GJB2基因p.V37I敲入小鼠缝隙连接蛋白的表达与功能研究

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.

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。

本研究主要观察了GJB2基因p.V37I突变小鼠听力损失情况，并对可能导致听力损失的潜在机制进行了一部分探索。首先，利用CRISPR/cas9技术构建了GJB2基因p.V37I突变小鼠模型，并通过设计杂交路线获得纯合突变小鼠。通过对突变小鼠定期ABR检测，发现突变小鼠随时间进展逐渐出现ABR反应阈值轻度升高，且以高频反应阈值（24 kHz）升高为主；然后分别对小鼠耳蜗石蜡切片进行HE染色和扫描电镜等实验观察GJB2基因p.V37I纯合突变小鼠（Homo）及对照小鼠（WT）的耳蜗形态结构，发现突变小鼠的耳蜗结构及毛细胞纤毛状态较对照小鼠无明显改变；使用耳蜗冰冻切片和基底膜铺片免疫荧光染色观察Connexin26蛋白的表达与定位，发现突变导致该蛋白表达与定位未出现明显改变。推测听力损失的主要原因，可能并非由耳蜗结构与Connexin26蛋白表达与定位异常改变引起。为了进一步探索GJB2基因p.V37I突变导致听力损失的潜在机制，通过采用听觉系统发育较好的1月龄与最早出现显著听力差异的8月龄作为时间点进行耳蜗转录组研究，发现1月龄和8月龄的突变小鼠较其同月龄的对照小鼠相比，存在242个共同下调的基因（Fold Change＞2），提示GJB2基因p.V37I突变可能是通过下调耳蜗内部分基因表达，如降低纤毛功能等改变耳蜗功能，从而进一步导致听力损失。通过本研究，我们进一步明确了GJB2基因p.V37I突变并不导致内耳明显结构异常，而是导致轻度功能损伤，其机制可能与内耳基因表达改变相关，突变通过下调耳蜗部分基因表达，如降低纤毛功能等方式导致耳蜗功能出现改变，进而造成听力损失。