一种高效小鼠骨髓红系造血岛富集方法的建立

doi:10.19586/j.2095-2341.2023.0156

生物技术进展 ›› 2024, Vol. 14 ›› Issue (3): 466-472.DOI: 10.19586/j.2095-2341.2023.0156

• 研究论文 • 上一篇

一种高效小鼠骨髓红系造血岛富集方法的建立

郑凌岳¹(), 王竟苇¹, 佟静媛¹(), 石莉红¹^,²()

^1.中国医学科学院血液病医院（中国医学科学院血液学研究所）北京协和医学院，血液与健康全国重点实验室，国家血液系统疾病临床医学研究中心，天津市血液病细胞治疗研究重点实验室，细胞生态海河实验室，天津 300020
^2.天津医学健康研究院，天津 301600

收稿日期:2023-12-07 接受日期:2024-02-05 出版日期:2024-05-25 发布日期:2024-06-18
通讯作者: 佟静媛,石莉红
作者简介:郑凌岳 E-mail: zhenglingyue@ihcams.ac.cn
基金资助:
国家自然科学基金项目(82225003);医科院医学与健康科技创新工程项目(2021-I2M-1-073);津门医学英才项目(TJSJMYXYC-D2-022);重点实验室课题项目(Z23-10)

The Establishment of A Novel Effective Method for the Enrichment of Murine Bone Marrow Erythroblastic Islands

Lingyue ZHENG¹(), Jingwei WANG¹, Jingyuan TONG¹(), Lihong SHI¹^,²()

^1.State Key Laboratory of Experimental Hematology，National Clinical Research Center for Blood Diseases，Tianjin Key Laboratory of Cell Therapy for Blood Diseases，Haihe Laboratory of Cell Ecosystem，Institute of Hematology & Blood Diseases Hospital，Chinese Academy of Medical Sciences & Peking Union Medical College，Tianjin 300020，China
^2.Tianjin Institutes of Health Science，Tianjin 301600，China

Received:2023-12-07 Accepted:2024-02-05 Online:2024-05-25 Published:2024-06-18
Contact: Jingyuan TONG,Lihong SHI

摘要/Abstract

摘要：

红系造血岛(erythroblastic island, EBI)是哺乳动物红细胞终末分化的独特微环境， EBI研究对于深入理解生理和病理条件下造血微环境的组成和变化至关重要。骨髓中EBI分布较为分散，现有富集方法的富集效果不尽如人意。研究旨在建立一种新的更高效的小鼠骨髓EBI富集方法。在前人研究的基础上进行了创新和优化，建立了以二次密度梯度沉降结合洗涤去除非EBI细胞团为核心的富集方法。通过细胞团形态观察、瑞氏吉姆萨染色、成像流式技术（imaging flow cytometry, IFC）等手段比较了该方法与现有富集方法的富集效果，在直接镜检和瑞氏吉姆萨染色中发现,方法4富集体系中少见单细胞，主要为以巨噬细胞为核心周围围绕红细胞的典型EBI结构。使用IFC技术对于F4/80、CD71以及CD11b进行分析，富集产物中约63.7％的细胞团为EBI，高出之前方法的最高标准（方法3），约38.4％。富集的EBI中发现巨噬细胞为CD11b^-,且除红细胞外，部分EBI中存在CD11b⁺细胞，与之前的研究相符。结果显示，该方法在没有造成EBI结构明显破坏的情况下，富集效果在去除单细胞以及非EBI细胞团等方面显著优于现有其他方法，适合对EBI纯度要求较高的研究，为造血微环境研究提供了有效的方法和手段。

关键词: 红系造血岛, 红细胞, 富集方法, 成像流式

Abstract:

Erythroblastic island （EBI） is the distinct microenvironment for mammalian erythroid terminal differentiation. Investigating EBI is essential for gaining a deep understanding of the cellular composition and phenotypic changes of hematopoietic microenvironment under both physiologic and pathologic conditions. The distribution of EBI in bone marrow is relatively scattered. Nowadays， existing enrichment methods exhibit unsatisfactory efficiency. Our research aimed to establish a novel and more efficient EBI enrichment method. Drawing on previous research， we introduced innovations and optimizations， developing a novel method centered on dual density gradient sedimentation combined with washing steps to remove non-EBI cell clusters. We compared the efficiency of our method with other existing methods using observing cell cluster structure and Wright-Giemsa staining and imaging flow cytometry （IFC）. Results showed that few single cells were found in the enriched product of Method 4 through direct microscopy and Wright-Giemsa staining， most of the products were typical EBI structures including a central macrophage and surrounding red blood cells. Using IFC technology to analysis F4/80， CD71 and CD11b in enriching products， about 63.7％ of the cell clusters enriched by our method were EBIs. This percentage was higher than the highest proportion obtained in previously reported Method 3， about 38.4％. As previously reported， EBI macrophages were CD11b^-. In some of the EBIs， there were CD11b⁺ cells other than erythrocytes and macrophages. These results showed that a significantly enhanced efficiency in our method， particularly in the removal of removing single cells and non-EBI clusters without causing significant destruction of EBI structures. Our modified method is suitable for research with high EBI purity requirements. Our research provided a more efficient method for researching hematopoietic microenvironment study.

Key words: erythroblastic island, red blood cell, enrichment method, imaging flow cytometry

中图分类号:

Q462

郑凌岳, 王竟苇, 佟静媛, 石莉红. 一种高效小鼠骨髓红系造血岛富集方法的建立[J]. 生物技术进展, 2024, 14(3): 466-472.

Lingyue ZHENG, Jingwei WANG, Jingyuan TONG, Lihong SHI. The Establishment of A Novel Effective Method for the Enrichment of Murine Bone Marrow Erythroblastic Islands[J]. Current Biotechnology, 2024, 14(3): 466-472.

图/表 5

图1 富集小鼠骨髓EBI方法的流程示意图A：方法1；B：方法2；C：方法3；D：方法4。

Fig. 1 Schematic diagram of methods for the enrichment of EBI from mouse bone marrow

图2 显微镜下观察富集产物A：方法3获得的富集产物；B：方法4获得的富集产物。标尺为50 μm。

Fig. 2 Microscopic analysis of enriched products

图3 光学显微镜观察瑞氏-吉姆萨染色的富集产物A：方法1；B：方法2；C：方法3；D：方法4。标尺为50 μm。

Fig. 3 Light microscopic analysis of Wright-Giemsa stained enriched products

图4 IFC分析小鼠骨髓EBI富集产物圈门策略A：方法1；B：方法2；C：方法3；D：方法4；E：R3门事件数占R1门的百分比（3次随机抽样，n=3）；F：方法3中F4/80+事件中CD71荧光强度分布图； G：方法4中F4/80+事件中CD71荧光强度分布图。**、***分别表示差异在P<0.01、P<0.001水平上有统计学意义。

Fig. 4 Gating strategy for analyzing enriched murine BM EBIs using IFC

图5 目检分析R3中EBI的形态和数量A：方法1； B：方法2；C：方法3；D：方法4；E：IFC后从R3中随机抽取300个细胞团。●：EBI细胞团；○：非EBI细胞团。计数其中EBI数量（3次随机抽样，n=3）。**、***分别表示差异在P<0.01、P<0.001水平上有统计学意义。

Fig. 5 Type and quantity in visual inspection of EBIs in R3

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[1]	吴静, 张英楠, 徐长禄, 刘金花, 石莉红. 碳酸酐酶在红细胞分化发育过程中的功能研究[J]. 生物技术进展, 2022, 12(4): 584-590.
[2]	徐长禄,赵艳红,马艺戈,王冰蕊,王鼎,郭青,佟静媛,高洁,李亚朴,刘金花,石莉红. 利用生物信息学方法解析自噬相关基因在人体红细胞终末分化各阶段的动态表达[J]. 生物技术进展, 2019, 9(3): 271-276.

一种高效小鼠骨髓红系造血岛富集方法的建立

The Establishment of A Novel Effective Method for the Enrichment of Murine Bone Marrow Erythroblastic Islands

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