人表皮干细胞在两种培养体系中生长增殖的比较研究  

Comparison of growth and proliferation of human epidermal stem cells cultured in nonfeeder layer and feeder layer systems

作　　者：张杰[1] 刘伟[1] 丛笑倩[1] 徐展远[2] 马东瑞[1] 滕晓进[1] 崔磊[1] 曹谊林[1] 

机构地区：[1]上海第二医科大学第九人民医院整复外科上海市组织工程研究与开发中心,上海200011 [2]上海市儿童医院泌尿外科

出　　处：《上海第二医科大学学报》2004年第4期284-287,295,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家重点基础研究发展规划资助项目("973"项目)(G1999054300);国家高技术研究发展计划资助项目("863"计划)(2003AA205122);上海市"重中之重"重点学科基金资助.

摘　　要：目的 比较表皮干细胞(KSC)在两种体外培养体系中的生长增殖状况。方法 用差速粘附法分离出的KSC,分别置于无饲养层、无血清培养基以及有饲养层、有血清培养基的培养条件下培养。比较KSC在两种培养体系中的生长增殖、克隆形成率、生长曲线和KSC相关标记物的表达等指标。结果 在第二种培养条件下,KSC在生长过程中不存在明显的接触抑制,在较长的体外培养时间内始终保持较高的增殖潜能。两种培养体系间KSC的克隆形成率有显著差异(P<0.01)。流式细胞仪检测两种培养体系间整合素β1阳性率无显著差异,而强阳性率则有显著性差异(P<0.05)。结论 体外培养和扩增KSC时,使用人成纤维制备的饲养层和含胎牛血清的培养基,较无饲养层、无血清的培养基,更有利于KSC的扩增及其表型的维持。Objective To compare effects of two culture systems on the cultivation and proliferation of epidermal keratinocyte stem cells (KSC) in vitro. Methods Human KSCs were isolated by adhering keratinocytes to collagen Type Ⅳ coated dishes. Cells that were adherent to Type Ⅳ collagen within 20 min were harvested. They were cultivated in two different conditions. In the first culture system, we used serum-free keratinocyte medium, containing rhEGF and bovine pituitary extract, and no feeder layer was used. In the second condition, the medium was made up of three parts of DMEM plus one part of Ham' s F12 medium, to which 10% fetal calf serum, adenine, insulin, hy-drocortisone, cholera toxin and EGF were added. A feeder layer of human dermal fibroblast pretreated with mitomycin C was also used. Colony-forming efficiency (CFE) , growth curve, and the expression of p, integrin were analysed in two different systems. Results The CFEs of KSC cultivated in the second systems was significantly higher than that in the first system (P <0. 01). Strong positive expression percentage of β1 integrin were significantly higher in KSCs cultivated in second culture system than that of first system ( P< 0. 05). Conclusion The results indicate that the use of feeder layer of human fibroblasts, serum and appropriate additives facilitates the growth and proliferation of keratinocyte stem cells in vitro. These factors may provide the stem cells with an environment relatively similar to the natural niche.

关 键 词：表皮干细胞 细胞培养 细胞增殖 差速粘附法 流式细胞仪 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]