摘要
目的寻找一种快递高效分离组织工程化皮肤种子细胞-皮肤干细胞的方法。方法取成人手术切除之包皮,利用I型胶原酶结合胰蛋白酶消化法,快速分离表皮干细胞,并对其进行鉴定及相关的生物学特性的研究。结果应用I型胶原酶结合胰蛋白酶,可在3小时内快速分离到表皮干细胞及成纤维细胞,经流式细胞仪及细胞免疫荧光鉴定,表皮细胞群体中角蛋白19(K19)阳性表达的细胞即表皮干细胞约占18％,但是其体外短期扩增仍较困难。结论利用胶原酶消化法可在短期内高效分离到用于构建组织工程化皮肤的种子细胞,这为快速构建组织工程化皮肤提供了可能。
Objectives To find a feasible method that can isolate seed cells-keratinocyte stem cell-for tissue-engineered skin efficiently. Methods The foreskin could be attained after operation, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2mm×2mm in size, then the pieces were submerged into a centrifuge tube containing collagenaseⅠin a oscillator. After 1-3 hours digestion at 37℃, the dermis was dissolved completely and the epidermis could be separated easily. With more 10 minutes digestion in trypsin at 37℃, the epidermal cells could be harvested. Then flowcytometry and FITC-immunofluorescence for cytokeratin 19 were conducted to identify that there were keratinocyte stem cells in the epidermal cells. Moreover, the epidermal cells were cultured in vitro to investigate their biological behavior. Results Using collagenaseⅠcombined with trypsin, epidermal cells could be isolated quickly and efficiently. Up to 18% cells demonstrated cytokeratin 19 positive in the epidermal cells, but the putative keratinocyte stem cells were difficult for subculture. Conclusions Seed cells for tissue-engineered skin could be harvested fast and efficiently, which made it possible to reconstruct very quickly tissue-engineered skin in vitro. But the amount of keratinocyte stem cell was difficult to be enlarged quickly.
出处
《组织工程与重建外科杂志》
2005年第5期268-271,共4页
Journal of Tissue Engineering and Reconstructive Surgery
基金
广东省"十五"科技计划组织工程重大专项(A302020301)广东省自然科学基金(A031704)。
关键词
组织工程
皮肤
表皮干细胞
分离
Tissue engineering
Skin
Epidermal stem cell
Isolation
