CAJ | 학술논문

目的探讨小于扰RNA(siRNA)抑制核因子κB(NF-κB)信号通路对细菌脂多糖(LPS)刺激的巨噬细胞释放肿瘤坏死因子α(TNF-α)的影响.方法人单核细胞白血病THP-1细胞经氟波酯诱导为巨噬细胞后分2组,分别感染应用分子克隆技术构建的针对NF-κB P65的siRNA逆转录病毒质粒(siRNA病毒组)及乱序(Scramble)对照序列逆转录病毒质粒(Scramble病毒组).用终浓度50μg/ml的LPS持续刺激2组巨噬细胞,分别应用RT-PCR技术检测LPS刺激不同时间巨噬细胞NF-κB P65 mRNA和TNF-α mRNA表达水平,蛋白质印迹法检测NF-κB P65蛋白表达水平,酶联免疫吸附试验检测巨噬细胞培养上清液TNF-α含量.结果 LPS刺激12和24 h,siRNA病毒组巨噬细胞NF-κB P65 mRNA表达水平(0.97±0.02,0.89±0.01)明显低于Scramble病毒组(1.01±0.03,0.97±0.01,均P＜0.05);LPS刺激24和72 h,siRNA病毒组NF-κB P65蛋白表达水平(0.95±0.04,0.94±0.01)明显低于Scramble病毒组(1.07±0.06,1.03±0.05,均P＜0.05).LPS刺激4、8、12和24 h的各时点,siRNA病毒组TNF-α mRNA表达水平均明显低于Scramble病毒组(0.92±0.02比0.98 ±-0.01,0.86±0.02比1.00±0.01,0.79±0.03比1.01±0.01,0.78±0.03比1.02±0.01,均P＜0.05).LPS刺激2、4、8、24、36、48、54和72 h的各时点,siRNA病毒组巨噬细胞培养上清液TNF-α含量均明显低于Sramble病毒组(均P＜0.01).结论针对NF-κB P65的siRNA可抑制NF-κB信号通路的功能活性,进而抑制内毒素刺激引起的巨噬细胞活化和TNF-α释放,提示RNA干扰技术在急性肺损伤早期过度炎症反应的防治中具有潜在应用价值.
목적 탐토소우우RNA(siRNA)억제핵인자κB(NF-κB)신호통로대세균지다당(LPS)자격적거서세포석방종류배사인자α(TNF-α)적영향.방법 인단핵세포백혈병THP-1세포경불파지유도위거서세포후분2조,분별감염응용분자극륭기술구건적침대NF-κB P65적siRNA역전록병독질립(siRNA병독조)급란서(Scramble)대조서렬역전록병독질립(Scramble병독조).용종농도50μg/ml적LPS지속자격2조거서세포,분별응용RT-PCR기술검측LPS자격불동시간거서세포NF-κB P65 mRNA화TNF-α mRNA표체수평,단백질인적법검측NF-κB P65단백표체수평,매련면역흡부시험검측거서세포배양상청액TNF-α함량.결과 LPS자격12화24 h,siRNA병독조거서세포NF-κB P65 mRNA표체수평(0.97±0.02,0.89±0.01)명현저우Scramble병독조(1.01±0.03,0.97±0.01,균P＜0.05);LPS자격24화72 h,siRNA병독조NF-κB P65단백표체수평(0.95±0.04,0.94±0.01)명현저우Scramble병독조(1.07±0.06,1.03±0.05,균P＜0.05).LPS자격4、8、12화24 h적각시점,siRNA병독조TNF-α mRNA표체수평균명현저우Scramble병독조(0.92±0.02비0.98 ±-0.01,0.86±0.02비1.00±0.01,0.79±0.03비1.01±0.01,0.78±0.03비1.02±0.01,균P＜0.05).LPS자격2、4、8、24、36、48、54화72 h적각시점,siRNA병독조거서세포배양상청액TNF-α함량균명현저우Sramble병독조(균P＜0.01).결론 침대NF-κB P65적siRNA가억제NF-κB신호통로적공능활성,진이억제내독소자격인기적거서세포활화화TNF-α석방,제시RNA간우기술재급성폐손상조기과도염증반응적방치중구유잠재응용개치.
Objective To explore the effect of small interfering RNA (siRNA) targeting NF-κB signal pathway on the expression level of tumor necrosis factor α (TNF-α) released by lipopolysaccharides (LPS)-stimulating-macrophages. Methods Human monocytic THP-1 cell was induced by phorbol myristate acetate (PMA) and transformed into macrophage. Two groups of macrophage were infected by siRNA retroviral expression vector specific to NF-κB functional subunit P65 (siRNA group) and Scramble control vector (Scramble control group) constructed by molecular cloning technology. Lipopolysaccharide (50 μg/ml)was used to treat the macrophages continuously. RT-PCR was performed to detect the expression level of NF-κB P65 mRNA and TNF-α mRNA at different time-points of LPS stimulation. Western blotting was used to analyze the protein level of NF-κB P65. Enzyme-linked immunosorbent assay was applied to analyze the expression level of TNF-α released by LPS-stimulated macrophages. Results At Hours 12 and 24 after LPSstimulation, the expression level of NF-κB P65 mRNA in siRNA group (0. 97 ± 0. 02, 0. 89 ± 0. 01 ) was significantly less than that in Scramble control group ( 1.01 ± 0. 03, 0. 97 ± 0. 01, both P ＜ 0. 05 ). At Hours 24 and 72 after LPS stimulation, the expression level of NF-κB P65 protein in siRNA group (0. 95 ±0. 04, 0.94 ±0. 01 ) was obviously less than that in Scramble control group ( 1.07 ± 0. 06, 1.03 ± 0. 05,both P ＜0. 05). At Hours 4, 8, 12 and 24 after LPS stimulation, TNF-α mRNA released by siRNA group macrophages was far less than that by Scramble control group macrophages ( 0.92 ± 0. 02 vs 0. 98 ± 0. 01,0.86±0.02 vs 1.00 ±0.01,0.79 ±0.03 vs 1.01 ±0.01,0.78 ±0.03 vs 1.02±0.01, all P＜0.05). At Hours 2, 4, 8, 24, 36, 48, 54 and 72 after LPS stimulation, the TNF-α content in culture medium supernatant in siRNA group macrophage was less than that in scramble control group (P ＜ 0. 05 ).Conclusion NF-κB P65 siRNA inhibits the functional activity of NF-κB signal pathway in PMA-induced macrophage. Then it blocks the activation of macrophage and the excessive release of TNF-α due to endotoxin stimulation. The RNA interference technology may be applied to prevent and treat excessive inflammatory reaction in acute lung injury.