血管纹内皮细胞无血清培养方法的建立及其氧化应激诱导的促炎分泌组变化。

IF 2.9 3区 医学 Q1 OTORHINOLARYNGOLOGY
Ying Yi, Xian-Ren Wang, Hui-Ting Chen, Wan-Yi Huang, Li-Xuan Feng, Shu-Bin Fang, Guan-Xia Xiong
{"title":"血管纹内皮细胞无血清培养方法的建立及其氧化应激诱导的促炎分泌组变化。","authors":"Ying Yi,&nbsp;Xian-Ren Wang,&nbsp;Hui-Ting Chen,&nbsp;Wan-Yi Huang,&nbsp;Li-Xuan Feng,&nbsp;Shu-Bin Fang,&nbsp;Guan-Xia Xiong","doi":"10.21053/ceo.2022.01172","DOIUrl":null,"url":null,"abstract":"<p><strong>Objectives: </strong>Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, and no feasible in vitro cell culture model exists for the functional study of SV-ECs.</p><p><strong>Methods: </strong>We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using β-galactosidase staining and flow cytometry. SV-ECs incubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 on their viability. The secretome of SV-ECs treated with or without H2O2 (100 μM or 500 μM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay.</p><p><strong>Results: </strong>We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 μM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 μM H2O2 was much higher than in those treated with 100 μM H2O2. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 μM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages.</p><p><strong>Conclusion: </strong>We successfully established an in vitro serum-free culture method, identified the differential proteins released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.</p>","PeriodicalId":10318,"journal":{"name":"Clinical and Experimental Otorhinolaryngology","volume":null,"pages":null},"PeriodicalIF":2.9000,"publicationDate":"2023-02-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cf/e2/ceo-2022-01172.PMC9985983.pdf","citationCount":"0","resultStr":"{\"title\":\"Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.\",\"authors\":\"Ying Yi,&nbsp;Xian-Ren Wang,&nbsp;Hui-Ting Chen,&nbsp;Wan-Yi Huang,&nbsp;Li-Xuan Feng,&nbsp;Shu-Bin Fang,&nbsp;Guan-Xia Xiong\",\"doi\":\"10.21053/ceo.2022.01172\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Objectives: </strong>Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, and no feasible in vitro cell culture model exists for the functional study of SV-ECs.</p><p><strong>Methods: </strong>We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using β-galactosidase staining and flow cytometry. SV-ECs incubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 on their viability. The secretome of SV-ECs treated with or without H2O2 (100 μM or 500 μM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay.</p><p><strong>Results: </strong>We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 μM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 μM H2O2 was much higher than in those treated with 100 μM H2O2. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 μM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages.</p><p><strong>Conclusion: </strong>We successfully established an in vitro serum-free culture method, identified the differential proteins released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.</p>\",\"PeriodicalId\":10318,\"journal\":{\"name\":\"Clinical and Experimental Otorhinolaryngology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.9000,\"publicationDate\":\"2023-02-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cf/e2/ceo-2022-01172.PMC9985983.pdf\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Clinical and Experimental Otorhinolaryngology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.21053/ceo.2022.01172\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"OTORHINOLARYNGOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Clinical and Experimental Otorhinolaryngology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.21053/ceo.2022.01172","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"OTORHINOLARYNGOLOGY","Score":null,"Total":0}
引用次数: 0

摘要

目的:耳蜗血管纹活性氧可能参与感音神经性听力损失的发病机制。然而,氧化应激对SV内皮细胞(SV- ecs)的影响在很大程度上仍然未知,也没有可行的体外细胞培养模型来研究SV- ecs的功能。方法:从新生小鼠SV中分离原代SV- ecs。采用β-半乳糖苷酶染色和流式细胞术检测纤维连接蛋白对无血清培养基培养的SV-ECs细胞凋亡的抑制作用。在无血清培养基中培养SV-ECs,用不同浓度的H2O2处理SV-ECs,评估H2O2对其活力的影响。采用高分辨率质谱法分析H2O2 (100 μM或500 μM)处理和不处理sv - ec的分泌组。通过巨噬细胞实验评估SV-EC分泌组的功能。结果:成功分离并鉴定了SV-ECs。浓度为500 μM的H2O2处理2小时后,再用无血清培养基在预包被纤维连接蛋白的培养皿中孵育,对细胞凋亡无显著影响。与对照组相比,500 μM H2O2刺激的SV-ECs分泌组中差异蛋白的数量明显高于100 μM H2O2处理的SV-ECs。京都基因与基因组百科和基因本体分析表明,500 μM H2O2处理下SV-ECs中差异表达的蛋白参与多种信号通路和细胞过程的调控。h2o2刺激的SV-ECs分泌组对巨噬细胞表现出显著的促炎作用。结论:我们成功建立了体外无血清培养方法,鉴定了氧化应激诱导的内皮细胞释放的差异蛋白及其功能,揭示了h2o2刺激的sv -内皮细胞分泌组的促炎作用。因此,SV-ECs可能对氧化应激诱导耳蜗微环境中的旁观者细胞,特别是耳蜗巨噬细胞,产生免疫调节作用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

Development of a Serum-Free Culture Method for Endothelial Cells of the Stria Vascularis and Their Pro-Inflammatory Secretome Changes Induced by Oxidative Stress.

Objectives: Reactive oxygen species in the stria vascularis (SV) of the cochlea may be involved in the pathogenesis of sensorineural hearing loss. However, the effects of oxidative stress on SV endothelial cells (SV-ECs) remain largely unknown, and no feasible in vitro cell culture model exists for the functional study of SV-ECs.

Methods: We isolated primary SV-ECs from the SV of neonatal mice. The apoptosis-reducing effects of fibronectin in SV-ECs cultured with serum-free medium were determined using β-galactosidase staining and flow cytometry. SV-ECs incubated in serum-free medium were treated with various H2O2 concentrations to evaluate the effects of H2O2 on their viability. The secretome of SV-ECs treated with or without H2O2 (100 μM or 500 μM) was analyzed using high-resolution mass spectrometry. The function of the SV-EC secretome was evaluated by a macrophage assay.

Results: We successfully isolated and characterized the SV-ECs. Treatment with H2O2 at concentrations up to 500 μM for 2 hours and further incubation with serum-free medium in plates precoated with fibronectin showed no significant effect on apoptosis. Compared to the control SV-ECs, the amount of differential proteins in the secretome of SV-ECs stimulated with 500 μM H2O2 was much higher than in those treated with 100 μM H2O2. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses suggested that the proteins differentially expressed in SV-ECs treated with 500 μM H2O2 were involved in the regulation of multiple signaling pathways and cellular processes. The secretome of H2O2-stimulated SV-ECs exhibited significant pro-inflammatory effects on macrophages.

Conclusion: We successfully established an in vitro serum-free culture method, identified the differential proteins released by oxidative stress-induced ECs and their functions, and revealed the pro-inflammatory effects of the secretome of H2O2-stimulated SV-ECs. Therefore, SV-ECs might elicit immunoregulatory effects on bystander cells in the microenvironment of oxidative stress-induced cochlea, especially cochlear macrophages.

求助全文
通过发布文献求助,成功后即可免费获取论文全文。 去求助
来源期刊
CiteScore
4.90
自引率
6.70%
发文量
49
审稿时长
6-12 weeks
期刊介绍: Clinical and Experimental Otorhinolaryngology (Clin Exp Otorhinolaryngol, CEO) is an international peer-reviewed journal on recent developments in diagnosis and treatment of otorhinolaryngology-head and neck surgery and dedicated to the advancement of patient care in ear, nose, throat, head, and neck disorders. This journal publishes original articles relating to both clinical and basic researches, reviews, and clinical trials, encompassing the whole topics of otorhinolaryngology-head and neck surgery. CEO was first issued in 2008 and this journal is published in English four times (the last day of February, May, August, and November) per year by the Korean Society of Otorhinolaryngology-Head and Neck Surgery. The Journal aims at publishing evidence-based, scientifically written articles from different disciplines of otorhinolaryngology field. The readership contains clinical/basic research into current practice in otorhinolaryngology, audiology, speech pathology, head and neck oncology, plastic and reconstructive surgery. The readers are otolaryngologists, head and neck surgeons and oncologists, audiologists, and speech pathologists.
×
引用
GB/T 7714-2015
复制
MLA
复制
APA
复制
导出至
BibTeX EndNote RefMan NoteFirst NoteExpress
×
提示
您的信息不完整,为了账户安全,请先补充。
现在去补充
×
提示
您因"违规操作"
具体请查看互助需知
我知道了
×
提示
确定
请完成安全验证×
copy
已复制链接
快去分享给好友吧!
我知道了
右上角分享
点击右上角分享
0
联系我们:info@booksci.cn Book学术提供免费学术资源搜索服务,方便国内外学者检索中英文文献。致力于提供最便捷和优质的服务体验。 Copyright © 2023 布克学术 All rights reserved.
京ICP备2023020795号-1
ghs 京公网安备 11010802042870号
Book学术文献互助
Book学术文献互助群
群 号:481959085
Book学术官方微信