【摘要】：生長抑素（somatostatin，SOM）是從下丘腦中分離提取出來的一種生長激素抑制因子，它作為一種神經(jīng)遞質(zhì)或神經(jīng)調(diào)質(zhì)，廣泛分布于哺乳動物中樞和外周神經(jīng)系統(tǒng)，對于疼痛具有調(diào)節(jié)作用。本實驗室前期在探討神經(jīng)病理性疼痛的發(fā)病機制研究中發(fā)現(xiàn)SOM對于外周神經(jīng)損傷（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，只能持續(xù)30分鐘，因此需要通過研究其類似物來探究其重要的功能。SOM受體廣泛分布于體內(nèi)各個靶器官，SOM主要和其受體相結(jié)合發(fā)揮其生物學(xué)效應(yīng)，其中生長抑素ⅡA受體（Somatostatin receptor-2A，SST2A）是與SOM具有高親和力的，普遍分布于各個組織器官的受體亞型，并且被廣泛報道具有抗腫瘤的作用，但它在抑制神經(jīng)病理性疼痛方面的作用至今沒有闡明。根據(jù)課題組前期研究結(jié)果顯示SST2A受體可能與SOM的鎮(zhèn)痛作用具有某種相關(guān)性，因此本論文運用神經(jīng)病理性疼痛的實驗動物模型，研究SST2A在外周神經(jīng)系統(tǒng)中的表達調(diào)控，并應(yīng)用其特異性的激動劑（Agonist）和拮抗劑（Antagonist）的藥理學(xué)作用所產(chǎn)生的相應(yīng)的動物行為學(xué)上的改變，繼而檢測信號轉(zhuǎn)導(dǎo)途徑中重要的蛋白分子、激酶的差異表達，旨在闡釋SOM通過SST2A受體發(fā)揮其鎮(zhèn)痛作用的分子機制。 本研究首先在小鼠中建立坐骨神經(jīng)分支選擇結(jié)扎損傷（spared nerve injury，SNI）動物模型，動物模型建立后，通過一系列的行為學(xué)檢測指標對模型進行評估，以確定實驗?zāi)Ｐ褪欠癯晒�。主要的行為學(xué)檢測包括通過機械刺激反應(yīng)(Von Frey Filaments Test），冷刺激反應(yīng)（Acetone Test）以及傷害性觸刺激反應(yīng)(Pin-prick Test)檢測其是否痛覺過敏或者超敏。實驗結(jié)果表明在SNI手術(shù)7天后，實驗小鼠即表現(xiàn)出疼痛異常的行為學(xué)特征，其疼痛閾值明顯低于未手術(shù)的對照組小鼠；此疼痛特征發(fā)展到14天達到嚴重程度，并且趨于穩(wěn)定一直持續(xù)6周以上，此結(jié)果表明SNI動物模型已成功建立。 在成功建立SNI動物模型的基礎(chǔ)上，本研究采用免疫組織化學(xué)方法（immunohistochemistry，IHC），Western blot技術(shù)檢測了SST2A蛋白在背根神經(jīng)節(jié)（dorsal root ganglia，DRG）和脊髓中的表達，結(jié)果表明幾乎所有的SST2A主要在CGRP型（98%）而不是IB-4型（1%）的中小型肽能神經(jīng)元中表達；神經(jīng)損傷后，表達SST2A蛋白的陽性神經(jīng)元的數(shù)目以及蛋白豐度，與正常對照側(cè)相比都明顯下調(diào)。同時雙重免疫組織化學(xué)結(jié)果表明，SST2A與神經(jīng)源性的一氧化氮還原酶（nNOS）、甘丙肽（galanin）和神經(jīng)肽受體1型（Y1R）等與疼痛相關(guān)的重要神經(jīng)肽和神經(jīng)分子有共存關(guān)系，但與其配體SOM不共存。 為了進一步確認SST2A的表達，本研究采用原位雜交技術(shù)（in situ hybridization，ISH）在脊髓和DRG中對SST2A mRNA的表達作了檢測。結(jié)果發(fā)現(xiàn)集中表達SST2A mRNA的區(qū)域，與用免疫組織化學(xué)方法得到的實驗結(jié)果相符，兩者相互佐證。 根據(jù)SOM對神經(jīng)病理性疼痛的抑制作用以及SST2A在神經(jīng)損傷后表達下調(diào)的特點，通過腹腔注射SOM的類似物奧曲肽即SST2A特異性的激動劑，劑量為40μg/kg，和拮抗劑CYN154806，劑量為6mg/kg，進行藥理學(xué)相關(guān)實驗，并通過行為學(xué)測試評估，分析SOM通過SST2A受體對疼痛行為的調(diào)節(jié)作用。研究結(jié)果表明注射奧曲肽后，相比起注射生理鹽水以及拮抗劑的小鼠，SNI小鼠的機械疼痛閾值明顯增高且對冷刺激和傷害性觸刺激誘發(fā)的疼痛反應(yīng)時間也顯著降低。 在藥理學(xué)實驗的基礎(chǔ)上，本研究采用分子生物學(xué)的相關(guān)技術(shù)檢測了不同藥物處理的實驗組小鼠DRG神經(jīng)元胞內(nèi)與SST2A相關(guān)神經(jīng)分子及其下游效應(yīng)物的表達差異情況，結(jié)果發(fā)現(xiàn)p38MAPK，這種參與調(diào)控疼痛的信號轉(zhuǎn)導(dǎo)通路激酶的表達活性與藥理學(xué)實驗的結(jié)果一致，受到藥物的顯著影響。 通過以上對SST2A在外周神經(jīng)系統(tǒng)中的表達分析、藥理學(xué)實驗及鎮(zhèn)痛活性的可能機制的研究分析，本論文的研究結(jié)果第一次揭示了SOM可能通過作用于SST2A受體介導(dǎo)胞內(nèi)p38MAPK信號通路，本研究有望為臨床治療神經(jīng)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.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R741

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