泛素羧基末端水解酶-1抑制剂对多巴胺能神经元的神经毒性作用  

Neurotoxicity of ubiquitin C-terminal hydrolase L1 inhibitor in dopaminergic neurons

作　　者：谭玉燕[1] 王志全[1] 周海燕[1] 陈生弟[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院神经科,上海交通大学医学院神经病学研究所,上海200025

出　　处：《中华神经医学杂志》2009年第6期541-545,共5页Chinese Journal of Neuromedicine

基　　金：国家重点基础研究发展计划（973计划）（2006CB500706）;国家自然科学基金（30570637,30471918）;上海市医学领军人才基金（LJ06003）

摘　　要：目的利用人神经母细胞瘤SK-N-SH细胞观察泛素羧基末端水解酶-1（UCH-L1）抑制剂对多巴胺能神经元的毒性作用并探讨其可能的毒性机制。方法用不同浓度（5、10、25、50、75、100μmol／L）的UCH—L1抑制剂作用SK-N—SH细胞24h，MTT法检测细胞活力、Hoechst染色检测凋亡的细胞核及Western blot检测UCH—L1蛋白、单个泛素分子及多聚化泛素蛋白的表达、荧光检测泛素蛋白酶体系统（UPS）的功能。结果经UCH—L1抑制剂处理24h后SK-N-SH细胞突起样结构消失，细胞体积变小、形态变圆；随着UCH—L1抑制剂浓度的增加，细胞活性进一步下降；与对照组比较，细胞活力在抑制剂浓度为50μmol／L时，作用24h后即出现明显下降，差异有统计学意义（P〈0．05）；Hoechst染色可见凋亡细胞碎裂的细胞核；Western blot检测到细胞内UCH-L1蛋白表达没有变化、单个泛素分子水平下降、多聚泛素化蛋白增加；荧光检测显示UPS功能下降。结论UCH—L1抑制剂在体外对多巴胺能神经元有毒性作用，可诱导细胞凋亡。在凋亡过程中。UPS功能下降、细胞内多聚泛素化蛋白堆积可能发挥了作用。Objective To investigate the neurotoxic effects of LDN-57444, a specific ubiquitin C-terminal hydrolase L1 （UCH-L1） inhibitor, on dopaminergic neurons and the possible mechanism. Methods The viability of SK-N-SH cells exposed to 5, 10, 25, 50, 75 or 100 μmol/L LDN-57444 for 24 h was assessed using MTT assay, and the cell apoptosis was detected with Hoechst staining. Western blot was performed to identify the expressions of UCH-L1 protein, ubiquitin monomer and polyubiquitinated proteins, and the activity of the ubiquitin-proteasome system （UPS） was evaluated with fluorometry. Results After exposure to UCH-L1 inhibitor for 24 h, the cell process-like structures of SK-N-SH cells diminished, and the cell body shrank and became spherical. Exposure to LDN-57444 resulted in concentration-dependent reduction of the cell viability, and the reduction became statistically significant following the exposure to 50 Ixmol/L LDN-57444, as compared with that in the control group （P〈0.05）. The exposure also resulted in obvious cell apoptosis as shown by nuclear fragmentation and presence of the apoptotic bodies. Western blot detected no obvious changes in UCH-L1 protein expression but identified reduced ubiquitin monomer and increased polyubiquitinated protein expression in the cells. Fluorometry showed reduced activity of UPS in the exposed cells. Conclusion UCH-L1 inhibitor produces neurotoxicity to dopaminergic neurons and induces cell apoptosis possibly as the result of impaired UPS activity and intracellular accumulation of polyubiquitinated proteins following the exposure.

关 键 词：UCH—L1抑制剂 帕金森病 泛素蛋白酶体系统 多聚泛素化蛋白 细胞凋亡 

分 类 号：R686[医药卫生—骨科学]