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目的:建立慢性癫痫发作SD大鼠模型并检测海马组织P2Y6受体表达改变,研究P2Y6受体-Ca2+信号通路改变。方法:采用氯化锂和匹罗卡品腹腔注射建立SD大鼠慢性癫痫发作模型(EPI组)并检测其脑电图,以腹腔注射生理盐水SD大鼠为对照(CON组)。采用免疫组织化学、RT-PCR和Western blot检测EPI组和CON组SD大鼠海马组织P2Y6受体mRNA和蛋白质表达并比较其差异。采用Fura-2/AM检测EPI组和CON组SD大鼠新鲜海马组织细胞内Ca2+浓度并对比其差异。结果:CON组正常SD大鼠脑电图波形主要以α波为主,并伴有低幅度的β波及少量的δ波,几乎未捕捉到棘波发放;EPI组SD大鼠的脑电图出现较为规律的棘波发放,其频率多集中于28Hz,波幅不高于200μV。EPI组慢性癫痫发作SD大鼠海马组织P2Y6受体表达、mRNA、蛋白质和细胞内Ca2+显著高于CON组正常SD大鼠(均P<0.05)。结论:P2Y6受体在慢性癫痫发作大鼠海马组织表达显著升高,激活下游信号传导通路导致细胞内Ca2+水平升高,可能在慢性癫痫发作的发病机制中具有重要作用。
OBJECTIVE: To establish a rat model of chronic epileptic seizures and detect the change of P2Y6 receptor expression in hippocampus to investigate the change of P2Y6 receptor-Ca2 + signal pathway. Methods: Chronic epileptic seizures model (EPI group) and electroencephalogram (EEG) were established by intraperitoneal injection of lithium chloride and pilocarpine into SD rats. CON rats were injected intraperitoneally with normal saline (SD) rats. Immunohistochemistry, RT-PCR and Western blot were used to detect the mRNA and protein expression of P2Y6 receptor in hippocampus of SD rats in EPI group and CON group. Fura-2 / AM was used to detect intracellular Ca2 + concentrations in fresh hippocampus of SD rats in EPI and CON groups. Results: The waveforms of EEG in CON group were mainly α wave with a small amount of β wave and a small amount of δ wave, almost no spike wave was found. The EEG of SD rats in EPI group appeared More regular spike distribution, the frequency of more concentrated in 28Hz, amplitude is not higher than 200μV. The expression of P2Y6 receptor, mRNA, protein and intracellular Ca2 + in hippocampus of SD rats with chronic epileptic seizure in EPI group were significantly higher than those in normal CON rats (all P <0.05). CONCLUSION: P2Y6 receptor is significantly increased in the hippocampus of chronic seizure rats. Activation of downstream signaling pathways leads to increased intracellular Ca2 + levels, which may play an important role in the pathogenesis of chronic seizures.