组织工程皮肤种子细胞的同期快速分离

FAST HARVEST OF SEED CELLS FOR COMPOSITE TISSUE ENGINEERED SKIN AT ONE TIME

目的寻找一种快速可行的同期分离组织工程皮肤种子细胞——表皮细胞及成纤维细胞的方法。方法取手术切除包皮,利用型胶原酶结合胰蛋白酶消化法,同期分离表皮细胞及成纤维细胞,观察细胞形态。对表皮细胞及表皮干细胞行PAN-FITC、K19-FITC荧光免疫染色及K19流式细胞仪鉴定;对成纤维细胞行波形蛋白FITC免疫荧光鉴定。并分别对两种细胞培养7d,观察其生长情况。结果应用型胶原酶结合胰蛋白酶,在3h内快速分离得到表皮细胞及成纤维细胞,免疫荧光染色表皮细胞PAN-FITC染色均为阳性、K19-FITC染色部分阳性,流式细胞仪鉴定其K19阳性表达的细胞接近17%。表皮细胞培养48h开始呈克隆样增长,至7d生长停滞,向终末的角细胞分化。成纤维细胞体外培养7d可扩增100倍,波形蛋白FITC免疫荧光染色呈阳性。结论利用胶原酶结合胰蛋白酶消化法可在3h内同期分离到用于构建复合型组织工程皮肤的种子细胞,为复合组织工程皮肤的快速构建提供了可能。

Objective To find a feasible method that can fast isolate seed cells, keratinocyte stem cell and fihrohlasts, for composite tissue engineered skin. Methods The foreskin could he attained from posthectomy, the subcutaneous tissue was removed completely, and the full-thick skin was cut into pieces, 2 mm× 2 mm in size, then the pieces were submerged into a centrifuge tube containing collagenase Ⅰ in a oscillator. After 3-hour digestion at 37℃, the dermis was dissolved completely with all the fihrohlasts in the digestion solution and the epidermis could he separated easily. With more than 10-minute digestion in trypsin at 37℃, the epidermal cells could he harvested. Then flowcytometry and FITC-immunofluorescence for cytokeratin 19 of epidermal cells and FITC-immunofluorescence for vimentin of fihrohlast were conducted to identify keratinocyte stem cells in the epidermal cells and fihrohlasts in the digestion solution. Moreover, epidermal cells and fibroblasts were cultured in vitro for 7 days to investigate their biological behavior. Results Using collagenase Ⅰ combined with trypsin, epidermal cells and fihroblasts could he isolated at one time within 3 hours. Up to 17% cells demonstrated cytokeratin 19 positive in the epidermal cells, with fihrohlast vimentin positive. The amount of fihrohlast could he enlarged to more than 100 times within 6 days, hut the putative keratinocyte stem cells were difficult for subculture. Conclusion Seed cells for composite tissue engineered skin could be harvested fast at one time, that made it possible to reconstruct composite tissue-engineered skin in vitro.