TLR4及MD2反义基因对内毒素诱导的肺泡Ⅱ型细胞活化的影响

TLR4 and MD2 antisense genes suppress activation of alveolar typeⅡ epithelial cells induced by lipopolysaccharide

目的观察Toll样受体4(Toll-like receptor4,TLR4)及myeloid differentiation protein2(MD2)反义基因对内毒素(lipopolysaccharide,LPS)诱导的肺泡Ⅱ型细胞NF-κB活化的影响。方法培养的肺泡Ⅱ型细胞分为:正常细胞(对照)组、LPS组、LPS+转染空载体组、LPS+转染TLR4反义基因组、LPS+转染MD2反义基因组、LPS+转染TLR4-MD2反义基因组(n=8)。Northern blot检测转染细胞的TLR4及MD2 mRNA表达,Western blot检测转染细胞TLR4及MD2蛋白表达,电泳迁移率改变法检测NF-κΒ的活性,ELISA测定细胞培养上清中TNF-α和IL-6含量。结果与对照组比较,LPS刺激后的细胞TLR4及MD2mRNA和蛋白表达、NF-κB活性、TNF-α和IL-6的生成均显著增加(P<0.01);而转染反义基因组细胞与其他LPS刺激组比较,TLR4及MD2mRNA和蛋白的表达、NF-κB活性及TNF-α和IL-6的含量均明显降低(P<0.01)。结论 TLR4及MD2反义基因能有效抑制LPS诱导的肺泡Ⅱ型细胞的活化。

Objective To observe effects of the antisense genes of Toll-like receptor 4 （TLR4 ） and myeloid differentiation protein-2 （MD2） on the activation of NF-κB in alveolar typeⅡepithelial cells （ATⅡcells） induced by lipopolysaccharide （LPS）. Methods Cultured ATⅡcells were randomized to normal cells （control） group,LPS group,LPS＋empty vector group,LPS＋TLR4 antisense gene group,LPS＋MD2 antisense gene group,LPS＋TLR4-MD2 antisense gene group. The expressions of TLR4 and MD2 mRNA and protein in ATⅡcells were detected by Northern blotting and Western blotting respectively. The activity of NF-κB in ATⅡcells was measured with electrophoretic mobility shift assay （EMSA）. The TNF-α and IL-6 concentrations in the supernatant of cultured ATⅡcells were tested with ELISA.Results Compared with control groups,the expressions of TLR4 and MD2 mRNA and protein,the NF-κΒ activity,the levels of TNF-α and IL-6 were increased remarkably in LPS group and LPS＋empty vector group （P0.01）. After stimulating with the antisense genes,the expressions of TLR4 and MD2 mRNA and protein,the NF-κΒ activity,the levels of TNF-α and IL-6 were decreased remarkably than in LPS and LPS＋empty vector group （P0.01）. Conclusion The antisense gene of TLR4 and MD2 can inhibit activation of ATⅡ cells induced by LPS.