發(fā)布時間：2018-04-29 13:16

  本文選題：阿爾茨海默病 + β-淀粉樣蛋白25-35　； 參考：《蘭州大學(xué)》2016年碩士論文

【摘要】：目的:阿爾茨海默病(Alzheimer's disease,AD)是一種老年人常見的癡呆類型,以進(jìn)行性認(rèn)知功能障礙和行為異常為特征的神經(jīng)系統(tǒng)退行性病變。腦組織里出現(xiàn)大量老年斑以及神經(jīng)原纖維纏結(jié)是AD主要的病理學(xué)特征。其中,β-淀粉樣蛋白(β-amyloid peptide,Aβ)作為老年斑的主要成分,具有非常嚴(yán)重的神經(jīng)毒性,所以影響著AD的形成和發(fā)展。有研究顯示內(nèi)質(zhì)網(wǎng)應(yīng)激的三個通路蛋白表達(dá)狀態(tài)變化,將導(dǎo)致細(xì)胞的凋亡,進(jìn)而引發(fā)疾病。本實驗通過建立AD細(xì)胞模型,研究Aβ25-35誘導(dǎo)HT22細(xì)胞內(nèi)質(zhì)網(wǎng)發(fā)生應(yīng)激時,介導(dǎo)內(nèi)質(zhì)網(wǎng)應(yīng)激的三個通路蛋白表達(dá)水平的變化及二苯乙烯苷(Tetrahydroxy stilbene glycoside,TSG)對其的保護(hù)作用。方法:將HT22細(xì)胞分為未分化組和分化組,未分化組加入完全培養(yǎng)基培養(yǎng)48小時,分化組為完全培養(yǎng)基貼壁生長24小時后加入分化液(Neurobasal培養(yǎng)基和N2添加劑)培養(yǎng)24小時后,Western blot檢測兩組細(xì)胞的ATF6、PERK、IRE1蛋白表達(dá)量的變化。不同濃度的Aβ25-35分別作用于兩組HT22細(xì)胞24小時,用MTT測定細(xì)胞活力變化。確定最佳AD細(xì)胞模型,將分化型HT22細(xì)胞分為對照組、Aβ處理組和Aβ+TSG組,最佳抑制濃度的Aβ25-35作用于HT22細(xì)胞24小時,加入TSG(0、100、300、500μmol/L)用MTT測定細(xì)胞活力變化,免疫細(xì)胞熒光檢測內(nèi)質(zhì)網(wǎng)應(yīng)激三個通路蛋白的表達(dá)位置,及Western blot檢測ATF6、PERK、IRE1蛋白表達(dá)量的變化。結(jié)果:1.ATF6、PERK、IRE1蛋白在未分化組的表達(dá)量明顯高于分化組的表達(dá)量。Aβ25-35對未分化組細(xì)胞雖有損傷作用,但各組間無明顯差異,且無劑量依賴關(guān)系。Aβ25-35可以誘導(dǎo)分化組HT22細(xì)胞發(fā)生損傷,且呈劑量依賴關(guān)系,Aβ25-35(20μmol/L、40μmol/L)作用于分化組HT22細(xì)胞24小時,細(xì)胞存活率分別為66%、60%,AD模型最佳。2.Aβ25-35可以誘導(dǎo)HT22細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激,且這種內(nèi)質(zhì)網(wǎng)應(yīng)激同Aβ的濃度具有一定的相關(guān)性。免疫細(xì)胞熒光檢測ATF6、PERK、IRE1在細(xì)胞核及細(xì)胞漿內(nèi)均有表達(dá)。ATF6、PERK蛋白的表達(dá)在Aβ處理組增加,在Aβ+TSG組減少。結(jié)論:Aβ25-35可誘導(dǎo)分化型HT22細(xì)胞建立AD細(xì)胞模型,且濃度為20μmol/L最佳。Aβ25-35可誘導(dǎo)HT22細(xì)胞發(fā)生內(nèi)質(zhì)網(wǎng)應(yīng)激,且TSG對其有保護(hù)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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R749.16

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