本文選題：硫化氫 + 同型半胱氨酸　； 參考：《南華大學(xué)》2014年碩士論文

【摘要】：【研究背景與目的】 同型半胱氨酸(Homocysteine, Hcy)具有神經(jīng)毒性，是老年性癡呆的獨(dú)立危險(xiǎn)因子。我們以往的研究結(jié)果顯示硫化氫(Hydrogen sulfide, H2S)具有抗Hcy神經(jīng)毒性作用，但作用機(jī)制有待深入闡明。過度的內(nèi)質(zhì)網(wǎng)(Endoplasmic reticulum, ER)應(yīng)激可導(dǎo)致神經(jīng)凋亡。沉默信息調(diào)節(jié)因子(Silent informationation regulator1, SIRT1)對(duì)神經(jīng)保護(hù)具有重要的調(diào)節(jié)作用。我們推測(cè)H2S通過調(diào)控SIRT1的表達(dá)拮抗Hcy所致的ER應(yīng)激而發(fā)揮其抗Hcy神經(jīng)毒性作用。 為此，我們以Hcy損傷PC12細(xì)胞為Hcy神經(jīng)毒性的細(xì)胞模型，探討H2S對(duì)Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激的拮抗作用以及SIRT1對(duì)H2S抗ER應(yīng)激和抗Hcy神經(jīng)毒性作用的介導(dǎo)效應(yīng)。 【方法】 Cell Counting Kit-8(CCK-8)法檢測(cè)PC12細(xì)胞存活率，碘化丙啶(propidiumiodide, PI)染色流式細(xì)胞儀(Flow cytometry, FCM)法檢測(cè)PC12細(xì)胞凋亡率，蛋白質(zhì)印跡法(Western blot)法檢測(cè)ER應(yīng)激相關(guān)蛋白(Glucose-regulated protein78, GRP78;Cleaved caspase-12, Cleaved caspase12)及SIRT1的表達(dá)情況。 【結(jié)果】 1. Hcy可促進(jìn)PC12細(xì)胞ER應(yīng)激并下調(diào)PC12細(xì)胞SIRT1表達(dá)以1.25,2.5,5mM的Hcy分別處理PC12細(xì)胞24h后，PC12細(xì)胞內(nèi)GRP78、Cleaved caspase12表達(dá)呈濃度依賴性上升，提示Hcy可誘導(dǎo)PC12細(xì)胞ER應(yīng)激而發(fā)揮其神經(jīng)毒性。 以1.25,2.5,5mM的Hcy分別處理PC12細(xì)胞24h后，PC12細(xì)胞內(nèi)SIRT1的表達(dá)呈濃度依賴性下降，提示Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激可能與其下調(diào)SIRT1的表達(dá)有關(guān)。 2. H2S拮抗Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激 200和400μM的NaHS (H2S供體)合用5mM的Hcy處理PC12細(xì)胞24h后，PC12細(xì)胞內(nèi)GRP78和Cleaved caspase12的表達(dá)水平較Hcy (5mM,24h)單用時(shí)明顯下降，表明H2S可拮抗Hcy誘導(dǎo)的PC12細(xì)胞ER應(yīng)激。 3. SIRT1介導(dǎo)H2S的抗Hcy誘導(dǎo)PC12細(xì)胞內(nèi)質(zhì)網(wǎng)應(yīng)激和神經(jīng)毒性的作用 3.1H2S能上調(diào)PC12細(xì)胞SIRT1的表達(dá) 100、200和400μM的NaHS處理PC12細(xì)胞24h，PC12細(xì)胞SIRT1表達(dá)水平顯著增加，表明H2S能促進(jìn)PC12細(xì)胞高表達(dá)SIRT1。 3.2H2S能減輕Hcy對(duì)SIRT1表達(dá)的抑制作用 400μM的NaHS合用5mM的Hcy處理PC12細(xì)胞24h后，Hcy對(duì)PC12細(xì)胞SIRT1表達(dá)的抑制作用明顯減輕。 3.3Sirtinol (SIRT1抑制劑)能阻斷H2S對(duì)Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激的拮抗作用 20nM的Sirtinol預(yù)處理PC12細(xì)胞2h后，H2S對(duì)Hcy誘導(dǎo)PC12細(xì)胞GRP78和Cleaved caspase12表達(dá)的抑制作用被阻斷，表明H2S可通過上調(diào)SIRT1的表達(dá)拮抗Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激。 3.4Sirtinol (SIRT1抑制劑)能阻斷H2S對(duì)Hcy誘導(dǎo)PC12細(xì)胞毒性和凋亡的拮抗作用 20nM的Sirtinol預(yù)處理PC12細(xì)胞2h后，H2S對(duì)Hcy誘導(dǎo)PC12細(xì)胞活力下降和凋亡增加的拮抗作用被阻斷，表明H2S可通過上調(diào)SIRT1的表達(dá)拮抗Hcy的神經(jīng)毒性。 【結(jié)論】 1. Hcy可通過下調(diào)SIRT1表達(dá)誘導(dǎo)PC12細(xì)胞ER應(yīng)激； 2. H2S可通過上調(diào)SIRT1的表達(dá)拮抗Hcy誘導(dǎo)PC12細(xì)胞ER應(yīng)激。
[Abstract]:[background and purpose of the study] Homocysteine (Hcyine) has neurotoxicity and is an independent risk factor for Alzheimer's disease. Our previous studies have shown that hydrogen sulfide Hydrogen sulfide (H _ 2S) has an anti-neurotoxic effect on Hcy, but the mechanism of action needs to be further elucidated. Excessive endoplasmic reticulum stress (ERS) may lead to neuronal apoptosis. Silent informationation regulator 1 (SIRT1) plays an important role in neuroprotection. We speculate that H2S can inhibit Hcy neurotoxicity by regulating the expression of SIRT1 and antagonizing ER stress induced by Hcy. Therefore, we used Hcy to damage PC12 cells as a cell model of Hcy neurotoxicity, to investigate the antagonistic effect of H2S on ER stress induced by Hcy and the mediated effect of SIRT1 on anti-ER stress and anti-Hcy neurotoxicity of PC12 cells induced by H2S. [methods] The survival rate of PC12 cells was detected by Cell Counting Kit-8 CCK-8 method, the apoptosis rate of PC12 cells was detected by flow cytometry, and the expression of ER stress-related proteins Glucose-regulated protein 78, GRP78Cleaved caspase-12, Cleaved caspase12 and SIRT1 were detected by Western blot. [results] 1. Hcy promoted ER stress in PC12 cells and down-regulated SIRT1 expression in PC12 cells treated with Hcy of 1.25 ~ 2.5mm for 24 h. The expression of GRP78-Cleaved caspase12 in PC12 cells increased in a concentration-dependent manner, suggesting that Hcy could induce ER stress in PC12 cells and exert its neurotoxicity. The expression of SIRT1 in PC12 cells decreased in a concentration-dependent manner after treated with 1.25 ~ 2.5mm Hcy for 24 h, suggesting that ER stress induced by Hcy might be related to the down-regulation of SIRT1 expression in PC12 cells. 2. H _ 2S antagonizes ER stress induced by Hcy in PC12 cells The expression levels of GRP78 and Cleaved caspase12 in PC12 cells treated with 200 渭 M and 400 渭 M NaHS / H2S + 5mM Hcy for 24 h were significantly lower than those in Hcy 5 mm M2s alone, indicating that H2S could antagonize ER stress induced by Hcy in PC12 cells. 3. Effects of SIRT1 mediated H2S on endoplasmic reticulum stress and neurotoxicity induced by Hcy in PC12 cells 3.1H2S can up-regulate the expression of SIRT1 in PC12 cells 100200 and 400 渭 M NaHS treatment increased the expression of SIRT1 in PC12 cells for 24 h, suggesting that H2S could promote the overexpression of SIRT1 in PC12 cells. 3.2H2S can attenuate the inhibitory effect of Hcy on the expression of SIRT1 After treated with 400 渭 M NaHS and 5mM Hcy for 24 h, the inhibitory effect of Hcy on SIRT1 expression in PC12 cells was significantly alleviated. 3.3Sirtinol inhibitor SIRT1 can block the antagonistic effect of H 2S on ER stress induced by Hcy in PC12 cells. After pretreatment of PC12 cells with Sirtinol of 20nM for 2 h, the inhibitory effect of H2S on GRP78 and Cleaved caspase12 expression induced by Hcy was blocked, which indicated that H2S could antagonize ER stress induced by Hcy by up-regulating SIRT1 expression. 3.4Sirtinol inhibitor SIRT1 can block the antagonistic effect of H 2S on the cytotoxicity and apoptosis of PC12 cells induced by Hcy After pretreatment of PC12 cells with Sirtinol of 20nM for 2 h, the antagonistic effect of H 2S on the decrease of PC12 cell viability and increase of apoptosis induced by Hcy was blocked, which indicated that H2S could antagonize the neurotoxicity of Hcy by up-regulating the expression of SIRT1. [conclusion] 1. Hcy can induce ER stress in PC12 cells by down-regulation of SIRT1 expression. 2. H _ 2S can antagonize ER stress of PC12 cells induced by Hcy by up-regulating the expression of SIRT1.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 李載權(quán),劉秉文;Ox-LDL和天然LDL誘導(dǎo)人動(dòng)脈平滑肌細(xì)胞增殖中蛋白激酶A作用的初步研究[J];中國生物化學(xué)與分子生物學(xué)報(bào);1999年03期

，

本文編號(hào)：1879326