【摘要】：目的:研究錳對人群和SH-SY5Y細胞中PARK2表達的影響。方法:⑴人群研究:選取鐵冶煉廠的20名工人為對照組,錳鐵合金冶煉廠的26名工人為錳暴露組。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)測定錳和鐵(iron,Fe)的濃度;實時熒光定量-聚合酶鏈反應(RT-PCR)檢測PARK2 mRNA表達水平。⑵SH-SY5Y細胞實驗:0、100、300、500μM濃度的MnCl_2染毒SH-SY5Y細胞24小時后,用MTT法測細胞存活率(線粒體損傷),AAS測定細胞內(nèi)錳濃度,高度水溶性四唑鹽(WST-1)法測定細胞內(nèi)超氧化物歧化酶(superoxide dismutase,SOD)活力,硫代巴比妥酸法(thibabituric acid,TBA)測定細胞內(nèi)丙二醛(malondialdehyde,MDA)含量,反相高效液相(reverse phase high performance liquid chromatography,RP-HPLC)-熒光法測定細胞內(nèi)多巴胺(dopamine,DA)含量,RT-PCR檢測PARK2 mRNA表達水平,蛋白免疫印跡法(western blot,WB)檢測Parkin蛋白表達水平。結(jié)果:⑴人群研究:與對照組比較,錳暴露組血漿、紅細胞(red blood cell,RBC)、唾液和累積錳暴露的錳濃度分別高出2.2,2.0,1.7和3.0倍(P0.05),錳暴露組血液中PARK2 mRNA的表達降低42%(P0.05)。相關性分析顯示,PARK2mRNA的表達與血漿、RBC、唾液以及累積錳暴露的錳濃度呈負相關,r值分別為-0.526(P0.05)、-0.317(P0.05)、-0.652(P0.05)、-0.737(P0.05)。⑵SH-SY5Y細胞實驗:與對照組比較,MnCl_2濃度為300、500μM時,細胞存活率降低(P0.05)。與對照組比較,染錳組細胞內(nèi)錳濃度增高(P0.05)。與對照組比較,MnCl_2濃度為300、500μM時,SOD活力和DA含量降低(P0.05),MDA含量升高(P0.05);細胞PARK2 mRNA表達和Parkin蛋白表達降低(P0.05)。相關性分析顯示,PARK2 mRNA表達與線粒體損傷、細胞內(nèi)錳含量、MDA含量呈負相關,r值分別為-0.902,(P0.05)、-0.880(P0.05)、-0.862(P0.05);PARK2 mRNA表達與SOD活力、DA含量、Parkin蛋白表達呈正相關,r值分別為0.879(P0.05)、0.859(P0.05)、0.809(P0.05)。結(jié)論:1錳暴露工人血液中PARK2 mRNA表達下降,并與血漿、RBC、唾液以及累積錳暴露的錳濃度呈負相關。2錳暴露引起SH-SY5Y細胞線粒體損傷、氧化應激、DA分泌減少和PARK2表達下降。
[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.
【學位授予單位】：遵義醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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