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目的观察蛋白激酶Cδ亚型(PKCδ)磷酸化在6羟基多巴胺(6-OHDA)引起的多巴胺能神经细胞死亡过程中的作用,探讨帕金森病中神经元缺失的分子机制。方法体外培养大鼠嗜铬细胞瘤细胞系PC12细胞,观察预先加入的PKC抑制剂(bisindolylmaleimide I,G6976及Rottlerin)和激动剂佛波酯对6-OHDA毒性作用的影响,噻唑蓝比色法检测细胞存活率,免疫印迹法观察磷酸化PKCδ的表达。结果PKCδ抑制剂Rottlerin(2μmol/L)可抑制PKCδ的磷酸化,减轻6-OHDA引起的细胞死亡,细胞存活率上升至69.6±2.63%(P<0.05)。PKCα抑制剂bisindolylmaleimide I和钙依赖性PKC抑制剂G6976对6-OHDA的毒性作用及PKCδ磷酸化均无显著影响,而PKCδ激活剂佛波酯(100nmol/L)能提高PKCδ磷酸化水平,加重6-OHDA的损害作用,使细胞存活率下降至单用6-OHDA组水平的49.8±5.06%(P<0.001)。结论Rottlerin能抑制PKCδ的磷酸化,进而减轻6-OHDA对多巴胺能神经细胞死亡的诱导作用,说明PKCδ505位点丝氨酸的磷酸化是6-OHDA发挥毒性作用的关键,提示PKCδ在帕金森病病人神经元缺失中起重要作用。
Objective To investigate the role of PKCδ phosphorylation in the death of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA), and to explore the molecular mechanism of neuronal loss in Parkinson’s disease. Methods The rat pheochromocytoma cell line PC12 was cultured in vitro. The effect of pretreatment with bisindolylmaleimide I (G6976 and Rottlerin) and agonist phorbol ester on the toxicity of 6-OHDA was observed. Thiazolyl blue colorimetry The cell viability was detected by Western blotting and the expression of phosphorylated PKCδ was observed by immunoblotting. Results PKCδ inhibitor Rottlerin (2μmol / L) inhibited the phosphorylation of PKCδ and attenuated the cell death induced by 6-OHDA. The cell viability increased to 69.6 ± 2.63% (P <0.05). PKCα inhibitor bisindolylmaleimide I and calcium-dependent PKC inhibitor G6976 had no significant effect on the toxicity of 6-OHDA and PKCδ phosphorylation, while PKCδ activator phorbol ester (100nmol / L) increased PKCδ phosphorylation, Increasing the damaging effect of 6-OHDA decreased cell survival to 49.8 ± 5.06% (P <0.001) at the level of 6-OHDA alone. Conclusion Rottlerin can inhibit the phosphorylation of PKCδ, and then reduce the induction of 6-OHDA on dopaminergic neuronal cell death, indicating that the serine phosphorylation of PKCδ505 is the key point of 6-OHDA play a toxic role, suggesting that PKCδ in Parkinson’s disease patients with nerve Meta missing play an important role.