【摘要】 目的体外合成针对人环氧化酶-2(hCOX-2)的小分子干扰RNA(siRNA),并转染入人滑膜成纤维细胞,通过比较不同siRNA抑制COX-2 mRNA表达、COX-2蛋白合成以及下游产物PGE2水平,旨在确定特异性阻断人滑膜成纤维细胞COX-2的siRNA。方法分离、培养和传代人类风湿关节炎(类风关)滑膜成纤维细胞。设计合成4条针对hCOX-2 mRNA siRNA(1#-4#siRNA),1条随机序列siRNA。设立1#-4#siRNA和随机序列siRNA组(NC)及未转染对照组(CTL组)。应用Lipofect AMINE2000将上述siRNA分别转染人滑膜成纤维细胞,4h后各培养孔加入终浓度为100nmol/L的佛波酯。转染36h、48h后,各组提取蛋白质和总RNA,应用RT-PCR检测hCOX-2 mRNA表达水平,通过Western Blot检测hCOX-2蛋白表达水平。采用ELISA方法检测各组上清液PGE2的水平。结果转染36h,PCR结果显示,CTL、NC、1#siRNA、2#RNA、3#RNA、4#siRNA、阳性对照HeLa细胞组hCOX-2 mRNA电泳条带密度值分别为1、0.72、0.3、0.25、0.4、0.04、2.1。WesternBlot结果提示上述各组hCOX-2蛋白密度值分别为1、1.04、0.52、0.39、0.9、0和2.48。转染48h后,各组hCOX-2蛋白条带密度值分别为0.05、0.52、0.51、0.9和0.15。转染24h、48h和72h后,4#siRNA组培养上清液PGE2的水平较其他各组低。结论4#siRNA能有效抑制人滑膜成纤维细胞COX-2 mRNA表达和COX-2蛋白的合成,且上清液中PGE2水平最低,证实4#siRNA能特异性阻断COX-2在滑膜成纤维细胞表达。更多还原

【Abstract】 Purpose To design, synthesize small interfering RNA(siRNA) targeting to human cyclooxygenase-2(hCOX-2) on human synovial fibroblasts and screen high efficient siRNA by measuring the mRNA and protein levels of hCOX-2 measured by RT-PCR and Western Blot respectively. Methods The human synovial fibroblasts from rheumatoid arthritis patients were isolated, cultured, and passed. 4 pairs of siRNA(1#-4#siRNA) directed to the human COX-2 mRNA were synthesized by utilizing RNA design software, while another random line was designed as control. The interfering groups were transfected in order as random siRNA(NC), 1#siRNA, 2#siRNA, 3#siRNA, and 4#siRNA.The negative control group was established and treated nothing. These siRNAs were differently transferred into human synovial fibroblasts by LipofectAMINE2000 package. 4 hours later, PMA(phorbol-12-myristate-13-acetate) with a final concentration of 100 nmol/L was added into each culture. The synovial fibroblasts of each group were collected after 36 and 48 hours of transfection, and the protein and total RNA were extracted. 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. Levels of the downstream product of PGE2 in the clear supernatant of the above groups were detected by ELISA. Results The ratios of COX-2 mRNA in NC, 1#-4#siRNA or HeLa group to CTL group were 0.72, 0.3, 0.25, 0.4, 0.04, 2.1 respectively by semi-quantitative RT-PCR. The ratios of protein level in NC,1#siRNA, 2#RNA, 3#RNA,4# group and HeLa cell group to CTL were 1.04、0.52、0.39、0.9、0和2.48 estimated by semi-quantitative method after 36h of transfection and the ratios of each positively treated group to CTL group were 1.05、0.52、0.51、0.9、0.15 after 48 h of transfection.respectively. The level of PGE2 in the clear supernatant was lower in the 4#siRNA group than in the other groups 24 h,36 h, 48 h after tranfectation. Conclusions 4#siRNA could effectively inhibited the expression of COX-2 mRNA and the synthesis of the COX-2 protein in human synovial fibroblasts. The level of PGE2 in 4#siRNA group was also found lowest. Thus 4#siRNA was confirmed specifically blocking the COX-2 in human synovial fibroblasts.更多还原