ADSCs构建皮肤复合组织修复皮肤溃疡实验研究

Experimental Study on Constructing Skin Complex Tissue by Adipose-derived Stem Cells to Repair Skin Ulcers

目的探索脂肪来源干细胞构建皮肤复合组织的方法及修复皮肤溃疡的效果。方法采用从人类包皮中分离出表皮细胞和成纤维细胞进行体外培养与鉴定,首先把成纤维细胞接种于牛胶原凝胶中培养生长构建活性真皮替代物,在裸鼠背部制做直径1cm全层皮肤缺损,将培养2周的活性真皮替代物移植于创面,观察其愈合情况;再接种表皮细胞于该活性真皮替代物中,经过气-液界面培养,表皮细胞成层并向基底上层分化形成表皮/真皮复合皮。取10 ml成人脂肪抽吸物,分离、培养和鉴定脂肪来源干细胞(ADSCs),将ADSCs作为种子细胞接种于牛胶原凝胶支架并行成脂诱导和扩增培养构建类"脂肪"组织,再把复合皮与所构建的"脂肪"组织按表皮、真皮、皮下脂肪的结构顺序"组合"培养以构建出具有多层结构的皮肤复合组织。在裸鼠背部两侧建立直径为10 mm的圆形皮肤创面模型,右侧(实验侧)创面缺损部位植入直径为10 mm的多层复合组织,左侧(对照侧)进行简单的创面包扎修复,观察两侧溃疡愈合速度和质量以及通过活检组织病理学检查比较各自的表皮、真皮、成纤维细胞、血管和脂肪组织再生情况。结果成纤维细胞接种于Ⅰ型牛胶原凝胶共同培养所构建的活性真皮替代物类似真皮,可修复皮肤缺损。在构建的活性真皮替代物上种植表皮细胞,经过气-液界面培养,表皮细胞成层分化,形成复合皮。从脂肪组织中可分离获取ADSCs,它具有多向分化潜能。ADSCs与Ⅰ型牛胶原凝胶支架在体内、外均可构建出脂肪样组织,复合皮与类"脂肪"组织参照正常皮肤结构顺序"组合"培养可构建出具有多层结构的皮肤复合组织。实验侧创面愈合率、肉芽组织厚度、真皮层厚度以及毛细血管密度显著高于对照侧。结论选择表皮细胞、成纤维细胞、脂肪来源干细胞(ADSCs)作为组织工程种子细胞,以牛胶原凝胶作为支架,能够构建出具有多层结构的皮肤复合组织,并可成功用于修复裸鼠背部皮肤全层组织缺损,具有提高愈合速度、增加愈合皮肤厚度的作用。

Objective To explore the method and effect for constructing skin complex tissue to repair skin ulcers with adult adipose-derived stem cells.Methods Epidermal cells and fibroblasts isolated from human foreskin were cultured and identified in vitro.The fibroblasts were inoculated firstly into bovine collagen gel in order to building dermal substitute.1 cm-2 whole layer defect was made on the back of nude mice.The active dermal substitute which had been cultured for 2 weeks was transplanted on the ulcers,observing the healing.The epidermal cells were inoculated secondly on the dermal substitute which the epidermal cells stratified and differentiated into the upper basement to build epidermis and dermis composite skin by gas-liquid interface culture.Adipose-derived stem cells were isolated,cultured and identified from10 ml adult liposuction.The adipose-derived stem cells as seed cells were inoculated into bovine collagen gel as scaffold.They were induced and differentiated into adipose tissue by continuous cultivation.The multi-layer composite skin tissue was assembled with the adipose tissue and compound skin containing epidermis and dermis according to normal skin structure order.10 mm round skin defect model was made on both sides of the nude mice back.The right skin defect was experimental side transplanted with 10 mm multi-layer composite skin tissue.The left skin defect was control side repaired by simple wound dressing.The speed and quality for every side heal was observed and the regeneration for epidermis,dermis,fibroblasts,vessel and adipose tissue was compared by histopathology.Results The active dermal substitute built by inoculating fibroblasts into bovine collagden gel was analogous to dermis and could be used to repair skin defect.The composite skin could be constructed by inoculating epidermal cells on the dermal substitute with gas-liquid interface culture.ADSCs could be isolated from adipose tissue and possessing multidirectional differentiation potential.The adipose tissue could be constructed successfully with ADSCs and bovine collagen gel as scaffold in vitro.The multi-layer composite skin tissue could be built by assembling with the adipose tissue and composite skin including epidermis and dermis according to normal skin structure order.The healing rate,granulation tissue thickness,derma thickness and capillary density for experimental side exceeded prominently those of control side.Conclusions The multi-layer composite skin tissue could be constructed successfully with a variety of cells as seed cells,such as epidermal cells,fibroblasts and adipose tissue-derived stem cells,and bovine collagen gel as scaffold.It could repair successfully whole layer skin defect on nude mice back with function for increasing healing speed and skin thickness.