应用载体介导小干扰RNA抑制小鼠巨噬细胞表达肿瘤坏死因子α

Inhibition of tumor necrosis factor-or expression in macrophages with vector-mediated RNA interference

目的研究特异性载体介导的小干扰RNA（siRNA）对小鼠巨噬细胞株表达肿瘤坏死因子（TNF）-α基Ⅲ的抑制作用。方法设计两段靶向TNF-α基因的特异性siRNA。合成相应寡核苷酸。插入载体H1启动子下游，构建表达干扰性发央状RNA（shRNA）载体pHS-A和pHS-B，转染小鼠巨噬细咆株（RAW264．7）。用实时荧光定量PCR和酶联免疫吸附法检测siRNA对TNF-amRNA和蛋白表达的抑制作用。结果内毒素脂多糖（LPS）刺激后6h。巨噬细胞表达TNF-α mRNA和合成的TNF-α量均增加。于9-12h达高峰。PHS-A转染后。LPS刺激巨噬细胞的TNF-α mRNA为0．00356±0．00187，TNF-α蛋白表达为（149．93±21．02）Pg／ml，比未转染组[分别为0．02134±0．00960、（1922．30±149．05）pg／m1]显著减少（P〈O．01），抑制率达83．3％。pHS-B及阴性对照组对基因及蛋白表达均无影响。结论LPS可刺激小鼠巨噬细胞合成TNF-α,pHS-A可抑制小鼠巨噬细胞TNF-α表达。

Objective To investigate the inhibitory effect of small interference RNA （siRNA） on the expression of tumor necrosis factor （TNF）-α in the LPS treated macrophages. Methods Two pairs of oligo nucleotide fragments were designed and synthesized according to the sequence of TNF-α gene and insert downstream to H1 promoter to construct plasmids pHS-A and pHS-B for expression of short hairpin RNA（shRNA）. Then the recombinant pSilence3. 1-TNF-α vectors were transfected into mouse macrophages （RAW264.7） by lipofectamin 2000. The inhibition of TNF-α mRNA and protein expression were examined by real time PCR and ELISA, respectively. Results After LPS stimulating 6 hours, TNF-α expression was increased in a specific time-dependent manner, and reached high peak at 9-12 hours. The macrophages treated with plasmids pHS-A showed a significant decrease in TNF-α mRNA（0. 021 34±0.00960）and protein （149. 93±21. 02 pg/ml, P〈0.01） compared with untransfected group rTNF-α mRNA： 0. 021 34±0. 009 60, protein： 1 922. 30±149. 051 pg/ml]. The relative rate of inhibition was 83.3 %. No inhibitory effects was found in plasrnids pHS-B and control group. Conclusions LPS stimulation results in a increasing expression of TNF-α. The plasmids pHS-A successfully inhilly the expression of TNF-α mRNA and protein in macrophages treated with LPS.