大上皮嵴支持细胞在哺乳动物耳蜗发育中的关键作用

Rodent has not hearing until 12 days after birth, which corresponds to the apoptosis of the greater epithelial ridge (GER) in the cochlea. Supporting cells within GER form a functional syncytium through connexin 26 and/or 30, however, how they promote cochlear maturation and its regulatory mechanism still remains unclear. We have long been engaged in the research of the relationship between ATP and cochlear development and are the first to get direct evidence of ATP release from supporting cells in GER. We found for the first time that the expression of mRNA and protein of apoptosis related factors is time-dependent, ATP-containing vesicles within marginal cells in the stria vascularis are lysosomes, and there are interactions among related proteins in the pathway of ATP synthesis, degradation, release and action in the cochlea. We found that not only apoptosis but also autophagy of the supporting cells exists in GER. We assume that supporting cells in GER play a crucial role in the development of the mammalian cochlea. To substantiate this assumption, the present research will observe the apoptosis or autophagy of supporting cells, morphological and functional changes in inner hair cells, synapsis and spiral ganglion neurons in Cx26sox10Cre and Cx30 (-/-) mice, the morphological and functional differences between the supporting cells adjacent to the inner hair cells and other cells within GER, and whether the ATP-containing vesicles in the supporting cell are lysosomes. The present research has profound significance for the further elucidation of the developmental mechanism of the peripheral auditory organ.

啮齿动物出生后12天才有听觉，与之对应的是耳蜗大上皮嵴消失。大上皮嵴的支持细胞通过缝隙连接蛋白（Cx26和/或Cx30）形成功能性合胞体，但其如何促进耳蜗发育成熟及其调控机制不明确。申请人长期致力于三磷酸腺苷（ATP）与耳蜗发育关系的研究，首次获得大上皮嵴支持细胞释放ATP的直接证据，证明耳蜗血管纹缘细胞内ATP贮存囊泡为溶酶体，耳蜗中ATP合成、降解、释放、作用通路上相关蛋白间存在相互作用，发现大上皮嵴支持细胞不仅有凋亡也有自噬。我们假设大上皮嵴支持细胞在哺乳动物耳蜗发育中发挥非常关键的作用，本项目拟观察模型动物Cx26sox10Cre和Cx30(-/-)小鼠大上皮嵴支持细胞凋亡/自噬，以及内毛细胞、突触、螺旋神经元形态功能的变化，大上皮嵴中邻近内毛细胞的支持细胞与其它细胞形态和功能有何不同，以及证明支持细胞内贮存ATP的囊泡是溶酶体。本研究对进一步阐明外周听器的发育机制有重要意义。

啮齿动物出生后12天才有听觉，与之对应的是耳蜗大上皮嵴（GER）消失。GER支持细胞如何促进耳蜗发育成熟及其调控机制不明确。（1）证明ATP贮存于GER支持细胞内溶酶体。观察到特异性标记ATP的细胞器是溶酶体，GPN处理增强了支持细胞ATP释放。支持细胞释放ATP受胞内和胞外Ca2+浓度变化的影响。抑制磷脂酶引起的细胞内Ca2+的变化影响支持细胞释放ATP。（2）GER支持细胞存在自噬。免疫组织化学染色显示，自噬标记物LC3-II、SQSTM1/p62和Beclin1在GER中表达。Western blot和实时PCR显示LC3-II的表达逐渐减少，而p62的表达逐渐增加。透射电镜观察到GER中有典型的自噬体和包装各种支持细胞细胞器的自噬溶酶体。（3）单细胞测序发现GER支持细胞的亚型。单细胞发育轨迹分析显示GER支持细胞的8种细胞亚型中，四种存在细胞增殖的亚型，具有转分化为内、外毛细胞的潜能，其中两种细胞亚型主要转分化为内毛细胞，另外两种细胞亚型最终转分化为外毛细胞。（4）Gjb2敲减小鼠的听力表型和自噬/凋亡。发现模型鼠表现出严重听力下降；HE染色显示Corti隧道未打开，Cx26表达下调，Cx30表达上调。敲减小鼠的GER支持细胞在P1即发生凋亡，活化型caspase 3表达上调，自噬标记物LC3-II及P62均表达下调。蛋白电泳分析与免疫荧光结果一致。Gjb2敲减小鼠的GER支持细胞总ATP浓度显著下降；培养后的原代细胞喹吖因染色显示ATP囊泡数量减少；ATP依赖性钙信号强度减弱，钙波消失。（5）Gjb6敲减小鼠的听力表型和自噬/凋亡。1、3和6个月龄的Cx30-/-小鼠表现出轻微的听力损失。免疫荧光和HE染色显示耳蜗微结构无显著改变，透射电镜显示血管纹有轻微的空洞样损伤。Cx30-/-小鼠中Cx30缺乏降低了耳蜗内电位（EP）的产生和ATP的释放，可能是导致听力损失的主要原因。（6）发现FGF22敲除通过影响内毛细胞带状突触的功能导致隐性听力损失。（7）饮食中摄入氧化氘会降低耳蜗代谢和年龄相关性听力损失小鼠模型中的氧化应激水平。（8）YTHDF1通过促进ATG14翻译激活自噬保护听觉毛细胞免受顺铂诱导的损伤。