ES细胞源性表皮干细胞及类真皮构成皮肤类似物分化.docVIP

ES细胞源性表皮干细胞及类真皮构成皮肤类似物分化 　 作者：刘爱军， 黄锦桃， 李海标 【摘要】 　　【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4 d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。 【关键词】 ES细胞； 表皮干细胞； 皮肤附属结构； 定向分化； 羊膜 Abstract： 【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 amnion. 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, immunohistochemical and di-labeled immunofluorescence methods to test the expression of β1 integrin, CK15, 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 squamous 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 β1 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 kerat