Yan Peng, Zhu Yeping, Zhao Hui, Lu Yanyan, Gao Yuzhong
Department of Orthopedics, The First Affiliated Hospital of Jinzhou Medical University, Jinzhou, Liaoning 121000, P.R. China.
Recovery Unit, Jinzhou Central Hospital, Jinzhou, Liaoning 121000, P.R. China.
Exp Ther Med. 2017 Jun;13(6):2900-2904. doi: 10.3892/etm.2017.4326. Epub 2017 Apr 12.
Currently, there is a lack of effective early screening and detection methods for femoral head necrosis. Current research on most orthopedic diseases focuses on proteomics in the preliminary stage. The recent fluorescence differential in gel electrophoresis (DIGE) has advantages such as a high reproducibility, high sensitivity, high throughput, and high dynamic range. It is currently one of the most widely used quantitative proteomic research means. We conducted this study to investigate the pathogenesis of non-traumatic femoral head necrosis using the fluorescence DIGE to screen non-traumatic femoral head necrosis based on proteomics and provide a theoretical basis for screening possible biomarkers and molecular targeted treatment. The DIGE technique was used to separate the protein. An electrophoretogram was established on the basis of scanning and analysis. Identification and a bioinformatics analysis were conducted for the differential protein. The protein with differential expression of over 2-fold was excavated and ionized by means of substrate assisted laser desorption. The flight time was identified with a mass spectrometer (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF/TOF). The formation on sequences, structures and functions of these proteins were obtained through database retrieval. Western blot analysis was used to verify the differential protein expression and the reliability of the DIGE result was verified. DIGE was used to successfully separate 1,500±40 protein spots. There were 252 significant differential protein spots. The Ettan™ Spot Picker automatic work station was used to excavate 49 significant differential protein spots with expression difference over 2-fold. The MALDI-TOF/TOF mass spectrometer was used to identify these differential protein spots. Six proteins were identified in total, which include apolipoprotein A1 (APOA1), fibrous protein original chain, fibrous protein original chain, serum albumin, sulfur-oxygen protein peroxiredoxin 2 (PRDX2) and actin. APOA1 and PRDX2 were subject to western blot analysis detection; results were consistent with the DIGE result. Based on an analysis of the biological information, these proteins may be associated with the incidence and progression of femoral head necrosis.
目前，股骨头坏死缺乏有效的早期筛查和检测方法。当前大多数骨科疾病的研究在初步阶段聚焦于蛋白质组学。最近的荧光差异凝胶电泳（DIGE）具有重现性高、灵敏度高、通量高和动态范围广等优点。它是目前应用最广泛的定量蛋白质组学研究手段之一。我们开展本研究，利用荧光DIGE基于蛋白质组学筛选非创伤性股骨头坏死，以探究其发病机制，并为筛选可能的生物标志物和分子靶向治疗提供理论依据。采用DIGE技术分离蛋白质。在扫描和分析的基础上建立电泳图谱。对差异蛋白质进行鉴定和生物信息学分析。挖掘表达差异超过2倍的蛋白质，并通过基质辅助激光解吸进行离子化。用质谱仪（基质辅助激光解吸/电离飞行时间质谱，MALDI-TOF/TOF）鉴定飞行时间。通过数据库检索获得这些蛋白质的序列、结构和功能信息。采用蛋白质免疫印迹分析验证差异蛋白质表达，验证DIGE结果的可靠性。DIGE成功分离出1500±40个蛋白质点。有252个显著差异蛋白质点。使用Ettan™ Spot Picker自动工作站挖掘出49个表达差异超过2倍的显著差异蛋白质点。用MALDI-TOF/TOF质谱仪鉴定这些差异蛋白质点。共鉴定出6种蛋白质，包括载脂蛋白A1（APOA1）、纤维蛋白原α链、纤维蛋白原β链、血清白蛋白、硫氧还蛋白过氧化物酶2（PRDX2）和肌动蛋白。对APOA1和PRDX2进行蛋白质免疫印迹分析检测；结果与DIGE结果一致。基于生物信息分析，这些蛋白质可能与股骨头坏死的发生和发展有关。
