摘 要：听力损失是一个普遍存在的全球健康问题，遍布所有地区和国家。听力损失分为传导性听力损失、感音神经性听力损失以及混合性听力损失。氧化应激和氧化还原稳态失衡是造成耳蜗损伤的重要因素，而NRF2是重要的抗氧化调节因子。在人体中，Nfe2l2基因普遍表达于中枢神经系统、听觉器官及代谢相关器官及组织中，在维持细胞氧化还原稳态，应对氧化应激，调节代谢、炎症、自噬及免疫反应等方面发挥重要作用。在噪声暴露试验中，Nrf2-KO小鼠相比于野生型小鼠表现出更严重的听力障碍。在斑马鱼中，nrf2a在胚胎发育早期即广泛表达，作为核心转录因子调控一系列抗氧化基因的表达，构成抵御化学毒物及重金属等外源氧化损伤的重要防御机制。除其抗氧化功能外，该基因对于斑马鱼多种器官（如肝脏、神经系统）的发育与稳态维持至关重要，并在调节炎症反应、代谢过程及抑制细胞凋亡等生理病理环节中发挥重要保护作用。为研究该基因在内耳及侧线毛细胞发育中的具体作用机制，本研究利用CRISPR/Cas9基因编辑技术构建斑马鱼nrf2a基因敲除品系。首先利用在线分析工具筛选nrf2a基因的敲除靶点，通过聚合酶链式反应扩增合成该基因的向导DNA，以此为模板进行转录，将转录得到的sgRNA与Cas9蛋白按5:1的比例混合后注入1 cell期的斑马鱼胚胎中，随后进行基因敲除的有效性鉴定。结果表明，nrf2a基因出现大片段缺失，翻译提前终止，该基因成功敲除。对其进行筛选后获得nrf2a基因的纯合突变体，这为研究该基因在内耳及侧线毛细胞发育中的作用奠定了基础。

关键词：斑马鱼；CRISPR/Cas9；nrf2a基因；听力损失；内耳发育
中图分类号：Q812                          文献标志码：A       DOI：10.3969/j.issn.1007-7146.2026.01.008

Abstract: Hearing loss is a widespread global health issue that affects all regions and countries. It is classified into conductive hearing loss, sensorineural hearing loss, and mixed hearing loss. Oxidative stress and redox homeostasis imbalance are important factors causing cochlear damage, and NRF2 is a crucial antioxidant regulatory factor. In the human body, the Nfe2l2 gene is widely expressed in the central nervous system, auditory organs, and metabolism-related organs and tissues, playing a significant role in maintaining cellular redox homeostasis, responding to oxidative stress, and regulating metabolism, inflammation, autophagy, and immune responses. In noise exposure experiments, Nrf2-KO mice showed more severe hearing impairment compared to wild-type mice. In zebrafish, nrf2a is widely expressed in the early stages of embryonic development and acts as a core transcription factor to regulate the expression of a series of antioxidant genes, constituting an important defense mechanism against exogenous oxidative damage from chemicals and heavy metals. Besides its antioxidant function, this gene is crucial for the development and homeostasis maintenance of various organs in zebrafish, such as the liver and nervous system, and plays an important protective role in regulating inflammatory responses, metabolic processes, and inhibiting apoptosis. To study the specific mechanism of this gene in the development of inner ear and lateral line hair cells, a zebrafish nrf2a gene knockout line was constructed using CRISPR/Cas9 gene editing technology. First, online analysis tools were used to screen the knockout target sites of the nrf2a gene, and the guide DNA of this gene was amplified and synthesized by polymerase chain reaction. This was then used as a template for transcription, and the transcribed sgRNA was mixed with Cas9 protein at a ratio of 5: 1 and injected into 1-cell stage zebrafish embryos. Subsequently, the effectiveness of the gene knockout was verified. The results showed that there was a large fragment deletion in the nrf2a gene, and translation was prematurely terminated, indicating successful knockout of the gene. After screening, a homozygous mutant of the nrf2a gene was obtained, laying the foundation for studying the role of this gene in the development of inner ear and lateral line hair cells.
Key words: zebrafish; CRISPR/Cas9; nrf2a gene; hearing loss; inner ear development
（Acta Laser Biology Sinica, 2026, 35(1): 072-080）