本文選題：脂肪酸結(jié)合蛋白 + 棕櫚酸��； 參考：《中南大學(xué)》2012年博士論文

【摘要】：目的：探討脂肪細(xì)胞脂肪酸結(jié)合蛋白(A-FABP)在棕櫚酸誘導(dǎo)巨噬細(xì)胞凋亡中的作用。 方法：體外培養(yǎng)人單核細(xì)胞白血病細(xì)胞系(Thp-1細(xì)胞),加入佛波酯(PMA)培養(yǎng)72小時(shí)使其分化為巨噬細(xì)胞后,用不同濃度棕櫚酸(50μM,100μM,200μM,500μM)處理,在不同時(shí)間點(diǎn)提取細(xì)胞mRNA和蛋白,用Real-Time PCR以及Western blot檢測(cè)A-FABP蛋白的表達(dá)。采用ELISA以及Western blot檢測(cè)Thp-1源巨噬細(xì)胞凋亡。同時(shí)用A-FABP siRNA轉(zhuǎn)染Thp-1源巨噬細(xì)胞,觀察A-FABP基因沉默對(duì)棕櫚酸誘導(dǎo)的上述指標(biāo)變化的影響。 結(jié)果：在棕櫚酸作用下,Thp-1源巨噬細(xì)胞中A-FABP mRNA水平和蛋白水平隨棕櫚酸濃度增加而上升；此外,隨著棕櫚酸處理時(shí)間的延長(zhǎng),A-FABP mRNA水平和蛋白水平也隨棕櫚酸干預(yù)時(shí)間延長(zhǎng)而上升。ELISA細(xì)胞凋亡檢測(cè)結(jié)果顯示,棕櫚酸呈時(shí)間依賴和濃度依賴方式誘導(dǎo)DNA鏈斷裂損傷加重；A-FABP siRNA可顯著減少棕櫚酸誘導(dǎo)的DNA鏈斷裂損傷。凋亡相關(guān)蛋白檢測(cè)結(jié)果顯示,棕櫚酸組的Bax蛋白表達(dá)明顯高于對(duì)照組,Bcl-2蛋白表達(dá)明顯低于對(duì)照組。A-FABP siRNA組予棕櫚酸培養(yǎng)24h,其Bax蛋白表達(dá)較棕櫚酸組明顯減少,Bcl-2蛋白表達(dá)明顯增加。 結(jié)論：棕櫚酸可誘導(dǎo)巨噬細(xì)胞凋亡,該作用通過A-FABP介導(dǎo)。 目的：①探討棕櫚酸對(duì)巨噬細(xì)胞線粒體功能和線粒體凋亡途徑的影響；②探討A-FABP在棕櫚酸誘導(dǎo)巨噬細(xì)胞線粒體功能障礙和線粒體凋亡途徑激活中的作用。 方法：采用熒光染色檢測(cè)細(xì)胞內(nèi)活性氧(ROS)含量；JC-1染色法檢測(cè)線粒體膜電位；ATP檢測(cè)試劑盒檢測(cè)細(xì)胞ATP含量；總丙二醛(MDA)檢測(cè)試劑盒檢測(cè)細(xì)胞總MDA含量；caspase3活性檢測(cè)試劑盒檢測(cè)caspase3活性；caspase9活性檢測(cè)試劑盒檢測(cè)caspase9活性；梯度離心法抽提線粒體；線粒體琥珀酸脫氫酶(SDH)檢測(cè)試劑盒檢測(cè)細(xì)胞線粒體琥珀酸脫氫酶活性；線粒體呼吸鏈復(fù)合物Ⅳ檢測(cè)試劑盒檢測(cè)線粒體呼吸鏈復(fù)合物Ⅳ活性；Western blot檢測(cè)細(xì)胞色素c表達(dá)。 結(jié)果：在棕櫚酸作用下,Thp-1源巨噬細(xì)胞內(nèi)ROS水平增加,線粒體膜電位下降,促進(jìn)線粒體細(xì)胞色素c釋放至胞漿,caspase3和caspase9活性增強(qiáng),線粒體凋亡途徑被激活；棕櫚酸還引起Thp-1源巨噬細(xì)胞呼吸鏈活性受抑、ATP生成減少、細(xì)胞脂質(zhì)過氧化加重,線粒體功能受損。而A-FABP siRNA轉(zhuǎn)染組予棕櫚酸培養(yǎng)24h后,與棕櫚酸組比較,ROS水平明顯減低,線粒體膜電位恢復(fù),細(xì)胞色素c釋放減少,caspase3和caspase9舌性下降,呼吸鏈復(fù)合物Ⅳ活性增加,ATP水平升高,脂質(zhì)過氧化減輕。 結(jié)論：棕櫚酸誘導(dǎo)巨噬細(xì)胞的線粒體呼吸鏈活性受抑,ATP生成減少,ROS生成增加,引起線粒體功能障礙并激活線粒體凋亡途徑。A-FABP基因沉默可部分逆轉(zhuǎn)這一過程。 目的：①探討棕櫚酸對(duì)巨噬細(xì)胞線粒體生物合成與抗氧化能力的影響；②探討A-FABP在棕櫚酸誘導(dǎo)線粒體生物合成與抗氧化能力變化中的作用。 方法：Real-Time PCR方法檢測(cè)線粒體DNA拷貝數(shù)；Western blot和Real-Time PCR檢測(cè)抗氧化酶和線粒體生物合成相關(guān)的蛋白和基因表達(dá)。Mito-Tracker Green熒光探針法標(biāo)記細(xì)胞線粒體,檢測(cè)線粒體數(shù)量。 結(jié)果：Thp-1源巨噬細(xì)胞經(jīng)棕櫚酸處理后,棕櫚酸誘導(dǎo)抗氧化酶包括錳超氧化物歧化酶(MnSOD)、銅鋅超氧化物岐化酶(Cu/ZnSOD)、解偶聯(lián)蛋白2(UCP2)的mRNA水平下降,同時(shí)生物合成相關(guān)蛋白過氧化物酶體增殖物激活受體γ輔激活因子1α(PGC-1α)、線粒體核呼吸因子(NRF-1)和雌激素相關(guān)受體α(ESRRA) mRNA水平和PGC-1α蛋白表達(dá)下調(diào),熒光探針法檢測(cè)棕櫚酸組的線粒體數(shù)量減少,而線粒體轉(zhuǎn)錄因子A (TFAM)和線粒體DNA拷貝數(shù)無明顯變化。A-FABP siRNA轉(zhuǎn)染組予棕櫚酸培養(yǎng)24h后,與棕櫚酸組比較,MnSOD、Cu/ZnSOD和UCP2的mRNA水平增加,PGC-1α、NRF-1和ESRRA表達(dá)上調(diào),熒光探針法檢測(cè)的線粒體數(shù)量增加,而TFAM和線粒體DNA拷貝數(shù)無明顯變化。 結(jié)論：A-FABP介導(dǎo)棕櫚酸引起的線粒體生物合成減少及抗氧化能力受損,從而調(diào)控細(xì)胞內(nèi)氧化應(yīng)激和線粒體功能。
[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.

【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前2條

1 杜巖青;活動(dòng)性肺結(jié)核血清代謝輪廓研究[D];天津醫(yī)科大學(xué);2014年

2 何廣深;百子蓮胚性愈傷組織超低溫保存中鈣離子的分布變化及逆境應(yīng)答機(jī)制初探[D];上海交通大學(xué);2014年

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本文編號(hào)：1762261