中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (17): 4309-4317.doi: 10.12307/2026.142

• 骨组织构建 bone tissue construction • 上一篇    下一篇

免疫微环境与激素性股骨头坏死囊性变炎症性的修复：单细胞测序分析

林  勇1，杨晓强2，林  锟2，杨  帆1，3，何敏聪1，3，魏秋实1，3   

  1. 1广州中医药大学第三附属医院关节中心，广东省广州市   510378；2广州中医药大学第三临床医学院，广东省广州市   510006；3广东省中医骨伤研究院，广东省广州市   510378 
  • 收稿日期:2025-07-09 接受日期:2025-08-08 出版日期:2026-06-18 发布日期:2025-11-26
  • 通讯作者: 魏秋实，博士，主任医师，博士生导师，广州中医药大学第三附属医院关节中心，广东省广州市 510378；广东省中医骨伤研究院，广东省广州市 510378
  • 作者简介:林勇，男，1977年生，广东省吴川市人，汉族，2002广州中医药大学毕业，主治中医师，从事髋、膝关节病、髋关节发育不良及股骨头坏死的临床研究。
  • 基金资助:
    国家自然科学基金面上项目(82274544)，项目负责人：魏秋实；广东省自然科学基金面上项目(2023A1515010551)，项目负责人：魏秋实

Immune microenvironment and inflammatory repair of cystic degeneration in steroid-induced osteonecrosis of the femoral head: a single-cell sequencing analysis

Lin Yong1, Yang Xiaoqiang2, Lin Kun2, Yang Fan1, 3, He Mincong1, 3, Wei Qiushi1, 3   

  1. 1Joint Center, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510378, Guangdong Province, China; 2The Third Clinical School of Clinical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510006, Guangdong Province, China; 3Guangdong Orthopedic and Traumatology Research Institute of Traditional Chinese Medicine, Guangzhou 510378, Guangdong Province, China
  • Received:2025-07-09 Accepted:2025-08-08 Online:2026-06-18 Published:2025-11-26
  • Contact: Wei Qiushi, PhD, Chief physician, Doctoral supervisor, Joint Center, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510378, Guangdong Province, China; Guangdong Orthopedic and Traumatology Research Institute of Traditional Chinese Medicine, Guangzhou 510378, Guangdong Province, China
  • About author:Lin Yong, Attending physician, Joint Center, The Third Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510378, Guangdong Province, China
  • Supported by:
    the National Natural Science Foundation of China (General Program), No. 82274544 (to WQS); Guangdong Natural Science Foundation (General Program), No. 2023A1515010551 (to WQS)

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摘要：


文题释义：
单细胞测序：一种高通量基因组学技术，能够在单个细胞水平上对DNA、RNA或表观遗传修饰进行测序分析。通过分离单个细胞并扩增其遗传物质，揭示细胞群体的异质性，识别传统“群体测序”无法观测的稀有细胞亚群或基因表达差异。
激素性股骨头坏死囊性变：激素性股骨头坏死进展至中晚期的一种病理改变，表现为坏死区骨小梁吸收、纤维组织增生及囊腔形成，影像学可见股骨头内不规则低密度/信号区。

背景：囊性变是激素性股骨头坏死的一个重要病理特征，存在不同寻常的修复反应。研究发现，囊性变区域免疫细胞的浸润和炎症因子的上调可能与修复过程中的骨组织再生现象密切相关。
目的：基于单细胞测序探讨激素性股骨头坏死囊性变中的免疫微环境，揭示炎症反应对囊性变修复的作用；体外细胞实验验证糖皮质激素对巨噬细胞极化及炎症反应的影响。
方法：①采用单细胞测序技术分析激素性股骨头坏死囊性变区域的免疫微环境，并进行囊性变标本的病理学观察。②建立体外细胞实验模型，体外培养RAW 264.7巨噬细胞分为对照组(RAW细胞正常培养)、脂多糖组(脂多糖干预24 h)，地塞米松+脂多糖组(地塞米松预处理72 h，脂多糖干预24 h)。RT-qPCR检测细胞M1极化标志物(诱导型一氧化氮合酶、肿瘤坏死因子α、白细胞介素6)、M2极化标志物(精氨酸酶1、白细胞介素10)表达，ELISA检测细胞培养上清中炎症因子水平，流式细胞术检测细胞活性氧水平。
结果与结论：①单细胞测序结果显示，囊性变区域存在多种免疫细胞浸润，且炎症因子如肿瘤坏死因子α、白细胞介素6显著上调。②病理标本观察显示，囊性变中存在多种炎症细胞浸润。③体外实验结果表明，地塞米松与脂多糖联合处理可显著诱导RAW 264.7巨噬细胞向促炎M1型极化，抑制抗炎M2表型表达，并促进活性氧的生成，增强局部的炎症反应(白细胞介素6、肿瘤坏死因子α及白细胞介素1β水平均显著升高)。上述结果说明，激素性股骨头坏死囊性变区域的免疫微环境对骨修复过程具有重要影响，免疫细胞的浸润与炎症因子的表达在修复过程中可能起到双重作用；提示过度的免疫激活可能导致修复失败，调控免疫反应，尤其是通过平衡M1型和M2型巨噬细胞的极化方向，可能成为激素性股骨头坏死囊性变修复的重要策略。
https://orcid.org/0009-0003-8002-4298 (林勇) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 激素性股骨头坏死, 囊性变, 修复, 炎症, 单细胞测序

Abstract: BACKGROUND: Cystic degeneration is a critical pathological feature of steroid-induced osteonecrosis of the femoral head and exhibits unusual reparative responses. Studies have shown that immune cell infiltration and upregulation of inflammatory cytokines in cystic areas may be closely related to bone regeneration during repair. Therefore, investigating the role of the immune microenvironment in cystic lesion repair has important clinical significance.
OBJECTIVE: To explore the immune microenvironment in cystic degeneration of steroid-induced osteonecrosis of the femoral head based on single-cell sequencing and to elucidate the role of inflammation in the repair process as well as to validate the effects of glucocorticoids on macrophage polarization and inflammatory responses via in vitro cellular experiments.
METHODS: (1) Single-cell RNA sequencing was used to analyze the immune microenvironment of cystic regions in steroid-induced osteonecrosis of the femoral head. Pathologic observation of cystic degeneration specimens was also performed. (2) In addition, an in vitro cell model was established. In vitro cultured RAW 264.7 macrophages were divided into control group (normal culture of RAW cells), lipopolysaccharide group (lipopolysaccharide intervention for 24 hours), and dexamethasone+lipopolysaccharide group (dexamethasone pretreatment for 72 hours and lipopolysaccharide intervention for 24 hours). RT-qPCR was used to detect the expression of cellular M1 polarization markers (inducible nitric oxide synthase, tumor necrosis factor α, and interleukin 6) and M2 polarization markers (arginase 1 and interleukin 10). ELISA was used to detect the levels of inflammatory factors in the cell culture supernatants. Flow cytometric assay was performed to detect the levels of cellular reactive oxygen species.
RESULTS AND CONCLUSION: (1) Single-cell sequencing results revealed a diverse infiltration of immune cells in the cystic region, with significant upregulation of inflammatory cytokines such as tumor necrosis factor α and interleukin 6. (2) Observation of the pathologic specimen showed the presence of multiple inflammatory cell infiltrates in the cystic degeneration. (3) In vitro experiments demonstrated that combined treatment with dexamethasone and lipopolysaccharide significantly induced M1 polarization of RAW 264.7 macrophages, inhibited the expression of anti-inflammatory M2 phenotype and promoted the generation of reactive oxygen species, thereby enhancing the local inflammatory response (significant upregulation of tumor necrosis factor α, interleukin 6 and interleukin 1β). Overall, the immune microenvironment in the cystic regions of steroid-induced osteonecrosis of the femoral head plays a critical role in bone repair. Immune cell infiltration and inflammatory cytokine expression may exert dual effects during the reparative process. Excessive immune activation may lead to repair failure. Thus, modulating immune responses—especially balancing M1/M2 macrophage polarization—may serve as a potential therapeutic strategy for enhancing cystic repair in steroid-induced osteonecrosis of the femoral head.


Key words: steroid-induced osteonecrosis of the femoral head, cystic degeneration, repair, inflammation, single-cell sequencing

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