PDCD5在类风湿关节炎成纤维样滑膜细胞凋亡中表达上调

Up-regulation of PDCD5 in Apoptosis of Rheumatoid Arthritis Fibroblast-like Synoviocytes

为了研究程序化细胞死亡因子5(PDCD5)在类风湿关节炎成纤维样滑膜细胞凋亡中的作用,在不同的时间加入有效剂量100nmol/L雷公藤内醇酯(triptolide)后,采用实时定量PCR、RT-PCR、Western印迹和直接免疫荧光染色方法检测体外分离培养的类风湿关节炎成纤维样滑膜细胞中PDCD5在mRNA和蛋白水平的表达及蛋白表达特征.在雷公藤内醇酯诱导类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5 mRNA表达水平明显地渐次增加,呈现一种明确的时间依赖性递增表达模式,而PDCD5蛋白有时间依赖性表达上调持续16h,并维持在相对恒定水平.直接免疫荧光染色结果显示,在正常体外培养的类风湿关节炎成纤维样滑膜细胞中,PDCD5蛋白的表达较弱,且主要分布在细胞浆.经雷公藤内醇酯处理4 h后,大多数细胞有PDCD5蛋白的聚集,直至12h,细胞核周围PDCD5蛋白聚集显著增强.36h后,PDCD5蛋白以核固缩的形式存在于凋亡的RA FLS中,细胞核染色质明显浓缩,片段化并出现了凋亡小体.上述结果表明,在类风湿关节炎成纤维样滑膜细胞凋亡的过程中,PDCD5表达上调并在凋亡早期出现核转位,PDCD5蛋白核转位要早于凋亡小体形成.PDCD5蛋白核转位是类风湿关节炎成纤维样滑膜细胞凋亡的早期事件,PDCD5不仅参与了类风湿关节炎成纤维样滑膜细胞的凋亡过程,而且在类风湿关节炎滑膜增生的凋亡调节中起到重要调节作用.

To investigate expression and effect of PDCD5（programmed cell death 5） gene in the apoptosis of rheumatoid arthritis（RA） fibroblast-like synoviocytes（FLS）,RA FLS were incubated with 100 nmol/L triptolide at the indicated times.The expressions of PDCD5 were evaluated at both mRNA and protein levels in RA FLS in vitro by real-time PCR,Western blot and immunofluorescence staining,respectively.Real-time PCR and RT-PCR revealed that PDCD5 mRNA expression increased gradually via a time-dependent pattern.Similarly,Western blot confirmed that PDCD5 protein also showed a time-dependent increased.Its expression peak appeared after treatment for 16 hours,then maintained at stable level with a tend towards decline.Immunofluorescence staining showed that PDCD5 protein was weakly expressed and mainly localized in the cytoplasm of untreated RA FLS.After stimulation 4 hours,most of cells exhibited a nuclear accumulating of PDCD5 protein,and eventually the protein was aggregated intensively around nucleus at 12 hours.Furthermore,cells were treated by triptolide for 36 hours,nuclear accumulation of PDCD5 and condensed nucleus clearly showed up,PDCD5 protein even appeared in the apoptotic bodies,the formation of apoptotic body preceded in the nucleus of RA FLS.Therefore,we proposed that PDCD5 was up-regulated in RA FLS undergoing apoptosis,nuclear translocation of PDCD5 protein followed by apoptosis due to the induction by triptolide.Nuclear translocation of PDCD5 may be an earlier event in apoptotic RA FLS.The study has important implications for understanding the major role of PDCD5 involved in the regulation of apoptosis in rheumatoid hyperplastic synovitis.