中国组织工程研究

中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (26): 5501-5510.doi: 10.12307/2025.738

• 骨组织构建 bone tissue construction •    下一篇

单细胞测序揭示骨质疏松症患者B细胞异质性及与成骨细胞的相互作用

唐  志1，邵  阳2，李绍烁2，戚树斌3，陆恒杨4，吴  毛2，杨俊锋2，王建伟2   

  1. 1南京中医药大学，江苏省南京市  210023；2无锡市中医医院，江苏省无锡市  214071；3青岛市中医医院，山东省青岛市  266500；4江南大学人工智能与计算机学院，江苏省无锡市  214112


  • 收稿日期:2024-07-24 接受日期:2024-09-14 出版日期:2025-09-18 发布日期:2025-02-20
  • 通讯作者: 王建伟，博士，教授，博士生导师，无锡市中医医院骨伤科，江苏省无锡市 214071
  • 作者简介:唐志，男，2000年生，江苏省宿迁市人，汉族，南京中医药大学在读硕士，主要从事中医骨伤科学方面的研究。
  • 基金资助:
    国家自然科学基金面上项目(82274546)，项目负责人：王建伟；无锡市“双百”中青年医疗卫生拔尖人才项目(HB2023072)，项目负责人：邵阳；无锡市“双百”中青年医疗卫生拔尖人才项目(HB2023074)，项目负责人：李绍烁；江苏省中医药科技发展计划青年人才项目(QN202322)，项目负责人：李绍烁；无锡市卫健委青年科研项目(Q202232)，项目负责人：李绍烁

Single-cell sequencing reveals heterogeneity of B cells in osteoporosis patients and their interactions with osteoblasts

Tang Zhi1, Shao Yang2, Li Shaoshuo2, Qi Shubin3, Lu Hengyang4, Wu Mao2, Yang Junfeng2, Wang Jianwei2   

  1. 1Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China; 2Wuxi Traditional Chinese Medicine Hospital, Wuxi 214071, Jiangsu Province, China; 3Qingdao Traditional Chinese Medicine Hospital, Qingdao 266500, Shandong Province, China; 4School of Artificial Intelligence and Computer Science, Jiangnan University, Wuxi 214112, Jiangsu Province, China
  • Received:2024-07-24 Accepted:2024-09-14 Online:2025-09-18 Published:2025-02-20
  • Contact: Wang Jianwei, MD, Professor, Doctoral supervisor, Wuxi Traditional Chinese Medicine Hospital, Wuxi 214071, Jiangsu Province, China
  • About author:Tang Zhi, Master’s candidate, Nanjing University of Chinese Medicine, Nanjing 210023, Jiangsu Province, China
  • Supported by:
     the National Natural Science Foundation of China, No. 82274546 (to WJW); Wuxi “Double Hundred” Young and Middle-aged Talents in Healthcare, Nos. HB2023072 (to SY) and HB2023074 (to LSS); Jiangsu Provincial Science and Technology Development Program of Traditional Chinese Medicine for Young Talents, No. QN202322 (to LSS); Wuxi Municipal Commission of Healthcare Youth Research Project, No. Q202232 (to LSS)

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摘要：


文题释义：
单细胞转录组测序：是在单个细胞水平上对单个细胞的转录组进行高通量测序，通过遗传信息比对反映细胞间遗传信息的差异，识别不同的细胞亚群，揭示单个细胞的基因结构和基因表达状态，反映细胞间的异质性，被广泛应用于生物和医学的各种领域，是探索疾病发生发展的有效技术。 
骨质疏松症：是一种以骨量低下、骨强度下降和骨折风险增加为特征的进展性全身性骨病。

背景：免疫与骨质疏松症的发病机制有着密切关系，全面、深入地研究两者之间的关系对于理解和治疗骨质疏松症具有重要意义。
目的：利用单细胞测序技术探究免疫细胞在骨质疏松症中的作用。
方法：从GEO数据库下载骨质疏松症和非骨质疏松症患者股骨头组织单细胞测序分析数据，利用R 4.3.0软件及Seurat v.4.3、monocle (2.28.0)、CellChat等软件包对测序数据进行细胞聚类分析、功能富集分析、拟时序分析、细胞互作等分析。获取行人工髋关节置换手术的股骨颈骨折患者股骨头组织，包括骨质疏松症患者2例、非骨质疏松症患者2例，采用免疫组化染色检测CCL13、CCL18蛋白表达，qPCR检测免疫球蛋白重常数γ-4、免疫球蛋白λ常数3、人Ⅱ类主要组织相容性复合体DRβ1、CD83 mRNA表达，Western Blot检测受体型酪氨酸蛋白磷酸酶C、CD22、CD99蛋白表达。
结果与结论：①转录组数据分析共识别了10个不同的细胞类型，包括破骨细胞、髓样细胞、T细胞、成骨细胞、巨噬细胞、单核细胞、红细胞、B细胞、骨髓间充质干细胞、肥大细胞，骨质疏松症组股骨头组织中破骨细胞与T细胞比例增加，成骨细胞与B细胞比例减少。在B细胞亚群中，骨质疏松症组股骨头组织中第1，3类群B细胞(BC1、BC3)比例高于非骨质疏松症组，第2类群B细胞(BC2)比例少于非骨质疏松症组。BC1富集显著的标签有适应性免疫反应调节、免疫受体的体细胞重组、调节淋巴细胞介导的免疫等，BC3富集显著的标签有免疫球蛋白产生的调节、对Ⅱ型干扰素的应答、半胱氨酸内肽酶参与的凋亡过程、细胞毒性等。骨质疏松症组股骨头组织中B细胞亚型BC1与成骨细胞间的通讯强度高于非骨质疏松症组，同时BC3与BC1之间的通讯强度也有所提高；BC3与BC1之间的通讯在CD22-受体型酪氨酸蛋白磷酸酶C通路富集显著，BC1与成骨细胞之间的通讯主要富集在CD99-CD99通路，并且BC3与成骨细胞之间的通讯在CD99-CD99通路中也有较高富集。②骨质疏松症组股骨头组织中CCL13、CCL18、受体型酪氨酸蛋白磷酸酶C、CD22、CD99蛋白表达及免疫球蛋白重常数γ-4、免疫球蛋白λ常数3、人Ⅱ类主要组织相容性复合体DRβ1、CD83 mRNA表达均高于非骨质疏松症组(P < 0.05)。③结果表明，骨质疏松症股骨头组织中B细胞特定亚群能够对成骨细胞的分化和凋亡产生影响。
https://orcid.org/0009-0004-2871-5603(唐志)

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

关键词: 单细胞测序, 骨质疏松症, B细胞, 成骨细胞, 细胞互作, 工程化组织构建

Abstract: BACKGROUND: The pathogenesis of osteoporosis is closely related to the immune system. A comprehensive and in-depth study of the relationship between immunity and osteoporosis is crucial for understanding and treating the disease. 
OBJECTIVE: To investigate the role of immune cells in osteoporosis using single-cell sequencing technology.
METHODS: Femoral head tissue samples from osteoporosis and non-osteoporosis patients were downloaded from GEO database and analyzed using single-cell sequencing. Data analysis, including cell clustering, functional enrichment, pseudotime trajectory, and cell interaction analyses, was performed using R4.3.0 and software packages such as Seurat v.4.3, monocle (2.28.0), and CellChat. The femoral head tissues of patients with femoral neck fracture who underwent artificial hip replacement surgery were obtained, including two cases of osteoporosis patients and two cases of non-osteoporosis patients. Immunohistochemical staining was used to detect the protein expression of CCL13 and CCL18. qPCR was used to detect the immunoglobulin heavy constant γ-4, immunoglobulin λ constant 3, human class II major histocompatibility complex DRβ1, and CD83 mRNA expression. Western blot was used to detect the protein expression of receptor-type tyrosine protein phosphatase C, CD22, and CD99.
RESULTS AND CONCLUSION: Transcriptomic analysis identified 10 cell clusters, including osteoclasts, myeloid cells, T cells, osteoblasts, macrophages, monocytes, erythrocytes, B cells, bone marrow mesenchymal stem cells, and mast cells. There was an increase in the ratio of osteoclasts to T cells and a decrease in the ratio of osteoblasts to B cells in the femoral head tissue of the osteoporosis group. Among the B-cell subpopulations, the proportion of B-cells of taxa 1,3 (BC1, BC3) in the femoral head tissue of the osteoporosis group was higher than that of the non-osteoporosis group, and the proportion of B-cells of taxa 2 (BC2) was less than that of the non-osteoporosis group. BC1 was enriched significantly for labels such as regulation of adaptive immune response, somatic recombination of immune receptors, and modulation of lymphocyte-mediated immunity, while BC3 was enriched significantly for labels such as regulation of immunoglobulin production, response to type II interferon, apoptotic processes involving cysteine endopeptidases, and cytotoxicity. The communication intensity between B-cell subtype BC1 and osteoblasts in the femoral head tissue of the osteoporosis group was higher than that of the non-osteoporosis group, while the communication intensity between BC3 and BC1 was also increased. The communication between BC3 and BC1 was significantly enriched in the CD22-receptor-type tyrosine protein phosphatase C pathway; the communication between BC1 and osteoblasts was mainly enriched in the CD99-CD99 pathway; and the communication between BC3 and osteoblasts was also highly enriched in the CD99-CD99 pathway. Protein expression of CCL13, CCL18, receptor-type tyrosine protein phosphatase C, CD22, CD99, immunoglobulin heavy constant γ-4, immunoglobulin λ constant 3, human class II major histocompatibility complex DRβ1, and CD83 mRNA were higher in femoral tissues of the osteoporosis group than those of the non-osteoporosis group (P < 0.05). To conclude, specific B cell subpopulations can influence the differentiation and apoptosis of osteoblasts in the femoral tissue of osteoporosis patients.

Key words: single-cell sequencing, osteoporosis, B cells, osteoblasts, cellular interactions, engineered tissue construction

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