蛋白酶体抑制剂lactacystin诱导PC12细胞凋亡以及半胱氨酸蛋白水解酶3活化的实验研究  

Proteasomal inhibitor lactacystin induces cell apoptosis and caspase 3 activation in PC12 cells

作　　者：杨卉[1] 陈生弟[1] 陆国强[1] 李彪[1] 梁梁[1] 徐洁懿[1] 

机构地区：[1]上海第二医科大学附属瑞金医院神经科帕金森病诊疗与研究中心,杭州市第一人民医院神经内科200025

出　　处：《中华医学杂志》2005年第29期2058-2061,共4页National Medical Journal of China

基　　金：国家自然科学基金资助项目(30471918);国家科技部973基金资助项目(G1999054008);上海市科委重大项目基金资助(04D214005);上海市卫生系统百名跨世纪优秀学科带头人培养计划资助项目(97BR001)

摘　　要：目的探讨蛋白酶体功能缺陷是否能诱导多巴胺能细胞发生凋亡及其可能的作用机理。方法应用高选择性的蛋白酶体抑制剂lactacystin(0、1μmol/L、5μmol/l或10μmol/L)处理大鼠嗜铬细胞瘤PC12细胞株24h,四甲基偶氮唑蓝(MTT)比色法检测细胞活力的改变。10μmol/Llactacystin作用24h后,Hoechst荧光染色观察凋亡细胞的核形态学改变,流式细胞仪测定细胞凋亡百分率。用Western印迹法检测10μmol/Llactacystin作用0、24或48h时PC12细胞内半胱氨酸蛋白水解酶3活化片段的动态变化。结果5μmol/L或10μmol/Llactacystin作用24h后,PC12细胞的活力(87%±6%和26%±6%)显著低于对照组(P<0.05)。Hoechst荧光染色显示10μmol/Llactacystin作用24h后部分细胞呈现凋亡细胞核形态学改变;流式细胞仪证实31.4%±4.0%的PC12细胞发生了凋亡,与对照组(7.5%±0.8%)比较差异有统计学意义(P<0.01)。Western印迹法显示对照组细胞内仅有极少量的半胱氨酸蛋白水解酶3活化片段表达;10μmol/Llactacystin作用48h的PC12细胞内半胱氨酸蛋白水解酶3活化片段的表达明显较多。结论蛋白酶体抑制剂lactacystin能诱导多巴胺能细胞PC12发生凋亡,半胱氨酸蛋白水解酶3蛋白酶的激活可能参与lactacystin诱导的PC12细胞凋亡过程;蛋白酶体的功能缺陷在帕金森病发病机制中可能发挥重要作用。Objective To explore whether proteasome dysfunction cam induce dopaminergic cell apoptosis and investigate the probable molecular mechanism. Methods MTT assay was applied to measure the cell vitality of rat pheochrom-ocytoma cells of the Line PC12 exposed to highly specific proteasomal inhibitor lactacystin （0, 1 μmol/L, 5 μmol/L or 10 μmol/L） for 24 hours. After the PC12 cells were treated with 10 μmol/L lactacystin for 24 hours, apoptosis was estimated by Hoechst fluorescence staining and flow cytometry. When the PC12 cells were treated with 10 μ mol/L lactacystin for 0,24 or 48 hours, the level of caspase 3 cleaved fragments were analyzed by Western blotting. Results The PC12 cells exposed to 5 μmol/L or 10 μmol/L lactacystin for 24 hours showed a significant decrease in cell vitality（P 〈0. 05）.Following treated with 10 p^mol/L lactacystin for 24 hours, PC12 cells were seen to be nuclear condensation and fragmentation consistent with an apoptotic nuclear morphology by were seen in the Hoechst fluorescence staining and confirmed to have a significant increase of apoptotic ceils （ about 31.4% ） by flow cytometry.Western blotting showed that there was a very low level of caspase 3 cleaved fragments （ 17 000） in control cells. But, after PC12 cells were exposed to 10 μmol/L lactacystin for 48 hours, the protein level of caspase 3 cleaved fragments （17 000） increased obviously. Conclusion Proteasomal inhibitor lactacystin leads to dopaminergic cell apoptosis. The activation of caspase 3 protease may contribute to the mechanism of lactacystin-induced apoptosis in PC12 cells. Proteasome dysfunction may play an important role in the pathogenesis of Parkinson＇s disease.

关 键 词：蛋白酶抑制药 PCI2细胞 脱噬作用 帕金森病 

分 类 号：R965[医药卫生—药理学]