GDF10在神經(jīng)病理性疼痛大鼠脊髓中的表達(dá)變化及機(jī)制研究
發(fā)布時(shí)間：2019-03-17 09:26
【摘要】：由中樞和周圍神經(jīng)系統(tǒng)損傷導(dǎo)致的神經(jīng)病理性疼痛(neuropathic pain,NP)是一種難治性疾病,目前的治療方法大都以暫時(shí)性的止痛和理療為主,但并沒(méi)有實(shí)現(xiàn)長(zhǎng)期的治療疼痛的目的,加強(qiáng)神經(jīng)病理性疼痛發(fā)生機(jī)制的研究對(duì)其開發(fā)新的治療藥物及方法有重要意義。目前關(guān)于神經(jīng)病理性疼痛的發(fā)生機(jī)制主要有神經(jīng)炎癥反應(yīng)以及中樞敏化。轉(zhuǎn)化生長(zhǎng)因子β(transforming growth factorβ,TGF-β)超家族是一類具有多功能并且具有環(huán)境依賴性的細(xì)胞因子,其亞家族包括TGF-βs、骨形態(tài)發(fā)生蛋白(bone morphogenetic proteins,BMPs)、活化素(activins)、生長(zhǎng)和分化因子(growth and differentiation factors,GDFs)等,其中許多成員(如TGF-β1)具有神經(jīng)炎癥抑制作用,可以減輕神經(jīng)病理性疼痛的發(fā)生。生長(zhǎng)和分化因子10(growth and differentiation factor 10,GDF10)是BMPs亞家族成員之一,它在神經(jīng)發(fā)生以及再生中起著重要作用,通過(guò)在GEO數(shù)據(jù)庫(kù)中查詢顯示神經(jīng)病理性疼痛大鼠的背根神經(jīng)節(jié)中GDF10的表達(dá)增加,而有關(guān)其在神經(jīng)病理性疼痛發(fā)生時(shí)在脊髓中的變化及作用尚未見(jiàn)報(bào)道。N-甲基-D-天冬氨酸(N-methyl-D-aspartate,NMDA)受體的活化而引起的脊髓中樞敏化在神經(jīng)病理性疼痛的發(fā)生中有著重要作用,由此我們進(jìn)一步研究GDF10是否與NMDA受體的活化間有關(guān)聯(lián),從而了解GDF10在神經(jīng)病理性疼痛中的相關(guān)機(jī)制。方法:(1)大鼠神經(jīng)病理性疼痛模型的建立。采用結(jié)扎雄性SD大鼠左側(cè)L5脊神經(jīng)的方法建立此模型。(2)藥物鞘內(nèi)注射。采用鞘內(nèi)注射的方法對(duì)大鼠注射NMDA使其產(chǎn)生神經(jīng)病理性疼痛,并對(duì)脊神經(jīng)結(jié)扎大鼠鞘內(nèi)注射NMDA受體的拮抗劑MK-801。(3)行為學(xué)檢測(cè)。用von Frey filaments對(duì)大鼠縮爪閾值進(jìn)行測(cè)量,以反應(yīng)后肢足底的機(jī)械痛敏的變化。(4)Western Blot。利用免疫印跡檢測(cè)脊神經(jīng)結(jié)扎大鼠術(shù)側(cè)脊髓在術(shù)后1d、3d、10d、21d GDF10蛋白表達(dá)的變化,并檢測(cè)NMDA鞘內(nèi)注射后脊髓內(nèi)GDF10的表達(dá)變化以及鞘內(nèi)注射MK-801對(duì)脊神經(jīng)結(jié)扎大鼠脊髓背角GDF10的表達(dá)。(4)免疫熒光。用免疫熒光檢測(cè)脊神經(jīng)結(jié)扎后脊髓背角中GDF10在術(shù)后3d、10d、21d的表達(dá)變化及NMDA鞘內(nèi)注射后GDF10的表達(dá),并通過(guò)免疫熒光雙標(biāo)檢測(cè)GDF10在脊髓中的細(xì)胞定位。主要結(jié)果:1.脊神經(jīng)結(jié)扎大鼠在術(shù)后第1 d縮爪閾值開始降低,3 d后與正常對(duì)照組相比,各時(shí)相點(diǎn)差異比較均有統(tǒng)計(jì)學(xué)意義(P0.05),到第10 d閾值下降最明顯,至21 d呈現(xiàn)持平狀態(tài),大鼠呈現(xiàn)出明顯的機(jī)械痛敏增加,表明成功建立了神經(jīng)病理性疼痛大鼠模型。2.免疫熒光雙標(biāo)檢測(cè)觀察到正常大鼠L5脊髓組織中GDF10與神經(jīng)元標(biāo)記物NeuN共表達(dá),而不與星形膠質(zhì)細(xì)胞標(biāo)記物GFAP或小膠質(zhì)細(xì)胞標(biāo)記物Iba-1共表達(dá)。Western blot證實(shí)脊神經(jīng)結(jié)扎后術(shù)側(cè)脊髓中GDF10蛋白的表達(dá)逐漸降低,至第10 d減少最為顯著,至21d呈現(xiàn)低水平表達(dá)狀態(tài),與假手術(shù)組相比差異具有統(tǒng)計(jì)學(xué)意義(P0.05)。免疫熒光顯示術(shù)側(cè)脊髓背角神經(jīng)元內(nèi)GDF10表達(dá)在3d、10d、21d逐漸降低,與western blot結(jié)果相一致。3.正常大鼠鞘內(nèi)注射NMDA,在連續(xù)3d的注射中,同一時(shí)間點(diǎn)與鞘內(nèi)注射生理鹽水組大鼠相比,大鼠縮爪閾值顯著降低(P0.05);western blot檢測(cè)顯示NMDA組大鼠脊髓背角內(nèi)GDF10表達(dá)較生理鹽水組顯著降低(P0.05);免疫熒光檢測(cè)發(fā)現(xiàn)NMDA鞘內(nèi)注射能顯著減少脊髓背角神經(jīng)元內(nèi)GDF10的表達(dá)。4.脊神經(jīng)結(jié)扎大鼠鞘內(nèi)注射MK-801,能顯著抑制脊神經(jīng)結(jié)扎引起的縮爪閾值的降低,與脊神經(jīng)結(jié)扎大鼠鞘內(nèi)注射生理鹽水組相比有統(tǒng)計(jì)學(xué)意義(P0.05)。western blot檢測(cè)發(fā)現(xiàn)脊神經(jīng)結(jié)扎大鼠鞘內(nèi)注射MK-801能顯著抑制脊髓內(nèi)GDF10的表達(dá)減少,與生理鹽水組相比有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:(1)采用左側(cè)L5脊神經(jīng)結(jié)扎成功建立了大鼠神經(jīng)病理性疼痛模型;(2)脊神經(jīng)結(jié)扎使大鼠機(jī)械痛敏增加,并使脊髓背角神經(jīng)元內(nèi)GDF10的表達(dá)減少;(3)鞘內(nèi)注射NMDA使大鼠機(jī)械痛敏顯著增加,并使大鼠脊髓背角神經(jīng)元內(nèi)GDF10表達(dá)減少,表明脊髓內(nèi)NMDA受體的活化與GDF10的表達(dá)減少相關(guān);(4)鞘內(nèi)注射MK-801抑制脊神經(jīng)結(jié)扎大鼠脊髓內(nèi)NMDA受體的活化可以減輕神經(jīng)病理性疼痛,并可抑制脊髓內(nèi)GDF10的表達(dá)減少。
[Abstract]:Neuropathic pain (NP), which is caused by the damage of the central and peripheral nervous system, is a refractory disease. The study of strengthening the pathogenesis of neuropathic pain is of great significance to the development of new therapeutic drugs and methods. At present, the mechanism of the occurrence of neuropathic pain is mainly the reaction of the nerve inflammation and the central sensitization. Transformed growth factor (TGF-1) superfamily is a class of cytokines with multi-function and environment-dependent, the subfamily of which includes TGF-Fass, bone morphogenic proteins (BMPs), activins, growth and differentiation factors. GDFs et al., many of which, such as TGF-(1), have a neuroinflammatory inhibitory effect and can reduce the occurrence of neuropathic pain. Growth and differentiation factor 10 (GDF10) is one of the members of the BMPs subfamily, which plays an important role in the genesis and regeneration of the nerve. The changes of the spinal cord in the spinal cord and its role in the occurrence of neuropathic pain have not been reported. The activation of N-methyl-D-aspartate (NMDA) receptor plays an important role in the pathogenesis of neuropathic pain, and we further study whether the GDF10 is associated with the activation of the NMDA receptor, So as to understand the relevant mechanism of the GDF10 in the neuropathic pain. Methods: (1) The model of neuropathic pain in rats was established. This model was established using a method of ligation of the left L5 spinal nerve in male SD rats. (2) Intra-drug injection. The rats were injected with NMDA to produce neuropathic pain and the antagonist MK-801 of the NMDA receptor was injected into the rat with spinal nerve ligation. (3) Behavior detection. The rat paw threshold was measured with von Frey film to respond to changes in mechanical pain in the foot of the hindlimb. (4)Western Blot. The expression of GDF10 and the expression of GDF10 in the spinal cord of rats were detected by immunoblotting. The expression of GDF10 in the spinal cord and the expression of the dorsal horn GDF10 of the spinal cord in the spinal cord of the rats were detected by the injection of MK-801 in the spinal cord. (4) immunofluorescence. The expression of GDF10 and the expression of GDF10 in the spinal dorsal horn of spinal cord after spinal nerve ligation were detected by immunofluorescence, and the cellular localization of the GDF10 in the spinal cord was detected by immunofluorescence double-label. Main results:1. The threshold of the first day after the operation of the spinal nerve was decreased, and the difference of the point-to-point difference was statistically significant after 3 days (P <0.05). The decrease of the threshold of the 10th day was the most obvious, and the level of 21 d was in the same state, and the rats exhibited a significant increase in the mechanical pain. It was shown that the model of neuropathic pain was successfully established. The co-expression of the GDF10 with the neuronal marker NeuN was observed in the normal rat L5 spinal cord tissue without co-expression with the astrocyte marker GFAP or the microglial cell marker Iba-1. Western blot confirmed that the expression of the GDF10 protein in the spinal cord after the spinal nerve ligation was gradually decreased, the most significant in the 10th day, and the expression of the low-level expression in the 21 d was statistically significant compared with the sham-operated group (P0.05). The results showed that the expression of GDF10 in the dorsal horn of the spinal dorsal horn of the lateral spinal cord decreased gradually in the 3-d,10-day, and 21-day, and was consistent with the western blot. In the normal rat, NMDA was injected into the rat, and the threshold of the rat's paw was significantly lower than that of the normal saline group (P <0.05), and the expression of GDF10 in the dorsal horn of the spinal cord of the NMDA group was significantly lower in the spinal dorsal horn of the rats (P0.05). The expression of GDF10 in the dorsal horn neurons of the spinal cord was significantly reduced by immunofluorescent assay. The MK-801 was injected into the rat with spinal nerve ligation, which can significantly inhibit the reduction of the threshold of the contraction claw caused by the ligature of the spinal nerve. Compared with the saline group, the expression of MK-801 in the rat spinal cord was significantly inhibited by Western blot, and the expression of GDF10 in the spinal cord was significantly inhibited. Conclusion: (1) The rat's neuropathic pain model was successfully established with the left L5 spinal nerve ligation; (2) the spinal nerve ligation increased the mechanical pain of the rat and decreased the expression of the GDF10 in the dorsal horn of the spinal cord; and (3) the intraventricular injection of NMDA increased the mechanical pain of the rat. and the expression of the GDF10 in the dorsal horn of the spinal cord of the rat is reduced, the activation of the NMDA receptor in the spinal cord is related to the reduction of the expression of the GDF10, and (4) the activation of the NMDA receptor in the spinal cord of the rat spinal cord is inhibited by the intra-spinal injection of the MK-801, and the neuropathic pain can be reduced, And the expression of the GDF10 in the spinal cord can be reduced.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R402
本文編號(hào)：2442154
[Abstract]:Neuropathic pain (NP), which is caused by the damage of the central and peripheral nervous system, is a refractory disease. The study of strengthening the pathogenesis of neuropathic pain is of great significance to the development of new therapeutic drugs and methods. At present, the mechanism of the occurrence of neuropathic pain is mainly the reaction of the nerve inflammation and the central sensitization. Transformed growth factor (TGF-1) superfamily is a class of cytokines with multi-function and environment-dependent, the subfamily of which includes TGF-Fass, bone morphogenic proteins (BMPs), activins, growth and differentiation factors. GDFs et al., many of which, such as TGF-(1), have a neuroinflammatory inhibitory effect and can reduce the occurrence of neuropathic pain. Growth and differentiation factor 10 (GDF10) is one of the members of the BMPs subfamily, which plays an important role in the genesis and regeneration of the nerve. The changes of the spinal cord in the spinal cord and its role in the occurrence of neuropathic pain have not been reported. The activation of N-methyl-D-aspartate (NMDA) receptor plays an important role in the pathogenesis of neuropathic pain, and we further study whether the GDF10 is associated with the activation of the NMDA receptor, So as to understand the relevant mechanism of the GDF10 in the neuropathic pain. Methods: (1) The model of neuropathic pain in rats was established. This model was established using a method of ligation of the left L5 spinal nerve in male SD rats. (2) Intra-drug injection. The rats were injected with NMDA to produce neuropathic pain and the antagonist MK-801 of the NMDA receptor was injected into the rat with spinal nerve ligation. (3) Behavior detection. The rat paw threshold was measured with von Frey film to respond to changes in mechanical pain in the foot of the hindlimb. (4)Western Blot. The expression of GDF10 and the expression of GDF10 in the spinal cord of rats were detected by immunoblotting. The expression of GDF10 in the spinal cord and the expression of the dorsal horn GDF10 of the spinal cord in the spinal cord of the rats were detected by the injection of MK-801 in the spinal cord. (4) immunofluorescence. The expression of GDF10 and the expression of GDF10 in the spinal dorsal horn of spinal cord after spinal nerve ligation were detected by immunofluorescence, and the cellular localization of the GDF10 in the spinal cord was detected by immunofluorescence double-label. Main results:1. The threshold of the first day after the operation of the spinal nerve was decreased, and the difference of the point-to-point difference was statistically significant after 3 days (P <0.05). The decrease of the threshold of the 10th day was the most obvious, and the level of 21 d was in the same state, and the rats exhibited a significant increase in the mechanical pain. It was shown that the model of neuropathic pain was successfully established. The co-expression of the GDF10 with the neuronal marker NeuN was observed in the normal rat L5 spinal cord tissue without co-expression with the astrocyte marker GFAP or the microglial cell marker Iba-1. Western blot confirmed that the expression of the GDF10 protein in the spinal cord after the spinal nerve ligation was gradually decreased, the most significant in the 10th day, and the expression of the low-level expression in the 21 d was statistically significant compared with the sham-operated group (P0.05). The results showed that the expression of GDF10 in the dorsal horn of the spinal dorsal horn of the lateral spinal cord decreased gradually in the 3-d,10-day, and 21-day, and was consistent with the western blot. In the normal rat, NMDA was injected into the rat, and the threshold of the rat's paw was significantly lower than that of the normal saline group (P <0.05), and the expression of GDF10 in the dorsal horn of the spinal cord of the NMDA group was significantly lower in the spinal dorsal horn of the rats (P0.05). The expression of GDF10 in the dorsal horn neurons of the spinal cord was significantly reduced by immunofluorescent assay. The MK-801 was injected into the rat with spinal nerve ligation, which can significantly inhibit the reduction of the threshold of the contraction claw caused by the ligature of the spinal nerve. Compared with the saline group, the expression of MK-801 in the rat spinal cord was significantly inhibited by Western blot, and the expression of GDF10 in the spinal cord was significantly inhibited. Conclusion: (1) The rat's neuropathic pain model was successfully established with the left L5 spinal nerve ligation; (2) the spinal nerve ligation increased the mechanical pain of the rat and decreased the expression of the GDF10 in the dorsal horn of the spinal cord; and (3) the intraventricular injection of NMDA increased the mechanical pain of the rat. and the expression of the GDF10 in the dorsal horn of the spinal cord of the rat is reduced, the activation of the NMDA receptor in the spinal cord is related to the reduction of the expression of the GDF10, and (4) the activation of the NMDA receptor in the spinal cord of the rat spinal cord is inhibited by the intra-spinal injection of the MK-801, and the neuropathic pain can be reduced, And the expression of the GDF10 in the spinal cord can be reduced.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R402
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 劉國(guó)凱,黃宇光,羅愛(ài)倫;神經(jīng)病理性疼痛動(dòng)物模型及其評(píng)價(jià)[J];中國(guó)臨床藥理學(xué)與治療學(xué);2005年06期
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