自体与异体细胞混合构建的活性皮肤替代物修复隐性营养不良型大疱性表皮松解症患者手部瘢痕挛缩一例

Construction of living skin equivalents using mixed autologous and allogeneic skin cells for repairing scar contracture of the hand in a patient with recessive dystrophic epidermolysis bullosa

目的观察以人羊膜为基质、自体与异体细胞混合构建的活性皮肤替代物（AM—LSE）修复大疱性表皮松解症患者手部瘢痕挛缩的疗效。方法采集1例隐性营养不良型大疱性表皮松解症（RDEB）患者及其母亲的皮肤组织，分别进行表皮角质形成细胞和真皮成纤维细胞分离、培养。将自体与异体细胞混合后构建AM—LSE并进行组织学检查和Ⅶ型胶原免疫荧光检查。移植AM-LSE皮片于RDEB患者手部瘢痕挛缩松解术后的皮肤缺损区域。术后观察皮片的成活以及创面修复效果。结果构建的AM-LSE具有真皮层、重层分化良好的表皮层和发育完好的基底膜。免疫荧光检查显示，Ⅶ型胶原沿基底膜线性分布。术后观察半年，移植的AM—LSE存活良好，未发现明显的排异反应，未发现受皮区再发水疱及破溃，瘢痕挛缩不显著，皮片色泽接近正常皮肤，质地柔软，患手可抓握，满足一般生活自理的要求。结论自体与异体细胞混合构建的AM-LSE具有良好的组织学形态，移植于RDEB患者瘢痕切除后的创面，无明显排异，不易因摩擦起水疱破溃或发生瘢痕挛缩。

Objective To evaluate the effect of living skin equivalents （LSE） constructed of mixed autologous and allogeneic skin cells and human amnion which served as a matrix on repairing scar contracture of the hand in a patient with recessive dystrophic epidermolysis bullosa （RDEB）. Methods Skin tissues were obtained from a patient with RDEB and her mother, and epidermal keratinocytes and dermal fibroblasts were isolated from these tissues and cultured in vitro separately. Human amnion was obtained from the placenta of an unrelated healthy parturient undergoing cesarean delivery, and served as a matrix of the LSE. The autologous and allogeneic fibroblasts were mixed and seeded on the stromal side of the amnion, and then the autologous and allogeneic keratinocytes were mixed and seeded on the epithelial side of the amnion, so as to construct the human amnion-LSE （AM-LSE）. Histological examination was performed to observe the structure of the skin tissues obtained from the patient and her mother, and immunofluorescence staining was conducted to detect the expression of type ~ collagen in the skin tissues of the patient and her mother and in the AM-LSE. The AM-LSE was grafted on the skin defects of the patient after release of scar contracture of the hand. After grafting, the survival status of the AM-LSE graft and repairing effect on the wounds were evaluated. Results The constructed AM-LSE consisted of dermis, multilayered and fully differentiated epidermis and well- developed basement membrane. Immunofluo-rescence examination revealed that type VII collagen was linearly distributed along the basement membrane. Half a year after grafting, the AM-LSE survived well, and no obvious rejection reaction was observed. No blisters or ulcers occurred at the recipient sites, and the scar eontracture was mild. The grafted area showed normal skin color with soft texture. The patient could grab and hold things, which had met self-care requirements of daily living. Conclusions The AM-LSE constructed of mixed autologous and allogeneic skin cells have good histological structures, and can be grafted on the wounds after resection of the scars in a RDEB patient, After grafting, no obvious rejection reaction was observed, and the skin was not liable to develop blisters, ulcers or scar contracture due to friction.