【摘要】：研究背景及目的： 急性腦缺血后會(huì)引發(fā)一系列的損傷級(jí)聯(lián)反應(yīng),包括鈣穩(wěn)態(tài)失衡、興奮性氨基酸的毒性作用、氧化應(yīng)激損傷、線粒體功能障礙等,以及隨之產(chǎn)生的蛋白酶激活、基因表達(dá)的改變,最終導(dǎo)致細(xì)胞壞死或凋亡。大量研究表明,氧化應(yīng)激損傷在腦缺血后神經(jīng)元損傷中起到了關(guān)鍵作用。氧化應(yīng)激時(shí)自由基的大量生成導(dǎo)致細(xì)胞內(nèi)Ca2+超載,細(xì)胞內(nèi)Ca2+濃度的增加能激活凋亡,使細(xì)胞產(chǎn)生不可逆的細(xì)胞的損傷。氧化應(yīng)激時(shí)可激活自噬溶酶體途徑,細(xì)胞內(nèi)自噬體的數(shù)量增加。自噬體吞噬細(xì)胞內(nèi)受損的線粒體和內(nèi)質(zhì)網(wǎng),并能控制線粒體的數(shù)量和質(zhì)量。對(duì)抗細(xì)胞內(nèi)的鈣超載,有細(xì)胞保護(hù)作用。但是缺血再灌注時(shí)高爾基體鈣泵SPCAl對(duì)細(xì)胞內(nèi)鈣超載的應(yīng)答、自噬對(duì)SPCAl的功能影響方面的研究鮮有人涉及。本實(shí)驗(yàn)將從神經(jīng)元受到缺血再灌注損傷時(shí)自噬的是否激活、激活的自噬對(duì)高爾基體的SPCAl的影響方面探討自噬對(duì)高爾基體的影響。尋求減少氧化應(yīng)激損傷的新途徑。 方法： 1.建立氧化應(yīng)激模型：選擇不同濃度的H202作用于N2a細(xì)胞,以MTT法檢測(cè)不同濃度的H202對(duì)N2a細(xì)胞活性的影響； 2.實(shí)驗(yàn)分為正常組H202處理組和3-MA預(yù)處理組；H202處理組和3-MA預(yù)處理組H202的濃度分別為20μM、50μM、80μM； 3.H202處理后MDC染色檢測(cè)自噬的活性變化； 4.Fura-2/am檢測(cè)各組細(xì)胞內(nèi)Ca2+濃度的變化； 5.RT-PCR檢測(cè)各組SPCAlmRNA表達(dá)變化； 6.Western blot檢測(cè)3-MA預(yù)處理前后,模型高爾基體蛋白SPCA1及自噬標(biāo)記物L(fēng)C3B表達(dá)變化。 實(shí)驗(yàn)結(jié)果： 1.MTT結(jié)果顯示隨著Ca2+濃度的增加,細(xì)胞損傷逐漸加重,H202對(duì)細(xì)胞的損害具有濃度依賴性,MDC染色觀察自噬數(shù)目發(fā)現(xiàn),自噬顆粒也隨著H202濃度的增加而增加。LC3B的western blot結(jié)果也表明自噬活性隨H202濃度的增加而增加(P0.05),同時(shí)證明自噬抑制劑3-MA可有效抑制自噬活性。 2.細(xì)胞經(jīng)H2O2處理后,Fura-2/am檢測(cè)到細(xì)胞內(nèi)Ca2+濃度較正常組明顯增加(P0.05),除20μM濃度時(shí)H202處理組和3-MA預(yù)處理組的細(xì)胞內(nèi)Ca2+濃度變化不明顯外(P0.05),其余各組3-MA預(yù)處理組均較H202處理組的細(xì)胞內(nèi)Ca2+濃度較H202組增加更顯著(P0.05)。 3.與正常組比較,H202處理組SPCAlmRNA和SPCAl蛋白表達(dá)明顯較少(P0.05),3-MA預(yù)處理組SPCAlmRNA和SPCAl蛋白表達(dá)較H202組表達(dá)更少(P0.05),但是20μM濃度的H202時(shí),兩組之間SPCAl變化不明顯(P0.05)。 結(jié)論 1.自噬溶酶體途徑在遭受氧化應(yīng)激損傷時(shí)可被激活； 2.氧化應(yīng)激可誘導(dǎo)自噬水平表達(dá)上調(diào)； 3.阻斷自噬溶酶體途徑可使細(xì)胞SPCAl表達(dá)減少,作用減弱,加重細(xì)胞內(nèi)鈣超載。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R743

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