【作者】 钱付平；

【导师】 柴人杰；

【作者基本信息】 东南大学 ， 生物学， 2021， 博士

【摘要】 听觉是我们最重要的感觉功能之一,听力下降或者耳聋将会大大影响我们的日常生活、降低我们的生活品质。在包括人类在内的哺乳动物中,听觉的主要器官是耳,耳可分为外耳、中耳和内耳。内耳中的耳蜗毛细胞是我们感知声音信号并将其传递到神经系统的关键成员。任何导致耳蜗毛细胞损伤的因素都会引起听力的下降,甚至耳聋。导致耳聋的因素主要包括噪声损伤、耳毒性药物处理和遗传因素等。遗传性耳聋是指由于基因突变所产生的可以遗传的耳聋,目前,已被发现的遗传性耳聋的致病基因超过200个,但是还有大量耳聋基因至今还没有被发现。因此,不断发现遗传性耳聋的致病基因对于更好的了解遗传性耳聋的致病机制、预防和治疗遗传性耳聋具有十分重要的作用。本文以模式动物斑马鱼为研究对象,通过对斑马鱼毛细胞进行单细胞RNA测序,筛选得到一批在斑马鱼毛细胞中特异性表达的候选基因,其中包括一些已经被证实的耳聋基因。斑马鱼solute carrier family 4,member 2b(slc4a2b)基因便是通过以上方法筛选得到的可能影响毛细胞发育和功能的候选基因之一,它是一种溶质转运蛋白的编码基因。通过基因保守性分析,我们发现slc4a2b基因在进化上高度保守。并且,原位杂交实验结果发现,slc4a2b基因特异表达于斑马鱼耳囊和侧线神经丘中。接下来,我们分别通过morpholino oligonucleotide(MO)介导的基因敲降技术和CRISPR/Cas9基因编辑技术构建了slc4a2b基因敲降和突变斑马鱼模型。研究结果发现,slc4a2b基因功能缺陷导致斑马鱼侧线神经丘毛细胞减少。进一步分析发现,slc4a2b基因功能缺陷引起毛细胞发生caspase-3介导的细胞凋亡。综上所述,slc4a2b基因对于斑马鱼的毛细胞发育及其功能具有重要的作用。这一结果可为临床上发现由于SLC4A2基因缺陷导致的毛细胞相关疾病提供实验依据、为耳聋基因的发现提供思路。更多还原

【Abstract】 Hearing is one of the most important sensory function.Hearing loss or deafness would greatly affect our daily life and lower the quality of our life.In mammals including humans,the organ of hearing is the ear,which can be divided into three parts,external ear,middle ear and inner ear.The cochlear hair cells,the key members in the inner ear,can sense the acoustic signals and transmit them to the nervous system.Any factors leading to cochlear hair cell damage would cause hearing loss,or even deafness.The main factors that cause hearing loss include acoustic trauma,ototoxic drug treatments and genetic factors.Hereditary deafness is caused by the gene mutations,and can be inherited to the next generation.At present,more than 200 deafness genes have been discovered,but there are still a large number of deafness genes waiting to be identified.Therefore,the continuous discovery of deafness genes plays a very important role for a better understanding of the pathogenic mechanism,prevention and treatment of hereditary hearing loss.Here,we took the zebrafish as the animal model,and obtained many of candidate genes which specifically expressed in the zebrafish hair cells using single-cell RNA-sequencing(sc RNA-seq)method,including the deafness genes that had been confirmed.The zebrafish solute carrier family 4,member 2b(slc4a2b)gene,which encoding a solute transport protein,is one of the many genes screened by this method.Through gene conservation analysis,we found that the slc4a2 b gene is highly conserved in evolution.In addition,in situ hybridization experiments revealed that the slc4a2 b gene was specifically expressed in the zebrafish otic vesicle and lateral line neuromasts.Next,we constructed slc4a2 b gene knockdown and mutant zebrafish models through morpholino oligonucleotide(MO)-mediated gene knockdown technology and CRISPR/Cas9 gene editing technology,respectively.According to the results,we found that loss of function of the slc4a2 b gene resulted in the decrease of the lateral line neuromast hair cells in zebrafish.Further analysis showed that loss of function of slc4a2 b gene led to caspase-3 mediated apoptosis in hair cells.In summary,the slc4a2 b gene plays an important role in the development and function of hair cells in zebrafish.These results can provide experimental evidence for the clinical discovery of hair cell-related diseases caused by SLC4A2 gene deficiency and provide ideas for the discovery of deafness genes.更多还原