复合型组织工程皮肤的体外快速构建

FAST RECONSTRUCTION OF COMPOSITE TISSUE-ENGINEERED SKIN IN VITRO

目的寻找一种体外快速构建复合型组织工程皮肤的方法,为大面积烧伤、创伤创面的覆盖提供足够的自体皮源。方法取人的包皮,进行表皮细胞及成纤维细胞的分离鉴定及体外培养扩增,对表皮细胞群行广谱角蛋白抗体-异硫氰酸荧光素（fluorescein isothiocyanate,FITC）、角蛋白19（cytokeratin19,K19）-FITC免疫荧光染色及K19流式细胞仪鉴定;对成纤维细胞行波形蛋白-FITC免疫荧光鉴定。将表皮细胞接种于异体脱细胞真皮（acellular dermalmatrix,ADM）乳头面,于气-液平面培养构建表皮层,实验分两组进行,实验组培养基中添加25ng/ml角化细胞生长因子（keratinocyte growth factor,KGF）,对照组不添加。6d后,接种成纤维细胞于两组ADM的另一面,继续培养24h。收集构建的组织工程皮肤,切片行苏木素-伊红染色,观察所构建皮肤的镜下结构,并对比添加KGF对其影响。结果免疫荧光染色培养扩增的表皮细胞群广谱角蛋白抗体-FITC染色均为阳性、K19-FITC染色部分阳性,流式细胞仪鉴定其中K19阳性表达的细胞接近17%。成纤维细胞体外培养7d可扩增超过100倍,波形蛋白-FITC免疫荧光染色呈阳性。经过7d的体外培养构建,可以得到复合型皮肤,经苏木素-伊红染色镜下观察显示,实验组表皮层连续,形成细胞层数2-3层的复层结构,真皮中含有成纤维细胞;对照组表皮层不连续,细胞层数少,两组比较差异有统计学意义（P〈0.01）。结论利用胶原酶结合胰蛋白酶消化法进行种子细胞的分离,应用添加KGF的条件培养基,采用分步接种构建的方法,可在7d内构建出复合型组织工程皮肤。

Objective To find a feasible method that can reconstruct the composite tissue-engineered skin fast in vitro and can provide enough skin as soon as possible for covering the surface of the large-area burn. Methods The foreskin was taken during the posthectomy. The epidermal cells and fibroblasts were isolated, identified and cultured. The cytokeratin 19 （K19） flow cytometry and the fluorescein isothiocyanate （FITC）-immunofluorescence for K19 and the FITC-immunofluorescence for PAN-cytokeratin of the epidermal cells and the FITC-immunofluorescence for vimentin of the fibroblasts were performed to identify the epidermal cells and the fibroblasts. Then, the epidermal cells were seeded onto the papillary surface of an acellular dermal matrix （ADM） and were submerged into the condition culture medium added with 25 ng/ml of the keratinocyte growth factor （KGF）. However, in the control group, no KGF was added. After 24 hours, the ADM was moved up to the air-fluid surface, and the culture was continued. After 6 days, the fibroblasts were seeded onto the other surface of the ADM. After a 24-hour culture, the ADM was harvested and fixed in formalin, and the hematoxylin-eosin staining was conducted. Then, the structure of the reconstructed skin was observed under the microscope and the cell count in the epidermal layer was also conducted. Results All the cultured and expanded epidermal cells stained by the immunofluorescence demonstrated a positive reaction for PAN- FITC, and a partially positive for K19-FITC, and 17% of the cells demonstrated a positive reaction for K19 identified by the flow cytometry. The fibroblasts could be expanded by more than 100 times after a 7-day culture in vitro, and they could demonstrate a positive reaction for vimentin-FITC. After a 7-day culture, a composite tissue-engineered skin could be attained. The hematoxylin-eosin staining of the reconstructed skin showed that there was one continuous layer of the epidermis on the papillary surface of the ADM and there were fibroblasts in the superficial layer of the other one, but the epidermal layer did not stick tightly to the ADM. The cell count demonstrated that KGF promoted the epidermal cells to proliferate better（P〈0.01）and to form more significantly continuous layers of the epidermis in the experimental group than in the control group （P 〈 0. 01）. Conclusion Through the seed-cell separation by the digestion of collagenase and trypsin combined with the use of the KGF-added condition culture medium, a composite tissueengineered skin can be reconstructed within 7 days.