本文選題：DRAM1 + 自噬��； 參考：《蘇州大學》2011年博士論文

【摘要】：目的:碩士期間的研究工作表明,大鼠紋狀體內(nèi)給予線粒體毒素3-硝基丙酸(3-NP)引起的紋狀體神經(jīng)元死亡伴有自噬和凋亡激活,p53誘導的DRAM1表達在自噬激活和細胞死亡中有重要作用。本論文研究DRAM1調(diào)節(jié)細胞線粒體失能引起的自噬激活和細胞死亡的分子機制。 方法:(1)建立3-NP所致A549細胞線粒體失能毒性模型。3-NP處理細胞24, 48和72h后,Western blot法測定DRAM1,BAX,p62和LC3等凋亡和自噬蛋白的表達和線粒體細胞色素C的釋放,免疫熒光法檢測LC3的表達,觀察自噬和凋亡通路的激活。(2)觀察自噬和凋亡過程在3-NP所致的細胞線粒體毒性引起的細胞死亡中的作用。Western blot法檢測Atg5的siRNA干擾效果。WST-1法檢測Atg5 siRNA干擾和Z-VAD-FMK對3-NP誘導的細胞死亡的影響。(3)檢測DRAM1在3-NP誘導的細胞自噬流量變化中的作用。Western Blot法檢測DRAM1的siRNA后觀察p62,LC3等自噬蛋白的表達。免疫組化法檢測DRAM1蛋白下調(diào)后3-NP所致的LC3表達的變化。(4)研究DRAM1調(diào)節(jié)3-NP誘導的細胞自噬流量的作用機制。免疫熒光雙標法觀察DRAM1和溶酶體標志物LysoTracker在細胞內(nèi)的共定位情況。為了觀察DRAM1在細胞清除自噬-溶酶體中的作用,野生型細胞和DRAM1 siRNA干擾細胞分別用雷帕霉素處理24h以及處理6h后撤去雷帕霉素,采用Western blot法檢測LC3的蓄積情況,并對LC3點狀聚集進行量化,以及用免疫組化法觀察LC3和Lamp2檢測在胞質(zhì)內(nèi)的降解情況。為了觀察DRAM1影響自噬體的降解機制,用Western blot法檢測Cathepsin D的激活以及使用分子探針觀察氫質(zhì)子泵和溶酶體的酸化情況。(5)驗證DRAM1是否是通過溶酶體來調(diào)節(jié)自噬流量。使用溶酶體抑制劑E64d和氯喹抑制溶酶體后觀察是否會產(chǎn)生LC3的蓄積,采用Western blot法觀察了抑制溶酶體后Cathepsin D的變化,以及用WST-1法觀察是否影響細胞活力。(6)檢測DRAM1在3-NP誘導的細胞凋亡過程中的作用。Western Blot法觀察DRAM1表達降低后BAX的表達和線粒體細胞色素C的釋放以及caspase-3的激活。(7)檢測DRAM1調(diào)節(jié)3-NP所致細胞凋亡的機制。Western Blot法驗證BAX siRNA干擾和過表達的效率。為了研究BAX在DRAM1調(diào)節(jié)凋亡中的作用機制,采用Western Blot法觀察BAX的表達降低后對于Cathepsin D和casepase 3的激活以及細胞色素C的釋放的影響。WST-1法檢測BAX表達降低后對3-NP引起的細胞死亡的影響。 結(jié)果: (1)在不同時間點3-NP所致A549細胞線粒體失能毒性模型中,Western blot和免疫熒光結(jié)果顯示DRAM1,LC3自噬相關(guān)蛋白在24-48h達到高峰(p0.01),自噬底物蛋白p62蓄積減少(p0.01)。同時,凋亡相關(guān)蛋白BAX和細胞色素C的釋放在48-72h達到高峰(p0.01)。表明3-NP導致了自噬和凋亡過程的激活。(2) Western blot結(jié)果顯示Atg5 siRNA干擾后,Atg5蛋白表達明顯降低(p0.01)。WST-1法結(jié)果顯示Atg5表達降低和Z-VAD-FMK處理對3-NP所致細胞死亡有顯著抑制作用。表明自噬和凋亡過程都參與了3-NP誘導的細胞死亡。(3) Western blot結(jié)果顯示DRAM1的siRNA干擾效果明顯(p0.01),并且DRAM1蛋白表達降低后對3-NP所致自噬相關(guān)蛋白LC3的升高有明顯抑制作用(p0.01),自噬底物蛋白p62表達升高(p0.01)。說明siRNA干擾DRAM1后細胞自噬流量降低。(4)免疫熒光雙標結(jié)果顯示DRAM1和溶酶體標志物LysoTracker在胞質(zhì)內(nèi)的共定位現(xiàn)象明顯。雷帕霉素處理24h組和處理6h后撤去雷帕霉素組的結(jié)果顯示,siRNA干擾DRAM1細胞組在撤去雷帕霉素后LC3和Lamp2的清除情況明顯比野生型細胞要緩慢。LC3的量化結(jié)果表明了siRNA干擾DRAM1組LC3-II在撤除雷帕霉素后下降減慢。Western blot結(jié)果顯示siRNA干擾DRAM1對3-NP所致Cathepsin D的激活有明顯抑制作用(p0.01)。說明siRNA干擾DRAM1細胞對比野生型細胞對胞內(nèi)自噬溶酶體的清除能力降低。溶酶體酸化指示劑和量化結(jié)果顯示siRNA干擾DRAM1對3-NP所致的溶酶體酸化程度降低(p0.01)。氫質(zhì)子泵結(jié)果顯示siRNA干擾DRAM1對3-NP所致的氫質(zhì)子泵的激活有抑制作用。說明siRNA干擾DRAM1的細胞溶酶體酸化作用降低,清除自噬溶酶體能力減弱。(5)免疫熒光雙染結(jié)果顯示,當線粒體毒性藥物3-NP處理后,LC3和Lamp2表達明顯增加,并且共定位現(xiàn)象明顯。E64d和氯喹處理后,LC3和Lamp2蓄積明顯增加。Western blot結(jié)果表明3-NP誘導的LC3Ⅱ在E64d處理后顯著上升(p0.05), 3-NP誘導的LC3Ⅱ在氯喹處理后也有顯著上升(p0.01)。Western blot結(jié)果表明在E64d和氯喹處理后3-NP誘導的細胞色素C從線粒體內(nèi)的釋放顯著減少(p0.01)。WST-1結(jié)果顯示W(wǎng)T細胞在溶酶體抑制劑處理后,細胞活力沒有很明顯的變化(p0.05),但溶酶體抑制劑顯著抑制了3-NP導致的細胞死亡(p0.05)。說明抑制了溶酶體就抑制了自噬的流量和3-NP誘導的細胞死亡,而DRAM1就是通過溶酶體來達到調(diào)節(jié)自噬流量的作用。(6) Western blot法結(jié)果顯示DRAM1蛋白siRNA干擾后對3-NP所致BAX蛋白升高有明顯抑制作用(p0.01),線粒體細胞色素C的釋放減少(p0.01), caspase-3的激活減少(p0.01)。(7) Western blot結(jié)果顯示BAX siRNA干擾后,BAX蛋白表達明顯下降(p0.01)。Western blot結(jié)果顯示,細胞內(nèi)BAX表達降低會抑制3-NP誘導的Cathepsin D和casepase-3的激活(p0.01)和3-NP誘導的細胞色素C的釋放(p0.01)。WST-1結(jié)果顯示BAX的siRNA對3-NP所致的細胞死亡有明顯抑制作用(p0.01)。說明DRAM1有可能通過BAX來調(diào)節(jié)3-NP誘導的凋亡過程。 結(jié)論:本文研究結(jié)果表明DRAM1通過促進溶酶體酸化和激活溶酶體酶來調(diào)節(jié)自噬流量,同時DRAM1通過上調(diào)BAX來調(diào)節(jié)線粒體介導的凋亡通路。 正在進行中的工作: 自噬體和溶酶體的融合對于自噬體的降解是一個很重要的過程,因此對于深刻的了解DRAM1在自噬體和溶酶體融合中的作用正在研究中。DRAM1通過BAX來調(diào)節(jié)線粒體信號凋亡通路是一個重要的發(fā)現(xiàn),DRAM1如何調(diào)節(jié)BAX的表達正在進一步研究中。
[Abstract]:Objective: the study during the master's period showed that the striatal neurons in the rat's striatum were killed by mitochondrial toxin 3- nitropropionic acid (3-NP) with autophagy and apoptosis activation. The expression of DRAM1 induced by p53 played an important role in autophagy activation and cell death. This paper studied the autophagy induced by DRAM1 to regulate the autophagy induced by mitochondria inactivation. The molecular mechanism of living and cell death.
Methods: (1) to establish the mitochondrial inability toxicity model of A549 cells induced by 3-NP,.3-NP treated cells 24, 48 and 72h, the Western blot method was used to determine the expression of apoptosis and autophagic proteins such as DRAM1, BAX, p62 and LC3 and the release of mitochondrial cytochrome C, the expression of LC3 was detected by immunofluorescence and the activation of autophagy and apoptosis pathway was observed. (2) autophagy and apoptosis were observed. The role of the process in cell death caused by mitochondrial toxicity induced by 3-NP.Western blot method to detect the siRNA interference effect of Atg5 by the.WST-1 method to detect the Atg5 siRNA interference and the effect of Z-VAD-FMK on the cell death induced by 3-NP. (3) detection of DRAM1 in the changes of the cell autophagic flow induced by 3-NP The expression of autophagic protein, such as p62, LC3 and so on, was observed after siRNA. Immunohistochemical method was used to detect the change of LC3 expression induced by DRAM1 protein down regulation. (4) the mechanism of DRAM1 regulating 3-NP induced autophagic flow was studied. The co localization of DRAM1 and lysosome markers LysoTracker in the cells was observed by the double immunofluorescent labeling method. In the cell removal of autophagy - lysosome, wild type and DRAM1 siRNA interfering cells treated 24h with rapamycin and removed rapamycin after 6h treatment. Western blot method was used to detect the accumulation of LC3, and LC3 dot aggregation was quantified, and the degradation of LC3 and Lamp2 in cytoplasm was observed by immunohistochemistry. In order to observe the effect of DRAM1 on the degradation mechanism of autophagosome, the activation of Cathepsin D and the acidification of the hydrogen proton pump and lysosome were observed by the Western blot method. (5) whether the DRAM1 was regulated by the lysosome to regulate the autophagic flow. Whether the lysosome inhibitor E64d and chloroquine were used to observe the lysosomes were observed. The accumulation of LC3 was produced, the changes of Cathepsin D after the inhibition of lysosomes were observed by Western blot method, and the effect of WST-1 on cell viability was observed. (6) the detection of DRAM1 in the process of 3-NP induced apoptosis was detected by.Western Blot method to observe the expression of BAX and the release of mitochondrial cytochrome as well as the release of mitochondrial cytochrome as well as the reduction of DRAM1 expression. (7) detect the mechanism of DRAM1 regulating 3-NP induced apoptosis by.Western Blot method to verify the efficiency of BAX siRNA interference and overexpression. In order to study the mechanism of BAX in DRAM1 regulation of apoptosis, Western Blot method was used to observe the effect of BAX expression on the activation of Cathepsin and 3 and the release of cytochrome. WST-1 assay was used to detect the effect of reduced expression of BAX on 3-NP induced cell death.
Results: (1) the results of Western blot and immunofluorescence showed that DRAM1, LC3 autophagy related proteins reached the peak in 24-48h (P0.01), and the accumulation of autophagic protein p62 decreased (P0.01), and the release of apoptotic phase protein BAX and cytochrome C reached the peak at Western blot and immunofluorescence results. The results showed that 3-NP resulted in the activation of autophagy and apoptosis. (2) Western blot results showed that Atg5 protein expression was significantly reduced after Atg5 siRNA interference (P0.01).WST-1 method showed that the decrease of Atg5 expression and Z-VAD-FMK treatment had significant inhibitory effect on 3-NP cell death. It showed that both the process of apoptosis and the process of apoptosis were involved in the cell death induced by 3-NP. (3) the results of Western blot showed that the siRNA interference effect of DRAM1 was obvious (P0.01), and the decrease of DRAM1 protein expression was significantly inhibited by 3-NP induced autophagic associated protein LC3 (P0.01), and the expression of the autophagic substrate protein p62 increased (P0.01). The co localization of the M1 and the lysosome marker LysoTracker in the cytoplasm was obvious. The results of rapamycin treatment group 24h and the withdrawal of rapamycin after the treatment of 6h showed that the clearance of LC3 and Lamp2 in the DRAM1 cell group after the withdrawal of rapamycin was significantly slower than that of the wild type cells. The quantitative results showed the siRNA interference DRAM1. Group LC3-II decreased.Western blot after withdrawal of rapamycin and showed a significant inhibitory effect of siRNA interference on the activation of Cathepsin D caused by 3-NP (P0.01). Indicating that siRNA interferes with DRAM1 cells to reduce the ability to scavenge the intracellular autophagic lysosomes of wild type cells. Soluble enzyme acidification indicator and quantitative results show siRNA interference. DRAM1 reduced the degree of lysosomal acidification (P0.01) caused by 3-NP. The results of hydrogen proton pump showed that siRNA interference DRAM1 inhibited the activation of the hydrogen proton pump caused by 3-NP. It indicated that the lysosome acidification of siRNA interfered DRAM1 cells decreased and the ability to clear the autophagosomes weakened. (5) the results of immunofluorescence double staining showed that the mitochondrial toxicity drug was a mitochondrial toxicity drug. After 3-NP treatment, the expression of LC3 and Lamp2 increased obviously, and the co localization phenomenon was obvious.E64d and chloroquine treatment. The accumulation of LC3 and Lamp2 significantly increased the result of.Western blot. The 3-NP induced LC3 II was significantly increased after E64d treatment (P0.05). The release of cytochrome C induced by 3-NP after treatment with 64D and chloroquine significantly reduced the release of cytochrome C from the mitochondria (P0.01).WST-1 results showed that the cell viability of WT cells was not significantly changed after the lysosome inhibitor treatment (P0.05), but the lysosome inhibitors significantly inhibited the cell death (P0.05) caused by 3-NP. It indicated that the lysosomes inhibited the inhibition of the lysosome. The flow of phagocytosis and 3-NP induced cell death, and DRAM1 was achieved through the lysosome to regulate autophagic flow. (6) Western blot results showed that DRAM1 protein siRNA interference significantly inhibited the increase of BAX protein induced by 3-NP (P0.01), mitochondrial cytochrome C release decreased (P0.01), caspase-3 activation decreased. 7) the results of Western blot showed that after BAX siRNA interference, the expression of BAX protein decreased significantly (P0.01).Western blot results showed that the decrease of BAX expression in the cells inhibited 3-NP induced Cathepsin D and activation. The obvious inhibitory effect (P0.01) indicates that DRAM1 may regulate 3-NP induced apoptosis through BAX.
Conclusion: the results of this study show that DRAM1 regulates autophagic flow by promoting lysosome acidification and activating lysosomal enzyme, and DRAM1 regulates mitochondrial mediated apoptosis pathway by up regulation of BAX.
Ongoing work:
The fusion of autophagosomes and lysosomes is an important process for the degradation of autophagosomes. Therefore, a profound understanding of the role of DRAM1 in the fusion of autophagosomes and lysosomes is being studied..DRAM1 is an important discovery in the regulation of mitochondrial signaling pathway through BAX. How to regulate the expression of BAX is further studied by DRAM1 Middle.
【學位授予單位】：蘇州大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R363

【共引文獻】

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