D4-F對THP-1巨噬細(xì)胞源性泡沫細(xì)胞ABCA1表達(dá)及膽固醇流出的影響
發(fā)布時間：2018-11-15 07:50
【摘要】： 目的:三磷酸腺苷結(jié)合盒轉(zhuǎn)運體A(ATP-binding cassette transporter A1, ABCA1)在細(xì)胞內(nèi)膽固醇流出中具有重要的作用。載脂蛋白A-1模擬肽D4-F是含有18氨基酸殘基的小分子肽,其-兩性螺旋基序是與脂質(zhì)結(jié)合重要的結(jié)構(gòu)基礎(chǔ)。目前,動物實驗證明,D4-F具有抗炎抗動脈粥樣硬化的作用,在D4-F喂養(yǎng)的小鼠血液中,其三酰甘油和低密度脂蛋白膽固醇水平較正常組降低。在細(xì)胞試驗中發(fā)現(xiàn)D4-F能夠上調(diào)ABCA1蛋白質(zhì)水平,但其具體的機(jī)制不清楚。 Cdc42是RhoGTP酶超家族成員,分子量為25KD。有研究表明Cdc42參與細(xì)胞內(nèi)膽固醇的流出。丹吉爾病患者的成纖維細(xì)胞和巨噬細(xì)胞中Cdc42表達(dá)下調(diào)。apoA-1與ABCA1結(jié)合后,能夠活化Cdc42。而本實驗組研究已經(jīng)證實apoA-1通過cAMP/PKA信號途徑影響ABCA1蛋白質(zhì)的表達(dá)及其介導(dǎo)的膽固醇的流出�；谶@些研究,我們以THP-1巨噬細(xì)胞源性泡沫細(xì)胞為模型,探討D4-F對ABCA1表達(dá)及其介導(dǎo)的膽固醇的影響,并進(jìn)一步觀察Cdc42/cAMP/PKA是否在這個過程中起作用。 第一部分D4-F對ABCA1表達(dá)及其介導(dǎo)的膽固醇流出的影響 目的: D4-F作為apoA-1的模擬肽,它是否影響THP-1巨噬細(xì)胞源性泡沫細(xì)胞中ABCA1表達(dá)及其介導(dǎo)的膽固醇流出?故本實驗的目的是觀察D4-F對THP-1巨噬細(xì)胞源性泡沫細(xì)胞中ABCA1表達(dá)及其介導(dǎo)的膽固醇流出的影響。 方法: THP-1單核細(xì)胞經(jīng)PMA誘導(dǎo)貼壁后,加入oxLDL誘導(dǎo)其轉(zhuǎn)化為泡沫細(xì)胞后,1.不同濃度(0μg/ml2μg/ml4μg/ml6μg/ml)D4-F處理細(xì)胞24小時; 2. 6μg/ml D4-F處理細(xì)胞不同時間(061224小時)和5μg/ml BSA處理細(xì)胞24小時;3. 6μg/ml D4-F與20μg/ml D4-F放線菌酮共處理細(xì)胞0,1,2小時。實時定量PCR和Western印跡分析法分別檢測ABCA1 mRNA與蛋白質(zhì)的表達(dá);液體閃爍計數(shù)器檢測細(xì)胞內(nèi)膽固醇流出,高效液相色譜分析細(xì)胞內(nèi)總膽固醇、游離膽固醇和膽固醇酯含量。 結(jié)果: D4-F不影響ABCA1m RNA水平; 6μg/ml D4-F和D4-F處理細(xì)胞24小時, ABCA1蛋白質(zhì)的表達(dá)至高峰;加入放線菌酮抑制蛋白質(zhì)合成之后,D4-F能夠明顯減緩ABCA1蛋白質(zhì)的降解;高效液相色譜法顯示D4-F降低了細(xì)胞內(nèi)總膽固醇,游離膽固醇和膽固醇酯的含量。液體閃爍法檢測D4-F能夠濃度依賴性和時間依賴性提高細(xì)胞內(nèi)膽固醇及其磷脂的流出。 第二部分D4-F增加ABCA1表達(dá)及其介導(dǎo)膽固醇流出的機(jī)制探討 目的:前期實驗結(jié)果顯示D4-F增加ABCA1蛋白質(zhì)表達(dá)及其介導(dǎo)的膽固醇流出,故本實驗主要探討D4-F影響ABCA1蛋白質(zhì)表達(dá)及其介導(dǎo)膽固醇流出的可能機(jī)制。 方法: THP-1單核細(xì)胞經(jīng)PMA誘導(dǎo)貼壁后,加入oxLDL誘導(dǎo)其轉(zhuǎn)化為泡沫細(xì)胞,1. 6μg/ml D4-F處理細(xì)胞0,15,20,25分鐘后,觀察PKA活性;2.轉(zhuǎn)染PKA-siRNA后,6μg/ml D4-F孵育24小時;3. 1.0mM PKA特異性激動劑dBcAMP與6μg/ml D4-F孵育24小時;4. 20μg/ml D4-F放線菌酮與PKA-siRNA干擾或dBcAMP和6μg/ml D4-F處理細(xì)胞0,1,2小時;5. 6μg/ml和100μg/l腺苷酸環(huán)化酶抑制劑SQ22536或25mM Cdc42抑制劑Scramine B在37℃孵育30分鐘;6. 6μg/ml D4-F處理細(xì)胞0,10,15,20分鐘后,觀察Cdc42活性。吸光度值檢測PKA活性和Cdc42活性檢測試劑盒檢測Cdc42活性;液體閃爍計數(shù)器檢測細(xì)胞內(nèi)膽固醇流出;Western印跡分析法檢測ABCA1蛋白質(zhì)的表達(dá)和ABCA1磷酸化;酶聯(lián)免疫試劑盒檢測cAMP水平。 結(jié)果: D4-F激活PKA;PKA-siRNA能下調(diào)PKA蛋白質(zhì)的表達(dá),與對照組比較表達(dá)下調(diào)達(dá)85%;PKA-siRNA和dBcAMP均不影響D4-F增加ABCA1蛋白質(zhì)的作用;PKA-siRNA,dBcAMP,SQ22536均能逆轉(zhuǎn)D4-F引起的ABCA1介導(dǎo)的膽固醇流出增加;同時,SQ22536能逆轉(zhuǎn)D4-F引起的cAMP水平,PKA活性及ABCA1磷酸化的增加;D4-F能影響Cdc42活性;Scramine B影響了D4-F引起的cAMP水平和PKA活性增加。 結(jié)論: 1 D4-F呈時間和濃度依賴上調(diào)ABCA1蛋白質(zhì)表達(dá); 2 D4-F可以通過Cdc42/cAMP/PKA途徑增加ABCA1絲氨酸磷酸化和ABCA1介導(dǎo)的膽固醇流出。
[Abstract]:Objective: The expression of ATP-binding cassette transporter A (ABCA1) in the 3-phosphate-binding cassette transporter A (ATP-binding cassette transporter A1, ABCA1) plays an important role in the efflux of cholesterol in the cells. Apolipoprotein A-1 mimetic peptide D4-F is a small molecular peptide containing 18 amino acid residues, and the-amphoteric helical motif is a structural basis that is important for lipid binding. At present, animal experiments show that D4-F has the effect of anti-inflammatory and anti-atherosclerosis, and in the blood of the mice fed with D4-F, the level of triglyceride and low-density lipoprotein cholesterol is lower than that of normal group. It was found that D4-F can increase the level of ABCA1 protein in cell test, but its specific mechanism is not clear. Cdc42 is a member of the RhoGTP enzyme superfamily with a molecular weight of 25KD. It was found that Cdc42 was involved in the cell liner. The outflow of alcohol. Cdc42 in fibroblasts and macrophages of patients with Tangier disease After the binding of apoA-1 and ABCA1, C can be activated dc42. The study of the experimental group has demonstrated that apoA-1 has the effect of the cAMP/ PKA signaling pathway on the expression of ABCA1 protein and its mediated cholineralization. Based on these studies, we studied the effect of D4-F on the expression of ABCA1 and its mediated cholesterol, and further observed whether Cdc42/ cAMP/ PKA was in this process. The effect of the first part D4-F on the expression of ABCA1 and its medium Effect of guided cholesterol efflux: D4-F as an analog peptide of apoA-1, and whether it affects the ABC of the THP-1 macrophage-derived foam cells The purpose of this experiment was to observe the ABCA1 table in the source foam cells of the THP-1 macrophage by D4-F. Dup and its effect on cholesterol efflux. Methods: THP-1 monocytes were induced by PMA, and then added. After the induction of oxLDL to the foam cells, 1. The different concentration (0. mu.g/ ml? 2. mu.g/ ml? 4. mu.g/ ml? 6. m g/ ml) D4-F treated cells for 24 h; 2.6. mu.g/ ml D4-F for different time (0? 6? 12? 24 h); and 5. mu.g/ ml BSA treated cells for 24 hours; 3.6. mu.g/ ml D4-F and 20.mu. g/ ml D The expression of ABCA1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. The contents of total cholesterol, free cholesterol and cholesteryl ester in the cell were determined. The results showed that D4-F did not affect the level of ABC1m RNA, and the expression of ABCA1 protein reached the peak at 6. m u.g/ ml of D4-F and D4-F. After the inhibition of protein synthesis, the D4-F could significantly slow the degradation of the ABCA1 protein.; High performance liquid chromatography to show D4-F The content of total cholesterol, free cholesterol and cholesteryl ester in cells is reduced. The concentration of D4-F can be detected by liquid scintillation. The dependence and time-dependence increase the efflux of cholesterol and its phospholipids in cells. Two-part D4-F increased the expression of ABCA1 and its mechanism to mediate the outflow of cholesterol. The effect of D4-F on the expression of ABCA1 protein and its possible mechanism to mediate the outflow of cholesterol were mainly discussed. after the THP-1 monocytes were induced to adhere by PMA, oxLDL was added to induce it to be converted into foam cells, 1. 6. mu. g/ ml of D4-F treated cells for 0, 15, 20, 25 minutes, observed PKA activity; 2. After the PKA-siRNA was transfected, 6. mu.g/ ml of D4-F was incubated for 24 hours; 3. 1. 0mM PKA specific agonist dBcAMP incubated with 6. mu.g/ ml D4-F for 24 hours; 4.20. mu.g/ ml of D4-F, and PKA-siRNA interference or dBcAMP and 6. mu. g/ ml D4-F treatment cells 0, 1, 2 hours; 5. 6. mu.g/ ml and 100.mu. g/ l of the adenoid acid cyclase inhibitor SQ 22536 or 25mM Cdc42 inhibitor, Sramine B, incubated at 37.degree. C.for 30 minutes; 6. 6. 6. mu. g/ ml of D4-F treated cells for 0, 10, 15 and 20 minutes. The activity of Cdc42 was observed. The activity of the Cdc42 was detected by the absorbance value, and the activity of Cdc42 was detected by the detection kit of the activity of the Cdc42. The liquid scintillation counter was used to detect the cholesterol efflux in the cells, and the Western blot analysis The expression of ABCA1 protein and the phosphorylation of ABCA1 were detected by enzyme-linked immunosorbent assay, and the level of cAMP was detected by enzyme-linked immunosorbent assay. Results: The expression of PKA and PKA-siRNA down-regulated the expression of PKA protein, and the expression of PKA-siRNA and dBcAMP did not affect the effect of D4-F on the increase of ABCA1 protein; and the PKA-siRNA At the same time, SQ22536 could reverse the increase of cAMP level, PKA activity and ABCA1 phosphorylation caused by D4-F, and D4-F can influence Cdc42 activity; Sramine B affects D4-F Conclusion: The time and concentration of 1D4-F are dependent on the up-regulation of ABCA1 protein.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363
本文編號：2332636
[Abstract]:Objective: The expression of ATP-binding cassette transporter A (ABCA1) in the 3-phosphate-binding cassette transporter A (ATP-binding cassette transporter A1, ABCA1) plays an important role in the efflux of cholesterol in the cells. Apolipoprotein A-1 mimetic peptide D4-F is a small molecular peptide containing 18 amino acid residues, and the-amphoteric helical motif is a structural basis that is important for lipid binding. At present, animal experiments show that D4-F has the effect of anti-inflammatory and anti-atherosclerosis, and in the blood of the mice fed with D4-F, the level of triglyceride and low-density lipoprotein cholesterol is lower than that of normal group. It was found that D4-F can increase the level of ABCA1 protein in cell test, but its specific mechanism is not clear. Cdc42 is a member of the RhoGTP enzyme superfamily with a molecular weight of 25KD. It was found that Cdc42 was involved in the cell liner. The outflow of alcohol. Cdc42 in fibroblasts and macrophages of patients with Tangier disease After the binding of apoA-1 and ABCA1, C can be activated dc42. The study of the experimental group has demonstrated that apoA-1 has the effect of the cAMP/ PKA signaling pathway on the expression of ABCA1 protein and its mediated cholineralization. Based on these studies, we studied the effect of D4-F on the expression of ABCA1 and its mediated cholesterol, and further observed whether Cdc42/ cAMP/ PKA was in this process. The effect of the first part D4-F on the expression of ABCA1 and its medium Effect of guided cholesterol efflux: D4-F as an analog peptide of apoA-1, and whether it affects the ABC of the THP-1 macrophage-derived foam cells The purpose of this experiment was to observe the ABCA1 table in the source foam cells of the THP-1 macrophage by D4-F. Dup and its effect on cholesterol efflux. Methods: THP-1 monocytes were induced by PMA, and then added. After the induction of oxLDL to the foam cells, 1. The different concentration (0. mu.g/ ml? 2. mu.g/ ml? 4. mu.g/ ml? 6. m g/ ml) D4-F treated cells for 24 h; 2.6. mu.g/ ml D4-F for different time (0? 6? 12? 24 h); and 5. mu.g/ ml BSA treated cells for 24 hours; 3.6. mu.g/ ml D4-F and 20.mu. g/ ml D The expression of ABCA1 mRNA and protein was detected by real-time quantitative PCR and Western blot analysis. The contents of total cholesterol, free cholesterol and cholesteryl ester in the cell were determined. The results showed that D4-F did not affect the level of ABC1m RNA, and the expression of ABCA1 protein reached the peak at 6. m u.g/ ml of D4-F and D4-F. After the inhibition of protein synthesis, the D4-F could significantly slow the degradation of the ABCA1 protein.; High performance liquid chromatography to show D4-F The content of total cholesterol, free cholesterol and cholesteryl ester in cells is reduced. The concentration of D4-F can be detected by liquid scintillation. The dependence and time-dependence increase the efflux of cholesterol and its phospholipids in cells. Two-part D4-F increased the expression of ABCA1 and its mechanism to mediate the outflow of cholesterol. The effect of D4-F on the expression of ABCA1 protein and its possible mechanism to mediate the outflow of cholesterol were mainly discussed. after the THP-1 monocytes were induced to adhere by PMA, oxLDL was added to induce it to be converted into foam cells, 1. 6. mu. g/ ml of D4-F treated cells for 0, 15, 20, 25 minutes, observed PKA activity; 2. After the PKA-siRNA was transfected, 6. mu.g/ ml of D4-F was incubated for 24 hours; 3. 1. 0mM PKA specific agonist dBcAMP incubated with 6. mu.g/ ml D4-F for 24 hours; 4.20. mu.g/ ml of D4-F, and PKA-siRNA interference or dBcAMP and 6. mu. g/ ml D4-F treatment cells 0, 1, 2 hours; 5. 6. mu.g/ ml and 100.mu. g/ l of the adenoid acid cyclase inhibitor SQ 22536 or 25mM Cdc42 inhibitor, Sramine B, incubated at 37.degree. C.for 30 minutes; 6. 6. 6. mu. g/ ml of D4-F treated cells for 0, 10, 15 and 20 minutes. The activity of Cdc42 was observed. The activity of the Cdc42 was detected by the absorbance value, and the activity of Cdc42 was detected by the detection kit of the activity of the Cdc42. The liquid scintillation counter was used to detect the cholesterol efflux in the cells, and the Western blot analysis The expression of ABCA1 protein and the phosphorylation of ABCA1 were detected by enzyme-linked immunosorbent assay, and the level of cAMP was detected by enzyme-linked immunosorbent assay. Results: The expression of PKA and PKA-siRNA down-regulated the expression of PKA protein, and the expression of PKA-siRNA and dBcAMP did not affect the effect of D4-F on the increase of ABCA1 protein; and the PKA-siRNA At the same time, SQ22536 could reverse the increase of cAMP level, PKA activity and ABCA1 phosphorylation caused by D4-F, and D4-F can influence Cdc42 activity; Sramine B affects D4-F Conclusion: The time and concentration of 1D4-F are dependent on the up-regulation of ABCA1 protein.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R363
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