以表皮干细胞与HaCaT细胞为种子细胞构建组织工程皮肤的比较研究

目的 比较人表皮干细胞(epidermal stem cells,ESCs)与人永生化角质细胞HaCaT作为种子细胞构建组织工程皮肤的差异.方法 Ⅳ型胶原快速黏附法分选表皮干细胞,无血清培养基DK-SFM培养,取第2代备用;应用DK-SFM培养HacaT细胞,取对数生长期的细胞备用;利用组织工程技术构建真皮等价物(dermal equivalent);将上述备用的两种种子细胞分别接种在真皮替代物的表面,构建组织工程皮肤,先后进行浸没培养和气-液面器官型培养,从形态学、物理特性、HE染色等方面观察比较培养物的差异.结果 以ESCs构建的组织工程皮肤收缩快,分层分化近似于正常皮肤,具有较好的张力和韧性.以HaCaT细胞构建的组织工程皮肤分层少,生长慢,张力韧性较差.两种皮肤的直径都随培养时间的增加而缩短,且在3～7 d各检测时相点间有显著性差异(P＜0.05).结论 ESCs比永生化的HaCaT细胞更适合作为种子细胞构建组织工程皮肤.

Comparison of epidermal stem cells and HaCaT cells as seed cells to construct tissue engineered skin

Objective To explore the differences of human epidermal stem cells ( ESCs) and human immortalized keratinocytes HaCaT as seed cells to construct tissue engineered skin. Methods Human ESCs were selected by rapid attachment to collagen Ⅳ , and fed with DK-SFM. The cells at passages 2 were ready to seed. Hacat cells were also fed with DK-SFM. ESCs and Hacat cells were respectively seeded on collagen matrices populated with increasing numbers of fibroblasts and cultured for 1 week at the air liquid interface. Morphology, physical properties observation, and HE staining were carried out to compare the differences of the 2 cells. Results ESCs were faster in constructing a contraction of the tissue-engineered skin, and had good tension and toughness and similar stratification as normal skin. HaCaT cells were less laminated, slow growth, and had low tension and toughness in tissue-engineered skin construction. The diameter of 2 types of constructed skin samples was shortened along with the increasing of culture time, and had significant difference in 3 to 7 d. Conclusion Epidermal stem cells are more suitable as seed cells for tissue engineering of skin than the HaCaT immortalized cells.