应力对颈椎后纵韧带骨化患者颈椎后纵韧带成纤维细胞蛋白表达的影响

Effect of stress on differential protein expression of fibroblast in postrerior longitudinal ligament of patients with ossification of cervical posterior longitudinal ligament

目的:寻找应力刺激前后颈椎后纵韧带骨化(OPLL)患者颈椎后纵韧带成纤维细胞表达差异的蛋白,探讨OPLL的发生机制。方法:从3例行颈前路减压融合内固定术的OPLL患者术中切取后纵韧带组织,清除硬化骨组织后采用组织块培养法培养成纤维细胞,采用机械应力装置对接种于Flexercell板的细胞施加10%、0.5Hz的机械应力,持续加载24h,以同样接种于Flexercell板未施加机械应力的细胞为对照组。提取细胞的总蛋白,采用双向差异凝胶电泳(DIGE)及MALDI-TOF/TOF质谱技术筛选并鉴定表达差异的蛋白,查阅相关文献资料,分析了解其功能特点及其在OPLL发展进程中的可能作用。结果:经过筛选和质谱分析共获得15个具有统计学意义的表达差异蛋白点,其中14个在应力刺激后的后纵韧带细胞中表达下调,1个表达上调。主要是代谢酶类、翻译相关蛋白、细胞骨架蛋白等相关的蛋白,查阅相关文献资料后提示4个表达差异的蛋白与骨化密切相关。结论:机械牵张应力可诱导颈椎后纵韧带成纤维细胞多个与骨化相关的蛋白发生差异性表达,其可能与OPLL发生有关。

Objective：To investigate the effect of stress on differential protein expression of fibroblast in postrerior longitudinal ligament of patients with ossification of cervical posterior longitudinal ligament（OPLL）and the inner mechanism.Method：Cervical posterior longitudinal ligaments were collected from three OPLL patients,and fibroblast was cultured by tissue fragment culturing method after removal of bone.Ligament fibroblast cells were subjected to mechanical strains using a Flexercell strain unit by 10%,0.5Hz for 24 hours,and the static cells were used as control.Protein lysates were extracted separately,and differential gel electrophoresis（DIGE） and MALDI-TOF/TOF mass spectrum was performed to identify the differential expression of proteins.According to papers published,the localization of the proteins,function and possible role in the developing of OPLL were reviewed.Result：15 differential expressed proteins were identified,among which 14 were down-regulated and 1 was up-regulated.The known differentially expressed proteins were classified as metabolic enzymes,translation related protein,cell cytoskeleton and other proteins,4 proteins played important role in the pathogenesis of ossification.Conclusion：Stress may induce differential expression of several ossification-related proteins in postrerior longitudinal ligament fibroblast,some of them may be involved in the developing of OPLL.