人肺泡上皮细胞A549中细胞间粘附分子-1的siRNA构建探讨  

Suppression of ICAM-1 in alveolar epithelial cells by small interfering RNA

作　　者：胡晓波[1] 张优仪[2] 程德云[3] 

机构地区：[1]成都市第一人民医院呼吸科,四川成都610016 [2]四川省人民医院放射科,四川成都610072 [3]四川大学华西医院呼吸科,四川成都610041

出　　处：《西部医学》2017年第11期1498-1503,共6页Medical Journal of West China

基　　金：四川省卫生厅科研课题(100051);成都市科技计划资助项目(12PPYB042SF-002)

摘　　要：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。10ng/ml的LPS组和100ng/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;10ng/ml LPS作用A549细胞1h后,ICAM-1mRNA的表达上调(P<0.05),而ICAM-1蛋白水平增加不明显;10ng/ml LPS作用A549细胞4h后,ICAM-1mRNA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ICAM-1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。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 A549 cells 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 hor so,there was no obvious damage to the cells.After treated with 10 ng/mL LPS for1 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.

关 键 词：细胞间黏附分子1 小干扰RNA 脂多糖 转染 

分 类 号：R56[医药卫生—呼吸系统] R392.11[医药卫生—内科学]