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.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-IinhititsLPS-inducedinflammatorycytokinesexpressioninmacrophage-derivedfoamcellsviaregulatingtristetraprolindephosphorylationABSTRACTObjective:To observethe effect ofapolipoproteinA1 (apoA-I)on the expression of inflammatorycytokines and explorethe role ofTTP-translational modification in the anti-inflammatoryeffect of apoA-Iin macrophage-derived foam cells.Methods:HumanTHP-1monocytesweretreatedin vitro with phorbol 12-myristate 13-acetate (PMA) (100μmol/L) for24h,and thenthe medium was replaced by serum-freemedium containingoxLDL(50μg/ml) to become fullydifferentiated macrophage foamcells beforetheir usein experiments. Cells werecultured and treated with apoA-Iand(or)LPS. Protein wasextracted and subjected to ELISA analysis forinterleukin-1β (IL-1β), tumor necrosisfactor-α(TNF-α),IL-6 andIL-8.Total protein extracts from cells weresubjected toWestern-Blot analyses for tristetraprolin (TTP), phospho-TTP, calmodulin (CaM), Calcineurin (CaN).TheAct D was added to stop thetranscription, RNAwereextractedand subjected to RT-PCR analysis forinflammatorycytokines mRNAanalysis. Cells weretreated with CaM antagonistW7, CaN antagonist FK506and siRNAofTTP. Protein and RNAwere extracted and subjected to ELISAor RT-PCR analysis forinflammatorycytokines. Protein was extracted and subjected tochromogenic substrate analysis forCaM and CaN activity.Results:HumanTHP-1macrophage-derived foam cells werepretreated byapoA-I3for2h,LPS-induced up-regulation ofIL-1β,TNF-α,IL-6