碳酸酐酶抑制剂对内耳液体平衡和内外淋巴离子转运机制的研究

The balance of inner ear lymph is foundation for maintain the shape of membranous labyrinth and normal auditory-vestibular sense. Break of the balance will lead to endolymphatic hydrops which is considered as a main pathological mechanism for Meniere’s disease（MD）.Clinical oral diuretics are widely used for control vertigo attacks in MD .The pharmacological mechanism behind diuretics is that it can down-regulate CAs activeness in varying degrees, thus CAIs is adopted to treat membranous labyrinth hydrops clinically ,but the exact role of CAIs in the inner ear is still unknown. CAIs are expressed in the inner ear according to our previous study, so we will discuss the effects produced by local administration and systemic use of CAIs on the balance of endolymph and the ion transport mechanism between endolymph and perilymph.①Observation :Evaluate the status of endolymphatic hydrops, hearing and vertigo change before and after CAIs administration through morphology and electrophysiology. ②Discussion on mechanism ：M-HPLC is used to detect the concentration distribution of significant ions and acetazolamide. Immunofluorescence staining for study the alternation of CAs, AQP2 and Na+-K+-ATPase expression. RNA-seq is applied for screening differential genes pre and post administration in different part of inner ear. This project aims to provide theoretical basis for using CAIs to cure endolymphatic hydrops, which is significant for MD treatment.

内耳淋巴液平衡是维持膜迷路形态及正常听觉和前庭觉的基础，其失衡引起内淋巴积水是梅尼埃病主要病理机制之一。临床口服利尿剂控制梅尼埃病眩晕发作已被广泛使用，这些药物不同程度抑制碳酸酐酶的活性，因此碳酸酐酶抑制剂可用于治疗内淋巴积水，但是其在内耳中的作用机制并不明确。我们前期研究证明碳酸酐酶在内耳有表达，因此，我们将探讨局部和全身使用碳酸酐酶抑制剂对内耳淋巴液平衡和内外淋巴离子转运机制的影响。①现象观察：通过形态学、电生理等评估碳酸酐酶抑制剂应用前后梅尼埃病豚鼠模型内淋巴积水、听力和眩晕改变情况；②机制探讨：微透析-高液相色谱技术检测内耳淋巴液中药物等重要离子的浓度分布；免疫荧光染色等研究碳酸酐酶、水通道蛋白、Na+-K+-ATP酶表达改变；RNA-seq筛选不同部位给药前后的差异基因探讨其分子机制。本项目将为内耳局部应用碳酸酐酶抑制剂治疗内淋巴积水提供理论基础，对梅尼埃病的治疗有重要意义。

内耳淋巴液平衡是维持膜迷路形态及正常听觉和前庭觉的基础，其失衡引起内淋巴积水是梅尼埃病主要病理机制之一。但是内淋巴积水发生的病理机制、积水程度与梅尼埃病的发生发展之间的关联尚不明确；此外，临床上应用利尿剂控制梅尼埃病眩晕发作已被广泛使用，这些药物不同程度抑制碳酸酐酶的活性，但其对内耳的作用机制并不明确。因此，为进一步探究这些问题的答案，本研究我们从梅尼埃病的临床患者及动物模型入手，（1）结合梅尼埃病患者的临床及影像学特征，进一步阐释了梅尼埃病可能的病理机制。（2）成功建立了动物内淋巴积水模型，并应用OCT、经静脉内耳钆造影技术更准确的对积水程度进行了检测，同时检测了内淋巴积水动物模型的听力及前庭功能的改变。（3）明确了碳酸酐酶不同的亚型、AQP4在内耳中的表达及分布，并明确了内淋巴积水动物模型中碳酸酐酶、AQP4表达水平的改变。（4）揭示了梅尼埃病患者的积水程度与听力、前庭功能之间的关系；探究了应用碳酸酐酶抑制剂对内淋巴积水的影响。本项目的研究结果不仅为梅尼埃病的钆造影的临床应用起到了一定的指导作用，而且探究了内耳积水的病理机制，同时为应用碳酸酐酶抑制剂治疗内淋巴积水提供了一定的理论基础，对梅尼埃病的诊治有重要意义。