特异性敲除内毛细胞肌球蛋白轻链激酶基因对小鼠听功能的影响  

Study on conditional myosin light chain kinase gene knockout mice resulting in hearing loss

作　　者：朱光洁[1] 马登滨[2] 钱晓云[1] 周函[1] 陈杰[1] 王芳[1] 高下[2] 

机构地区：[1]南京大学医学院附属鼓楼医院耳鼻咽喉头颈外科,210008 [2]南京医科大学附属鼓楼临床学院

出　　处：《中华耳鼻咽喉头颈外科杂志》2013年第1期42-47,共6页Chinese Journal of Otorhinolaryngology Head and Neck Surgery

基　　金：国家自然科学基金（30973302）

摘　　要：目的建立特异性敲除内毛细胞肌球蛋白轻链激酶（myosinlightchainkinase，MLCK）基因小鼠模型，初步评价敲除该基因对小鼠听功能的影响。方法从DNA水平对Mlck基因敲除小鼠作基因型鉴定并验证敲除效率；以短声和8、16、32kHz短纯音作为刺激音对基因敲除组及对照组小鼠行听性脑干反应（ABR）测试，分析两组小鼠反应阈值及波形差异。结果根据鼠尾DNAPCR结果能够初步判定小鼠基因型，分离内毛细胞提取DNA，PCR结果证实目的基因被剔除。Mlck基因敲除组小鼠短声及短纯音各频率ABR平均阈值较对照组均有升高，差异具有统计学意义（P值均〈0．05）；其中高频阈值升高更为显著，在32kHz两组平均阈值相差〉20dB。基因敲除组小鼠16kHz短纯音60、50、40dBSPL声强下Ⅰ波幅值明显小于对照组，差异具有统计学意义（P值均〈0．05）。两组小鼠16kHz短纯音在60dBSPL下的Ⅰ／Ⅱ、Ⅰ／Ⅲ波幅比差异无统计学意义（P值均〉0．05）。结论内毛细胞特异性敲除Mlck小鼠成功将目的基因剔除，敲除后小鼠听力受损，尤以高频明显。Objective To investigate the function of myosin light chain kinase （MLCK） in hearing in mouse by generating inner hair cell-specific Mlck knockout mice and analyze the effect on their hearing. Methods Cross Mlck floxed mice with IHC-Cre mice, the genotype and knockout efficiency were confirmed by PCR. We used auditory brain stem response （ABR） to evaluate mice hearing function at different frequencies. Results Mlck konckout mice were selected by mice tail DNA genotyping and confirmed the deletion of the target gene by isolated inner hair cell DNA genotyping. Mlck-deficient mice showed impaired hearing with a rise in ABR threshold response to click and three different pure tones （8 kHz, 16 kHz, 32 kHz）, and the rise was over 20 dB at high-frequency（32 kHz）. Further analyses of waveforms showed that wave- I amplitudes on 60 dB SPL, 50 dB SPL and 40 dBSPL in response to tone （ 16 kHz） were less than control group（P 〈0. 05） on average, but the ratio of wave Ⅰ / Ⅱ/ and Ⅰ/Ⅲ were not difference （P 〉 0.05）. Conclusions Mlck is successfully deleted in inner hair cell-specific Mlck knockout mice. Mlck knockout mice display a significantly higher threshold in response to click and tones, especially in high- frequencies.

关 键 词：肌球蛋白轻链激酶 小鼠 基因敲除 毛细胞 内 诱发电位 听觉 脑干 

分 类 号：R764[医药卫生—耳鼻咽喉科]