TERT线粒体移位调控Kölliker器支持细胞ATP释放在出生后耳蜗结构重塑中的作用及机制研究

The mammal cochlea is immature at birth. Postnatally, structural remodeling happens as auditory nerve circuits were established through the refinement of synaptic connections and eventually hearing appears. Studies had shown that ATP released from Kölliker supporting cells is crucial for cochlear development and maturation. But the mechanism which regulate ATP release is still unclear. In our previous study, we confirmed the significant expression of TERT in the cochlear progenitor cells and Kölliker supporting cells during early postnatal period. And for the first time, we observed mitochondrial transposition of TERT during progenitor cells differentiation and postnatal cochlear development. Thus we hypothesized that during the postnatal development, TERT may shift into mitochondria, which may enhance its function. The mitochondria then mediated ATP release of Kölliker supporting cells which promote postnatal cochlear remodeling and auditory circuits formation. The conditioned knockout Tertfl/fl andTert-S227E point mutant mice and Kolliker support cells cultured in vitro were used as models to verify this hypothesis. The effects of TERT knockout or inhibition of TERT extranuclear expression on cochlear structure and hearing were evaluated, and then mechanism of TERT mitochondrial migration in regulating ATP release was explored. These studies will reveal new mechanisms for the development of auditory system, and provide theoretical basis and key targets for the treatment strategies of sensorineural hearing loss.

哺乳动物出生时耳蜗尚未发育成熟，出生后将经历结构重塑，并通过突触连接的精细化建立听觉神经回路最终产生听力。研究表明Kölliker器支持细胞释放ATP对耳蜗发育成熟至关重要，但其上游调控机制尚不清楚。我们已证实TERT在耳蜗前体细胞及出生后Kölliker器中明显表达，并首次观察到随着前体细胞分化和耳蜗发育TERT逐渐向线粒体移位。因此提出假说：在出生后TERT发生线粒体移位，通过线粒体介导Kölliker器支持细胞ATP释放调控耳蜗结构重塑和听觉神经回路形成。本研究拟以Tertfl/fl条件性敲除和Tert-S227E点突变小鼠及体外培养的Kölliker器支持细胞为模型，对这一假说进行验证。明确敲除TERT或抑制TERT核外表达对耳蜗结构及听力的影响，探讨TERT线粒体移位对ATP释放的调控机制。这些研究将揭示调控内耳听觉系统发育的新机制，为感音神经性聋治疗策略提供理论依据和关键靶点。

哺乳动物出生时耳蜗尚未发育成熟，出生后将经历结构重塑，并通过突触连接的精细化建立听觉神经回路最终产生听力。研究表明Kölliker器支持细胞释放ATP对耳蜗发育成熟至关重要，但其上游调控机制尚不清楚。本研究在前期研究基础上，进一步在C57小鼠中验证耳蜗Kölliker器支持细胞内TERT线粒体移位的情况，然后我们通过设计携带不同表型的TERT基因不的质粒并转染体外培养的耳蜗支持细胞，观察调控耳蜗支持细胞内TERT不同亚细胞定位表达的影响，最后通过条件性基因敲除小鼠观察对TERT敲除后对小鼠听力及耳蜗发育的影响。本研究结果提示TERT在出生后早期Kölliker器支持细胞及外培养的支持细胞中存在由核向核外移位的趋势，且在核外主要表达于线粒体中，同时伴有细胞内钙电流的升高，线粒体膜电位的升高，和ATP释放的增加。同时，在孕晚期和出生后早期不同时间前激活TERT基因敲除，均可导致明显的听力下降，但耳蜗结构未见明显发育异常。本研究结果明确了TERT在小鼠听力产生过程中的重要作用，并为阐明TERT通过调节线粒体功能调控ATP释放这一机制提供了初步的证据。

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