ES细胞源性表皮干细胞与类真皮构成皮肤类似物的分化

Differentiation of Skin Analogs Reconstructed with ES Cell-Derived Epidermal Stem Cells and Dermal Analog

【目的】以ES细胞源性表皮干细胞为种子细胞构建皮肤类似物,探讨其在皮下的分化潜能。【方法】Hoechst33342标记E14-ES细胞,经羊膜诱导4d后,形成表皮干细胞克隆,并以其作为种子细胞,取129胎鼠成纤维细胞与复合凝胶-明胶海绵复合,形成皮肤类似物,埋植于129小鼠皮下组织。术后2周、4周、6周、8周取材,做HE染色观察,β1整合素、CK15、CK19、CEA、CK18免疫组化荧光双标和免疫组化观察。【结果】术后2周和4周,植块内可见单层立方或柱状上皮构成的管状和泡状结构,6周和8周后,可见角化复层上皮、毛囊样、汗腺样、皮脂腺样等结构,免疫双标结果显示,植入2周和4周,带有蓝色核标记的细胞形成的管状或泡状结构呈β1整合素、CK15、CK19、CEA和CK18阳性,6周和8周后,HE染色中汗腺样结构呈CEA、CK18阳性。【结论】ES细胞源性表皮干细胞与类真皮构建的皮肤类似物,在同种小鼠皮下有分为角化复层上皮、汗腺样、毛囊样、皮脂腺样等结构的潜能。

[ Objective ] To explore the differentiation potency of skin analog reconstructed with ES cell-derived epidermal stem cells and dermal analog in hypodermis. [Methods] E14-ES cells, labeled with Hoechst 33342, were cocultured with human anmion. Four days later, epidermal stem cell clones were formed. The skin analog was reconstructed with ES-derived epidermal stem cells and dermal analog, which reconstructed with 129 mice fetus fibroblasts and compound gel-gelatin sponge. The skin analogs were transplanted into 129 mice hypodermis. The differentiation tissues of skin analog were sampled at 2w, 4w, 6w, and 8w. Sections were observed with HE staining, and di-labeled irnmunofluoreseence methods to test the expression of 61 integrin, CK1S, CK19, CEA, and CK18. [Results] Sections showed tubular or follicular like structure formed with simple or stratified epithelium at 2 and 4 weeks. Keratinized stratified squamons epithelium, sweat gland-like, sebaceous gland-like and hair follicle-like structures were observed at 6 week and 8 week after transplantation. Cells labeled by Hoechst 33342, formed tubular or follicular-like structure, expressed 61 integrin, CK15, CK19, CEA and CK18 positive respectively at 2w and 4w. The sweat glands-like structure expressed CEA and CK18 positive respectively at 6w and 8w. [Conclusion] Skin analogs reconstructed with ES-derived epidermal stem cells and dermal analogs can differentiate into keratinized stratified squamous epithelium, sweat gland-like, sebaceous gland-like and hair follicle-like structures in hypodermis.