人羊膜活性皮肤替代物修复巨痣切除术后创面的可行性及疗效研究

Application of amniotic membrane-living skin equivalent in repairing skin defect after removal of congenital giant nevus

目的探讨临床应用以人羊膜为基质的活性皮肤替代物(amniotic membrane-living skin equivalent,AM-LSE)修复皮肤缺损的可行性。方法 2016年7月收治1例5岁颈肩背部先天性巨痣患儿。首先取其正常皮肤分离扩增表皮角化细胞和真皮成纤维细胞,再以自愿捐赠的人去上皮AM为基质,经气液面培养10 d构建含有真皮基质和重层分化表皮的AM-LSE。对AM-LSE行大体及组织学观察。手术切除巨痣至深筋膜层,遗留创面面积为20 cm×15 cm;覆盖胶原蛋白海绵人工真皮,2周后改用AM-LSE贴敷于肉芽创面,之后常规换药。术后观察AM-LSE成活以及创面修复效果。结果气液面培养10 d后,成功构建具有真皮层以及重层分化良好的表皮层的AM-LSE。临床修复术后见AM-LSE贴敷良好,逐渐扩大融合,至术后6个月其色泽接近正常皮肤,质地柔软,皮片间隙为淡红色瘢痕。组织学观察,可见呈柱状紧凑排列的基底层细胞,以及分化的棘细胞层、颗粒层以及角质层。真皮层内分布大量成纤维细胞以及血管,未见明显炎性细胞浸润。结论采用自体皮肤细胞构建的AM-LSE移植修复创面可行,术后能长期存活,且无排斥反应。

Objective To investigate the feasibility of human amniotic membrane-living skin equivalent （AM- LSE） in repairing the skin defect. Methods A 5-year-old boy with giant nevus at neck, shoulder, and back was admitted in July 2016. Normal skin tissue of the patient was harvested and keratinocytes and dermal fibroblasts were separated and expanded in vitro. Human AM was donated from a normal delivery and de-epithelialized for constructing an LSE as a matrix. Keratinocytes were seeded on the epithelial side of the AM which was previously seeded with fibroblasts on the stromal side and then the complex was lifted for air-liquid surface cultivation for 10 days and observed under naked eyes and sampled for histological study. The nevus was excised to deep fascia and the skin defect in size of 20 cm×15 cm was covered with artificial skin of collagen sponge for 2 weeks to enhance granulation tissue formation, and then the AM-LSE grafts of stamp size were grafted on. The dressing was changed until the wound healed. Results After 10 days of air- liquid surface cultivation, the AM-LSE developed a multilayered and differentiated epidermis with the fibroblasts- populated amnion as the dermal matrix. The LSE stamps survived and expanded to cover the whole wound. The grafted area showed normal skin color and soft contexture at 6 months after operation, and histological study showed welldeveloped epidermis with compactly aligned basal cells, stratified and well differentiated squamous, granular layers and stratum corneum and well vascularized dermal compartment without inflammatory cells infiltration. Conclusion The cultivated AM-LSE with autologous cells can repair skin defect and survive for a long term without reiection.