ADSCs转染GH和Tβ4复合微孔结构羊皮支架促进创面愈合的机制研究

Biological dressing is widely used in wound healing. Currently, our group is always committed to the development and application of the acelluar dermal matrix, and we have achieved some good results during early experimental study. Cell therapy has become a new strategy for the treatment of many diseases in clinical and research work. Adipose-derived stem cells(ADSCs) have the ability to self-renewal and multi-directional differentiation potential, and it is easy to take. In addition, further studies have shown that growth hormone(GH) and thymosin β4(Tβ4) has obvious promote wound healing. In this study, ADSCs are isolated and cultured in vitro. On the one hand, ADSCs are differentiated to epidermal cells (ECs); on the other hand, construction of eukaryotic expression vector of human GH and Tβ4 and establishment of its stable transfected ADSCs cell lines.And then, the ECs and stable transfected ADSCs cell lines were cultured on the acellular dermal matrix, constructing a complex dressing capable of secreting growth factors to promote wound healing. Choosing full-thickness cutaneous damage model, the composite dressing are transplanted in nude mice that full-thickness cutaneous damage, to observe the repair effects of the composite dressing, as well as ADSCs implanted in vivo wound environment, cell survival, growth and differentiation .

严重大面积烧伤救治过程中，临床上多选用生物敷料对创面进行修复。本课题组多年来致力于脱细胞羊皮的开发与应用，前期实验研究中取的了较好的效果。细胞疗法在临床及科研工作中已成为治疗多种疾病的新策略。ADSCs具备自我更新能力与多向分化潜能，同时具有自体取材容易、来源广泛的优势。此外有研究证明，GH和Tβ4具有明显促进创面愈合的作用。本课题拟在体外分离培养脂肪干细胞，一方面将ADSCs诱导成为ECs；另一方面构建hGH和Tβ4的真核表达载体，转染ADSCs，建立转染稳定的ADSCs细胞系。本课题将ECs以及转染的ADSCs细胞系种植于微孔羊皮支架中，构建一种能够分泌促进创面愈合生长因子的复合生物敷料。选用全层损伤模型，将复合羊皮敷料移植于裸鼠皮肤全层损伤处，观察其对皮肤全层损伤创面修复的情况，以及植入的干细胞在体内创伤环境下，细胞存活、生长和分化情况。

严重大面积烧伤救治过程中，临床上多选用生物敷料对创面进行修复。本课题组多年来致力于脱细胞羊皮的开发与应用，前期实验研究中取的了较好的效果。细胞疗法在临床及科研工作中已成为治疗多种疾病的新策略。本项目通过组织工程技术和细胞工程技术，通过低渗的方法制备无细胞毒性的sADM，将sADM作为载体可与细胞复合培养。分离培养ADSCs，通过慢病毒转染技术构建过表达GH和Tβ4细胞系，分别将过表达GH和Tβ4细胞系及ADSC与sADM复合培养，制备成sADM-GH敷料、sADM- Tβ4敷料和sADM-ADSC敷料，并通过动物试验检测敷料对创面愈合的作用效果，研究表明sADM-ADSC敷料、sADM-GH敷料和sADM- Tβ4敷料对裸小鼠皮肤创面的愈合有积极作用，sADM-GH敷料和sADM- Tβ4敷料效果更加明显。在观察创面愈合过程中，分别在第7天、14天、21天三个时间段取创面组织，提取RNA和蛋白并分别检测第7天、14天、21天三组裸小鼠创面bcl-2、Bax、MMP9、VEGF、bFGF、LN、Collagen I、Collagen Ⅲ基因的相对表达情况以及VEGF、bFGF、LN的蛋白表达情况。研究表明sADM-GH敷料和sADM-Tβ4敷料较sADM-ADSC敷料对裸小鼠皮肤创面的愈合有积极作用，可明显抑制创面细胞凋亡，促进细胞增殖，上调VEGF、bFGF、LN、Collagen Ⅲ的表达，促进创面新生血管形成，加强新生组织黏附作用，促进创面愈合。sADM-GH敷料和sADM-Tβ4敷料组MMP9表达量升高，具有潜在的瘢痕抑制作用。期望本研究制备的敷料能够为临床治疗提供额外的治疗策略来治疗烧创伤、手术损伤等疾病。