Tan Bing, Li Yu-yuan, Nie Yu-qiang, DU Yan-lei
Department of Gastroenterology, First Municipal People's Hospital of Guangzhou, Guangzhou Medical College, Guangzhou 510180, China.
Zhonghua Yi Xue Za Zhi. 2007 Aug 14;87(30):2140-3.
To investigate the effects of siRNAs targeting mouse tumor necrosis factor (TNF)-alpha gene on the expression of TNF-alpha mRNA and protein in the LPS activated macrophages.
Mouse macrophages of the line RAW264.7 were cultured. Three siRNA sequences targeting different sites of mouse TNF-alpha gene (siRNA 1 - 3) and a fluorescein-labeled double-stranded RNA (dsRNA) oligomer (siRNA 4), as negative control, were designed and chemically synthesized. All siRNAs were transfected into the mouse macrophages. 0, 3, 6, 9, 12, 15, and 18 hours after the transfection samples of supernatant were collected. Inverse microscopy was used to observe the efficacy of transfection. Then the macrophages were co-incubated with LPS for 9 hours. Macrophages stimulated by LPS only and macrophages without transfection and LPS stimulation were used as controls. Then samples of supernatant were collected. TNF-alpha protein expression was detected by ELISA. Real time PCR was used to detect the TNF-alpha mRNA expression.
The transfection rate was 72% - 80%. The TNF-alpha mRNA expression levels of the LPS + siRNA2 and LPS + siRNA3 groups were 0.158 +/- 0.031 and 0.114 +/- 0.028 respectively, both significantly lower than that of the LPS control group (0.294 +/- 0.147, P < 0.05 and P < 0.01 respectively), however, the TNF-alpha mRNA expression level of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group. The TNF-alpha mRNA expression inhibition rates of the LPS + siRNA and LPS + siRNA3 groups were 46.0% and 61.2% respectively. The TNF-alpha protein expression levels of the LPS + siRNA2 and LPS + siRNA3 group were (1358 +/- 348) pg/ml and (817 +/- 138) pg/ml, both significantly lower than that of the LPS control group [(2104 +/- 32) pg/ml, P < 0.05 and P < 0.01], and the TNF-alpha mRNA expression levels of the LPS + siRNA1 and LPS + siRNA4 groups were not significantly different from that of the LPS control group.
LPS time-dependently increases the expression of TNF-alpha in macrophages. SiRNAs targeting TNF-alpha inhibit the expression levels of TNF-alpha mRNA and protein in macrophages treated with LPS.
研究靶向小鼠肿瘤坏死因子（TNF）-α基因的小干扰RNA（siRNA）对脂多糖（LPS）激活的巨噬细胞中TNF-α mRNA和蛋白表达的影响。
培养RAW264.7小鼠巨噬细胞系。设计并化学合成了靶向小鼠TNF-α基因不同位点的3条siRNA序列（siRNA 1 - 3）以及作为阴性对照的荧光素标记双链RNA（dsRNA）寡聚物（siRNA 4）。将所有siRNA转染至小鼠巨噬细胞。转染后0、3、6、9、12、15和18小时收集上清液样本。采用倒置显微镜观察转染效果。然后将巨噬细胞与LPS共孵育9小时。仅用LPS刺激的巨噬细胞以及未转染和未用LPS刺激的巨噬细胞作为对照。随后收集上清液样本。采用酶联免疫吸附测定（ELISA）检测TNF-α蛋白表达。采用实时聚合酶链反应（PCR）检测TNF-α mRNA表达。
转染率为72% - 80%。LPS + siRNA2组和LPS + siRNA3组的TNF-α mRNA表达水平分别为0.158±0.031和0.114±0.028，均显著低于LPS对照组（0.294±0.147，分别为P < 0.05和P < 0.01），然而，LPS + siRNA1组和LPS + siRNA4组的TNF-α mRNA表达水平与LPS对照组无显著差异。LPS + siRNA2组和LPS + siRNA3组的TNF-α mRNA表达抑制率分别为46.0%和61.2%。LPS + siRNA2组和LPS + siRNA3组的TNF-α蛋白表达水平分别为（1358±348）pg/ml和（817±138）pg/ml，均显著低于LPS对照组[（2104±32）pg/ml，P < 0.05和P < 0.01]，LPS + siRNA1组和LPS + siRNA4组的TNF-α mRNA表达水平与LPS对照组无显著差异。
LPS可使巨噬细胞中TNF-α的表达呈时间依赖性增加。靶向TNF-α的siRNA可抑制LPS处理的巨噬细胞中TNF-α mRNA和蛋白的表达水平。
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