表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的实验研究

Experimental Study on Promotion of Wound Healing with Artificial Skin Constructed with Epidermal Stem Cells and Acellular Dermal Matrix

目的探讨表皮干细胞联合脱细胞真皮构建人工皮肤促进创面愈合的可行性。方法(1)用胰蛋白酶和EDTA联合消化法分离表皮,用Ⅳ型胶原快速黏附法分离、纯化人表皮干细胞,以含表皮生长因子、角质细胞无血清培养液等组成人表皮干细胞培养基进行体外培养,测定克隆形成率。(2)将培养人表皮干细胞接种于制备的脱细胞真皮支架中,构建组织工程人工皮肤,移植治疗兔全层皮肤缺损创面,观察创面修复效果。(3)取新西兰白兔常规制作背部全层皮肤缺损创面,随机分为4组:A、B、C组分别用含表皮干细胞的组织工程皮肤、含角质细胞的组织工程皮肤和单纯脱细胞真皮移植于皮肤缺损创面;D组用创面空置为对照。观察创面修复情况、局部炎症反应,并记录创面愈合时间。结果体外培养的人表皮干细胞增殖稳定,克隆形成率明显高于角质细胞对照组(P<0.05)。移植后A组创面愈合良好,局部炎症反应轻微,无出血、积脓、坏死,创面愈合时间较B、C、D组明显缩短(P<0.05或P<0.01)。结论以表皮干细胞作为种子细胞联合脱细胞真皮构建人工皮肤可用于皮肤缺损创面的修复治疗。

Objective To construct the artificial skin with epidermal stem cells and acellular dermal matrix and to repair the rabbit full thickness dermal wounds. Methods （1） Human fetal epidermis was isolated and then dispersed by Trypsin-EDTA, and the human epidermal stem cells were isolated and purified by type IV collagen adhering method. The cells were cultured in kerati- nocyte serum free medium supplemented with 10% fetal bovine serum and 10 μg/L epidermal growth factor. The non-attaching keratinocytes were cultured as the control. The colony forming efficiency was examined by Giemsa staining. （2）The human epidermal stem cells were inoculated into the acellular dermal matrix to construct tissue-engineered skin,which was used for transplan- tation in treatment of rabbit full thickness skin wounds. （3）The wounds were produced on the back of New Zealand white rabbits. The rabbits were randomly divided into four groups： group A （tissue-engineered skin graft with epidermal stem cells） ; group B（tissue-engineered skin graft with keratinocytes）;group C（simple acellular dermal matrix graft） and group D（vacant wounds as the control）. The repairing effect of wounds,the local inflammatory response and the wound healing time were determined. Results The human epidermal stem cells proliferated steadily in vitro and the colony forming efficiency was significantly higher than that in cultured keratinocyte group（P〈0.05）. After the artificial skin transplantation, the woulds in group A healed well,accompanied by slight local inflammatory response without bleeding, empyema and necrosis. The wound healing time in group A significantly shortened compared with group B,C and D, respectively. （P〈0.05 or P〈0.01）. Conclusion The construction of artificial skin with epidermal stem cells and acellular dermal matrix can be used to repair and treat the skin defect wounds.