老年性聋小鼠内耳突触调控钾通道基因的筛选与鉴定  

Screening and identification of synaptic regulatory genes in the inner ear of mice with presbycusis

作　　者：陈小玲 唐峰 贾立峰 李海 李静雅 袁伟 CHEN Xiaoling;TANG Feng;JIA Lifeng;LI Hai;LI Jingya;YUAN Wei(Department of Otolaryngology,Head and Neck Surgery,First Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400038,China)

机构地区：[1]陆军军医大学(第三军医大学)第一附属医院耳鼻咽喉-头颈外科,重庆400038

出　　处：《第三军医大学学报》2020年第8期765-771,共7页Journal of Third Military Medical University

基　　金：国家自然科学基金(81873702,81470694)。

摘　　要：目的通过筛选内耳突触调控基因,探索老年性聋发生过程中突触损伤特点。方法利用转录组测序技术检测成年组(4周)和老年组(40周)C57BL/6J小鼠内耳基底膜,筛选差异表达基因(differentially expression genes,DEGs)。分析DEGs基因本体(GO)功能富集,京都基因和基因组百科全书(KEGG)通路富集情况,并筛选突触调控差异基因,最后利用实时荧光定量PCR及免疫荧光染色验证结果。结果共筛选出3267个DEGs,其中下调1521个,上调1746个(FC≥1.5,P<0.05)。功能富集显示,下调基因主要参与电压门控离子通道活性调控和突触调控,上调基因主要参与免疫调控。筛选出突触调控基因64个,其中6个钾通道基因Kcnq2,Kcnk3,Kcnmb4,Kcnk9,Kcnh5,Kcnc2富集度最高。qRT-PCR验证了6个调控突触的钾通道基因在不同年龄组内耳的差异表达;免疫荧光显示KCNQ2、KCNMB4在老年鼠内耳表达明显下降。结论筛选了老年性聋突触调控基因,其中6个钾通道基因表达明显下降,可能是导致老年性聋突触改变的重要因素。Objective To screen synaptic regulatory genes in the inner ear and explore the mechanism of synaptic damage in presbycusis.Methods We examined the mRNA expression of the genes in the basilar membrane of the inner ear of adult(4-week-old)mice and aged(40-week-old)mice using transcriptome sequencing.Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the differentially expressed genes(DEGs)were analyzed to identify synaptic regulatory DEGs,and the results were verified using real-time quantitative PCR and immunofluorescence staining.Results We identified a total of 3267 DEGs,including 1521 down-regulated genes and 1746 up-regulated genes(FC≥1.5,P<0.05).GO function enrichment analysis showed that the down-regulated DEGs mainly participated in the regulation of voltage-gated cation channel activity and synaptic function,and the up-regulated DEGs were mostly responsible for regulation of immune functions.We screened 64 synaptic regulatory genes,among which 6 down-regulated potassium channel genes(Kcnq2,Kcnk3,Kcnmb4,Kcnk9,Kcnh5,and Kcnc2)had the highest enrichment.qRT-PCR verified the differential expression of the 6 genes in the inner ear of mice at different ages;immunofluorescence assay demonstrated that the expression of KCNQ2 and KCNMB4 were significantly decreased in the inner ear of aged mice.Conclusion Among the synaptic regulatory genes screened in mice with presbycusis,6 potassium channel genes show significantly decreased expressions in the inner ear of aged mice,suggesting their important involvement in synaptic changes in presbycusis.

关 键 词：老年性聋 内耳 突触 转录组测序 钾离子通道 

分 类 号：R322.923[医药卫生—人体解剖和组织胚胎学] R394.3[医药卫生—基础医学]