發(fā)布時間：2018-05-20 10:12

  本文選題：A型肉毒毒素 + 三叉神經痛��； 參考：《鄭州大學》2016年博士論文

【摘要】：三叉神經痛是臨床上最為痛苦和常見的神經病理性疼痛之一。臨床表現(xiàn)為局限于三叉神經分布區(qū)域的單側短暫性電擊樣或刀割樣劇痛。它的首選治療為口服藥物治療,在口服藥物治療無效的情況下可以考慮手術治療,然而有不少患者經過多種治療后效果仍不理想。A型肉毒毒素是革蘭氏陽性厭氧芽孢肉毒桿菌釋放的一種外毒素,廣泛應用于肌張力障礙治療和美容領域。近年來,它在疼痛治療方面的研究越來越多,2012年我們改進了A型肉毒毒素治療三叉神經痛的技術路線,在國際上首次用隨機、雙盲、安慰劑對照的試驗方法證實了A型肉毒毒素能夠長期有效的緩解三叉神經痛的疼痛且不良反應輕微。隨后的多項臨床研究也得出了相似的結論。但是A型肉毒毒素治療三叉神經痛的機制仍不清楚。為此,我們建立大鼠眶下神經慢性縮窄環(huán)術三叉神經痛模型,探討A型肉毒毒素對三叉神經痛的治療作用、作用部位和作用機制。為A型肉毒毒素治療三叉神經痛和其他疼痛性疾病提供基礎理論依據(jù)。第一部分大鼠眶下神經慢性縮窄環(huán)術建立三叉神經痛模型的行為學和組織學研究目的建立大鼠眶下神經慢性縮窄環(huán)術三叉神經痛模型,模擬經典三叉神經痛,觀察術后大鼠的疼痛行為學和眶下神經組織學改變。方法實驗組大鼠經口建立眶下神經慢性縮窄環(huán)術三叉神經痛模型,對照組只暴露眶下神經而不結扎。用Von Frey filaments對兩組術前和術后每2天大鼠的疼痛學行為進行評價;采用HE染色和MBP免疫組織化學染色對術前和術后14天眶下神經組織學改變進行評價。結果實驗組術后第4天出現(xiàn)一明顯的不應期,同側面部疼痛閾值顯著上升,第6天疼痛閾值達到高峰,為24.6±8.6g,與術前相比有統(tǒng)計學差異(P0.05)。隨后疼痛閾值急劇下降,在術后第14天達到5.5±2.3g(P0.05),這一趨勢至少持續(xù)至術后34天。對照組術前和術后各時間點疼痛閾值相比無統(tǒng)計學差異(P0.05)。實驗組和對照組術前疼痛閾值分別為13.5±3.6g和12.6±5.5g,兩者相比無統(tǒng)計學差異(P0.05)。兩組間疼痛閾值在術后第10天分別為5.5±3.2g和13.6±4.8g,組間比較有統(tǒng)計學差異(P0.05),這一差異至少持續(xù)至術后第34天。HE染色和MBP免疫組織化學染色表明實驗組術后第14天眶下神經存在明顯的脫髓鞘改變,而對照組與術前相比無顯著變化。結論大鼠眶下神經慢性縮窄環(huán)術三叉神經痛模型符合經典三叉神經痛的臨床特點和組織學改變。該模型簡單易行,我們可以利用該模型探討三叉神經痛的發(fā)病機制,研究新的治療方法。第二部分A型肉毒毒素對大鼠三叉神經痛模型的抗傷害感受作用研究目的用A型肉毒毒素對三叉神經痛模型進行干預,觀察大鼠疼痛行為學的改變。方法大鼠眶下神經慢性縮窄環(huán)術建立三叉神經痛模型術后第14天,實驗組在手術同側面部須墊部皮下注射A型肉毒毒素(3U/Kg和10U/Kg),對照組在同樣部位注射等量的生理鹽水。用Von Frey filaments對各組術前和術后不同時間點的疼痛學行為進行評價。結果A型肉毒毒素治療后第4天(術后第18天),3U組和10U組痛覺閾值均顯著升高,與對照組相比有統(tǒng)計學差異(P0.05),這一差異至少持續(xù)至治療后20天(術后34天)。10U組同一時間點疼痛閾值高于3U組,但兩組間痛覺閾值對比無統(tǒng)計學差異(P0.05)。結論A型肉毒毒素須墊部皮下注射可以顯著升高大鼠三叉神經痛模型的疼痛閾值,提高其抗傷害感受能力。第三部分A型肉毒毒素治療大鼠三叉神經痛的作用部位研究目的在前兩部分研究的基礎上,通過對A型肉毒毒素的特異性標記物c SNAP-25的檢測,研究A型肉毒毒素治療三叉神經痛的作用部位,探討軸突運輸在A型肉毒毒素治療三叉神經痛中的作用。方法大鼠眶下神經慢性縮窄環(huán)術后13.5天,實驗組在三叉神經節(jié)內注射軸突運輸抑制劑秋水仙堿,對照組注射等量生理鹽水。術后14天在手術同側面部須墊部皮下注射A型肉毒毒素(10U/Kg)或生理鹽水溶液。觀察不同時間點各組大鼠對疼痛刺激的變化,探討軸突運輸在A型肉毒毒素治療三叉神經痛中的作用。用Western blot和免疫熒光對A型肉毒毒素的特異性標志物c SNAP-25進行檢測,觀察A型肉毒毒素在三叉神經痛模型大鼠的作用部位。結果實驗組用秋水仙堿阻斷軸突運輸降低了A型肉毒毒素的抗傷害感受作用,A型肉毒毒素治療后第4天實驗組疼痛閾值顯著低于對照組,差異有統(tǒng)計學意義(P0.05),這一差異持續(xù)至實驗結束。A型肉毒毒素治療后7天,通過western-blot和免疫熒光檢測到大鼠三叉神經脊束核尾側亞核c SNAP-25水平明顯增高。三叉神經節(jié)注射秋水仙堿阻斷軸突運輸后,可以阻斷其增高。Western blot表明三叉神經節(jié)注射秋水仙堿阻斷軸突運輸后三叉神經脊束核尾側亞核c SNAP-25較對照組顯著降低,差異有統(tǒng)計學意義(P0.05)。結論秋水仙堿阻斷軸突運輸后使A型肉毒毒素的抗傷害感受作用顯著減弱。A型肉毒毒素通過軸突運輸至三叉神經脊束核尾側亞核發(fā)揮作用。第四部分TRPs在A型肉毒毒素治療大鼠三叉神經痛中的作用機制研究目的探討痛覺相關TRPs(TRPA1、TRPV1、TRPV2及TRPM8)在A型肉毒毒素對大鼠三叉神經痛模型發(fā)揮抗傷害感受作用的機制。方法TRPA1、TRPV1、TRPV2和TRPM8是TRPs家族中可能與疼痛相關的離子通道,為了進一步明確A型肉毒毒素對三叉神經痛抗傷害感受的機制,采用Western blot對三叉神經痛模型三叉神經脊束核尾側亞核不同時間點的TRPA1、TRPV1、TRPV2和TRPM8蛋白表達進行檢測,觀察這些蛋白在三叉神經痛模型中的動態(tài)變化。同時用Western blot檢測A型肉毒毒素同側面部須墊部皮下注射(3U/Kg和10U/Kg)對三叉神經痛模型三叉神經脊束核尾側亞核中TRPA1、TRPV1、TRPV2和TRPM8蛋白表達的影響。結果Western blot表明三叉神經脊束核尾側亞核中TRPA1和TRPV1的表達水平在眶下神經慢性縮窄環(huán)術后第14天明顯增高(P0.05),在術后第28天仍顯著增高(P0.05)。TRPV2在術后第7天表達明顯增高(P0.05),術后第28天仍然持續(xù)增高(P0.05)。TRPM8在術后第7天開始增高,在術后第14天達到高峰,術后第28天與對照組相比仍有統(tǒng)計學差異(P0.05)。A型肉毒毒素治療后第14天,Western blot表明大鼠三叉神經脊束核尾側亞核中TRPA1和TRPV1的表達較對照組顯著降低(P0.05),A型肉毒毒素降低TRPA1和TRPV1的表達有劑量依賴的效果,10U組的TRPA1和TRPV1的表達與3U相比有統(tǒng)計學差異(P0.05)。A型肉毒毒素(10U/Kg)治療后第14天顯著降低了TRPV2的表達(P0.05),3U組TRPV2的表達水平與對照組無統(tǒng)計學差異(P0.05)。與對照組相比,A型肉毒毒素(3U/Kg和10U/Kg)不影響TRPM8的表達(P0.05)。結論大鼠眶下神經慢性縮窄環(huán)術后三叉神經脊束核尾側亞核TRPA1、TRPV1、TRPV2和TRPM8的表達顯著升高,且有一動態(tài)變化過程。A型肉毒毒素治療三叉神經痛的機制可能是抑制三叉神經脊束核尾側亞核中TRPA1、TRPV1和TRPV2的高表達,降低中樞敏化,從而發(fā)揮抗傷害感受作用。
[Abstract]:Trigeminal neuralgia is one of the most painful and common neuropathic pain in the clinic. Clinical manifestations are unilateral transient electric shock or knife cut pain limited to the trigeminal distribution area. Its first choice is oral medication, and surgical treatment can be considered in the case of ineffective oral medication. However, there are many patients. After a variety of treatments, the effect of botulinum toxin type.A is a kind of exotoxin released by Gram positive anaerobic bacillus botulinum. It is widely used in the field of dystonia and Cosmetology. In recent years, more and more studies have been made on the treatment of pain. In 2012, we changed the technology of botulinum toxin type A to treat trigeminal neuralgia. Route, for the first time, a randomized, double blind, placebo-controlled trial for the first time confirmed that botulinum A can effectively alleviate the pain and slight adverse reactions of trigeminal neuralgia for a long time. Several subsequent clinical studies have drawn similar conclusions. However, the mechanism of A botulinum toxin for the treatment of trigeminal neuralgia is still unclear. We established a rat model of trigeminal neuralgia with chronic suborbital coarctation of the suborbital nerve to explore the therapeutic effect, location and mechanism of botulinum toxin A on trigeminal neuralgia. It provides the basis for the treatment of trigeminal neuralgia and other painful diseases by botulinum toxin type A in the first part of the rats. The behavior and histology of the trigeminal neuralgia model aim to establish a rat model of trigeminal neuralgia with chronic constrictive ring of the suborbital nerve, to simulate the classical trigeminal neuralgia, to observe the pain behavior and the histological changes of the suborbital nerve in the rats after the operation. Von Frey filaments was used to evaluate the pain behavior of the two groups of rats before and after the operation. HE staining and MBP immunohistochemical staining were used to evaluate the histological changes of the suborbital nerve 14 days before and after the operation. The results showed an obvious refractory period in the experimental group fourth days after operation. The threshold of pain in the same side was increased significantly, the sixth day pain threshold reached a peak of 24.6 + 8.6g, which was significantly different from that before the operation (P0.05). Then the threshold of pain decreased sharply and reached 5.5 + 2.3g (P0.05) fourteenth days after the operation. This trend was at least 34 days after the operation. The pain threshold of the group before and after the operation was not statistically significant. The pain threshold of the experimental group and the control group was 13.5 + 3.6g and 12.6 + 5.5G, respectively. There was no statistical difference between the two groups (P0.05). The pain threshold between the two groups was 5.5 + 3.2g and 13.6 + 4.8g after the operation, and there was a statistically significant difference between the groups (P0.05). The difference lasted to at least thirty-fourth days after the operation,.HE and MBP immunity. Histochemical staining showed that there were obvious demyelination changes in the suborbital nerve in the experimental group fourteenth days after operation, but there was no significant change in the control group. Conclusion the model of trigeminal neuralgia for the chronic suborbital constriction ring operation of the rat's suborbital nerve conforms to the clinical characteristics and histological changes of the classical trigeminal neuralgia. This model studies the pathogenesis of trigeminal neuralgia and studies new treatment methods. Second part of the anti nociceptive effect of botulinum toxin type A on the trigeminal neuralgia model in rats. Aim to intervene the trigeminal neuralgia model with A botulinum toxin and observe the change of pain behavior in rats. Method the chronic constriction ring of the suborbital nerve in rats. Fourteenth days after the operation of the trigeminal neuralgia model, the experimental group was subcutaneously injected with botulinum toxin A (3U/Kg and 10U/Kg) and the control group was injected with equal amount of physiological saline in the same part. Von Frey filaments was used to evaluate the pain and pain behavior of each group before and after the operation. Results the treatment of A botulinum toxin was found. Fourth days after treatment (eighteenth days after operation), the pain threshold of both group 3U and group 10U was significantly higher than that of the control group (P0.05). This difference lasted for at least 20 days after the treatment (34 days after the operation) and the pain threshold of the.10U group was higher than that of the 3U group, but there was no statistical difference between the two groups (P0.05). Conclusion A botulinum toxin must be found. Subcutaneous injection can significantly increase the pain threshold of the rat trigeminal neuralgia model and improve its anti nociceptive ability. Third Research on the role of botulinum toxin type A in the treatment of trigeminal neuralgia in rats, based on the study of the first two parts and the detection of C SNAP-25, a specific marker for botulinum toxin type A, study A The role of botulinum toxin in the treatment of trigeminal neuralgia and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A. Methods 13.5 days after the operation of the chronic constrictive ring of the suborbital nerve in rats, the experimental group injected the axon transport inhibitor colchicine in the trigeminal ganglion and injected the same amount of normal saline to the group for 14 days after operation. A botulinum toxin (10U/Kg) or saline solution was injected subcutaneously on the same side of the facial facial pad. The changes of pain stimulation in each group of rats at different time points were observed and the role of axon transport in the treatment of trigeminal neuralgia by botulinum toxin type A was investigated. C SNAP-25, a specific marker for botulinum toxin type A, was examined by Western blot and immunofluorescence. Test, observe the role of botulinum toxin type A in the rat model of trigeminal neuralgia. Results the experimental group used colchicine blocking axonal transportation to reduce the anti nociceptive effect of type A botulinum toxin. The pain threshold of the experimental botulinum toxin type A was significantly lower than that of the control group at fourth days after the treatment, and the difference was statistically significant (P0.05). The difference continued to be sustained. 7 days after the treatment of botulinum toxin type.A, the level of C SNAP-25 in the nucleus caudal subnucleus of the trigeminal nucleus of the rat was significantly increased by Western-blot and immunofluorescence. After blocking the axon transport by injection of colchicine, the trigeminal ganglion could block the.Western blot Biao Mingsan ganglion injection of colchicine to block axonal transportation The C SNAP-25 in the posterior trigeminal nucleus caudal subnucleus was significantly lower than that in the control group (P0.05). Conclusion colchicine inhibited the anti nociceptive effect of A type botulinum toxin by colchicine blocking axon transport and the effect of.A type botulinum toxin on the nucleus caudal subnucleus of the trigeminal nucleus through axon. Fourth part TRPs was observed. Study on the mechanism of A botulinum toxin in the treatment of trigeminal neuralgia in rats. Objective to explore the mechanism of pain related TRPs (TRPA1, TRPV1, TRPV2 and TRPM8) in the anti nociceptive effect of botulinum toxin type A on rat trigeminal neuralgia model. Methods TRPA1, TRPV1, TRPV2 and TRPM8 are the ion channels associated with pain in the TRPs family, for the purpose of The mechanism of the anti nociceptive sensitivity of botulinum toxin A to trigeminal neuralgia was further clarified, and the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of trigeminal neuralgia model was detected by Western blot, and the dynamic changes of these proteins in the trigeminal neuralgia model were observed. At the same time, Western blot was used. The effects of subcutaneous injection (3U/Kg and 10U/Kg) on the expression of TRPA1, TRPV1, TRPV2 and TRPM8 in the trigeminal nucleus caudal nucleus of the trigeminal neuralgia model of botulinum toxin type A botulinum toxin (3U/Kg and 10U/Kg). Results Western blot showed that the expression level of TRPA1 and TRPV1 in the caudal nucleus of the trigeminal nucleus of the spinal trigeminal tract was performed by chronic constrictive ring of the orbital nerve After fourteenth days (P0.05), the expression increased significantly (P0.05) (P0.05) at the seventh day after operation (P0.05), and continued to increase on the twenty-eighth day after operation (P0.05).TRPM8 began to increase on the seventh day after the operation, and reached the peak at the fourteenth day after the operation, and the twenty-eighth day after the operation was still statistically different (P0.05).A type botulinum toxin compared to the control group (P0.05). On the fourteenth day after treatment, Western blot showed that the expression of TRPA1 and TRPV1 in the nucleus caudal nucleus of the trigeminal nucleus of the rat was significantly lower than that of the control group (P0.05). The expression of TRPA1 and TRPV1 of the A type botulinum toxin was dose-dependent. The expression of TRPA1 and TRPV1 in the 10U group was statistically different from that of 3U. The expression of TRPV2 was significantly reduced in the last fourteenth days (P0.05), and the expression level of TRPV2 in group 3U was not significantly different from that in the control group (P0.05). Compared with the control group, A type botulinum toxin (3U/Kg and 10U/Kg) did not affect the expression of TRPM8 (P0.05). Conclusion the caudal nucleus of the trigeminal nucleus of the trigeminal nucleus after the operation of the suborbital nerve of the rat is TRPA1, TRPV1, and The mechanism of botulinum toxin type.A in the treatment of trigeminal neuralgia may be to inhibit the high expression of TRPA1, TRPV1 and TRPV2 in the caudal nucleus of the trigeminal nucleus, and reduce the central sensitization, thus exerting anti nociceptive effect.
【學位授予單位】：鄭州大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R745.11
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本文編號：1914210