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目的研究左旋多巴诱发异动症(levodopa-induced dyskinesias,LID)大鼠纹状体内前强啡肽原(prodynorphin,PDyn)基因表达与DARPP-32蛋白磷酸化状态的变化,探讨LID时直接通路过度活化的机制。方法6-羟多巴胺(6-OHDA)大鼠模型应用左旋多巴治疗28 d诱发LID大鼠模型。采用原位杂交技术检测PDyn mRNA水平,逆转录-聚合酶链反应(RT-PCR)及免疫印迹技术检测LID大鼠纹状体内总DARPP-32的mRNA与蛋白表达及其Thr-34位点磷酸化水平。结果LID大鼠毁损侧PDyn mRNA表达(0.3662±0.0625)较对照组(0.2085±0.0573)及L-dopa治疗组(0.2235±0.0582)明显增高,差异有统计学意义(P<0.01)。LID大鼠毁损侧Thr-34位点磷酸化的DARPP-32蛋白水平(1075.2±103.3)较对照组(198.7±49.5)及L-dopa治疗组(213.9±58.9)明显增高,差异均有统计学意义(P<0.01)。结论DARPP-32蛋白的Thr-34位点的磷酸化水平的改变是LID时多巴胺D1受体介导的直接通路异常活化的关键因素之一。
Objective To investigate the changes of the expression of PDyn gene and phosphorylation of DARPP-32 in the striatum of levodopa-induced dyskinesias (LID) rats, Activation mechanism. Methods 6-hydroxydopamine (6-OHDA) rat model was induced by levodopa for 28 days to induce LID rat model. PDyn mRNA level was detected by in situ hybridization. The mRNA and protein expression of total DARPP-32 in the striatum of LID rats and the expression of Thr-34 phosphorylation sites were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting Level Results The PDyn mRNA expression (0.3662 ± 0.0625) in LID rats was significantly higher than that in control group (0.2085 ± 0.0573) and L-dopa treatment group (0.2235 ± 0.0582) (P <0.01). The level of phosphorylated DARPP-32 protein at the Thr-34 site of LID rats (1075.2 ± 103.3) was significantly higher than that of control group (198.7 ± 49.5) and L-dopa treatment group (213.9 ± 58.9), both of which were statistically significant Significance (P <0.01). Conclusion The change of Thr-34 phosphorylation level in DARPP-32 is one of the key factors in the abnormal activation of the direct pathway mediated by dopamine D1 receptor in LID.