ANALYSIS OF GENES AND PROTEINS RELATED TO COCHLEAR DAMAGE USING DNA MICROARRAY AND PROTEOMICS

我们建立了一只PolgD257A/D257A(D257A)突变小鼠(将点突变引入DNA聚合酶γ的核酸外切酶结构域(校对活性结构域)的转基因小鼠)作为老年性耳聋动物模型。D257A小鼠在9月龄时ABR阈值平均漂移41-52dBSPL，而野生型小鼠在9月龄时未观察到ABR阈值漂移，表明D257A小鼠随着年龄的增长而出现听力损失。组织学上，该突变小鼠表现出耳蜗毛细胞和螺旋神经节细胞的严重退化。DNA微阵列分析显示，与声音感知、神经传递、离子代谢、能量代谢、DNA合成和DNA修复相关的基因减少，与炎症和细胞凋亡相关的基因增加。我们还通过给CBA小鼠喂食二氧化锗，建立了另一种显示线粒体功能障碍的动物模型。在启动Ge Fe…的3个月内此外，这只小鼠出现了严重的听力损失，血管纹和Corti器官严重退化，DNA微阵列分析显示基因表达变化与D257A小鼠相似。在另一项实验中，我们检测了热量限制(CR)是否可以预防老年性耳聋模型小鼠C57BL/6的老年性耳聋。未接受CR的C57BL/6小鼠在17个月龄时出现听力损失和耳蜗组织变性，而CR直到17月龄才出现听力损失和耳蜗变性的表现。对接受和未接受CR的C57BL/6小鼠耳蜗组织的DNA微阵列分析表明，CR阻止了与年龄相关的基因表达的变化，如与声音感知、神经传递和能量代谢相关的基因减少，与炎症和细胞凋亡相关的基因增加。通过实时定量RT-PCR分析确认了几个重要基因的表达变化，并通过免疫组织化学方法证实了相关蛋白的表达变化。最后，我们对暴露在强声中的小鼠进行了DNA微阵列分析，目前正在对几个被认为是噪声性听力损失表现关键的基因进行详细分析。较少

We generated a PolgD257A/D257A (D257A) mutant mouse (a transgenic mouse in which point mutations were introduced into exonuclease domain (Proof Reading activity domain) of DNA polymerase gamma) as a presbycusis animal model. The D257A mouse exhibited ABR threshold shifts of an average of 41-52 dBSPL at the age of 9 month, whereas no ABR threshold shifts were observed in a wild type mouse at this age, indicating that hearing loss appeared by aging early in D257A mouse. Histologically, this mutant mouse exhibited severe degeneration of the cochlear hair cells and spiral ganglion cells. DNA microarray analysis of their cochleae showed a decrease in genes related to sound perception, neurotransmission, ion metabolism, energy metabolism, DNA synthesis, and DNA repair and an increase in genes related to inflammation and apoptosis. We also generated another animal model showing mitochondria dysfunction by feeding CBA mouse with germanium dioxide. Within 3 months from starting the germanium fe … More eding, this mouse developed profound hearing loss and severe degeneration of the stria vascularis and an organ of Corti, and DNA microarray analysis revealed gene expression changes similar to those observed in D257A mouse. In another experiment, we examined whether caloric restriction (CR) could prevent presbycusis in C57BL/6 mouse, a presbycusis model mouse. The C57BL/6 mouse not subjected to CR developed hearing loss and degeneration of the cochlear tissues by the age of 17 months, and CR prevented the manifestation of hearing loss and cochlear degeneration until this age. DNA microarray analysis of the cochlea in C57BL/6 mouse both subjected and not subjected to CR revealed that CR prevented age-related changes of gene expression, such as a decrease of genes related to sound perception, neurotransmission, and energy metabolism, and an increase of genes related to inflammation and apoptosis. We confirmed the expression changes of several important genes by means of real time RT-PCR analysis and their related proteins by performing immunohistochemistry. Finally, we did DNA microarray analysis for mouse exposed to intense sound and are currently doing detailed analysis for several genes that are considered keys in the manifestation of noise0-induced hearing loss. Less