不同月龄BALB/c小鼠耳蜗miR-96表达与ABR阈值及耳蜗形态的关系

Relationship between the miR-96 expression in the cochlea and ABR threshold and cochlear morphology of BALB/c mice

目的研究不同月龄BALB/c小鼠ABR阈值、耳蜗形态学改变及miR-96的表达,明确miR-96对老年性聋小鼠听力的调节作用。方法通过听性脑干反应测试、荧光染色、扫描电镜,观察3、6、12、18月龄BALB/c小鼠8 kHz听反应阈值及耳蜗形态学改变。实时定量PCR定量检测miR-96在各月龄BALB/c小鼠耳蜗内的表达。SPSS13.0统计软件进行统计分析。结果 3、6、12月龄组小鼠8 kHz听反应阈值分别为(18.5±8.3)、(45.8±7.8)、(85.6±15.6)dB SPL,18月龄组120 dB SPL刺激声基本测不出听觉反应。荧光染色及扫描电镜发现,自6月龄起毛细胞出现显著缺失,静纤毛出现不同程度缺失、倒伏、融合、变短和转位等改变,并随月龄增大病变逐渐加重。miR-96在3、6、12、18月龄BALB/c小鼠耳蜗中的相对表达量(2^(-△CT))分别为0.0225±0.0073、0.0162±0.0048、0.0116±0.0048和0.0050±0.0014,与3月龄小鼠相比,差异有统计学意义(P<0.05)。结论 BALB/c小鼠听力损失、耳蜗毛细胞缺失及纤毛损害随月龄增长而逐渐加重,小鼠耳蜗中miR-96表达随月龄增加而减少,提示miR-96可能在老年性聋的发病机制中起重要用。

OBJECTIVE Through researching the ABR threshold, the cochlear morphology and miR- 96 expression in the cochlear of BALB/c mice at different month＇s age, to find out if the miR-96 can regulate the agerelated hearing loss of BALB/c mice. METHODS ABR testing, AO/PI staining and scanning electron microscope were used to observe the ABR threshold and cochlear morphology of the BALB/c mice at the ages of 3 months, 6 months, 12 months and 18 months. Real-time PCR was used to detect the expression of miR-96 in the cochlea of BALB/c mice at the ages of 3 months, 6 months, 12 months and 18 months. RESULTS The ABR thresholds of BALB/ c mice were （18.5 ± 8.3）, （45.8 ±7.8）, and （85.6 ± 15.6） dB SPL separately at the age of 3, 6 and 12 months. At the age of 18 months, no response was observed in the ABR testing with 120 dB SPL acoustic stimulation. In the AO/PI staining, we found that the outer hair cells was apparently lost since the age of 6 months and the loss of hair cells aggravated as the month＇s age increased. At the age of 12 months, no outer hair cells was left, inner hair cells was lost apparently too. With the scanning electron microscope, we found the changes of deficiency, lodging, fusion, shortening and inversion in the hair cell cilia. And these changes were aggravated as the month＇s ages increased. At the age of 3 months, the relative expression of miR-96 （2-△CT） was 0.0225 ± 0.0073. The relative expression of miR-96 （2-△CT） in the cochlea were 0.0162±0.0048, 0.0116±0.0048, and 0.0050±0.0014 at the age of 6 months, 12 months and 18 months separately, comparing with the relative expression of miR-96 at the age of 3 months, the differences were significant（P〈0.05）. CONCLUSION The hearing loss, hair cells loss, and cilia damage aggravated as the month＇s age increased, but the miR-96 expression in the cochlea decreased. Which suggest that miR-96 might play an important role in the age related hearing loss.