细胞共培养人体皮肤类似物超微结构观察及其渗透性测试

Ultrastructure and penetration of cell co-culture model of human skin equivalent

目的观察人体皮肤类似物模型形态结构，对其屏障功能进行初步评价。方法通过胶原蛋白包被成纤维细胞，制备真皮层，真皮层表面接种角质细胞，浸没培养1周后，气一液界面培养以促进角质细胞的分化形成角质层，3周后收获皮肤模型；通过肉眼观察、电镜观察皮肤类似物的宏观结构和超微结构；以吲哚美辛作为模型药物，进行体外透皮实验考察细胞共培养皮肤类似物模型的屏障功能。结果皮肤类似物形成与人类皮肤相似的真皮层和表皮层，表皮层表面形成完整的角质层，具有与人类皮肤类似的超微结构；24h内渗透性实验结果显示皮肤类似物屏障功能略低于大鼠腹部皮肤，但22—24h，皮肤类似物对模型药的透过速率远小于大鼠腹部皮肤（约为其28％），且2种皮肤模型对吲哚美辛经皮渗透的稳态流速没有显著性差异（P〉0．05）。结论皮肤类似物有与人体皮肤相类似的形态结构特征，与大鼠腹部皮肤相比对吲哚美辛有相似的屏障作用，有望替代动物皮肤成为经皮给药系统评价的新型皮肤模型。

Objective To observe the structure of human skin equivalent （HSE） model in vitro, and to preliminarily assess the barrier function of HSE. Methods The HSE model was established by two steps ： preparation of dermal layer and preparation of full-thickness skin. Keratinocytes were inoculated on the surface of dermal layer prepared by type 1 collagen coating fibroblasts. After submerged cultured for one week, air-liquid interface cultured for three weeks to enhance the proliferation of keratinocytes, the HSE model was obtained. Then the macroscopical structure and electon microscopical structure of HSE were studied. To investigate the barrier function of HSE model, indometacin was selected as the model drug for the penetration testing in vitro. Results The structures similar to dermal layer and epidermal layer of human skin were developed in HSE model, furthermore, complete straum corneum with similar uhrastructure of human skin was developed on the surface of epidermal layer. The penetration testing showed that the barrier function of HSE was slightly lower than that of rat abdominal skin within 24 h, but the cumulative content （ Q24 ） of indometacin of HSE was far slower （ only 28% ） than rat abdominal skin from 22 to 24 h. However, the steady state flux of indometacin showed no significant difference in HSE model and rat abdominal skin （ P 〉 0.05 ）. Conclusion The morphology and structure of HSE model were close to human skin; and in HSE model, the barrier function against indometacin was almost as the same as the rat abdominal skin. It is expected that HSE model be an alternative option of animal skin to evaluate the effect of transdermal drug delivery system.