生长抑素ⅡA受体在外周神经系统的表达及镇痛机制研究

发布时间：2018-09-06 15:51

【摘要】：生长抑素（somatostatin，SOM）是从下丘脑中分离提取出来的一种生长激素抑制因子，它作为一种神经递质或神经调质，广泛分布于哺乳动物中枢和外周神经系统，对于疼痛具有调节作用。本实验室前期在探讨神经病理性疼痛的发病机制研究中发现SOM对于外周神经损伤（peripheral nerve injury，，PNI）所造成的疼痛具有抑制作用。但是天然的SOM半衰期短，只能持续30分钟，因此需要通过研究其类似物来探究其重要的功能。SOM受体广泛分布于体内各个靶器官，SOM主要和其受体相结合发挥其生物学效应，其中生长抑素ⅡA受体（Somatostatin receptor-2A，SST2A）是与SOM具有高亲和力的，普遍分布于各个组织器官的受体亚型，并且被广泛报道具有抗肿瘤的作用，但它在抑制神经病理性疼痛方面的作用至今没有阐明。根据课题组前期研究结果显示SST2A受体可能与SOM的镇痛作用具有某种相关性，因此本论文运用神经病理性疼痛的实验动物模型，研究SST2A在外周神经系统中的表达调控，并应用其特异性的激动剂（Agonist）和拮抗剂（Antagonist）的药理学作用所产生的相应的动物行为学上的改变，继而检测信号转导途径中重要的蛋白分子、激酶的差异表达，旨在阐释SOM通过SST2A受体发挥其镇痛作用的分子机制。本研究首先在小鼠中建立坐骨神经分支选择结扎损伤（spared nerve injury，SNI）动物模型，动物模型建立后，通过一系列的行为学检测指标对模型进行评估，以确定实验模型是否成功建立。主要的行为学检测包括通过机械刺激反应(Von Frey Filaments Test），冷刺激反应（Acetone Test）以及伤害性触刺激反应(Pin-prick Test)检测其是否痛觉过敏或者超敏。实验结果表明在SNI手术7天后，实验小鼠即表现出疼痛异常的行为学特征，其疼痛阈值明显低于未手术的对照组小鼠；此疼痛特征发展到14天达到严重程度，并且趋于稳定一直持续6周以上，此结果表明SNI动物模型已成功建立。在成功建立SNI动物模型的基础上，本研究采用免疫组织化学方法（immunohistochemistry，IHC），Western blot技术检测了SST2A蛋白在背根神经节（dorsal root ganglia，DRG）和脊髓中的表达，结果表明几乎所有的SST2A主要在CGRP型（98%）而不是IB-4型（1%）的中小型肽能神经元中表达；神经损伤后，表达SST2A蛋白的阳性神经元的数目以及蛋白丰度，与正常对照侧相比都明显下调。同时双重免疫组织化学结果表明，SST2A与神经源性的一氧化氮还原酶（nNOS）、甘丙肽（galanin）和神经肽受体1型（Y1R）等与疼痛相关的重要神经肽和神经分子有共存关系，但与其配体SOM不共存。为了进一步确认SST2A的表达，本研究采用原位杂交技术（in situ hybridization，ISH）在脊髓和DRG中对SST2A mRNA的表达作了检测。结果发现集中表达SST2A mRNA的区域，与用免疫组织化学方法得到的实验结果相符，两者相互佐证。根据SOM对神经病理性疼痛的抑制作用以及SST2A在神经损伤后表达下调的特点，通过腹腔注射SOM的类似物奥曲肽即SST2A特异性的激动剂，剂量为40μg/kg，和拮抗剂CYN154806，剂量为6mg/kg，进行药理学相关实验，并通过行为学测试评估，分析SOM通过SST2A受体对疼痛行为的调节作用。研究结果表明注射奥曲肽后，相比起注射生理盐水以及拮抗剂的小鼠，SNI小鼠的机械疼痛阈值明显增高且对冷刺激和伤害性触刺激诱发的疼痛反应时间也显著降低。在药理学实验的基础上，本研究采用分子生物学的相关技术检测了不同药物处理的实验组小鼠DRG神经元胞内与SST2A相关神经分子及其下游效应物的表达差异情况，结果发现p38MAPK，这种参与调控疼痛的信号转导通路激酶的表达活性与药理学实验的结果一致，受到药物的显著影响。通过以上对SST2A在外周神经系统中的表达分析、药理学实验及镇痛活性的可能机制的研究分析，本论文的研究结果第一次揭示了SOM可能通过作用于SST2A受体介导胞内p38MAPK信号通路，本研究有望为临床治疗神经病理性疼痛提供新的研究思路。
[Abstract]:Somatostatin (SOM) is a growth hormone inhibitor isolated from the hypothalamus. As a neurotransmitter or neuromodulator, it is widely distributed in the mammalian central and peripheral nervous system and has a regulatory effect on pain. Previous studies in this laboratory were conducted to explore the pathogenesis of neuropathic pain. SOM is found to inhibit pain caused by peripheral nerve injury (PNI). However, natural SOM has a short half-life, lasting only 30 minutes. Therefore, it is necessary to study its analogues to explore its important function. SOM receptors are widely distributed in various target organs in the body, and SOM is mainly associated with its receptors. Somatostatin receptor-2A (SST2A) is a receptor subtype with high affinity to SOM and widely distributed in various tissues and organs. It has been widely reported to have anti-tumor effects, but its role in inhibiting neuropathic pain has not been elucidated until now. The results showed that SST2A receptor may be related to the analgesic effect of SOM. Therefore, in this study, we used the experimental animal model of neuropathic pain to study the expression and regulation of SST2A in peripheral nervous system, and applied its specific agonist (Agonist) and antagonist (Antagonist) to produce the corresponding pharmacological effects. The purpose of this study is to elucidate the molecular mechanism by which SOM exerts its analgesic effect through SST2A receptors.
In this study, we first established an animal model of selective ligation injury (SNI) of sciatic nerve branches in mice. After the establishment of the animal model, the model was evaluated by a series of behavioral tests to determine whether the experimental model was successfully established. The results showed that 7 days after SNI operation, the experimental mice showed abnormal behavioral characteristics of pain, and the pain threshold was significantly lower than that of the control group. The results showed that the animal model of SNI was successfully established.
Based on the successful establishment of SNI animal model, the expression of SST2A protein in dorsal root ganglia (DRG) and spinal cord was detected by immunohistochemistry (IHC) and Western blot. The results showed that almost all of the SST2A proteins were mainly expressed in CGRP type (98%) rather than IB-4 type (1%). The number and abundance of SST2A positive neurons were significantly decreased after nerve injury compared with the control group. The results of double immunohistochemistry showed that SST2A was associated with neurogenic nitric oxide reductase (nNOS), galanin and neuropeptide receptor type 1 (Y1R) and pain. The related important neuropeptides have coexistence with nerve molecules, but they do not coexist with their ligand SOM.
In order to further confirm the expression of SST2A, in situ hybridization (ISH) was used to detect the expression of SST2A mRNA in the spinal cord and DRG. The results showed that the regions where SST2A mRNA was concentrated were consistent with the experimental results obtained by immunohistochemistry.
According to the inhibitory effect of SOM on neuropathic pain and the down-regulation of SST2A expression after nerve injury, pharmacological experiments were carried out by intraperitoneal injection of SOM analogue octreotide, a specific agonist of SST2A, at a dosage of 40 ug/kg, and antagonist CYN154806, at a dosage of 6 mg/kg. The results showed that the mechanical pain threshold of SNI mice was significantly higher than that of normal saline and antagonist mice after octreotide injection, and the response time to cold and noxious touch stimulation was also significantly reduced.
On the basis of pharmacological experiments, we used molecular biology techniques to detect the differences in the expression of intracellular and SST2A-related neuronal molecules and their downstream effectors in DRG neurons of mice treated with different drugs. The results of pharmacological experiments were consistent and were significantly influenced by drugs.
Through the above analysis of the expression of SST2A in peripheral nervous system, pharmacological experiments and the possible mechanism of analgesic activity, the results of this study reveal for the first time that SOM may mediate intracellular p38 MAPK signaling pathway by acting on SST2A receptors, which is expected to provide new research for clinical treatment of neuropathic pain. Thinking.
【学位授予单位】：哈尔滨工业大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741

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