人表皮干细胞的分离及其生物学特性的评价

J4 ›› 2011, Vol. 37 ›› Issue (2): 357-360.

人表皮干细胞的分离及其生物学特性的评价

马英智^1,2|高航¹|邓方阁³|张丽红¹|李玉林¹

1. 吉林大学白求恩医学院　病理生物学教育部重点实验室|吉林　长春　130021；2. 空军航空大学飞行基础训练基地门诊部|吉林　长春　130022；3. 广州医学院第一附属医院中科院呼吸疾病研究所|广东　广州　510120

收稿日期:2011-01-02 出版日期:2011-03-28 发布日期:2011-03-28
通讯作者: 李玉林（Tel: 0431-85619481，E-mail: ylli@mail.jlu.edu.cn） E-mail:ylli@mail.jlu.edu.cn
作者简介:马英智(1974-)|女|吉林省敦化市人|主治医师|医学博士|主要从事干细胞组织工程学研究。
基金资助:
国家自然科学基金资助课题（30700872）；国家863重大专项基金资助课题(2004AA205020)；吉林省科技厅重大科研项目资助课题（20076023）

Isolation of human keratinocyte stem cells and evaluation of |their biological characteristics

MA Ying-zhi^1,2,GAO Hang¹,DENG Fang-ge³,ZHANG Li-hong¹,LI Yu-lin¹

1. Key Laboratory of Pathobiology,Ministry of Ednacation|Norman Bethune College of Medicine|Jilin University,Changchun 130021,China;2. Depantment of Policlinic|Basic Flying Training Base, Air Force
Aviation University, Changchun 130022,China;3. |Institute of Respiratory Disease, First Affiliated Hospital| Guangzhou Medical College,Guangzhou 510120,China

Received:2011-01-02 Online:2011-03-28 Published:2011-03-28

摘要/Abstract

摘要：

［摘要］目的:分离人表皮干细胞（hKSCs），并评价其生物学特性。方法：取包皮环切术的正常皮肤，采用酶消化、快速贴壁筛选法分离hKSCs，采用倒置相差显微镜及透射电镜观察细胞形态，采用细胞免疫组织化学法和间接免疫荧光法检测细胞的分子标志物，利用流式细胞术检测细胞周期。结果:倒置相差显微镜下显示，细胞为多角形，体积小、核大，呈铺路石样排列，形态一致；透射电镜显示，胞质内细胞器稀少，细胞核大，核内具有丰富的异染色质；免疫细胞化学、间接免疫荧光和流式细胞术结果显示，KSCs相对特异性标志物CK15、CK19、P63和CD29呈阳性表达，成纤维特异性标志物Vimentin和表皮终末分化标志物CK10呈阴性表达，RT-PCR结果与上述结果一致；细胞周期检测结果显示，大多数细胞处于G0/G1期，符合hKSCs的慢周期特性。 结论:快速贴壁筛选法可分离出纯度较高的hKSCs，可为后续的实验研究提供细胞来源。

关键词: 表皮干细胞；分离；生物学特性

Abstract:

Abstract:Objective To isolate the human keratinocyte stem cells(hKSCs) in vitro and evaluate their biological characteristics.
Methods The normal skin was taken by circum cision and the hKSCs were digested by enzyme and isolated by rapid adherence screening method. The morphology and ultrastructure of hKSCs were observed by phase contrast microscope and transmission electronic microscope,and the molecular markers were identified with immunocytochemistry and indirect immunofluorescence and the cell cycles were detected by flow cytometry.Results With phase contrast microscope,the rapidly adherent cells were uniformed small, large nucleus and epithelial-like. There were positive expressions of cytokeratin 19,cytokeratin 15,p63 and CD29 by immunocy tochemistry,immunofluorescence and flow cytometry, while there were negative expressions of vimentin and cytokeratin 10,which were the specific markers of fibroblasts and epidermal terminal differentiation respectively. The result of RT-PCR was the same to the above. Most cells were in G0/G1 stage by cell cycle analysis which suggested that hKSCs had slow cell cycling nature. Conclusion hKSCs can be isolated and cultivated in vitro successfully and they can be used as cell sources in the further experiments.

Key words: keratinocyte stem cells；isolation；biological characteristics

中图分类号:

Q813.1

马英智|高航|邓方阁|张丽红|李玉林. 人表皮干细胞的分离及其生物学特性的评价[J]. J4, 2011, 37(2): 357-360.

MA Ying-zhi,GAO Hang,DENG Fang-ge,ZHANG Li-hong,LI Yu-lin. Isolation of human keratinocyte stem cells and evaluation of |their biological characteristics[J]. J4, 2011, 37(2): 357-360.