电针治疗骨癌痛吗啡耐受模型的阿片受体机制研究

More than half advanced cancer patients suffer from severe pain. It is an imminent task to control cancer pain in clinical medicine. Opioid is the overwhelming drug in cancer pain management. But its clinical utility is severely hindered by the development of drug tolerance. It is implicated that opioid tolerance mechanism is correlated with Mu-opioid receptor (MOR) abnormal internalization desensitization and resensitization. Other opioid receptors as delta and kappa receptors and pain neurotransmitters as substance P (SP) and calcitonin - gene related peptide (CGRP) also play important roles in MOR internalization. Due to the ambiguous mechanism of opioid tolerance, there has not been elucidated until now, especially lack of the efficacy treatment strategy.Cancer induced bone pain (CIBP) is the most common type of cancer pain and morphine is the representative drug of opioid. This research selects CIBP - morphine tolerance rat model which highly imitates clinical pathology. Basing on the preliminary experiment, we explore to find the effect of electroacunpuncture by mechanical pain threshold measure. We propose to explore the three opioid receptors expression in the major neucleus participating in pain modulation. These nucleus (periaqueductal gray, neucleus centromedian, nucleus parafasciculas，spinal cord horn, dorsal root ganglion) will be determined by protein assay and mRNA analyses. We will also assay the content of endorphin, enkephalin and dynorphins at plasma and spinal level. The relationship between endogenous opioid peptides and opioid receptors will be determined further. This research would at first provide scientific evidence of electroacupuncture as a new strategy alleviating morphine tolerance in CIBP.

中晚期癌症患者多数伴有剧烈疼痛，控制癌痛是临床医学亟待解决的任务。阿片以无可替代的优势成为癌痛治疗最常用和最有效的药物，但容易出现的阿片耐受严重影响了治疗。阿片耐受与μ阿片受体不能及时内吞脱敏和复敏有关，δ、κ阿片受体以及疼痛神经递质P物质(SP)、降钙素基因相关肽(CGRP)共同影响μ受体内吞。但这些研究尚未得到确切结论，治疗对策尤其缺乏。骨癌痛是最常见的癌痛类型，吗啡是阿片研究的代表药物。本课题在模拟临床病理状态的骨癌痛-吗啡耐受大鼠模型上，基于预实验结果，通过机械痛阈测定，进一步了解电针治疗吗啡耐受疗效；通过动态观察阿片受体(μ、δ、κ)在疼痛相关神经核团(中脑导水管周围灰质、中央中核、束旁核、脊髓背角、背根神经节)的蛋白表达和mRNA表达，测定血浆和脊髓内啡肽、脑啡肽、强啡肽水平及与阿片受体相互关系，首次探索电针治疗骨癌痛-吗啡耐受的阿片受体机制。

吗啡为代表的阿片药物是癌痛治疗最常用和最有效的药物，但容易出现的阿片耐受现象严重限制了临床使用，阿片耐受成为癌痛治疗中最棘手和亟待解决的重要课题。本研究按照计划建立50例骨癌痛-吗啡耐受大鼠模型，采用随机对照方法实施电针治疗，通过机械刺激下50%缩足阈值证实电针能够减轻骨癌痛，减缓吗啡耐受发生。通过免疫组化分析重要疼痛递质免疫阳性成分在脊髓背角和背根神经节分布特征，明确神经递质SP、CGRP在骨癌痛模型中过度反应，电针有助于减少其表达。通过血清学测定内啡肽、脑啡肽和强啡肽三种阿片肽，了解内源性阿片肽在骨癌痛吗啡耐受过程中的变化，明确电针能够提高大鼠β-内啡肽和脑啡肽含量，但对强啡肽并无影响。在研究过程中我们确定吗啡耐受根本原因是产生中枢神经系统产生抗阿片肽物质,于是成功分离并纯化出具有拮抗吗啡镇痛作用的蛋白，序列测定表明它们分别是已知的pDBI（猪-苯二氮卓结合抑制剂），又称抗阿片肽99A (anti-opioid peptide-99A，AOP-99A)。我们分离合成出这种抗阿片肽的活性肽段(99A-16,99A-19)，发现能够拮抗吗啡镇痛。我们将在后续课题继续研究抗阿片肽作用机制和靶点，并合成抗阿片肽抗体，这将为解决吗啡耐受提供更直接有效的方法。