自噬—溶酶体途径在氧化应激时对SPCA1活性的影响
发布时间：2019-03-30 15:35
【摘要】：研究背景及目的： 急性脑缺血后会引发一系列的损伤级联反应,包括钙稳态失衡、兴奋性氨基酸的毒性作用、氧化应激损伤、线粒体功能障碍等,以及随之产生的蛋白酶激活、基因表达的改变,最终导致细胞坏死或凋亡。大量研究表明,氧化应激损伤在脑缺血后神经元损伤中起到了关键作用。氧化应激时自由基的大量生成导致细胞内Ca2+超载,细胞内Ca2+浓度的增加能激活凋亡,使细胞产生不可逆的细胞的损伤。氧化应激时可激活自噬溶酶体途径,细胞内自噬体的数量增加。自噬体吞噬细胞内受损的线粒体和内质网,并能控制线粒体的数量和质量。对抗细胞内的钙超载,有细胞保护作用。但是缺血再灌注时高尔基体钙泵SPCAl对细胞内钙超载的应答、自噬对SPCAl的功能影响方面的研究鲜有人涉及。本实验将从神经元受到缺血再灌注损伤时自噬的是否激活、激活的自噬对高尔基体的SPCAl的影响方面探讨自噬对高尔基体的影响。寻求减少氧化应激损伤的新途径。 方法： 1.建立氧化应激模型：选择不同浓度的H202作用于N2a细胞,以MTT法检测不同浓度的H202对N2a细胞活性的影响； 2.实验分为正常组H202处理组和3-MA预处理组；H202处理组和3-MA预处理组H202的浓度分别为20μM、50μM、80μM； 3.H202处理后MDC染色检测自噬的活性变化； 4.Fura-2/am检测各组细胞内Ca2+浓度的变化； 5.RT-PCR检测各组SPCAlmRNA表达变化； 6.Western blot检测3-MA预处理前后,模型高尔基体蛋白SPCA1及自噬标记物LC3B表达变化。 实验结果： 1.MTT结果显示随着Ca2+浓度的增加,细胞损伤逐渐加重,H202对细胞的损害具有浓度依赖性,MDC染色观察自噬数目发现,自噬颗粒也随着H202浓度的增加而增加。LC3B的western blot结果也表明自噬活性随H202浓度的增加而增加(P0.05),同时证明自噬抑制剂3-MA可有效抑制自噬活性。 2.细胞经H2O2处理后,Fura-2/am检测到细胞内Ca2+浓度较正常组明显增加(P0.05),除20μM浓度时H202处理组和3-MA预处理组的细胞内Ca2+浓度变化不明显外(P0.05),其余各组3-MA预处理组均较H202处理组的细胞内Ca2+浓度较H202组增加更显著(P0.05)。 3.与正常组比较,H202处理组SPCAlmRNA和SPCAl蛋白表达明显较少(P0.05),3-MA预处理组SPCAlmRNA和SPCAl蛋白表达较H202组表达更少(P0.05),但是20μM浓度的H202时,两组之间SPCAl变化不明显(P0.05)。 结论 1.自噬溶酶体途径在遭受氧化应激损伤时可被激活； 2.氧化应激可诱导自噬水平表达上调； 3.阻断自噬溶酶体途径可使细胞SPCAl表达减少,作用减弱,加重细胞内钙超载。
[Abstract]:Background & objective: acute cerebral ischemia can induce a series of damage cascade reactions, including calcium homeostasis imbalance, toxicity of excitatory amino acids, oxidative stress damage, mitochondrial dysfunction, and so on. And the resulting protease activation, gene expression changes, eventually leading to cell necrosis or apoptosis. A large number of studies have shown that oxidative stress plays a key role in neuronal injury after cerebral ischemia. During oxidative stress, the production of free radicals leads to the overload of intracellular Ca2, and the increase of intracellular Ca2 concentration can activate apoptosis and induce irreversible cell damage. Oxidative stress can activate autophagy lysosome pathway and increase the number of autophagy in cells. The damaged mitochondria and endoplasmic reticulum in autophagy phagocytes can control the quantity and quality of mitochondria. Anti-intracellular calcium overload, has cell protection. However, the response of Golgi calcium pump SPCAl to intracellular calcium overload during ischemia-reperfusion is rarely involved in the study of the effect of autophagy on the function of SPCAl. In this study, the effects of autophagy on the SPCAl of Golgi apparatus were investigated in terms of the activation of autophagy during neuronal ischemia-reperfusion injury and the effect of activated autophagy on Golgi apparatus. To find a new way to reduce oxidative stress damage. Methods: 1. The oxidative stress model was established: different concentrations of H2O2 were selected to act on N2a cells, and the effects of different concentrations of H2O2 on N2a cell viability were detected by MTT assay. The experiment was divided into normal group and 3-MA pretreatment group, the concentration of H2O2 in H2O2 group and 3-MA pretreatment group were 20 渭 M, 50 渭 M and 80 渭 M, respectively, and the changes of autophagy activity were detected by MDC staining after 3.H202 treatment. 4.Fura-2/am was used to detect the change of intracellular Ca2 concentration, 5.RT-PCR was used to detect the expression of SPCAlmRNA in each group. The expression of Golgi protein SPCA1 and autophagy marker LC3B were detected by 6.Western blot before and after 3-MA pretreatment. Results: the results of 1.MTT showed that with the increase of Ca2 concentration, the cell damage was gradually aggravated, and H2O2 had a concentration-dependent effect on the cell damage. MDC staining showed that the number of autophagy was observed. The western blot results of LC 3B also showed that the autophagy activity increased with the increase of H 202 concentration (P0.05), and the autophagy inhibitor 3-MA could effectively inhibit the autophagy activity. 2. After treated with H2O2, the intracellular Ca2 concentration in the cells was significantly higher than that in the normal group (P0.05), except that the intracellular Ca2 concentration in the H2O2 treatment group and the 3-MA pretreatment group did not change significantly at the concentration of 20 渭 M (P0.05). The intracellular Ca2 concentration in 3-MA pretreatment group was higher than that in H2O2 group (P0.05). 3. Compared with the normal group, the expression of SPCAlmRNA and SPCAl protein in H2O2 treated group was significantly lower than that in H202 group (P0.05), and the expression of SPCAlmRNA and SPCAl protein in 3-MA pretreatment group was lower than that in H2O2 group (P0.05), but at 20 渭 M concentration of H2O2, the expression of SPCAlmRNA and SPCAl protein was lower than that of H2O2 group (P0.05). There was no significant change in SPCAl between the two groups (P0.05). Conclusion 1. Autophagy lysosome pathway can be activated by oxidative stress. Oxidative stress can induce up-regulation of autophagy expression; Blocking autophagy lysosome pathway could decrease the expression of SPCAl, weaken the effect and aggravate the intracellular calcium overload.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
[Abstract]:Background & objective: acute cerebral ischemia can induce a series of damage cascade reactions, including calcium homeostasis imbalance, toxicity of excitatory amino acids, oxidative stress damage, mitochondrial dysfunction, and so on. And the resulting protease activation, gene expression changes, eventually leading to cell necrosis or apoptosis. A large number of studies have shown that oxidative stress plays a key role in neuronal injury after cerebral ischemia. During oxidative stress, the production of free radicals leads to the overload of intracellular Ca2, and the increase of intracellular Ca2 concentration can activate apoptosis and induce irreversible cell damage. Oxidative stress can activate autophagy lysosome pathway and increase the number of autophagy in cells. The damaged mitochondria and endoplasmic reticulum in autophagy phagocytes can control the quantity and quality of mitochondria. Anti-intracellular calcium overload, has cell protection. However, the response of Golgi calcium pump SPCAl to intracellular calcium overload during ischemia-reperfusion is rarely involved in the study of the effect of autophagy on the function of SPCAl. In this study, the effects of autophagy on the SPCAl of Golgi apparatus were investigated in terms of the activation of autophagy during neuronal ischemia-reperfusion injury and the effect of activated autophagy on Golgi apparatus. To find a new way to reduce oxidative stress damage. Methods: 1. The oxidative stress model was established: different concentrations of H2O2 were selected to act on N2a cells, and the effects of different concentrations of H2O2 on N2a cell viability were detected by MTT assay. The experiment was divided into normal group and 3-MA pretreatment group, the concentration of H2O2 in H2O2 group and 3-MA pretreatment group were 20 渭 M, 50 渭 M and 80 渭 M, respectively, and the changes of autophagy activity were detected by MDC staining after 3.H202 treatment. 4.Fura-2/am was used to detect the change of intracellular Ca2 concentration, 5.RT-PCR was used to detect the expression of SPCAlmRNA in each group. The expression of Golgi protein SPCA1 and autophagy marker LC3B were detected by 6.Western blot before and after 3-MA pretreatment. Results: the results of 1.MTT showed that with the increase of Ca2 concentration, the cell damage was gradually aggravated, and H2O2 had a concentration-dependent effect on the cell damage. MDC staining showed that the number of autophagy was observed. The western blot results of LC 3B also showed that the autophagy activity increased with the increase of H 202 concentration (P0.05), and the autophagy inhibitor 3-MA could effectively inhibit the autophagy activity. 2. After treated with H2O2, the intracellular Ca2 concentration in the cells was significantly higher than that in the normal group (P0.05), except that the intracellular Ca2 concentration in the H2O2 treatment group and the 3-MA pretreatment group did not change significantly at the concentration of 20 渭 M (P0.05). The intracellular Ca2 concentration in 3-MA pretreatment group was higher than that in H2O2 group (P0.05). 3. Compared with the normal group, the expression of SPCAlmRNA and SPCAl protein in H2O2 treated group was significantly lower than that in H202 group (P0.05), and the expression of SPCAlmRNA and SPCAl protein in 3-MA pretreatment group was lower than that in H2O2 group (P0.05), but at 20 渭 M concentration of H2O2, the expression of SPCAlmRNA and SPCAl protein was lower than that of H2O2 group (P0.05). There was no significant change in SPCAl between the two groups (P0.05). Conclusion 1. Autophagy lysosome pathway can be activated by oxidative stress. Oxidative stress can induce up-regulation of autophagy expression; Blocking autophagy lysosome pathway could decrease the expression of SPCAl, weaken the effect and aggravate the intracellular calcium overload.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743
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