LXR激動劑調(diào)節(jié)小鼠巨噬細(xì)胞脂質(zhì)內(nèi)穩(wěn)態(tài)及其炎癥因子分泌的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-09-19 07:31
【摘要】： 【目的】肝X受體(liver X receptors, LXRs)是配體依賴性的核轉(zhuǎn)錄因子,可調(diào)控多個(gè)與脂質(zhì)代謝及炎癥反應(yīng)有關(guān)的重要蛋白質(zhì)的表達(dá),在維持膽固醇內(nèi)穩(wěn)態(tài)和調(diào)節(jié)炎癥反應(yīng)中發(fā)揮重要作用。LXRs由DNA結(jié)合域及配體結(jié)合域構(gòu)成。LXRs與維甲酸X受體(retinoid X receptors, RXRs)形成的異源二聚體可被LXRs配體和/或RXRs配體激活,通過與靶基因上的肝X受體反應(yīng)元件(LXR response elements, LXRE)結(jié)合,調(diào)節(jié)靶基因轉(zhuǎn)錄。LXRs的配體包括天然的氧化甾醇(oxysterol)如:20(S)-羥基膽甾醇、22(R)-羥基膽甾醇、24(S)-羥基膽甾醇、27-羥基膽甾醇、24(S),25-環(huán)氧膽固醇以及人工合成的T-1317、GW3965。LXRs激動劑一方面可調(diào)節(jié)膽固醇和脂肪酸代謝,另一方面可調(diào)節(jié)機(jī)體先天性免疫反應(yīng)。LXRs激動劑通過上調(diào)巨噬細(xì)胞ABCA1、ApoE的表達(dá),促進(jìn)巨噬細(xì)胞中膽固醇的外流;并可下調(diào)巨噬細(xì)胞內(nèi)一些促炎癥因子如:iNOS、COX-2、MMP-9的表達(dá)。第三軍醫(yī)大學(xué)藥學(xué)教研室采用高立體選擇性化學(xué)合成法,以豬去氧膽酸為原料高效合成了LXRs激動劑3β-羥基-5α, 6α-環(huán)氧膽酸甲酯(methyl 3β-hydroxy- 5α,6α-epoxycholante, MHEC)。本實(shí)驗(yàn)采用兩種LXR激動劑(MHEC和T-1317)為實(shí)驗(yàn)用藥,對培養(yǎng)的小鼠巨噬細(xì)胞樣細(xì)胞株RAW 264.7細(xì)胞進(jìn)行體外實(shí)驗(yàn)研究。探討LXR激動劑對小鼠巨噬細(xì)胞ABCA1蛋白、清道夫受體以及TNF-α、IL-6的影響。 【方法】應(yīng)用免疫組化S-P法檢測LXR激動劑對RAW 264.7細(xì)胞ABCA1蛋白表達(dá)的影響。用熒光標(biāo)記的ac-LDL(Dil-Ac-LDL)孵育細(xì)胞,通過流式細(xì)胞術(shù)檢測細(xì)胞內(nèi)熒光強(qiáng)度,分析LXR激動劑對RAW 264.7細(xì)胞清道夫受體活性的影響。應(yīng)用夾心法ELISA檢測細(xì)胞培養(yǎng)上清中TNF-α及IL-6水平,分析LXR激動劑對ox-LDL誘導(dǎo)的TNF-α及IL-6產(chǎn)生和分泌的影響�！窘Y(jié)果】 1. MHEC和T-1317兩種LXRs激動劑均可顯著促進(jìn)RAW 264.7細(xì)胞ABCA1蛋白的表達(dá);9-cRA與LXRs激動劑聯(lián)合作用時(shí),ABCA1蛋白表達(dá)的增加更為顯著。 2. MHEC和T-1317兩種LXRs激動劑均可使RAW 264.7細(xì)胞的清道夫受體活性顯著降低。RXRs激動劑9-cRA與T-1317聯(lián)合作用時(shí),清道夫受體活性有更顯著的降低;
[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 偽, 6 偽 -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 偽 -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- 偽 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- 偽 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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R363
本文編號：2249454
[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 偽, 6 偽 -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 偽 -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- 偽 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- 偽 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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前2條
1 鄭輝,徐長榮,周向東;新型LXRs配體——環(huán)氧膽烷酸甲酯一類物的化學(xué)合成[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2001年09期
2 王克勤,何錦麟;一次密度梯度超速離心分離人血清的VLDL、LDL、HDL_2、HDL_3及無脂血清[J];生物化學(xué)雜志;1986年01期
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