基础医学与临床 ›› 2013, Vol. 33 ›› Issue (11): 1418-1421.

• 间充质干细胞与皮肤损伤修复专题 • 上一篇    下一篇

人2型糖尿病皮肤角质形成细胞的无血清培养及纯化

张殿宝1,王竟2,庞希宁1,施萍3,苏航4   

  1. 1. 中国医科大学
    2. 中国医科大学附属第一医院肛肠外科
    3. 中国医科大学附属第一医院
    4. 中国医科大学 附属第一医院
  • 收稿日期:2013-08-26 修回日期:2013-09-16 出版日期:2013-11-05 发布日期:2013-10-28
  • 通讯作者: 庞希宁 E-mail:pangxining@126.com

Primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium

  • Received:2013-08-26 Revised:2013-09-16 Online:2013-11-05 Published:2013-10-28

摘要: 目的 体外原代无血清培养人2型糖尿病皮肤角质形成细胞,并进行纯化。方法 采用中性蛋白酶-胰蛋白酶两步消化法分离人2型糖尿病皮肤角质形成细胞,使用无血清培养基进行原代培养,通过两步消化传代纯化细胞,使用倒置相差显微镜观察细胞生长状态,WST-8法测定细胞增殖曲线,流式细胞术检测其表型和纯度。结果 无血清培养并纯化的角质形成细胞呈典型的上皮形态,第3代角质形成细胞增殖曲线为S形,对数增长期的平均倍增时间为36.9 hours,角蛋白阳性率为98.9 %。结论 建立高纯度的人2型糖尿病皮肤角质形成细胞的无血清培养方法。

关键词: 角质形成细胞, 2型糖尿病, 原代培养

Abstract: Objective To improve the methods for primary culture and purification of type 2 diabetic human keratinocytes in serum-free medium in vitro. Methods Type 2 diabetic human keratinocytes was isolated by 2 step digestion of neutral protease and trypsin, cultured in serum-free medium and purified by two-step subculture, the keratinocytes were observed using inverted phase contrast microscope, the growth curve was obtained by the WST-8 assay and phenotype and purity were analyzed by flow cytometry. Results Type 2 diabetic human keratinocytes in serum-free medium showed typical epithelial morphology, the growth curve of the keratinocyte at passage 3 is S-shaped, the average doubling time is 36.9 hours during logarithmic growth phase and the keratin-positive rate was 98.9%. Conclusion The methods for culture of high-purity type 2 diabetic human keratinocytes in serum-free media have been established.

Key words: keratinocytes, type 2 diabetes, primary culture