基于小鼠白血病模型探究不同定居部位对白血病细胞干性的影响

doi:10.19586/j.2095-2341.2024.0177

生物技术进展 ›› 2025, Vol. 15 ›› Issue (2): 349-354.DOI: 10.19586/j.2095-2341.2024.0177

• 研究论文 • 上一篇

基于小鼠白血病模型探究不同定居部位对白血病细胞干性的影响

王尚尚¹^,²(), 薛贞雅¹^,², 邢海燕¹^,², 杨雪¹^,²^,³, 王敏¹^,², 饶青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
^3.复旦大学附属中山医院，上海 200032

收稿日期:2024-11-12 接受日期:2024-12-27 出版日期:2025-03-25 发布日期:2025-04-29
通讯作者: 饶青
作者简介:王尚尚 E-mail: wangshangshang@ihcams.ac.cn；
基金资助:
国家重点研发计划项目(2022YFA12058031);国家自然科学基金项目(82200176)

Influence of Different Lodging Sites on the Stemness of Leukemia Cells in a Mouse Leukemia Model

Shangshang WANG¹^,²(), Zhenya XUE¹^,², Haiyan XING¹^,², Xue YANG¹^,²^,³, Min WANG¹^,², Qing RAO¹^,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
^3.Department of Hematology，Zhongshan Hospital，Fudan University，Shanghai 200032，China

Received:2024-11-12 Accepted:2024-12-27 Online:2025-03-25 Published:2025-04-29
Contact: Qing RAO

摘要/Abstract

摘要：

自我更新、静息及对化疗药物的抵抗是白血病干细胞的重要特征，也是白血病化疗后复发及预后不良的重要原因之一。骨髓及脾脏是白血病干细胞的定居部位，探索不同微环境下的白血病细胞干性特征对阐明其干性维持机制具有重要意义。利用AML1-ETO9a (AE9a)小鼠白血病模型，探讨骨髓不同区域和脾脏中定居的白血病细胞干性特征；通过转录组测序（RNA sequencing，RNA-seq）及基因富集分析（gene set enrichment analysis, GSEA），阐明干性相关的基因表达特征；通过DAPI和Ki67联合标记及流式细胞仪分析测定G0期细胞比例；通过集落形成实验确定集落形成能力，逆转录实时荧光定量PCR(reverse transcription quantitative real-time PCR, RT-qPCR )验证相关分子表达。研究结果发现，与脾脏相比，定居在骨内膜的白血病细胞具有更显著的干性基因表达特征、更高的G0期比例和更强的集落形成能力。骨内膜白血病细胞高表达的基因在白细胞介素-1（interleukin-1,IL-1）通路显著富集，其中相关分子IL-1β的表达显著升高。结果表明骨内膜定居的白血病细胞具有最强的干性特征，IL-1β可能通过IL-1通路介导骨髓微环境对白血病干细胞干性的维持，IL-1通路有望作为急性髓系白血病（acute myelogenous leukemia, AML）的治疗靶点。

关键词: 白血病干细胞, 骨髓微环境, 静息, 自我更新

Abstract:

Self-renewal， quiescence， and resistance to chemotherapy are critical properties of leukemia stem cells， which contribute to disease relapse and poor prognosis after treatment. Bone marrow and spleen were identified as primary lodging sites for leukemia stem cells. Investigating the stemness properties of leukemia cells in these distinct microenvironments facilitated elucidation of the regulatory mechanisms underlying stemness maintenance. In this study， the AML1-ETO9a （AE9a） mouse leukemia model was used to evaluate the stemness of leukemia cells residing in bone marrow （endosteal region） and spleen. Stemness-related gene expression signatures were identified through RNA sequencing （RNA-seq） and gene set enrichment analysis （GSEA）. The proportion of cells in the G0 phase of the cell cycle was determined via DAPI/Ki67 labeling and flow cytometry， while colony-forming ability was assessed using a colony formation assay. Stemness-related molecules were validated by reverse transcription quantitative real-time PCR （RT-qPCR）. GSEA revealed that upregulated genes in endosteal leukemia cells， compared to spleen-resident leukemia cells， were significantly enriched in stemness-related gene sets， including quiescence. Leukemia cells in the endosteal region exhibited a higher proportion of G0-phase cells and enhanced colony-forming capacity. Genes highly expressed in endosteal leukemia cells showed significant enrichment in the interleukin-1 （IL-1） pathway. Specifically， IL-1β expression was markedly elevated in endosteal leukemia cells. These findings suggest that leukemia cells lodged in the endosteal region retain stemness properties， and the IL-1β-mediated IL-1 pathway may contribute to stemness maintenance within the bone marrow microenvironment. The IL-1 pathway represents a potential therapeutic target for acute myeloid leukemia （AML）.

Key words: leukemia stem cell, bone marrow microenvironment, quiescence, self-renewal

中图分类号:

王尚尚, 薛贞雅, 邢海燕, 杨雪, 王敏, 饶青. 基于小鼠白血病模型探究不同定居部位对白血病细胞干性的影响[J]. 生物技术进展, 2025, 15(2): 349-354.

Shangshang WANG, Zhenya XUE, Haiyan XING, Xue YANG, Min WANG, Qing RAO. Influence of Different Lodging Sites on the Stemness of Leukemia Cells in a Mouse Leukemia Model[J]. Current Biotechnology, 2025, 15(2): 349-354.

图/表 5

表1 实时荧光定量PCR的引物序列

Table 1 Primer sequences for quantitative real-time PCR

扩增基因	引物序列（5'→3'）
GAPDH	F：5'-ACTCCACTCACGGCAAATTCAAC-3'
GAPDH	R：5'-GACACCAGTAGACTCCACGACAT-3'
IL-1β	F：5'-AATGCCACCTTTTGACAGTGATG-3'
IL-1β	R：5'-GGAAGGTCCACGGGAAAGAC-3'

图1 骨内膜、骨髓腔及脾脏白血病细胞的干细胞特征基因富集分析A: EBM vs SP组白血病细胞基因表达的静息和低氧特征富集分析；B: CBM vs SP和EBM vs SP组在不同干细胞特征下的基因差异分析气泡图

Fig. 1 Stemness related gene set enrichment analysis on leukemia cell in EBM, BM and SP

图2 脾脏和骨内膜白血病细胞的G0期细胞比例A: 脾脏和骨内膜中白血病细胞周期流式图; B: 脾脏和骨内膜中白血病细胞的G0期细胞比例柱状统计图

Fig. 2 Proportion of G0 phase cells in leukemia cells located in endosteal and spleen

图3 脾脏和骨内膜中白血病细胞的集落形成数量

Fig. 3 Number of colony-forming units of leukemia cells in endosteal and spleen

图4 脾脏和骨内膜白血病细胞中IL-1通路富集及IL-1β的基因表达A: EBM vs SP组白血病细胞IL-1信号通路的富集分析; B: 脾脏和骨内膜白血病细胞中IL-1β基因的表达水平

Fig. 4 GSEA on IL-1 pathway and IL-1β gene expression of leukemia cells of endosteal and spleen

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基于小鼠白血病模型探究不同定居部位对白血病细胞干性的影响

Influence of Different Lodging Sites on the Stemness of Leukemia Cells in a Mouse Leukemia Model

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