正常和退变腰椎间盘蛋白质的双向电泳和质谱鉴定

Identification of the proteins in the normal and degenerative intervertebral discs by two-dimensional electrophoreses and mass spectrometry

背景:近年来对于退变腰椎间盘组织蛋白质成分改变的研究取得了很大进展,但是对于正常和退变腰椎间盘整体蛋白质成分改变的分离和差异鉴定却鲜见报道。目的:建立正常和退变腰椎间盘蛋白质的双向电泳图谱,鉴定差异表达蛋白,探讨腰椎间盘退变的生物学机制。设计、时间及地点:对比观察,于2007-06/2008-12在南方医科大学病理生理实验室完成。材料:由自愿者提供的正常和退变腰椎间盘组织各6例,年龄21～45岁,其中男9例,女3例;取材范围L3～L5。方法:氯化铯梯度离心去除正常和退变腰椎间盘组织蛋白多糖,通过固相pH梯度(IPG)等电聚焦和梯度聚丙烯酰胺双向电泳分离蛋白质,分析电泳图像,使用基质辅助激光解吸附飞行时间质谱仪对差异蛋白质点进行分析鉴定。主要观察指标:正常腰椎间盘及退变腰椎间盘组织IPG等电聚焦双向凝胶电泳结果。结果:双向电泳显示正常和退变腰椎间盘蛋白质图谱差异显著,从中选取16个差异明显的位点进行质谱仪分析,确定了6种具有显著性意义的差异蛋白质。结论:双向电泳可有效地分离正常和退变腰椎间盘组织蛋白质,通过质谱仪鉴定可以确定其之间差异蛋白质的性质。

BACKGROUND： It has made a great progress in the proteomics research of degenerative lumbar disc tissues, however, it is rarely reported that separation and identification of the composition and difference as a whole between normal and degenerative lumbar intervertebral disc. OBJECTIVE： To establish a two-dimensional electrophoreses system for identifying the proteins in normal and degenerative intervertebral disc, in addition, to explore the biological mechanism of lumbar disc degeneration. DESIGN, TIME AND SETTING： The comparative observation was performed in the Pathological and Physiological Laboratory of Southern Medical University between June 2007 and December 2008. MATERIALS： Six normal and six degenerative lumbar intervertebral disc tissues were provided by volunteers, including 9 male and 3 female, which aged between 21 45 years. L35 of the lumbar intervertebral disc was harvested. METHODS： Proteins were extracted from normal and degenerative lumbar intervertebral disc, the aggrecan was removed by cesium chloride gradient centrifugation, and proteins were separated by two-dimensional electrophoreses and identified by matrix assisted laser adsorption/ionization time-of flight mass spectrometry （MALDI-TOF-MS）. MAIN OUTCOME MEASURES： IPG two-dimensional gel electrophoresis of the normal and degenerative lumbar intervertebral disc. RESULTS： The two-dimensional electrophoresis results showed that there was an obvious difference between the normal and degenerative lumbar interverfebral disc. A total of 6 differential proteins with statistical significance, were identified by MALDI-TOF-MS between the 16 differential protein spots. CONCLUSION： The different proteins in normal and degenerative lumbar intervertebral disc can be separated effectively by two-dimensional electrophoresis, and identified by using mass spectrometry.