中华神经医学杂志
中華神經醫學雜誌
중화신경의학잡지
CHINESE JOURNAL OF NEUROMEDICINE
2009年
6期
541-545
,共5页
谭玉燕%王志全%周海燕%陈生弟
譚玉燕%王誌全%週海燕%陳生弟
담옥연%왕지전%주해연%진생제
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
UCH-L1抑製劑%帕金森病%汎素蛋白酶體繫統%多聚汎素化蛋白%細胞凋亡
UCH-L1억제제%파금삼병%범소단백매체계통%다취범소화단백%세포조망
Ubiquitin C-terminal hydrolase L1%Parkinson's disease%Ubiquitin proteasome pathway%Polyubiquitinated proteins%Apoptosis
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(OCH-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、100 μmol/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24 h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.
目的 利用人神經母細胞瘤SK-N-SH細胞觀察汎素羧基末耑水解酶-1(OCH-L1)抑製劑對多巴胺能神經元的毒性作用併探討其可能的毒性機製. 方法 用不同濃度(5、10、25、50、75、100 μmol/L)的UCH-L1抑製劑作用SK-N-SH細胞24h,MTT法檢測細胞活力、Hoechst染色檢測凋亡的細胞覈及Western blot檢測UCH-L1蛋白、單箇汎素分子及多聚化汎素蛋白的錶達、熒光檢測汎素蛋白酶體繫統(UPS)的功能. 結果經UCH-L1抑製劑處理24 h後SK-N-SH細胞突起樣結構消失,細胞體積變小、形態變圓;隨著UCH-L1抑製劑濃度的增加,細胞活性進一步下降;與對照組比較,細胞活力在抑製劑濃度為50μmol/L時.作用24h後即齣現明顯下降,差異有統計學意義(P<0.05);Hoechst染色可見凋亡細胞碎裂的細胞覈;Western blot檢測到細胞內UCH-L1蛋白錶達沒有變化、單箇汎素分子水平下降、多聚汎素化蛋白增加;熒光檢測顯示UPS功能下降.結論 UCH-L1抑製劑在體外對多巴胺能神經元有毒性作用,可誘導細胞凋亡.在凋亡過程中,UPS功能下降、細胞內多聚汎素化蛋白堆積可能髮揮瞭作用.
목적 이용인신경모세포류SK-N-SH세포관찰범소최기말단수해매-1(OCH-L1)억제제대다파알능신경원적독성작용병탐토기가능적독성궤제. 방법 용불동농도(5、10、25、50、75、100 μmol/L)적UCH-L1억제제작용SK-N-SH세포24h,MTT법검측세포활력、Hoechst염색검측조망적세포핵급Western blot검측UCH-L1단백、단개범소분자급다취화범소단백적표체、형광검측범소단백매체계통(UPS)적공능. 결과경UCH-L1억제제처리24 h후SK-N-SH세포돌기양결구소실,세포체적변소、형태변원;수착UCH-L1억제제농도적증가,세포활성진일보하강;여대조조비교,세포활력재억제제농도위50μmol/L시.작용24h후즉출현명현하강,차이유통계학의의(P<0.05);Hoechst염색가견조망세포쇄렬적세포핵;Western blot검측도세포내UCH-L1단백표체몰유변화、단개범소분자수평하강、다취범소화단백증가;형광검측현시UPS공능하강.결론 UCH-L1억제제재체외대다파알능신경원유독성작용,가유도세포조망.재조망과정중,UPS공능하강、세포내다취범소화단백퇴적가능발휘료작용.
Objective To investigate the neurotoxic effects ofLDN-57444, a specific ubiquitin C-termiual 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-LI 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 μmol/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 apoptotie 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 dopaminergie neurons and induces cell apoptosis possibly as the result of impaired UPS activity and intracellular accumulation of polyubiquitinated proteins following the exposure.
