研究背景

滑膜炎、痤疮、脓疱病、骨质增生和骨炎综合征（Synovitis, Acne, Pustulosis, Hyperosteogeny and Osteoarthritis Syndrome，SAPHO综合征）是一种罕见的慢性炎症性疾病，具有常累及前胸壁和中轴骨的骨炎、滑膜炎、骨质疏松、骨质增生等骨关节表现，和掌跖脓疱病、痤疮等皮肤表现。骨关节表现是SAPHO综合征的特征，其造成的疼痛，活动受限及病程后期导致的关节僵硬，变型甚至塌陷严重影响患者的生活质量。SAPHO综合征病程迁延不愈，活动期与稳定期交替，发病机制尚不清楚。

SAPHO综合征的慢性炎症表现和骨关节症状，提示免疫异常和骨代谢失衡在SAPHO综合征的发生发展中扮演重要角色。近年来，随着对细胞外囊泡（Extracellular Vesicles，EVs）的研究越来越深入，其在调节免疫和骨代谢中的作用逐步展现。研究表明细胞外囊泡在生理和疾病状态下参与骨代谢的调节，同时也参与炎症过程，细胞外囊泡内的许多炎症相关细胞因子同时也是调节骨代谢的重要调节因子。包含各种蛋白成分又肩负细胞通讯功能的细胞外囊泡，可能作为免疫和骨代谢的“桥梁”在SAPHO综合征中发挥作用。而目前针对SAPHO综合征的血清来源细胞外囊泡（Serum-derived Extracellular Vesicles，SEVs）的研究尚属空白。

研究方法

本研究通过超速离心法分离SEVs，经电镜检测证明SEVs的成功分离后，采用定量蛋白质组学方法对SAPHO患者和健康志愿者的SEVs进行分析，明确SAPHO患者SEVs的蛋白表达谱和差异表达蛋白。通过生物信息学分析发现参与骨代谢的SAPHO患者SEVs差异表达蛋白和潜在的SAPHO疾病标志物。

利用酶联免疫吸附实验（Enzyme Linked Immunosorbent Assay，ELISA）检测患者和健康志愿者的血清和试剂法提取SEVs中潜在标志物的表达量，验证其辅助SAPHO诊断的能力。进一步用ELISA比较SAPHO患者，风湿性关节炎（Rheumatic Arthritis，RA）患者和强直性脊柱炎（Ankylosing Spondylitis，AS）患者的SEVs中潜在标志物含量，明确其能否用于鉴别诊断SAPHO综合征，RA和AS。

为了验证SAPHO患者SEVs对骨代谢的调节作用，通过试剂法提取SEVs，经免疫印迹实验（Western Blot，WB）和纳米颗粒跟踪分析（Nanoparticle Tracking Analysis，NTA）证明SEVs的成功分离后，分别在人源和鼠源细胞进行SEVs刺激下的体外破骨细胞（Osteoclasts，OCs）生成和骨生成实验。对不同SEVs刺激下发生表型变化的细胞，进行定量蛋白质谱，结合生物信息学分析，寻找差异表达蛋白，即可能是形成表型差异的原因。

研究结果

结果表明SAPHO患者SEVs与健康人群SEVs蛋白表达谱有明显差异，其差异表达蛋白中有17个（35%）参与炎症和骨代谢。通过对54例SAPHO患者和84例健康志愿者的血清和试剂法提取SEVs进行ELISA检测，发现差异表达蛋白中血清淀粉样蛋白A-1（Serum Amyloid A-1，SAA1），在SAPHO患者血清和SEVs中高表达，具有辅助SAPHO综合征诊断的潜力。但54例SAPHO患者，22例RA患者和29例AS患者SEVs中SAA1表达量无显著差异。

骨生成实验表明，SAPHO患者SEVs对小鼠和健康志愿者来源细胞的成骨过程和破骨形成过程无显著影响。但在诱导36例SAPHO患者来源的外周血单个核细胞（Peripheral Blood Mononuclear Cells，PBMCs）向破骨细胞分化的实验中，有24例其OCs分化过程被SAPHO患者SEVs抑制。统计分析提示，抑制的发生与患者处于活动期相关。进一步利用定量蛋白质组学检测健康志愿者及SAPHO患者SEVs刺激后生成的OCs蛋白组成的变化，发现SAPHO患者SEVs刺激下，患者破骨细胞中核仁素（Nucleolin，NCL）表达上调，并处于差异表达蛋白相互作用网络的关键节点。

研究意义

本研究首次报导了SAPHO患者SEVs的蛋白表达谱和其中的差异表达蛋白。差异表达蛋白中，上调蛋白SAA1是一种有助于SAPHO综合征诊断的炎症标志物。SAPHO患者SEVs能够抑制活动期患者破骨细胞形成和功能，可能具有负调节免疫应答的作用。NCL是潜在的调控活动期SAPHO患者破骨细胞生成的关键调节因子。

Background

Synovitis, acne, pustulosis, hyperosteogeny and osteoarthritis syndrome (SAPHO syndrome) is a rare chronic inflammatory disease. It has osteoarthritis, synovitis, osteoporosis and hyperosteogeny which often involve the anterior chest wall and axial bone, and skin manifestations such as palmoplantar pustulosis and acne. Bone and joint manifestations are the characteristics of SAPHO syndrome, which causes pain, limited activity and stiffness of joints in the later stage of the course, the morphological changes and even collapses seriously affect the quality of life of patients. The pathogenesis of SAPHO syndrome is still unclear.

The chronic inflammatory manifestations and bone and joint symptoms of SAPHO syndrome suggest that immune abnormalities and bone metabolic imbalance play an important role in the occurrence and development of SAPHO syndrome. In recent years, with the more and more in-depth study of extracellular vesicles, its role in the regulation of immune and bone metabolism is gradually revealed. Studies have shown that extracellular vesicles are involved in the regulation of bone metabolism and inflammation in physiological and disease conditions. Many inflammatory cytokines are also important regulators of bone metabolism. Extracellular vesicles, which contain various protein components and shoulder the function of cell communication, may play a role as a "bridge" between immunity and bone metabolism in SAPHO syndrome. At present, the research on serum-derived extracellular vesicles (SEVs) of SAPHO syndrome is still blank.

Methods

In this study, SEVs were isolated by ultracentrifugation. After the successful isolation of SEVs was proved by electron microscopy, the quantitative proteomics method was used to analyze the SEVs of SAPHO patients and healthy volunteers, so as to clarify the protein expression profile and differential expression proteins (DEPs) of SEVs in SAPHO patients. Bioinformatics analysis identified DEPs and potential SAPHO disease biomarkers involved in bone metabolism .

Enzyme linked immunosorbent assay (ELISA) was used to detect the expression of potential biomarkers in serum and SEVs isolated by reagents of patients and healthy volunteers, and to verify its ability to assist the diagnosis of SAPHO. Further, the levels of SAA1 in SEVs of SAPHO, rheumatoid arthritis (RA) and ankylost spondylitis (AS) patients were compared by ELISA to determine whether SAA1 can be used for the differential diagnosis of SAPHO syndrome, RA and AS.

In order to verify the regulatory effect of SEVs on bone metabolism in SAPHO patients, SEVs were extracted by reagent. Western Blot (WB) and Nanoparticle Tracking Analysis (NTA) confirmed the successful isolation of SEVs. The osteoclastogenesis and osteogenesis experiments were conducted with human and mouse cells stimulated by SEVs respectively. The cells with phenotypic changes under the stimulation of different SEVs were analyzed by quantitative proteomics and bioinformatics to find the DEPs, that is, the possible reasons for the phenotypic differences.

Results

The results showed that there were significant differences in SEVs protein expression profiles between SAPHO patients and healthy volunteers, and 17 (35%) of the DEPs were involved in inflammation and bone metabolism. SEVs were extracted from 54 SAPHO patients and 84 healthy volunteers by reagents. Serum Amyloid A-1 (SAA1) was found to be highly expressed in the serum and SEVs of SAPHO patients. It has the potential to assist in the diagnosis of SAPHO syndrome. However, there was no significant difference in the expression of SAA1 in SEVs among 54 SAPHO patients, 22 RA patients and 29 AS patients.

Bone formation experiments showed that SEVs in SAPHO patients had no significant effect on the osteogenesis and osteoclastogenesis processes of cells derived from mice and healthy volunteers. However, in the experiment of inducing differentiation of peripheral blood mononuclear cells (PBMCs) from patients with SAPHO into OCs, the differentiation process was inhibited by SAPHO SEVs in 24 out of 36 cases. Statistical analysis indicated that the occurrence of inhibition was related to the active stage of the patients. Furthermore, quantitative proteomics was used to detect the protein composition changes of OCs stimulated by SEVs from SAPHO pateints and healthy volunteers. It was found that the expression of Nucleolin (NCL) was upregulated in OCs stimulated by SAPHO SEVs, and it was the key node of the interaction network of DEPs.

Significance

In this study, we reported the protein expression profile and DEPs of SEVs in SAPHO patients for the first time. Among the DEPs, the up-regulated protein SAA1 is an inflammatory marker which is helpful for the diagnosis of SAPHO syndrome. SEVs in SAPHO patients could inhibit the formation and function of OCs in patients in active stage, which may have a negative role in regulating the immune response. NCL is a potential key regulator of OCs generation in SAPHO patients in active stage.