Lactacystin诱导PC12细胞致帕金森病的实验研究

The experimental study of lactacystin inducing PC12 cells to Parkinson's disease

目的应用蛋白酶体抑制剂Lactacystin构建帕金森病细胞模型,从泛素-蛋白酶体功能角度探讨帕金森病的发病机制。方法Lactacystin(0、5、10、15和20μmol/L)分别处理PC12细胞24h,MTT法检测细胞活力;HE染色观察包涵体生成;免疫组织化学法观察α-synuclein在胞内聚集情况;AO/EB双染及电镜检测细胞凋亡。结果10μmol/L Lactacystin作用24h后细胞活力开始显著低于对照组(P<0.01),随着浓度大,细胞活力进一步下降,呈浓度依赖性(P<0.01);HE染色显示胞浆核周出现圆形或椭圆形嗜伊红的包涵体;免疫组化显示包涵体α-synuclein染色呈强阳性;10μmol/L Lactacystin作用细胞24h后AO/EB双染提示细胞早期凋亡;电镜显示细胞核变小偏位,部分胞核固缩、趋边、凝聚。结论Lactacystin对多巴胺能神经元有毒性作用,可形成胞浆内包涵体并诱导细胞凋亡。蛋白酶体功能异常可能在帕金森病发病机制中发挥重要作用。

Objective To establish a cell model of Parkinson＇s disease using proteasome inhibitor lactacystin and to approach its patbogenesy from ubiquitin-proteasome pathway. Methods PC12 cells were treated with different concentrations of lactacystin （0,5,10 and 20μmoL/L）for 24h and cell viability was measured by MTT assay. HE staining and immunohistochemistry were used to observe inclusion body and the accumulation of α-synuclein in cytoplasm. AO/EB double staining and electron microscope were also adopted to test apoptosis. Results Cell viability began to decline significantly at the concentration of 10μmol/L （P 〈 0.01 ）, as concentrations grew, cell viability declined in the dose dependent manner （P 〈0. 01 ）. HE staining showed eosinophilic materials closed to nucleus. Immunohistoehemistry demonstrated a brown round α-synuclein immunoactive mass in cytoplasm. AO/EB staining showed viable apoptotic and electron microscope showed nucleus shrinked and dyssymmetred,some karyopycnosis and aggregated. Conclusion Lactacystin has toxic effect on dopaminergic neurons, which form inclusion body and lead apoptosis. Ubiquitin-proteasome dysfunction might play an important role in the pathogenisis of Parkinsong＇s disease.