| 郝峻,郝彦哲,王丽,等.ACE2在细胞膜表面分布及其与 S蛋白片段聚合状态的原位单分子超显微观察[J].安徽医药,2026,30(4):722-727. |
| ACE2在细胞膜表面分布及其与 S蛋白片段聚合状态的原位单分子超显微观察 |
| In-situ single molecule ultra-microscopic observation of ACE2 distribution and its clusterization with S protein on cell surface |
| |
| DOI：10.3969/j.issn.1009-6469.2026.04.017 |
| 中文关键词: 新型冠状病毒感染 血管紧张素转化酶 2(ACE2) 刺突蛋白(S蛋白) 环境扫描电子显微镜 共定位 |
| 英文关键词: Coronavirus infection disease Angiotensin-converting enzyme 2 (ACE2) Spike protein (S protein) Environmental scanning electron microscopy Colocalization |
| 基金项目:北京市教委基金科技一般项目( KM201910005004) |
| 作者 | 单位 | E-mail | | 郝峻 | 北京工业大学, 化学与生命科学学院, | | | 郝彦哲 | 中国疾病预防控制中心病毒病预防控制所,北京 100052 | | | 王丽 | 固体微结构与性能研究所,北京 100124 | | | 李长硕 | 固体微结构与性能研究所,北京 100124 | | | 韩晓东 | 固体微结构与性能研究所,北京 100124 | | | 盛望 | 北京工业大学, 化学与生命科学学院, | | | 王明连 | 北京工业大学, 化学与生命科学学院, | | | 李劲涛 | 北京工业大学, 化学与生命科学学院, | ljt2008@bjut.edu.cn |
|
| 摘要点击次数: 152 |
| 全文下载次数: 104 |
| 中文摘要: |
| 目的利用单分子可视化超高分辨分析平台观察分析细胞膜表面血管紧张素转化酶 2(ACE2)在不同组织来源细胞上的单分子定位和分布特点，验证并明确细胞膜表面 ACE2与严重急性呼吸综合征冠状病毒 2型( SARS-CoV-2)病毒 S1蛋白在单分子水平聚合状态，解读 ACE2与外源的新型冠状病毒刺突蛋白( S蛋白)片段的分子聚合特点。方法研究起止时间为 2022年 1月至 2023年 12月。人胚胎肾细胞 HEK293、非小细胞肺癌细胞 A549、食管鳞癌细胞 EC9706、人宫颈癌肠转移细胞 CaSki，以及慢病毒包装所需的慢病毒穿梭质粒 plvx-puro、慢病毒骨架质粒 PsPAX2和 PMD.2G均来源自中国疾病预防控制中心病毒病预防控制所。在氧化铟锡( ITO)导电玻璃上培养 ACE2过表达的 HEK293细胞，上述不同组织来源的癌细胞，以及用特定外源 S1片段处理过的 ACE2过表达的 HEK293细胞，经多聚甲醛固定后分别标记特定的纳米金颗粒，并利用扫描电子显微镜对细胞膜表面 ACE2的分布和聚合状态以及 ACE2与外源 S1蛋白片段的结合情况进行纳米尺度下的超高分辨单分子成像。结果 ACE2在其过表达的 HEK293细胞表面主要以单体存在，可形成极少数的二聚体和多聚体(由 3个以上单体构成)。在选取的 5张同一 HEK293细胞表面纳米金颗粒标记的 ACE2分子聚合状态照片视野中，总分子数为( 502.00±80.84)个，单体分子数为( 383.80±71.33)个，二聚体分子数为( 82.80±10.16)个，多聚体分子数为( 35.40±11.26)个。 ACE2在不同组织来源的细胞中表达量存在明显差异，肺源细胞中表达量明显高于其他来源细胞，且 ACE2表达量极低的情况下依旧可形成单分子二聚体。外源 S1蛋白片段可与 ACE2在细胞膜表面以不同比例结合，且 S1片段可能存在除 ACE2以外其他膜受体。结论通过环境扫描电子显微镜单分子可视化超高分辨分析平台初步揭示了 ACE2在不同细胞膜表面的表达状况，聚合状态以及与外源 S1蛋白的结合情况，为抑制 SARS-CoV-2病毒感染细胞的相关策略的研究提供了理论基础。 |
| 英文摘要: |
| Objective To observe and analyze the single-molecule location and distribution characteristics of angiotensin-converting enzyme 2 (ACE2) on the surface of cell membranes in cells from different tissues by applying single-molecule visualization and ultra-high-resolution analysis platform, to verify and confirm the clusterization states of ACE2 on the surface of cell membranes with severeacute respiratory syndrome coronavirus 2 (SARS-CoV-2) S1 protein at the single-molecule level, and to interpret the molecular cluster-ization characteristics of ACE2 with exogenous SARS-CoV-2 virus spike protein (S protein) fragments. Methods The research spanned from January 2022 to December 2023. Human embryonic kidney cells HEK293, non-small cell lung cancer cells A549,esophageal squamous carcinoma cells EC9706, human cervical cancer cells CaSki, as well as the lentiviral shuttle plasmid plvx-puro,lentiviral backbone plasmids PsPAX2 and PMD.2G were provided by National Institute for Viral Disease Control and Prevention, Chi-nese Center for Disease Control and Prevention. ACE2-overexpressed HEK293 cells with or without exogenous S1 protein fragmenttreatment and cancer cells from different human tissues were cultured on indium tin oxide (ITO) coated glass, then fixed with parafor-maldehyde and specifically labeled with gold nanoparticles. The distribution and polymerization state of ACE2 on the surface of cellmembrane and the binding of ACE2 with exogenous S1 protein fragment were studied by scanning electron microscopy. Super-resolu-tion single-molecule imaging was performed at the nanoscale.Results ACE2 mainly exists as monomers, with very few dimers and high-order clusters (consisting of 3 or more monomers) on the surface of ACE2 over-expressed HEK293 cells. In five randomly select-ed photos of ACE2 aggregation states on the surface of single HEK293 cell, the total count of labled ACE2 was 502.00±80.84, withACE2 monomer count of 383.80±71.33, ACE2 dimer count of 82.80±10.16, and ACE2 high-order cluster count of 35.40±11.26. There was significant difference in ACE2 expression in cell lines from different tissues. The expression level in lung source cell line was sig-nificantly higher than that in other cell lines. ACE2 could still form homodimers in ACE2 low expression circumstances. S1 proteinfragments could bind to ACE2 on cell surface in different proportions, and S1 fragment might have other membrane receptors besidesACE2.Conclusion In this study, single-molecule visualization with ultra-high-resolution analysis platform based on environmentalscanning electron microscopy was used to preliminarily reveal ACE2 expression pattern, clusterization status, and its binding to exoge-nous S1 protein fragment on different cell membrane surfaces, which provided a theoretical basis for further studies of relevant virusinfection-inhibiting strategies. |
|
查看全文
查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
