高迁移率族蛋白1在骨关节炎滑膜细胞中的表达及意义  

EXPRESSION AND FUNCTION OF HIGH MOBILITY GROUP BOX CHROMOSOMAL PROTEIN 1 IN SYNOVIOCYTES OF PATIENTS WITH OSTEOARTHRITIS

作　　者：宋登新[1] 曹云[1] 丁徐[1] 何小文[1] 黄涛[1] 赵毅[1] 祁军[2] 

机构地区：[1]上海市第一人民医院宝山分院骨科,上海200940 [2]华中科技大学同济医学院附属同济医院骨科

出　　处：《中国修复重建外科杂志》2015年第11期1376-1380,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：上海市科技委员会课题资助项目(124119b0500)~~

摘　　要：目的比较OA与正常膝关节滑膜细胞中高迁移率族蛋白1(high mobility group box chromosomal protein 1,HMGB1)的差异,探讨HMGB1在OA中的作用。方法取OA与正常膝关节滑膜组织,采用组织块培养法分离培养关节滑膜细胞,倒置相差显微镜下观察细胞形态,应用免疫荧光染色、免疫组织化学染色以及Western blot检测比较两种滑膜细胞HMGB1表达差异。构建HMGB1小干扰RNA(small interfering RNA,si RNA)的真核表达载体Pgenesil-1/HMGB1 si RNA,转染至OA滑膜细胞中(si RNA转染组),以Pgenesil-1质粒转染至OA滑膜细胞(空载体转染组),以OA滑膜细胞(未转染组)作为对照。Western blot检测各组OA滑膜细胞中HMGB1蛋白的表达,ELISA法检测各组滑膜细胞培养上清液中IL-1β及TNF-α含量。结果原代培养的OA与正常膝关节滑膜细胞呈长梭状,为成纤维细胞样细胞。免疫组织化学染色及免疫荧光染色观察示,OA滑膜细胞中HMGB1以细胞质分布为主、细胞核中较少,而正常滑膜细胞则相反。Western blot检测示,OA滑膜细胞中HMGB1高表达,表达量为0.687±0.025,显著高于正常滑膜细胞的0.172±0.030(t=32.159,P=0.000)。酶切鉴定显示成功构建si RNA表达载体Pgenesil-1/HMGB1 si RNA。Western blot检测显示,si RNA转染组HMGB1蛋白表达为0.134±0.048,显著低于空载体转染组的0.581±0.032及未转染组的0.514±0.069(P<0.05)。ELISA法检测si RNA转染组IL-1β及TNF-α含量均显著低于空载体转染组及未转染组(P<0.05)。结论膝关节滑膜细胞中HMGB1表达的上调和表达位置的改变(从细胞核移位至细胞质)与OA密切相关,应用si RNA技术抑制HMGB1表达可明显降低OA滑膜细胞分泌IL-1β和TNF-α的能力,延缓OA的炎性反应。Objective To explore the pathological role of high mobility group box chromosomal protein 1（HMGB1） in osteoarthritis（OA） by comparing the difference of HMGB1 in the synoviocytes between OA and normal knees. Methods Synoviocyte lines from OA and normal knees were collected and cultured. Immunohistochemistry and Western blot were applied to identify the difference of HMGB1 between the OA and normal synoviocyte lines. The eukaryotic expression vector containing human Pgenesil-1/HMGB1 small interfering RNA（si RNA） were constructed and identified. The synoviocyte lines were transfected with the eukaryotic expression vector of Pgenesil-1/HMGB1 si RNA（Pgenesil-1/HMGB1 si RNA group） and with Pgenesil-1 plasmid（Pgenesil-1 group） and were not transfected as a control（untransfected group）. Western blot was applied to identify the difference of HMGB1 among groups, and the levels of interleukin 1β（IL-1β） and tumor necrosis factor α（TNF-α） protein synthesis in the supernatants were measured by ELISA. Results Primary knee synoviocytes cultured in vitro were fibroblast-like cells with longspindle shape. The immunohistochemistry and immunofluorescence results showed positive staining for HMGB1 in cytoplasm and weak positive staining in the nucleus in the OA synoviocyte line, but positive staining for HMGB1 in the nucleus and weak positive staining in the cytoplasm in the synoviocyte line of normal knee. The level of HMGB1 in the OA synoviocytes（0.687±0.025） was significantly higher than that of normal synoviocytes（0.172±0.030）（t=32.159, P=0.000） by Western blot. The recombinant plasmid Pgenesil-1/HMGB1 si RNA was successfully constructed. The expression of HMGB1 protein in Pgenesil-1/HMGB1 si RNA group（0.134±0.048） was significantly lower than that of Pgenesil-1 group（0.581±0.032） and untransfected group（0.514±0.069）（P〈0.05）. ELISA results showed that IL-1β and TNF-α in supernatants of Pgenesil-1/HMGB1 si RNA group were significantly lower than those of Pgen

关 键 词：骨关节炎 滑膜细胞 小干扰RNA 高迁移率族蛋白1 

分 类 号：R684.3[医药卫生—骨科学]