不同血清型腺相关病毒经枕大池注射转染小鼠内毛细胞的比较

doi:10.3969/j.issn.1674-8115.2026.07.008

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (7): 896-905.doi: 10.3969/j.issn.1674-8115.2026.07.008

• 论著 · 基础研究 • 上一篇

不同血清型腺相关病毒经枕大池注射转染小鼠内毛细胞的比较

安益冉¹^,², 冯宝怡¹^,², 孙怡琳¹^,², 吴皓¹^,², 陶永¹^,²()

^1.上海交通大学医学院附属第九人民医院耳鼻咽喉头颈外科，上海 200011
^2.上海交通大学医学院耳科学研究所，上海市耳鼻疾病转化医学重点实验室，上海 200125

收稿日期:2026-03-03 接受日期:2026-04-17 出版日期:2026-07-28 发布日期:2026-07-28
通讯作者: 陶　永，副研究员，博士；电子信箱：taoyent@sjtu.edu.cn。
基金资助:
国家自然科学基金(82371145);上海交通大学医学院“双百人”项目(20191821)

Comparison of inner hair cell transduction by different serotypes of adeno-associated virus via cisterna magna injection in mice

An Yiran¹^,², Feng Baoyi¹^,², Sun Yilin¹^,², Wu Hao¹^,², Tao Yong¹^,²()

^1.Department of Otolaryngology-Head and Neck Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Ear Institute, Shanghai Jiao Tong University School of Medicine; Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200125, China

Received:2026-03-03 Accepted:2026-04-17 Online:2026-07-28 Published:2026-07-28
Contact: Tao Yong, E-mail: taoyent@sjtu.edu.cn.
Supported by:
National Natural Science Foundation of China(82371145);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191821)

摘要/Abstract

摘要：

目的·探究经枕大池注射携带人巨细胞病毒立即早期启动子（human cytomegalovirus immediate-early promoter，CMV）的不同血清型的腺相关病毒（adeno-associated virus，AAV）对于听觉系统及中枢神经系统的安全性，以及对内毛细胞（inner hair cells，IHCs）与中枢神经系统的转染差异。方法·将8种带有增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）的不同血清型（AAV1、AAV2、AAV8、AAV9、AAV-rh10、AAV-DJ、AAV-PHP.B、AAV-PHP.eB）的AAV-EGFP，经枕大池注射入4周龄野生型小鼠脑脊液内。采用听性脑干反应（auditory brainstem response，ABR）评价枕大池注射前及注射2周后的听力阈值变化。于注射2周后取小鼠耳蜗，通过免疫荧光染色比较注射8种血清型AAV‑EGFP后耳蜗IHCs的转染效率及数量；对其中转染效率较高的3种血清型在IHCs中表达的荧光信号强度行进一步分析，并通过对小鼠脑组织冰冻切片进行DNA荧光染料染色比较3种不同血清型AAV‑EGFP对中枢神经系统脑区的转染情况；采用旷场实验评估枕大池注射该3种血清型AAV‑EGFP对小鼠中枢神经系统安全性的影响。结果·ABR显示枕大池注射前、注射2周后小鼠全频率听力阈值无明显差异。免疫荧光染色的结果显示注射不同血清型AAV‑EGFP 2周后小鼠耳蜗32 kHz处IHCs均实现转染，其中AAV2-CMV-EGFP［（83.33±14.79）%］、AAV-DJ-CMV-EGFP［（82.52±5.18）%］、AAV-PHP.B-CMV-EGFP［（96.61±1.87）%］这3种病毒对IHCs 32 kHz处的转染效率较高；且注射后组间IHCs数量无显著差异。针对转染效率位于前三的血清型的荧光信号强度进行分析，结果显示AAV-PHP.B-CMV-EGFP和AAV2-CMV-EGFP在IHCs 32 kHz处的荧光信号较强。脑部冰冻切片显示，3种血清型AAV‑EGFP经枕大池注射后在中枢神经系统呈现不同的转染特征，其中AAV2-CMV-EGFP转染水平较低，AAV-DJ-CMV-EGFP可在小脑皮层、延髓背侧及脑室周围区域检测到转染，AAV-PHP.B-CMV-EGFP在小脑皮层分子层、颗粒层及脑室周围存在转染。旷场实验的结果显示经枕大池注射3种血清型病毒后，小鼠自主活动未见异常。结论·经枕大池注射不同血清型AAV-EGFP不会造成小鼠听力下降与耳蜗IHCs损伤，不同血清型AAV‑EGFP在耳蜗及中枢神经系统的转染效率存在明显区别。

关键词: 基因治疗, 腺相关病毒, 枕大池注射, 内毛细胞

Abstract:

Objective ·To evaluate the safety of cisterna magna-delivered adeno-associated virus (AAV) of different serotypes carrying the human cytomegalovirus immediate-early promoter (CMV) in the auditory and central nervous systems, as well as their transduction differences in inner hair cells (IHCs) and the central nervous system. Methods ·Eight AAV serotypes (AAV1, AAV2, AAV8, AAV9, AAV-rh10, AAV-DJ, AAV-PHP.B, and AAV-PHP.eB) carrying enhanced green fluorescent protein (EGFP) were injected into the cerebrospinal fluid of 4-week-old wild-type mice via cisterna magna. Auditory brainstem response (ABR) was measured to evaluate changes in auditory thresholds before injection and 2 weeks after injection. At 2 weeks post-injection, cochleae were collected and subjected to immunofluorescence staining to compare the transduction efficiency and number of cochlear IHCs among the eight AAV-EGFP serotypes. The EGFP fluorescence signal intensity of the three serotypes with higher transduction efficiency was further analyzed in IHCs. Fluorescent DNA dye staining on frozen brain sections was used to compare the central nervous system transduction characteristics of the three serotypes. Open-field test was performed to assess the effects of cisterna magna injection of three AAV-EGFP serotypes on central nervous system safety in mice. Results ·ABR tests showed no significant differences in auditory thresholds at all tested frequencies before and 2 weeks after cisterna magna injection. Immunofluorescence staining demonstrated that all eight AAV-EGFP serotypes successfully transduced IHCs in the 32 kHz cochlear region at 2 weeks after in jection. Among them, AAV2-CMV-EGFP [(83.33±14.79)%], AAV-DJ-CMV-EGFP [(82.52±5.18)%], and AAV-PHP.B-CMV-EGFP [(96.61±1.87)%] exhibited relatively high transduction efficiency in IHCs at 32 kHz, and no significant intergroup difference was observed in the number of IHCs after injection. Analysis of EGFP fluorescence intensity in the three serotypes with the highest transduction efficiency revealed stronger fluorescence signals in IHCs transduced by AAV-PHP.B-CMV-EGFP and AAV2-CMV-EGFP at the 32 kHz region. Staining of frozen brain sections revealed distinct transduction patterns of the three AAV-EGFP serotypes in the central nervous system following cisterna magna injection. AAV2-CMV-EGFP exhibited relatively low transduction levels; AAV-DJ-CMV-EGFP showed detectable transduction in the cerebellar cortex, dorsal medulla, and periventricular regions; AAV-PHP.B-CMV-EGFP showed transduction in the molecular and granular layers of the cerebellar cortex and periventricular regions. Open-field test results indicated that spontaneous locomotor activity of mice remained normal following cisterna magna injection of the three tested viral serotypes. Conclusion ·Cisterna magna injection of different AAV-EGFP serotypes causes neither hearing impairment nor IHCs injury in mice. Different AAV-EGFP serotypes exhibit distinct transduction efficiencies in the cochlea and central nervous system.

Key words: gene therapy, adeno-associated virus (AAV), cisterna magna injection, inner hair cell (IHC)

中图分类号:

R764.3

安益冉, 冯宝怡, 孙怡琳, 吴皓, 陶永. 不同血清型腺相关病毒经枕大池注射转染小鼠内毛细胞的比较[J]. 上海交通大学学报（医学版）, 2026, 46(7): 896-905.

An Yiran, Feng Baoyi, Sun Yilin, Wu Hao, Tao Yong. Comparison of inner hair cell transduction by different serotypes of adeno-associated virus via cisterna magna injection in mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(7): 896-905.

图/表 4

图1 枕大池注射示意及AAV-EGFP注射2周后小鼠听力安全性评价Note： A. Workflow of ABR detection. B. Anatomical location of cisterna magna. CB—cerebellum; CM—cisterna magna. C. Detection of ABR thresholds in mice receiving eight AAV-EGFP serotypes before injection and 2 weeks post-injection at different frequencies.

Fig 1 Schematic illustration of cisterna magna injection and auditory safety evaluation in mice 2 weeks after AAV-EGFP administration

图2 8种血清型AAV-EGFP经枕大池注射2周后小鼠32 kHz区域IHCs转染效率和数量分析Note： A. Representative images of IHCs transduced by different AAV-EGFP serotypes in the 32 kHz cochlear region (×40). Green fluorescence represents EGFP expression, and red fluorescence represents MYO7A expression. B. Statistical results of viral transduction efficiency in wild-type mouse IHCs at 32 kHz. C. Statistical results of the number of IHCs per 100 μm at 32 kHz.

Fig 2 Analysis of transduction efficiency and number of IHCs in the 32 kHz cochlear region of mice at 2 weeks after cisterna magna injection of 8 AAV-EGFP serotypes

图3 AAV2-CMV-EGFP、AAV-DJ-CMV-EGFP以及AAV-PHP.B-CMV-EGFP 经枕大池注射后在小鼠耳蜗IHCs中的荧光信号强度及转染效率比较Note： A. Representative images of the basal cochlear turn showing specific transduction in SGNS (×20). Green fluorescence represents EGFP expression, red fluorescence represents MYO7A expression, magenta fluorescence represents TUJ1 expression, and blue fluorescence represents DAPI nuclear staining. B. Quantitative analysis of EGFP fluorescence intensity in IHCs of the 32 kHz cochlear region for the three AAV-EGFP serotypes. C. Statistical results of viral transduction efficiency of three AAV-EGFP serotypes in IHCs from apical, middle, and basal cochlear whole mounts of wild-type mice.

Fig 3 Comparison of fluorescence intensity and transduction efficiency of AAV2-CMV-EGFP, AAV-DJ-CMV-EGFP, and AAV-PHP.B-CMV-EGFP in mouse cochlear IHCs after cisterna magna injection

图4 枕大池注射AAV2-CMV-EGFP、AAV-DJ-CMV-EGFP和AAV-PHP.B-CMV-EGFP对中枢神经系统脑区的转染情况及其对中枢神经系统安全性的影响Note： A. Representative images of central nervous system regions showing the transduction patterns of the three AAV-EGFP serotypes (×20). Green fluorescence represents EGFP expression, and blue fluorescence represents DAPI nuclear staining. B. Open-field test trajectory maps of wild-type mice after cisterna magna injection of the three AAV-EGFP serotypes. C. Detection of general activity indicators in the open-field test in the wild-type mice after cisterna magna injection of the three AAV-EGFP serotypes.

Fig 4 Transduction patterns in central nervous system regions and safety effects of AAV2-CMV-EGFP, AAV-DJ-CMV-EGFP, and AAV-PHP.B-CMV-EGFP following cisterna magna injection

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不同血清型腺相关病毒经枕大池注射转染小鼠内毛细胞的比较

Comparison of inner hair cell transduction by different serotypes of adeno-associated virus via cisterna magna injection in mice

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