锰对人群和SH-SY5Y细胞中PARK2表达的影响

发布时间：2018-11-29 08:24

【摘要】：目的:研究锰对人群和SH-SY5Y细胞中PARK2表达的影响。方法:⑴人群研究:选取铁冶炼厂的20名工人为对照组,锰铁合金冶炼厂的26名工人为锰暴露组。收集工人的血液和唾液,采用原子吸收分光光度法(atomic absorption spectrometry,AAS)测定锰和铁(iron,Fe)的浓度;实时荧光定量-聚合酶链反应(RT-PCR)检测PARK2 mRNA表达水平。⑵SH-SY5Y细胞实验:0、100、300、500μM浓度的MnCl_2染毒SH-SY5Y细胞24小时后,用MTT法测细胞存活率(线粒体损伤),AAS测定细胞内锰浓度,高度水溶性四唑盐(WST-1)法测定细胞内超氧化物歧化酶(superoxide dismutase,SOD)活力,硫代巴比妥酸法(thibabituric acid,TBA)测定细胞内丙二醛(malondialdehyde,MDA)含量,反相高效液相(reverse phase high performance liquid chromatography,RP-HPLC)-荧光法测定细胞内多巴胺(dopamine,DA)含量,RT-PCR检测PARK2 mRNA表达水平,蛋白免疫印迹法(western blot,WB)检测Parkin蛋白表达水平。结果:⑴人群研究:与对照组比较,锰暴露组血浆、红细胞(red blood cell,RBC)、唾液和累积锰暴露的锰浓度分别高出2.2,2.0,1.7和3.0倍(P0.05),锰暴露组血液中PARK2 mRNA的表达降低42%(P0.05)。相关性分析显示,PARK2mRNA的表达与血浆、RBC、唾液以及累积锰暴露的锰浓度呈负相关,r值分别为-0.526(P0.05)、-0.317(P0.05)、-0.652(P0.05)、-0.737(P0.05)。⑵SH-SY5Y细胞实验:与对照组比较,MnCl_2浓度为300、500μM时,细胞存活率降低(P0.05)。与对照组比较,染锰组细胞内锰浓度增高(P0.05)。与对照组比较,MnCl_2浓度为300、500μM时,SOD活力和DA含量降低(P0.05),MDA含量升高(P0.05);细胞PARK2 mRNA表达和Parkin蛋白表达降低(P0.05)。相关性分析显示,PARK2 mRNA表达与线粒体损伤、细胞内锰含量、MDA含量呈负相关,r值分别为-0.902,(P0.05)、-0.880(P0.05)、-0.862(P0.05);PARK2 mRNA表达与SOD活力、DA含量、Parkin蛋白表达呈正相关,r值分别为0.879(P0.05)、0.859(P0.05)、0.809(P0.05)。结论:1锰暴露工人血液中PARK2 mRNA表达下降,并与血浆、RBC、唾液以及累积锰暴露的锰浓度呈负相关。2锰暴露引起SH-SY5Y细胞线粒体损伤、氧化应激、DA分泌减少和PARK2表达下降。
[Abstract]:Objective: to study the effect of manganese on the expression of PARK2 in human and SH-SY5Y cells. Methods: 1 crowd study: 20 workers in iron smelter were selected as control group and 26 workers in manganese alloy smelter as exposed group. The blood and saliva of the workers were collected and the concentrations of manganese and iron (iron,Fe) were determined by atomic absorption spectrophotometry (atomic absorption spectrometry,AAS). Real-time fluorescence quantification-polymerase chain reaction (RT-PCR) was used to detect the expression of PARK2 mRNA. 2SH-SY5Y cell experiment: after 0100300500 渭 M MnCl_2 was exposed to SH-SY5Y cells for 24 hours, cell survival rate (mitochondrial damage) was measured by MTT assay. The concentration of manganese in cells was measured by AAS, the activity of superoxide dismutase (superoxide dismutase,SOD) by high water-soluble tetrazolium salt (WST-1), and the content of malondialdehyde (malondialdehyde,MDA) by thiobarbituric acid (thibabituric acid,TBA). The levels of dopamine (dopamine,DA), PARK2 mRNA and Parkin were detected by reversed-phase high performance liquid phase (reverse phase high performance liquid chromatography,RP-HPLC) -fluorescence assay, RT-PCR and Western blotting (western blot,WB) respectively. Results: 1Compared with the control group, manganese concentrations in plasma, erythrocyte (red blood cell,RBC, saliva and accumulative manganese exposure in manganese exposure group were 1.7 and 3.0 times higher than those in control group, respectively (P0.05). The expression of PARK2 mRNA in blood of manganese exposure group was decreased by 42% (P0.05). Correlation analysis showed that the expression of PARK2mRNA was negatively correlated with the concentrations of manganese in plasma, RBC, saliva and cumulative manganese exposure. The r values were-0.526 (P0.05), -0.317 (P0.05), -0.652 (P0.05), respectively. -0.737 (P0.05). 2SH-SY5Y cell experiment: compared with the control group, when MnCl_2 concentration was 300500 渭 M, the cell survival rate decreased (P0.05). Compared with the control group, the intracellular manganese concentration in the exposed group was higher than that in the control group (P0.05). Compared with the control group, when MnCl_2 concentration was 300500 渭 M, the activity of SOD and the content of DA decreased (P0.05) (P0.05), and the expression of PARK2 mRNA and Parkin protein decreased (P0.05). Correlation analysis showed that the expression of PARK2 mRNA was negatively correlated with mitochondrial damage, intracellular manganese content and MDA content, r values were -0.902, (P0.05), -0.880 (P0.05), -0.862 (P0.05), respectively. The expression of PARK2 mRNA was positively correlated with the activity of SOD, the content of DA and the expression of Parkin protein (r = 0.879 (P0.05), 0.859 (P0.05), 0.809 (P0.05) respectively). Conclusion: 1 the expression of PARK2 mRNA in blood of workers exposed to manganese decreased, and was negatively correlated with the concentrations of plasma, RBC, saliva and manganese exposed to accumulative manganese. 2 Manganese exposure induced mitochondrial damage and oxidative stress in SH-SY5Y cells. The secretion of DA and the expression of PARK2 decreased.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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