Objective To construct the small interfering RNA of ICAM-1 cell ICAM-1,and to explore whether specific siRNA could inhibit the expression of ICAM-1 induced by LPS in A549 cells.Methods Human type 2-like alveolar epithelial cells (A549 cells) were examined for expression of ICAM-1 after treated with lipopolysaccharide (LPS).SiRNA to target human ICAM-1 was synthesized.After transfected with non viral siRNA,A549 cells were treated with LPS.ICAM-1 expression was measured by flow cytometry and RT-PCR.Results The expression of ICAM-1 on A549cells was induced by LPS.But cells were damaged in high LPS concentration.Compared with the OD value of 10 ng/mL LPS group and 100 ng/mL LPS group,there were significant differences between the two groups (P＜0.001).After treated with 10 ng/mL LPS for 8 h or so,there was no obvious damage to the cells.After treated with 10 ng/mL LPS for 1 h,the expression of ICAM-1 mRNA was upregulated (P＜0.05).But the expression of ICAM-1 protion was no significant difference.After treated with 10 ng/mL LPS for 4 h,the expression of ICAM-1 mRNA and protein were upregulated too(P＜0.05).Non-transfected cells showed a strong increase of ICAM-1 expression following LPS stimulation.SiRNA-mediated gene suppression of ICAM-1 was also reflected by corresponding decreases in protein and transcript levels.Conclusion The expression of ICAM-1 on A549 cells can be effectively inhibited by specific siRNAs using a non-viral transfection approach.%目的 构建A549细胞中细胞间粘附分子-1(ICAM-1)的小干扰RNA,探讨ICAM-1特异性siRNA能否抑制LPS诱导A549细胞ICAM-1的表达.方法 采用化学合成法构建A549细胞ICAM-1的小干扰RNA.LPS诱导A549细胞ICAM-1表达,用RT-PCR和流式细胞仪分别从mRNA和蛋白水平检测ICAM-1的表达.使用阳离子脂质体Lipofectamin2000转染ICAM-1siRNA到A549细胞,再用LPS刺激A549细胞,RT-PCR和流式细胞仪分别检测RNA干扰A549细胞后ICAM-1的mRNA和蛋白表达.结果 LPS可以诱导A549细胞ICAM-1的表达,但LPS浓度过大损伤细胞.10 ng/ml的LPS组和100 ng/ml的LPS组OD值相比,两组OD值之间差异有显著性(P＜0.001).10ng/ml的LPS作用A549细胞8h左右,细胞无明显损伤;10 ng/ml LPS作用A549细胞1h后,ICAM-1 mRNA的表达上调(P＜0.05),而ICAM-1蛋白水平增加不明显;10 ng/ml LPS作用A549细胞4h后,ICAM-1 mRNA表达水平明显增加(P ＜0.05),ICAM-1蛋白水平也增加明显.通过化学合成法构建ICAM1的siRNA,筛选出最优siRNA.转染ICAM-1siRNA后,LPS刺激A549细胞的ICAM-1的mRNA和蛋白表达水平增加不明显.结论 通过化学合成法构建的ICAM-1siRNA,可以有效地干扰ICAM-1的表达,ICAM-1siRNA降低了LPS诱导的ICAM-1的表达.
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