溶酶体保护蛋白/组织蛋白酶A基因敲除小鼠听力和耳形态学初步研究

Morphological and Functional Alterations of Ear in Lysosomal Protective Protein/Cathepsin A Gene Knock Out Mouse

目的研究溶酶体保护蛋白／组织蛋白酶A（protective protein／cathepsin A，PPCA）基因敲除小鼠听功能和耳形态学改变，探讨半乳糖唾液酸沉积症听力损害的病理生理机制。方法应用听性脑干反应（ABR）测试和颞骨连续切片，观察1月和2月龄的PPCA基因敲除纯合子（PPCA-／-）小鼠ABR反应阈和光镜下外耳、中耳及内耳形态改变，并以野生型（PPCA＋／＋）小鼠作对照。结果1月龄PPCA-／-小鼠ABR反应阈和耳形态无明显改变；2月龄时，短声和短音8、16、32kHz反应阈较PPCA＋／＋提高40—45dB SPL，中耳黏膜增厚、听骨细胞囊泡化、变形和关节腔融合，血管纹增厚、螺旋神经节细胞、螺旋缘纤维细胞、前庭膜、基底膜及沿前庭阶外淋巴隙的间皮细胞囊泡化，但Corti器毛细胞及支持细胞正常。结论溶酶体保护蛋白／组织蛋白酶A缺乏可导致听力损害、中耳及耳蜗形态改变、中耳炎、听骨改变以及耳蜗螺旋神经节、血管纹、螺旋缘、前庭膜和基底膜等细胞的溶酶体储积，可能分别是传导性聋和感觉神经性聋的形成机制。

Obiective Galactosialidosis （GS） is an autosomal recessive lysosomal storage disease caused by a combined deficiency of lysosomal β- galactosidase and neuraminidase as a result of a primary defect in the protective protein/cathepsin A （PPCA） .Mouse model of GS has been generated by targeted deletion of PPCA gene and closely resembled the phenotypes in human conditions. However, it remains to be determined whether hearing loss observed in human also occurs in the mouse model. In this study, we observed their alterations of the auditory function and morphology of the ear, and explored pathophysiological mechanisms of hearing impairment. Methods PPCA homozygons （PPCA - / - ） mice at 1 and 2 months of age, and their wildtype littermates （PPCA ＋ / ＋ ） were examined for auditory thresholds through auditory brainstem responses （ABR） to click, tone pips 8, 16, and 32 kHz stimuli. Morphological analyses in ears were performed by series temporal bone section and light microscopy. Results PPCA- / - mice at 1 month of age showed a normal threshold and the morphology of ears. Up to 2months of age, their thresholds were elevated 4-45 dB SPL above those of PPCA ＋ / ＋ mice. There were distinct pathological changes of middle and inner ear in PPCA - / - mice of 2 months old. The severe otitis media and the vacuolation associated with lysosomal storage were observed within ossicles and cochlear bone cells, stria vascularis cells, spiral ganglion neurons, spiral limbus, Reissner＇ s membrane cells, and the mesothelial cells of the perilyrnphatic scala and basilar membrane, but not within the organ of Corti. Vestibular organ did not show vacuolation. Conclusion The deficiency of lysosomal protective protein/cathepsin A may result in hearing loss and morphological alterations of ear. The otitis media and ossicle changes, and the defects in lysosomal storage of neurons, stria vascularis, spiral limbns, Reissner＇ s membrane and basilar membrane cells may contribute to the conductive and sensorineural hearing loss respectively.