利用听力缺陷荣昌猪研究Mitf基因在听觉发育与形成中的作用

Recently, the applicants reported the first big animal model of spontaneous deafness —Hearing loss Rongchang pigs. We mapped the causative mutation in Mitf gene as a loss-of-function mutation (Mitfr). In order to elucidate the pathogenic mechanism of Mitf, we make a scientific hypothesis based on some of our previous findings: Mitf directly transcriptional activates certain cochlear ion channels, and consequently regulates the biogenesis of endolymphatic potential and hearing function. Firstly, differential expression of ion channels will be screened on transcript and protein level. Then, reporter gene assay and DNA-protein binding assay will be employed to verify Mitf’s transcriptional activation on the target ion channels. Finally, specific small-molecule inhibitor and conditional knock-out of ion channels will be used to explore their function in hearing development. Our results may provide theoretical basis to the early intervention, hearing reconstruction and gene therapy of the hereditary sensorineural deafness. In addition, we may provide new potential therapeutic targets to certain types of age-related hearing loss and noise-induced hearing loss which related on the endolymphatic potential anomalies.

申请人此前发掘了国内外首个大动物自发性耳聋模型听力缺陷荣昌猪，并证明了Mitf功能缺失性突变（Mitfr突变）导致了该表型。为阐明Mitf在哺乳动物中突变致聋的分子机制，申请人在前期研究的基础上提出科学假设：Mitf通过调控耳蜗离子通道转录直接参与内淋巴电位生成与听觉形成。课题组拟首先利用基因、蛋白差异表达分析技术筛选Mitf的下游离子通道基因；随后利用转录活性分析及DNA-蛋白互做分析技术找到直接受到Mitf转录激活的通道；最后利用特异性药物抑制和条件性基因敲除的方式在活体动物水平产生离子通道功能性缺失效应，从而验证Mitf及其调控的离子通道在听觉发育与形成中的作用。本研究成果将为遗传性感音神经性耳聋的早期干预、人工听力重建、基因治疗等提供基础数据和理论依据，并为与耳蜗内淋巴电位异常相关的老年性耳聋和噪声性耳聋提供潜在的治疗靶点。

项目组基于具有自主知识产权的自发性耳聋模型听力缺陷荣昌猪，深入研究了Mitf基因在哺乳动物中突变致聋的分子机制。课题组研究发现Mitf直接激活钾离子通道Kcnj13，该通道特异性表达于血管纹中间层细胞。利用Kcnj13的特异性小分子抑制剂VU590进行的功能缺失分析结果表明，Kcnj13失活大幅降低了中间层细胞的内向整流电位。Mitf基因突变导致的耳聋主要导致Kcnj13的缺失表达，并进一步导致中间层细胞缺失。上述结构表明Mitf通过调控Kcnj13转录直接参与内淋巴电位生成与听觉形成。本研究成果为遗传性感音神经性耳聋的早期干预、人工听力重建、基因治疗等提供了基础数据和理论依据，并为与耳蜗内淋巴电位异常相关的老年性耳聋和噪声性耳聋提供潜在 的治疗靶点。