β-淀粉样蛋白25-35诱导HT22细胞发生内质网应激及二苯乙烯苷的保护作用

发布时间：2018-04-29 13:16

本文选题：阿尔茨海默病 + β-淀粉样蛋白25-35　；参考：《兰州大学》2016年硕士论文

【摘要】：目的:阿尔茨海默病(Alzheimer's disease,AD)是一种老年人常见的痴呆类型,以进行性认知功能障碍和行为异常为特征的神经系统退行性病变。脑组织里出现大量老年斑以及神经原纤维缠结是AD主要的病理学特征。其中,β-淀粉样蛋白(β-amyloid peptide,Aβ)作为老年斑的主要成分,具有非常严重的神经毒性,所以影响着AD的形成和发展。有研究显示内质网应激的三个通路蛋白表达状态变化,将导致细胞的凋亡,进而引发疾病。本实验通过建立AD细胞模型,研究Aβ25-35诱导HT22细胞内质网发生应激时,介导内质网应激的三个通路蛋白表达水平的变化及二苯乙烯苷(Tetrahydroxy stilbene glycoside,TSG)对其的保护作用。方法:将HT22细胞分为未分化组和分化组,未分化组加入完全培养基培养48小时,分化组为完全培养基贴壁生长24小时后加入分化液(Neurobasal培养基和N2添加剂)培养24小时后,Western blot检测两组细胞的ATF6、PERK、IRE1蛋白表达量的变化。不同浓度的Aβ25-35分别作用于两组HT22细胞24小时,用MTT测定细胞活力变化。确定最佳AD细胞模型,将分化型HT22细胞分为对照组、Aβ处理组和Aβ+TSG组,最佳抑制浓度的Aβ25-35作用于HT22细胞24小时,加入TSG(0、100、300、500μmol/L)用MTT测定细胞活力变化,免疫细胞荧光检测内质网应激三个通路蛋白的表达位置,及Western blot检测ATF6、PERK、IRE1蛋白表达量的变化。结果:1.ATF6、PERK、IRE1蛋白在未分化组的表达量明显高于分化组的表达量。Aβ25-35对未分化组细胞虽有损伤作用,但各组间无明显差异,且无剂量依赖关系。Aβ25-35可以诱导分化组HT22细胞发生损伤,且呈剂量依赖关系,Aβ25-35(20μmol/L、40μmol/L)作用于分化组HT22细胞24小时,细胞存活率分别为66%、60%,AD模型最佳。2.Aβ25-35可以诱导HT22细胞发生内质网应激,且这种内质网应激同Aβ的浓度具有一定的相关性。免疫细胞荧光检测ATF6、PERK、IRE1在细胞核及细胞浆内均有表达。ATF6、PERK蛋白的表达在Aβ处理组增加,在Aβ+TSG组减少。结论:Aβ25-35可诱导分化型HT22细胞建立AD细胞模型,且浓度为20μmol/L最佳。Aβ25-35可诱导HT22细胞发生内质网应激,且TSG对其有保护作用。
[Abstract]:Objective: Alzheimer's disease (AD) is a common type of senile dementia characterized by progressive cognitive impairment and behavioral disorders. A large number of senile plaques and neurofibrillary tangles are the main pathological features of AD. Among them, 尾 -amyloid peptide A 尾, as the main component of senile plaque, has very serious neurotoxicity, so it affects the formation and development of AD. Studies have shown that changes in the expression of three pathway proteins in endoplasmic reticulum stress may lead to apoptosis and disease. In this study, AD cell model was established to investigate the changes of three pathway proteins mediated by endoplasmic reticulum stress in HT22 cells induced by A 尾 25-35 and the protective effect of Tetrahydroxy stilbene glycoside TSGs on them. Methods: HT22 cells were divided into undifferentiated group and differentiation group. In the differentiation group, the expression of ATF6 PERKN IRE1 protein was detected by Western blot after 24 hours of adherent growth of the complete medium and addition of differentiation medium and N2 additive. Two groups of HT22 cells were treated with different concentrations of A 尾 25-35 for 24 hours. The changes of cell viability were measured by MTT. The best AD cell model was established. The differentiated HT22 cells were divided into A 尾 treated group and A 尾 TSG group. The cells were treated with A 尾 25-35 at the best inhibitory concentration for 24 hours, and TSG(0100300500 渭 mol / L was added to the cells. The changes of cell viability were measured by MTT. Immunofluorescence was used to detect the expression of three pathway proteins in endoplasmic reticulum (ER) stress, and Western blot was used to detect the expression of ATF6 PERKN IRE1 protein. Results: 1. The expression of PERKN IRE1 protein in the undifferentiated group was significantly higher than that in the undifferentiated group. Although there was no significant difference between the two groups, there was no significant difference among the three groups, and no dose-dependent relationship could induce HT22 cell damage in the differentiated group. In a dose-dependent manner, A 尾 25-35 ~ 20 渭 mol / L ~ (40 渭 mol 路L ~ (-1) could induce endoplasmic reticulum stress (ER) in HT22 cells after 24 hours of treatment. The survival rate of the cells was 660.The survival rate was 660.2.A 尾 _ (25-35) could induce endoplasmic reticulum stress in HT22 cells, and the endoplasmic reticulum stress was correlated with the concentration of A 尾. The expression of ATF6 PERKN IRE1 in nucleus and cytoplasm was detected by immunofluorescence. The expression of ATF6 PERK protein was increased in A 尾 treated group and decreased in A 尾 TSG group. Conclusion A 尾 25-35 can induce differentiated HT22 cells to establish AD cell model. The best concentration of 20 渭 mol/L. A 尾 25-35 can induce endoplasmic reticulum stress in HT22 cells, and TSG has protective effect on AD cells.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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