LXR激动剂调节小鼠巨噬细胞脂质内稳态及其炎症因子分泌的实验研究

发布时间：2018-09-19 07:31

【摘要】： 【目的】肝X受体(liver X receptors, LXRs)是配体依赖性的核转录因子,可调控多个与脂质代谢及炎症反应有关的重要蛋白质的表达,在维持胆固醇内稳态和调节炎症反应中发挥重要作用。LXRs由DNA结合域及配体结合域构成。LXRs与维甲酸X受体(retinoid X receptors, RXRs)形成的异源二聚体可被LXRs配体和/或RXRs配体激活,通过与靶基因上的肝X受体反应元件(LXR response elements, LXRE)结合,调节靶基因转录。LXRs的配体包括天然的氧化甾醇(oxysterol)如:20(S)-羟基胆甾醇、22(R)-羟基胆甾醇、24(S)-羟基胆甾醇、27-羟基胆甾醇、24(S),25-环氧胆固醇以及人工合成的T-1317、GW3965。LXRs激动剂一方面可调节胆固醇和脂肪酸代谢,另一方面可调节机体先天性免疫反应。LXRs激动剂通过上调巨噬细胞ABCA1、ApoE的表达,促进巨噬细胞中胆固醇的外流;并可下调巨噬细胞内一些促炎症因子如:iNOS、COX-2、MMP-9的表达。第三军医大学药学教研室采用高立体选择性化学合成法,以猪去氧胆酸为原料高效合成了LXRs激动剂3β-羟基-5α, 6α-环氧胆酸甲酯(methyl 3β-hydroxy- 5α,6α-epoxycholante, MHEC)。本实验采用两种LXR激动剂(MHEC和T-1317)为实验用药,对培养的小鼠巨噬细胞样细胞株RAW 264.7细胞进行体外实验研究。探讨LXR激动剂对小鼠巨噬细胞ABCA1蛋白、清道夫受体以及TNF-α、IL-6的影响。【方法】应用免疫组化S-P法检测LXR激动剂对RAW 264.7细胞ABCA1蛋白表达的影响。用荧光标记的ac-LDL(Dil-Ac-LDL)孵育细胞,通过流式细胞术检测细胞内荧光强度,分析LXR激动剂对RAW 264.7细胞清道夫受体活性的影响。应用夹心法ELISA检测细胞培养上清中TNF-α及IL-6水平,分析LXR激动剂对ox-LDL诱导的TNF-α及IL-6产生和分泌的影响。【结果】 1. MHEC和T-1317两种LXRs激动剂均可显著促进RAW 264.7细胞ABCA1蛋白的表达;9-cRA与LXRs激动剂联合作用时,ABCA1蛋白表达的增加更为显著。 2. MHEC和T-1317两种LXRs激动剂均可使RAW 264.7细胞的清道夫受体活性显著降低。RXRs激动剂9-cRA与T-1317联合作用时,清道夫受体活性有更显著的降低;
[Abstract]:[objective] liver X receptor (liver X receptors, LXRs) is a ligand dependent nuclear transcription factor that regulates the expression of several important proteins related to lipid metabolism and inflammation. LXRs play an important role in maintaining cholesterol homeostasis and regulating inflammatory response. LXRs, composed of DNA binding domain and ligand binding domain, can be activated by LXRs ligands and / or RXRs ligands. By binding to the liver X receptor response element (LXR response elements, LXRE) on the target gene, Ligands regulating target gene transcription. LXRs include natural oxosterol (oxysterol) such as: 20 (S)-hydroxyl cholerosterol 22 (R)-hydroxy choleride, 24 (S)-hydroxyl cholestanosterol, 24 (S) 25-epoxide cholesterol and synthetic T-1317 GW3965. LXRs agonist can regulate gallbladder on the one hand. Steroid and fatty acid metabolism, On the other hand, it can modulate the innate immune response. LXRs agonist can promote cholesterol efflux by up-regulating the expression of ABCA1,ApoE in macrophages, and down-regulate the expression of pro-inflammatory factors such as: iNOS, COX-2, MMP-9 in macrophages. LXRs agonist 3 尾 -hydroxy-5 伪, 6 伪 -epoxycholante (MHEC).) was synthesized from porcine deoxycholic acid by high-stereoselective chemical synthesis method in the Department of Pharmacy, third military Medical University. Methyl 3 尾 -hydroxy-5 伪 -epoxycholante (MHEC).) was synthesized efficiently. In this experiment, two LXR agonists (MHEC and T-1317) were used as experimental drugs to study the culture of murine macrophage like cell line RAW 264.7 in vitro. To investigate the effect of LXR agonist on ABCA1 protein in mouse macrophages. [methods] the effects of LXR agonists on the expression of ABCA1 protein in RAW 264.7 cells were detected by immunohistochemical S-P method. Fluorescence labeled ac-LDL (Dil-Ac-LDL) was used to incubate the cells. The intracellular fluorescence intensity was detected by flow cytometry. The effect of LXR agonist on the scavenger receptor activity of RAW 264.7 cells was analyzed. The levels of TNF- 伪 and IL-6 in the supernatant of cell culture were detected by sandwich ELISA. The effects of LXR agonists on the production and secretion of TNF- 伪 and IL-6 induced by ox-LDL were analyzed. [results] 1. Both MHEC and T-1317 LXRs agonists could significantly promote the expression of ABCA1 protein in RAW 264.7 cells. Both MHEC and T-1317 LXRs agonists could significantly reduce the scavenger receptor activity of RAW 264.7 cells. The scavenger receptor activity of RAW 264.7 cells was significantly decreased when the combination of 9-cRA and T-1317.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R363

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