协同调控NICD和Math1基因促进内耳毛细胞的再生

Permanent hearing and balance dysfunctions are due to the fact that mammals, including humans, have extremely limited ability to regenerate sensory HCs spontaneously after being damaged. Regulating the genes and signaling pathways which involved in HCs proliferation and differentiation, might be an ideal way to activate HCs regeneration. Notch signaling pathway controls inner ear development and sensory cells regeneration. Math1 gene plays a vital role in sensory cells direct differentiation and HCs formation. Activation Notch signaling pathway by over expression NICD, could promote the auditory epithelium and auditory organ development and the ectopic sensory epithelium formation. This subject aimed to explore the method by activating Notch signaling pathway and Math1 gene, to promote sensory precursor cells re-enter the cell cycle, to proliferate and differentiate to new HCs. Our previous results showed that by overexpression NICD to activate Notch signaling could promote sensory precursor cells re-entering the cell cycle and many support cells proliferation. Based on our preliminary results, we will overexpression NICD and math1 gene by using transgene mice in vitro and in vivo, to activate sensory precursor cells proliferation and differentiation to new HCs. Which would shed light on treatment of mammalian hearing and balance disorder.

哺乳动物包括人类的内耳毛细胞自发再生能力极其有限，毛细胞受损后将导致永久性听力减退或前庭功能障碍；调控与细胞增殖和分化相关基因和信号通路，是诱导毛细胞再生的理想途径。 Notch信号通路在内耳发育和毛细胞再生中有着重要的作用，Math1基因决定着感觉细胞的定向分化及毛细胞的形成。激活Notch信号通路，可促进听觉上皮和听觉器官的发育及异位感觉上皮的形成。本课题将探索通过激活Notch信号通路，同时调控Math1基因，达到促进感觉前体细胞重新进入细胞周期、增殖分化为新生毛细胞，促进毛细胞再生。预实验显示：在内耳感觉上皮中上调NICD基因激活Notch通路，可以促进感觉前体细胞进入细胞周期，支持细胞发生增殖；在前期工作的基础上，使用转基因小鼠模型，通过体外组织培养和体内内耳局部给药，试图通过上调NICD基因、同时过量表达Math1基因，激活毛细胞的再生；为人类听力和平衡障碍的治疗奠定实验基础。

为探索同时调控Notch信号通路和Math1基因以诱导小鼠内耳感觉前体细胞的增殖和毛细胞再生，为促进哺乳动物内耳毛细胞的再生寻求新的方法。本研究通过配制R26-M2rtTA/ tetO-NotchIC转基因小鼠，体外和体内上调NICD激活Notch信号通路，可见小鼠椭圆囊感觉上皮上明显的支持细胞增殖和少量毛细胞的增殖；配制成的新生转基因小鼠Sox2CreERT2/ROSA26-rtTA/tetO-NochIC/CAG-LOXP-STO-LOXP-ATOH1-HA+的内耳基底膜体外培养，给予Tamoxifen、Dox上调NICD基因激活Notch信号通路的同时上调Math1基因，可见基底膜上大量异位新生毛细胞，并见支持细胞和毛细胞的增殖，且分布于基底膜感觉上皮的全程。并通过调控Notch信号通路及Wnt、Shh信号通路，促进了小鼠耳蜗和椭圆囊感觉前体细胞的增殖和毛细胞的再生，使用DAPT抑制Notch通路的同时用QS11激活Wnt通路，并进一步使用Sox2-CreER、Notch1-flox (exon 1)和Catnb-flox (exon 3) 转基因小鼠，抑制Notch 信号通路同时在Sox2+的细胞里激活Wnt信号通路，发现均诱导了小鼠基底膜和椭圆囊上皮上大量前体细胞的增殖和毛细胞的再生；并且在体外培养的新生小鼠基底膜上同时给予DAPT抑制Notch通路、QS11激活Wnt通路和Shh激活Shh通路，可见促进的感觉细胞增殖较同时调控Notch和Wnt通路更明显，并且诱导的基底膜上增殖的支持细胞不仅分布在毛细胞区域，而且在大上皮嵴区域也有明显的分布。可见调控Notch信号通路和Math1、或同时调控Notch信号通路和Wnt及Shh信号通路，可以有效的诱导小鼠内耳毛细胞的再生，这将为人类听力和平衡障碍的治疗奠定实验基础。