Nrf2信号通路在铅致SH-SY5Y细胞毒性和凋亡中保护作用的研究

发布时间：2018-11-11 22:19

【摘要】：铅是广泛存在于环境中的有毒重金属，铅暴露所致的神经细胞凋亡是神经毒性的重要表现之一。核因子E2相关因子2（Nuclear factor erythroid2-realated factor2，Nrf2）信号通路是机体对抗外源性氧化应激和毒效应的非常重要的防御体系之一，但在铅神经毒性中的作用和机制还研究甚少。因此，本实验拟用不同剂量的醋酸铅对SH-SY5Y细胞进行染毒，以观察Nrf2转录活性的变化，并探讨Nrf2是否对铅所致SH-SY5Y细胞的毒性和凋亡发挥保护作用及相关的机制。本研究主要由以下二部分组成第一部分醋酸铅对SH-SY5Y细胞Nrf2转录活性的影响目的：探讨醋酸铅对核转录因子Nrf2转录活性的影响。方法：用不同剂量的醋酸铅溶液（5μmol/L、25μmol/L、125μmol/L）对体外培养的人神经母细胞瘤SH-SY5Y细胞染毒，用凝胶迁移试验（EMSA）测染毒后6h、12h、24h细胞核内Nrf2-ARE结合能力；用MTT测染毒后12h、24h细胞活力。结果：与对照组相比，，醋酸铅染毒染毒使Nrf2-ARE的结合能力明显升高（P 0.05），并呈现时间剂量依赖效应；不同浓度醋酸铅处理细胞后，细胞存活率降低（P 0.05），增殖受到不同程度的抑制，并呈现剂量反应关系。结果提示，醋酸铅激活了SH-SY5Y细胞Nrf2的转录活性，随染毒剂量的升高胞核内Nrf2-ARE结合能力增强。结论：醋酸铅可激活SH-SY5Y细胞Nrf2的转录活性。第二部分Nrf2在铅致SH-SY5Y细胞毒性和凋亡中保护作用的研究目的：研究Nrf2对铅致SH-SY5Y细胞毒性和凋亡中的保护作用，并从分子水平上探讨Nrf2对铅致SH-SY5Y细胞凋亡保护作用的机理。方法：用0μmol/L，0.2μmol/L，1μmol/L，5μmol/L，10μmol/L姜黄素干预细胞24h后，提取核蛋白，用凝胶迁移试验（EMSA）测细胞核内Nrf2-ARE结合能力；用前述试验确定好的姜黄素浓度预处理细胞24h后，再对细胞进行醋酸铅染毒，用MTT检测细胞活力，用流式细胞术和TUNEL法检测细胞凋亡，用western法检测凋亡相关蛋白的表达。结果：SH-SY5Y细胞经醋酸铅(125μmol/L)处理24h后，MTT实验表明，与单纯铅染毒组相比较，姜黄素预处理+醋酸铅染毒组细胞存活率都明显升高（P 0.05）；流式细胞术和TUNEL结果显示，醋酸铅导致神经细胞的凋亡增加（P 0.05），姜黄素预处理+醋酸铅染毒组细胞凋亡率明显降低（P 0.05）；western结果显示，铅染毒使胞浆中Caspase-3、Bax、Cytochrome C的表达升高，Bcl-2的表达降低；姜黄素预处理+醋酸铅染毒组的Caspase-3、Bax、Cytochrome C的表达低于单纯醋酸铅染毒组（P 0.05）。结论：Nrf2对铅致神经细胞的毒性和凋亡有保护作用；Nrf2对凋亡的保护作用可能与其对凋亡相关蛋白的调控有关。
[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.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R135.11

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