摘要
目的比较人表皮干细胞在两种体外培养体系下细胞的生长增殖情况。方法运用中性蛋白水解酶和胰蛋白酶两步法分离表皮层和真皮层,将得到的表皮细胞悬液差速黏附法分离角质形成细胞,在无血清角质细胞培养基中培养并免疫荧光法鉴定P63和K19的表达。鉴定后的细胞分别在无血清及有血清有饲养层条件下培养,MTT法检测两种条件下细胞的增殖情况,荧光定量PCR鉴定两种培养条件下不同代次细胞P63和K19的表达。结果在有血清有饲养层培养条件下表皮干细胞增殖较快。与无血清培养条件相比较,有血清培养条件下P3代及P5代K19 mRNA的表达水平显著提高,P5代P63 mRNA的表达水平显著提高(P<0.05)。结论表皮干细胞在有血清、有饲养层的培养条件下生长增殖较快且能更好地保持表皮干细胞的特性。
Objective To compare effects of two culture systems on the cultivation and proliferation of human keratinocyte stem cells( HKCs) in vitro. Methods Epidermis and single epidermis cells were obtained by digesting human skin with Dispase II and Trypsin. The rapidly adherent cells were cultured in K- SFM and the expression of P63 and K19 was identified by immumofluorescence method. The cells identified were cultured in serum- free keratinocyte medium and feeder layer with serum medium respectively. The proliferation of the cells under those two culture conditions were detected by MTT and the expression of P63 and K19 of different generation cells were detected by Q- PCR. Results The cells cultured in feeder cells with serum medium proliferated more rapidly. Compared with serum- free keratinocyte medium,the mRNA expression of K19 in P3 and P5 HKC were all increased significantly and also the mRNA expression of P63 in P5 HKC was increased significantly in feeder cells with serum medium( P < 0. 05). Conclusion HKCs grow and proliferate more quickly and can keep the character of stem cell better when cultured in feeder cells with serum medium.
出处
《宁夏医科大学学报》
2013年第9期971-974,1069,共5页
Journal of Ningxia Medical University
基金
宁夏医科大学校级课题(XQ2011022)
关键词
表皮干细胞
分离
培养
鉴定
keratinocyte stem cells
isolate
culture
identify
