MicroRNA-182对内耳干细胞增殖分化的影响及机制研究

It is a complex procedure from inner ear progenitor cells to mature hair cells. miRNAs，21-23 nucleotides in length that bind to the 3' untranslated region of target mRNAs to suppress their translation，are believed to play important roles in inner ear development and hair cells differentiation and matureation. Our preliminary data suggest that miRNA-182 is conserved and specific expressed in inner ear neurosensory cells and promotes inner ear progenitor cells differentiation into hair cel-like cell. In the same time we identi?ed Tbx1 as a target of miR-182. The mechanism underlying miR-182-induced development of neurosensory epithelia could be through transcriptional regulation of Tbx1 expression. Tbx1 is thought to be a critical gene in the pathogenesis of del22q11/DiGeorge syndrome. Tbx1 function is required in the otic epithelium cell autonomously and required for expansion of a subpopulation of otic epithelial cells.so we hypothesize that miRNA-182 affect inner ear stem cells differentiation into hair cells through Tbx1 regulating expansion of a subpopulation of otic epithelial cells. To confirm the presumption, we using in situ hybridization and immunofluorescence methods double labelled miRNA-182 and some otic development key protein, such as Tbx1, sox2, Ngn1, explore their expression pattern, identify their potential relationship; we also imply otic transplant methods to explore miRNA-182 functions on subpopulation of otic epithelial cell, especially focus on speci?cation of anterior/posterior (A/P) axis; we also utilize our setup inner ear stem/progenitor cell culture model found out the signal pathway of miRNA-182 promotion inner ear stem cells differentiation.

内耳前体干细胞通过一系列不对称分裂最终增殖分化成毛细胞。microRNA被认为在此过程中扮演重要作用。我们前期研究发现miRNA-182持续特异性地表达在内耳感觉上皮细胞, 体外实验证实miRNA-182促进内耳前体干细胞分化为毛细胞，这一作用可能与其靶基因Tbx1相关。Tbx1是调控内耳前后轴形成的重要因子，即具有调控内耳亚细胞群数量作用。我们研究同时发现miRNA-182调控细胞分化时，总细胞数量没有改变，因此我们推测miRNA-182通过调控Tbx1影响内耳亚细胞群增殖分化而发挥作用。因此项目拟利用原位杂交与免疫荧光双标法观察miRNA-182与内耳早期发育相关基因的关系，通过听泡移植实验观察miRNA-182对内耳亚细胞群增殖分化的影响；应用我们建立的内耳前体干细胞培养模型研究miRNA-182、Tbx1参与内耳早期发育的机制，探明miRNA-182在内耳干细胞增殖分化中作用机制。

内耳前体干细胞通过一系列不对称分裂最终增殖分化成毛细胞。microRNA被认为在此过程中扮演重要作用。我们研究发现miRNA-182持续特异性地表达在内耳感觉上皮细胞, 体外实验证实miRNA-182促进内耳前体干细胞分化为毛细胞，这一作用与其靶基因Tbx1相关。Tbx1是调控内耳前后轴形成的重要因子，即具有调控内耳亚细胞群数量作用。抑制Tbx1表达促进内耳干细胞分化成毛细胞，而抑制神经元分化。在斑马鱼模型中也证实这一结果。.羊水中分离的细胞可以体外传代扩增培养，培养的细胞中可以分离出干细胞特性的细胞。羊水干细胞可以诱导分化成神经元。鼠内耳干细胞作为滋养层细胞，可以促进羊水干细胞向神经元分化成熟，这一现象是否与内耳干细胞分泌miRNAs有关，仍在进一步研究当中。.在本项目资助下，项目负责人有幸去美国交流学习。交流学习期间研究了线粒体钙离子转运蛋白MCU，找出了MCU在内耳中的表达规律，发现噪声损伤条件下MCU在毛细胞中的表达增加，提示MCU参与了毛细胞的损伤和保护机制。并发现这一机制可能与MCU参与了毛细胞突触功能有关。MCU是miRNA-25的靶基因之一，miRNA-25在其中的作用机制仍需进一步研究。