多氯联苯醌诱导细胞发生自噬以及自噬—凋亡转换的机制分析
发布时间：2018-07-25 19:32
【摘要】：多氯联苯(PCBs)是一类无所不在的持久性有机污染物,由于其多种毒性效应如内分泌毒性、免疫毒性、神经毒性等,在二十世纪七十年代已被禁止使用,但是由于其特殊的理化及生物学特性,PCBs仍然广泛存在于我们的环境包括陆地和水生系统。细胞自噬是细胞内受损衰老的蛋白质或者细胞器包裹起来形成自噬体,自噬体再与溶酶体融合进而进行消化降解的过程,也常被作为细胞的自我保护机制。该研究的目的是为了分析PCB29-pQ激活自噬的具体机制,以及探讨细胞自噬与细胞凋亡间的转换机制。第一部分:该部分研究考察了多氯联苯醌通过mTOR/p70S6k诱导细胞自噬发生的分子机制。选用HepG2和MDA-MB-231细胞作为研究对象,并通过实验发现PCB29-pQ诱导的自噬没有细胞特异性。首先,两种细胞在5μM PCB29-pQ处理24 h后透射电镜检测细胞超微结构显示出自噬泡显著特征,并通过AO染色、MDC染色观察到明显的自噬泡形成,由此先从形态学和生化特征表明PCB29-pQ作用后可以引起细胞发生自噬。然后从自噬体形成的3个阶段证明诱导自噬发生的分子机制。第一阶段从浓度和时间梯度上分别检测了对自噬开关负调控的蛋白mTOR和p70S6k的表达,在PCB29-pQ作用后其蛋白表达水平降低,表明激活了自噬的诱导阶段。第二阶段检测了自噬体形成过程中自噬相关蛋白ATG5、ATG12、LC3的表达,并用RT-PCR分析LC3 mRNA水平。与对照组相比,PCB29-pQ处理的细胞蛋白和基因的表达水平都显著增加,且在5μM PCB29-pQ处理24 h有最大值。而自噬降解底物p62蛋白水平随时间梯度降低,在5μM PCB29-pQ处理24 h表达水平最低。这些结果说明PCB29-pQ能够激活自噬体的形成阶段,促进自噬体降解底物的能力。第三阶段通过加入CQ抑制LC3的降解后检测LC3表达的净通量,结果表明PCB29-pQ作用后提高了LC3净通量并在5μM PCB29-pQ作用24 h时最明显。并且通过转染GFP-LC3质粒,免疫荧光观察了自噬体标记LC3与溶酶体标记LAMP2的共定位,结果表明促进了自噬体与溶酶体融合的阶段。接着阐明了自噬的发生在PCB29-pQ诱导细胞毒性中的作用,在加入不同阶段自噬抑制剂3-MA和CQ后,通过CCK8、流式细胞仪、细胞凋亡标志蛋白caspase3的检测,表明抑制自噬促进了PCB29-pQ诱导的细胞毒性的增加。最后用加入ROS清除剂NAC的方法考察PCB29-pQ引起自噬水平升高的原因,结果表明,PCB29-pQ诱导的细胞自噬的激活受ROS水平的调节。根据以上研究可知,PCB29-pQ可以诱导HepG2和MDA-MB-231细胞发生自噬,并受细胞内ROS水平的调节。PCB29-pQ引起的自噬受mTOR/p70S6k和ATG5/ATG12/LC3信号通路的调控,且作为一种存活机制保护细胞。第二部分:该部分初步探讨了多氯联苯醌在低浓度诱导自噬,高浓度诱导凋亡时,钙蛋白酶活性的作用对自噬向凋亡信号传递的影响。首先通过JC-1荧光探针检测HepG2细胞线粒体膜电位,免疫印迹法检测凋亡相关蛋白caspase9/caspase3等表达,TUNEL法检测细胞凋亡,免疫荧光检测自噬标志蛋白LC3焦点,结果发现,与对照组相比,随着PCB29-pQ处理浓度的升高细胞凋亡水平逐渐增加,而在低浓度5μM PCB29-pQ处理后自噬LC3焦点最多,随着浓度升高,自噬LC3焦点数目降低。随后用流式细胞仪检测PCB29-pQ诱导的细胞钙离子水平的变化,荧光分光光度计检测钙蛋白酶活性,结果表明,PCB29-pQ可以诱导HepG2细胞钙离子水平以及钙蛋白酶活性的升高,以及在加入钙离子螯合剂BAPTA-AM后抑制了calpain蛋白表达,表明,calpain的表达依赖于Ca2+水平的调节。文献报道Beclin1、ATG5可经钙蛋白酶切割形成Beclin1-c、tATG5易位至线粒体将自噬向凋亡信号传递,而在本研究中PCB29-pQ没有引起Beclin1、ATG5切割易位至线粒体。以上研究可知,PCB29-pQ可以在低浓度时诱导HepG2细胞自噬,高浓度时诱导细胞凋亡,并且伴随着细胞内钙离子水平和钙蛋白酶活性的升高,但钙蛋白酶活性并不引起Beclin1、ATG5产生切割将信号从自噬传递至凋亡。第三部分:该部分进一步探讨了多氯联苯醌诱导细胞自噬与细胞凋亡转换的机制,分析选取了对自噬和凋亡有双重调节作用的p53/HMGB1蛋白。Western Blot和免疫荧光实验表明了PCB29-pQ作用后诱导HepG2细胞p53/HMGB1易位到细胞质。免疫共沉淀实验表明了5μM PCB29-pQ作用后促进了HepG2细胞p53/HMGB1在细胞核中的结合,15μM PCB29-pQ作用后促进了HepG2细胞p53/HMGB1在细胞质中的结合。由于HMGB1/p53可以作为核转录因子调控下游靶基因的表达,影响细胞自噬与凋亡。我们干扰了p53蛋白表达后检测了p53下游靶基因DRAM、ULK1、Bax表达,同时干扰了HMGB1蛋白表达后Western Blot检测了HMGB1的下游蛋白HSPB1表达,结果表明,在抑制了p53/HMGB1的作用后,抑制了该蛋白调控的靶基因的表达。针对上述检测结果,我们进一步研究p53/HMGB1诱导细胞自噬和细胞凋亡的作用机制。干扰p53基因后提取了核质蛋白,Western Blot表明p53 siRNA后促进了PCB29-pQ诱导的HMGB1进一步易位至细胞质,细胞存活率实验表明了p53 siRNA抑制了PCB29-pQ诱导的细胞凋亡,免疫荧光LC3焦点实验表明p53 siRNA促进了PCB29-pQ诱导的细胞自噬,而在加入HMGB1抑制剂EP后,细胞凋亡增加,细胞自噬水平降低。这些结论表明,易位到细胞质中的HMGB1发挥着抑制凋亡、促进自噬的作用。在干扰了HMGB1蛋白后相同的实验方法证明HMGB1 siRNA后促进了PCB29-pQ诱导的p53进一步易位至细胞质,促进了PCB29-pQ诱导的细胞凋亡,以及抑制了PCB29-pQ诱导的细胞自噬,而在加入p53抑制剂PFT-α后,细胞凋亡减少,细胞自噬水平升高。这些结论表明,易位到细胞质中的p53发挥着促进凋亡、抑制自噬的作用。以上研究表明,PCB29-pQ诱导HepG2细胞自噬与细胞凋亡的过程中,是通过p53/HMGB1在细胞核与细胞质定位以及对靶基因表达的不同发挥其在PCB29-pQ引起细胞自噬与细胞凋亡间的调控作用。
[Abstract]:Polychlorinated biphenyl (PCBs) is a ubiquitous persistent organic pollutant. Due to its many toxic effects, such as endocrine toxicity, immunotoxicity, neurotoxicity, and so on, it has been banned in 1970s, but because of its special physicochemical and biological characteristics, PCBs still exists in our environment including land and aquatic products. Autophagy is the process of cell autophagy or cell organelle wrapped up to form autophagosome, and the autophagosome is fused with lysosomes for digestion and degradation, and is often used as a self-protection mechanism for cells. The purpose of this study is to analyze the specific mechanism of PCB29-pQ activation and to explore the autophagy of cells. The transformation mechanism with cell apoptosis. Part 1: this part studied the molecular mechanism of polychlorinated biphenyquinone induced autophagy through mTOR/p70S6k. HepG2 and MDA-MB-231 cells were selected as the research object, and the experiment found that the autophagy induced by PCB29-pQ had no cell specificity. First, two cells were treated in 5 mu M PCB29-pQ. After 24 h, the ultrastructure of the cell was detected by the transmission electron microscope. The obvious autophagy was observed by AO staining and MDC staining was used to observe the formation of autophagic vesicles. From the morphological and biochemical characteristics, the cell autophagy could be caused by the action of PCB29-pQ. Then, the molecular mechanism of inducing autophagy was proved from the 3 stages of autophagic formation. In the first stage, the expression of protein mTOR and p70S6k negatively regulated by autophagic switch was detected from the concentration and time gradient, and the expression level of the protein decreased after the action of PCB29-pQ, indicating the activation of the induction stage of autophagy. The second stage detected the expression of autophagic related protein ATG5, ATG12, LC3 and RT-PCR in the process of autophagic formation. Analysis of LC3 mRNA level. Compared with the control group, the expression level of cell protein and gene in PCB29-pQ treatment increased significantly, and the maximum value was 24 h at 5 M PCB29-pQ treatment. The level of the autophagy degradation substrate p62 protein decreased with the time gradient, and the lowest expression level in the 5 u M PCB29-pQ processing 24 h. These results indicate that PCB29-pQ can activate autophagy. The formation stage of the body promotes the ability of the autophagic to degrade the substrate. The third phase detects the net flux of LC3 expression by adding CQ to inhibit the degradation of LC3. The results show that the net flux of LC3 is enhanced after the action of PCB29-pQ and is most obvious at the action of 24 h by 5 M PCB29-pQ. And the expression of the autophagic marker LC3 is observed by the transfection of the GFP-LC3 particles. The co localization of lysosome labeled LAMP2 showed that the fusion of autophagosomes and lysosomes was promoted. Then the role of autophagy in PCB29-pQ induced cytotoxicity was clarified. After adding different stages of autophagy inhibitor 3-MA and CQ, the detection of the inhibition of autophagy through the detection of CCK8, flow cytometry, and apoptosis marker protein Caspase3. The increase of cytotoxicity induced by PCB29-pQ was promoted. Finally, the reasons for the increase of autophagy induced by PCB29-pQ were investigated by the addition of ROS scavenger NAC. The results showed that the activation of autophagy induced by PCB29-pQ was regulated by the ROS level. According to the above study, PCB29-pQ could induce autophagy in HepG2 and MDA-MB-231 cells and was finer. The autophagy induced by the intracellular ROS level of.PCB29-pQ is regulated by the mTOR/p70S6k and ATG5/ATG12/LC3 signaling pathways and protects the cells as a survival mechanism. The second part: this part preliminarily discussed the effect of the activity of calcium protease on the autophagy to the apoptosis signal when the autophagy induced by the low concentration of polychlorinated biphenyquinone. The JC-1 fluorescence probe was used to detect the mitochondrial membrane potential of HepG2 cells, and the expression of apoptosis related protein caspase9/caspase3 was detected by immunoblotting. Apoptosis was detected by TUNEL method and the focal point of autophagic marker protein LC3 was detected by immunofluorescence. The results were found to be compared with the control group, and the apoptotic water was increased with the concentration of PCB29-pQ. The level of autophagy was increased gradually, and the focus of autophagy LC3 was most in the low concentration of 5 M PCB29-pQ. With the increase of concentration, the number of autophagic LC3 focal points decreased. Then the level of calcium ion induced by PCB29-pQ was detected by flow cytometry, and the activity of calsin was detected by the fluorescence spectrophotometer. The results showed that PCB29-pQ could induce the calcium ion of HepG2 cells. Levels and the increase of calsin activity and the inhibition of calpain protein expression after adding calcium ion chelating agent BAPTA-AM showed that the expression of calpain depended on the regulation of Ca2+ level. The literature reported that Beclin1, ATG5 can be cut through calsin to form Beclin1-c, tATG5 translocation to mitochondria to transmit autophagy to apoptosis signal, and in this study PCB29-pQ did not cause Beclin1, ATG5 cut translocation to mitochondria. Above study, PCB29-pQ can induce autophagy at low concentration of HepG2 cells, induce apoptosis at high concentration, and increase the intracellular calcium level and calsin activity, but the activity of calcinease does not cause Beclin1, ATG5 produces cutting signal. From autophagy to apoptosis. The third part: this part further explored the mechanism of polychlorinated biphenyquinone induced autophagy and cell apoptosis, and selected the p53/HMGB1 protein.Western Blot and immunofluorescence test, which had dual regulation on autophagy and apoptosis, and showed that PCB29-pQ could induce p53/HMGB1 translocation to HepG2 cells after PCB29-pQ action. Cytoplasm. The immunoprecipitation experiment showed that the interaction of 5 M PCB29-pQ promoted the binding of p53/HMGB1 in the nucleus and promoted the binding of p53/HMGB1 in the cytoplasm after the action of 15 mu M PCB29-pQ. Because HMGB1/p53 could be used as a nuclear transcription factor to regulate the expression of the target gene of the downstream target and affect the autophagy and apoptosis of the cell. After interfering with the expression of p53 protein, the downstream target gene of p53 was detected, DRAM, ULK1, Bax expression, and Western Blot detected the downstream protein HSPB1 expression of HMGB1 after the expression of HMGB1 protein. The results showed that the expression of the target gene was suppressed after the inhibition of p53/HMGB1 action. We further studied the results of the above detection. The mechanism of p53/HMGB1 induced autophagy and apoptosis of cells. After interfering with the p53 gene, the nuclear protein was extracted, and Western Blot showed that p53 siRNA promoted the PCB29-pQ induced HMGB1 to be further translocated to the cytoplasm. The cell survival rate experiment showed that p53 siRNA inhibited the apoptosis of PCB29-pQ induced cells, and the immunofluorescent LC3 focus experiment table P53 siRNA promoted the autophagy induced by PCB29-pQ, and the apoptosis increased and the level of autophagy decreased after the addition of HMGB1 inhibitor EP. These conclusions suggest that the HMGB1 in the cytoplasm may play a role in inhibiting apoptosis and promoting autophagy. After the interference of HMGB1 protein, the same experimental method proved that HMGB1 siRNA promoted PCB2. 9-pQ induced p53 further translocation to cytoplasm, promoting apoptosis induced by PCB29-pQ and inhibiting autophagy induced by PCB29-pQ, and the decrease of cell apoptosis and the increase of autophagy after adding the p53 inhibitor PFT- alpha. These conclusions suggest that p53 in the translocation of the cytoplasm plays a role in promoting apoptosis and inhibiting autophagy. The study shows that PCB29-pQ induces autophagy and apoptosis in HepG2 cells, which regulates the role of p53/HMGB1 in cell autophagy and cell apoptosis through the localization of the nucleus and cytoplasm and the difference in the expression of the target gene.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2144848
[Abstract]:Polychlorinated biphenyl (PCBs) is a ubiquitous persistent organic pollutant. Due to its many toxic effects, such as endocrine toxicity, immunotoxicity, neurotoxicity, and so on, it has been banned in 1970s, but because of its special physicochemical and biological characteristics, PCBs still exists in our environment including land and aquatic products. Autophagy is the process of cell autophagy or cell organelle wrapped up to form autophagosome, and the autophagosome is fused with lysosomes for digestion and degradation, and is often used as a self-protection mechanism for cells. The purpose of this study is to analyze the specific mechanism of PCB29-pQ activation and to explore the autophagy of cells. The transformation mechanism with cell apoptosis. Part 1: this part studied the molecular mechanism of polychlorinated biphenyquinone induced autophagy through mTOR/p70S6k. HepG2 and MDA-MB-231 cells were selected as the research object, and the experiment found that the autophagy induced by PCB29-pQ had no cell specificity. First, two cells were treated in 5 mu M PCB29-pQ. After 24 h, the ultrastructure of the cell was detected by the transmission electron microscope. The obvious autophagy was observed by AO staining and MDC staining was used to observe the formation of autophagic vesicles. From the morphological and biochemical characteristics, the cell autophagy could be caused by the action of PCB29-pQ. Then, the molecular mechanism of inducing autophagy was proved from the 3 stages of autophagic formation. In the first stage, the expression of protein mTOR and p70S6k negatively regulated by autophagic switch was detected from the concentration and time gradient, and the expression level of the protein decreased after the action of PCB29-pQ, indicating the activation of the induction stage of autophagy. The second stage detected the expression of autophagic related protein ATG5, ATG12, LC3 and RT-PCR in the process of autophagic formation. Analysis of LC3 mRNA level. Compared with the control group, the expression level of cell protein and gene in PCB29-pQ treatment increased significantly, and the maximum value was 24 h at 5 M PCB29-pQ treatment. The level of the autophagy degradation substrate p62 protein decreased with the time gradient, and the lowest expression level in the 5 u M PCB29-pQ processing 24 h. These results indicate that PCB29-pQ can activate autophagy. The formation stage of the body promotes the ability of the autophagic to degrade the substrate. The third phase detects the net flux of LC3 expression by adding CQ to inhibit the degradation of LC3. The results show that the net flux of LC3 is enhanced after the action of PCB29-pQ and is most obvious at the action of 24 h by 5 M PCB29-pQ. And the expression of the autophagic marker LC3 is observed by the transfection of the GFP-LC3 particles. The co localization of lysosome labeled LAMP2 showed that the fusion of autophagosomes and lysosomes was promoted. Then the role of autophagy in PCB29-pQ induced cytotoxicity was clarified. After adding different stages of autophagy inhibitor 3-MA and CQ, the detection of the inhibition of autophagy through the detection of CCK8, flow cytometry, and apoptosis marker protein Caspase3. The increase of cytotoxicity induced by PCB29-pQ was promoted. Finally, the reasons for the increase of autophagy induced by PCB29-pQ were investigated by the addition of ROS scavenger NAC. The results showed that the activation of autophagy induced by PCB29-pQ was regulated by the ROS level. According to the above study, PCB29-pQ could induce autophagy in HepG2 and MDA-MB-231 cells and was finer. The autophagy induced by the intracellular ROS level of.PCB29-pQ is regulated by the mTOR/p70S6k and ATG5/ATG12/LC3 signaling pathways and protects the cells as a survival mechanism. The second part: this part preliminarily discussed the effect of the activity of calcium protease on the autophagy to the apoptosis signal when the autophagy induced by the low concentration of polychlorinated biphenyquinone. The JC-1 fluorescence probe was used to detect the mitochondrial membrane potential of HepG2 cells, and the expression of apoptosis related protein caspase9/caspase3 was detected by immunoblotting. Apoptosis was detected by TUNEL method and the focal point of autophagic marker protein LC3 was detected by immunofluorescence. The results were found to be compared with the control group, and the apoptotic water was increased with the concentration of PCB29-pQ. The level of autophagy was increased gradually, and the focus of autophagy LC3 was most in the low concentration of 5 M PCB29-pQ. With the increase of concentration, the number of autophagic LC3 focal points decreased. Then the level of calcium ion induced by PCB29-pQ was detected by flow cytometry, and the activity of calsin was detected by the fluorescence spectrophotometer. The results showed that PCB29-pQ could induce the calcium ion of HepG2 cells. Levels and the increase of calsin activity and the inhibition of calpain protein expression after adding calcium ion chelating agent BAPTA-AM showed that the expression of calpain depended on the regulation of Ca2+ level. The literature reported that Beclin1, ATG5 can be cut through calsin to form Beclin1-c, tATG5 translocation to mitochondria to transmit autophagy to apoptosis signal, and in this study PCB29-pQ did not cause Beclin1, ATG5 cut translocation to mitochondria. Above study, PCB29-pQ can induce autophagy at low concentration of HepG2 cells, induce apoptosis at high concentration, and increase the intracellular calcium level and calsin activity, but the activity of calcinease does not cause Beclin1, ATG5 produces cutting signal. From autophagy to apoptosis. The third part: this part further explored the mechanism of polychlorinated biphenyquinone induced autophagy and cell apoptosis, and selected the p53/HMGB1 protein.Western Blot and immunofluorescence test, which had dual regulation on autophagy and apoptosis, and showed that PCB29-pQ could induce p53/HMGB1 translocation to HepG2 cells after PCB29-pQ action. Cytoplasm. The immunoprecipitation experiment showed that the interaction of 5 M PCB29-pQ promoted the binding of p53/HMGB1 in the nucleus and promoted the binding of p53/HMGB1 in the cytoplasm after the action of 15 mu M PCB29-pQ. Because HMGB1/p53 could be used as a nuclear transcription factor to regulate the expression of the target gene of the downstream target and affect the autophagy and apoptosis of the cell. After interfering with the expression of p53 protein, the downstream target gene of p53 was detected, DRAM, ULK1, Bax expression, and Western Blot detected the downstream protein HSPB1 expression of HMGB1 after the expression of HMGB1 protein. The results showed that the expression of the target gene was suppressed after the inhibition of p53/HMGB1 action. We further studied the results of the above detection. The mechanism of p53/HMGB1 induced autophagy and apoptosis of cells. After interfering with the p53 gene, the nuclear protein was extracted, and Western Blot showed that p53 siRNA promoted the PCB29-pQ induced HMGB1 to be further translocated to the cytoplasm. The cell survival rate experiment showed that p53 siRNA inhibited the apoptosis of PCB29-pQ induced cells, and the immunofluorescent LC3 focus experiment table P53 siRNA promoted the autophagy induced by PCB29-pQ, and the apoptosis increased and the level of autophagy decreased after the addition of HMGB1 inhibitor EP. These conclusions suggest that the HMGB1 in the cytoplasm may play a role in inhibiting apoptosis and promoting autophagy. After the interference of HMGB1 protein, the same experimental method proved that HMGB1 siRNA promoted PCB2. 9-pQ induced p53 further translocation to cytoplasm, promoting apoptosis induced by PCB29-pQ and inhibiting autophagy induced by PCB29-pQ, and the decrease of cell apoptosis and the increase of autophagy after adding the p53 inhibitor PFT- alpha. These conclusions suggest that p53 in the translocation of the cytoplasm plays a role in promoting apoptosis and inhibiting autophagy. The study shows that PCB29-pQ induces autophagy and apoptosis in HepG2 cells, which regulates the role of p53/HMGB1 in cell autophagy and cell apoptosis through the localization of the nucleus and cytoplasm and the difference in the expression of the target gene.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
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