中国动脉硬化杂志2011，Vol.19Issue(1)：6-12,7.
小干扰RNA沉默Toll样受体4表达对慢性温和应激ApoE-/-小鼠腹腔巨噬细胞TLR4/NF-κB途径基因表达的影响
Effects of Silencing Toll-Like Receptor 4 Expression on the Expression of Toll-Like Receptor 4/ Nuclear Factor-κB Signaling Pathway in Peritoneal Macrophages of Chronic Mild Stress ApoE-/-Mice
摘要
Abstract
Aim To examine the effect of Toil-like receptor 4(TLR4) gene silencing by small interfering RNA (siRNA) on the expression of cytokines produced by peritoneal macrophages in chronic mild stress (CMS) ApeE -/- mice.Methods Two siRNA sequences and one negative control sequence were designed targeting the mouse TLR4 gene.Two complementary single-strand DNA were designed and synthesized based on siRNA sequences.The DNA fragments were annealed and ligated to the GFP expression vector pRNAT-H1, 1/Adeno.One siRNA with higher interference efficiency than the other was found after siRNA plasmid transfection into 293A cells mediated by liposome.After adenovirus partical packaging and production, the 293A cells were infected, and the single cell clone was acquired and cultured to establish the stable cell strain.The titer of concentrated virus was detected by hole-by-dilution titer assay.One hundred twenty male ApoE-/- mice were divided into groups of CMS control, CMS + empty vector and CMS + siRNA (tail vein injection, 10 μL/mouse; n = 40 per group).All mice were fed a high-fat ( 5 ％, wt/w), high-cholesterol ( 1％, wt/wt) diet and subjected to daily CMS for 0, 4, 12 weeks, respectively.Peritoneal macrophages were prepared from APoE -/- mice and then total proteins from cells were extracted.Western Blotting was used to determine the expressions of TLR4 and nuclear factor-κB (NF-κB) of peritoneal macrophages.The supernatants of cultured peritoneal macrophages were collected.and then used for detection of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) levels by ELISA.Results Compared with the blank control group, real-time PCR showed that the expression of TLR4 mRNA in 293A cells was decreased by 56％ and 67％ after 48 h transfection with siRNA1 and siRNA2, respectively.The hole-by-dilution titer assay showed that viral titer was 3.4 × 1014 TU/L.After exposure to CMS for 4 and 12 weeks, there was no difference in serum corticosterone levels between siRNA group and the CMS control group( P ＞ 0.05 ), and siRNA group mice demonstrated markedly decrease in TLR4 ( P ＜ 0.05 ) and NF-κB ( P ＜ 0.05 ) levels of peritoneal macrophages compared with the corresponding control group, respectively.IL-1β ( P ＜ 0.01 ) and TNF-α ( P ＜ 0.01 ) levels in supernatants of cultured peritoneal macrophages of siRNA group were significantly lower than those of the CMS group.Conclusion These results showed that siRNA effectively inhibited the expressions of TLR4/NF-κB-IL-1β and TNF-α of peritoneal macrophages in CMS ApoE -/- mice, which suggested that the activation of TLR4 pathway of macrophages might play an important role in chronic inflammation response induced by CMS.关键词
小干扰RNA/Toll样受体4/慢性温和应激/核因子κB/肿瘤坏死因子α分类
医药卫生引用本文复制引用
顾洪丰,唐朝克,唐雅玲,覃丽,杨永宗..小干扰RNA沉默Toll样受体4表达对慢性温和应激ApoE-/-小鼠腹腔巨噬细胞TLR4/NF-κB途径基因表达的影响[J].中国动脉硬化杂志,2011,19(1):6-12,7.基金项目
国家自然科学基金(81000946)和湖南省教育厅基金(09C835) （81000946）
