摘要
目的比较不同酶浓度作用后进行分选的表皮干细胞的生长增殖状况。方法用不同浓度的酶消化表皮,再用差速黏附法分离KSC,置于低钙无血清培养基中培养。比较KSC在两种酶浓度作用后生长增殖、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0.05%胰酶和0.02%EDTA(1∶1)混合的酶作用下,能分选出较高比例的呈高克隆形成率的KSC,在较长的体外培养时间内始终保持较高的增殖潜能。两种酶作用下KSC的克隆形成率有显著差异(P<0.01)。角蛋白K19和整粘蛋白β1有强阳性表达。结论体外培养KSC时,使用低浓度胰酶加EDTA消化表皮,更有利于KSC在Ⅳ型胶原上的筛选,也有利于KSC的体外扩增及其表型的维持。
Objective To compare effects of different enzyme concentration on isolating mouse epidermal stem cells by adhering to type Ⅳ collage.Methods Mouse epidermis were digested by different enzyme concentrations. Type Ⅳ collagen coated dishes were used to enrich KSC.Cells were cultured in serum-free keratinocyte medium ,containing low calcium concentration, rhEGF and bovine pituitary extract. Colony-forming efficiency,growth curve,and the expression of β1 integrin and keratin 19 were analyzed in two different methods.Results Morphologically,epidermal stem cells are smaller and have fewer organelles.The CFE in group A was higher than that in group B(P<0.01). The expression of integrin β1 and K19 is a high level.Conclusion The results indicate that the use of low concentration trypsin with EDTA facilitates selective adhesion of KSC to type Ⅳ collagen.
出处
《重庆医学》
CAS
CSCD
2005年第7期1032-1034,共3页
Chongqing medicine
关键词
表皮干细胞
分选
胰酶
keratinocyte stem cells
isolate
comparison
