复方健耳剂干预老年性耳蜗神经细胞凋亡关键分子机制研究

Age-related hearing loss belong to a type of sensorineural deafness. Along with aging of population, its morbidity is obviously increasing year by year，which is common stubborn disease in impairing or influencing elder people's healthy and quality of life. So far there are still a lot of aspects in etiology and pathology to be unclear, furthermore, owing to sensorineural cellular physiological and pathological property, at present its prevention and therapy still are very difficult. Jianer capsule, a kind of preparation from traditional chinese medical comound, is our clinical experiential formula that is showed to have better effect on preventing or treating sensorineural deafness in the course of long time of applying to many clinical cases. The results from our previous research also showed that it possessed the effect on preventing animal cochlea spiral ganglion neuron, nerve fiber and hair cell from age-related damage. It indicated that Jianer capsule had the potential pharmacological effect on preventing presbycusis. The project is to study the active mechanism of the traditional chinese medical preparation on intervening in age-related cochlea nerve cells apoptosis by applying animal model of age-related cochlea damage and multi-overlap immunofluorescence technique, selecting related factors that regulate and control cell apoptosis in molecular biology, thus provide further scientific experimental confirmation for future application of the traditional Chinese medicine in clinic or development new preparation from traditional chinese medical comound that its therapeutic effect will be more affirmative, and its effective mechanism will be more clear, in the meantime, also provide essential experience for developing the research of same field.

老年性聋属感音神经性聋之一，随着人口老龄化，发病率呈逐年大量增加趋势，是危害老年人健康,严重影响老年人生活质量的常见顽疾。由于该病病因病机许多方面尚未明了，加之感音神经性细胞的生理病理特性，临床防治甚是棘手。因此，寻找有效防治方法意义重大。中药复方健耳剂系我们长期临床治疗大量感音神经性耳鸣耳聋总结出的有效验方，经前期动物试验发现其具有较好对抗动物老年性耳蜗螺旋神经节神经元和毛细胞凋亡、神经纤维损害的作用，从而提示其具有潜在的防治老年性聋的药理效应。本项目拟继续通过老年性耳蜗损害动物模型，利用多重免疫荧光新方法为主，选择研究调控神经细胞生长与凋亡各种相关因子分析技术，从分子生物学角度研究耳蜗神经细胞生长发育与凋亡规律，揭示中药制剂干预老年性耳蜗损害的作用机制，为复方健耳剂的临床应用提供科学依据，也为今后开发疗效肯定，机理明确的防聋治聋中成药提供实验依据，同时为深化该领域研究提供必要经验。

随着我国人口老年化，老年性聋患者急剧增加，因此，开展药物防治老年性聋研究意义重大。本项目主要研究内容：（1）复方健耳剂优化与研制；（2）老年性聋动物模型建立；（3）探索多重免疫荧光技术在神经细胞领域应用研究；（4）耳蜗铺片、切片技术中细胞染色方法的研究；（5）老年性聋动物模型耳蜗毛细胞和螺旋神经节神经元形态学、听力学及中药干预作用的研究；（6）老年性聋动物模型多个相关分子机制及中药干预作用的研究。.通过研究，确立了7月龄C57BL/6J、6月龄DBA小鼠可作为老年性聋短期观察模型，发现该龄动物耳蜗基底部毛细胞和螺旋神经节神经元已经出现明显凋亡现象，并呈现由底部向顶部逐渐发展的趋势，听力减退也呈现由高频向低频逐渐发展的规律。通过比较，进一步优化复方健耳剂配方及制备方法。发现耳蜗铺片、切片技术中应用琥珀酸脱氢酶、甲苯胺蓝进行耳蜗毛细胞和螺旋神经节神经元染色具有更加清晰、定位准确等明显优点。完成了多重免疫荧光技术在神经细胞领域应用研究探索，通过观察DAPI、NeuN、BrdU、GFAP等表达与重叠，可明确是否有神经元或其它特定神经细胞增殖，今后通过这项技术应用，它们不仅能够辨认神经细胞种类、性质、功能，还可应用于各种不同的基因或蛋白靶向表达与筛选，更好、更直观揭示神经细胞生理和病理机制和它们之间的关系，可作为研究神经细胞一种新的手段和方法。通过RT-PCR或分光光度计研究，探索发现老年性聋细胞形态学凋亡属性及由ROS积累、通过FasL、cycs启动细胞膜途径和线粒体途径、由capase介导的细胞程序性死亡即凋亡的分子机制；探索发现复方健耳剂防治老年性聋显著作用，及通过调控caspase-3、cycs、FasL、txnrd1、BDNF、MDA等互相影响的不同因子，最终起到抑制细胞凋亡，保护听力的分子作用机制。通过实验，发现论证复方健耳剂以中医肝脾肾瘀立论，以中西医结合为指导新的科学依据。这些原创性研究结果对于今后类似研究借鉴，发明可靠防聋治聋中药，继承和发展中医防聋治聋新理论新方法具有极其重要意义。