类固醇激素20-羟基蜕皮酮通过增加细胞内钙离子水平促进自噬向凋亡转化

发布时间：2018-06-10 14:12

本文选题：棉铃虫 + 中肠　；参考：《山东大学》2017年博士论文

【摘要】：研究背景及存在的科学问题程序性细胞死亡(PCD)主要包括有自噬和凋亡。自噬是指当细胞受到压力或营养缺乏时,细胞将自身的一些损伤蛋白或细胞器降解掉从而产生能量让自己存活下来的过程。凋亡是指机体通过自身基因及其产物主动而有序的调控机体死亡的过程,称为Ⅰ型PCD。对于自噬的功能目前主要存在两种观点:一种是自噬导致细胞存活,在众多的哺乳动物细胞中,如在人类的成纤维和T细胞中,自噬是一种存活过程。另一种是自噬是一种死亡过程,既Ⅱ型PCD,如在果蝇中肠的降解。因此,自噬究竟是存活还是死亡、自噬与凋亡之间究竟存在着一种什么关系以及自噬转向凋亡的分子机制是当今亟待解决的科学问题。昆虫在经历幼虫到蛹的转变过程中,旧的中肠会发生PCD降解,包括自噬与凋亡,因此昆虫中肠PCD为研究自噬和凋亡以及二者的关系提供了理想模型。双翅目昆虫果蝇中肠PCD是一种自噬性死亡,没有凋亡性死亡,而鳞翅目昆虫家蚕中肠PCD中却是先发生自噬,然后发生凋亡,但家蚕中肠PCD自噬转化成凋亡的机制尚不清楚。因此,不同昆虫中肠的PCD的机制有所不同。昆虫PCD的发生依赖于蜕皮激素(20-Hydroxyecdysone,20E),在家蚕脂肪体中,20E可以通过上调自噬相关基因和凋亡相关基因的表达来促进脂肪体PCD。在家蚕丝腺中,20E可以引起胞内Ca2+水平的升高最终活化下游的Caspase-3导致PCD。钙离子的变化和平衡对细胞行使多种功能有着重要的作用,胞质中的钙离子水平相对较低,而胞外可以维持较高的钙离子水平。在一些细胞中,胞内钙离子的增加可以促进自噬。另外还有一些细胞中,胞内钙离子的增加可以活化钙依赖蛋白酶Calpain,Calpain一旦活化后,就会引起自噬相关蛋白5(ATG5)切割成氨基端ATG5(NtATG5),这样自噬的过程就会受阻,凋亡就会增强。因此,我们推测20E增加的细胞内Ca2+在自噬与凋亡的转化中发挥重要作用。为了验证我们的假设以及解决自噬研究领域存在的科学问题,我们以鳞翅目昆虫棉铃虫的中肠PCD作为模型,并以棉铃虫的表皮细胞系(HaEpi)作为平台开展研究,以回答自噬的功能究竟是一种存活还是死亡,自噬与凋亡究竟是一种什么关系以及自噬转向凋亡的分子机制等科学问题。研究结果在鳞翅目昆虫棉铃虫中,自噬相关基因(ATGs)和凋亡相关基因(Caspases)都在变态期高表达,而且都响应20E的诱导。大部分自噬相关基因的表达要先于凋亡相关基因。20E可以诱导微管轻链3蛋白-磷脂酰乙醇胺(LC3-Ⅱ,又名ATG8)的形成,表明自噬受20E的诱导,但是转染pIEx-RFP-GFP-LC3-His荧光双标签质粒进一步检测自噬发现,20E诱导的自噬不能持续下去,这表明自噬在20E诱导的晚期呈现出下降趋势,而凋亡却显著增加,说明20E诱导的自噬最终转向了凋亡。在鳞翅目昆虫棉铃虫中肠程序性细胞死亡过程中(PCD),既存在自噬也存在凋亡而且自噬发生在前凋亡的发生在后。LC3-Ⅱ形成的增加预示自噬的水平的增加;活化的Caspase-3和流式细胞仪用于检测凋亡的水平。凋亡发生时Caspase-3被活化同时自噬相关蛋白ATG5被切割产生氨基端ATG5(NtATG5)。在棉铃虫的表皮细胞系(HaEpi)中,相对较低浓度20E情况下,LC3-Ⅱ的形成增多预示着自噬会逐渐增强,在相对较高浓度的20E情况下,ATG5被切割成为NtATG5,Caspase-3的活性逐渐增强,预示着自噬转向了凋亡。当干扰ATG5或施加自噬的抑制剂3-甲基嘌呤(3-MA)时,20E诱导的自噬和凋亡会受到抑制。但是,当施加凋亡的抑制剂Ac-DEVD-CHO时,可以抑制20E诱导的凋亡,但没有阻止20E诱导的自噬。这暗示了自噬控制并决定凋亡。另外,高浓度20E可以诱导胞内Ca2+水平升高促进ATG5的切割成NtATG5,Caspase-3的活化,进而将自噬转向凋亡。阻止20E诱导的胞内Ca2+的升高将会导致NtATG5以及活化的Caspase-3下降,细胞会维持自噬存活。在人类乳腺癌细胞中,20E可诱导自噬的发生,但是当施加高浓度的Ca2+时,自噬会转化成凋亡。这些结果表明胞内高浓度的Ca2+将细胞自噬性存活转为凋亡性死亡,而且20E诱导了胞内高浓度的Ca2+促进了自噬向凋亡的转化。结论1.自噬是一种存活过程,凋亡是一种细胞死亡过程。当凋亡受到抑制时,细胞将处于自噬的存活状态。这与果蝇中肠的自噬死亡不一样,可能是物种间调控机制的差异。2.20E诱导中肠自噬和凋亡先后发生并促使自噬向凋亡转化。自噬是凋亡的前提,凋亡终结自噬。阐明了中肠中自噬与凋亡的关系不是简单的拮抗关系。3.20E通过增加细胞内钙离子浓度促进自噬向凋亡转化。高浓度的20E诱导胞内Ca2+增加,高水平的Ca2+使ATG5被切割成NtATG5进而活化Caspase-3引起凋亡。抑制Ca2+通道可以阻止自噬向凋亡的转化。在没有钙离子时自噬不能被转化成凋亡。该结果在人乳腺癌细胞中可以得到验证。确定了自噬转向凋亡的关键因子是钙离子。科学意义阐明了 20E调控钙离子增加,介导自噬转化为凋亡是中肠PCD的分子机制;自噬是一种存活过程而凋亡是一种细胞死亡过程;Ca2+离子的浓度决定了自噬向凋亡的转化。丰富了昆虫中肠PCD的理论,为防治病虫害提供了新的靶标。为自噬的功能及在细胞存活与死亡中的机制提供了新的理论依据。
[Abstract]:The research background and existing scientific problems of programmed cell death (PCD) mainly include autophagy and apoptosis. Autophagy refers to the process that cells degrade some of their damaged proteins or organelles when they are subjected to pressure or nutritional deficiency to generate energy for their own survival. Apoptosis refers to the body through its own genes and their products. The active and orderly process of regulating the death of the body, known as type I PCD., has two main views on autophagy: one is autophagy that leads to cell survival. In many mammalian cells, autophagy is a survival process, such as in human fibroblasts and T cells. The other is that autophagy is a death process, both type II PC. D, such as the degradation of the midgut in the Drosophila. Therefore, whether autophagy is alive or dead, what is the relationship between autophagy and apoptosis and the molecular mechanism of autophagy to apoptosis is a scientific problem to be solved today. In the course of the transition from larva to pupae, the insect is degraded in the old midgut, including autophagy and apoptosis, including autophagy and apoptosis. Therefore, the midgut PCD in insects provides an ideal model for the study of autophagy and apoptosis and the relationship between the two. The midgut PCD of the Diptera Insect is a autophagy death and no apoptotic death. However, autophagy occurs in the midgut PCD of the Lepidoptera silkworm, and then apoptosis occurs, but the mechanism of PCD autophagy in the midgut of the Bombyx mori is not yet a mechanism of apoptosis. It is clear that the mechanism of PCD in the midgut of different insects is different. The occurrence of insect PCD is dependent on the ecdysone (20-Hydroxyecdysone, 20E). In the fat body of the silkworm, 20E can promote the fat body PCD. in the silk gland by up regulation of autophagy related genes and apoptosis related genes, and 20E can cause the increase of intracellular Ca2+ level most. The final activation of the downstream Caspase-3 leads to the change and balance of PCD. calcium ions to play an important role in the exercise of various functions. The level of calcium ions in the cytoplasm is relatively low, while the extracellular calcium levels can maintain high levels of calcium ions. In some cells, the increase of intracellular calcium can promote autophagy. In addition, some cells, intracellular calcium, are also available. The increase of ions activates calcium dependent protease Calpain, when Calpain is activated, it causes autophagy related protein 5 (ATG5) to cut into the amino terminal ATG5 (NtATG5), so the process of autophagy will be blocked and apoptosis will increase. Therefore, we speculate that the intracellular Ca2+ in 20E has an important role in the transformation of autophagy and apoptosis. On the basis of our hypothesis and the scientific problems that exist in the field of autophagy, we use the midgut PCD of the Lepidoptera cotton bollworm as a model, and take the epidermal cell line (HaEpi) of the cotton bollworm as a platform to study whether the function of autophagy is a survival or death, and what is the relationship between autophagy and apoptosis. The molecular mechanism of autophagy turned to apoptosis. In the Lepidoptera cotton bollworm, the autophagy related genes (ATGs) and apoptosis related genes (Caspases) are highly expressed in the metamorphosis stage, and all respond to the induction of 20E. Most autophagy related genes are expressed first by apoptosis related gene.20E to induce microtubule light chain 3 protein - The formation of phosphatidylethanolamine (LC3- II, also known as ATG8) showed that autophagy was induced by 20E, but the transfection of pIEx-RFP-GFP-LC3-His fluorescence double label plasmid further detected autophagy found that the autophagy induced by 20E could not continue, indicating that autophagy showed a downward trend in the late stage of 20E induction, while apoptosis was significantly increased, indicating 20E induced autophagy. Apoptosis. In the middle intestinal programmed cell death (PCD) of Lepidoptera (Lepidoptera, Helicoverpa armigera), there is both autophagy and apoptosis and the occurrence of autophagy occurring before the apoptosis of.LC3- II indicates an increase in the level of autophagy; activated Caspase-3 and flow cytometry are used to detect the level of apoptosis. When Caspase-3 was activated and autophagy associated protein ATG5 was cut to produce amino terminal ATG5 (NtATG5). In the epidermis cell line (HaEpi) of Helicoverpa armigera (HaEpi), relatively low concentration 20E, the increase of LC3- II indicates that autophagy increases gradually. In relatively high concentration of 20E, ATG5 is cut into NtATG5, Caspase-3 activity gradually Enhancement indicates autophagy to apoptosis. When 3- methyl purine (3-MA) is used to interfere with ATG5 or autophagy, the autophagy and apoptosis induced by 20E can be inhibited. However, when the apoptosis inhibitor Ac-DEVD-CHO is applied, the apoptosis induced by 20E can be inhibited, but the autophagy induced by 20E is not prevented. This implies autophagy control and determines apoptosis. In addition, high concentration of 20E can induce the increase of intracellular Ca2+ level and promote the cleavage of ATG5 into NtATG5, the activation of Caspase-3, and then the autophagy to apoptosis. To prevent 20E induced intracellular Ca2+ rise will lead to the decrease of NtATG5 and activated Caspase-3, and the cell will maintain autophagy. In human breast cancer cells, 20E can induce autophagy. However, when high concentration of Ca2+ was applied, autophagy could be transformed into apoptosis. These results suggest that high intracellular concentration of Ca2+ turns autophagic survival to apoptotic death, and 20E induces high intracellular concentration of Ca2+ to promote the transformation of autophagy to apoptosis. Conclusion 1. autophagy is a survival process and apoptosis is a cell death process. At the time of inhibition, the cell will be in the state of autophagy. This is different from the autophagy death in the midgut of the Drosophila. It may be the difference between the regulation mechanism of the species..2.20E induces autophagy and apoptosis in the midgut and promotes autophagy to apoptosis. Autophagy is the precondition of apoptosis. Apoptosis terminates autophagy. The relationship between autophagy and apoptosis is clarified. Not a simple antagonistic relationship.3.20E promotes autophagy by increasing intracellular calcium concentration to promote autophagy to apoptosis. High concentration of 20E induces intracellular Ca2+ increase, high level Ca2+ causes ATG5 to be cut into NtATG5 and activates Caspase-3 to induce apoptosis. Inhibition of Ca2+ channels can prevent autophagy from transforming into apoptosis. Autophagy can not be used in the absence of calcium ions. Transformation into apoptosis. This result can be verified in human breast cancer cells. The key factor for autophagy to apoptosis is calcium ion. Scientific significance clarifies that 20E regulates the increase of calcium ions and mediates the transformation of autophagy into apoptosis as a molecular mechanism of midgut PCD; autophagy is a survival process and a cell death process; Ca2+ ions The concentration determines the transformation of autophagy to apoptosis. It enriches the theory of PCD in the midgut of insects and provides a new target for the prevention and treatment of diseases and insect pests. It provides a new theoretical basis for the function of autophagy and the mechanism of cell survival and death.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q25

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