PPARδ激动剂GW501516在oxLDL诱导THP-1源性巨噬细胞凋亡及脂质蓄积中的作用

发布时间：2018-03-06 20:35

本文选题：动脉粥样硬化　切入点：过氧化物酶体增殖物激活型受体δ　出处：《南华大学》2009年硕士论文　论文类型：学位论文

【摘要】：研究背景：过氧化物酶体增殖物激活型受体δ(peroxisome Proliferator- activated receptorδ, PPARδ)是过氧化物酶体增殖物激活型受体家族中的一员,广泛表达于多种器官和组织,主要控制脂肪组织和肌肉中脂肪酸氧化和能量解偶联,抑制巨噬细胞诱导的炎症,改善动脉粥样硬化,并参与许多疾病的发生和发展过程。PPARδ的配体分为天然配体和合成配体。GW501516是PPARδ的合成配体,属于苯氧乙酸衍生物,PPARδ与GW501516结合后,活化的PPARδ与9-顺视黄酸类受体(9-cisretinoid X receptor, RXR)形成异二聚体,然后识别并于靶基因启动子上游的过氧化物酶体增殖物反应元件(peroxisome proliferator response element, PPRE)结合,调节靶基因下游的基因转录、翻译及生物学效应。本课题观察oxLDL对THP-1源性巨噬细胞PPARδ表达的影响。同时以PPARδ激动剂GW501516来探讨PPARδ对巨噬细胞增殖、凋亡的影响及在巨噬细胞荷脂过程的作用。第一部分oxLDL对THP-1源性巨噬细胞PPARδ表达的影响目的：观察oxLDL对THP-1源性巨噬细胞PPAR 8表达的影响。方法：1.用不同浓度(0mg/L、25mg/L、50mg/L、100mg/L)的oxLDL与THP-1源性巨噬细胞共孵育24小时；用RT-PCR及Western blotting检测细胞PPARδmRNA和蛋白的表达。2.50mg/L oxLDL与THP-1源性巨噬细胞共孵育0h、12h、24h、48h等不同时间,用RT-PCR及Western blotting检测细胞PPAR 8 mRNA和蛋白的表达。结果：不同浓度oxLDL可诱导THP-1源性巨噬细胞PPAR 8的表达。随着oxLDL浓度的增加,细胞PPAR8的mRNA水平及蛋白表达逐渐增加,差别有显著性,且在浓度为50mg/L时PPAR 8 mRNA的表达达峰值(P0.05)。而时效试验结果显示50mg/L oxLDL与THP-1源性巨噬细胞孵育孵育24h时,PPARδmRNA水平及蛋白表达增加明显,且差别有统计学意义(P0.05)。而在孵育48h后,表达较24h时有所减少,但与24h相比差别无统计学意义。结论：oxLDL可上调THP-1源性巨噬细胞PPARδ的表达第二部分PPARδ对THP-1源性巨噬细胞增殖、凋亡的影响目的：通过PPARδ激动剂GW501516活化PPARδ来观察PPARδ在oxLDL诱导THP-1源性巨噬细胞增殖、凋亡中的作用。方法：THP-1单核细胞经PMA诱导分化成为巨噬细胞后,以50mg/L oxLDL孵育THP-1源性巨噬细胞24h为阳性对照组,50mg/L oxLDL与不同浓度的PPARδ激动剂GW501516 (1、10、100nmol及1μmol)共同孵育THP-1源性巨噬细胞24h,MTT法检测THP-1源性巨噬细胞增殖活性,Hoechst33258染色、Annexin V/PI双染后细胞流式仪检测空白组、oxLDL组及1μmol GW501516组细胞凋亡情况,分光光度法测定细胞内caspase 3活性变化。结果：PPARδ激动剂GW501516可阻抑oxLDL对THP-1源性巨噬细胞增殖的抑制作用,且能减少oxLDL诱导THP-1源性巨噬细胞的凋亡。MTT及Hoechst33258染色结果显示在GW501516浓度达100nmol时作用显著,且1μmol浓度时作用更明显(P0.05)。流式分析结果也同样表明1μmol GW501516能显著抑制oxLDL诱导THP-1源性巨噬细胞的凋亡(P0.05)。分光光度法检测细胞内Caspase 3活性显示GW501516能呈浓度依赖性下调caspase 3的活性(P0.05) 结论：PPARδ激动剂GW501516可下调caspase 3活性,抑制oxLDL诱导的THP-1源性巨噬细胞凋亡。第三部分PPARδ对THP-1源性巨噬细胞脂质蓄积的影响目的：通过PPARδ激动剂GW501516活化PPARδ来观察PPARδ对THP-1源性巨噬细胞脂质蓄积的影响的影响。方法：THP-1单核细胞经PMA诱导分化成为巨噬细胞后,以50mg/L oxLDL孵育THP-1源性巨噬细胞24h为阳性对照组,50mg/L oxLDL与不同浓度的PPARδ激动剂GW501516 (1、10、100nmol及1μmol)共同孵育THP-1源性巨噬细胞24h,油红O染色观察细胞内脂质蓄积情况,高效液相色谱检测细胞内总胆固醇、和胆固醇酯含量。结果：PPARδ激动剂GW501516可增加THP-1源性巨噬细胞内脂质蓄积,油红0染色显示100nmol GW501516处理后可见细胞内脂滴颗粒体积明显增大且脂滴数增多,当GW501516浓度达1μmol时作用更为明显(P0.05)。高效液相色谱检测结果显示阳性对照组总胆固醇含量为483.10±12.70,胆固醇酯/总胆固醇(%)为49.60%。1、10、100nmol和1μmolGW501516处理组总胆固醇含量分别为501.53±15.73,497.69±14.25,647.42±18.62和696.55±20.35；胆固醇酯/总胆固醇(%)分别为56.7%,53.90%,64.48%和66.26%,100nmol和1μmol GW501516组与阳性对照组比较差别有显著性(P0.05)。结论：PPARδ激动剂GW501516可增加THP-1源性巨噬细胞内脂质蓄积。总结： (1) oxLDL可上调THP-1源性巨噬细胞PPARδ的表达。 (2) PPARδ激动剂GW501516可下调caspase 3活性,抑制oxLDL诱导的THP-1源性巨噬细胞凋亡。 (3) PPARδ激动剂GW501516可增加THP-1源性巨噬细胞内脂质蓄积。
[Abstract]:Background: peroxisome proliferator activated receptor delta (peroxisome Proliferator- activated receptor 8, PPAR 8) is a member of the peroxisome proliferator activated receptor family, is widely expressed in a variety of organs and tissues, mainly in adipose tissue and muscle control in fatty acid oxidation and energy uncoupling, inhibit inflammation, improve atherosclerosis induced by macrophages and the occurrence and development of.PPAR delta ligands and are involved in many diseases are divided into natural and synthetic ligand ligand.GW501516 is a synthetic ligand of PPAR 8, which belongs to the Phenoxy Acetic acid derivatives, PPAR 8 when combined with GW501516, PPAR and delta 9- activated CIS retinoic acid receptor (9-cisretinoid X receptor, RXR) the formation of poly two then, to identify and target gene promoter of peroxisome proliferator response element upstream of the peroxisome proliferator response (sub element, PPRE ) with the regulation of transcription downstream target genes, translation and biological effects. This study observed the effect of oxLDL on the expression of THP-1 in macrophages of PPAR 8. At the same time with the PPAR delta agonist GW501516 on PPAR 8 on macrophage proliferation, apoptosis and function in lipid loaded macrophages.
The effect of part one oxLDL on the expression of PPAR Delta in THP-1 derived macrophages
Objective: To observe the effect of oxLDL on the expression of PPAR 8 in THP-1 derived macrophages.
Methods: 1. different concentrations (0mg/L, 25mg/L, 50mg/L, 100mg/L) oxLDL and THP-1 derived macrophages were incubated for 24 hours; with the expression of.2.50mg/L oxLDL and THP-1 RT-PCR and Western derived macrophages were detected by blotting PPAR and delta mRNA protein co incubated with 0h, 12h, 24h, 48h etc. with different time. The expression of RT-PCR and Western were detected by blotting PPAR and mRNA 8 protein.
Results: different concentrations of oxLDL can induce expression of THP-1 derived macrophage PPAR 8. With the increase of oxLDL concentration, the expression level of mRNA and protein in PPAR8 cells gradually increased, there was significant difference in expression, and when the concentration of 50mg/L PPAR 8 mRNA peak (P0.05). While the aging test results show 50mg/L and oxLDL THP-1 derived macrophages were incubated and incubated for 24h, the expression of PPAR Delta mRNA and protein levels increased significantly, and the difference was statistically significant (P0.05). While after 48h incubation, the expression of 24h was decreased, but compared with 24h, the difference was not statistically significant.
Conclusion: oxLDL can increase the expression of PPAR Delta in THP-1 derived macrophages
The effect of second part PPAR Delta on the proliferation and apoptosis of THP-1 derived macrophages
Objective: To observe the role of PPAR Delta in oxLDL induced THP-1 derived macrophage proliferation and apoptosis through the activation of PPAR Delta PPAR delta agonist GW501516.
Methods: THP-1 mononuclear cells differentiated into macrophages induced by PMA, 50mg/L oxLDL were incubated in THP-1 derived macrophages 24h as positive control group, 50mg/L oxLDL with different concentrations of PPAR delta agonist GW501516 (1,10100nmol and 1 mol) were incubated with THP-1 macrophage 24h, Hoechst33258 staining method was used to detect the MTT THP-1 source macrophage proliferation, Annexin activity, V/PI staining and flow cytometry were detected in blank group, oxLDL group and 1 mol group GW501516 cells apoptosis, spectrophotometric determination of intracellular caspase activity in 3.
Results: PPAR delta agonist GW501516 inhibited the inhibitory oxLDL of THP-1 derived macrophage proliferation, apoptosis of.MTT and Hoechst33258 and can reduce THP-1 derived macrophages by oxLDL staining showed that in GW501516 at the concentration of 100nmol significantly, and 1 mol concentration by more obvious (P0.05). Flow cytometry analysis results it also shows that the apoptosis of 1 mol GW501516 can significantly inhibit oxLDL induced by THP-1 derived macrophages (P0.05). Spectrophotometric detection of intracellular Caspase 3 activity showed that GW501516 showed a concentration dependent downregulation of caspase 3 activity (P0.05)
Conclusion: PPAR delta agonist GW501516 can down regulate the activity of caspase 3 and inhibit the apoptosis induced by oxLDL in THP-1 derived macrophages.
The effect of the third part PPAR Delta on the lipid accumulation of THP-1 derived macrophages
Objective: To observe the effect of PPAR Delta on the lipid accumulation of THP-1 derived macrophages by activating the PPAR delta agonist GW501516 to activate PPAR Delta.
Methods: THP-1 mononuclear cells differentiated into macrophages induced by PMA, 50mg/L oxLDL were incubated in THP-1 derived macrophages 24h as positive control group, 50mg/L oxLDL with different concentrations of PPAR delta agonist GW501516 (1,10100nmol and 1 mol) were incubated with THP-1 macrophage 24h, observe the cellular lipid accumulation of oil red O staining, total cholesterol were determined by HPLC, and the content of cholesterol ester.
Results: PPAR delta agonist GW501516 can increase THP-1 derived macrophage lipid accumulation, oil red staining showed that 0 100nmol after GW501516 treatment showed intracellular lipid droplets increased and the particle size of lipid droplets increased when GW501516 concentration was 1 mol when the effect is more obvious (P0.05). High performance liquid chromatography detection results positive control group total cholesterol content was 483.10 + 12.70, cholesterol / total cholesterol (%) were 49.60%.1,10100nmol and 1 molGW501516 treatment group total cholesterol content were 501.53 + 15.73497.69 + 14.25647.42 + 18.62 and 696.55 + 20.35; cholesterol ester / total cholesterol (%) were 56.7%, 53.90%, 64.48% and 66.26% 100nmol, and 1 mol GW501516 group compared with the positive control group had significant difference (P0.05).
Conclusion: PPAR delta agonist GW501516 can increase the accumulation of lipid in THP-1 derived macrophages.
Summary:
(1) oxLDL can increase the expression of PPAR Delta in THP-1 derived macrophages.
(2) PPAR delta agonist GW501516 can down regulate the activity of caspase 3 and inhibit the apoptosis induced by oxLDL in THP-1 derived macrophages.
(3) PPAR delta agonist GW501516 can increase the accumulation of lipid in THP-1 derived macrophages.

【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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