前庭感觉上皮重度损伤与修复机制及Atoh1基因治疗研究

The occurrence of vertigo is closely associated with impairments of hair cells, supporting cells and innervations in the vestibular sensory epithelium. In our previous study, we found a type of vestibular sensory epithelial severe lesion, which was characterized by the loss of normal cytoarchitecture of both vestibular hair cells and supporting cells, and the replacement of monolayer flat cells. However, limited spontaneous regeneration of hair cells was found in the severely damaged vestibular sensory epithelium. So far, there is no systematic study on its damage and repair mechanism, and its intervention research has not been reported. Based on our previous studies, we hypothesize these new hair cells and flat epithelial cells may trans-differentiated from supporting cells or cells in the surrounding transitional epithelium, and Notch signaling and the downstream factor, Atoh1, may play a role in the repair process. In this study, we will firstly assess pathological features and changes of the innervation in the severely damaged vestibular sensory epithelium. Then we will study the mechanism of the repair, including Notch signaling factors, Atoh1 and cell mitosis. We will use transgenic mice for lineage tracing of supporting cells to identify the dynamic changes of supporting cells and the origin of new hair cells. Finally, we will over-express Atoh1 by using an adeno-associated virus, to induce hair cell regeneration in the severely damaged vestibular sensory epithelium. Our study will provide clues for illuminating the mechanisms of the damage and repair and developing interventions in the vestibular sensory epithelium after a sever lesion.

眩晕的发生与前庭毛细胞、支持细胞及神经支配受损密切相关。在前期动物实验中，我们发现了一类前庭感觉上皮严重损伤的病理类型：前庭毛细胞和支持细胞正常结构均消失，呈现为单层扁平上皮，但仍出现少量的新生毛细胞。目前对于其病理特点和修复机制尚缺乏系统研究，其干预研究也未见报道。结合我们既往研究，我们推测这些新生毛细胞和扁平上皮细胞可能由支持细胞或感觉上皮周围的移行上皮细胞分化而来，而Notch信号通路和下游因子Atoh1参与了此修复过程。本课题中，我们首先将评估其病理特点和神经支配改变；然后探讨Notch信号通路和Atoh1以及细胞有丝分裂在修复过程中的作用；并利用转基因小鼠进行支持细胞谱系示踪，以明确支持细胞的动态变化及新生毛细胞的来源；最后我们探索利用腺相关病毒介导Atoh1过表达，在重度损伤后的前庭感觉上皮中诱导毛细胞再生的可行性，为阐明前庭感觉上皮重度损伤的修复机制和研发其干预措施提供线索。

眩晕的发生与前庭毛细胞、支持细胞及神经支配受损密切相关。在前期动物实验中，我们发现了一类前庭感觉上皮严重损伤的病理类型：前庭毛细胞和支持细胞正常结构均消失，呈现为单层扁平上皮，但仍出现少量的新生毛细胞。目前对于其病理特点和修复机制尚缺乏系统研究，其干预研究也未见报道。在本研究中，我们首先通过免疫荧光染色、树脂切片、扫描电镜观察了前庭扁平上皮的形态特点，发现扁平上皮的形态与正常椭圆囊相比出现显著变化，神经支配表现为延迟性的退化。然后我们对Notch通路信号因子表达情况进行了观察，发现Jagged1和Notch1在扁平上皮细胞中的表达与正常椭圆囊相比减弱。采用基因芯片检测了正常椭圆囊和前庭扁平上皮的基因表达谱，发现两者具有显著差异，上皮间质转化可能参与扁平上皮的形成过程。采用EdU染色显示前庭感觉上皮重度损伤后有丝分裂细胞数目先增加后减少。细胞谱系示踪结果发现前庭扁平上皮中的毛细胞部分来源于残留的毛细胞，前庭扁平上皮细胞可能来源于支持细胞或移形上皮细胞。最后，我们将腺相关病毒导入小鼠内耳，发现AAV8-GFP及AAV8-Atoh1-GFP对前庭扁平上皮的转染效率较低，然而在导入AAV8-Atoh1-GFP的围手术期给予组蛋白去乙酰化酶抑制剂辛二酸苯胺异羟肟酸（suberoylanilide hydroxamic acid，SAHA）能够提高病毒的转染效率，并提高前庭扁平上皮myosin VIIa阳性细胞的数量和目的基因的表达水平，但游泳实验显示动物的前庭功能没有明显恢复。本研究为阐明前庭感觉上皮重度损伤的修复机制和研发其干预措施提供了理论基础。