内耳放射性损伤中耳毛细胞prestin蛋白的作用及其miRNA调控机制的研究

Radiotherapy for head and neck tumors will induce radioactive injury in the inner ear, which clinical manifestation is the sensorineural hearing loss after radiotherapy, and its high incidence seriously affects the life quality of patients. Research shows that the main lesion of radioactive injury in the inner ear induced by radiotherapy is ear hair cells, and moreover, the prestin protein is the biological basis of ear hair cells electroluminescent movement. But currently, the change and regulation mechanism of the radiation induced inner ear hair cell injury is still unknown. We have preliminarily established the animal model of radioactive injury in the rat inner ear, and observed the expresstion changes in prestin protein; whereas it is observed by the microarray screening that the miRNA expression file significantly changes before and after radiotherapy. Therefore, in this project, on the basis of preliminary work, we will observe the change of ear hair cell morphology, biological functions and electroluminescent movement after radiotherapy through the vivo and vitro experiments; verify the presence of radiation injury related miRNA in ear hair cells and its regulation mechanism to the prestin protein by QRT-PCR, transfection, interference and other methods. Thus we can reveal the mechanism for controlling the degree of the radioactive injury in ear hair cells from miRNA target to prestin protein, and provide the experimental basis of searching for the prevention strategies from inner ear radioactive injury after radiotherapy and therapeutic targets.

头颈肿瘤的放疗引起内耳放射性损伤最终导致放疗后感音神经性耳聋，严重影响患者生存质量。研究证明放疗致内耳放射性损伤主要病变部位为内耳毛细胞，而prestin蛋白是耳毛细胞运动功能的生物学基础。但目前内耳毛细胞放射损伤后prestin改变及调控机制不明。我们前期已建立了大鼠内耳放射损伤动物模型并观察到prestin蛋白的变化；而通过芯片筛查发现miRNA表达谱在耳毛细胞株放射前后有显著改变。故本课题拟在前期工作基础上通过体内、外实验，观察放射损伤后耳毛细胞形态学、生物学功能及电生理运动的改变；并通过QRT-PCR、转染、干扰等方法，验证耳毛细胞中放射损伤相关miRNA的存在及其对prestin蛋白的调控作用，从而揭示一个由miRNA调控下游靶基因靶向prestin蛋白进而调节内耳毛细胞放射性损伤程度的作用机制，为寻找放疗后内耳放射性损的防治策略和治疗靶点提供实验依据。

头颈肿瘤的放疗引起内耳放射性损伤最终导致放疗后感音神经性耳聋，严重影响患者生存质量。研究证明放疗致内耳放射性损伤主要病变部位为内耳毛细胞，但是内耳毛细胞放射性损伤分子机制不明。我们建立了大鼠内耳放射损伤动物模型以及耳毛细胞株HEI-OC1放射性损伤体外模型，通过芯片筛查发现miRNA表达谱在耳毛细胞株放射前后有显著改变。进一步通过实验验证，发现上调miR-207会加重放射性损伤，而下调miR207可减轻耳毛细胞株放射性损伤；接下来我们证实了AKT3激酶是miR-207的直接靶点，下调AKT3与上调miR-207具有相似的生物学功能，通过激活凋亡通路加重耳毛细胞放射性损伤，至此，我们阐明了一条由miR-207靶向AKT3调控细胞凋亡最终调节耳毛细胞放射敏感性的分子通路，为放疗后感音神经性耳聋的防治提供可能的基因治疗靶点。并且我们将实验深入至研究放射性神经损伤的具体发生机制及防护策略，建立了斑马鱼放射性神经损伤模型，通过行为学的评估来判定神经的放射性损伤。在进一步实验中，我们通过对斑马鱼放射损伤模型的形态学及行为学研究，发现丙戊酸钠能够减轻斑马鱼的放射性脑损伤，并且这一作用机制与丙戊酸钠对nrf-2/HO-1通路的调节相关，相关实验结果已发表，为我们深入了解放射性神经损伤发生机制及制定临床防护策略提供了更多的实验数据及理论依据。