无血清培养条件下人表皮干细胞的生物学特性研究

Study on the biological features of human epidermal stem cells cultured in serum-free medium

目的建立人表皮干细胞的无血清培养法并观察其生物学特性。方法从幼儿包皮中分离表皮细胞,用Ⅳ型胶原纯化表皮干细胞,分别在常规(血清组)、无血清(无血清1组)和无血清但添加牛垂体提取物(无血清2组)条件下进行培养。20d后观察比较3组细胞形态学变化、克隆计数、传代计数、α6及CD71表达情况,并进行细胞周期分析,检测细胞角蛋白(CK)19、CK5/8、CK10的阳性细胞百分比。结果无血清1组与血清组细胞形态相近,均可形成43个左右克隆,可传代10次,α6briCD71dim细胞百分比为(48±6)%,(72.7±6.2)%的细胞处于G0/G1期,CK19、CK5/8、CK10表达情况与血清组相近,差异无统计学意义(P>0.05)。无血清2组除细胞克隆数、CK10阳性细胞百分比高于前两组外(P<0.05),其他检测指标均偏低(P<0.05)。结论无血清培养基能选择性培养人表皮干细胞,这一模型可用于进行表皮干细胞生物学特性的相关研究。

Objective To establish a methodology of serum-free culture of human epidermal stem cells in vitro, and observe their biological characteristics. Methods Epidermal stem cells were isolated from child foreskin and purified with type Ⅳ collagen. They were then cultured in regular medium （S group）, serum-free medium （SF1 group） and bovine pituitary extract but serum-free medium （SF2 group） ,respectively. The morphological changes, clone counting, passage time, expression of surface α6 and CD71 ,cell cycle analysis, and the expression of cytokeratin （CK） 19, CK5/8, CK10 in each group were determined 20 days after culture. Results There was no significant difference in morphology, CK19, 5/8 and 10 positive cells between SF1 group and S group. The cells in these two groups could both form about 43 clones and be passaged for 10 times. About （48 ＋6）% cells were α6^bri CD71^dim positive, （72. 2 ＋6. 2）% cells were at G0- G1 phase, with similar expression of CK19,CK5/8, CK10. Despite more clone numbers were found in SF2 group, the levels of all other parameters were lower than those in S and SF1 groups.Conclusion Human epidermal stem cells can be cultured successfully in serum-free culture for the study of their biological characteristics.