Wei Zhenyuan, Guo Shang, Wang Hongwei, Zhao Yang, Yan Jiren, Zhang Chi, Zhong Biao
Department of Orthopedic Surgery, And Shanghai Institute of Microsurgery on Extremities, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai 200233, China.
Department of Medicine, the University of Chicago. Chicago, IL 60637, USA.
J Orthop Translat. 2022 May 14;34:42-59. doi: 10.1016/j.jot.2022.04.003. eCollection 2022 May.
Traumatic Heterotopic Ossification (tHO) is one of complications of elbow fractures to the detriment of patients' rehabilitation, and the severity of tHO corresponds to the size of ectopic bone. It has yet to be elucidated which proteins and pathways underlying the progression of tHO, and biomarkers to predict the severity of tHO at early stage of the disease also need further investigation.
In this study, a new rat model with distinct volume of ectopic bone was established first. Then a data-independent acquisition proteomics approach was used to investigate injured site tissues sequentially obtained from these rats (2, 7, 14, and 28 days post-injury). Differentially expressed analysis, functional annotation and co-expression analysis and protein-protein interaction network were performed to explore the pathways and hub proteins in the tHO progression. Clinical samples from a nest case-control study were used to validate the selected proteins for predicting the severity of tHO.
The Achilles Tenotomy (AT) induced significantly larger sizes of ectopic bone compared to Partial Achilles Tenotomy (PAT) in rat models. A total of 3547 quantifiable proteins were screened for differential expression analysis among the AT, PAT and control groups. The hierarchical clustering and expression pattern analysis revealed more apparent difference in the pathways such as oxidative phosphorylation, mitochondrial function, and sirtuin signaling between AT and PAT group at the early stage (2 dpi) of tHO. The co-expression analysis identified five hub proteins, UBA1, EIF3E, RPL17, RPL27, and RPS28. qPCR assay, immunoblot assay and immunohistochemistry assay verified that these proteins had higher expression level in the tissue samples of clinically relevant HO patients and clinically irrelevant HO patients than HO negative patients.
The new established animal model and proteome profile could serve as a solid foundation for the comprehensive investigation of the progression of traumatic heterotopic ossification. And the identified 5 proteins (UBA1, EIF3E, RPL17, RPL27, and RPS28) may serve as potential biomarkers to predict the severity of tHO.
The proteins identified in this study may be the potential biomarkers and therapeutic targets for predicting and treating the tHO at early stage.
创伤性异位骨化（tHO）是肘部骨折的并发症之一，不利于患者康复，且tHO的严重程度与异位骨的大小相关。tHO进展背后的蛋白质和信号通路尚未阐明，预测疾病早期tHO严重程度的生物标志物也需要进一步研究。
在本研究中，首先建立了一种具有不同异位骨体积的新型大鼠模型。然后采用数据非依赖采集蛋白质组学方法，对这些大鼠在损伤后2、7及14和28天依次获取的损伤部位组织进行研究。进行差异表达分析、功能注释、共表达分析和蛋白质-蛋白质相互作用网络分析，以探索tHO进展中的信号通路和枢纽蛋白。利用巢式病例对照研究的临床样本对所选用于预测tHO严重程度的蛋白质进行验证。
在大鼠模型中，跟腱切断术（AT）诱导的异位骨大小明显大于部分跟腱切断术（PAT）。在AT、PAT和对照组之间共筛选出3547种可定量蛋白质用于差异表达分析。层次聚类和表达模式分析显示，在tHO早期（损伤后2天），AT组和PAT组在氧化磷酸化、线粒体功能和沉默调节蛋白信号等信号通路方面存在更明显差异。共表达分析确定了5种枢纽蛋白，即泛素激活酶1（UBA1）、真核翻译起始因子3E（EIF3E）、核糖体蛋白L17（RPL17）、核糖体蛋白L27（RPL27）和核糖体蛋白S28（RPS28）。定量聚合酶链反应（qPCR）检测、免疫印迹检测和免疫组织化学检测证实，这些蛋白质在临床相关HO患者和临床不相关HO患者的组织样本中的表达水平高于HO阴性患者。
新建立的动物模型和蛋白质组图谱可为全面研究创伤性异位骨化的进展提供坚实基础。所鉴定的5种蛋白质（UBA1、EIF3E、RPL17、RPL27和RPS28）可能作为预测tHO严重程度的潜在生物标志物。
本研究中鉴定的蛋白质可能是预测和治疗早期tHO的潜在生物标志物和治疗靶点。
