p115在氧化应激中介导高尔基体应激机制研究
发布时间：2018-11-10 07:35
【摘要】：目的：利用体外细胞氧化应激模型探索在缺血性脑损伤中,高尔基体囊泡转运蛋白p115表达及裂解变化,及其与凋亡信号因子激活的关系,探讨p115介导高尔基体应激的机制。 方法： 1.培养小鼠神经瘤N2a细胞,通过不同浓度H2O2干预建立氧化应激模型,采用MTT法、流式细胞术等评价氧化应激模型。 2.实验分为氧化应激组、保护性药物干预后氧化应激组和正常对照组；其中氧化应激H2O2干预浓度分为40umol/l、80umol/l两个浓度梯度；保护性药物为抗氧化剂丙酮酸钠,可以抑制caspase-3的激活,减少p115裂解。 3. Western blot法检测各组中p115、p115C末端片段、p53、p-p53(ser15)的蛋白表达变化； 4.流式细胞术测定各组凋亡率的变化(Annexin V-FITC/PI); 5.MTT法测定并计算各组存活率的变化； 6.免疫荧光共聚焦方法观察p115的亚细胞定位及在应激中碎裂情况。 实验结果： 1.H2O2干预氧化应激后细胞形态的变化、细胞存活率及凋亡率检测显示：随着H2O2浓度的增加,干预组小鼠N2a细胞的MTT值与正常对照组相比,MTT值显著减少(P0.05),凋亡率明显增加(P0.05),表明不同浓度H2O2造成的氧化应激引起了细胞损伤,导致了细胞凋亡的发生,H2O2的浓度可以造成不同程度的氧化应激损伤。 2.药物干预后再予以H2O2处理,24小时后观察细胞变化细胞存活率及凋亡率检测显示：N2a细胞形态变化较非药物干预组变化不明显,MTT值高于非药物干预组(P0.05),凋亡率小于非药物干预组(P0.05),表明抗氧化预处理可以减轻H2O2诱发的氧化应激损伤。 3.p115、p53表达的变化：H2O280umol/l干预后的2小时、4小时、8小时、12小时和24小时,随着时间的延长,p115(115KD)全长表达逐渐减低,p115-CTF表达持续上升,p53表达较对照组无明显变化,而p-p53(ser15)表达上升并在4小时达到峰值,此后下降。说明在H2O2的干预下,p115发生了持续性的裂解,p53发生了磷酸化；在H2O240umol/l,80umol/l及药物处理4小时后,高浓度H2O2干预下,p115C末端片段、p-p53(ser15)的表达变化一致,均较低浓度组增加(P0.05),p115的表达较低浓度组减少(P0.05),而p53表达无显著差异(P0.05),在药物干预组p115、p115C末端片段、p53及p-p53(ser15)的表达均无显著差异。结果提示p115C末端片段的生成与p53在ser15位点上的磷酸化有关,药物干预减少了p115的裂解,继而减少了p53的磷酸化。 4.p115细胞内定位及形态变化：免疫共聚焦显微镜下观察到随着H2O2浓度的增加,p115的红色荧光由正常状态下的环绕于核周的紧凑囊泡样结构逐渐向胞浆散开,部分呈点状散布于整个细胞质中。结果提示在氧化应激模型中,随着应激程度的加重,p115C末端片段表达上调,形态上发生裂解,与高尔基体碎裂发生相同的形态变化。 结论： 1.p115是高尔基体应激相关蛋白,在氧化应激中p115裂解,高尔基体发生碎裂。 2.p115介导的高尔基体应激性碎裂,参与了凋亡信号的扩大,可能与磷酸化p53蛋白有关。
[Abstract]:Objective: To explore the mechanism of p115-mediated Golgi stress in ischemic brain injury by using in vitro oxidative stress model to explore the expression and change of p115 and its relationship with the activation of apoptosis signal. Methods: 1. The N2a cells of mouse neuroma were cultured. The oxidative stress model was established by different concentration of H2O2, and the oxygen was evaluated by MTT and flow cytometry. 2. The experiment was divided into the oxidative stress group, the oxidative stress group after the protective drug intervention and the normal control group, wherein the concentration of the oxidative stress H2O2 intervention is divided into the concentration gradient of 40uml/ l and 80umol/ l; the protective medicine is an antioxidant pyruvate, and the activation of the caspase-3 can be inhibited, p115, p115C terminal fragment, p53, p-p53 (se) were detected by Western blot. r15) protein expression changes; 4. Flow cytometry to determine the change in apoptotic rates in each group (Ann exin V-FITC/ PI); 5. The change of the survival rate of each group was determined by the MTT method, and the method of fluorescence confocal microscopy was used. tp115 The results of the experiment were as follows: 1. The changes of cell morphology, cell survival rate and apoptosis rate after oxidative stress in the intervention group showed that, with the increase of the concentration of H2O2, the MTT value of the N2a cells in the intervention group was compared with that of the normal control group. The apoptosis rate was significantly increased (P0.05), and the apoptosis rate was significantly increased (P0.05). The results showed that the concentration of H2O2 could lead to different degree of oxidative stress injury. 2. After the intervention of the drug, the H2O2 treatment was carried out, and the cell survival rate and the apoptosis rate of the cells were observed after 24 hours. The change of the cell morphology of the N2a cells was less obvious than that of the non-drug intervention group, and the MTT value was higher than that of the non-drug. In the intervention group (P0.05), the apoptosis rate was lower than that of the non-drug intervention group (P0. The changes of the expression of p115 and p53: 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the intervention of H2O280umol/ l, the full-length expression of p115 (115KD) was gradually reduced, and the expression of p115-CTF continued to increase, and the expression of p53 was higher in the control group. No significant change The expression of p-p53 (ser15) and p-p53 (ser15) increased and reached the peak at 4 h, and then decreased. The results showed that under the intervention of H2O2, p115 had persistent lysis, and p53 was phosphorylated; after 4 hours of treatment with H2O240umol/ l, 80umol/ l and drug, the end fragment of p115C, p-p The expression of 53 (ser15) was consistent with that of low-concentration group (P0.05). The expression of p115 was lower than that of low-concentration group (P0.05), while the expression of p53 was not significant (P0.05). There was no significant difference in the expression of the terminal fragment, p53 and p-p53 (ser15) at the end of the 5C. The results suggested that the formation of the terminal fragment of p115C and the expression of p53 at the site of ser15 acidification-related, drug intervention reduced the lysis of p115, which in turn reduced the phosphorylation of p53. 4. p115 intracellular localization and morphological changes: the red fluorescence of p115 was observed under normal conditions as the concentration of h2o2 increased under an immunocofocus microscope. The compact vesicle-like structure around the core was gradually spread to the cytoplasm and partially dispersed throughout the cytoplasm. The results suggest that in the oxidative stress model, the p11 5C鏈，
本文编号：2321815
[Abstract]:Objective: To explore the mechanism of p115-mediated Golgi stress in ischemic brain injury by using in vitro oxidative stress model to explore the expression and change of p115 and its relationship with the activation of apoptosis signal. Methods: 1. The N2a cells of mouse neuroma were cultured. The oxidative stress model was established by different concentration of H2O2, and the oxygen was evaluated by MTT and flow cytometry. 2. The experiment was divided into the oxidative stress group, the oxidative stress group after the protective drug intervention and the normal control group, wherein the concentration of the oxidative stress H2O2 intervention is divided into the concentration gradient of 40uml/ l and 80umol/ l; the protective medicine is an antioxidant pyruvate, and the activation of the caspase-3 can be inhibited, p115, p115C terminal fragment, p53, p-p53 (se) were detected by Western blot. r15) protein expression changes; 4. Flow cytometry to determine the change in apoptotic rates in each group (Ann exin V-FITC/ PI); 5. The change of the survival rate of each group was determined by the MTT method, and the method of fluorescence confocal microscopy was used. tp115 The results of the experiment were as follows: 1. The changes of cell morphology, cell survival rate and apoptosis rate after oxidative stress in the intervention group showed that, with the increase of the concentration of H2O2, the MTT value of the N2a cells in the intervention group was compared with that of the normal control group. The apoptosis rate was significantly increased (P0.05), and the apoptosis rate was significantly increased (P0.05). The results showed that the concentration of H2O2 could lead to different degree of oxidative stress injury. 2. After the intervention of the drug, the H2O2 treatment was carried out, and the cell survival rate and the apoptosis rate of the cells were observed after 24 hours. The change of the cell morphology of the N2a cells was less obvious than that of the non-drug intervention group, and the MTT value was higher than that of the non-drug. In the intervention group (P0.05), the apoptosis rate was lower than that of the non-drug intervention group (P0. The changes of the expression of p115 and p53: 2 hours, 4 hours, 8 hours, 12 hours and 24 hours after the intervention of H2O280umol/ l, the full-length expression of p115 (115KD) was gradually reduced, and the expression of p115-CTF continued to increase, and the expression of p53 was higher in the control group. No significant change The expression of p-p53 (ser15) and p-p53 (ser15) increased and reached the peak at 4 h, and then decreased. The results showed that under the intervention of H2O2, p115 had persistent lysis, and p53 was phosphorylated; after 4 hours of treatment with H2O240umol/ l, 80umol/ l and drug, the end fragment of p115C, p-p The expression of 53 (ser15) was consistent with that of low-concentration group (P0.05). The expression of p115 was lower than that of low-concentration group (P0.05), while the expression of p53 was not significant (P0.05). There was no significant difference in the expression of the terminal fragment, p53 and p-p53 (ser15) at the end of the 5C. The results suggested that the formation of the terminal fragment of p115C and the expression of p53 at the site of ser15 acidification-related, drug intervention reduced the lysis of p115, which in turn reduced the phosphorylation of p53. 4. p115 intracellular localization and morphological changes: the red fluorescence of p115 was observed under normal conditions as the concentration of h2o2 increased under an immunocofocus microscope. The compact vesicle-like structure around the core was gradually spread to the cytoplasm and partially dispersed throughout the cytoplasm. The results suggest that in the oxidative stress model, the p11 5C鏈，
本文编号：2321815
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