| 基于MDLC-MS/MS技术的股骨头缺血性坏死的组织蛋白质组分析 |
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Received:October 13, 2010
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| 期刊信息：《中国骨伤》2011年24卷，第3期，第213-217页 |
| DOI：10.3969/j.issn.1003-0034.2011.03.011 |
| 基金项目:浙江省医药卫生科学研究基金(编号：2007B207) |
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目的：应用组织蛋白质组学方法探寻与成人非创伤性股骨头缺血性坏死(ONFH)相关的蛋白质。
方法：自2008年7月至2009年2月，从非创伤性ONFH(男7例，女3例)和新鲜股骨颈骨折(男、女各5例)而行人工髋关节置换术的患者中随机选取10例，平均年龄分别为52岁和63岁。术中取ONFH患者股骨头中坏死骨组织为试验组，取股骨颈骨折患者股骨头中的正常骨组织样品为对照组。用四步溶剂法提取骨组织总蛋白，经SDS-PAGE分离和酶解，应用多维液相色谱与串联质谱联用技术分离鉴定组织蛋白，以TPP软件检测两组蛋白质的差异表达水平，并用Turbo SEQUEST软件鉴定差异表达蛋白质。同时Western免疫印迹验证质谱检测的准确性。
结果：试验组和对照组样品中分别鉴定二肽段以上的高可信度蛋白质数量1 233个和 999 个，假阳性率0.8%.试验组中共鉴定差异表达蛋白质192个，其中表达量上调3倍以上的蛋白点107个，下调3倍以上53个，其中34种蛋白质与ONFH密切相关。Western免疫印迹证实试验组样品中GPCR26和CHST2表达量下调，与质谱结果一致。
结论：非创伤性ONFH存在复杂的病理生理学过程，本研究发现的差异表达蛋白极有可能成为ONFH早期临床诊断的候选生物学标志物。 |
| [关键词]:股骨头坏死 蛋白质组学 色谱法 光谱分析，质量 |
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| High throughout proteomic analysis of non-traumatic osteonecrosis of the femoral head |
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Abstract:
Objective: To explore proteins associated with non-traumatic osteonecrosis of the femoral head in adults by using high throughput proteomic approach.
Methods: From July 2008 to February 2009,10 patients with non-traumatic osteonecrosis of the femoral head (7 males,3 females) and 10 patients with fresh femoral head fracture (5 males,5 females) who underwent total hip arthroplasty were enrolled randomizedly in the study. The median age was 52 years and 63 years respectively. Necrotic bone tissues were harvested as experimental group from the femoral head of patients with osteonercrosis of the femoral head,and normal bone tissues were collected as the control group from the patients with femoral head fracture. After bone tissues were demineralized,total bone proteins were extracted sequentially by using four different lysis buffers. Thereafter,SDS-PAGE,in-gel digestion and subsequent multi-dimensional liquid chromatography-tandem mass spectrometry(MDLC-MS/MS) were applied to separate and identify the extracted proteins. Different expression level of the proteins in both groups was investigated by TPP software,and different expression protein analysis was then carried out by Turbo SEQUEST. Western blot was applied finally to evaluate the accuracy of mass spectrometry.
Results: The in-depth proteome analysis in experimental and control groups revealed 1 233 and 999 high-confidence proteins with two or more peptides respectively,and the false-positive rate was 0.8%. In total,192 differentially expressed proteins were identified in experimental samples. At compared with the control,107 and 53 proteins were identified with the level upregulated and downregulated at least 3 folds respectively. Among them,34 proteins were found highly associated with osteonecrosis of the femoral head. Downregulated expression of GPCR26 and CHST2,similar with results of mass spectrometry analysis,were confirmed by Western blot in the necrotic bone tissues.
Conclusion: The pathogenesis of the non-traumatic osteonecrosis of the femoral head has been demonstrated a complicated process and remained unknown. These identified differentially expressed proteins might be promising biomarker candidates for early diagnosis of the devastating disease. This study sheds new light on novel approach to understand the pathogenesis of osteonecrosis of the femoral head. |
| KEYWORDS:Femoral head necrosis Proteomics Chromatography Spectrum analysis,mass |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 张雷,杨国敬,王珺,严世贵.基于MDLC-MS/MS技术的股骨头缺血性坏死的组织蛋白质组分析[J].中国骨伤,2011,24(3):213~217 |
| 英文格式: | ZHANG Lei,YANG Guo-jing,WANG Jun,YAN Shi-gui.High throughout proteomic analysis of non-traumatic osteonecrosis of the femoral head[J].zhongguo gu shang / China J Orthop Trauma ,2011,24(3):213~217 |
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