【作者】 何志军；

【导师】 易光辉；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2009， 硕士

【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(P<0.05)。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(P<0.05)。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(P<0.05)。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(P<0.05)。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(P<0.05)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(P<0.05)。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(P<0.05)。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。更多还原

【Abstract】 BACKGROUND:PPARδis one of the three isoforms of peroxisome proliferator-activated receptors (PPARs) which is expressed ubiquitously. PPARδcan enhance fatty acid catabolism and energy uncoupling in adipose tissue and muscles, suppress macrophage-induced inflammation, decrease atherosclerosis and is involved in the progression of many diseases. PPARδligands are divided into two kinds, natural ligands and synthetic ligands. GW501516 is a synthetic ligand which belongs to the phenoxy acetic acid derivative. When it binds to PPARδ, PPARδis activated and heterodimers will be formed together with retinoid X receptors (RXRs). The heterodimer binds to peroxisome proliferator response element (PPRE) to modulate transcription and translation of the target genes downstream and regulate biological functions such as controlling weight gain, enhancing physical endurance, and improving insulin sensitivity. We investigated the effect of oxLDL on the PPARδexpression in THP-1 derived macrophage and observed the effect of PPARδagonist GW501516 on macrophage proliferation, apoptosis and foam cells formation.PartⅠ. Effect of oxLDL on PPARδExpression Induced by oxLDL in THP-1 Derived MacrophagesObjective:To identify the effect of oxLDL on PPARδexpression in THP-1-derived macrophages.Method:1. THP-1 derived macrophages were incubated with different concentrations (0 mg/L,25 mg/L,50 mg/L,100 mg/L) of oxLDL for 24 hours. RT-PCR and Western blotting were applied to detect PPAR 8 mRNA and protein expression, respectively.2. THP-1 derived macrophages were treated with 50mg/L oxLDL in different time (Ohour, 12 hours,24 hours h,48 hours). RT-PCR and Western blotting were used to detect PPARδmRNA and protein expression in cellsResults:Different concentrations of oxLDL increased the mRNA and protein expression of PPARδgene in THP-1 derived macrophage. With increasing in the concentration of oxLDL the mRNA and protein expression of PPARδwas gradually increased with statistical significance At the concentration of 50mg/L oxLDL the expression level of PPARδmRNA was peaked (P<0.05). The time-course experiments showed that after 50mg/L oxLDL incubation with THP-1 derived macrophages for 24 hours, the expression levels of PPARδboth mRNA and protein significantluy increased, (P<0.05). In the time duration of 48 hours incubation, the expression of PPARδdecreased, but there was no significant difference compared to that in 24 hours.Conclusions:oxLDL may increase THP-1 derived macrophages PPARδexpression.PartⅡ. Effect of PPARδAgonist GW501516 on Oxidized LDL-induced Cell Proliferation and Apoptosis of THP-1 Derived MacrophagesObjective:To explore the effect of PPARδagonist GW501516 on proliferation and apoptosis induced by oxLDL in THP-1 derived macrophages.Methods:THP-1 monocytes were stimulated with PMA to differentiate into macrophage. Then the cells were incubated with 50mg/L oxLDL for 24 hours, also designed as the positive control group. Other groups were co-incubated 50mg/L of oxLDL and different concentrations of PPARδagonists GW501516 (1,10, 100nmol and 1μmol, respectively) for 24h. MTT assay was used to determine THP-1 derived macrophage proliferation activity. The dye Hoechst33258 staining, Annexin V/PI double staining and flow cytometer were applied to detect cell apoptosis in control group, oxLDL group and lumol GW501516 group of cells, Spectrophotometry determined caspase 3 activity in cells. Results:PPARδagonist GW501516 down-regulated oxLDL inhibition effect on proliferation of THP-1 derived macrophage, and reduced the oxLDL-induced THP-1 derived macrophage apoptosis. MTT and Hoechst33258 staining results showed that 100nmol GW501516 was effective and in 1μmol such effect was more significantly (P <0.05). Flow cytometry analysis results also showed that 1μmol GW501516 significantly inhibited oxLDL-induced THP-1 derived macrophage apoptosis (P<0.05). Spectrophotometry results showed GW501516 reduced the activity of apoptotic executive enzyme, caspase 3, in a concentration-dependent manner (P<0.05).Conclusion:PPAR 8 activation may reduce the activity of caspase 3 and inhibit oxLDL induced apoptosis of THP-1 derived macrophage.PartⅢ. Effect of PPARδAgonist GW501516 on lipid accumulation in THP-1 Derived MacrophagesObjective:To observe the effect of PPARδagonist GW501516 on the lipid accumulation in THP-1 derived macrophage induced by treatment of oxidized LDL.Methods:THP-1 derived macrophages were from monocytes pretreated with PMA. Then, THP-1-derived macrophages were incubated with 50mg/L of oxLDL for 24 hours, that was assigned as the positive control. In other experiments, the cells were co-incubated with 50mg/L of oxLDL and different concentrations of PPARδagonists GW501516 (1,10,100 nmol and 1μmol, respectively) for same time as in control. The harvested cells were stained with oil red to show the lipid droplets under microscope. High performancel iquid chromatography (HPLC) was applied to analyze and evaluate the contents of intracellular cholesterol and cholestryl ester.Results:Oil red O staining showed that PPARδagonist GW501516 increased lipid accumulation in THP-1 derived macrophages. 100nmol of GW501516 significantly increased intracellular lipid droplets both in size and in number (P<0.05). HPLC data demonstrated that the contents of cholesterol and cholesteryl ester in cells were also significantly increased. the data showed in positive group, total cholesterol was 483.10±12.70, cholesterol ester/total cholesterol(%) was 49.60%, and in 1,10,100 nmol and 1μmol GW501516 treatment groups, the total cholesterol levels were 501.53±15.73, 497.69±14.25,647.42±18.62 and 696.55±20.35 respectively; cholesterol ester/total cholesterol(%)respectively,56.7%,53.90%,64.48% and 66.26%. The cholesterol ester/total cholesterol(%) in 100nmol and 1μmol GW501516 group were both significantly increased compared with the positive control group (P<0.05)Conclusion:PPARδagonist GW501516 increases the THP-1 derived macrophage lipid accumulation.Summary(1) oxLDL may up-regulate both mRNA and protein expression in THP-1 derived macrophages.(2) PPARδagonist GW501516 may inhibit oxLDL-induced cell apoptosis of THP-1 derived macrophages, and reduce the activity of caspase 3.(3) PPARδagonist GW501516 may increase the lipid accumulation of THP-1 derived macrophages.更多还原