GJB2基因p.V37I敲入小鼠缝隙连接蛋白的表达与功能研究

GJB2基因是遗传性耳聋最常见的致病基因，其突变导致的听力损失程度取决于GJB2基因编码的Connexin26缝隙链接蛋白的损害程度。研究证实，GJB2基因p.V37I突变为非截断突变，在中国汉族人群中高达6.2%，p.V37I纯合突变与轻中度听力损失有关，其不完全外显率的致病机制尚不清楚。本研究拟建立p.V37I敲入小鼠模型，对突变敲入小鼠与野生小鼠每4周进行1次听性脑干反应（ABR）测试，观察其1年听力损失模式；分期处死小鼠，分离培养耳蜗组织，通过免疫荧光染色及蛋白免疫印迹等技术，观察Connexin26蛋白的定位及定量表达；分离培养基底膜上皮细胞，应用细胞内微注射技术，肌醇三磷酸（IP3）-与钙成像及荧光恢复后光漂白（FRAP）技术，检测Connexin26缝隙连接蛋白对miRNA及IP3的渗透活性，旨在探讨p.V37I突变敲入小鼠Connexin26蛋白可能的致聋机制。

GJB2 gene is the most common pathogenic gene for nonsyndromic hearing loss, Hearing phenotype caused by the mutations varies greatly, such as the age of onset ranges from birth to adult, degree of hearing loss ranges from mild to profound. The degree of hearing loss depends on the degree of damage of the connexin26 protein encoded by the GJB2 gene. The p.V37I mutation of GJB2 gene is a nontruncating mutation with a high prevalence in Southeast Asia, especially in Chinese Han population as high as 6.2%. The p.V37I homozygous mutation has been shown to be closely associated with mild to moderate hearing loss, and the p.V37I mutation reduces the penetrance of the GJB2 gene by 17%, whereas the pathogenesis of mild phenotype of the p.V37I homozygous mutation and incomplete penetrance is not clear, which is a hot research topic areas..In this study, p.V37I mutant knock-in mice model was established and regular auditory brainstem response (ABR) was detected in p.V37I mutant knock-in mice and wild type mice, then observe the longitudinal hearing loss mode of p.V37I mutant knock-in mice. Cochlear tissues were isolated and cultured. Immunofluorescence staining, western blot and quantitative polymerase chain reaction(PCR) were used to observe the localization and quantitative expression of connexin26 in the cochlea. In the record of patch clamp ,using the cell microinjection technique, immunofluorescence labeled miRNA (miRNA-F) was injected intracellularly to detect the connexin26 for its penetration activity to miRNA .Application of inositol trisphosphate (IP3) and calcium imaging and fluorescence recovery after photobleaching (FRAP) detection technology, to detect connexin26 for its penetration activity to miRNA . To investigate the deafness mechanism of connexin26 in p.V37I mutant knock-in mice.