论文部分内容阅读
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 investigate the effects of dysfunction of ubiquitin proteasome pathway on the viability of dopaminergic cells and the formation of cytoplasmic inclusion bodies. Methods PC12 cells were treated with proteasome inhibitor lactacystin (5μmol / L, 10μmol / L, 15μmol / L and 20μmol / L) for 24 h. MTT assay was used to determine the cell viability. Western blotting was used to determine the level of ubiquitinated protein. Immunofluorescence cytochemistry Dyeing was used to observe the formation of ubiquitin immunopositive inclusion bodies. Results The viability of PC12 cells was significantly decreased after treatment with 5μmol / L, 10μmol / L, 15μmol / L and 20μmol / L of lactacystin for 24 hours (cell viability were 81.5% ± 3.6%, 75.4% ± 2.4%, 70.2% ± 2.7% And 60.4% ± 3.9%, respectively) in a dose-dependent manner. Western Blot showed that no relatively high molecular weight ubiquitinated protein was detected in the control cells. As the concentration of lactacystin increased, the content of relatively high molecular weight ubiquitinated protein gradually increased. Immunofluorescence staining showed that only a few cells in the control group contained ubiquitin-positive inclusion bodies. The number of cells with ubiquitin-positive inclusion bodies in 20μmol / Llactacystin treatment group increased significantly (P <0.01). Conclusion The loss of function of ubiquitin proteasome pathway can induce the death of dopaminergic cells, cause the accumulation of ubiquitinated protein in cells and promote the formation of ubiquitin-positive inclusion bodies, which may result in the degeneration of dopaminergic neurons in Parkinson’s disease And Lewy bodies play an important role.