小鼠MRSA慢性假体周围感染诱导成熟三级淋巴结构形成及骨溶解

doi:10.12122/j.issn.1673-4254.2026.07.09

摘要/Abstract

摘要：

目的探讨耐甲氧西林金黄色葡萄球菌（MRSA）诱导的小鼠膝关节假体周围感染（PJI）模型中三级淋巴结构（TLS）的结构与功能特征及其与局部骨溶解的关系。方法构建MRSA诱导的小鼠膝关节PJI模型，将C57BL/6J小鼠随机分为假体组（PBS）和PJI组（1×10⁴CFU MRSA USA300），观察60 d。检测外周血白细胞计数（WBC）、红细胞沉降率（ESR）及C反应蛋白（CRP）等炎症指标；HE染色评估假体周围组织TLS形成及成熟程度；多重免疫荧光分析T/B细胞分区（CD3/B220）、滤泡树突状细胞（CD21）及TLS功能活性（Ki67、PNAd）；TRAP染色进行骨形态计量学分析，评估破骨细胞数量（N.Oc/BS）。结果术后21d，PJI组外周血炎症指标显著高于假体组（P<0.0001）。组织学显示，PJI组假体周围形成大量成熟TLS，截面积达17212.10±7624.28 μm²，显著高于假体组（P<0.01）。多重免疫荧光证实PJI组呈现“T细胞环绕B细胞”的成熟TLS典型空间结构，并伴有FDC网络；TLS内存在大量Ki67阳性增殖细胞，边缘及实质内可见PNAd阳性高内皮微静脉。TRAP分析显示，PJI组单位骨表面破骨细胞数量为（28.75±4.13）/mm，显著高于假体组的（1.63±0.74）/mm（P<0.0001）。结论 MRSA慢性感染可在小鼠假体周围诱导形成具备典型结构分区及功能活性的成熟TLS，并伴随显著骨溶解。

关键词: 耐甲氧西林金黄色葡萄球菌, 假体周围感染, 三级淋巴结构, 小鼠模型, 免疫微环境

Abstract:

Objective To investigate the histomorphological and functional characteristics of tertiary lymphoid structures (TLS) in a mouse model of chronic knee periprosthetic joint infection (PJI) with methicillin-resistant Staphylococcus aureus (MRSA) and explore their association with local osteolysis. Methods C57BL/6J mice receiving prosthesis implantation in the knee joint were randomized into control group and PJI group for injection of sterile PBS and of MRSA USA300 (1×10⁴ CFU) into the knee joint cavity, respectively. Peripheral blood leukocyte counts, erythrocyte sedimentation rates, and C-reactive protein levels were monitored regularly. Sixty days after the injections, periprosthetic tissues were harvested for HE staining to measure the cross-sectional area of mature TLS. Multiplex immunofluorescence staining was used to analyze spatial segregation of T and B cells (CD3/B220) and the network of follicular dendritic cells (FDCs) and detect Ki67 and PNAd expressions for assessing TLS functionality. The number of osteoclast per unit of bone surface (N.Oc/BS) in the periprosthetic region was determined using histomorphometric analysis with TRAP staining. Results At 21 days post-surgery, the mice in PJI group showed significant elevation of all the peripheral blood inflammatory markers. Numerous mature TLS were observed in the periprosthetic tissues in PJI group with an average cross-sectional area significantly greater than that in the control group. The TLS in PJI group exhibited a typical spatial organization, characterized by a B-cell-centered core surrounded by T cells with an FDC network; abundant Ki67-positive proliferating cells were observed within the TLS, and PNAd-positive high endothelial venules were visible at the periphery and within the parenchyma. The N.Oc/BS was significantly greater in PJI group than in the control group (28.75±4.13 vs 1.63±0.74 mm^-1, P<0.0001). Conclusion Chronic MRSA infection induces formation of mature TLS with well-defined structural organization and functional activity in the periprosthetic region of mice, accompanied by significant bone loss.

Key words: methicillin-resistant Staphylococcus aureus, periprosthetic infection, tertiary lymphoid structures, mouse model, immune microenvironment

木尼拉·麦合木提, 黄利思, 李家楷, 蔡如龙, 冯棋, 林于迪, 黄泽泓, 伍思佳, Umar Muhammad, 方航. 小鼠MRSA慢性假体周围感染诱导成熟三级淋巴结构形成及骨溶解[J]. 南方医科大学学报, 2026, 46(7): 1553-1562.

Maihemuti Munila, Lisi HUANG, Jiakai LI, Rulong CAI, Qi FENG, Yudi LIN, Zehong HUANG, Sijia WU, Muhammad Umar, Hang FANG. Chronic MRSA periprosthetic joint infection induces formation of mature tertiary lymphoid structures and osteolysis in mice[J]. Journal of Southern Medical University, 2026, 46(7): 1553-1562.

图/表 9

图1 小鼠膝关节微型假体的设计与制备

Fig.1 Design and manufacturing of the murine knee prosthesis. A: The micro-prosthesis model designed via CAD software, tailored to the distal femoral anatomy of C57BL/6J mice. B: Macroscopic view of the final Ti6Al4V implant produced by selective laser melting.

表1 Ti6Al4V粉末的化学成分

Tab.1 Chemical composition of Ti6Al4V powder (wt%)

Powder Ti Al V O N C H Fe Zn P Cu Ag
Ti6Al4V powder	- 5.5-6.5 3.5-4.5 <0.13 <0.03 <0.08 <0.012 <0.25 - - - -

图2 小鼠膝关节PJI模型构建流程示意图

Fig.2 Schematic diagram of construction workflow for establishing mouse models of chronic knee periprosthetic joint infection (PJI) with methicillin-resistant Staphylococcus aureus (MRSA). Left: Experimental grouping and modeling procedure. Right: Schematic illustration of the periprosthetic microenvironment in the presence of infection (indicated by the red box).

图3 小鼠膝关节PJI模型建立及术后评价

Fig.3 Establishment and postoperative evaluation of the mouse knee PJI model. A: Surgical procedures of the PJI model. B: X-ray images of mouse knee joints at 28 days post-surgery. C: Quantitative analysis of peripheral blood WBC, ESR, and CRP levels. Data are presented as Mean±SD, n=6. *P<0.05, ****P<0.0001.

图4 小鼠膝关节假体周围软组织HE染色及TLS定量分析

Fig.4 HE staining and quantitative analysis of tertiary lymphoid structures (TLS) in periprosthetic soft tissues of mouse knee joints (n=6). A-C: HE staining showing massive dense inflammatory cell infiltration and formation of typical lymphoid follicle-like aggregate structures in PJI group. D-F: HE staining showing only sparse inflammatory cells in the prosthesis (control) group. Scale bar=100 μm. Data are presented as Mean±SD. **P<0.01.

图5 小鼠假体周围组织免疫细胞HE染色及mIF染色

Fig.5 HE staining and multiplex immunofluorescence (mIF) staining of periprosthetic tissues. A-D: HE staining and merged mIF images in PJI group (CD21, green; CD45, red; CD3, yellow). E, F: HE staining and merged mIF image in prosthesis group (Scale bar=50 μm).

图6 小鼠假体周围组织中T细胞、B细胞及FDC的组织学特征与空间分布

Fig.6 Histological features and spatial organization of T cells, B cells, and follicular dendritic cells (FDCs) in periprosthetic tissues of the mice, using spleen tissue as the positive control. A, C, E: Representative HE staining images of the PJI group (A), prosthesis group (C), and the spleen (E). B, D, F: Corresponding merged mIF images showing immune cell distribution in PJI group (B), prosthesis group (D), and the spleen (F). The markers are colored as follows: CD3 (red), B220 (yellow), CD21 (green), and nuclei are counterstained with DAPI (blue) (Scale bar=50 μm).

图7 小鼠假体周围组织中Ki67与PNAd的表达特征

Fig.7 Expression of Ki67 and PNAd in periprosthetic tissues of the mice (Scale bar=50 μm). A: Representative mIF images of Ki67 and B220 in PJI group (top two rows) and prosthesis group (bottom row). B: Representative mIF images of PNAd and B220 in PJI group (top two rows) and prosthesis group (bottom row). The markers are colored as follows: Ki67 or PNAd (green), B220 (red), and nuclei are counterstained with DAPI (blue).

图8 小鼠假体周围骨组织TRAP染色及破骨细胞定量分析

Fig.8 TRAP staining and quantitative analysis of osteoclasts in periprosthetic bone tissues of the mice in PJI and prosthesis groups (Scale bar=100 μm). Data are presented as Mean±SD (n=6). ****P<0.0001.

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