论文部分内容阅读
目的:评价内侧前额叶皮质(mPFC)小胶质细胞P2Xn 7受体在大鼠神经病理性痛中的作用及其与自噬的关系。n 方法:SPF级健康成年雄性SD大鼠64只，6~8周龄，体重200~250 g，采用随机数字表法分为4组(n n＝16)：假手术组(S组)、神经病理性痛组(NP组)、假手术+P2Xn 7受体阻断组(SP组)和神经病理性痛+P2Xn 7受体阻断组(NP+P组)。采用坐骨神经干结扎法制备大鼠神经病理性痛模型，14 d后通过脑立体定位仪于内侧前额叶皮质置入套管，SP组和NP+P组第14天开始连续3 d双侧mPFC注射P2Xn 7受体阻断剂A-740003 0.5 μg/0.5 μl，S组和NP组第14天开始连续3 d双侧mPFC注射二甲基亚砜0.5 μl。于造模后3、7、10 d及14、15、16 d给药后30 min时，测定机械缩足反应阈(MWT)和热缩足潜伏期(TWL)。随后处死大鼠取mPFC，采用qRT-PCR法检测P2Xn 7受体mRNA的表达，采用Western blot法检测P2Xn 7受体、自噬相关蛋白Beclin1和LC3Ⅱ/Ⅰ的表达，采用免疫荧光法检测P2Xn 7受体与Iba-1共表达情况，透射电镜下观察并计数mPFC自噬体。n 结果:与S组比较，NP组和NP+P组造模后3、7和10 d时MWT降低，TWL缩短，造模后16 d给药后30 min时P2Xn 7受体及其mRNA、Beclin1和LC3Ⅱ/Ⅰ表达上调，P2Xn 7受体与Iba-1共表达细胞数和自噬体数增加(n P0.05)；与NP组比较，NP+P组造模后14、15和16 d给药后30 min时MWT升高，TWL延长，造模后16 d给药后30 min时P2Xn 7受体及其mRNA、Beclin1和LC3Ⅱ/Ⅰ表达下调，P2Xn 7受体与Iba-1共表达细胞数和自噬体数减少(n P<0.05)。n 结论:mPFC小胶质细胞P2Xn 7受体表达上调参与了大鼠神经病理性痛的过程，与促进自噬有关。n “,”Objective:To evaluate the role of P2Xn 7 receptor in microglia in the medial prefrontal cortex (mPFC) in neuropathic pain (NP) and the relationship with autophagy in rats.n Methods:Sixty-four healthy SPF male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-250 g, were divided into 4 groups (n n=16 each) using a random number table method: sham operation group (S group), NP group, sham operation+ P2Xn 7 receptor blocking group (SP group), and NP+ P2Xn 7 receptor blocking group (NP+ P group). The NP model was established by ligation of the sciatic nerve.Fourteen days later a cannula was placed in the mPFC with a brain stereotactic instrument, P2Xn 7 receptor blocker A-740003 0.5 μg/0.5 μl was injected into bilateral mPFC for 3 consecutive days starting from the 14th day in SP and NP+ P groups, and DMSO 0.5 μl was injected instead of A-740003 in S and NP groups.The mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured at 3, 7 and 10 days after establishing the model and 14, 15 and 16 days after administration.Then the rats were sacrificed, and the mPFC was removed for determination of the expression of P2X n 7 receptor and mRNA and autophagy-related proteins Beclin1 and LC3Ⅱ/Ⅰ (by quantitative real-time polymerase chain reaction or by Western blot) and co-expression of P2Xn 7R and microglia (by immunofluorescence) and the number of autophagosomes in mPFC (with a transmission electron microscope).n Results:Compared with group S, MWT was significantly decreased, and TWL was shortened at 3, 7 and 10 days after establishing the model, the expression of P2Xn 7 receptor and mRNA, Beclin1 and LC3Ⅱ/Ⅰ was up-regulated at 30 min after administration on 16 days after establishing the model, and the number of cells co-expressing P2Xn 7 receptor and IBA-1 and the number of autophagosomes were increased in NP and NP+ P groups (n P0.05). Compared with group NP, MWT was significantly increased, and TWL was prolonged at 30 min after administration on 14, 15 and 16 days after establishing the model, the expression of P2Xn 7 receptor and mRNA, Beclin1 and LC3Ⅱ/Ⅰ was down-regulated, and the number of cells co-expressing P2Xn 7 receptor and Iba-1 and the number of autophagosome were decreased in group NP+ P (n P<0.05).n Conclusion:Up-regulation of P2Xn 7 receptor expression in microglia in mPFC is involved in the process of NP in rats, which is associated with the promotion of autophagy.n