Math1诱导的哺乳动物再生毛细胞与螺旋神经节形成的传入突触的形态学和功能学研究

Cochlear hair cell loss or damage causes permanent sensorineural hearing loss, hair cell regeneration is of great significance for the treatment of deafness. In recent years, it has been a great breakthrough that newly regenerated hair cells could be successfully induced in different ways. For the studies of function of these newly regenerated hair cells, however, is still in infancy. In pilot studies, we had got the adenovirus to transfer Math1 into inner ear end organs and got the extra hair cells successfully; we also did a series studies on the morphological and functional properties of newly formed hair cells. We found that these newly formed hair cells had the similar morphological features as developmental hair cells; and their functional features in line with the developmental hair cells. We could record IK, ICa, INa, action potentials and mechanoelectrical transduction from these newly formed hair cells, which were basis of sound signal transduct. We hypothesize that these newly formed hair cells could form synapse with spiral ganglions base on our pilot studies. In the project, our goals include: 1) we intend to take advantage of Ad-EGFP-Math1 to induced the afferent synapse formation of new hair cells and spiral ganglion in vitro and in vivo; 2) immunofluorescence, transmission electron microscopy (TEM) were performed to identify morphological features of the synapses; 3)whole-cell patch-clamp technique will be used to record the miniature excitatory / inhibitory postsynaptic currents from spiral ganglions to detect the function of the new afferent synapses. It is of great importance to get the goal for the therapeutic treatment of deafness.

耳蜗毛细胞缺失或损伤是造成永久感音神经性聋的关键原因，耳蜗毛细胞再生对于治疗耳聋意义重大。目前，包括Math1过表达等多种方式可以成功诱导内耳毛细胞的再生，但是对于再生毛细胞功能的研究尚处于初级阶段。本课题组已经成功利用腺病毒载体在体外培养的新生大鼠耳蜗过表达Math1诱导出了再生毛细胞，发现其具有与正常毛细相似的形态学特征；并且，再生毛细胞的功能符合发育中的毛细胞的功能的特点，具备声信号传递的功能基础，包括IK,ICa,INa，动作电位和机械门控通道电流。据此推断再生毛细胞可以与螺旋神经节形成突触。本课题拟利用Math1诱导的再生毛细胞与螺旋神经节在体外/体内形成传入突触，采用免疫荧光、透射电镜对其进行形态学鉴定，并采用全细胞膜片钳技术在形成突触的螺旋神经节记录兴奋性/抑制性微小突触后电流，研究再生毛细胞是否能与螺旋神经节细胞形成有功能的传入突触，这对于再生毛细胞的临床应用具有深远意义。

在前期研究中，本课题组已经成功构建了腺病毒载体，并在在体外培养的新生大鼠耳蜗过表达Math1诱导出再生毛细胞，发现其具有与正常毛细相似的形态学特征；并且，再生毛细胞的功能符合发育中的毛细胞的功能的特点，具备声信号传递的功能基础，包括IK,ICa,INa,自发/诱发的动作电位和机械门控通道电流。据此我们推断再生毛细胞可以与螺旋神经节形成突触。本资助项目以研究Math1诱导产生的异位再生毛细胞与螺旋神经节形成突触相关研究为核心内容，并与展开相关研究。研究中发现，在体外培养条件下，Math1诱导的新生SD大鼠耳蜗基底膜产生的异位再生毛细胞可以诱导螺旋神经节和其发生突触连接。通过免疫荧光染色成功标记了突触前、突触后标记物。通过全细胞膜片钳技术记录到了再生突触的突触后电流，包括抑制性和兴奋性的突触后电流。利用透射电镜寻找到再生毛细胞中的大量囊泡聚集体，未找到典型的带状突触结构。在体条件下，中阶入路转染腺病毒到成年豚鼠耳蜗，可诱导产生再生毛细胞，但是听力无明显改善。同时，在展开的相关研究中我们发现，Math1的表达水平对于再生毛细胞形成的时间和形成数量至关重要，并且，我们通过联合Notch信号抑制DAPT诱导产生了原位再生毛细胞，并证实了Math1诱导产生的毛细胞是一个直接转分化的过程。