Toll样受体2基因小干扰RNA对高氧所致氧化应激状态下肺泡上皮细胞功能的影响

Inhibitory effect of special siRNA targeting toll-like-receptor 2 gene on the inflammatory factor expression of alveolar epithelial cells under hyperoxia-induced oxidative stress

目的探讨Toll样受体2（TLR2）基因小干扰RNA（siRNA）对体外高氧暴露条件下A549细胞TLR2基因、蛋白表达及其下游信号通路功能的影响。方法肺泡上皮细胞株A549置于6孔培养板培养，完全随机分为4组：N组（正常培养组）、H组（高氧组）、T组（高氧±TLR2-siRNA转染组，转染化学合成TLR2-siRNA）和C组（高氧±阴性对照siRNA转染组，转染化学合成含对照序列的阴性对照-siRNA）。运用脂质体转染技术介导化学合成siRNA转染，其中H组、T组和C组细胞高氧培养6h后，流式细胞术检测转染后A549细胞5．羧基荧光素双醋酸盐的表达率，实时定量荧光聚合酶链反应方法检测各组A549细胞TLR2mRNA的表达水平，流式细胞术方法检测各组A549细胞TLR2蛋白表达变化，凝胶电泳迁移率实验检测各组细胞核因子KB核转录水平，酶联免疫吸附测定方法检测各组上清液中白细胞介素6（IL-6）和IL-8含量。结果转染后A549细胞5-羧基荧光素双醋酸盐的表达率为（92．3±9．6）％；经高氧处理后，H组和C组的TLR2mRNA、TLR2蛋白水平、核因子KB核转录水平较N组均明显上调[（1．68±0．02）、（1．52±0．06）比（1．26±0．02），（9．1±0．8）、（7．8±1．4）比（5．9±2．8），（484±64）、（378±38）比（46±3）]（P〈0．05），各组上清液IL-6、IL-8含量较N组也明显升高（P〈0．05）；而T组TLR2mRNA表达（0．87±0．06）、TLR2蛋白表达（2．8±0．4）、核因子KB核转录水平（228±29）和上清液IL-6、IL-8含量较H组和C组均明显下调[IL-6：（48．5±6．3）ng／L比（66．7±7．8）、（59．6±6．5）ng／L，IL-8：（125±19）ng／L比（773±96）、（971±149）ng／L]（均P〈0．05）；T组与N组相比，各项检测指标表达差异也有统计学意义（P〈0．05）。结论针对TLR2mRNA的小干扰RNA可以部分抑制高氧诱导的A549细胞的炎症反应，但是并不能完全阻断核因子KB-炎症因子信号通路。

Objective To study the function of toll-like receptor 2 （TLR2） gene and to observe if the intracellular reactive oxygen species （ ROS ） can activate the signal transduction pathway of TLR2-nuclear factor （NF）-KB-inflammatory mediators resulting in inflammatory response during hyperoxia-induced acute lung injury. Methods The A549 was cultured in six-well plates and randomly divided into group N （air group）, group H （hyperoxia group）, group T（ hyperoxia ± TLR2-siRNA transfected group ） and group C （hyperoxia ± control-siRNA transfected group）. The siRNA was transfected into A549 cells mediated by lipofectamine 2000. Group H, group T and group C were cultured in hyperoxia condition for 6 h. The expression of FAM was detected by flow eytometry to observe the efficiency of transfection. Real-time fluorescence polymerase chain reaction （ Real-time PCR ） method was used to detect the level of mRNA of TLR2. TLR2 protein expression was observed by flow cytometry. The activity of NF-KB in A549 cells was measured with eleetrophoretic mobility shift assay （EMSA） and enzyme-linked immunosorbent assay（ELISA） was used to test the level of IL-6 and IL-8 in the supematants. Results The expression of FAM was （92.3 ±9.6）% after transfection. Compared with group N, the TLR2 mRNA,TLR2 protein level, activity of NF-KB and the IL-6 and IL-8 quantity in group H and group C increased after the hyperoxia treatment[（1.68 ±0.02） ,（1.52 ±0.06） vs （1.26 ±0.02） ;（9.1 ±0.8）, （7.8 ± 1.4） vs （5.9 ±2.8） ;（484 ± 64）, （ 378 ± 38 ） vs （46 ± 3 ） ] （ P 〈 0.05 ）. While the expression of the TLR2 mRNA （ 0.87 ± 0.06 ）, TLR2 protein level（ 2.8 ± 0.47 ） , activity of NF-KB and the IL-6 and IL-8 quantity in the group T were decreased compared with group H and group C [ L-6 ： （48.5 ± 6.3 ） ng/L vs （ 66.7 ± 7.8 ）, （ 59.6 ± 6.5 ） ng/L, IL-8 ： （ 125 ± 19 ） ng/L vs （773 ± 96） , （971 ± 149 ） ng/L ] （ all P 〈 0, 05 ）, The group T had a significant difference from the group N regarding the expression of detection indexes （P 〈 0.05）. Conclusion The siRNA targeting TLR2 mRNA can inhibit the inflammatory reaction.