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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 the effect of Toll-like receptor 4 (TLR4) and myeloid differentiation protein2 (MD2) antisense gene on the activation of NF-κB in alveolar type Ⅱ cells induced by lipopolysaccharide (LPS). The cultured alveolar type Ⅱ cells were divided into normal cells (control group), LPS group, LPS + transfected empty vector group, LPS + transfected TLR4 antisense gene group, LPS + transfected MD2 antisense gene group, LPS + transfected TLR4-MD2 anti Genome (n = 8). The expression of TLR4 and MD2 mRNA in transfected cells was detected by Northern blot. The expressions of TLR4 and MD2 protein in transfected cells were detected by Western blot. The activity of NF-κB was detected by electrophoretic mobility shift assay. The levels of TNF-α and IL- 6 content. Results Compared with the control group, the mRNA and protein expressions of TLR4 and MD2, the activities of NF-κB, the production of TNF-α and IL-6 in LPS-stimulated cells increased significantly (P <0.01) LPS stimulation group, TLR4 and MD2 mRNA and protein expression, NF-κB activity and TNF-α and IL-6 levels were significantly lower (P <0.01). Conclusion The antisense gene TLR4 and MD2 can effectively inhibit the activation of alveolar type Ⅱ cells induced by LPS.