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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 investigate the age-related hearing performance of NKCCl +/- and wild-type NKCCl + / + mice with different copies of genes and to investigate the role of NKCCl gene and its ion channels in age-related hearing loss loss, AHL), and to explore its possible mechanism. Methods NKCCl +/- heterozygous mice and NKCCl + / + wild type mice were used as platforms to detect the auditory brainstem response (ABR) and intracochlear in mice at various ages during their growth. The endocochlear potential (EP) was measured. The morphology of NKCCl channel in the cochlea of mice of different age was detected by scanning electron microscopy and immunoblotting. Results Compared with NKCCl + / + wild-type mice, the short-circuit threshold of ABR in NKCCl +/- heterozygous mice increased at each test frequency in each age group, and the difference was statistically significant among all age groups (P All <0.05). The hearing ability of NKCCl +/- heterozygous half-copy gene mice gradually decreased with age, and the ABR threshold of aged mice was significantly higher than that of the younger mice (all P <0.05). The EP of NKCCl +/- mice also showed a decreasing trend with age, and the EP value of aged mice was significantly lower than that of the younger mice (P <0.05). The content of NKCCl protein in the cochlea of aged mice was lower than that of young mice. In contrast to aged NKCCl + / + mice, scattered punctate deletions occurred in the basal gyrus outer hair cells of aged NKCCI +/- mice. Conclusion When the NKCCl gene shows half copy replication, the mouse can present AHL. NKCCl gene may play a role in the pathogenesis of AHL. The pathogenesis of AHL may be related to the genetic characteristics and function of NKCCl channel protein, and may also be related to the absence of outer hair cells in the bottom of the cochlea of NKCCl +/- mice.