本文選題：神經(jīng)病理性疼痛　切入點(diǎn)：膠質(zhì)細(xì)胞　出處：《南京大學(xué)》2014年博士論文　論文類型：學(xué)位論文

【摘要】：[目的] 在外周神經(jīng)損傷后,位于脊髓背角的感覺(jué)神經(jīng)元興奮性增強(qiáng),成為傳遞和調(diào)控痛覺(jué)的重要位點(diǎn)。而背角神經(jīng)元周圍的星形膠質(zhì)細(xì)胞在神經(jīng)元興奮性增強(qiáng),痛覺(jué)敏感傳遞,尤其是神經(jīng)病理性疼痛的維持中也起到重要作用。雖然已知星形膠質(zhì)細(xì)胞之間信號(hào)傳遞的一個(gè)重要途徑是縫隙連接,但脊髓膠質(zhì)細(xì)胞間縫隙連接在維持神經(jīng)病理性疼痛中的作用并不清楚,尤其是特異性表達(dá)在星形膠質(zhì)細(xì)胞之間的縫隙連接蛋白43(Connexin43,Cx43)在神經(jīng)病理性疼痛中的作用。甘珀酸(Carbenoxolone, CBX)是非特異性縫隙連接阻滯劑,可非特異性阻滯縫隙連接的功能。利用小干擾RNA (Small interfering RNA, siRNA)技術(shù)可以特異性降低星形膠質(zhì)細(xì)胞間縫隙連接蛋白Cx43的表達(dá)。本研究旨在觀察用藥物(如甘珀酸)阻滯縫隙連接功能和siRNA下調(diào)脊髓Cx43蛋白表達(dá)能否抑制神經(jīng)病理性疼痛或降低脊髓背角廣動(dòng)力范圍神經(jīng)元的活性。 [材料與方法] 1.建立大鼠脊神經(jīng)結(jié)扎(Spinal nerve ligation, SNL)神經(jīng)病理性疼痛模型后,使用甘珀酸(CBX)鞘內(nèi)注射。首先在大鼠持續(xù)異氟烷吸入麻醉下,單次注射不同劑量的CBX,采用清醒動(dòng)物行為學(xué)觀察大鼠機(jī)械痛敏和熱痛敏的變化。其次,進(jìn)一步觀察和對(duì)比相同劑量的CBX、甘草酸(Glycyrrhizic acid, GCA,是CBX的類似物,但不阻滯縫隙連接)、甲氟喹(Mefloquine, MFQ,是特異性神經(jīng)元縫隙連接阻滯劑)對(duì)大鼠機(jī)械痛敏的不同影響。最后,檢測(cè)連續(xù)注射CBX對(duì)大鼠機(jī)械痛敏是否有長(zhǎng)期影響。 2.建立大鼠SNL神經(jīng)病理性疼痛模型后,動(dòng)物在麻醉狀態(tài)下在體記錄脊髓背角廣動(dòng)力范圍(Wide dynamic range, WDR)神經(jīng)元的活性,并觀察記錄脊髓表面給予CBX藥物處理后,WDR神經(jīng)元對(duì)外周機(jī)械性刺激反應(yīng)的變化。此外,在WDR神經(jīng)元上記錄反復(fù)電刺激外周感受野所誘發(fā)的C纖維傳導(dǎo)的動(dòng)作電位增強(qiáng)的變化,并做出windup曲線。觀察脊髓表面應(yīng)用CBX對(duì)WDR神經(jīng)元C-componer動(dòng)作電位windup的影響。 3.免疫熒光方法檢測(cè)和比較假手術(shù)組與SNL組腰段脊髓膠質(zhì)纖維酸性蛋白(Glial fibrillary acidic protein, GFAP,星形膠質(zhì)細(xì)胞標(biāo)志)、Cx43和OX42(小膠質(zhì)細(xì)胞標(biāo)志)蛋白的表達(dá)分布。 4.構(gòu)建Cx43siRNA和Control siRNA,將大鼠分為假手術(shù)組、Cx43siRNA組和Control siRNA組。觀察鞘內(nèi)反復(fù)注射siRN垢,SNL大鼠機(jī)械痛敏的變化。同時(shí)采用Western blot檢測(cè)雙側(cè)SNL大鼠腰段脊髓Cx43, Cx36,和GFAP蛋白表達(dá)水平的變化。 [結(jié)果] 1.在大鼠SNL術(shù)后2-3周,鞘內(nèi)注射CBX (0.1μig、0.5μg、5μg、25μg、50μg,10μl注射量)可劑量依賴性地抑制SNL誘導(dǎo)的機(jī)械痛敏。鞘內(nèi)注射CBX5μg也明顯抑制了大鼠熱痛敏。與對(duì)照組相比,在相同劑量(0.81nmol、8.1nmol、81nmol)下,CBX明顯抑制了大鼠機(jī)械痛敏,而GCA和MFQ無(wú)明顯鎮(zhèn)痛作用。雖然GCA在最大劑量81nmol下似乎對(duì)機(jī)械痛敏有抑制作用,但大鼠表現(xiàn)出明顯藥物副作用。連續(xù)注射CBX(25μg/天,連續(xù)3天)過(guò)程中,對(duì)大鼠機(jī)械痛敏的抑制作用表現(xiàn)出了明顯的累加效應(yīng),即在第2天和第3天給藥前所測(cè)得的機(jī)械縮足閾值均明顯比第1天給藥前高。 2.脊髓表面應(yīng)用CBX可以明顯減少SNL大鼠WDR神經(jīng)元對(duì)遞增機(jī)械刺激的反應(yīng),并明顯抑制WDR神經(jīng)元所表現(xiàn)的對(duì)電刺激誘導(dǎo)C-component的windup現(xiàn)象。 3.與假手術(shù)組相比,免疫熒光實(shí)驗(yàn)顯示大鼠腰段脊髓背角星形膠質(zhì)細(xì)胞(GFAP)和小膠質(zhì)細(xì)胞(OX42)在SNL后均明顯激活；脊髓背角Cx43表達(dá)在SNL后明顯減少。星形膠質(zhì)細(xì)胞標(biāo)志GFAP與Cx43的表達(dá)有共同定位,而小膠質(zhì)細(xì)胞標(biāo)志OX42與Cx43無(wú)明顯共同定位。 4.在大鼠SNL術(shù)后14-17天,鞘內(nèi)注射Cx43siRNA明顯抑制了SNL誘導(dǎo)的大鼠機(jī)械痛敏,并下調(diào)了腰段脊髓Cx43的表達(dá),而對(duì)GFAP表達(dá)無(wú)明顯影響。 [結(jié)論] 1.鞘內(nèi)注射CBX可以劑量依賴性的抑制SNL誘導(dǎo)的機(jī)械性痛敏,其機(jī)制可能通過(guò)抑制膠質(zhì)細(xì)胞間的縫隙連接功能。 2.脊髓表面應(yīng)用CBX可以抑制WDR神經(jīng)元對(duì)遞增機(jī)械刺激的反應(yīng),也可以減少電刺激誘導(dǎo)WDR神經(jīng)元C-component的windup,表明CBX對(duì)機(jī)械性痛敏的抑制作用可能由于其對(duì)脊髓WDR神經(jīng)元興奮性的抑制作用。 3.神經(jīng)損傷后,位于脊髓的星形膠質(zhì)細(xì)胞和小膠質(zhì)細(xì)胞均被激活,但Cx43表達(dá)下降。而SNL術(shù)后14-17天,采用鞘內(nèi)注射Cx43siRN進(jìn)一步下調(diào)Cx43表達(dá)后,機(jī)械性痛敏明顯減輕。這些結(jié)果表明神經(jīng)損傷后,雖然Cx43蛋白表達(dá)量減少,但縫隙連接的功能或通透性可能增強(qiáng)了。 4.綜上所述,本研究發(fā)現(xiàn)脊髓膠質(zhì)細(xì)胞之間縫隙連接的功能增強(qiáng),尤其是Cx43所構(gòu)成的縫隙連接,可能在維持SNL誘導(dǎo)的神經(jīng)病理性疼痛中起到重要作用。這些新發(fā)現(xiàn)為神經(jīng)病理性疼痛的機(jī)制研究提供了新線索,也為臨床治療神經(jīng)病理性疼痛提供了重要靶點(diǎn)。
[Abstract]:[Objective]
In peripheral nerve injury, located in the dorsal horn of the spinal cord excitability of sensory neurons increased, become an important site for transmission and modulation of pain. And around the dorsal horn neurons astrocytes increase in neuronal excitability, pain sensitive transfer, especially the maintenance of neuropathic pain also play an important role. Although an important way the signal transduction between known astrocytes are gap junctions, but the spinal glial cell gap junctions is not clear in the maintenance of neuropathic pain in the role, especially the specific expression of gap between glial cell connexin 43 (Connexin43, Cx43) in neuropathic pain. The effect of carbenoxolone (Carbenoxolone, CBX) non specific gap junction blockers can block the non-specific gap junction function. Using small interfering RNA (siRNA Small interfering RNA) technology In order to reduce the specific expression of astrocyte gap junction protein Cx43. The purpose of this study was to observe the drug (such as carbenoxolone) block gap junction function and down-regulation of siRNA expression of Cx43 protein in the spinal cord can inhibit neuropathic pain or reduce spinal dorsal horn wide dynamic range of neuronal activity.
[materials and methods]
Established in 1. rats with spinal nerve ligation (Spinal nerve, ligation, SNL) model of neuropathic pain, the use of carbenoxolone (CBX) intrathecal injection. First in rats anesthetized with isoflurane, a single injection of different doses of CBX, the conscious animal behavior changes of rats were observed mechanical and thermal hyperalgesia. Secondly, further observation and comparison of the same dose of CBX, glycyrrhizic acid (Glycyrrhizic acid, GCA CBX, is similar, but not to block the gap junction), mefloquine (Mefloquine, MFQ, is a specific neuronal gap junction blocker) effect on mechanical hyperalgesia in rats. Finally, whether there is long term effects of detection of continuous injection of CBX on mechanical pain rat sensitivity.
2. establish the SNL rat model of neuropathic pain after animal under anesthesia in vivo recording of spinal dorsal horn wide dynamic range (Wide dynamic, range, WDR) neurons, and observe and record the spinal cord surface given CBX after treatment, changes of WDR neurons to the peripheral mechanical stimulus response. In addition, the WDR neurons recorded repeated electrical stimulation of action potential changes of peripheral receptive fields induced by C fiber conduction enhancement, and make windup curve. To observe the effect of application of CBX on the WDR C-componer spinal cord neurons action potential windup.
3. immunofluorescence method was used to detect and compare the expression and distribution of Glial fibrillary acidic protein (GFAP), astrocyte markers, Cx43 and OX42 (microglia markers) protein in lumbar spinal cord of sham operation group and SNL group.
4. construction of Cx43siRNA and Control siRNA, the rats were divided into sham operation group, Cx43siRNA group and Control siRNA group. Observation of intrathecal repeated injections of siRN scale, SNL changes the mechanical hyperalgesia in rats. At the same time using Western blot to detect rat bilateral SNL lumbar spinal cord Cx43, Cx36, and the expression of GFAP protein.
[results]
In 1. rats 2-3 weeks after SNL, intrathecal injection of CBX (0.1 Ig, 0.5 g, 5 g, 25 g, 50 g, 10 L injection volume) dose dependently inhibited SNL induced mechanical hyperalgesia. Intrathecal injection of CBX5 g also significantly inhibited the rats of thermal hyperalgesia. Compared with the control group at the same dose (0.81nmol, 8.1nmol, 81nmol), the mechanical hyperalgesia in rats was inhibited by CBX, GCA and MFQ had no significant analgesic effect. Although the GCA in the maximum dose of 81nmol seems to have inhibitory effect on mechanical hyperalgesia, but the rats showed obvious the side effects of the drug. The continuous injection of CBX (25 g/ day for 3 consecutive days) in the process of mechanical hyperalgesia in rats of inhibition showed significant additive effect, namely in the second and third days before the administration of the measured mechanical withdrawal thresholds were significantly more than first days before administration high.
2., the application of CBX on the spinal cord surface can significantly reduce the response of WDR neurons to incremental mechanical stimulation in SNL rats, and significantly inhibit the windup phenomenon of WDR induced by electrical stimulation.
3. compared with sham operation group, immunofluorescence experiments showed that rat spinal cord dorsal horn astrocytes (GFAP) and microglia (OX42) after SNL were significantly activated; spinal Cx43 expression decreased obviously after SNL. Astrocyte marker expression of GFAP and Cx43 are Co located, and small glial cell marker OX42 and Cx43 were Co located.
4., on the 14-17 day after SNL, intrathecal injection of Cx43siRNA significantly inhibited SNL induced mechanical hyperalgesia in rats, and down regulated the expression of Cx43 in lumbar spinal cord, but had no significant effect on GFAP expression.
[Conclusion]
The 1. intrathecal injection of CBX can inhibit SNL induced mechanical pain sensitization in a dose-dependent manner, and its mechanism may be mediated by the inhibition of gap junction function between glial cells.
2., the application of CBX on the spinal cord can inhibit the response of WDR neurons to incremental mechanical stimulation, and also reduce the windup of WDR neurons induced by electrical stimulation, indicating that the inhibitory effect of CBX on the mechanical pain sensitivity may be due to its inhibitory effect on the excitability of the spinal WDR neurons.
3. after nerve injury in spinal cord astrocytes and microglia were activated, but decrease the expression of Cx43 and SNL. After 14-17 days, the intrathecal injection of Cx43siRN by down-regulation of Cx43 expression after mechanical hyperalgesia significantly reduced. These results indicate that after nerve injury, although the expression of Cx43 protein decreased. But the gap junction function or permeability may enhance.
4. in summary, this study found that the gap junction function between spinal cord glial cells increased, especially the gap formed by the Cx43 connection, may play an important role in the maintenance of neuropathic pain induced by SNL. These new findings for the mechanism study of neuropathic pain and provided new clues, but also provides an important target for neuropathic pain in clinic the treatment.

【學(xué)位授予單位】：南京大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741;R614

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5 唐聰;急性脊髓損傷后pERK、PCNA、GFAP的表達(dá)和意義以及U0126對(duì)其表達(dá)的影響[D];福建醫(yī)科大學(xué);2011年

6 朱慧;轉(zhuǎn)錄因子Pax3對(duì)膠質(zhì)纖維酸性蛋白基因的負(fù)性轉(zhuǎn)錄調(diào)控研究[D];南通大學(xué);2010年

7 郭亮;新型番荔枝酰胺衍生物FLZ對(duì)去血清培養(yǎng)損傷星形膠質(zhì)細(xì)胞的保護(hù)作用[D];北京協(xié)和醫(yī)學(xué)院;2011年

8 謝中;促紅細(xì)胞生成素聯(lián)合骨髓間充質(zhì)干細(xì)胞治療脊髓損傷的實(shí)驗(yàn)研究[D];中南大學(xué);2011年

9 郭志慧;星形膠質(zhì)細(xì)胞活化與表皮生長(zhǎng)因子受體表達(dá)的相關(guān)性研究[D];南華大學(xué);2011年

10 張程;輸合配穴針刺法對(duì)腦癱大鼠神經(jīng)行為能力發(fā)育及腦內(nèi)GFAP含量的影響[D];遼寧中醫(yī)藥大學(xué);2011年

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