半拷贝基因NKCC1杂合小鼠与年龄相关性听力下降的关系

Association of age-related hearing loss with mice which expressed only one copy of gene encoding NKCC1 co-transporter

目的培育杂合子NKCCl+／-和野生型NKCCl+／+小鼠,观察携带不同拷贝基因的NKCCl小鼠的年龄相关性听力表现,研究NKCCl基因及其离子通道在年龄相关性听力下降(age- related hearing loss,AHL)中的作用,并初步探讨其可能发生的机制。方法运用NKCCl+／-杂合子小鼠和NKCCl+／+野生型小鼠为平台,于小鼠生长过程中的各个年龄段分别检测小鼠的听性脑干反应(auditory brainstem response,ABR)和耳蜗内电位(endocochlear potential,EP);同时采用扫描电镜进行耳蜗形态学观察和免疫印迹杂交检测不同鼠龄小鼠耳蜗的NKCCl通道蛋白的含量。结果NKCCl+／-杂合子小鼠与NKCCl+／+野生型小鼠相比较,其ABR检测短声阈值在每一年龄组的各测试频率均升高,各年龄组相比较差异有统计学意义(P值均<0．05)。NKCCl+／-杂合子半拷贝基因鼠的听力随年龄增长而逐渐下降,与低龄鼠相比,老龄鼠的ABR阈值显著性升高(P值均< 0．05)。NKCCl+／-小鼠的EP也随年龄增长而呈下降趋势,老龄小鼠的EP值与其低龄鼠相比明显降低(P<0．05)。老龄小鼠的耳蜗NKCCl蛋白含量低于低龄鼠。与老龄NKCCl+／+鼠相比较,老龄NKCCI+／-鼠的耳蜗底回外毛细胞出现散在的点状缺失。结论当NKCCl基因呈现半拷贝复制时小鼠可呈现AHL,NKCCl基因可能在AHL的发病中起一定的作用。AHL的发病可能与NKCCl通道蛋白的遗传特性及功能有关,还可能与NKCCl+／-鼠出现的耳蜗底回外毛细胞缺失有关。

Objective To generate transgenic mice of NKCC1 ＋/- （heterozygous） and NKCC1 ＋/＋ （wild-type） that have a targeted disruption in the NKCC1 gene in order to investigate the relationship of one copy of NKCCI gene（ NKCCI ＋/- ） and age-related hearing loss （AHL） and to study the possible pathogenesis of AHL. Methods Auditory function of NKCC1 ＋/- mice was detected regularly by auditory brain response （ABR） and endocochlear potential （EP）. Morphology of cochlea was observed by scanning electron microscope and content of NKCC1 protein was detected by Western blot. Results The mean value for ABR thresholds was elevated in NKCC1 ＋ / - mice more than that of NKCC1 ＋ / ＋ mice （ P 〈 0. 01 ）. A progression of age-related hearing loss was found in NKCC1 ＋/- mice. Compared with younger NKCC1 ＋/- mice, the mean value for ABR thresholds in aged NKCC1 ＋/- mice was significantly increased （P〈0. 05）. The EP of NKCC1 ＋/- aged mice was also significantly decreased more than that of the younger NKCC1 ＋/＋ mice （P〈0.05）. And content of NKCC1 protein were reduced with the growth of the age. The scanning electron microscope showed a kind of scattered punctiform absence of outer hair cells in elder NKCC1 ＋/- mice cochlea. Conclusions NKCC1 gene maybe takes part in the pathogenesis of AHL. Mice that expressed only one copy of NKCC1 could lead to AHL. AHL may be correlative with the amounts of NKCC1 protein and its function and also with the loss of outer hair cells perhaps.