【摘要】 目的体外合成和筛选特异性阻断人滑膜成纤维细胞（RASF）环氧合酶-2（hCOX-2）的小分子干扰RNA（siRNA）,同时了解COX-2抑制对炎症因子表达水平的影响。方法设计4条针对人COX-2 mRNA siRNA（1#～4#siRNA）,1条随机序列作为对照。分成A～H组,A组为空白阴性对照,B～F组处理依次为随机siRNA、1#～4#siRNA。应用LipofectAMINE 2000将上述siRNA转染入RASF,各培养孔加入100 nmol/L的佛波酯。转染48h后,分别应用反转录聚合酶链式反应（RT-PCR）和Western Blot检测hCOX-2 mRNA和蛋白表达水平。采用酶联免疫吸附（ELISA）方法检测各组上清液中前列腺素E₂（PGE₂）和白细胞介素（IL）-1β、IL-6、肿瘤坏死因子（TNF）-α、血管内皮生长因子（VEGF）水平。结果4#siRNA转染的RASF表达hCOX-2 mRNA和蛋白水平明显低于其他siRNA干预组和阴性对照,且其上清液PGE₂和IL-1β、IL-6、TNF-α、VEGF水平较其他各组明显下降。结论4#siRNA能有效抑制COX-2 mRNA表达和COX-2蛋白的合成,且上清液中PGE₂和IL-1β、IL-6、TNF-α及VEGF水平最低。更多还原

【Abstract】 Objective To design,synthesize and screen high efficient small interfering RNA（siRNA） targeting to cyclooxygenase-2（COX-2）on rheumatoid arthritis synovial fibroblasts（RASF）.To further study the effect of specific COX-2 siRNA interfering on mediators of inflammatory cytokines.Methods Four pairs of siRNA for human COX-2 mRNA were synthesized by utilizing RNA design software,while another random sequence was designed as control.They were divided into group A to H.Among them,group A was used as the negative control（CTL）,and group B to F were transfected as random siRNA（NC）,1#～4#siRNA in order. These siRNAs were transferred into RASF by LipofectAMINE2000 package and PMA（phorbol-12-myristate- 13-acetate）was added into each culture and with a final concentration of 100 nmol/l.RASF was collected 48 hours after transfection.The expression of hCOX-2 at mRNA level was determined by reverse transcription- polymerase chain reaction（RT-PCR）and hCOX-2 protein level by Western Blot.The supernatant levels of PGE₂,IL-1β,IL-6,TNF-αand vascular endothelial growth factor（VEGF）of the above groups were detected by ELISA.Results The levels of hCOX mRNA and protein in RASF treated with 4-#siRNA were significantly lower than those of the negative control and other groups.The level of PGE₂ and cytokines like IL-1β,IL-6, TNF-αand VEGF in the supernatant were lower in the 4#siRNA group than in other groups.Conclusion 4#siRNA can effectively inhibit the expression of COX-2 mRNA and the synthesis of the COX-2 protein in human synovial fibroblasts.The level of PGE₂,IL-1β,IL-6,TNF-αand VEGF is the lowest in the super- natant.Thus 4#siRNA has been confirmed to specifically block the COX-2 in human synovial fibroblasts.更多还原