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表皮干细胞体外分离培养、鉴定The Isolation, Culture and Identification of Epidermal Stem Cell 昆明医学院第一附属医院皮肤科何 黎 教授 The First Affiliated Hospital of Kunming Medical College Prof. He Li 课题来源：云南省重大攻关项目《人工皮肤制备及临床应用》 参加单位：昆明医学院第一附属医院皮肤性病科 香港中文大学威尔斯亲王医院 Source of Project: The key research of Yunnan Province《The Fabrication and Clinical Application of Artificial skin 》 Member of project: Dermatology Department of The First Affiliated Hospital Of Kunming Medical College Prince of Wales Hospital in Hong Kong 一、Preface（背景） The Diseases Applicated Artificial Skin （二）Evolution of Wound Cover Strategies for Management Goals（目标） Rapid Healing （迅速愈合） Minimum Scar （减少疤痕形成） Minimum Pigmentation Sequelae （减少色素沉着） The Ideal Wound Cover理想的创面覆盖 Relieves Pain （减轻疼痛） Debrides Burn（清创） Prevents infection （预防感染） Promotes Healing through REGENERATION （通过再生促进创面愈合） Superficial Partial Thickness Deep Partial Thickness/Full Thickness Deep Partial Thickness/Full Thickness Cell Culture （一）Material （材料） The foreskin from donor aged 6-26 years old. 6-26岁男性包皮组织 （二）Culture medium（试剂） PBS and D-hank’s keratinocyte serum-free medium （培养基） Dispase （重型蛋白酶） Trypsin （胰蛋白酶） monoclone antibody of cytokeratin （角蛋白单克隆抗体） monoclone antibody of cytokeratin 19（角蛋白19单克隆抗体） Culture Method (培养方法） 1、 Epidermal sheet were separated from the dermit. 2、 The cell pellet resuspended in the completed K-SFM. 3、 Count the cell 4、 Cells were plated in 25 cm2 culture flasks 1、分离表皮、真皮 2、离散细胞悬浮于培 养基中 3、细胞计数 4、培养瓶中培养细胞 （三）Culture Method（培养方法） The foreskin was cut into halves of 0.5×0.5cm2 , the pieces were rinsed in 75% alcohol for three times and then placed in PBS, containing gentamycin for 10min. The pieces were transferred to a