阻断泛素-蛋白酶体通路诱导PC12细胞死亡和泛素阳性包涵体生成  

The block of ubiquitin-proteasome pathway induces cell death and the formation of ubiquitin-immunoreactive inclusions in PC12 cells

作　　者：杨卉[1] 陈生弟[1] 李彪[1] 陆国强[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,200025

出　　处：《中华神经科杂志》2005年第7期430-433,共4页Chinese Journal of Neurology

基　　金：国家科技部"973"计划资助项目(G1999054008);国家自然科学基金资助项目(30471918;30171025);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：目的探讨泛素蛋白酶体通路的功能障碍对于多巴胺能细胞的活力以及胞质内包涵体生成的影响。方法应用蛋白酶体抑制剂lactacystin(5μmol/L、10μmol/L、15μmol/L和20μmol/L)处理PC12细胞24h,MTT方法检测细胞活力,WesternBlot方法测定细胞内泛素化蛋白质水平,免疫荧光细胞化学染色观察泛素免疫阳性包涵体的生成。结果经5μmol/L、10μmol/L、15μmol/L和20μmol/Llactacystin处理24h后,PC12细胞的活力显著降低(细胞存活率分别为81.5%±3.6%、75.4%±2.4%、70.2%±2.7%和60.4%±3.9%),呈现剂量依赖性。WesternBlot证实对照组细胞内未检测到相对高分子质量的泛素化蛋白质;随着lactacystin作用浓度的增加,细胞内相对高分子质量泛素化蛋白质的含量逐渐增高。免疫荧光染色显示对照组中仅有极少数细胞内含有泛素阳性包涵体;20μmol/Llactacystin处理组中含有泛素阳性包涵体的细胞数目显著增多(P<0.01)。结论泛素蛋白酶体通路的功能缺失能诱导多巴胺能细胞死亡,造成细胞内泛素化蛋白质积聚,促进胞质内泛素阳性包涵体的生成,可能在帕金森病黑质多巴胺能神经元变性死亡和Lewy小体形成中发挥重要作用。Objective To explore the effects of blocking the ubiquitin-proteasome pathway on cell vitality and the formation of cytoplasmic inclusions in dopaminegic cells. Methods PC12 ceils were exposed to proteasomal inhibitor, lactacystin （5μmol/L, 10 μmol/L, 15 μmol/ and 20μmol/L）, for 24 hours.The cell vitality was measured by MTT assay. The expression of high molecular weight ubiquitinated proteins was analyzed by Western Blot. The formation of ubiquitin immunoreactive inclusions was determined with immunofluorescence cytochemistry. Results After exposing to lactacystin （5 μmol/L.10 μmol/L.15μmol/L and 20μmol/L） for 24 hours, PC12 cells showed a dose-dependent decrease in cell vitality （81.5%±3.6% .75.4%± 2. 4% .70. 2%± 2. 7% and 60. 4%± 3.9% ）. Western Blot confirmed that no high molecular weight ubiquitinated proteins were found in control cells and an obvious dose-dependent accumulation of high molecular weight ubiquitinated proteins was detected in cells treated with lactacystin.Immunofluorescence staining showed that ubiquitin immunoreactive inclusions were only found in very few control cells. In PC12 cells treated with 20 p.mol/L lactacystin for 24 hours, the number of cells including cytoplasmic ubiquitin immunoreactive inclusions increased significantly （ P 〈 0. 01 ）. Conclusion The dysfunction of ubiquitin-protcasome pathway might induce cell death, cause accumulation of ubiquitinated proteins and promote the formation of cytoplasmic ubiquitin immunoreactive inclusions in dopaminergic cells,which might contribute to the degeneration of dopaminergic neurons and Lewy body formation in substantia nigra of patients with Parkinson＇ s disease.

关 键 词：泛素 蛋白酶抑制药 PCI2细胞 帕金森病 

分 类 号：R742.5[医药卫生—神经病学与精神病学]