一种胶原基质全层皮肤模型的构建和表征

Construction and characterization of a full- thickness skin model with collagen matrix

目的构建一种体外维持时间长、有利于后续测试研究的胶原基质皮肤模型。方法将猪胶原和正常人皮肤成纤维细胞混合接种于培养皿，培养得到真皮层结构后，将第3～7代正常人角质形成细胞接种于真皮层表面，培养得到双层皮肤模型。通过HE和Masson染色评估皮肤模型的形态和组织结构，使用电镜观察模型的超微结构，并通过免疫组化和免疫荧光染色观察模型中各层的主要标记物表达情况。结果HE和Masson染色发现，皮肤模型与正常人皮肤组织有相近的真皮及表皮结构，且模型收获后仍具有稳定良好的真表皮结构，可在体外继续培养14d。电镜下显示，皮肤模型中可见角质层中脂质、颗粒层中透明角质颗粒、角化桥粒、桥粒、基底膜等超微结构。免疫组化及免疫荧光显示，皮肤模型在表皮层中具有与正常皮肤一致的转谷氨酰胺酶、丝聚合蛋白、角蛋白10、Ki67的表达，基底膜具有与正常皮肤一致的Ⅳ型胶原和层黏连蛋白5表达，真皮层具有一致的Ⅰ型胶原、Ⅲ型胶原及原纤维蛋白表达。结论本研究构建的三维皮肤模型组织结构和多种蛋白的表达与正常人体皮肤相似，模型收获后体外可继续稳定培养至少14d。

Objective To construct a collagen matrix-based skin model that can last a long time in vitro for potential application of subsequent tests and studies. Methods The porcine collagen and normal human skin fibroblasts were mixed and seeded into the culture dish, which were cultured for 3 - 4 days to obtain a dermal structure. Then, the third- to seventh-passage normal human keratinocytes were seeded onto the dermal surface and cultured for 14 days to obtain a double-layer skin model. Hematoxylin and eosin （HE） staining and Masson staining were performed to evaluate the morphology and structure of the skin model, and electron microscopy was conducted to observe the ultrastructure of the skin model. Immunohistochemical study and immunofluorescence staining were conducted to determine the expression of major markers in each layer of the skin model. Results HE and Masson staining showed that the skin model and normal human skin tissues showed very similar dermal and epidermal structures. After harvest of the skin model, it can be cultured in vitro for another 14 days with favorable and stable dermal and epidermal structures. Electron microscopy showed lipids in the stratum corneum, keratohyalin granules in the stratum granulosum, corneodesmosomes, desmosomes and basal membrane in the skin model. Immuno- histochemical and immunofluorescence staining showed the consistent expression of transglutaminase, filaggrin, keratin 10 and Ki67 in the epidermis in both the skin model and normal skin. Moreover, the expression of type IV collagen and laminin-5 in the basal membrane, as well as that of type I collagen, type m collagen and fibrillin in the dermis were both consistent between the skin model and normal skin. Conclusion The constructed three-dimensional （3D） skin model is highly analogous to normal human skin in the aspect of tissue structure and various protein expression, and the skin model can be stably cultured in vitro for at least 14 days after harvest.