用于替代皮肤刺激试验的组织工程表皮模型的构建研究

EXPERIMENTAL STUDY ON CONSTRUCTING A TISSUE ENGINEERED EPIDERMIS FOR REPLACEMENT OF SKIN IRRITATION IN VITRO

目的探讨人表皮细胞与聚碳酸脂膜构建组织工程表皮模型的方法,建立可用于皮肤刺激试验的组织工程表皮模型。方法应用组织工程方法,以聚碳酸脂膜为支架,以人皮肤角质形成细胞为细胞来源,构建组织工程表皮模型。气液面培养13 d,通过HE染色、角蛋白10(keratin 10,K10)和K13抗体、K14抗体、层粘连蛋白抗体、角化细胞交联外膜蛋白抗体、中间丝相关蛋白抗体免疫荧光染色、透射电镜观察组织工程表皮模型组织结构。采用快速渗透试验方法,SDS分别作用于培养基中添加脂质物(实验组)和未添加脂质物(对照组)的组织工程表皮模型18 h,测定组织活性减小50%所需化学物质的浓度(half maximal inhibitory concentration of a substance,IC50)值。结果 HE染色、免疫荧光染色及透射电镜观察显示,构建的组织工程表皮模型分化良好,具有与正常表皮相似的结构:基底膜、棘层、颗粒层和角化层。实验组和对照组组织工程表皮模型的IC50值分别为0.183%(6.00 mmol/L)和0.072%(2.36 mmol/L)。结论在聚碳酸酯膜上构建的组织工程表皮模型具有与正常表皮相似的组织结构,并且具有一定的屏障功能。

Objective To investigate the method of constructing a tissue engineered epidermis with human epidermal cells and polycarbonate membrane,and to establish a tissue engineered epidermis with barrier function which is intended to be the replacing model in vitro of skin irritation test.Methods The tissue engineered epidermis was constructed by using polycarbonate membrane as scaffold and stratified differentiated epidermis derived from human keratinocytes.The tissue engineered epidermis was cultured on an inert polycarbonate filter at the air-liquid interface.After 13 days of culture,the composition and structure of tissue engineered epidermis were observed by HE staining,immunofluorescence staining of keratin 10（K10） ＆ K13,K14,laminin,involucrin,and filaggrin,and transmission electronic microscope.The half maximal inhibitory concentration of a substance（IC50） of SDS was determined in the penetration test of tissue engineered epidermis cultured in the absence（control group） or the presence（experimental group） of lipid supplement for 18 hours.Results The constructed epidermis was similar to normal epidermis,which was consisted of a proliferating basal layer,differentiated spinous layer,granular layer,and stratum corneum.The IC50 values of tissue engineered epidermis cultured in the control group and experimental group were 0.072%（2.36 mmol/L） and 0.183%（6.00 mmol/L ）, respectively. Conclusion The tissue engineered epidermis constructed on polycarbonate membrane has normal composition and structure and barrier function corresponding to the normal epidermis.