联合调控Gfi1、Pou4f3和Atoh1促进成年小鼠耳蜗毛细胞再生和功能成熟

激活耳蜗毛细胞再生从而重建听觉功能是治疗感音神经性聋的理想途径。耳蜗新生毛细胞不能终末分化为具有功能的毛细胞是该领域的难点和亟待解决的问题。上调Atoh1表达水平能够促进耳蜗支持细胞转分化为毛细胞，但是新生毛细胞缺少成熟毛细胞的形态学和电生理特征。在预实验中，我们在幼年期支持细胞中同时上调Gfi1、Pou4f3和Atoh1（简称GPA）的表达水平不仅能够提高Atoh1诱导的支持细胞转分化效率，而且能够促进新生毛细胞的亚型分化和功能成熟。本项目拟借助条件性转基因小鼠和耳聋模型，在不同亚型的耳蜗支持细胞中上调GPA表达水平，研究联合调控GPA能否促进成年小鼠耳蜗毛细胞再生和听觉修复。拟采用单细胞测序方法分析不同成熟阶段毛细胞的转录组差异，鉴定与毛细胞功能成熟相关的关键基因和信号通路，阐明联合调控GPA促进新生毛细胞成熟的机制，为临床上应用毛细胞再生策略治疗耳聋奠定实验基础。

Sensorineural hearing loss is permanent because the mammalian cochlea cannot spontaneously regenerate functional hair cells (HCs) after damage. Multiple studies have shown that ectopic upregulation of Atoh1 expression alone or combined with Notch inhibition, Wnt activation, p27Kip1 deletion, and Myc/NICD co-activation can convert supporting cells (SCs) into HCs. However, none of the newly generated HCs converted by Atoh1 expression express the mature/terminal inner HC (IHC) marker vesicular glutamate transporter 3 (vGlut3) or the mature/terminal outer HC marker molecular motor Prestin, nor do they show biophysical maturation as evaluated by electrophysical recordings. In the preliminary experiment, we found that upregulating the expression levels of Gfi1, Pou4f3 and Atoh1 (GPA) in the supporting cells from young postnatal mice could not only enhance the Atoh1-induced SC-HC trans-differentiation, but also promote the subtype differentiation and maturation of newborn hair cells. The electrophysiological characteristics of these newborn hair cells and the possibility of promoting adult hair cell regeneration and hearing repair by upregulation of GPA require further research. In this project, we will overexpress GPA in different subtypes of cochlear support cells using several supporting cell tracing mouse lines and hearing loss mouse model, examine the quantity, morphological features, and synapse formation of new hair cells by immunofluorescence staining, detect the biophysical features of new hair cells by electrophysical recording, and explore the hearing function recovery of mouse upon damage. This study has profound scientific value and shed light on the treatment of sensorineural hearing loss by providing solid experimental evidence.