基于TMT技术的激素性股骨头坏死硬化带修复模式探索及临床验证  

Exploration and clinical validation of the repair mode of the sclerotic zone of steroid-induced osteonecrosis of the femoral head based on Tandem Mass Tags technology

作　　者：庄至坤 何敏聪 林天烨 吴荣凯 郭金花 吴昭克 魏秋实 Zhuang Zhikun;He Mincong;Lin Tianye;Wu Rongkai;Guo Jinhua;Wu Zhaoke;Wei Qiushi(Department of Joint Diseases,Quanzhou Orthopedic-Traumatological Hospital,Quanzhou 362000,Fujian Province,China;Guangdong Research Institute for Orthopedics&Traumatology of Chinese Medicine,Guangzhou 510000,Guangdong Province,China;Department of Joint Diseases,The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510000,Guangdong Province,China)

机构地区：[1]泉州市正骨医院关节科,福建省泉州市362000 [2]广东省中医骨伤研究院,广东省广州市510000 [3]广州中医药大学第三附属医院关节科,广东省广州市510000

出　　处：《中国组织工程研究》2024年第14期2191-2196,共6页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(82274544),项目负责人:魏秋实;泉州市科技计划项目(2022NS011),项目负责人:庄至坤。

摘　　要：背景:股骨头内硬化带是激素性股骨头坏死疾病发展过程中重要的影像学特征并与预后相关。过氧化物酶体增殖物活化受体γ协同刺激因子1α(peroxisome proliferator-activated receptor gamma coactivator 1α,PGC-1α)被证明具有成骨、促血管形成、抗线粒体凋亡等生物活性,与激素性股骨头坏死的骨修复可能存在紧密关联。目的:筛选激素性股骨头坏死硬化带区与正常区的差异蛋白,筛选出硬化带的关键蛋白,并验证关键蛋白在激素性股骨头坏死股骨头标本中的差异表达;探索激素性股骨头坏死硬化带修复模式。方法:取激素性股骨头坏死行人工全髋关节置换术取出的股骨头标本,通过串联质谱标签技术筛选硬化带区与正常区的差异表达基因,并进行GO与KEGG信号通路分析,构建关键靶点的蛋白互作网络、筛选关键基因。观察关键蛋白在激素性股骨头坏死硬化带中的表达,通过Western blotting、免疫组化验证关键蛋白在硬化带中的表达。结果与结论:①通过串联质谱标签定量蛋白质谱检测发现:相比于正常区骨组织,股骨头硬化带骨组织中具有显著差异表达(Log_(2)FC>1.20、Log_(2)FC<0.84和P<0.05)的蛋白质有609个,其中发生上调的蛋白质290个,发生下调的蛋白质319个;②通过GO与KEGG通路富集分析,发现在前10位的富集通路中,Wnt信号通路与生命周期调控通路与骨修复关系密切;在生命周期调控通道中,PGC-1α是重要的蛋白之一;③与激素性股骨头坏死标本正常区相比,Western blotting验证了PGC-1α、NRF-1在硬化带中低表达,Cleaved Caspase-3在硬化带中高表达;④光镜下免疫组化结果显示,股骨头组织标本中硬化区及正常区的PGC-1α、NRF1及Cleaved Caspase-3阳性染色分布,可以发现骨小梁、成骨细胞和骨髓均有表达存在;硬化带区Cleaved Caspase-3表达更为明显;⑤结论:激素性股骨头坏死硬化带中存在包含Wnt等成骨相关通路BACKGROUND:The sclerotic zone in the femoral head is an important imaging feature in the progression of steroid-induced femoral head necrosis,which is associated with disease prognosis.Peroxisome proliferator-activated receptorγcoactivator 1α(PGC-1α)has been shown to possess biological activities such as osteogenesis,angiogenesis and anti-mitochondrial apoptosis,which may be closely related to bone repair of steroid-induced femoral head necrosis.OBJECTIVE:To screen for the differential proteins in the sclerotic zone of steroid-induced osteonecrosis of the femoral head versus the normal zone,to screen for hub proteins in the sclerotic zone,and to verify the differential expression of hub proteins in the femoral head specimens following steroid-induced femoral head necrosis,and to to explore the repair pattern of the sclerotic zone following steroid-induced femoral head necrosis.METHODS:Femoral head samples were collected from patients with steroid-induced osteonecrosis of the femoral head receiving total hip arthroplasty.The differentially expressed genes in the sclerotic zone and the normal zone were screened by Tandem Mass Tags and analyzed by GO and KEGG signaling pathways to construct a protein-protein interaction network and screen hub genes.In addition,the expression of hub genes in the sclerotic zone was verified by immunohistochemistry and western blot.RESULTS AND CONCLUSION:Quantitative protein profiling by Tandem Mass Tags revealed that 609 proteins were significantly differentially expressed(Log_(2)FC>1.20,Log_(2)FC<0.84 and P<0.05)in the sclerotic zone of the femoral head compared with the normal zone,of which 290 proteins were upregulated and 319 proteins were downregulated.The GO and KEGG pathway enrichment analyses revealed that among the top 10 enriched pathways,Wnt signaling pathway and life-cycle regulatory pathway were closely related to bone repair;in the life-cycle regulatory pathway,PGC-1αwas one of the important proteins.In addition,western blot results verified the low expression of PGC

关 键 词：串联质谱标签 激素性股骨头坏死 PGC-1Α 线粒体氧化凋亡 骨修复 

分 类 号：R496[医药卫生—康复医学] R318[医药卫生—临床医学] R446