上皮癌来源细胞外囊泡亚群蛋白质谱分析

doi:10.19438/j.cjoms.2023.04.002

中国口腔颌面外科杂志 ›› 2023, Vol. 21 ›› Issue (4): 326-331.doi: 10.19438/j.cjoms.2023.04.002

上皮癌来源细胞外囊泡亚群蛋白质谱分析

吴若怡¹, 王晓宁², 翟培淞¹, 张建军^1,*, 陈万涛^1,*

1.上海交通大学医学院附属第九人民医院口腔颌面-头颈肿瘤科,2.口腔病理科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海市口腔医学研究所,上海 200011

收稿日期:2023-02-07 修回日期:2023-03-27 出版日期:2023-07-20 发布日期:2023-08-16
通讯作者: 张建军,E-mail: zjjshuobo@163.com;陈万涛,E-mail: chenwantao196323@sjtu.edu.cn。^*共同通信作者
作者简介:吴若怡（1997-）,女,硕士研究生,E-mail: wuruoyimiki@163.com
基金资助:
国家自然科学基金重点项目（82230090）; 上海地方高水平大学创新团队（SHSMU-ZLCX20212301）; 上海市科学技术委员会科研项目（21DZ2292000）

Protein profiling analysis of oral squamous cancer derived extracellular vesicle subgroups

WU Ruo-yi¹, WANG Xiao-ning², ZHAI Pei-song¹, ZHANG Jian-jun¹, CHEN Wan-tao¹

1. Department of Oromaxillofacial Head and Neck Oncology, 2. Department of Oral Pathology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology. Shanghai 200011, China

Received:2023-02-07 Revised:2023-03-27 Online:2023-07-20 Published:2023-08-16

摘要/Abstract

摘要： 目的：揭示通过不同免疫标志物分离获得的细胞外囊泡（extracellular vesicles,EVs）亚群在蛋白质内容物方面的共性和异质性,为后续研究选择合适的分选方案提供数据支持。方法：以口腔癌CAL27细胞为主、胰腺癌细胞PANC-1以及胃癌细胞AGS为辅作为研究对象,分别通过4种EVs分子标志物CD9、CD63、CD81和EpCAM,对不同类型细胞来源的EVs进行免疫富集沉淀,通过蛋白质谱技术对不同EVs亚群进行内容物检测,通过funrich3.1.3软件分析评价EVs亚群之间的蛋白质共性和异质性。采用SPSS 19.0软件包对数据进行统计学分析。结果：在相同培养条件下,AGS细胞产生的EVs数量最多,其次是CAL27细胞,最少的是PANC-1细胞。在4种不同的EVs标志物分选方案中,基于EpCAM标志物的分选方案效能最强。主成分分析结果显示,本研究中涉及的EVs亚群在细胞来源层面以及分选标志物层面均存在异质性。蛋白质谱细胞成分分析结果表明,在口腔癌细胞系CAL27 来源的EVs亚群中,CD63⁺ EVs富集的蛋白中有86.11%位于外泌体生成相关途径,显著高于CD9⁺、CD81⁺和EpCAM⁺ EVs亚群的占比—70.92%、70.90%和63.68%;而CD63⁺ EVs亚群中的富集蛋白有37.96%位于细胞膜,低于CD9⁺、CD81⁺和EpCAM⁺ EVs亚群的占比—53.20%、61.19%和49.53%,表明基于不同分子标志物的分选方案获得的EV亚群可能具有不同的来源。结论：本研究中涉及的EVs亚群在细胞来源层面、免疫标志物层面均存在异质性。其中CD63⁺ EVs可能更多来源于内体途径,而CD9⁺、CD81⁺和EpCAM⁺ EVs更多来源于质膜出芽途径。基于不同分选方案获得的EV亚群,在细胞生物学功能方面具有差异性。

关键词: 细胞外囊泡, 肿瘤, 异质性, 分选标志物, 外颗粒体

Abstract: PURPOSE: To reveal the commonality and heterogeneity in protein contents of extracellular vesicles (EVs) subgroups obtained by different immunomarkers, and provide data support for researchers to select appropriate isolation protocols. METHODS: In this study, oral cancer cells CAL27, pancreatic cancer cells PANC-1 and gastric cancer cells AGS were used, and EVs derived from them were isolated by 4 EVs markers CD9, CD63, CD81 and EpCAM. Then the proteins from these 4 different EVs subgroups were detected by mass spectrometry. The commonality and heterogeneity between EVs subgroups were evaluated by funrich3.1.3 software. SPSS 19.0 software package was used for statistical analysis. RESULTS: Under the same culture conditions, AGS cells produced the highest number of EVs, followed by CAL27 cells and the least by PANC-1 cells. Among the 4 different EVs isolation methods, the EpCAM marker-based isolation method was the most efficacious. The results of principal component analysis showed that the subgroups of EVs involved in this study were heterogeneous at the level of cell origin as well as at the level of isolation markers. The results of the cellular component analysis of the protein profile showed that 86.11% of the proteins enriched by CD63⁺ EVs derived from CAL27 were related to exosomes, which was significantly higher than the percentage of the CD9⁺, CD81⁺ and EpCAM⁺ EVs subgroups(70.92%, 70.90% and 63.68%); while 37.96% of the enriched proteins in the CD63⁺ EVs subgroup were related with cell membrane, which was lower than the percentage of CD9⁺, CD81⁺, and EpCAM⁺ EVs subgroups (53.20%, 61.19% and 49.53%), indicating that EVs subgroups isolated by different immunomarkers had different origins. CONCLUSIONS: The subgroups of EVs involved in this study are heterogeneous at the cellular origin level and at the immunomarker level. Among them, CD63⁺ EVs may originate more from the endosomal pathway, while CD9⁺, CD81⁺ and EpCAM⁺ EVs originate more from the plasma membrane outgrowth pathway. The EVs subgroups obtained based on different isolation markers were differential in terms of cell biological functions.

Key words: Extracellular vesicles, Tumor, Heterogeneity, Isolation markers, Ectosome

中图分类号:

R739.8

吴若怡, 王晓宁, 翟培淞, 张建军, 陈万涛. 上皮癌来源细胞外囊泡亚群蛋白质谱分析[J]. 中国口腔颌面外科杂志, 2023, 21(4): 326-331.

WU Ruo-yi, WANG Xiao-ning, ZHAI Pei-song, ZHANG Jian-jun, CHEN Wan-tao. Protein profiling analysis of oral squamous cancer derived extracellular vesicle subgroups[J]. China J Oral Maxillofac Surg, 2023, 21(4): 326-331.

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上皮癌来源细胞外囊泡亚群蛋白质谱分析

Protein profiling analysis of oral squamous cancer derived extracellular vesicle subgroups

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