中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (1): 96-99.doi: 10.3969/j.issn.1673-8225.2011. 01.021

• 干细胞基础实验 basic experiments of stem cells • 上一篇    下一篇

糖尿病大鼠表皮干细胞的分离培养及特性

朱飞滨1，2，刘德伍1，张红艳1，彭  燕1，钟清玲2，李勇铁1，2   

  1. 1南昌大学第一附属医院烧伤研究所，江西省南昌市   330006
    2南昌大学医学院，江西省南昌市  330006
  • 收稿日期:2010-09-09 修回日期:2010-10-08 出版日期:2011-01-01 发布日期:2011-01-01
  • 通讯作者: 刘德伍，博士，教授，主任医师，博士生导师，南昌大学第一附属医院烧伤研究所，江西省南昌市 330006 dewuliu@126.com
  • 作者简介:朱飞滨★，男，1984年生，江西省抚州市人，汉族，南昌大学医学院在读硕士，主要从事创面修复研究。 Shi78080808@163.com
  • 基金资助:

    国家自然科学基金资助项目(30560058)。

Separation, culture and characteristics of epidermal stem cells in diabetes mellitus rats

Zhu Fei-bin1,2, Liu De-wu1, Zhang Hong-yan1, Peng Yan1, Zhong Qing-ling2, Li Yong-tie1,2   

  1. 1Institute of Burn, First Affiliated Hospital of Nanchang University, Nanchang  330006, Jiangxi Province, China
    2Medical College of Nanchang University, Nanchang  330006, Jiangxi Province, China
  • Received:2010-09-09 Revised:2010-10-08 Online:2011-01-01 Published:2011-01-01
  • Contact: Liu De-wu, Doctor, Professor, Chief physician, Doctoral supervisor, Institute of Burn, First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China dewuliu@126.com
  • About author:Zhu Fei-bin★, Studying for master’s degree, Institute of Burn, First Affiliated Hospital of Nanchang University, Nanchang 330006, Jiangxi Province, China; Medical College of Nanchang University, Nanchang 330006, Jiangxi Province, China Shi78080808@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30560058

PDF

441

被引次数

9

摘要：

背景：表皮干细胞作为皮肤组织特异性干细胞，在创面修复中发挥关键作用。但有关糖尿病皮肤来源的表皮干细胞体外分离培养及生物特性研究较少。
目的：探索糖尿病大鼠表皮干细胞体外分离培养的方法及其生物特性，为糖尿病难愈创面的防治及机制研究提供实验依据。
方法：SD大鼠随机分成糖尿病组和正常对照组。糖尿病组采用一次性腹腔注射链脲佐菌素制备糖尿病大鼠模型，正常对照组不作处理。分别取成模糖尿病和正常大鼠背部全层皮肤，采用酶消化联合Ⅳ型胶原黏附法分离培养大鼠表皮干细胞。倒置相差显微镜下观察细胞形态变化和细胞克隆形成，细胞计数绘制生长曲线，计算克隆形成率，免疫细胞化学染色和图像分析软件鉴定K19、β1-integrin阳性表达和测定阳性细胞的积分吸光度(IA)值。
结果与结论：糖尿病组大鼠表皮干细胞原代贴壁数量较少，其克隆形成率明显低于正常对照组(P < 0.01)。表皮干细胞的K19、β1-integrin均呈阳性表达，糖尿病组阳性细胞的IA值均低于正常对照组(P < 0.01)。结果提示，运用酶消化联合Ⅳ型胶原黏附法可以实现糖尿病大鼠表皮干细胞的体外分离培养；糖尿病大鼠表皮干细胞体外增殖能力较正常皮肤增殖能力弱，这可能是导致糖尿病创面难愈合的重要因素之一。

关键词: 表皮干细胞, 糖尿病, 大鼠, 体外培养, 创面愈合

Abstract:

BACKGROUND: Epidermal stem cells (ESCs) serve as skin tissues specific stem cells, it plays critical roles in wound repairing, but the study about separation and culture of diabetes mellitus skin-derived ESCs in vitro and biological characteristics is fewer. 
OBJECTIVE: To explore the methods of separation and culture of diabetes mellitus rat ESCs, investigate basic biological characteristics, and provide the experimental evidences for diabetes mellitus mechanism research and non-healing wound treatment.
METHODS: Sprague Dawley rats were randomly divided into diabetes mellitus group and normal control group. Diabetes mellitus rat model was made by intraperitoneal injection of streptozocin, and the normal control group was not treated. Full-thickness skins of the back of diabetes mellitus group rats and normal rats were taken respectively. Enzyme digestion combined with type IV collagen attachment method was used to separate, culture and choose rat ESCs. Morphologic change and cell colony formation were observed under inverted phase contrast microscope. Cell growth curve was detected by cell counting and cell colony formation rates were measured. The positive expressions of K19, β1-integrin were identified by immunocytochemical stain and picture analysis software. The integral absorbance value of positive cells was detected.
RESULTS AND CONCLUSION: The number of primary anchorage-dependent ESCs in diabetes mellitus group was decreased, the colony forming efficiency of ESCs in diabetes mellitus group was significantly lower than that in the normal control group (P < 0.01). ESCs of both groups expressed K19 and β1-integrin. The integral absorbance values of positive cells for K19 and β1-integrin in ESCs of diabetes mellitus group were significantly lower than those in the normal control group (P < 0.01). Results indicate that fast separation and stable culture in vitro of diabetes mellitus ESCs could be achieved by enzyme digestion and type IV collagen attachment method. The multiplication capacity in vitro of rat ESCs in diabetes mellitus group was slower than normal skin and it may be one of important factors that diabetes mellitus wound is hard to heal.

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