ES细胞源性表皮干细胞与类真皮构建组织工程皮肤的体内分化

目的以ES细胞源性表皮干细胞为种子细胞与类真皮构建组织工程皮肤,探讨其在体内的分化。方法胎鼠皮肤成纤维细胞和大鼠骨髓基质干细胞(BMSCs),分别与复合凝胶-明胶海绵构建类真皮(类真皮Ⅰ、Ⅱ),植入小鼠全层皮肤缺损创面,以生物膜为载体,把羊膜诱导后带有核标记的表皮干细胞覆盖在类真皮上,术后1～8周连续取材,苏木精-伊红染色,β1整合素、CK15、CK19、CK10和CEA免疫荧光双标和免疫组化观察。结果两组组织工程皮肤植入皮肤缺损3～4周后,创面完全长合,较厚新生皮完全覆盖创面,基底层细胞增生,形成短的细胞柱突向真皮层。新生表皮中可见核标记的细胞呈β1整合素、CK15、CK19阳性,真皮中的管腔样结构呈核荧光和CEA免疫组化双标阳性,4～8周新生表皮基底层细胞呈CK19、CK10阳性,新生表皮下可见毛囊样、皮脂腺样结构。结论ES细胞源性表皮干细胞为种子细胞与类真皮构建的两种组织工程皮肤在体内具有修复缺损皮肤及分化为表皮及毛囊样、皮脂腺样和汗腺样等皮肤附属结构的潜能。

Differentiation of tissue-engineered skin reconstructed with ES cell-derived epidermal stem cells and dermal analogs in vivo

Objective To explore the differentiation potency of tissue-engineered skin reconstructed with ES cell-derived epidermal stem cells and dermal analogs in vivo. Methods The dermal scaffolds were reconstructed by gel-gelatin sponge compounded with mice fetus skin fibroblasts or rat bone BMSCs (tissue-engineered skin Ⅰ,Ⅱ). First, the scaffolds were transplanted into mice full thickness skin defects. And then, ES cell-derived epidermal stem cells, labeled with Hoechst 33342, carried by a layer of biomembrane, were transplanted onto the dermal scaffolds. The differentiation tissue of the tissue-engineered skin was sampled each week. The sections were observed with HE staining, immunohistochemical and dilabeled immunofluorescence methods to test β1 integrin, CK15, CK19, CK10 and CEA. Results The full thickness skin defects, covered with the two types of tissue-engineered skin, were healed at 3-4 weeks. The newborn skin was thicker than normal skin. There were short bulky cellular poles towards dermis. The cells labeled by Hoechst 33342 located in the epidermis expressed β1 integrin, CK15 and CK19. And the tubular or follicular structure in dermis showed nucleus fluorescence and CEA positive. The basal cells of keratinized stratified squamous epithelium expressed CK19 and CK10 positive respectively. There were sebaceous glands-like and hair follicles-like structures in newborn skin. Conclusion The two types of tissue-engineered skin reconstructed with ES cell-derived epidermal stem cells and dermal analogs can restore the skin defects and have the potency to differentiate into sweat glands-like, sebaceous glands-like and hair follicles-like structures in vivo.