【摘要】：鉛是廣泛存在于環(huán)境中的有毒重金屬，鉛暴露所致的神經(jīng)細(xì)胞凋亡是神經(jīng)毒性的重要表現(xiàn)之一。核因子E2相關(guān)因子2（Nuclear factor erythroid2-realated factor2，Nrf2）信號(hào)通路是機(jī)體對(duì)抗外源性氧化應(yīng)激和毒效應(yīng)的非常重要的防御體系之一，但在鉛神經(jīng)毒性中的作用和機(jī)制還研究甚少。因此，本實(shí)驗(yàn)擬用不同劑量的醋酸鉛對(duì)SH-SY5Y細(xì)胞進(jìn)行染毒，以觀察Nrf2轉(zhuǎn)錄活性的變化，并探討Nrf2是否對(duì)鉛所致SH-SY5Y細(xì)胞的毒性和凋亡發(fā)揮保護(hù)作用及相關(guān)的機(jī)制。 本研究主要由以下二部分組成 第一部分醋酸鉛對(duì)SH-SY5Y細(xì)胞Nrf2轉(zhuǎn)錄活性的影響 目的：探討醋酸鉛對(duì)核轉(zhuǎn)錄因子Nrf2轉(zhuǎn)錄活性的影響。 方法：用不同劑量的醋酸鉛溶液（5μmol/L、25μmol/L、125μmol/L）對(duì)體外培養(yǎng)的人神經(jīng)母細(xì)胞瘤SH-SY5Y細(xì)胞染毒，用凝膠遷移試驗(yàn)（EMSA）測(cè)染毒后6h、12h、24h細(xì)胞核內(nèi)Nrf2-ARE結(jié)合能力；用MTT測(cè)染毒后12h、24h細(xì)胞活力。 結(jié)果：與對(duì)照組相比，，醋酸鉛染毒染毒使Nrf2-ARE的結(jié)合能力明顯升高（P 0.05），并呈現(xiàn)時(shí)間劑量依賴(lài)效應(yīng)；不同濃度醋酸鉛處理細(xì)胞后，細(xì)胞存活率降低（P 0.05），增殖受到不同程度的抑制，并呈現(xiàn)劑量反應(yīng)關(guān)系。結(jié)果提示，醋酸鉛激活了SH-SY5Y細(xì)胞Nrf2的轉(zhuǎn)錄活性，隨染毒劑量的升高胞核內(nèi)Nrf2-ARE結(jié)合能力增強(qiáng)。 結(jié)論：醋酸鉛可激活SH-SY5Y細(xì)胞Nrf2的轉(zhuǎn)錄活性。 第二部分Nrf2在鉛致SH-SY5Y細(xì)胞毒性和凋亡中保護(hù)作用的研究 目的：研究Nrf2對(duì)鉛致SH-SY5Y細(xì)胞毒性和凋亡中的保護(hù)作用，并從分子水平上探討Nrf2對(duì)鉛致SH-SY5Y細(xì)胞凋亡保護(hù)作用的機(jī)理。 方法：用0μmol/L，0.2μmol/L，1μmol/L，5μmol/L，10μmol/L姜黃素干預(yù)細(xì)胞24h后，提取核蛋白，用凝膠遷移試驗(yàn)（EMSA）測(cè)細(xì)胞核內(nèi)Nrf2-ARE結(jié)合能力；用前述試驗(yàn)確定好的姜黃素濃度預(yù)處理細(xì)胞24h后，再對(duì)細(xì)胞進(jìn)行醋酸鉛染毒，用MTT檢測(cè)細(xì)胞活力，用流式細(xì)胞術(shù)和TUNEL法檢測(cè)細(xì)胞凋亡，用western法檢測(cè)凋亡相關(guān)蛋白的表達(dá)。 結(jié)果：SH-SY5Y細(xì)胞經(jīng)醋酸鉛(125μmol/L)處理24h后，MTT實(shí)驗(yàn)表明，與單純鉛染毒組相比較，姜黃素預(yù)處理+醋酸鉛染毒組細(xì)胞存活率都明顯升高（P 0.05）；流式細(xì)胞術(shù)和TUNEL結(jié)果顯示，醋酸鉛導(dǎo)致神經(jīng)細(xì)胞的凋亡增加（P 0.05），姜黃素預(yù)處理+醋酸鉛染毒組細(xì)胞凋亡率明顯降低（P 0.05）；western結(jié)果顯示，鉛染毒使胞漿中Caspase-3、Bax、Cytochrome C的表達(dá)升高，Bcl-2的表達(dá)降低；姜黃素預(yù)處理+醋酸鉛染毒組的Caspase-3、Bax、Cytochrome C的表達(dá)低于單純醋酸鉛染毒組（P 0.05）。 結(jié)論：Nrf2對(duì)鉛致神經(jīng)細(xì)胞的毒性和凋亡有保護(hù)作用；Nrf2對(duì)凋亡的保護(hù)作用可能與其對(duì)凋亡相關(guān)蛋白的調(diào)控有關(guān)。
[Abstract]:Lead is a toxic heavy metal widely found in the environment. Apoptosis of nerve cells induced by lead exposure is one of the important manifestations of neurotoxicity. Nuclear factor E2 related factor 2 (Nuclear factor erythroid2-realated factor2,Nrf2) signaling pathway is one of the most important defense systems against exogenous oxidative stress and toxic effects. In order to observe the changes of Nrf2 transcriptional activity and to explore whether Nrf2 plays a protective role in the toxicity and apoptosis of lead-induced SH-SY5Y cells and its related mechanisms, the present study intends to use different doses of lead acetate to treat SH-SY5Y cells. This study consists of two parts: the first part is the effect of lead acetate on the Nrf2 transcription activity of SH-SY5Y cells. Objective: to investigate the effect of lead acetate on the transcription activity of nuclear transcription factor (Nrf2). Methods: human neuroblastoma SH-SY5Y cells were exposed to different doses of lead acetate (5 渭 mol/L,25 渭 mol/L,125 渭 mol/L) in vitro. Gel migration assay (EMSA) was used to determine the dose of 5 渭 mol/L,25 渭 mol/L,125 渭 mol/L for 6 h and 12 h after exposure. 24 h nuclear Nrf2-ARE binding ability; The cell viability was measured by MTT at 12 h and 24 h after exposure. Results: compared with the control group, lead acetate exposure significantly increased the binding ability of Nrf2-ARE (P 0.05), and showed a time-dose dependent effect. After treated with lead acetate at different concentrations, the cell survival rate was decreased (P 0.05), the proliferation was inhibited in different degree, and the dose-response relationship was presented. The results suggest that lead acetate activates the transcription activity of Nrf2 in SH-SY5Y cells and increases the binding ability of Nrf2-ARE in the nucleus with the increase of exposure dose. Conclusion: lead acetate can activate the transcriptional activity of Nrf2 in SH-SY5Y cells. The second part of the study on the protective effect of Nrf2 on lead-induced SH-SY5Y cell toxicity and apoptosis objective: to study the protective effect of Nrf2 on lead-induced SH-SY5Y cell toxicity and apoptosis. The protective mechanism of Nrf2 on lead-induced apoptosis of SH-SY5Y cells was discussed at molecular level. Methods: after treated with 0 渭 mol/L,0.2 渭 mol/L,1 渭 mol/L,5 渭 mol/L,10 渭 mol/L curcumin for 24 hours, the nucleoprotein was extracted and the intracellular Nrf2-ARE binding ability was measured by gel migration test (EMSA). The cells were pretreated with curcumin concentration for 24 hours, then treated with lead acetate. The cell viability was detected by MTT, apoptosis was detected by flow cytometry and TUNEL, and the expression of apoptosis-related protein was detected by western. Results: after SH-SY5Y cells were treated with lead acetate (125 渭 mol/L) for 24 hours, MTT test showed that the survival rate of curcumin pretreated with lead acetate was significantly higher than that of lead acetate pretreated with curcumin alone (P 0.05). The results of flow cytometry and TUNEL showed that the apoptosis of neurons was increased by lead acetate (P0. 05), and the apoptosis rate was significantly decreased in the group treated with curcumin pretreated with lead acetate (P0. 05). Western results showed that lead exposure increased the expression of Caspase-3,Bax,Cytochrome C and decreased the expression of Bcl-2 in the cytoplasm. The expression of Caspase-3,Bax,Cytochrome C in curcumin pretreated lead acetate group was lower than that in pure lead acetate group (P 0. 05). Conclusion: Nrf2 has protective effect on the toxicity and apoptosis of nerve cells induced by lead, and the protective effect of Nrf2 on apoptosis may be related to its regulation of apoptosis-related proteins.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R135.11

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