脂肪酸结合蛋白在棕榈酸诱导巨噬细胞凋亡中的作用及机制

发布时间：2018-04-17 05:21

本文选题：脂肪酸结合蛋白 + 棕榈酸　；参考：《中南大学》2012年博士论文

【摘要】：目的：探讨脂肪细胞脂肪酸结合蛋白(A-FABP)在棕榈酸诱导巨噬细胞凋亡中的作用。方法：体外培养人单核细胞白血病细胞系(Thp-1细胞),加入佛波酯(PMA)培养72小时使其分化为巨噬细胞后,用不同浓度棕榈酸(50μM,100μM,200μM,500μM)处理,在不同时间点提取细胞mRNA和蛋白,用Real-Time PCR以及Western blot检测A-FABP蛋白的表达。采用ELISA以及Western blot检测Thp-1源巨噬细胞凋亡。同时用A-FABP siRNA转染Thp-1源巨噬细胞,观察A-FABP基因沉默对棕榈酸诱导的上述指标变化的影响。结果：在棕榈酸作用下,Thp-1源巨噬细胞中A-FABP mRNA水平和蛋白水平随棕榈酸浓度增加而上升；此外,随着棕榈酸处理时间的延长,A-FABP mRNA水平和蛋白水平也随棕榈酸干预时间延长而上升。ELISA细胞凋亡检测结果显示,棕榈酸呈时间依赖和浓度依赖方式诱导DNA链断裂损伤加重；A-FABP siRNA可显著减少棕榈酸诱导的DNA链断裂损伤。凋亡相关蛋白检测结果显示,棕榈酸组的Bax蛋白表达明显高于对照组,Bcl-2蛋白表达明显低于对照组。A-FABP siRNA组予棕榈酸培养24h,其Bax蛋白表达较棕榈酸组明显减少,Bcl-2蛋白表达明显增加。结论：棕榈酸可诱导巨噬细胞凋亡,该作用通过A-FABP介导。目的：①探讨棕榈酸对巨噬细胞线粒体功能和线粒体凋亡途径的影响；②探讨A-FABP在棕榈酸诱导巨噬细胞线粒体功能障碍和线粒体凋亡途径激活中的作用。方法：采用荧光染色检测细胞内活性氧(ROS)含量；JC-1染色法检测线粒体膜电位；ATP检测试剂盒检测细胞ATP含量；总丙二醛(MDA)检测试剂盒检测细胞总MDA含量；caspase3活性检测试剂盒检测caspase3活性；caspase9活性检测试剂盒检测caspase9活性；梯度离心法抽提线粒体；线粒体琥珀酸脱氢酶(SDH)检测试剂盒检测细胞线粒体琥珀酸脱氢酶活性；线粒体呼吸链复合物Ⅳ检测试剂盒检测线粒体呼吸链复合物Ⅳ活性；Western blot检测细胞色素c表达。结果：在棕榈酸作用下,Thp-1源巨噬细胞内ROS水平增加,线粒体膜电位下降,促进线粒体细胞色素c释放至胞浆,caspase3和caspase9活性增强,线粒体凋亡途径被激活；棕榈酸还引起Thp-1源巨噬细胞呼吸链活性受抑、ATP生成减少、细胞脂质过氧化加重,线粒体功能受损。而A-FABP siRNA转染组予棕榈酸培养24h后,与棕榈酸组比较,ROS水平明显减低,线粒体膜电位恢复,细胞色素c释放减少,caspase3和caspase9舌性下降,呼吸链复合物Ⅳ活性增加,ATP水平升高,脂质过氧化减轻。结论：棕榈酸诱导巨噬细胞的线粒体呼吸链活性受抑,ATP生成减少,ROS生成增加,引起线粒体功能障碍并激活线粒体凋亡途径。A-FABP基因沉默可部分逆转这一过程。目的：①探讨棕榈酸对巨噬细胞线粒体生物合成与抗氧化能力的影响；②探讨A-FABP在棕榈酸诱导线粒体生物合成与抗氧化能力变化中的作用。方法：Real-Time PCR方法检测线粒体DNA拷贝数；Western blot和Real-Time PCR检测抗氧化酶和线粒体生物合成相关的蛋白和基因表达。Mito-Tracker Green荧光探针法标记细胞线粒体,检测线粒体数量。结果：Thp-1源巨噬细胞经棕榈酸处理后,棕榈酸诱导抗氧化酶包括锰超氧化物歧化酶(MnSOD)、铜锌超氧化物岐化酶(Cu/ZnSOD)、解偶联蛋白2(UCP2)的mRNA水平下降,同时生物合成相关蛋白过氧化物酶体增殖物激活受体γ辅激活因子1α(PGC-1α)、线粒体核呼吸因子(NRF-1)和雌激素相关受体α(ESRRA) mRNA水平和PGC-1α蛋白表达下调,荧光探针法检测棕榈酸组的线粒体数量减少,而线粒体转录因子A (TFAM)和线粒体DNA拷贝数无明显变化。A-FABP siRNA转染组予棕榈酸培养24h后,与棕榈酸组比较,MnSOD、Cu/ZnSOD和UCP2的mRNA水平增加,PGC-1α、NRF-1和ESRRA表达上调,荧光探针法检测的线粒体数量增加,而TFAM和线粒体DNA拷贝数无明显变化。结论：A-FABP介导棕榈酸引起的线粒体生物合成减少及抗氧化能力受损,从而调控细胞内氧化应激和线粒体功能。
[Abstract]:Objective: To investigate the role of adipocyte fatty acid binding protein (A-FABP) in the apoptosis of macrophages induced by palmitic acid.
Methods: human monocytic leukemia cell line (Thp-1 cells) in vitro, with phorbol ester (PMA) cultured for 72 hours. The differentiation of macrophages, palmitic acid with different concentrations (50 M, 100 M, 200 M, 500 M) treatment, extraction of mRNA and protein of the cells at different time with Real-Time, PCR and Western blot to detect the expression of A-FABP protein. Using ELISA and Western blot detection of Thp-1 derived macrophages apoptosis. At the same time with the A-FABP siRNA transfection of Thp-1 derived macrophages, to observe the effect of A-FABP gene silencing on the changes of the above indexes by palmitic acid.
Results: the effect of palmitic acid, Thp-1 in A-FABP derived macrophages mRNA and protein level increased with the increase of the concentration of palmitic acid; in addition, along with the prolonging of treatment time of palmitic acid, A-FABP mRNA and protein level with palmitic acid intervention time increased.ELISA cell apoptosis assay showed that palmitic acid was time dependent the concentration dependent manner and DNA strand breaks induced injury; A-FABP siRNA significantly reduced DNA strand breaks induced by palmitic acid. The apoptosis related protein detection results showed that the expression of Bax protein in palmitic acid group was significantly higher than the control group, the expression of Bcl-2 protein was significantly lower than the control group.A-FABP siRNA group was treated with palmitic acid in cultured 24h, the protein expression of Bax was obviously less than that of palmitic acid group, the expression of Bcl-2 protein increased significantly.
Conclusion: palmitic acid can induce apoptosis of macrophages, which is mediated by A-FABP.
Objective: To investigate the effects of palmitic acid on mitochondrial function and mitochondrial apoptosis pathway in macrophages, and explore the role of A-FABP in palmitate induced mitochondrial dysfunction and mitochondrial apoptosis pathway activation.
Methods: using fluorescent staining was used to detect intracellular reactive oxygen species (ROS) content; JC-1 staining was used to detect the mitochondrial membrane potential; cell ATP content detection ATP kit; the total MDA (MDA) detection kit to detect the content of total MDA cells; Caspase3 assay Caspase3 activity detection kit; caspase9 assay caspase9 activity detection kit; gradient centrifugation from mitochondria; mitochondrial succinate dehydrogenase (SDH) kit to detect the activity of succinate dehydrogenase in mitochondria; mitochondrial respiratory chain complex IV kit to detect the mitochondrial respiratory chain complex IV activity; Western blot detected the expression of cytochrome c.
Results: the effect of palmitic acid, the level of ROS in macrophages Thp-1 source increased, decreased the mitochondrial membrane potential, promote the release of cytochrome c from mitochondria to the cytoplasm, enhanced Caspase3 and caspase9 activity by activation of the mitochondrial apoptosis pathway; palmitic acid also induced Thp-1 macrophage respiratory chain inhibition, ATP decreased cell lipid peroxidation oxidation weight, impaired mitochondrial function. A-FABP siRNA transfection group was treated with palmitic acid after 24h, compared with the palmitic acid group, ROS was significantly reduced, mitochondrial membrane potential, cytochrome c release decreased, Caspase3 and caspase9 tongue decreased, increased respiratory chain complex IV activity, increased the level of ATP, reduce lipid peroxidation.
Conclusion: palmitic acid induces the inhibition of the mitochondrial respiratory chain activity of macrophages, the decrease of ATP production, the increase of ROS production, the mitochondrial dysfunction and the activation of mitochondrial apoptotic pathway..A-FABP gene silencing can partially reverse this process.
Objective: To explore the effect of palmitic acid on mitochondrial biosynthesis and antioxidant capacity of macrophages, and to explore the role of A-FABP in the biosynthesis and antioxidant capacity of mitochondria induced by palmitic acid.
Methods: Real-Time PCR method was used to detect the copy number of mitochondrial DNA. Western blot and Real-Time PCR were used to detect antioxidant enzymes and mitochondrial biosynthesis related protein and gene expression..Mito-Tracker Green fluorescence probe was used to label cell mitochondria and detect the number of mitochondria.
Results: Thp-1 derived macrophages treated by palmitic acid, palmitic acid induced antioxidant enzymes including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (Cu/ZnSOD), uncoupling protein 2 (UCP2) mRNA level decreased, while protein related to biosynthesis of peroxidase proliferator activated receptor gamma. The activation of factor 1 alpha (PGC-1 alpha), mitochondrial nuclear respiratory factor (NRF-1) and estrogen related receptor (ESRRA) expression level of mRNA and PGC-1 alpha protein decreased number of mitochondria fluorescence detection of palmitic acid group is reduced, and the mitochondrial transcription factor A (TFAM) and mitochondrial DNA copy number of.A-FABP had no obvious change in siRNA transfection group to palmitic acid after 24h, compared with palmitic acid group of MnSOD, Cu/ZnSOD and UCP2 increase in the levels of mRNA, PGC-1 alpha, upregulation of NRF-1 and ESRRA, the number of mitochondria fluorescence probe method, TFAM and mitochondrial DNA copy number There was no obvious change.
Conclusion: A-FABP mediated decrease of mitochondrial biosynthesis and impaired antioxidant capacity induced by palmitic acid, which regulate oxidative stress and mitochondrial function in cells.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【引证文献】