人脐带间充质干细胞减缓小鼠年龄相关性听力损失

摘要/Abstract

摘要： 目的研究人脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUC-MSCs)延缓小鼠耳蜗衰老的机制。方法自人脐带分离、培养出稳定的hUC-MSCs,饲养近交系C57BL/6J雄性小鼠至对应月龄。对照组小鼠自3月龄起,每间隔1个月采用微量注射器经尾静脉注射生理盐水0.2 ml;MSCs组小鼠自3月龄起,每间隔1个月经尾静脉注射悬液浓度为5×10⁶个/mL的第4代hUC-MSCs。分别于3、6、10、17、21月龄对小鼠行听性脑干反应(auditory brainstem response, ABR)检测,于6、10、21月龄解剖小鼠耳蜗柯蒂氏器并行基底膜免疫荧光染色,通过激光共聚焦显微镜观察并记录耳蜗外毛细胞形态学变化及带状突触数量,采集并分析小鼠血清MDA、SOD、GSH等氧化还原指标。结果分离培养的hUC-MSCs呈典型长梭形贴壁生长,传代后形态稳定,流式检测符合间充质干细胞鉴定标准。听力学结果显示,小鼠听力随月龄增长呈进行性减退,高频听力最先受损,并逐步累及中、低频;MSCs组全年龄段ABR阈值低于对照组(P<0.01),在32 kHz高频听阈(6月龄)及Click、8、16 kHz中频听阈(10、21月龄)差异有统计学意义(P<0.05)。耳蜗形态学结果显示,MSCs组外毛细胞全年龄段总丢失率均低于对照组(P<0.01),6、10、21月龄分别在底回、顶回、顶中回两组差异有统计学意义(P<0.05);带状突触结果显示,6月龄时MSCs组较对照组在16~22 kHz频段耳蜗区域突触数量分布更多(P<0.05),21月龄时在4、22 kHz中高频区域突触数量更多(P<0.05)。血清氧化应激指标发现,6 月龄两组 MDA、SOD、GSH 水平无统计学差异;10、21 月龄时,hUC-MSCs 组丙二醛含量显著降低,超氧化物歧化酶、还原型谷胱甘肽水平升高(P<0.05)。结论 hUC-MSCs可以减少小鼠内耳外毛细胞及带状突触的丢失,延缓小鼠耳蜗基底膜衰老,hUC-MSCs具有减缓小鼠年龄相关性听力损失的作用。

Abstract: Objective To investigate the mechanism of prevention and treatment of presbycusis, human umbilical cord mesenchymal stem cells(hUC-MSCs)were used to delay the aging of mouse cochlea. Methods Stable human umbilical cord-derived mesenchymal stem cells(hUC-MSCs)were isolated and cultured, followed by breeding C57BL/6J male mice of corresponding age. In the control group, mice received 0.2 mL of normal saline via tail vein injection using a microsyringeevery month starting at 3 months of age. The MSCs group received injections of fourth-generation hUC-MSCs at a concentration of 5×10⁶ cells/mL via tail vein every month starting at 3 months of age. Auditory brainstem response(ABR)threshold tests were performed at 3, 6, 10,17, and 21 months of age, with threshold data recorded. At 6, 10, and 21 months, the Corti's apparatus in the inner ear was dissected, and the basilar membrane was immunofluorescently stained. Morphological changes in cochlear outer hair cells and the number of zonal synapses were observed and documented using laser confocal microscopy. Serum levels of oxidative stress markers such as MDA, SOD, and GSH were measured and analyzed. Results The hUC-MSCs isolated and cultured in this study exhibited typical elongated, spindle-shaped adhesion growth with stable morphology after passage; flow cytometry analysis met the identification criteria for mesenchymal stem cells. Audiological results demonstrated progressive hearing decline in mice with increasing age, with high-frequency hearing impairment occurring earliest and gradually progressing to middle and low-frequency ranges. The ABR hearing thresholds in the MSCs group were significantly lower than those in the control group across all age groups(P<0.01), with significant differences observed at 32 kHz(6 months), as well as at Click frequency and 8/16 kHz(10 and 21 months, respectively)(P<0.05). Cochlear morphological findings revealed significantly lower total loss rates of outer hair cells in the MSCs group compared to the control group across all age groups(P<0.01), with significant differences observed in the basal, apical, and apical-medial turns at 6, 10, and 21 months(P<0.05). Synaptic banding analysis demonstrated that the MSCs group exhibited a higher density of synapses in the cochlear region at 16-22 kHz compared to the control group at 6 months(P<0.05), and increased synaptic density at medium-high frequencies of 4 kHz and 22 kHz at 21 months(P<0.05). Serum oxidative stress markers revealed no significant differences between the two groups at 6 months for MDA, SOD, or GSH levels; however, at 10 and 21 months, the hUC-MSCs group exhibited significantly lower malondialdehyde levels and significantly higher levels of superoxide dismutase and reduced glutathione(P<0.05). Conclusion Human umbilical cord mesenchymal stem cells(hUC-MSCs)can reduce the loss of outer hair cells and ribbon synapses in the inner ear of mice, as well as delay the aging of the basilar membrane in the cochlea. This suggests that hUC-MSCs effectively mitigate age-related hearing loss in mice and exhibit therapeutic potential for presbycusis.

Key words: Mesenchymal stem cells, Presbycusis, Aging, Hearing protection, Cochlear hair cells

中图分类号:

R764.43+6

吴满满,廖康,麦伟园,周浩,范君琪,周志汉,许禄华,姜鸿彦. 人脐带间充质干细胞减缓小鼠年龄相关性听力损失[J]. 山东大学耳鼻喉眼学报, 2026, 40(6): 100-108.

WU Manman, LIAO Kang, MAI Weiyuan, ZHOU Hao, FAN Junqi, ZHOU Zhihan, XU Luhua, JIANG Hongyan. Human umbilical cord mesenchymal stem cellsattenuateage-related hearing loss in mice[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2026, 40(6): 100-108.

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