高迁移率蛋白-1在大鼠腰椎软骨终板退变中的作用及相关机制  

The role and mechanism of high mobility group box-1 protein in the degeneration of lumbar cartilage endplate in rats

作　　者：李磊 邢文华[1] 李峰[1] 吉德民 胡宝阳 辛大齐 祝勇[1] 杨学军[1] LI Lei;XING Wenhua;LI Feng(Department of Spine,the Second Affiliated Hospital of Inner Mongolia Medical University,Holahot,010030,China)

机构地区：[1]内蒙古医科大学第二附属医院骨科,呼和浩特市010030 [2]内蒙古医科大学研究生学院,呼和浩特市010050

出　　处：《中国脊柱脊髓杂志》2018年第11期1026-1033,共8页Chinese Journal of Spine and Spinal Cord

基　　金：国家自然科学基金地区基金项目(编号:81360278)

摘　　要：目的 :观察高迁移率蛋白-1(high mobility group box-1 protein,HMGB1)在大鼠腰椎软骨终板退变中的作用,探讨其相关机制。方法:选取4周龄SD大鼠处死,在显微镜下取出腰椎软骨终板,消化后提取细胞,再进行培养、分离、纯化,免疫荧光染色检测Ⅱ型胶原鉴定软骨终板细胞(cartilage endplate cell,CEC)。用不同浓度的FBS分别培养CEC 24h、48h,MTT法检测细胞活性。在第三代CEC中加入HMGB1后,分别在3h、6h、12h、24h时检测金属基质蛋白酶-3 (proteins of the matrix metalloproteinase,MMP-3)、血管内皮生长因子(vascular endothelial growth factor,VEGF)及白细胞介素-10(interleukin-10,IL-10)的m RNA表达,6h、12h、24h时检测蛋白表达量;CEC中加入100ng/ml HMGB1后分别在0、5、10、30、60和120min,使用Western blot检测细胞外信号调节激酶(extracellular regulated protein kinases,ERK)磷酸化的表达;继而将CEC分为6组:对照组、HMGB1(100ng/ml)组、FPSMZ1(HMGB1抑制剂)组、HMGB1+FPSMZ1组、U0126(ERK抑制剂)组、HMGB1+U0126组),使用Western blot分别检测MMP-3、VEGF和ERK信号转导途径磷酸化的表达及Elisa检测IL-10的表达。全部结果均为重复独立3次实验,计算其均数±标准差。结果:分离纯化的细胞Ⅱ型胶原抗体表达呈阳性,为CEC细胞;10%FBS培养48h为CEC增殖最佳的时间点和浓度;HMGB1能够诱导ERK和JNK信号转导途径磷酸化,从开始到10min逐渐升高,10min时达峰值。HMGB1上调了MMP-3 (P=0.039)和VEGF的m RNA (P=0.042)表达,下调IL-10 (P=0.025)的m RNA表达,三种因子蛋白表达与m RNA有相同的规律;HMGB1+FPSZM1组与HMGB1组相比,ERK信号转导途径的磷酸化明显减少;在CEC中加入U0126后,MMP-3(P=0.041)和VEGF(P=0.042)蛋白表达明显降低,而IL-10(P=0.004)蛋白表达明显增高;在CEC中加入FPSMZ1后,HMGB1对ERK信号转导途径磷酸化表达的作用明显降低(P=0.031)。结论:HMGB1可增加大鼠腰椎CEC中的MMP-3和VEGF的表达,降低IL-10的表达,其可能是通过EObjectives:To explore the effect and mechanism of high mobility group box-1protein(HMGB1) on the degeneration of lumbar cartilage endplate in rats.Methods:Four-week-old SD rats were sacrificed and the lumbar cartilage endplates were taken out under the microscope.The supematant was extracted after digestion.After culture,isolation and purification,immunofluorescence staining was used to detect type Ⅱ collagen and cartilage endplate cell (CEC).Different concentrations of FBS were used to culture CEC for 24h and 48h respectively.MTTassay was used to detect cell viability.The expressions of MMP-3,vascular endothelial growth factor and interleukin were detected at 3,6,12and 24hours after adding human HMGB1 to the third generation CEC.The expression of extracellular signal-regulated kinase phosphorylation was detected by Western blot at 0,5,10,30,60and 120rain after adding 100ng/ml HMGB1to CEC.CEC was di-vided into six groups:control group,HMGBI(100ng/ml)group,FPSMZI(HMGB1inhibitor)group,HMGBI+ FPSMZ1group,U0126(ERK inhibitor)group and HMGB1+U0126group.The expressions of MMP-3,VEGF and ERK signal transduction pathway phosphorylation and Elisa IL-10were detected by Western blot.All results were repeated independently for three experiments,and their mean +standard deviation was calculated. Results:The expression of type Ⅱ collagen antibody was positive in CEC cells,and the best time and concentration for proliferation of CEC was 48hours on 10%FBS.HMGB1could induce phosphorylation of ERK signal transduction pathway,which gradually increased from the beginning to 10min and reached its peak at 10min.HMGB1up-regulated the expressions of MMP-3(P=0.039)and VEGF(P=0.042),but down-regulated the expression of IL-10(P=0.025).The expression of three factors was the same as that of gene.Compared with HMGB group,phosphorylation of ERK signal transduction pathway was significantly reduced in HMGBI+ FPSMZ1group.After ERK signal transduction pathway inhibitor (U0126)added to CEC,the expressions of MMP-3(P=0.041)and VEGF(P=0.042)dec

关 键 词：高迁移率蛋白-1 ERK信号转导途径 软骨终板 大鼠 

分 类 号：R681.5[医药卫生—骨科学] R363[医药卫生—外科学]