人肺上皮细胞感染呼吸道合胞病毒后核内活性因子κB与诱导型一氧化氮合酶的表达

Expression of nuclear factor kappa B and inducible nitric oxide synthase in the respiratory syncytial virus infection on human lung epithelial cells

目的:呼吸道合胞病毒感染呼吸道上皮细胞后,诱导细胞过度表达活性递质。实验拟观察呼吸道合胞病毒感染人肺上皮细胞A549后核内活性核因子κB,诱导型一氧化氮合酶mRNA和蛋白、一氧化氮和丙二醛表达的变化。方法:实验于2006-12/2007-05在安徽医科大学基础医学院病原与免疫学实验中心完成。①实验过程:将冻存的病毒株复苏后经Hep-2细胞增殖。用呼吸道合胞病毒感染体外培养的A549细胞,分别于感染4,8,16,24h后收集细胞和细胞培养上清。以未感染病毒的细胞为正常对照组。②实验评估:反转录聚合酶链反应检测诱导型一氧化氮合酶mRNA表达的变化;免疫印迹法(Western-Blot)分别检测核内活性核因子κB、诱导型一氧化氮合酶蛋白的变化;用硝酸还原酶法、硫代巴比妥酸法分别检测细胞培养上清一氧化氮和丙二醛的表达。结果:①A549细胞中核内活性核因子κB有基础表达,经由呼吸道合胞病毒感染后,核内活性核因子κB表达升高,且随着感染时间的延长而增加。②呼吸道合胞病毒感染A549细胞后,诱导型一氧化氮合酶mRNA和蛋白的表达均升高且有时间依赖性,24h的诱导型一氧化氮合酶mRNA表达量是基础表达量的近12倍,诱导型一氧化氮合酶蛋白的表达亦显著高于正常对照组。核内活性核因子κB与诱导型一氧化氮合酶的mRNA和蛋白升高表达呈正相关。③呼吸道合胞病毒感染能促进A549细胞培养上清中一氧化氮和丙二醛的表达升高,24h内细胞上清中的一氧化氮呈缓慢升高变化。结论:在呼吸道合胞病毒感染A549细胞的炎性反应中核内活性核因子κB升高,与诱导型一氧化氮合酶基因和蛋白的上升表达呈正相关。

AIM： After human lung epithelial cells （A549） infected with respiratory syncytial virus （RSV）, inducer cells over-express active transmitter. The study investigated the expression changes in nuclear factor κB （NF- κB）, inducible nitric oxide synthase （iNOS） mRNA and protein, nitric oxide （NO） and malonaldehyde （MDA） on human lung epithelial cells （A549） infected with RSV. METHODS： Experiments were performed at the Laboratory of Etiology and Immunology, Anhui Medical University between December 2006 and May 2007. （1） Gyopreserved virus broliferated in Hep-2 cells after recovery. A549 cells infected with RSV in vitro were used to collect cells and cellular culture superuatant at hours 4, 8, 16 and 24. Cells non-infected with RSV were served as controls. （2） Reverse transcription-polymerase chain reaction （RT-PCR） was used to evaluate the expression of iNOS mRNA. The expression of iNOS protein and NF- κB was detected by Western-Blot, The concentrations of NO and MDA were measured by nitrate reductase method and thiobarbituric acid method. RESULTS： （1）The basal expression of NF- κB was increased with the prolongation of RSV infection to A549 cells.（2）RSV infection to A549 increased the amounts of mRNA and protein of iNOS in a time-dependent manner. The expression of iNOS mRNA was about 12 times as many as basal expression, RSV infection promoted the expression of iNOS protein, which was higher than the control group, The activities of NF- κB significantly were positively correlated with the mRNA and protein expression of iNOS. （3） RSV could improve A549 cells secreting NO and MDA, but the NO levels rose slowly during the 24 hours of infection. CONCLUSION： The inflammatory response of RSV can increase the activity of NF-κB and it is positive correlated with the increase in iNOS mRNA and protein levels.