联合调控Shh和Wnt/β-catenin信号通路促进小鼠耳蜗毛细胞再生的作用及其机制研究

Unlike lower vertebrates, mammalian cochlear hair cells cannot regenerate spontaneously after damage, although the vestibular system maintains limited hair cell regeneration capacity. As a result, hair cell loss is the major cause of permanent sensorineural hearing loss. In situ cochlear hair cell regeneration should be explored as one of the main approaches to restore hearing that is lost due to hair cell loss. Shh and Wnt/β-catenin signaling play important roles in the proliferation, differentiation, and cell fate decision of embryonic inner ear progenitor cells. Our in vitro experiment study showed that Shh could effectively promote the proliferation and differentiation of mouse neonatal Lgr5 positive progenitor cells. Up-regulation of Wnt/beta-catenin signal in mouse neonatal cochlear support cells could promote precursor cells to proliferate, but a very low proportion of the proliferating cells differentiated into hair cells, and while up-regulation of Shh signaling induced few proliferating cells, but the higher percentage of proliferating cells differentiated into hair cells. In this project, we aim to explore whether co-regulation of Shh and Wnt signaling could promote cochlear hair cell regeneration by using transgenic mice, agonists, and antagonists of Shh and Wnt/β-catenin signaling. The mechanism of hair cell regeneration and the interaction of Shh and Wnt signaling will also be investigated. This study has important scientific value and application prospect for the treatment of sensorineural hearing loss by providing experimental evidence.

哺乳动物耳蜗毛细胞不能自发再生，一旦毛细胞丢失将造成永久性耳聋。激活耳蜗毛细胞再生是治疗感音神经性聋的理想途径。Shh和Wnt/β-catenin信号在胚胎期内耳前体细胞的增殖、分化和命运决定过程中发挥重要作用。毛细胞损伤后在新生小鼠耳蜗支持细胞中单上调Wnt/β-catenin信号能够促进前体细胞增殖，但增殖细胞分化为毛细胞的比例很低；而单上调Shh信号时虽然增殖细胞较少，但增殖细胞分化为毛细胞的比例较高。本项目拟在前期工作基础上，借助Shh和Wnt/β-catenin信号条件性基因高表达小鼠，结合Shh和Wnt信号的激动剂和抑制剂，研究联合调控Shh和Wnt/β-catenin信号对新生小鼠内耳前体细胞和耳蜗毛细胞再生的作用。拟采用高通量测序方法分析耳蜗前体细胞基因表达水平，阐明联合调控Shh和Wnt/β-catenin信号促进毛细胞再生的机制，为感音神经性耳聋的防治提供实验依据。

本项目在前期工作基础上，借助Shh和Wnt/β-catenin信号条件性基因高表达小鼠，结合Shh和Wnt信号的激动剂和抑制剂，研究联合调控Shh和Wnt/β-catenin信号对新生小鼠内耳前体细胞和耳蜗毛细胞再生的作用。研究结果显示：1）在内耳前体细胞球成球和分化实验中，shh 信号能够促进wnt诱导的增殖的Lgr5+细胞分化为毛细胞。2）在新霉素损伤后的耳蜗感觉上皮中，相对于Wnt或Hedgehog单上调组，同时上调Wnt和Hedgehog信号促进了支持细胞增殖，并且增殖细胞分化为毛细胞的比例显著增加，表明联合调控Shh和Wnt/β-catenin 信号能够激活耳蜗支持细胞增殖和促进毛细胞分化。此外，我们对Atoh1诱导的新生毛细胞与原有毛细胞的转录组差异进行分析研究，发现新生毛细胞不能成熟这种现象可能与毛细胞成熟因子（如Gfi1）缺乏有关。我们通过联合调控Gfi1、Pou4f3和Atoh1在耳蜗支持细胞中的表达，提高了支持细胞转分化为耳蜗毛细胞的效率，并促进了新生毛细胞走向成熟。这些研究成果为临床上感音神经性耳聋的治疗提供了新颖的治疗策略和坚实的实验依据。.在本项目的支持下，发表SCI论文7篇，其中影响因子大于5的2篇。申请人和项目组成员的研究成果“基因重编程策略调控内耳干细胞增殖与分化”获得1项教育部自然科学奖一等奖（2020）。