基于对DRG阿片受体的调控研究华蟾素治疗骨癌痛的机制

Bone cancer pain is one common and uneasily controlled symptom which is caused by malignant tumor metastasis to multiple bone tissues. It seriously undermines the quality of life of cancer patients. Cinobufacini had been widely used in the treatment of carcinoma and played an important role in the relief of cancer pain. But the involved mechanism remains unclear. Former studies of our group had verified its analgesic effect on cancer pain, and confirmed its peripheral analgesic mechanism, that is by increasing the synthesis and release of β-endorphin in immune cells, thereby increasing the combination of β-endorphin with μ-opioid receptor in the tumor and surrounding tissue. Based on it, this project intends to investigate the roles of μ-opioid receptor (MOR)and δ-opioid receptor (DOR)in Cinobufacini analgesic effect on bone cancer pain, by establishing bone cancer pain rats model. Thermal and mechanical hyperalgesia werer measured,such techniques as RTqPCR,Western blot,and immunohistochemical analysis were applied to measure and analyze the expression of MOR and DOR at dorsal root ganglion (DRG) and in the tumor and surrounding tissue, patch-clamp technique was used to study characteristics of voltage-gated calcium channel currents in rat DRG neurons. Besides, opioid receptor antagonists were used to determine the role and use of MOR and DOR. Neurotransmitter such as substance P(SP)and calcitonin-gene related peptide(CGRP) were also observed. The object of this study is to clarify the two kinds of opioid receptors in the status and role of Cinobufacini in the treatment of bone cancer pain, and reveal Cinobufacini affect cell membrane ion channels and the regulation of neurotransmitter when opioid receptors releasing from DRG to the outer circumference. It will provide more sufficient theoretical foundations for Cinobufacini’s appliance for cancer pain patients in clinic.

骨癌痛是恶性肿瘤转移至骨组织引起的常见的、不易控制的症状之一，严重影响癌症患者的生存质量。临床研究显示华蟾素具有较好的镇痛作用，课题组前期研究验证了华蟾素抗癌痛的的效应，并证实其主要机制之一是通过增加免疫细胞β-内啡肽的合成与释放，从而增加其与阿片受体在肿瘤及周围组织中的结合而发挥外周镇痛机制。基于此，本项目以骨癌痛模型大鼠为研究对象，采用 RTqPCR、Western blot、免疫组化、膜片钳等技术，通过疼痛行为学、阿片受体的表达、背跟神经节（DRG）细胞膜电位改变等指标检测，并采用阿片受体拮抗剂来进一步研究μ和δ阿片受体在华蟾素镇痛机制中的作用。以阐明两种阿片受体在华蟾素治疗骨癌痛中的地位和作用，并揭示华蟾素对阿片受体从DRG释放到外周时对细胞膜离子通道和神经递质调控的影响。本项目的开展为华蟾素临床治疗癌痛提供更充分的理论依据，同时为中医药抗癌痛机制探讨提供思路与方法。

骨癌痛是中晚期恶性肿瘤患者常见临床症状之一，传统阿片类受体激动剂如吗啡对其控制效果不佳，中医药能有效弥补“三阶梯药物止痛法”不足。华蟾素及其原药材蟾蜍皮具有解毒散结、消肿止痛的功效，课题组前期发现华蟾素可增加免疫细胞中POMC表达来提高β-END的合成，并通过增加细胞因子的含量来促进β-END的释放，从而增加其与μ-阿片受体在肿瘤病变组织的结合机会而发挥外周镇痛作用。. 本课题成功建立大鼠骨癌痛动物模型，进一步深入探讨华蟾素对骨癌痛大鼠DRG及瘤周组织中的MOR表达、NGF及其受体，以及神经递质CGRP表达情况；在体外DRG神经元细胞培养鉴定后，运用膜片钳技术检测华蟾素对体外培养DRG神经元细胞细胞膜钙离子电位的影响。研究发现：华蟾素能抑制骨癌痛模型大鼠的热痛敏与机械痛敏；华蟾素可增加骨癌痛大鼠DRG中μ阿片受体的表达，对δ阿片受体也有一定促进作用，同时能增加β-内啡肽的表达；华蟾素可抑制NGF及其受体TrkA 、p75NTR的表达，减少神经递质CGRP与SP的释放；华蟾素能减少L-VGCC电流密度，但不影响L-VGCC激活及失活的电学特征。. 在前期研究基础上，本课题进一步证实了华蟾素能促进DRG中μ阿片受体的合成，可能通过抑制NGF及其下游受体TrkA 、p75NTR的表达，促进MOR的激活，减少神经递质CGRP与SP的释放，最终参与骨癌痛的镇痛过程；并在体外实验证实华蟾素能通过调节DRG神经元L-VGCC电流起抑制神经兴奋作用和保护神经元的效应。.本项目为癌痛的机制探讨及临床上癌痛的治疗提供新的方法和研究范例，同时也为华蟾素更广泛的应用于癌痛病人的临床治疗提供充分的理论依据。

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