核转录因子Atoh1联合多种诱导因子促进真皮多潜能干细胞成内耳毛细胞分化的作用研究

Cochlear hair cells cannot spontaneous regeneration is the key to prophylaxis and treatment for sensorineural deafness. Application of positive regulatory factors of hair cell combined with stem cell transplantation is an ideal way to treatment of sensorineural deafness. Research shows that the multipotential mesenchymal stem cells are also exist in dermis, which can differentiated into neurons and spongiocyte. What's more, they're extracted easily and could become a possibility of autologous transplantation. Atoh1 gene is a indispensable positive regulatory gene in the progress of progenitor cells eventually transdifferentiated into hair cells. The research subject of this study is adult SD rats, to investigation the biological characteristics of follicular papilla cells from back skin of actual SD Rats are separated and cultured in vitro, identification neural differentiation potential after induced by beta-mercaptoethanol. Lentivirus vector mediated overexpression of Atoh1 gene in dermal papillary cells of hair follicle, observation the cellular morphology of hair cells which transformed from dermis-derived stem cells capacity of attract axons. By using cell transplants technology, the construction of dermis-derived stem cells modified by Atoh1 gene are transplanted into the deaf animal model's cochlea which hair cells had damaged. induce the transplanted cells differentiated into hair cells and spiral ganglion neurons by using sound motivate. Based on analysis of transplanted cells migration and differentiation the to directional situation, discusses the function of dermal stem cells-derived hair cells transplanted into cochlear and the degree of restoration of auditory function in deafness rats model, attempt to provide a foundation for gene therapy of sensorineural deafness.

毛细胞不能自发再生是感音神经性聋防治难题的关键，联合应用毛细胞正性调控因子及干细胞移植是可能治疗该疾病的理想途径。研究显示真皮中同样具向神经元和胶质细胞分化的多潜能间充质干细胞，且易于取材，存在自体移植的可能性。Atoh1是祖细胞最终转分化为毛细胞不可缺少的正性调控基因。本项目以成年SD大鼠为研究对象，考察体外分离培养的成年SD大鼠背部毛囊真皮乳头细胞的生物学特性，经β-巯基乙醇诱导后鉴定其成神经分化潜能。以慢病毒为载体介导Atoh1基因在毛囊真皮乳头细胞过量表达，观察真皮源性干细胞转化为毛细胞样细胞及具有吸引神经轴突能力，并通过细胞移植技术将构建的Atoh1基因修饰的干细胞移植到毛细胞损伤的动物模型耳蜗，采用声刺激诱导其向毛细胞及螺旋神经节神经元定向分化，对移植细胞迁移、分化情况进行分析，探讨耳蜗植入真皮干细胞源性毛细胞功能以及对耳聋鼠模型听觉功能修复程度，为基因治疗神经性耳聋提供依据。

毛细胞不能自发再生是感音神经性聋防治难题的关键，联合应用毛细胞正性调控因子及干细胞移植是可能治疗该疾病的理想途径。研究显示真皮中同样具向神经元和胶质细胞分化的多潜能间充质干细胞，且易于取材，存在自体移植的可能性。Atoh1 是祖细胞最终转分化为毛细胞不可缺少的正性调控基因。本项目以成年 SD 大鼠为研究对象，考察体外分离培养的成年 SD 大鼠背部毛囊真皮乳头细胞的生物学特性，经β-巯基乙醇诱导后鉴定其成神经分化潜能。以慢病毒为载体介导 Atoh1 基因在毛囊真皮乳头细胞过量表达，观察真皮源性干细胞在大鼠皮下转化为毛细胞样细胞及具有吸引神经轴突能力，为基因干细胞治疗神经性耳聋提供依据。研究工作自2013年1月至2013年9月完成SD大鼠毛囊真皮乳头细胞的体外分离和培养，流式鉴定细胞周期，免疫染色检测细胞表面标志物的表达。体外诱导真皮乳头细胞进行成神经分化，于不同时相点在置显微镜下观察细胞的形态学改变，染色检测细胞表面神经元标志物的表达。2013年10月至2013年12月完成构建可分泌表达pEF1a–Atoh1–IRES–GFP的通用表达载体和重组慢病毒，观察过量表达Atoh1基因对真皮毛乳头细胞增殖和分化的影响。Atoh1过表达促进毛囊真皮乳头细胞SD大鼠皮下转分化的实验研究未按照预期研究计划进行。