C57BL／6J小鼠耳蜗钾离子转运蛋白KCNQ1和NKCC1的年龄相关性表达及其与听力的关系

Association of age-related hearing loss with ion transporter KCNQ1 and NKCC1 in cochlea of C57BL/6J mice

目的观察钾离子转运蛋白KCNQ1和Na-K-2C1联合转运子1（Na—K-2C1CO—transporter1，NKCC1）在C57BL／6J小鼠耳蜗侧壁的年龄相关．陛表达，探讨这两种转运蛋白在年龄相关性听力减退发病中的作用。方法通过听性脑干反应（ABR）检测C57BL／6J小鼠各年龄段（4、8、14、24、40周龄）的听力变化。应用免疫组化法确定KCNQ1和NKCC1在C57BL／6J小鼠耳蜗中的表达定位，并检测各年龄段小鼠耳蜗中两种转运蛋白含量随着年龄增加的表达变化。采用反转录聚合酶链反应（RT-PCR）分别检测KCNQ1和NKCC1的mRNA在不同鼠龄小鼠耳蜗中的表达水平。结果随着C57BL／6J小鼠年龄的增加，其ABR反应阈值逐渐升高，4周龄小鼠的短声（click）和4kHz、8kHz的ABR反应阈值与8周龄小鼠相比，差异均无统计学意义（P值均〉0．05）；14周龄后，三种刺激声的ABR反应阈逐渐升高，与低龄组小鼠相比其差异均具有统计学意义（P值均〈0．05）。KCNQ1蛋白主要表达于耳蜗血管纹的边缘细胞顶膜，而NKCC1蛋白主要表达于血管纹边缘细胞、螺旋韧带的Ⅱ型纤维细胞及螺旋缘下的纤维细胞。随着年龄的增加，血管纹KCNQ1和NKCC1蛋白的表达逐渐减少。KCNQ1和NKCC1mRNA的表达量也随着年龄的增加呈现减少的趋势。结论C57BL／6J小鼠随着年龄的增加其听力逐渐下降。KCNQ1和NKCC1两种钾离子转运蛋白随着小鼠年龄的增加逐渐减少；KCNQ1和NKCC1mRNA表达水平亦呈现随年龄增加而逐渐减少的趋势。耳蜗侧壁KCNQ1和NKCC1可能在年龄相关性听力减退的发生中起一定作用。

Objective To investigate the age-related expression of KCNQ1 and NKCC1 ion transporters in the stria vaseularis in the cochlea of C57BL/6J mice, and to analyze the relationship between the two ion transporters and age-related hearing loss. Methods Auditory function of C57BL/6J mice was measured by auditory brainstem response（ABR） at the ages of 4, 8, 14, 24, 40 weeks old respectively. The location of KCNQ1 and NKCC1 ion transporters in the cochlea of C57BL/6J mice was detected by immunohistoehemistry staining. Reverse transcription polymerase chain reaction （RT-PCR） was used to detect the levels of KCNQ1 and NKCC1 mRNA in the cochlea of C57BL/6J mice at different ages. Results The mean values for ABR thresholds in response to click, 4 kHz and 8 kHz sound stimulus of C57BL/6J mice gradually increased with age. The ABR thresholds of the mice of over 14 weeks age were significantly elevated in comparison with lower ages （ P 〈 0. 05 ）. In the lateral wall of C57BL/6J mice cochlea, theKCNQ1 protein was mainly expressed at the apical membrane of the strial marginal cells. The localization of NKCC1 protein was mainly present at the basolateral membrane of the stria marginal cells, spiral ligament and the fibrocytes in the inferior portion of spiral limbus. Expression of KCNQ1 and NKCC1 protein in cochlea of C57BL/6J mice showed age-related decreasing. The level of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J also showed a age-related decreasing trend. There was a significant reducing of KCNQ1 mRNA level between C57BL/6J mice of 40 and 4 weeks old （P 〈 0. 05 ）. In comparison with the C57BL/6J mice of 4 weeks old, the NKCCI mRNA levels of 24 and 40 weeks old also showed significiant reducing （ P 〈 0. 05 ）. Conclusions The mean value for ABR thresholds of C57BE/6J mice gradually increased with age. Expression of KCNQ1 and NKCC1 protein in the stria vascularis of C57BL/6J mice decreases with age. The levels of KCNQ1 and NKCC1 mRNA in cochlea of C57BL/6J showed a age-related reducing trend. Regulating after post-translation may also participate in the adjusting of the age-related decreasing of KCNQ1 and NKCC1 protein in the cochlea of C57BL/6J mice. KCNQ1 and NKCC1 ion transporters may play a critical role in maintaining normal hearing function of inner ear.