中文摘要：

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。

英文摘要：

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