Jiang Pei, Ma Xiangyu, Wang Xinlin, Huang Jingyuan, Wang Yintao, Ai Jingru, Xiao Hairong, Dai Mingchen, Lin Yanqin, Shao Buwei, Tang Xujun, Tong Wei, Ye Zixuan, Chai Renjie, Zhang Shasha
State Key Laboratory of Digital Medical Engineering, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, School of Life Sciences and Technology, Advanced Institute for Life and Health, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, Southeast University, Nanjing, 210096, China.
Southeast University Shenzhen Research Institute, Shenzhen, 1518063, China.
Adv Sci (Weinh). 2024 Dec;11(48):e2408964. doi: 10.1002/advs.202408964. Epub 2024 Nov 5.
Small extracellular vesicles (sEVs) act as a critical mediator in intercellular communication. Compared to sEVs derived from in vitro sources, tissue-derived sEVs can reflect the in vivo signals released from specific tissues more accurately. Currently, studies on the role of sEVs in the cochlea have relied on studying sEVs from in vitro sources. This study evaluates three cochlear tissue digestion and cochlear tissue-derived sEV (CDsEV) isolation methods, and first proposes that the optimal approach for isolating CDsEVs using collagenase D and DNase І combined with sucrose density gradient centrifugation. Furthermore, it comprehensively investigates CDsEV contents and cell origins. Small RNA sequencing and proteomics are performed to analyze the miRNAs and proteins of CDsEVs. The miRNAs and proteins of CDsEVs are crucial for maintaining normal auditory function. Among them, FGFR1 in CDsEVs may mediate the survival of cochlear hair cells via sEVs. Finally, the joint analysis of single CDsEV sequencing and single-cell RNA sequencing data is utilized to trace cellular origins of CDsEVs. The results show that different types of cochlear cells secrete different amounts of CDsEVs, with Kölliker's organ cells and supporting cells secrete the most. The findings are expected to enhance the understanding of CDsEVs in the cochlea.
小细胞外囊泡（sEVs）是细胞间通讯的关键介质。与源自体外来源的sEVs相比，组织来源的sEVs能更准确地反映特定组织释放的体内信号。目前，关于sEVs在耳蜗中作用的研究一直依赖于对体外来源sEVs的研究。本研究评估了三种耳蜗组织消化和耳蜗组织来源的sEV（CDsEV）分离方法，并首次提出使用胶原酶D和脱氧核糖核酸酶І结合蔗糖密度梯度离心法分离CDsEVs的最佳方法。此外，还全面研究了CDsEV的内容物和细胞来源。进行了小RNA测序和蛋白质组学分析以分析CDsEVs的微小RNA（miRNAs）和蛋白质。CDsEVs的miRNAs和蛋白质对于维持正常听觉功能至关重要。其中，CDsEVs中的成纤维细胞生长因子受体1（FGFR1）可能通过sEVs介导耳蜗毛细胞的存活。最后，利用单个CDsEV测序和单细胞RNA测序数据的联合分析来追踪CDsEVs的细胞来源。结果表明，不同类型的耳蜗细胞分泌不同量的CDsEVs，其中柯蒂氏器细胞和支持细胞分泌最多。这些发现有望增进对耳蜗中CDsEVs的理解。
