摘要
目的 探讨泛素蛋白酶体(ubiquitin proteasome,UP)功能失调诱发的内质网应激反应(endoplasmic reticulum stress response,ERS)机制在多巴胺(dopamine,DA)能神经元变性死亡中的作用.方法 通过MTT及流式细胞仪检测UPS抑制剂Lactacystin对NGF诱导的PC12细胞的神经毒性作用,应用RT-PCR和Western blot技术观察Lactacystin处理后ERS通路中XBP1、Grp78、CHOP表达水平的变化及利血平耗竭胞内DA水平后的影响.结果 不同浓度的Lactacystin 5~20μmol/L处理PC12细胞后,细胞活力呈浓度依赖性下降,其中Lactacystin 10μmo1/L作用24h使细胞活力下降51%.在相同浓度条件下(10μmol/L)流式细胞仪显示的细胞凋亡率在4h、8h、1 6h、24h内逐渐增高(6.1%、14.1%、24.9%、30.9%)(P<0.01).RT-PCR及免疫印迹检测显示UPS抑制剂处理后XBP1、Grp78和CHOP的基因及蛋白表达水平明显增加,分别在8h、16~24h达到高峰(P<0.05).Caspase-12基因水平也在Lactacystin诱导后16h显著升高(P<0.05).利血平预处理则使CHOP、caspase-12基因表达减弱(P<0.05).结论 UPS功能抑制诱发的内质网应激和相关凋亡途径机制是DA能神经元选择性变性死亡的内在环节之一.
Objective To explore the mechanism of endoplasmic reticulum stress response(ERS) induced by ubiquitin proteasome(UP) dysfunction in dopaminergic neurons death. Methods The neurotoxicity of UPS inhibitor,lactacystin,on NGF treated-PC12 cells was measured by using MTT assay and flow cytometry. The expression of ERS-related gene XBP1,Grp78,CHOP,caspase-12 in lactacystin-treated group and reserpine preincubation group was determined with RT-polymerase chain reaction (RT-PCR)and Western blot. Results After exposed to different concentration of lactacystin(5~20μmol/L),PC12 cells showed dose-dependent decrease in cell vitality,51 decline at 10μmol/L concentration for 24 hours. FCM assay confirmed time-dependent cell apoptosis for 4h,8h,16h,24h under 10μmol/L lactacystin treated(6.1,14.1,24.9,30.9,respectively)(P< 0.01 ). The gene and protein expression of XBP1 and Grp78 in lactacystin treated group were significantly increased and reached peak at 8h (P< 0.05 ). The expression level of CHOP and transcription of caspase-12 were increased at 16~24h (P< 0.05 ),but alleviated by reserpine preincubayion(P< 0.05 ). Conclusion The dysfunction of UPS might induce dopaminergic neuron selective death resulted from excessive ERS and relative activated cell apoptosis pathway.
出处
《中风与神经疾病杂志》
CAS
CSCD
北大核心
2006年第5期516-519,共4页
Journal of Apoplexy and Nervous Diseases
基金
国家自然科学基金资助项目(30570627)
关键词
泛素蛋白酶体
内质网应激
细胞凋亡
帕金森病
Ubiquitin proteasome
Endoplasmic reticulum stress
Apoptosis
Parkinson’s disease