Nrf2信號通路在鉛致SH-SY5Y細胞毒性和凋亡中保護作用的研究
發(fā)布時間：2018-11-11 22:19
【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）信號通路是機體對抗外源性氧化應(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)毒性中的作用和機制還研究甚少。因此，本實驗擬用不同劑量的醋酸鉛對SH-SY5Y細胞進行染毒，以觀察Nrf2轉(zhuǎn)錄活性的變化，并探討Nrf2是否對鉛所致SH-SY5Y細胞的毒性和凋亡發(fā)揮保護作用及相關(guān)的機制。 本研究主要由以下二部分組成 第一部分醋酸鉛對SH-SY5Y細胞Nrf2轉(zhuǎn)錄活性的影響 目的：探討醋酸鉛對核轉(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）對體外培養(yǎng)的人神經(jīng)母細胞瘤SH-SY5Y細胞染毒，用凝膠遷移試驗（EMSA）測染毒后6h、12h、24h細胞核內(nèi)Nrf2-ARE結(jié)合能力；用MTT測染毒后12h、24h細胞活力。 結(jié)果：與對照組相比，，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)時間劑量依賴效應(yīng)；不同濃度醋酸鉛處理細胞后，細胞存活率降低（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。 結(jié)論：醋酸鉛可激活SH-SY5Y細胞Nrf2的轉(zhuǎn)錄活性。 第二部分Nrf2在鉛致SH-SY5Y細胞毒性和凋亡中保護作用的研究 目的：研究Nrf2對鉛致SH-SY5Y細胞毒性和凋亡中的保護作用，并從分子水平上探討Nrf2對鉛致SH-SY5Y細胞凋亡保護作用的機理。 方法：用0μmol/L，0.2μmol/L，1μmol/L，5μmol/L，10μmol/L姜黃素干預(yù)細胞24h后，提取核蛋白，用凝膠遷移試驗（EMSA）測細胞核內(nèi)Nrf2-ARE結(jié)合能力；用前述試驗確定好的姜黃素濃度預(yù)處理細胞24h后，再對細胞進行醋酸鉛染毒，用MTT檢測細胞活力，用流式細胞術(shù)和TUNEL法檢測細胞凋亡，用western法檢測凋亡相關(guān)蛋白的表達。 結(jié)果：SH-SY5Y細胞經(jīng)醋酸鉛(125μmol/L)處理24h后，MTT實驗表明，與單純鉛染毒組相比較，姜黃素預(yù)處理+醋酸鉛染毒組細胞存活率都明顯升高（P 0.05）；流式細胞術(shù)和TUNEL結(jié)果顯示，醋酸鉛導(dǎo)致神經(jīng)細胞的凋亡增加（P 0.05），姜黃素預(yù)處理+醋酸鉛染毒組細胞凋亡率明顯降低（P 0.05）；western結(jié)果顯示，鉛染毒使胞漿中Caspase-3、Bax、Cytochrome C的表達升高，Bcl-2的表達降低；姜黃素預(yù)處理+醋酸鉛染毒組的Caspase-3、Bax、Cytochrome C的表達低于單純醋酸鉛染毒組（P 0.05）。 結(jié)論：Nrf2對鉛致神經(jīng)細胞的毒性和凋亡有保護作用；Nrf2對凋亡的保護作用可能與其對凋亡相關(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)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R135.11
本文編號：2326304
[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)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R135.11
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