体外培养表皮干细胞构建组织工程皮肤的研究

Construction of tissue-engineered skin by using in vitro cultured epidermal stem cells

目的探讨应用表皮干细胞构建组织工程皮肤修复皮肤缺损的可行性。方法运用黏附实验将人表皮干细胞分选出来后大量培养,将成纤维细胞和表皮干细胞转移到真皮底物上构建组织工程皮肤,并将之移植于裸鼠皮肤缺损模型,于术后第1、2、4周处死动物,收集标本,进行组织学、透射电镜及免疫组织化学检查。结果组织工程皮肤移植后4周具备完整的表皮层和真皮层。表皮层中具备基底层、棘层、颗粒层和角质层。表皮细胞间还可以看到有桥粒、半桥粒、角质透明颗粒和粗张力微丝束。表、真皮层间有连续的、完整的基底膜。真皮网架破坏明显,大量的成纤维细胞增殖活跃,胶原纤维排列整齐,有丰富的毛细血管。结论本研究所构建的组织工程皮肤组织学形态和超微结构上已接近于正常的皮肤,从而为临床修复皮肤缺损的研究打下了坚实的理论和实践基础。

Objective To study the feasibifity of using epidermal stem cells to construct the tissueengineered skin for repairing the skin defect. Methods After the epidermal stem cells were selected di- rectly by rapid adhesion to type IV collagen within 10 rain from keratinoeytes isolated from foreskin epidermis, the tissue-engineered skin was constructed by seeding large-scale cultured epidermal stem cells in combination with fibroblasts to the artifical dermis substrate, then grafted onto athymie immunodefieient mice. All specimens were harvested on the 1st, 2nd and 4th week after transplantation to evaluate its potential to reconstitute a full-thiekness skin substitute in vivo by gross, histological, transmission electron microscopic examinations and immunohistoehemical technique. Results After 28 days of transplantation, the transplanted skin developed a well differentiated epidermis and dermis with the morphological features of intact skin. The well-stratified differentiated epidermis was composed of stratum basale, prickle cell layer, granular layer and stratum eorneum. The desmosomes, hemidesmosomes, keratohyaline granules and Keratin filaments were seen at the epidermis. A continuous basement membrane with a well-defined lamina densa was established and both collagen filaments and anchoring fibril connections to the lamina densa were numerous on the dermal side. The collagen of dermal substitute was degraded gradually and fibroblast lined up in order. Capillaries and little inflammation cells were seen. Conclusion The constructed tissue-engineered skin was similar to the uninjured skin in morphological features, suggesting the reconstructed tissue engineering skin by combining cultured epidermal stem cells as the seed cells with the artifical dermis substrate could satisfy the need for clinics to radically settle the problems of repairment of skin defects.