小鼠耳蜗Lgr5阳性干细胞再生毛细胞过程中SEC信号通路的调控作用及其机制研究

Irreversible damage of hair cells in mammalian cochlea is the main cause of sensorineural hearing loss. Previous studies have reported that Lgr5+ cells are cochlear stem cells with the ability of regenerating hair cells. It is documented that SEC signaling plays an important role in the transcription regulation of embryonic stem cells. Our previous studies have confirmed that SEC is highly expressed in the cochlear Organ of corti, but its founction and mechanism of regulating Lgr5+ cochlear stem cells and its are not clear. This project intends to achieve the following objectives: firstly, to investigate the expression and localization of main factors of SEC signaling including AFF1, AFF4 and ELL3 in the mice cochlear at different time points;secondly, to study the mechanism of proliferation and differentiation regulated by the SEC signaling in the sorted Lgr5+ cochlear stem cells; thirdly, to study the mechanism of regeneration of hair cells regulated by the SEC signaling and the morphology and function of new regenerated hair cells in the damaged cochlea caused by neomycin in ELL3 knockout mice;lastly, to illuminate the specific molecular mechanism of regeneration of hair cells regulated by the SEC signaling. This project can provide experimental foundation for the clinical application of regenerating hair cells to treat hearing loss.

哺乳动物耳蜗毛细胞的不可逆损伤是导致感音性聋的主要原因。研究表明耳蜗中Lgr5阳性细胞是具有再生毛细胞潜能的干细胞。已有文献报道SEC信号通路可调控胚胎干细胞的增殖和分化，申请人前期研究证实SEC在耳蜗corti’s器中也有较高表达，但其对Lgr5阳性干细胞的调控作用及其机制尚不清楚。本项目将进一步研究SEC信号通路对耳蜗Lgr5阳性干细胞的调控作用及其机制，拟实现目标：一、研究SEC信号关键因子AFF1、AFF4、ELL3在内耳中的时空表达；二、研究SEC信号对于耳蜗Lgr5阳性干细胞增殖、分化及再生毛细胞的作用；三、构建SEC信号通路关键因子ELL3敲除小鼠，在活体新霉素损伤模型中研究SEC信号对Lgr5阳性干细胞再生毛细胞的调控作用及新生毛细胞的形态和功能；四、阐明SEC信号调控Lgr5阳性干细胞的具体分子机制。本课题将为临床上应用内耳干细胞再生有功能的毛细胞奠定实验基础。

据报道，超级延伸复合物（SEC）在小鼠胚胎干细胞的增殖和分化中起关键作用。 但是，尚未研究内耳中SEC的表达模式和功能。 在这里，我们研究了三个关键SEC成分AFF1，AFF4和ELL3的内耳表达模式，发现这三种蛋白均在耳蜗毛细胞和支持细胞中表达。 我们还培养了Lgr5 +内耳祖细胞，用于在SEC抑制剂flavopiridol存在的情况下进行球形成测定和分化测定。 我们发现flavopiridol处理降低了Lgr5 +祖细胞的增殖能力，而对Lgr5 +祖细胞的分化能力没有影响。 我们的结果表明，SEC可能在调节内耳祖细胞从而调节毛细胞再生中起重要作用。 因此，进一步研究SEC信号传导途径在体内内耳中的详细作用对于开发对感音神经性听力损失的有效治疗方法将是非常有意义的。