内耳中Wnt和Notch信号通路对Wnt受体Frizzled 9阳性细胞调控机制的研究

The main reason for sensorineural hearing loss is the irreversible loss of sensory hair cells. According to our current findings, Wnt receptor Frizzled9 positive cells could act as cochlear stem cells. Wnt and Notch are two basic signaling pathways regulating proliferation and cell fate of inner ear precursor cells. This project has been designed to systematically investigate the role of Wnt and Notch signaling pathway in regulating the proliferation, differentiation and hair cell regeneration of the Frizzled9 positive cochlear stem cells by in vitro cell culture and in vivo mice experiments, with the goal to promote hair cell regeneration from the Frizzled9 positive cochlear stem cells, thus establish the experimental foundation for the clinical treatment of sensorineural hearing loss by stem cell therapy. In this project we propose to fulfill the following aims: Aim1: Characterize the temporal and spatial expression of Frizzled9 in the inner ear in wild-type mouse at embryonic, neonatal, mature stages. Aim2: Characterize the proliferation, differentiation and hair cell regeneration capacity of the Frizzled9 positive cochlear stem cells in neonatal cochlea hair cell damage model and investigate hair cell regeneration capacity of neonatal mice in Frizzled9 positive cells damaged model. Aim3: Investigate the role of Wnt and Notch signaling in regulating the proliferation, differentiation and hair cell regeneration of the Frizzled9 positive cochlear stem cells, and then evaluate the function of newly regenerated hair cells and the relative hearing recovery.

感音神经性聋主要由耳蜗毛细胞不可逆损失造成。根据我们目前的研究结果，在耳蜗中Wnt受体Frizzled9阳性细胞可能是干细胞。内耳中Wnt和Notch信号通路是调节前体细胞增殖和决定细胞命运的基本通路。本项目主要是在体外细胞培养和小鼠活体实验中研究这两个通路如何调控Frizzled 9阳性耳蜗干细胞的增殖和分化，并促进其再生毛细胞，为在临床上通过耳蜗干细胞治疗听力障碍奠定实验基础。拟实现以下研究目标：一，报道Frizzled9在野生型小鼠胚胎期、新生期、成熟期耳蜗中的表达。二，在新生小鼠离体和活体毛细胞损伤模型中，研究Frizzled9阳性耳蜗干细胞增殖，分化及再生毛细胞的能力，以及研究破坏Frizzled9阳性细胞后小鼠的毛细胞再生能力；三，研究 Wnt和Notch通路如何调控这些耳蜗干细胞的增殖，分化，从而促进毛细胞再生，同时研究新生毛细胞的功能，及检测再生毛细胞所导致的听觉功能恢复。

Lgr5+耳蜗支持细胞的一个亚群已被报道为内耳祖细胞,具有新生小鼠耳蜗毛细胞再生的能力,可通过Wnt信号通路调控Frizzled9是Wnt受体之一,已被报道是人胎盘和骨髓中神经元干细胞和间充质干细胞的标记物。我们使用Fzd9-creER小鼠来谱系追踪在出生后耳蜗中Fzd9阳性细胞的时空表达。谱系追踪数据显示Fzd9主要高表达在包裹着内毛细胞的两个支持细胞,内指细胞和内缘细胞中,第三排deiter细胞中也有一定的表达,Fzd9的表达量随着小鼠年龄的增加逐渐减少,P14 小鼠耳蜗内几乎检测不到Fzd9的表达, 细胞成球实验表明,流式细胞仪对Fzd9+细胞进行分选,体外培养后,Fzd9+细胞可形成球,且Fzd9阳性细胞在体外培养情况下具有分化并再生毛细胞的能力，在体外组织培养条件下,Wnt信号激活促进了 Fzd9阳性细胞的增殖能力,Notch信号抑制促进了内耳Fzd9阳性细胞增殖能力,说明内耳Fzd9阳性细胞受到Wnt和Notch信号通路的双调控。这些数据均提示Fzd9可以作为内耳前体细胞的标记物，这为在临床治疗上应用这些毛细胞前体细胞恢复听力功能的长期目标前迈进了一大步。