apoai调节lps处理泡沫细胞锌指蛋白36去磷酸化的抗炎作用及其机制分析word格式论文.docxVIP

处理细胞，以ELISA、实时定量 PCR 等方法分别检测巨噬细胞炎症因子蛋白质和 mRNA 的表达；发色底物法检测 CaM 和 CaN 的活性。结果：相对于 LPS 单独处理组，apoA-I 预处理 THP-1 巨噬细胞源性泡沫细胞 2h后 显著降低 LPS 诱导的炎症因子 IL-1β、TNF-α、IL-6 和 IL-8 表达水平； Act D 终止转录后，apoA-I 促进 IL-1β mRNA 的降解；apoA-I 促进 LPS 刺激 THP-1 巨噬细胞源性泡沫细胞 TTP 去磷酸化，对 CaN 和 CaM 表达没有影 响，但增强 CaM 和 CaN 活性。TTP siRNA下调 TTP 的表达，并明显抑制 apoA-I促进 THP-1 巨噬细胞源性泡沫细胞 IL-1β mRNA 降解的效应。W7 和 FK506分别抑制 CaM 和 CaN 活性，并明显抑制 apoA-I 对 phospho-TTP 表达 的下调作用，也抑制了apoA-I 促进 IL-1β mRNA 降解的效应。结论：在 THP-1 巨噬细胞源性泡沫细胞中，apoA-I 通过激活细胞内 CaM/CaN 信号转导途径，促进 TTP 去磷酸化，增强 TTP 功能，从而促进炎症因子 mRNA 的降解。关键词：锌指蛋白 36；载脂蛋白 A-I；泡沫细胞；炎症；动脉粥样硬化2ApoA-I inhitits LPS-induced inflammatory cytokines expression in macrophage-derived foam cells via regulating tristetraprolin dephosphorylationABSTRACTObjective: To observe the effect of apolipoprotein A1 (apoA-I) on the expression of inflammatory cytokines and explore the role of TTP-translational modification in the anti-inflammatory effect of apoA-I in macrophage-derived foam cells.Methods: Human THP-1 monocytes were treated in vitro with phorbol 12-myristate 13-acetate (PMA) (100μmol/L) for 24h, and then the medium was replaced by serum-free medium containing oxLDL (50 μg/ml) to become fully differentiated macrophage foam cells before their use in experiments. Cells were cultured and treated with apoA-I and (or) LPS. Protein was extracted and subjected to ELISA analysis for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6 and IL-8.Total protein extracts from cells were subjected to Western-Blot analyses for tristetraprolin (TTP), phospho-TTP, calmodulin (CaM), Calcineurin (CaN). The Act D was added to stop the transcription, RNA were extracted and subjected to RT-PCR analysis for inflammatory cytokines mRNA analysis. Cells were treated with CaM antagonist W7, CaN antagonist FK506 and siRNA of TTP. Protein and RNA were extracted and subjected to ELISA or RT-PCR analysis for inflammatory cytokines. Protein was extracted and subjected to chromogenic substrate analysis for C