MSCs构建的类真皮对ES细胞源性表皮干细胞分化潜势的影响

Influence of dermal analog composed with MSCs upon the differentiation potency of ES cell-derived epidermal stem cells

目的探讨以MSCs构建类真皮对ES细胞源性表皮干细胞分化潜能的影响。方法大鼠骨髓MSCs与复合凝胶-明胶海绵组成类真皮,Hoechst33342标记E14-ES细胞,经羊膜诱导4 d后,形成表皮干细胞克隆,将二者构成皮肤类似物,埋植于129小鼠皮下。术后2、4、6和8周取材,做HE染色,β1整合素、CK15、CK19、CEA、CK18免疫荧光双标和免疫组化观察。结果术后2和4周,植块中以形成单层立方或柱状上皮构成的管状和泡状结构为主,6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,皮脂腺样结构多见。免疫双标结果显示,植入2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和8周后,HE染色汗腺样结构呈CEA、CK18阳性。结论ES细胞源性表皮干细胞与骨髓MSCs构建的类真皮,在同种小鼠皮下可分化出角化复层上皮、汗腺样、毛囊样、皮脂腺样等结构。

Objective To explore the differentiation potency of ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs in hypodermis. Methods The dermal analogs were reconstructed with rat bone MSCs and compound gel-gelatin sponge. E14-ES cells, labeled with Hoechst 33342, were cocuhured with human amnion. Four days later, epidermal stem cell clones were formed. The skin analogs, reconstructed with ES cell-derived epidermal stem cells and dermal analogs, were transplanted into 129 mice hypodermis. The differentiation tissue of skin analogs was sampled at 2, 4, 6, 8 weeks. The sections were observed with HE staining, immunohistochemical and di-labeled immunofluorescence methods to test the expression of β1 integrin, CK15, CK19, CEA, CK18. Results The sections were showed tubular or follicular like structures formed with simple or stratified epithelium at 2 and 4 weeks. Keratinized stratified squamous epithelium, sweat glands-like, sebaceous glandslike and hair follicles-like structures were observed at 6 week and 8 week after transplantation. The cells labeled by Hoeehst 33342, formed tubular or follicular like structures, expressedβ1 integrin, CK15, CK19, CEA and CK18 positive respectively at 2 and 4 weeks. The sweat glands-like structure expressed CEA and CK18 positive respectively at 6 and 8 weeks. There were more sebaceous glands-like structures. Conclusion ES cell-derived epidermal stem cells compounded by dermal analogs reconstructed with bone MSCs can differentiate into keratinized stratified squamous epithelium, sweat glands-like, sebaceous glands-like and hair follicles-like structures in hypodermis.