp specimens were intact,and showed an inverted pear-like shape, and residual dermal sheaths could be observed around some hair papillae. However, no hair papilla was isolated by one-step enzyme digestion. With the two-step enzyme digestion method, the hair papilla separation rate was 60.8% ± 2.1%, the adherence rate of the dermal papilla cells at 72 hours was 86.6% ± 3.9%, the time for cells to emigrate out of hair papillae was 0.5 - 3.0 days, the total operation duration was 2.0 - 3.0 hours, and the actual operation duration after subtraction of digestion duration was 1.0 - 1.5 hours. The dermal papilla cells isolated by the two-step enzyme digestion method could grow in an aggregative pattern in early stage, but grew in a non-aggregative pattern after 8 passages. Conclusion The two-step enzyme digestion of small specimens is a simple and efficient method for isolating human scalp dermal papilla cells.

Key words: Hair follicle, Cell separation, Cell culture techniques, Scalp, Dermal papilla cells