【作者】 李晶晶；

【导师】 王公明；

【作者基本信息】 山东大学 ， 麻醉学（专业学位）， 2022， 硕士

【摘要】 背景骨癌痛(Bone cancer pain,BCP)是原发或转移性骨肿瘤引起的慢性疼痛,包括感觉、情绪、认知等多种维度。BCP伴随的厌恶情绪不仅加剧疼痛,还可能导致自杀等严重后果。研究表明,疼痛持续时间超过6个月的骨癌患者中55%伴有心理障碍。因此,探讨BCP相关厌恶情绪的发生机制对于BCP及相关精神疾病的治疗具有重要意义。前扣带回皮层吻侧部(Rostral anterior cingulate cortex,rACC)是机体产生疼痛厌恶体验的关键脑区,rACC中谷氨酸能突触传递增强,参与疼痛诱导的厌恶行为的表达。此外,脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)通过结合酪氨酸受体激酶B(Tropomyosin receptor kinase B,TrkB)发挥增强谷氨酸传递的作用。因此我们推测,rACC神经元中,BDNF/TrkB这条通路可能参与骨癌痛相关厌恶情绪的调节。目的本研究拟采用右胫骨注射MRMT-1乳腺癌细胞大鼠骨癌痛模型,探讨在rACC神经元中BDNF/TrkB通路在骨癌痛相关厌恶情绪形成的作用方式及潜在的分子机制。方法选取健康成年雄性SD大鼠(200-220g),分为空白对照组(Naive)、假手术组(Sham)和骨癌痛组(BCP),使用右胫骨注射MRMT-1乳腺癌细胞的方法建立BCP大鼠模型,分别于不同时间点检测各组大鼠机械缩足反应阈值(Paw withdrawal threshold,PWT)和位置逃避/回避实验(Place escape/avoidance paradigm,PEAP)的变化,确保建立能够反应大鼠疼痛相关情绪的模型,并选取痛情绪稳定的时间点进行后续研究。通过rACC脑区注射鹅膏蕈氨酸(神经元损毁剂)损毁大鼠rACC脑区,利用qRT-PCR和Western blotting等技术从核酸和蛋白质水平上完成BDNF/TrkB通路各相关基因的定量和半定量检测。通过应用各种拮抗剂验证rACC脑区BDNF/TrkB通路参与癌痛厌恶情绪产生的可能机制,并通过条件性位置回避(Conditioned place avoidance,CPA)实验探究外源性BDNF对情绪的影响。结果(1)rACC脑区在骨癌痛厌恶情绪形成中起重要作用:与Sham组相比,BCP大鼠术后7天PWT、暗室停留时间均显著降低,说明癌痛厌恶情绪造模成功。与未损毁rACC的BCP大鼠相比,损毁rACC的BCP大鼠PWT无显著变化,暗室停留时间显著延长,说明损毁rACC缓解了厌恶情绪但不影响痛阈。(2)rACC脑区BDNF/TrkB信号通路参与癌痛厌恶情绪的形成:与Sham组相比,术后7天BCP大鼠rACC中BDNF mRNA含量及蛋白表达显著增加。TrkB受体阻断剂CTX-B减轻BCP大鼠厌恶情绪,而不改变疼痛阈值。外源性BDNF使大鼠CPA实验训练后对BDNF条件室产生回避行为,CTX-B预处理可以改善回避行为的产生。(3)NR2B参与rACC脑区BDNF/TrkB介导的癌痛厌恶情绪形成过程:与Sham组相比,术后7天BCP大鼠rACC中NR2B mRNA含量显著增加。特异性拮抗NR2B减轻了大鼠的厌恶情绪,但疼痛阈值无改变。特异性拮抗BDNF/TrkB能够抑制BCP大鼠rACC脑区NR2B的蛋白表达。特异性拮抗NR2B可以改善BDNF诱导的CPA实验中大鼠的回避行为。(4)ERK-CREB信号通路参与rACC脑区BDNF/TrkB介导的癌痛厌恶情绪形成过程:与Sham组相比,BCP大鼠rACC脑区磷酸化ERK(pERK)、磷酸化CREB(pCREB)表达显著增加,且这种增加能够被CTX-B抑制,表明BDNF/TrkB通路介导ERK-CREB的激活。特异性阻断ERK减轻了大鼠的厌恶情绪,但疼痛阈值无改变。此外,特异性阻断ERK可以改善BDNF诱导的大鼠回避行为。结论(1)rACC脑区BDNF/TrkB通路参与骨癌痛相关厌恶情绪形成过程;(2)BDNF/TrkB信号通路通过NR2B和ERK-CREB信号通路在癌性疼痛相关厌恶情绪的产生中发挥作用。更多还原

【Abstract】 BackgroundBone cancer pain(BCP)is a chronic pain caused by primary or metastatic bone tumors.Patients with BCP have changes in feeling,emotion,cognition and so on.Negative emotions such as anxiety and fear caused by pain not only aggravate pain,but also lead to serious consequences such as suicide.Studies have shown that 55%of patients with BCP lasting more than 6 months are accompanied by psychological disorders.Therefore,to explore the mechanism and prevention of BCP related negative emotions is of great significance for the treatment of BCP and related mental diseases.Rostral anterior cingulate cortex(rACC)is the key to pain aversion.Recent studies have found that there is emotion related glutamatergic synaptic transmission in rACC,and its changes contribute to the development of pain induced aversion.In addition,brain derived neurotrophic factor(BDNF)enhances glutamate transmission by binding to tropomyosin receptor kinase B(TrkB).Therefore,we speculate that rACC neurons may participate in the regulation of bone cancer pain and negative emotion through BDNF/TrkB signaling pathway.ObjectiveThe aim of this study was to explore the effect and influence of rACC neuron BDNF/TrkB pathway on the formation of cancer pain related aversion,by injecting breast cancer cells into the right tibia to establish a rat model of BCP.MethodsHealthy adult male Sprague-Dawley rats(200-220g)were selected and divided into blank control group(Naive),sham operation group(Sham)and bone cancer pain group(BCP).BCP rat model was established by injecting MRMT-1 breast cancer cells into right tibia.To ensure the model we established can respond to pain aversion in rats,the changes of paw withdrawal threshold(PWT)and place escape/avoidance paradigm(PEAP)were detected at different time points in each group,and selected the time point of stable pain emotions for follow-up research.The rACC of rats were damaged by injection of ibotenic acid(neuron damaging agent)into rACC.The quantitative and semiquantitative detection of relevant genes of BDNF/TrkB pathway were completed at the nucleic acid and protein level by qRT-PCR and Western blotting.The possible mechanism of BDNF/TrkB pathway involved in cancer pain aversion was verified by using various antagonists,and the effect of exogenous BDNF on emotion was explored by conditional place avoidance(CPA)experiment.Results(1)rACC plays an important role in the formation of BCP aversion:Compared with sham group,the PWT and time spent in the dark area of BCP rats were significantly reduced from the 7th day postoperatively,indicating that the BCP aversion model was successfully established.Compared with BCP rats without rACC damage,there was no significant change in PWT in BCP rats with rACC damage,and the time spent in the dark area was significantly prolonged,suggesting that rACC damage alleviated aversion,but had no effect on pain threshold.(2)BDNF/TrkB signaling pathway in rACC is involved in the formation of cancer pain aversion:Compared with sham group,BDNF mRNA content and protein expression in rACC of BCP rats increased significantly on the 7th day postoperatively.TrkB receptor blocker CTX-B alleviated the aversion of BCP rats without changing the PWT.Exogenous BDNF can induce avoidance behavior in BDNF-conditioned room after CPA experimental training.CTX-B pretreatment can improve avoidance behavior.(3)NR2B is involved in the formation of cancer pain aversion mediated by BDNF/TrkB in rACC:Compared with sham group,the content of NR2B mRNA in rACC of BCP rats increased significantly on the 7th day after operation.The specific antagonism of NR2B alleviated aversion in rats,but the PWT did not change.Specifically antagonizing BDNF/TrkB can inhibit the protein expression of NR2B in rACC of BCP rats.Specific antagonism of NR2B can improve the avoidance behavior of rats in BDNF induced CPA experiment.(4)ERK-CREB signaling is involved in the formation of cancer pain aversion mediated by BDNF in rACC:Compared with sham group,the expression of phosphorylated ERK(pERK)and phosphorylated CREB(pCREB)in rACC of BCP rats increased significantly,and could be inhibited by CTX-B,which indicating that BDNF/TrkB signaling pathway mediates the activation of ERK-CREB signalling.Specific blocking of ERK alleviated aversion in rats,but the PWT did not change.In addition,blocking of ERK can improve BDNF induced avoidance behavior in rats.Conclusion(1)BDNF/TrkB signaling pathway of rACC is involved in the generation of cancer pain related aversion:(2)BDNF/TrkB signaling pathway plays a role in the generation of cancer pain related aversion through NR2B and ERK-CREB signaling pathway.更多还原