DRAM1通过溶酶体调节自噬和细胞凋亡

发布时间：2018-07-01 11:27

本文选题：DRAM1 + 自噬　；参考：《苏州大学》2011年博士论文

【摘要】：目的:硕士期间的研究工作表明,大鼠纹状体内给予线粒体毒素3-硝基丙酸(3-NP)引起的纹状体神经元死亡伴有自噬和凋亡激活,p53诱导的DRAM1表达在自噬激活和细胞死亡中有重要作用。本论文研究DRAM1调节细胞线粒体失能引起的自噬激活和细胞死亡的分子机制。方法:(1)建立3-NP所致A549细胞线粒体失能毒性模型。3-NP处理细胞24, 48和72h后,Western blot法测定DRAM1,BAX,p62和LC3等凋亡和自噬蛋白的表达和线粒体细胞色素C的释放,免疫荧光法检测LC3的表达,观察自噬和凋亡通路的激活。(2)观察自噬和凋亡过程在3-NP所致的细胞线粒体毒性引起的细胞死亡中的作用。Western blot法检测Atg5的siRNA干扰效果。WST-1法检测Atg5 siRNA干扰和Z-VAD-FMK对3-NP诱导的细胞死亡的影响。(3)检测DRAM1在3-NP诱导的细胞自噬流量变化中的作用。Western Blot法检测DRAM1的siRNA后观察p62,LC3等自噬蛋白的表达。免疫组化法检测DRAM1蛋白下调后3-NP所致的LC3表达的变化。(4)研究DRAM1调节3-NP诱导的细胞自噬流量的作用机制。免疫荧光双标法观察DRAM1和溶酶体标志物LysoTracker在细胞内的共定位情况。为了观察DRAM1在细胞清除自噬-溶酶体中的作用,野生型细胞和DRAM1 siRNA干扰细胞分别用雷帕霉素处理24h以及处理6h后撤去雷帕霉素,采用Western blot法检测LC3的蓄积情况,并对LC3点状聚集进行量化,以及用免疫组化法观察LC3和Lamp2检测在胞质内的降解情况。为了观察DRAM1影响自噬体的降解机制,用Western blot法检测Cathepsin D的激活以及使用分子探针观察氢质子泵和溶酶体的酸化情况。(5)验证DRAM1是否是通过溶酶体来调节自噬流量。使用溶酶体抑制剂E64d和氯喹抑制溶酶体后观察是否会产生LC3的蓄积,采用Western blot法观察了抑制溶酶体后Cathepsin D的变化,以及用WST-1法观察是否影响细胞活力。(6)检测DRAM1在3-NP诱导的细胞凋亡过程中的作用。Western Blot法观察DRAM1表达降低后BAX的表达和线粒体细胞色素C的释放以及caspase-3的激活。(7)检测DRAM1调节3-NP所致细胞凋亡的机制。Western Blot法验证BAX siRNA干扰和过表达的效率。为了研究BAX在DRAM1调节凋亡中的作用机制,采用Western Blot法观察BAX的表达降低后对于Cathepsin D和casepase 3的激活以及细胞色素C的释放的影响。WST-1法检测BAX表达降低后对3-NP引起的细胞死亡的影响。结果: (1)在不同时间点3-NP所致A549细胞线粒体失能毒性模型中,Western blot和免疫荧光结果显示DRAM1,LC3自噬相关蛋白在24-48h达到高峰(p0.01),自噬底物蛋白p62蓄积减少(p0.01)。同时,凋亡相关蛋白BAX和细胞色素C的释放在48-72h达到高峰(p0.01)。表明3-NP导致了自噬和凋亡过程的激活。(2) Western blot结果显示Atg5 siRNA干扰后,Atg5蛋白表达明显降低(p0.01)。WST-1法结果显示Atg5表达降低和Z-VAD-FMK处理对3-NP所致细胞死亡有显著抑制作用。表明自噬和凋亡过程都参与了3-NP诱导的细胞死亡。(3) Western blot结果显示DRAM1的siRNA干扰效果明显(p0.01),并且DRAM1蛋白表达降低后对3-NP所致自噬相关蛋白LC3的升高有明显抑制作用(p0.01),自噬底物蛋白p62表达升高(p0.01)。说明siRNA干扰DRAM1后细胞自噬流量降低。(4)免疫荧光双标结果显示DRAM1和溶酶体标志物LysoTracker在胞质内的共定位现象明显。雷帕霉素处理24h组和处理6h后撤去雷帕霉素组的结果显示,siRNA干扰DRAM1细胞组在撤去雷帕霉素后LC3和Lamp2的清除情况明显比野生型细胞要缓慢。LC3的量化结果表明了siRNA干扰DRAM1组LC3-II在撤除雷帕霉素后下降减慢。Western blot结果显示siRNA干扰DRAM1对3-NP所致Cathepsin D的激活有明显抑制作用(p0.01)。说明siRNA干扰DRAM1细胞对比野生型细胞对胞内自噬溶酶体的清除能力降低。溶酶体酸化指示剂和量化结果显示siRNA干扰DRAM1对3-NP所致的溶酶体酸化程度降低(p0.01)。氢质子泵结果显示siRNA干扰DRAM1对3-NP所致的氢质子泵的激活有抑制作用。说明siRNA干扰DRAM1的细胞溶酶体酸化作用降低,清除自噬溶酶体能力减弱。(5)免疫荧光双染结果显示,当线粒体毒性药物3-NP处理后,LC3和Lamp2表达明显增加,并且共定位现象明显。E64d和氯喹处理后,LC3和Lamp2蓄积明显增加。Western blot结果表明3-NP诱导的LC3Ⅱ在E64d处理后显著上升(p0.05), 3-NP诱导的LC3Ⅱ在氯喹处理后也有显著上升(p0.01)。Western blot结果表明在E64d和氯喹处理后3-NP诱导的细胞色素C从线粒体内的释放显著减少(p0.01)。WST-1结果显示WT细胞在溶酶体抑制剂处理后,细胞活力没有很明显的变化(p0.05),但溶酶体抑制剂显著抑制了3-NP导致的细胞死亡(p0.05)。说明抑制了溶酶体就抑制了自噬的流量和3-NP诱导的细胞死亡,而DRAM1就是通过溶酶体来达到调节自噬流量的作用。(6) Western blot法结果显示DRAM1蛋白siRNA干扰后对3-NP所致BAX蛋白升高有明显抑制作用(p0.01),线粒体细胞色素C的释放减少(p0.01), caspase-3的激活减少(p0.01)。(7) Western blot结果显示BAX siRNA干扰后,BAX蛋白表达明显下降(p0.01)。Western blot结果显示,细胞内BAX表达降低会抑制3-NP诱导的Cathepsin D和casepase-3的激活(p0.01)和3-NP诱导的细胞色素C的释放(p0.01)。WST-1结果显示BAX的siRNA对3-NP所致的细胞死亡有明显抑制作用(p0.01)。说明DRAM1有可能通过BAX来调节3-NP诱导的凋亡过程。结论:本文研究结果表明DRAM1通过促进溶酶体酸化和激活溶酶体酶来调节自噬流量,同时DRAM1通过上调BAX来调节线粒体介导的凋亡通路。正在进行中的工作: 自噬体和溶酶体的融合对于自噬体的降解是一个很重要的过程,因此对于深刻的了解DRAM1在自噬体和溶酶体融合中的作用正在研究中。DRAM1通过BAX来调节线粒体信号凋亡通路是一个重要的发现,DRAM1如何调节BAX的表达正在进一步研究中。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R363

【共引文献】