过表达parkin基因对蛋白酶体抑制剂致SH—SY5Y细胞损伤的影响  

Overexpression of parkin fails to protect SH-SYSY cells against the selective toxicity of the proteasome inhibitor

作　　者：周海燕[1] 杨卉[1] 范国华[1] 王刚[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帕金森病诊疗与研究中心,200025

出　　处：《中华老年医学杂志》2007年第1期56-59,共4页Chinese Journal of Geriatrics

基　　金：国家自然科学基金资助项目（30471918）;国家重点基础研究发展973计划（2006cb500700）

摘　　要：目的 探讨过表达parkin基因能否抵抗蛋白酶体抑制剂lactacystin对SH—SY5Y细胞的特异性损伤。方法 不同浓度的lactacystin（1、5、10、15、20μmol／L）分别作用于人多巴胺能SH—SY5Y细胞和人胶质瘤U251细胞24h，用二甲基噻唑二苯基四唑溴盐（MTT）法检测细胞活力。选取10μmol／L和20μmol／L lactacystin处理SH—SY5Y细胞，Western印迹法检测细胞内多泛素化蛋白的含量。瞬时转染parkin至SH-SY5Y细胞，并以MTT法检测10μmol／L和20μmol／L lactacystin作用下的细胞活力，Western印迹法检测细胞内多泛素化蛋白的含量，以及用免疫荧光法检测泛素阳性包涵体的形成。结果 lactacystin呈剂量依赖性地损伤多巴胺能SH—SY5Y细胞（细胞活力分别为104％、82％、72％、60％、50％），而对胶质瘤U251细胞则无毒性作用。parkin过表达不能缓解lactacystin的毒性作用，也没有增加细胞内多泛素化蛋白生成，但却促进泛素阳性包涵体的形成（增加5％）。结论 蛋白酶体功能障碍有可能在多巴胺能神经元的选择性死亡中起关键作用，而Parkin蛋白在路易小体的形成过程中可能起重要作用。Objective To explore whether the overexpression of parkin protects human dopaminergic neuroblastoma SH-SY5Y cells against the selective toxicity of lactacystin, the selective inhibitor of the 26S proteasome. Methods Both human dopaminergic SH-SY5Y cells and human glioma U251 cells were treated with different concentrations of lactacystin （1μmol/L, 5 μmol/L, 10 μmol/L, 15 μmol/L, 20 μmol/L） for 24 h, then the cellular viability was assessed by the MTT assay. 10 μmol/L and 20 μmol/L lactacystin were chosen for treating SH-SY5Y ceils, and the cellular polyubiquitinated proteins were detected by Western blot. 10μmol/L and 20μmol/L lactacystin were also chosen for treating SH-SY5Y cells transfected transiently by parkin gene, then the cellular viability was evaluated by the mono-nuclear cell direct cytotoxicity （MTT） assay, the cellular polyubiquitinated proteins were detected by Western blot, and the cellular ubiquitin-positive inclusions were detected by immunofluorescence. Results Lactacystin injured the dopaminergic SH-SY5Y cells in a dose-dependent manner, while did no harm to the glioma U251 cells. Overexpression of parkin did not protect SH-SY5Y cells against the toxicity of lactacystin, nor increase the amount of the cellular ubiquitinated proteins, but promoted the formation of the ubiquitin-positive inclusions. Conclusions Ubiquitin-proteasome system dysfunction may be the key factor involving in the selective loss of the dopaminergic neurons, and the parkin protein may contribute to the formation of the Lewy bodies.

关 键 词：蛋白酶体内肽酶复合物 泛素类 多巴胺 基因表达 细胞死亡 

分 类 号：R686[医药卫生—骨科学]