萊菔硫烷對(duì)脂肪細(xì)胞的代謝調(diào)控作用及分子機(jī)制研究
發(fā)布時(shí)間：2019-04-13 16:58
【摘要】：目的:以3T3-L1成熟脂肪細(xì)胞為模型,研究萊菔硫烷(sulforaphane,SFN)對(duì)3T3-L1成熟脂肪細(xì)胞糖、脂代謝的影響及其潛在的分子機(jī)制。方法:經(jīng)典激素雞尾酒(methylisobutylxanthine,dexamethasone and insulin,MDI)法誘導(dǎo)3T3-L1前脂肪細(xì)胞分化成為成熟脂肪細(xì)胞。油紅O染色法直接觀察3T3-L1前脂肪細(xì)胞在誘導(dǎo)分化過(guò)程中的脂質(zhì)積聚情況,2-N[7-硝基苯-2-乙二酸,34羥氨基]-2-脫氧葡萄糖(2-NBDG)染色法檢測(cè)誘導(dǎo)分化過(guò)程中脂肪細(xì)胞的葡萄糖攝取變化,以探索SFN干預(yù)的最佳時(shí)點(diǎn)。低濃度(0-10.0μmol/L)SFN干預(yù)3T3-L1成熟脂肪細(xì)胞48h,經(jīng)2-NBDG染色后,分別用共聚焦和多功能酶標(biāo)儀定性、定量檢測(cè)SFN處理的成熟脂肪細(xì)胞內(nèi)的葡萄糖攝取變化,蛋白印跡法和實(shí)時(shí)熒光定量PCR法分別檢測(cè)SFN干預(yù)48h對(duì)葡萄糖攝取和代謝相關(guān)蛋白GLUT-4、GCK、CS及基因ACC、FAS表達(dá)的影響;甘油GPO-POD酶法檢測(cè)成熟脂肪細(xì)胞經(jīng)SFN處理后培養(yǎng)基中的甘油含量,間接評(píng)價(jià)SFN對(duì)白色脂肪細(xì)胞脂質(zhì)水解的影響,蛋白印跡法檢測(cè)SFN干預(yù)48h對(duì)白色脂肪細(xì)胞脂質(zhì)分解代謝相關(guān)蛋白ATGL、HSL、p-HSL~(Ser563)、p-HSLSer565、p-HSL~(Ser660)、Perilipin、CPT1A、CS、DGAT-1表達(dá)的影響,綜合評(píng)價(jià)SFN處理對(duì)3T3-L1成熟脂肪細(xì)胞葡萄糖和脂質(zhì)代謝的影響,探討SFN調(diào)控白色脂肪細(xì)胞的糖、脂代謝作用及分子機(jī)制。結(jié)果:誘導(dǎo)分化過(guò)程中的3T3-L1前脂肪細(xì)胞葡萄糖攝取和脂質(zhì)積累逐漸增加,到分化第10天逐漸趨于穩(wěn)定,故本課題選擇誘導(dǎo)分化第10天的3T3-L1脂肪細(xì)胞作為本研究對(duì)象。2-NBDG染色顯示,SFN可顯著增強(qiáng)3T3-L1成熟脂肪細(xì)胞的葡萄糖攝取。甘油GPO-POD酶法檢測(cè)提示,SFN干預(yù)可明顯增加脂肪細(xì)胞的脂質(zhì)水解作用。蛋白印跡法和實(shí)時(shí)熒光定量PCR法檢測(cè)表明,SFN可上調(diào)葡萄糖攝取相關(guān)蛋白GLUT-4,葡萄糖有氧氧化相關(guān)蛋白GCK、CS,脂質(zhì)水解相關(guān)蛋白HSL、p-HSL~(Ser563)、p-HSL~(Ser660)和脂肪酸β氧化相關(guān)蛋白CPT1A的表達(dá),同時(shí)下調(diào)脂肪酸合成相關(guān)基因ACC、FAS,脂質(zhì)水解相關(guān)蛋白p-HSLSer565、Perilipin,甘油三酯合成相關(guān)蛋白DGAT-1的表達(dá)。結(jié)論:低濃度(0-10μmol/L)SFN干預(yù)可通過(guò)上調(diào)葡萄糖攝取和代謝相關(guān)蛋白GLUT-4、GCK、CS的表達(dá),增強(qiáng)3T3-L1成熟脂肪細(xì)胞的葡萄糖攝取和有氧氧化;上調(diào)脂質(zhì)水解和脂肪酸β氧化相關(guān)蛋白HSL及磷酸化p-HSL~(Ser563)、p-HSL~(Ser660)和CPT1A水平,增強(qiáng)成熟脂肪細(xì)胞的脂質(zhì)代謝,下調(diào)ACC、FAS基因表達(dá)和DGAT-1蛋白水平,抑制葡萄糖合成脂肪酸以及甘油三酯的再合成。上述結(jié)果表明,低濃度SFN干預(yù)可改善白色脂肪細(xì)胞糖、脂代謝作用,為以白色脂肪細(xì)胞為靶點(diǎn),探索肥胖的防治策略提供科學(xué)依據(jù)。
[Abstract]:Aim: to study the effects of sulforaphane (sulforaphane,SFN) on glucose and lipid metabolism in 3T3-L1 mature adipocytes and its potential molecular mechanism using 3T3-L1 mature adipocytes as a model. Methods: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by classical hormone cocktail (methylisobutylxanthine,dexamethasone and insulin,MDI). Oil red O staining was used to observe the lipid accumulation of 3T3-L1 preadipocytes in the process of induced differentiation, 2N [7-nitrobenzene-2-oxalic acid], 34 hydroxyamino]-2-deoxyglucose (2-NBDG) staining was used to detect the changes of glucose uptake in adipocytes during induction and differentiation in order to explore the optimal time point of SFN intervention. 3T3-L1 mature adipocytes were treated with low concentration (0-10.0 渭 mol / L) SFN) for 48 h. After 2-NBDG staining, the glucose uptake in mature adipocytes treated with SFN was quantitatively detected by confocal and multi-functional enzyme labeling apparatus. Protein blot and real-time fluorescence quantitative PCR were used to detect the effects of SFN intervention for 48 h on glucose uptake and metabolism-related protein GLUT-4,GCK,CS and gene ACC,FAS expression. Glycerol GPO-POD method was used to detect the glycerol content in the medium of mature adipocytes treated with SFN, and the effect of SFN on lipid hydrolysis of white adipocytes was evaluated indirectly. Western blot assay was used to detect the effects of SFN on the expression of lipid catabolism-related proteins ATGL,HSL,p-HSL~ (Ser563), pJHSLSer565, pHSLSer660 and Perilipin,CPT1A,CS,DGAT-1 in white adipocytes after 48 h intervention. To evaluate the effects of SFN treatment on glucose and lipid metabolism in mature adipocytes of 3T3-L1, and to explore the effects and molecular mechanisms of SFN on glucose and lipid metabolism in white adipocytes. Results: glucose uptake and lipid accumulation of 3T3-L1 preadipocytes increased gradually during differentiation, and became stable on the 10th day of differentiation. In this study, 3T3-L1 adipocytes on the 10th day of differentiation were selected as the object of this study. 2-NBDG staining showed that SFN significantly enhanced glucose uptake of 3T3-L1 mature adipocytes. Glycerol GPO-POD enzyme assay suggested that SFN intervention could significantly increase the lipid hydrolysis of adipocytes. Protein blot and real-time fluorescence quantitative PCR assay showed that SFN could up-regulate glucose uptake-related protein GLUT-4, glucose oxidation-related protein GCK,CS, lipid hydrolysis-associated protein HSL,p-HSL~ (Ser563). The expression of CPT1A, a fatty acid 尾-oxidation-related protein, and the expression of lipid hydrolysis-related protein (ACC,FAS,) related to fatty acid synthesis genes ACC,FAS, perilipin and DGAT-1 were also down-regulated. The expression of Ser660 and 尾-oxidation-related protein DGAT-1 of fatty acid synthesis was also down-regulated. Conclusion: low concentration (0-10 渭 mol / L) SFN) can enhance glucose uptake and aerobic oxidation of 3T3-L1 mature adipocytes by up-regulating glucose uptake and GLUT-4,GCK,CS expression. By up-regulating lipid hydrolysis and fatty acid 尾-oxidation-associated protein HSL and phospho-hsl ~ (Ser563), phospho-HSL ~ (Ser660) and CPT1A levels, enhancing lipid metabolism of mature adipocytes and down-regulating ACC,FAS gene expression and DGAT-1 protein level, the expression of ACC,FAS gene and DGAT-1 protein were up-regulated in mature adipocytes. Inhibition of glucose synthesis of fatty acids and triglyceride re-synthesis. These results suggest that low concentration of SFN can improve glucose and lipid metabolism of white adipocytes and provide scientific basis for the prevention and treatment of obesity by targeting white adipocytes.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R589.2
[Abstract]:Aim: to study the effects of sulforaphane (sulforaphane,SFN) on glucose and lipid metabolism in 3T3-L1 mature adipocytes and its potential molecular mechanism using 3T3-L1 mature adipocytes as a model. Methods: 3T3-L1 preadipocytes were induced to differentiate into mature adipocytes by classical hormone cocktail (methylisobutylxanthine,dexamethasone and insulin,MDI). Oil red O staining was used to observe the lipid accumulation of 3T3-L1 preadipocytes in the process of induced differentiation, 2N [7-nitrobenzene-2-oxalic acid], 34 hydroxyamino]-2-deoxyglucose (2-NBDG) staining was used to detect the changes of glucose uptake in adipocytes during induction and differentiation in order to explore the optimal time point of SFN intervention. 3T3-L1 mature adipocytes were treated with low concentration (0-10.0 渭 mol / L) SFN) for 48 h. After 2-NBDG staining, the glucose uptake in mature adipocytes treated with SFN was quantitatively detected by confocal and multi-functional enzyme labeling apparatus. Protein blot and real-time fluorescence quantitative PCR were used to detect the effects of SFN intervention for 48 h on glucose uptake and metabolism-related protein GLUT-4,GCK,CS and gene ACC,FAS expression. Glycerol GPO-POD method was used to detect the glycerol content in the medium of mature adipocytes treated with SFN, and the effect of SFN on lipid hydrolysis of white adipocytes was evaluated indirectly. Western blot assay was used to detect the effects of SFN on the expression of lipid catabolism-related proteins ATGL,HSL,p-HSL~ (Ser563), pJHSLSer565, pHSLSer660 and Perilipin,CPT1A,CS,DGAT-1 in white adipocytes after 48 h intervention. To evaluate the effects of SFN treatment on glucose and lipid metabolism in mature adipocytes of 3T3-L1, and to explore the effects and molecular mechanisms of SFN on glucose and lipid metabolism in white adipocytes. Results: glucose uptake and lipid accumulation of 3T3-L1 preadipocytes increased gradually during differentiation, and became stable on the 10th day of differentiation. In this study, 3T3-L1 adipocytes on the 10th day of differentiation were selected as the object of this study. 2-NBDG staining showed that SFN significantly enhanced glucose uptake of 3T3-L1 mature adipocytes. Glycerol GPO-POD enzyme assay suggested that SFN intervention could significantly increase the lipid hydrolysis of adipocytes. Protein blot and real-time fluorescence quantitative PCR assay showed that SFN could up-regulate glucose uptake-related protein GLUT-4, glucose oxidation-related protein GCK,CS, lipid hydrolysis-associated protein HSL,p-HSL~ (Ser563). The expression of CPT1A, a fatty acid 尾-oxidation-related protein, and the expression of lipid hydrolysis-related protein (ACC,FAS,) related to fatty acid synthesis genes ACC,FAS, perilipin and DGAT-1 were also down-regulated. The expression of Ser660 and 尾-oxidation-related protein DGAT-1 of fatty acid synthesis was also down-regulated. Conclusion: low concentration (0-10 渭 mol / L) SFN) can enhance glucose uptake and aerobic oxidation of 3T3-L1 mature adipocytes by up-regulating glucose uptake and GLUT-4,GCK,CS expression. By up-regulating lipid hydrolysis and fatty acid 尾-oxidation-associated protein HSL and phospho-hsl ~ (Ser563), phospho-HSL ~ (Ser660) and CPT1A levels, enhancing lipid metabolism of mature adipocytes and down-regulating ACC,FAS gene expression and DGAT-1 protein level, the expression of ACC,FAS gene and DGAT-1 protein were up-regulated in mature adipocytes. Inhibition of glucose synthesis of fatty acids and triglyceride re-synthesis. These results suggest that low concentration of SFN can improve glucose and lipid metabolism of white adipocytes and provide scientific basis for the prevention and treatment of obesity by targeting white adipocytes.
【學(xué)位授予單位】：寧波大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R589.2
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 Surapon Tangvarasittichai;;Oxidative stress,insulin resistance,dyslipidemia and type 2 diabetes mellitus[J];World Journal of Diabetes;2015年03期
2 謝述瓊;何s，
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