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摘要 目的：采用蛋白质组学方法筛选痛记忆模型大鼠ACC脑区差异蛋白，并进一步筛选与电针干预痛记忆可能相关的差异蛋白，为电针干预痛记忆的深入研究提供理论支持。方法：将18只健康雄性SD大鼠随机分为空白组（生理盐水对照组）、模型组和电针组，每组6只。模型组及电针组大鼠通过二次角叉菜胶足跖注射建立痛记忆模型。对照组注射同等剂量的生理盐水。电针组于首次注射后5 h、1～5 d进行电针治疗。模型组与空白组仅作与电针组相同的束缚处理。分别检测3组大鼠造模前，首次注射后4 h、5 d和二次注射后4 h、72 h的机械痛阈。二次注射后第3天，大鼠处死后取ACC脑组织。以双向凝胶电泳分离，双向电泳后经不同波长光激发扫描得到不同样品的蛋白质组图谱，经Image Master2D 6.0软件进行分析后，筛选相差在1.5倍以上的蛋白作为差异表达的蛋白质，并进行质谱鉴定。结果：1）与空白组比较，模型组二次未注射角叉菜胶足跖痛阈显著下降（P<0.05）;与模型组比较，电针组二次未注射角叉菜胶足跖痛阈显著上升（P<0.05）。2）经蛋白质组学分析共筛选出18个差异蛋白质。其中模型组有明显差异者有11个，4个表达呈现下调，分别为微管蛋白α-1A链、DAB2相互作用蛋白、NADH脱氢酶的辅酶黄素蛋白2、转凝蛋白-3;7个表达呈现上调，分别为微管蛋白β-3链、肌动蛋白1、磷酸甘油酸激酶1、类固醇激素合成急性蛋白、肌动蛋白2、细胞色素c氧化酶6A1亚基、泛素40S核糖体蛋白S27a。与空白组比较，电针组有明显差异者有15个，3个表达呈现下调，分别为微管蛋白α-1C链、Rho GDP解离抑制因子、NADH脱氢酶的辅酶黄素蛋白2;12个表达呈现上调，分别为微管蛋白β-2A链、微管蛋白β-3链、肌动蛋白1、乌头酸水合酶、丙酮酸激酶同工酶、异柠檬酸脱氢酶、磷酸甘油酸激酶1、Ras相关Rab-19蛋白、类固醇激素合成急性蛋白、肌动蛋白2、细胞色素c氧化酶6A1亚基、泛素40S核糖體蛋白S27a。电针组与模型组比较后呈现明显差异的有8个差异蛋白，2个呈现下调，分别是Rho GDP解离抑制因子、肌动蛋白2。6个呈现上调，分别是微管蛋白α-1A链、微管蛋白β-2A链、DAB2相互作用蛋白、乌头酸水合酶、异柠檬酸脱氢酶、Ras相关Rab-19蛋白。结论：经蛋白质组学分析发现，有11个差异蛋白参与痛记忆的唤醒过程;有8个差异蛋白参与电针干预痛记忆的过程。提示电针对痛记忆的干预可能与稳定神经细胞骨架蛋白，进而抑制神经突触可塑性改变有关。
关键词 电针;痛记忆;前扣带皮层;蛋白质组学;差异蛋白;角叉菜胶;全景式;大鼠
Abstract Objective：To screen the differential protein of ACC brain region in pain memory model rats by proteomics method，to further screen the differential proteins related to electroacupuncture intervention pain memory，so as to provide theoretical support for the further study of electroacupuncture intervention pain memory.Methods：A total of 18 healthy male Sprague Dawley rats were divided into three groups，with 6 rats in each group，including a control group，a model group and an EA group.The pain memory model was made by twice cross-injection carrageenan in EA and model groups.The control group was given the same dose of normal saline in the same time point as the model and EA group.EA was applied to bilateral Zusanli（ST36）and given at 5 h，1～5 days after the first injection for 30 min per time.The model group was taken the same measures except for EA.The pain withdrawal thresholds were detected before modeling and at 4 h，120 h after the first injection and before second injection and at 4 h，72 h after the second injection.Rats were killed at 3rd day after the second injection and taking the ACC of rats for preparation.Samples were separated by 2-D gel electrophoresis（2-DE）.Then we got different samples of proteomic map by different excitation wavelength light scan.The gels were respectively image analyzed by Image Master 2D 6.0，to screen the protein changed in abundance more than 1.5 folder up or down compared with which in constitution of the control group as differential proteomics，and then had the mass spectrum identification.Results：1）Compared with the blank group，the pain threshold of carrageenan ankle was significantly decreased in the model group（P<0.05）.Compared with the model group，the ankle pain threshold of electroacupuncture group without twice cross-injection carrageenan was rised significantly（P<0.05）.）2）After proteomic analysis，18 differential proteins were screened out.Among them，there were 11 with significant difference in the model group，4 of them had down expression，including Tubulin alpha-1C chain，DAB2 interacting protein，NADH dehydrogenase [ubiquinone] flavoprotein 2，Transglutinin-3; and 7 of them had up expression，including Tubulin beta-3，Phosphoglycerate kinase 1，Steroidogenic acute regulatory protein，Actin cytoplasmic 2，Cytochrome c oxidase subunit 6A1，Ubiquitin-40S ribosomal protein S27a.Compared with the blank group，EA group had 15 proteins with significant differences，3 of them had down expression，including Tubulin alpha-1C chain，Rho GDP-dissociation inhibitor 1，NADH dehydrogenase [ubiquinone] flavoprotein 2，and 12 of them had up expression，including Tubulin beta-2A chain，Tubulin beta-3A chain，Actin 1，aconitate hydratase，pyruvate kinase isoenzyme，isocitrate dehydrogenase，phosphoglycerate kinase 1，Ras-related Rab-19 protein，steroid hormone synthesis of acute protein，actin 2，cells Pigment c oxidase 6A1 subunit，ubiquitin 40S ribosomal protein S27a.There were 8 differentially expressed proteins in the EA group compared with the model group，and 2 were down-regulated，which were Rho GDP dissociation inhibitor and actin 2.6 were up-regulated，respectively，tubulin α-1A Chain，tubulin β-2A chain，DAB2 interacting protein，aconitate hydratase，isocitrate dehydrogenase，Ras-related Rab-19 protein.Conclusion：There were 11 differential proteomics involved in pain memory of awakening process through proteomics analysis.And 8 differential proteomics were involved in the intervention of EA on pain memory.The intervention effect of electroacupuncture（EA）pretreatment on pain memory may be related to inhibition of synaptic plasticity.And the inhibition may be related to reinforce nerve cytoskeleton structure.