邻近标记技术对活体小鼠耳蜗毛细胞表面蛋白进行特异性标记

doi:10.6040/j.issn.1673-3770.0.2024.667

山东大学耳鼻喉眼学报 ›› 2026, Vol. 40 ›› Issue (1): 6-12.doi: 10.6040/j.issn.1673-3770.0.2024.667

邻近标记技术对活体小鼠耳蜗毛细胞表面蛋白进行特异性标记

王暖^1,2,王欣阳^1,2,吉长皓^1,2,刘敏²,高建刚^2,3,孙锦^1,2

山东省医学科学院)实验动物学院, 山东济南 250117;
2.山东第一医科大学(山东省医学科学院)医学科技创新中心, 山东济南 250117;
3.山东大学生命科学学院/实验畸形学教育部重点实验室, 山东济南 250100

出版日期:2026-01-20 发布日期:2026-02-13
通讯作者: 孙锦. E-mail:sunjin@sdfmu.edu.cn
基金资助:
山东省自然科学基金(ZR2023MC207)

In vivo proximity labeling of cell-surface proteins in mouse cochlear hair cells

WANG Nuan^1,2, WANG Xinyang^1,2, JI Changhao^1,2, LIU Min², GAO Jiangang^2,3, SUN Jin^1,2

1. School of Laboratory Animal, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, Shandong, China2. Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250117, Shandong, China3. School of Life Science, Key Laboratory of the Ministry of Education for Experimental Teratology, Shandong University, Jinan, 250100, Shandong, China

Online:2026-01-20 Published:2026-02-13

摘要/Abstract

摘要： 目的开发标记小鼠耳蜗毛细胞的细胞表面蛋白的方法,为测定耳蜗毛细胞表面蛋白质组提供依据。方法构建在毛细胞中特异性表达一种辣根过氧化物酶融合蛋白(HRP fused with a transmembrane domain, HRP-TM)的重组腺相关病毒(recombinant adeno-associated virus, rAAV),HRP-TM可以定位到毛细胞膜的外表面。通过显微注射将病毒注射到2 d新生C57BL/6小鼠的后半规管中,30 d时检测听觉脑干反应(auditory brainstem response, ABR),并通过基底膜免疫荧光染色检测耳蜗中HRP表达和蛋白标记情况。结果 ABR结果显示,注射病毒的小鼠和未注射病毒的对照小鼠相比,听力无明显差异。基底膜免疫荧光染色结果显示,HRP-TM在毛细胞中特异性表达,内毛细胞的病毒感染效率超过95%,外毛细胞的病毒感染效率达到80%左右。而且,毛细胞的形态和排列与对照相比无明显差异。用荧光偶联链霉亲和素进行免疫荧光染色,只有表达HRP-TM的毛细胞的细胞膜表面有明显荧光信号。结论本研究中构建的rAAV病毒可以高效特异性的标记耳蜗毛细胞的细胞表面蛋白,并且无明显的细胞毒性,不影响毛细胞的正常生理功能,为下一步分离耳蜗毛细胞的细胞表面蛋白并进行质谱分析奠定了基础。

关键词: 耳蜗毛细胞, 邻近标记, 重组腺相关病毒, 辣根过氧化物酶, 细胞表面蛋白质组

Abstract: Objective To develop a method for labeling cell-surface proteins of cochlear hair cells in mice and establish conditions for profiling the cell-surface proteome of these cells. Methods A recombinant adeno-associated virus(rAAV)was engineered to express a horseradish peroxidase fusion protein(HRP-TM)specifically in hair cells, targeting the outer surface of the cell membrane. The virus was microinjected into the posterior semicircular canal of 2-day-old C57BL/6 mice. At 1 month of age, auditory brainstem response(ABR)was measured, and HRP expression and protein labeling were assessed via basilar membrane immunofluorescence staining. Results ABR results showed no significant hearing differences between virus-injected and control mice. Immunofluorescence revealed HRP-TM was specifically expressed in hair cells, with >95% transduction efficiency in inner hair cells and ~80% transduction efficiency in outer hair cells. Hair cell morphology and arrangement remained normal. Fluorescent streptavidin staining confirmed membrane-specific labeling exclusively in HRP-TM-expressing hair cells. Conclusion The rAAV-HRP-TM system efficiently and specifically labels cochlear hair cell surface proteins without cytotoxicity or functional impairment, providing a foundation for isolating and analyzing these proteins via mass spectrometry.

Key words: Cochlear hair cells, Proximity labeling, rAAV, HRP, Cell-surface proteome

中图分类号:

Q95-3

王暖,王欣阳,吉长皓,刘敏,高建刚,孙锦. 邻近标记技术对活体小鼠耳蜗毛细胞表面蛋白进行特异性标记[J]. 山东大学耳鼻喉眼学报, 2026, 40(1): 6-12.

WANG Nuan, WANG Xinyang, JI Changhao, LIU Min, GAO Jiangang, SUN Jin. In vivo proximity labeling of cell-surface proteins in mouse cochlear hair cells[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2026, 40(1): 6-12.

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邻近标记技术对活体小鼠耳蜗毛细胞表面蛋白进行特异性标记

In vivo proximity labeling of cell-surface proteins in mouse cochlear hair cells

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