组织工程皮肤的体外构建及用于大鼠皮肤损伤修复的效果分析

In vitro construction of tissue-engineered skin and the effect on damage repair

目的探讨由人表皮干细胞、成纤维细胞和纤维蛋白支架构建组织工程皮肤(TES)及用于皮肤缺损大鼠损伤修复的效果。方法通过胰蛋白酶消化法分离人表皮干细胞和真皮成纤维细胞,传代培养并与纤维蛋白支架构建TES。取60只适龄无特定病原体级雄性大鼠,建立皮肤缺损模型并随机分成TES组、表皮干细胞支架组、成纤维细胞支架组、支架组和对照组,每组12只。术后2、4、6周观察大鼠移植部位和整体情况,获取组织标本行免疫组织化学检查,并统计各组的皮片成活率、创面愈合率、移植物收缩率、微血管数目等指标。比较各组大鼠术后6周新生皮肤厚度及创面完全愈合时间。结果所有大鼠术后饮食、饮水、排便及排尿正常,均无感染情况出现,顺利完成实验。TES组术后2周便完成新生皮肤生长,术后6周新生皮肤质地、韧性和光滑程度均较好,其余4组术后6周观察创面凹陷、显著收缩、新生皮肤欠平整,各组均无肿瘤性组织生长或炎性组织。术后6周,TES组移植物收缩率、创面愈合率、微血管数目、皮肤厚度均明显高于其余4组,且创面完全愈合时间明显短于对照组和支架组[(28±4)d比(44±6)、(42±7)d](均P<0.05)。各组大鼠术后2、4、6周皮片成活率比较差异均无统计学意义(均P>0.05)。组织学观察发现,术后6周TES组棘细胞层达3~4层,表皮干细胞达8~10层,可见少量淋巴细胞,血管和成纤维细胞排列有序,数目较多。其余4组术后6周开始出现颗粒层、棘层、基底层,角质层不连续;真皮层成纤维细胞排列无序,数目较多,并可见少量淋巴细胞和血管。结论采用人表皮干细胞、成纤维细胞和纤维蛋白支架构建TES可以帮助缺损皮肤快速修复,并改善愈合质量。

Objective To explore the effect of tissue-engineered skin(TES)constructed by human epidermal stem cells,fibroblasts and fibrin scaffolds on skin defect repair in rats.Methods Human epidermal stem cells and dermal fibroblasts were isolated by trypsinization and cultured to construct TES with fibrin scaffolds.Sixty specific pathogen free male rats were made skin defect model and divided into TES group,epidermal stem cell scaffold group,fibroblast scaffold group,scaffold group and control group,with 10 rats in each group.At 2,4 and 6 weeks after skin grafting,repair site and general condition of rats were observed;tissue specimen was obtained for immunohistochemistry examination.The skin graft survival rate,wound healing rate,graft contraction rate and the number of microvessels were measured.The 6-week neonatal skin thickness and wound healing time were recorded.Results All rats successfully survived from operation and had normal postoperative diet,water drinking,defecation and urination,and no infection occurred.In TES group,neonatal skin grew completely in 2 weeks after operation;skin texture,toughness and smoothness grew well after 6 weeks.In the other 4 groups,skin wound sank and contracted at 6 weeks after operation and neonatal skin were uneven.No tumorous tissue or inflammatory tissue developed.At 6 weeks after operation,the skin graft contraction rate,wound healing rate,number of microvessels and skin thickness in TES group were significantly higher than those in the other 4 groups;wound healing time in TES group was significantly shorter than that in control group and scaffold group[(28±4)d vs(44±6),(42±7)d](all P<0.05).There were no significant differences in the 2-,4-and 6-week skin graft survival rates among groups(all P>0.05).Histological examination showed that prickle cells reached 3-4 layers and epidermal stem cells reached 8-10 layers in TES group at 6 weeks after operation;a small amount of lymphocytes infiltrated;a large amount of blood vessels and fibroblasts distributed orderly.In the other 4 groups there were uncontinuous layers of granular cells,prickle cells,basal cells and keratinized cells;a large amount of fibroblasts distributed irregularly and a small amount of lymphocytes and vessels were observed.Conclusion Using human epidermal stem cells,fibrin scaffolds and fibroblasts to construct TES can help repair skin defect quickly and improve wound healing quality.