摘要
目的建立佐剂型关节炎大鼠滑膜成纤维细胞(AAFLS)原代培养方法及其特性分析,探讨AA-FLS作为RA体外研究细胞模型的可行性。方法采用热杀死结核分枝杆菌H37Ra(Mtb)免疫SD大鼠,建立佐剂型关节炎大鼠模型,取大鼠双侧膝关节滑膜组织进行原代细胞培养,显微镜下观察细胞形态;免疫细胞化学方法鉴定细胞;CCK-8法检测细胞增殖活性;ELISA方法测定细胞上清中TNF-α和IL-1β水平;Hochest 33258法检测细胞凋亡状态;Western bolt方法检测AA FLS细胞线粒体凋亡通路重要调控蛋白Bcl-2和Bax及Pro-caspase-3和Cleave-caspase-3水平的表达。结果胶原酶法分离获得的滑膜成纤维细胞经传代纯化,呈梭形的细胞占0.95以上,免疫细胞化学鉴定显示滑膜成纤维细胞vimentin表达呈阳性,CD68表达为阴性;AA模型大鼠滑膜成纤维细胞增殖活性较正常大鼠滑膜成纤维细胞增殖活性明显提高,细胞凋亡率降低;在培养的细胞上清中TNF-α和IL-1β水平明显升高;线粒体凋亡通路调控蛋白Bcl-2水平明显升高,而Bax蛋白表达水平明显降低,同时AA-FLS细胞中pro-caspase-3表达量增加。结论 AA-FLS具有增殖活跃、凋亡抑制和分泌高水平促炎性细胞因子的生物特性,可作为体外筛选抗RA药物的细胞模型。
Aims To establish the methods of primary culture of fibroblast-like synoviocytes in rats with adjuvant arthritis( AA-FLS) and analyze the feature and to investigate the possibility of AA-FLS as the model for the RA in vitro. Methods The synovial cells obtained from the SD rats were immunized by the Mtb and identified by morphology and immunocytochemistry. The viability of AA-FLS was assessed by Cell Counting Kit-8. ELISA was applied to detect TNF-α and IL-1β in cell media. Apoptosis was measured by Hochest33258. The expressions of mitochondrial apoptosis-related molecules,including Bcl-2,Bax,Pro-caspase-3and Cleave-caspase-3 were determined by Western Blot. Result In isolated primary synovial cells,more than 95% of AA-FLSs were fusiform. Immunocytochemistry result showed a positive expression of vimentin and a negative expression of CD68 in AA-FLS. Cell proliferation of AA-FLS was higher than FLS and cell apoptosis of AA-FLS was curbed. Western blot data demonstrated that the protein expressions of Bcl-2,Bax were regulated and the expression of caspase-3 was activated in AA-FLS. Conclusions AA-FLS is biologically characterized by high level proliferation activity and inflammatory cytokines and apoptosis suppression.AA-FLS can be used as the model for the RA in vitro.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2015年第12期1693-1698,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 31240045)
