目的·通过构建线粒体内膜转位酶8A（translocase of inner mitochondrial membrane 8A，Timm8a）基因敲除小鼠（即Timm8a^-/- 小鼠），探究其听力表型及该基因在内耳的功能。方法·设计并构建Timm8a^-/- 小鼠，采用PCR及蛋白质印迹法（Western blotting）验证是否构建成功。观察并比较1月龄时Timm8a^-/- 小鼠与野生型（wild type，WT）小鼠的体型及体质量。采用免疫荧光染色观察WT小鼠耳蜗组织中TIMM8A的表达分布。通过听性脑干反应（auditory brainstem response，ABR）比较Timm8a^-/- 小鼠和WT小鼠的听力阈值。采用甲苯胺蓝染色法观察该2种小鼠内耳螺旋器、螺旋神经节与血管纹形态。通过透射电镜观察该2种小鼠的内耳毛细胞、螺旋神经节细胞与血管纹细胞中的线粒体超微结构，并统计异常线粒体所占比例。结果·PCR结果显示Timm8a基因已成功敲除，Western blotting结果显示Timm8a^-/- 小鼠的耳蜗、大脑、心肌、骨骼肌组织中已无TIMM8A表达，表明Timm8a^-/- 小鼠构建成功。免疫荧光染色结果提示TIMM8A在WT小鼠耳蜗组织中呈泛表达，且高表达于螺旋器、螺旋神经节与血管纹区域。与WT小鼠相比，1月龄时的Timm8a^-/- 小鼠发育较慢，体质量明显偏轻，且ABR阈值有所升高（均P<0.05）；该小鼠的ABR的Ⅰ波波幅出现下降、Ⅰ波潜伏期有所延长。甲苯胺蓝染色结果显示，与WT小鼠相比，Timm8a^-/- 小鼠的螺旋神经节细胞的形态与数量均无明显改变，耳蜗中圈及底圈的血管纹厚度减薄（均P<0.05）。透射电镜的观察结果显示，与WT小鼠相比，Timm8a^-/- 小鼠内耳毛细胞、螺旋神经节细胞与血管纹细胞中线粒体结构出现异常，且异常线粒体比例明显增多（均P<0.05）。结论·该研究成功构建了Timm8a^-/- 小鼠，其听力阈值的升高可能与其内耳线粒体超微形态结构异常有关。

Objective ·To explore the hearing phenotype of Timm8a^-/- mice and the function of translocase of inner mitochondrial membrane 8A(Timm8a)gene in inner ear by constructing Timm8a gene knockout mice. Methods ·Timm8a^-/- mice were designed and constructed. PCR and Western blotting were used to verify whether the construction was successful. The body size and weight of Timm8a^-/- mice and wild type (WT) mice aged one-month were observed and compared. Immunofluorescence staining was used to observe the expression and distribution of TIMM8A protein in the cochlea of WT mice. Auditory brainstem response (ABR) was used to compare the hearing threshold of Timm8a^-/- mice and WT mice. Toluidine blue staining was performed to observe the morphology of organ of Corti (OC), spiral ganglion neuron (SGN) and stria vascularis (SV) in the inner ear of the two kinds of mice. Transmission electron microscope was used to observe the ultrastructure of mitochondria in inner ear hair cells (HCs), SGN cells and SV cells of the two kinds of mice, and the proportion of abnormal mitochondria was counted. Results ·The results of PCR showed that Timm8a gene had been successfully knocked out, and the results of Western blotting showed that there was no TIMM8A proteins in the cochlea, brain, heart and skeletal muscle tissues. The both results indicated that Timm8a^-/- mice were successfully constructed. The results of immunofluorescence staining showed that TIMM8A was abundantly expressed in the cochlea of WT mice, and was highly expressed in the OC, SGN and SV. Compared with WT mice, Timm8a^-/- miceaged one-month developed slower, had lighter body weight and significantly higher hearing threshold (all P<0.05), their amplitude of Ⅰ wave of ABR was decreased and the latency was prolonged. The results of toluidine blue staining showed that compared with WT mice, there was no significant change in the shape and number of SGN cells in the inner ear, but the thickness of SV in the middle turn and basal turn of the cochlea was reduced (both P<0.05). The results of transmission electron microscope showed that compared with WT mice, the structure of mitochondria in inner ear HCs, SGN cells and SV cells of Timm8a^-/- mice was abnormal, and the proportion of abnormal mitochondria was significantly increased (all P<0.05). Conclusion ·Timm8a^-/- mice are successfully constructed in this study, and the elevated hearing threshold of Timm8a^-/- mice may be related to the abnormal ultrastructure of mitochondria in inner ear.