人羊膜上皮细胞复合去表皮的真皮构建组织工程皮肤修复裸鼠皮肤缺损的实验研究

目的 探索由人羊膜上皮细胞(hAEC)、成纤维细胞与去表皮的真皮(DED)构建组织工程皮肤修复裸鼠全层皮肤缺损的可行性.方法 采用低浓度胰蛋白酶多步消化分离法提取hAEC,并用两步酶消化法处理健康小儿包皮,获得成纤维细胞悬液,传代培养.将体外扩增培养至第3～5代的成纤维细胞和第2代hAEC分别接种在DED真皮面和基底膜面,体外器官培养构建组织工程皮肤.取3～4周龄健康雄性裸鼠20只,抽签法随机分为2组,在所有小鼠背部正中制备全层皮肤缺损模型.组织工程组用构建的组织工程全层皮肤覆盖创面;对照组创面仅覆盖凡士林油纱.分别于术后第7、14、21、28天对裸鼠全身及移植部位大体观察,比较两组间创面愈合时间及创面愈合率,并对移植部位行组织学观察.结果 hAEC具有干细胞特征,免疫荧光显示其表达结合转录因子4(0CT-4)、阶段特异性胚胎抗原4(SSEA-4).器官培养2周后,体外构建的组织工程皮肤形成4～9层复层表皮,且表皮的组织结构形态与正常皮肤类似.移植组织工程皮肤至裸鼠创面后肉眼观察显示,在移植后第7、14、21天,组织工程组创面愈合率分别为57.49％±6.11％、92.80％±3.10％、98.83％±0.25％,均明显高于对照组(22.93％±4.26％、54.57％±7.94％、91.16％±4.79％),差异均有统计学意义(n=10,t值分别为27.36、32.23、11.80,均P＜0.001),组织工程组创面愈合时间[(21.51±1.51)d]明显短于对照组[(28.80±1.14)d](n=10,￡=42.23,P＜0.001),且在移植后第28天移植物颜色与自体皮肤颜色接近.组织学观察显示,组织工程组上皮层次清晰,角化明显,真皮层细胞生长良好;对照组移植区上皮较薄且有部分缺损,真皮层次欠佳,且可见炎症细胞浸润.结论 用hAEC、成纤维细胞复合DED构建的组织工程皮肤移植后能在裸鼠体内存活,且创面愈合更佳,可望成为一种较理想的组织工程皮肤.

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human deepidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED).Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs,and a two-step collagenase digestion was used to treat healthy children's prepuce tissues to prepare fibroblast suspensions.When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2,they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissueengineered skin.A total of 20 heahhy male nude mice aged 3-4 weeks were enrolled into this experiment,and full-thickness skin defects were made on the middle of the back of mice.Then,these mice were randomly divided into 2 groups by using a lottery method,and reconstructed full-thickness tissueengineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively.The whole body and transplantation sites of the nude mice were observed on day 7,14,21 and 28 after transplantation,the wound healing time and rate were compared between the above two groups,and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination.Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen4 (SSEA-4).After 2-week organ culture,the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium,and the histological structure of the epidermis was similar to that of the normal human skin.Compared with the control group,the tissue-engineered skin group showed significantly higher wound healing rates on day 7,14 and 21 after transplantation (57.49％ ± 6.11％ vs.22.93％ ± 4.26％,92.80％ ± 3.10％ vs.54.57％ ± 7.94％,98.83％ ± 0.25％ vs.91.16％ ± 4.79％,respectively;n =10,t =27.36,32.23,11.80,respectively,all P ＜ 0.001),shorter wound healing time [(21.51 ± 1.51) d vs.(28.80 ± 1.14) d,n =10,t =42.23,P ＜ 0.001],with the color of skin grafts closer to that of autologous skin on day 28 after transplantation.Histological examination revealed distinct stratification of the epithelium,obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group,but thin epithelium with some defects,indistinct stratification of the dermis,and inflammatory cell infiltration in the control group.Condusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation,resulting in a more favorable healing of wounds,and is expected to serve as a kind of ideal tissue-engineered skin.