Hippo-YAP信号通路对小鼠耳蜗毛细胞再生的调控作用研究

Sensory hair cells are of importance in the hearing, and the injury of the hair cells can lead to the hearing loss directly. Some studies have shown that Lgr5-positive inner ear stem cells can regenerate hair cells after hair cells damage, and this progress can be regulated by the Wnt signaling. It was reported that the Hippo signaling and the Wnt signaling can interact with each other in many organs; but the mechanism of the regeneration of hair cells regulated by the Hippo signaling remain unclear. This project would devote to study the role of the Hippo signaling in the inner ear stem cells, and further promote the regeneration of functional hair cells, and this can recover the structure and function of the inner ear. There are some aims in this project: firstly, to study the mechanism of proliferation and differentiation regulated by the Hippo signaling in the sorted inner ear stem cells; secondly, to study the mechanism of proliferation and differentiation regulated by both of the Hippo and Wnt signaling in the sorted inner ear stem cells; thirdly, to study the mechanism of regeneration of hair cells regulated by the Hippo signaling in the damaged cochlea caused by neomycin. This research would provide experimental base for recovering of the hearing function treated with inner ear stem cells clinically.

感觉毛细胞在听觉功能中起着重要作用，毛细胞的损伤将直接导致听力损失。研究表明，毛细胞损伤后Lgr5阳性内耳干细胞可以再生毛细胞，并且该过程受到Wnt信号调控。在多种器官中已有报导Hippo信号和Wnt信号存在相互作用；但是Hippo信号对毛细胞再生的调控机制还不清楚。本项目将研究Hippo信号对内耳干细胞的调控机制，以促进其再生有功能的毛细胞，从而重建内耳结构和功能。拟实现以下目标：一、在离体实验中研究Hippo信号对流式分选出的内耳干细胞增殖分化的调控机制；二、在离体实验中研究Hippo和Wnt信号对内耳干细胞的协同调控机制；三、在活体毛细胞新霉素损伤模型中研究Hippo信号如何调控内耳干细胞，从而促进毛细胞再生。本课题将为临床上应用内耳干细胞恢复听觉功能奠定实验基础。

毛细胞的不可逆损伤是感音神经性耳聋的主要致病因素。在哺乳动物中，新生儿毛细胞损伤后其再生能力存在容量限制，成年后由于毛细胞再生能力的不足，将导致毛细胞的永久性损伤。研究表明，新生小鼠耳蜗中的Lgr5阳性支持细胞可作为内耳毛细胞再生的祖细胞。因此，促进小鼠Lgr5阳性内耳干细胞的毛细胞再生能力可能是听力恢复治疗的一种潜在策略。Hippo信号通路对细胞的增殖、分化、死亡、生长都具有调控作用。因此本课题旨在探究Hippo-YAP信号通路对小鼠耳蜗毛细胞再生的调控作用。我们通过RT-PCR、Western blot及免疫荧光验证Hippo-YAP信号通路在小鼠耳蜗中的表达情况。利用新生小鼠Lgr5阳性内耳干细胞的成球实验、分化实验，耳蜗组织体外培养实验进行了系统性研究。结果显示：Hippo-YAP信号通路在小鼠耳蜗中高度表达。激活 YAP可以促进新生小鼠Lgr5阳性内耳干细胞的增殖及分化，进而提高了毛细胞再生能力。相反地，抑制YAP使小鼠Lgr5阳性内耳干细胞的增殖、分化能力、及毛细胞再生均被削弱。这可能与YAP激活后促进了细胞 G1-S期的转换，激活了YAP下游增殖相关靶基因(ctgf, cyr61)的表达，提高了Wnt/β-catenin的转录活性有关。本课题研究可能对探索调节哺乳动物内耳毛细胞再生机制有一定帮助，有望为未来临床上听力恢复治疗提供新的策略。