复合汗腺的组织工程三维皮肤模型的构建

Construction of a three dimensional skin model containing sweat gland cells with tissue-engineering technique

目的:运用组织工程化方法在体外建立复合汗腺的三维皮肤模型,阐明汗腺体外再生的可行性,并为初步建立含有汗腺的仿生化功能性组织工程皮肤奠定实验基础。方法:分离培养人表皮细胞、成纤维细胞和汗腺细胞,将表皮细胞与汗腺细胞按1:1的比例共培养,在培养体系中分别加入含表皮细胞生长因子(EGF)的微球,噻唑蓝(MTT)法观察细胞增殖情况,并与培养时直接加入EGF及不加EGF的对照组进行比较。将共培养的细胞接种于复合成纤维细胞的胶原基质并通过三维培养方式构建组织工程皮肤模型,在模型中加入复合EGF的微球释放载体促进汗腺再生和上皮化进程,HE染色和免疫组织化学方法检测所构建组织工程皮肤以及体外再生汗腺的结构,并与模型中直接加入EGF和未加EGF的对照组进行比较。结果:MTT检测结果显示,与两对照组相比较,通过共培养方式获得的表皮与汗腺细胞团在复合EGF微球作用下生长良好,有明显增殖作用;组织学观察结果显示所构建组织工程皮肤模型具有和正常皮肤相似的结构,并在真皮浅层形成了类似汗腺结构的细胞密集区域,存在大量汗腺特异性标志--角蛋白19(CKl9)和癌胚抗原(CEA)阳性的细胞,这种现象在单纯EGF组仅有微弱表现,在空白对照组则无此现象出现。结论:构建具有汗腺结构的体外皮肤模型具有可行性,这为汗腺再生和功能性组织工程皮肤的研究开创了新的途径。

Objective： To reproduce a three-dimensional engineered skin construct with incorporation of sweat glands in vitro with tissue engineering technique and to verify the feasibility of sweat gland regeneration in vitro, in order to provide the foundation of development of the biomimetic and functional tissue engineering skin containing sweat glands. Methods： Human keratinocytes, fibroblasts and sweat gland cells （SGCs） were isolated and cultured respectively, and then keratinocytes and SGCs （1 ： 1 ） were co-cultured with the sustained-releasing epidermal growth factor （EGF）-loaded microspheres. The growth and proliferation of the cells was estimated by MTT assay. EGF directly added medium and EGF free medium were as control. Finally, the engineered skin construct was completed by inoculating the co-cultured keratinocytes and SGCs on top of a fibroblast-embedded collagen-based matrix in an organotypic co-culture model. The structure of engineered skin constructs and formed sweat glands was evaluated by HE staining and immunohistochemical method. Results： MTT assay showed that the co-cultured keratinocytes and SGCs grew better and proliferated more eminently under the effect of EGF released from sustained-release EGF-loaded microspheres. Histological examination showed that the engineered skin construct manifested a similar structure of the natural skin,and aggregations of sweat gland-like cells expressing both CK19 and earcino-embryonic antigen （CEA） were found in the superficial layer of dermis. This effect was less obvious in the specimens in which EGF was directly added, and it was not observed in EGF free specimens. Conclusion： The result suggests that the reproduction of engineered skin model containing sweat glands in vitro is feasible and it can be used as a novel and promising strategy for the development of functional tissue engineering skin with sweat glands regeneration.