本文選題：動脈粥樣硬化　切入點：過氧化物酶體增殖物激活型受體δ　出處：《南華大學》2009年碩士論文　論文類型：學位論文

【摘要】：研究背景：過氧化物酶體增殖物激活型受體δ(peroxisome Proliferator- activated receptorδ, PPARδ)是過氧化物酶體增殖物激活型受體家族中的一員,廣泛表達于多種器官和組織,主要控制脂肪組織和肌肉中脂肪酸氧化和能量解偶聯(lián),抑制巨噬細胞誘導(dǎo)的炎癥,改善動脈粥樣硬化,并參與許多疾病的發(fā)生和發(fā)展過程。PPARδ的配體分為天然配體和合成配體。GW501516是PPARδ的合成配體,屬于苯氧乙酸衍生物,PPARδ與GW501516結(jié)合后,活化的PPARδ與9-順視黃酸類受體(9-cisretinoid X receptor, RXR)形成異二聚體,然后識別并于靶基因啟動子上游的過氧化物酶體增殖物反應(yīng)元件(peroxisome proliferator response element, PPRE)結(jié)合,調(diào)節(jié)靶基因下游的基因轉(zhuǎn)錄、翻譯及生物學效應(yīng)。本課題觀察oxLDL對THP-1源性巨噬細胞PPARδ表達的影響。同時以PPARδ激動劑GW501516來探討PPARδ對巨噬細胞增殖、凋亡的影響及在巨噬細胞荷脂過程的作用。 第一部分oxLDL對THP-1源性巨噬細胞PPARδ表達的影響 目的：觀察oxLDL對THP-1源性巨噬細胞PPAR 8表達的影響。 方法：1.用不同濃度(0mg/L、25mg/L、50mg/L、100mg/L)的oxLDL與THP-1源性巨噬細胞共孵育24小時；用RT-PCR及Western blotting檢測細胞PPARδmRNA和蛋白的表達。2.50mg/L oxLDL與THP-1源性巨噬細胞共孵育0h、12h、24h、48h等不同時間,用RT-PCR及Western blotting檢測細胞PPAR 8 mRNA和蛋白的表達。 結(jié)果：不同濃度oxLDL可誘導(dǎo)THP-1源性巨噬細胞PPAR 8的表達。隨著oxLDL濃度的增加,細胞PPAR8的mRNA水平及蛋白表達逐漸增加,差別有顯著性,且在濃度為50mg/L時PPAR 8 mRNA的表達達峰值(P0.05)。而時效試驗結(jié)果顯示50mg/L oxLDL與THP-1源性巨噬細胞孵育孵育24h時,PPARδmRNA水平及蛋白表達增加明顯,且差別有統(tǒng)計學意義(P0.05)。而在孵育48h后,表達較24h時有所減少,但與24h相比差別無統(tǒng)計學意義。 結(jié)論：oxLDL可上調(diào)THP-1源性巨噬細胞PPARδ的表達 第二部分PPARδ對THP-1源性巨噬細胞增殖、凋亡的影響 目的：通過PPARδ激動劑GW501516活化PPARδ來觀察PPARδ在oxLDL誘導(dǎo)THP-1源性巨噬細胞增殖、凋亡中的作用。 方法：THP-1單核細胞經(jīng)PMA誘導(dǎo)分化成為巨噬細胞后,以50mg/L oxLDL孵育THP-1源性巨噬細胞24h為陽性對照組,50mg/L oxLDL與不同濃度的PPARδ激動劑GW501516 (1、10、100nmol及1μmol)共同孵育THP-1源性巨噬細胞24h,MTT法檢測THP-1源性巨噬細胞增殖活性,Hoechst33258染色、Annexin V/PI雙染后細胞流式儀檢測空白組、oxLDL組及1μmol GW501516組細胞凋亡情況,分光光度法測定細胞內(nèi)caspase 3活性變化。 結(jié)果：PPARδ激動劑GW501516可阻抑oxLDL對THP-1源性巨噬細胞增殖的抑制作用,且能減少oxLDL誘導(dǎo)THP-1源性巨噬細胞的凋亡。MTT及Hoechst33258染色結(jié)果顯示在GW501516濃度達100nmol時作用顯著,且1μmol濃度時作用更明顯(P0.05)。流式分析結(jié)果也同樣表明1μmol GW501516能顯著抑制oxLDL誘導(dǎo)THP-1源性巨噬細胞的凋亡(P0.05)。分光光度法檢測細胞內(nèi)Caspase 3活性顯示GW501516能呈濃度依賴性下調(diào)caspase 3的活性(P0.05) 結(jié)論：PPARδ激動劑GW501516可下調(diào)caspase 3活性,抑制oxLDL誘導(dǎo)的THP-1源性巨噬細胞凋亡。 第三部分PPARδ對THP-1源性巨噬細胞脂質(zhì)蓄積的影響 目的：通過PPARδ激動劑GW501516活化PPARδ來觀察PPARδ對THP-1源性巨噬細胞脂質(zhì)蓄積的影響的影響。 方法：THP-1單核細胞經(jīng)PMA誘導(dǎo)分化成為巨噬細胞后,以50mg/L oxLDL孵育THP-1源性巨噬細胞24h為陽性對照組,50mg/L oxLDL與不同濃度的PPARδ激動劑GW501516 (1、10、100nmol及1μmol)共同孵育THP-1源性巨噬細胞24h,油紅O染色觀察細胞內(nèi)脂質(zhì)蓄積情況,高效液相色譜檢測細胞內(nèi)總膽固醇、和膽固醇酯含量。 結(jié)果：PPARδ激動劑GW501516可增加THP-1源性巨噬細胞內(nèi)脂質(zhì)蓄積,油紅0染色顯示100nmol GW501516處理后可見細胞內(nèi)脂滴顆粒體積明顯增大且脂滴數(shù)增多,當GW501516濃度達1μmol時作用更為明顯(P0.05)。高效液相色譜檢測結(jié)果顯示陽性對照組總膽固醇含量為483.10±12.70,膽固醇酯/總膽固醇(%)為49.60%。1、10、100nmol和1μmolGW501516處理組總膽固醇含量分別為501.53±15.73,497.69±14.25,647.42±18.62和696.55±20.35；膽固醇酯/總膽固醇(%)分別為56.7%,53.90%,64.48%和66.26%,100nmol和1μmol GW501516組與陽性對照組比較差別有顯著性(P0.05)。 結(jié)論：PPARδ激動劑GW501516可增加THP-1源性巨噬細胞內(nèi)脂質(zhì)蓄積。 總結(jié)： (1) oxLDL可上調(diào)THP-1源性巨噬細胞PPARδ的表達。 (2) PPARδ激動劑GW501516可下調(diào)caspase 3活性,抑制oxLDL誘導(dǎo)的THP-1源性巨噬細胞凋亡。 (3) PPARδ激動劑GW501516可增加THP-1源性巨噬細胞內(nèi)脂質(zhì)蓄積。
[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.

【學位授予單位】：南華大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R363

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