表皮细胞、成纤维细胞和几丁质-胶原蛋白复合人工皮的体外构建

Construction of a composite skin graft composed of cultured human keratinocytes and fibroblasts and a chitosan-collagen matrix in vitro

目的 探讨几丁质 胶原蛋白膜作为表皮细胞、成纤维细胞培养支架的可能性 ,体外构建包含双层细胞的复合皮。方法 取健康成人环切包皮 ,分离成纤维细胞、表皮细胞 ,分别制成单细胞悬液 ,将几丁质 胶原蛋白膜 (有孔面向上 )平铺于 60mm培养皿中 ,首先接种成纤维细胞 ,培养 2d后 ,将含 10 %小牛血清的DMEM更换成完全型DMEM ,翻转膜 ,使光滑面朝上 ,再种植表皮细胞 ,每日换液。定期观察细胞与材料的粘附、细胞贴壁及其生长增殖情况。结果 几丁质 胶原蛋白膜对成纤维细胞、表皮细胞无明显毒性作用 ,成纤维细胞培养 1d后 ,细胞贴附于材料支架 ,细胞胞体较大 ,呈典型的梭形。加入表皮细胞复合培养 2d示表皮细胞贴壁生长 ,并分化、增殖。复合皮构建 2周 ,网格支架及孔内均有大量细胞生长 ,膜表面细胞融合成片 ,表皮细胞分化形成复层。结论 几丁质 胶原蛋白膜对细胞无毒性 ,有利于培养细胞的粘附、生长。在体外可以构建成功类似生理性皮肤的人工皮肤。

Objective To investigate the behaviour of humankeratinocytes (HKs) and human fibroblasts (HFs) cultivated on the chitosan-collagen matrix and to reconstitute the two-layer composite skin involving viable cells in vitro.Methods Proliferative human fibroblasts and keratinocytes were isolated from surgical discard foreskin and cultured respectively.The chitosan-collagen membranes (the side with more pores being upwards) were removed from storage in phosphate buffered saline (PBS),transferred into 60 mm diameter Petri dishes.Subconfluent human fibroblasts were inoculated on the porous surface of chitosan-collagen membranes.After 2 days,supplemented Dulbecco's modified Eagle's medium (DMEM) was replaced with serum-free DMEM.Inverting the membrane,subconfluent human keratinocytes were inoculated on the smooth surface,and were incubated continuously.Biologic attachment,growth,and differentiation of cultured human keratinocytes and fibroblasts were observed daily.Results No toxicity to HKs and HFs was found during the culture course.After one day,subconfluent HFs attached to the membranes and HFs were shuttle and bigger.Two days after HKs cultured,HKs attached to the chitosan-collagen membranes,grew and differentiated.After two weeks,there were many cells in the scaffold fibrils and pores of chitosan-collagen membranes.HKs became confluent to form a continuous sheet of epithelium.Conclusion No toxicity to HKs and HFs in the chitosan-collagen membranes was found.The chitosan-collagen membranes provide superior cells adherence,growth and differentiation.A composite skin graft composed of cultured human keratinocytes and fibroblasts and the chitosan-collagen matrix is feasible in vitro.