非创伤性股骨头缺血性坏死差异表达蛋白的鉴定和分析

Proteomic analysis of non-traumatic osteonecrosis of the femoral head

目的:应用组织蛋白质组学方法寻找与成人非创伤性股骨头缺血性坏死相关的蛋白质。方法:分别于非创伤性股骨头缺血性坏死患者和新鲜股骨颈骨折患者的股骨头中采集坏死骨组织样本与正常骨组织样本8份,采用四种不同的溶剂提取骨组织中的总蛋白,应用多维液相色谱与串联质谱联用技术对组织蛋白进行分离鉴定和生物信息学分析。结果:坏死组织样本和正常组织样本中鉴定二肽段以上的高可信度蛋白质数量分别为1233个和999个,假阳性率为0.9%。共鉴定154种3倍以上的差异蛋白质。基因本体论蛋白功能分析和文献检索显示34种蛋白质与股骨头缺血性坏死密切相关。Western免疫印迹证实了ONFH患者骨组织中GPCR26和ChST2表达量下调,与质谱结果一致。结论:非创伤性股骨头缺血性坏死存在复杂的病理生理学过程。研究发现的34个差异表达蛋白可能成为股骨头缺血性坏死早期临床诊断的候选生物学标志物。

Objective：To explore proteins associated with non-traumatic osteonecrosis of the femoral head in adults by using high throughput proteomic approach.Methods：Eight necrotic bone tissue samples were harvested from the femoral head of patients with osteonercrosis of the femoral head,and 8 normal bone tissue samples were collected as the control group in the same patients.After bone tissues were demineralized by HCl solution,total bone proteins were extracted sequentially by using four different lysis buffers.Thereafter,multi-dimensional liquid chromatography-tandem mass spectrometry（MDLC-MS/MS） were applied to separate and identify the extracted proteins,and bioinformatics analysis was carried out then by the software tools such as TurboSEQUEST and GoMiner.Results：The in-depth proteome analysis of necrotic and normal bone tissue revealed 1233 and 999 high-confidence proteins respectively based on two peptides minimum,with the a false-positive rate of 0.9%.154 differential expressed proteins,the level of which were upregulated or downregulated at least 3 fold were identified by spectral counting,as compared with the normal samples.Enrichment analysis in Gene Ontology categories of the differentially expressed gene found that 34 differential expressed proteins were highly associated with osteonecrosis of the femoral head.Downregulated expression of GPCR26 and ChST2 was confirmed by Western Blot on the samples of ONFH.Conclusion：The pathogenesis of the non-traumatic osteonecrosis of the femoral head has been proved complicated and remains unknown.These identified differential expressed proteins might be promising biomarker candidates for early diagnosis of the devastating disease.The results of this study will affirmatively provide a new approach to understand the etiopathogenesis of the osteonecrosis of the femoral head.