表皮干细胞体外培养体系的初步研究

The initial study of culture system in vitro about epidermal stem cells

目的探讨以成纤维细胞Ⅰ型胶原网格模型体外培养表皮干细胞的可行性。方法酶消化法获取细胞悬液,Ⅳ型胶原快速贴壁法分选表皮干细胞,将体外培养的表皮干细胞和成纤维细胞接种于0.01%的戊二醛交联的Ⅰ型胶原上,气-液界面培养2周,免疫组化法、流式细胞仪和细胞周期来鉴定贴壁细胞,共聚焦显微镜和扫描电镜观察细胞材料。结果贴壁细胞CK19和P63阳性表达,β1整合(CD29)表达率为98.57%,CD71表达率为9.05%,94.99%细胞处于静息期,以上均表明该细胞是表皮干细胞,并在Ⅰ型胶原上呈鱼鳞状排列。结论表皮干细胞在Ⅰ型胶原上生长良好,可以生成全层组织工程皮肤。

Objective To investigate the feasibility of the culture in vitro about epidermal stem cells on the netshaped model constructed by desmocyte and collagen type Ⅰ . Methods The single epidermal cell suspension was obtained by enzyme digestion, and then adherent cell selected by adherence rapidly on collagen type Ⅳ. Epidermal stem cells and fibroblasts were inoculated on the collagen type Ⅰ cross-linked with 0.01% glutaraldehyde and cultivated two weeks under aer-liquor surface. The adherent cells were identified with the immunohistochemical methods, flow cytometry and cell cycle. The collagen type Ⅰ with cells was observed by confocal microscopy and scanning electron microscope （SEM）. Results The CK19 和 P63 were positive on adherent ceils, the expression rate of β1 integrin （CD29） was 98.57%, and CD71 was 9.05%. About 94.99% cells were during the period of proliferation, all indicate that they were ESCs. The epidermal stem cells and fibroblasts were scale-work on the the collagen type Ⅰ. Conclusion The adherent cells grow well on the collagen type Ⅰ and could constitute tissue engineering skin.