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目的分析 siRNA 对前列腺素 E2(PGE2)、白介素-1β(IL-1β)、IL-6、肿瘤坏死因子(TNF-α)和血管内皮生长因子(VEGF)合成的影响。方法设计4条(1~#～4~#)环氧化酶-2(hCOX-2)mRNA 的小分子干扰 RNA(siRNA),设立空白阴性(CTL)和1条随机序列(NC)对照。应用LipofectAMINE 2000将 siRNA 转染入滑膜成纤维细胞(RASF),4 h 后,再加入100 nmol/L 的佛波酯。应用 RT-PCR 和 Western 印迹分别检测 hCOX-2 mRNA 和蛋白表达水平。采用 ELISA 法检测各组上清液中致炎因子水平。结果转染48 h 后,NC、1~#～4~# siRNA 组与 CTL 组 mRNA 条带吸光度比值分别为0.72、0.3、0.25、0.4、0.04。转染36和48 h 后,各组与 CTL 组 hCOX-2蛋白吸光度比值分别为1.04、0.52、0.39、0.9、0和1.05、0.52、0.51、0.9、0.15;4~#siRNA 组上清液 PGE2、IL-1β、TNF-α、IL-6和VEGF 水平较其他各组为低,且与其他处理组相比死亡细胞率差异无统计学意义(P 均>0.05)。结论4~#iRNA 组能有效抑制 COX-2mRNA 和蛋白表达,其上清液中致炎因子水平最低,死亡细胞较其他各组无明显增加。
Objective To analyze the effect of siRNA on the synthesis of PGE2, IL-1β, IL-6, TNF-α and VEGF. Methods Four small interfering RNAs (siRNAs) encoding 4 (1 ~ # ~ 4 ~) cyclooxygenase-2 (hCOX-2) mRNAs were designed and used to establish a blank negative (CTL) and a random sequence (NC) SiRNA was transfected into synovial fibroblasts (RASF) using LipofectAMINE 2000, and after 4 h, 100 nmol / L phorbol ester was added. The expression of hCOX-2 mRNA and protein were detected by RT-PCR and Western blot respectively. The levels of inflammatory cytokines in supernatants of each group were detected by ELISA. Results After 48 h of transfection, the ratio of mRNA bands of NC, 1 ~ # ~ 4 ~ # siRNA group and CTL group were 0.72,0.3,0.25,0.4,0.04 respectively. After 36 and 48 h of transfection, the ratio of hCOX-2 protein absorbance in each group was 1.04, 0.52, 0.39, 0.9, 0 and 1.05, 0.52, 0.51, 0.9 and 0.15 respectively; , IL-1β, TNF-α, IL-6 and VEGF levels were lower than those in other groups. There was no significant difference in the number of dead cells between the two groups (P> 0.05). Conclusion 4 ~ # iRNA group can effectively inhibit the expression of COX-2 mRNA and protein, the lowest level of inflammatory cytokines in the supernatant and no significant increase in dead cells compared with other groups.