摘要
目的:研究二价金属离子转运蛋白1(DMT1)在乳胞素(lactacystin)诱导的SH-SY5Y细胞中的表达改变,从而进一步了解DMT1在帕金森病(PD)神经元损伤中的可能作用机制。方法:建立lactacystin损伤的SH-SY5Y细胞模型,用免疫荧光、Western blotting等方法检测细胞DMT1表达水平的变化;在高亚铁环境下,荧光探针DCFH-DA检测胞内氧化应激水平的变化,免疫组织化学法、Western blotting检测胞内α-突触核蛋白(α-SYN)聚合体的改变。结果:Lactacystin处理后,细胞活力呈浓度依赖性降低。与正常对照组相比,lactacystin处理组DMT1表达增加(P<0.01)。正常对照组、lactacystin处理组及Fe2+处理组3组比较,其细胞活力逐渐降低,胞内氧化应激反应逐渐增强,胞浆α-SYN低聚体(43-55 kD)表达量逐渐增多(P<0.05)。结论:Lactacystin诱导SH-SY5Y细胞高表达DMT1,增强细胞摄铁能力,这可能是铁直接或者通过氧化应激反应促进胞内α-SYN的错误折叠和聚集、最终导致PD神经元损伤的关键因素。
AEM: To observe the expression of divalent metal transporter 1 ( DMT1 ) in SH - SY5Y cells with lactacystin- induced injury, and to investigate the possible role of DMT1 in the degeneration of dopaminergic neuron in Parkinson disease(PD). METHODS: An in vitro model of cell injury was established in SH - SYSY cells induced by lactacystin. The protein expression of DMT1 was detected by immunofluorescence and Western blotting. Under the environ- ment of high iron level, the cellular oxidative stress was observed by fluorescent probe DCFH - DA. The level of ot - synu- clein was determined by immunohistochemistry and Western blotting. RESULTS: The cell viability rate was reduced by lactacystin in a concentration - dependent pattern. Compared with the control, the protein level of DMT1 was obviously increased in lactacystin - treated cells. The arrangements of the changes from high to low in decreased cell viability, increased intracellular oxidative stress and increased aggregation of α -synuclein (43 -55 kD) were Fe2 + treatment group 〉 lactacystin treatment group 〉 control group( P 〈 0.05 ). CONCLUSION: Lactacystin up - regulates the protein expres- sion of DMTI. By this way, the increased function of DMTI - mediated iron uptake may play an important role in iron or i- ron - catalyzed oxidative reactions to enhance a - synuclein aggregation, leading to the degeneration of neurons in PD.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2011年第2期350-356,共7页
Chinese Journal of Pathophysiology
基金
国家自然科学基金资助项目(No.30670748)
浙江省自然科学基金资助项目(No.Y207557)
浙江省中医药局资助项目(No.2010ZB103)