锰对PC12细胞功能的影响与PARK2产物的关系研究

发布时间：2018-05-08 08:43

本文选题：锰 + PC12细胞　；参考：《遵义医学院》2013年硕士论文

【摘要】：目的：研究锰在不同剂量和不同时间下对鼠嗜铬神经瘤细胞(PC12)功能和PARK2(Parkin基因)表达的影响,并进一步探讨锰致PARK2表达与PC12细胞功能损害的关系。方法：鼠嗜铬神经瘤细胞(PC12)中分别加入①0、100、300、500μmol/L浓度的Mnc12分别染毒24h；②500μmol/L Mncl2染毒6、24、48h。采用MTT法检测线粒体功能损伤；化学比色法测定细胞内丙二醛(MDA)含量、超氧化物歧化酶(SOD)活性和谷胱甘肽过氧化物酶(GSH-PX)活性；反相高效液相(RP-HPLC)-荧光法测定细胞内多巴胺(DA)含量；实时荧光定量PCR (real time quantitative PCR)检测PARK2mRNA水平；蛋白免疫印迹法(Western blot)检测Parkin蛋白含量。结果-MTT实验结果显示,锰可诱导PC12细胞线粒体损伤,与对照组相比,300-500μmol/L MnCl2作用6、24、48h对线粒体有明显损伤(P0.01)。比色法结果显示：随着染锰浓度的增高,MDA的含量逐渐升高,SOD、GSH-PX活性逐渐降低。与对照组相比,300-500μmol/L MnCl2作用24h, PC12细胞内MDA、SOD及GSH-PX水平差异有显著性(P0.01);500μmol/L MnCl2作用6、24、48h,MDA含量、SOD及GSH-PX活性差异有显著性(P0.01)。RP-HPLC-荧光法检测结果显示：随着染锰浓度的增高,多巴胺含量呈下降趋势,与对照组比较,300μmol/L或以上锰浓度所致多巴胺含量降低有统计学意义(P0.01)。500μmol/L锰暴露6h,24h,48h,多巴胺含量明显降低(P0.01)。实时荧光定量PCR及Western blot结果显示：与对照组比较,300μmol/L或以上锰浓度作用下,PARK2mRNA及蛋白表达下调(P0.05)。500μmol/L锰暴露6h,24h,48h, PARK2mRNA及蛋白表达下调(P0.05)。结论：锰可诱导PC12细胞功能损害,该作用过程可能与锰致PARK2的表达下调有关。
[Abstract]:Aim: to study the effects of manganese on the function of rat pheochromocytoma cell line (PC12) and the expression of PARK2(Parkin gene at different dose and time, and to explore the relationship between the expression of PARK2 and the damage of PC12 cell function induced by manganese. Methods: rat pheochromocytoma cells (PC12) were exposed to 10100300500 渭 mol/L Mnc12 for 24 h and 2 500 渭 mol/L Mncl2 for 48 h. The contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) in the cells were determined by MTT assay, and the contents of dopamine butadiene (DAA) in the cells by RP-HPLCI-fluorescence method were measured by reversed-phase high performance liquid phase (RP-HPLCI-fluorescence) method, and the activity of glutathione peroxidase (GSH-PX) was determined by chemical colorimetry. The level of PARK2mRNA was detected by real-time fluorescence quantitative PCR real time quantitative PCR, and the content of Parkin protein was detected by Western blotting. Results the results of -MTT assay showed that manganese could induce mitochondrial damage in PC12 cells. Compared with the control group, 300-500 渭 mol/L MnCl2 could significantly damage the mitochondria for 48 h. The results of colorimetry showed that the activity of GSH-PX decreased with the increase of manganese concentration. Compared with the control group treated with 300-500 渭 mol/L MnCl2 for 24 h, the levels of MDA-SOD and GSH-PX in PC12 cells were significantly higher than those in the control group. There was a significant difference in the activity of MDA-SOD and GSH-PX in PC12 cells treated with P0.01-500 渭 mol/L MnCl2 for 48h. The results of P0.01- RP-HPLC- fluorescence assay showed that the content of dopamine decreased with the increase of mn concentration. Compared with the control group, the dopamine content was significantly decreased by manganese concentration of 300 渭 mol/L or above. The content of dopamine was significantly decreased after exposure to manganese for 6 h, 24 h and 48 h respectively, and the content of dopamine was significantly lower than that of the control group (P 0. 01%, P 0. 01%, P 0. 01, P 0. 01). The results of real-time fluorescence quantitative PCR and Western blot showed that the expression of PARK2 mRNA and protein was down-regulated at the concentration of 300 渭 mol/L or more than that of the control group. The expression of PARK2 mRNA and protein was down-regulated (P 0.05), and the expression of PARK2mRNA and protein was down-regulated (P 0.05). Conclusion: manganese can induce the damage of PC12 cell function, which may be related to the down-regulation of PARK2 expression induced by mn.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

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