本文關(guān)鍵詞： A型肉毒毒素 突觸體相關(guān)蛋白SNAP-25 逆向軸漿運輸 中樞效應(yīng)　出處：《浙江大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景：A型肉毒毒素(Botulinum toxin type A,BTX-A)是革蘭陽性厭氧產(chǎn)孢肉毒桿菌產(chǎn)生的一種神經(jīng)外毒素,可特異性裂解運動終板中運動神經(jīng)元突觸前膜內(nèi)突觸體相關(guān)蛋白SNAP-25(synaptosomal-associated protein of25-kDa)使得遞質(zhì)囊泡無法與突觸前膜融合,從而抑制神經(jīng)末梢乙酰膽堿的釋放而松弛肌纖維,因而一直被用于眼肌痙攣、面肌痙攣等肌張力障礙性疾病的治療。臨床應(yīng)用也日漸增多。既往普遍認(rèn)為臨床局部肌肉注射BTX-A,其作用只局限于注射點以及附近的突觸,但近年來有越來越多的證據(jù)提示BTX-A的局部注射后,不僅在注射部位發(fā)揮作用,并且對遠(yuǎn)隔的神經(jīng)肌肉接頭也有作用,甚至還能通過逆向軸漿運輸?shù)姆绞竭M(jìn)入高一級神經(jīng)系統(tǒng)。這一現(xiàn)象打破了人們一直以來教條式的認(rèn)識。現(xiàn)今BTX-A的中樞效應(yīng)已被逐步揭示,但對其機制的研究仍然十分匱乏。 研究目的：外周肌肉注射BTX-A并檢測BTX-A局部注射后逆向軸漿運輸?shù)街袠械那闆r,以及遠(yuǎn)隔對側(cè)神經(jīng)肌肉接頭的播散情況,闡明BTX-A產(chǎn)生中樞效應(yīng)的途徑,將為應(yīng)用BTX-A后出現(xiàn)的中樞效應(yīng)給出合理解釋,從而能為臨床注射BTX-A后可能出現(xiàn)的中樞效應(yīng)提供更多有用的信息。 研究方法： 1)對成年小鼠的單側(cè)腓腸肌注射,低于臨床應(yīng)用劑量(1U/kg)、臨床應(yīng)用劑量(5U/kg)和小鼠耐受的最大安全劑量(30U/kg)三種不同劑量的BTX-A后,不同時間點通過免疫印跡檢測BTX-A水解底物cleaved SNAP-25(cl.SNAP-25),分析BTX-A局部肌肉注射后播散并且影響的范圍。BTX-A在動物體內(nèi)的播散和作用特點。 2)檢測支配注射肌肉的神經(jīng)和相應(yīng)的高一級運動神經(jīng)元內(nèi)的cl.SNAP-25,從而了解是否真的存在BTX-A逆向軸漿運輸?shù)街袠械默F(xiàn)象。 結(jié)果： 1)單側(cè)注射三個不同梯度的劑量后,均能在對側(cè)肌肉中發(fā)現(xiàn)cl.SNAP-25的存在。 2)單側(cè)注射最大耐受安全劑量后,均不能在支配該肌肉的神經(jīng)以及脊髓運動區(qū)發(fā)現(xiàn)cl.SNAP-25的存在。 3)不同注射劑量下,機體對BTX-A削減功能性的SNAP-25進(jìn)行不同程度的代償。 結(jié)論： 1)即使是低于臨床應(yīng)用劑量的BTX-A可通過血液循環(huán)達(dá)到對側(cè)非注射肌肉,并水解神經(jīng)肌肉接頭內(nèi)的SNAP-25。 2)即使是應(yīng)用了小鼠最大耐受安全劑量的BTX-A后,在中樞神經(jīng)系統(tǒng)內(nèi),仍然不能檢測到cl.SNAP-25,說明其逆向軸漿運輸?shù)默F(xiàn)象極其微弱或并不存在。 3)機體在接受BTX-A注射后,對注射側(cè)肌肉的神經(jīng)及接頭和支配該肌肉的神經(jīng)內(nèi)的SNAP-25進(jìn)行代償補充。
[Abstract]:Background Botulinum toxin type (BTX-A) is a neurotoxin produced by Gram-positive Botulinum Botulinum type. The synaptosome associated protein (SNAP-25(synaptosomal-associated protein of 25-kDa) in motor neuron presynaptic membrane in motor terminal plate can specifically dissociate the neurotransmitter vesicle from fusion with presynaptic membrane, thus inhibiting the release of acetylcholine from nerve endings and relaxing muscle fibers. Therefore, it has been used in the treatment of ocular spasm, hemifacial spasm and other muscular dystonia diseases. Clinical application is also increasing. It was generally thought that the clinical local intramuscular injection of BTX-A was limited to the injection point and the nearby synapses. However, there is more and more evidence in recent years that the local injection of BTX-A not only works at the injection site, but also on the distant neuromuscular junction. It can even enter the higher nervous system by reverse axonal transport. This phenomenon has broken the dogmatic understanding. Now the central effect of BTX-A has been gradually revealed, but the study of its mechanism is still very scarce. Objective: to investigate the mechanism of central effect of BTX-A by intramuscular injection of BTX-A and the transmission of contralateral neuromuscular junctions to the center after local injection of BTX-A. It will provide a reasonable explanation for the central effect after the application of BTX-A, which can provide more useful information for the possible central effect after BTX-A injection. Research methods:. 1) after unilateral gastrocnemius injection in adult mice, the dose of BTX-A was lower than that of clinical dose of 1 U / kg, clinical dose of 5 U / kg and maximum safe dose of 30 U / kg). BTX-A hydrolyzed substrate cleaved SNAP-25 / cl.SNAP-25 was detected by Western blotting at different time points. The dissemination and action characteristics of BTX-A were analyzed after local intramuscular injection of BTX-A. 2) Detection of cl.SNAP-25 in the nerves innervating the injected muscles and the corresponding higher-level motor neurons to determine whether there is a real phenomenon of BTX-A reverse axonal transport to the central nervous system. Results:. 1) the presence of cl.SNAP-25 was found in contralateral muscles after unilateral injection of three different gradient doses. 2) after unilateral injection of the maximum tolerated safe dose, the presence of cl.SNAP-25 could not be found in the nerve and spinal motor area innervating the muscle. 3) at different injection doses, the BTX-A reduced functional SNAP-25 was compensated to varying degrees. Conclusion:. 1) even if the dose of BTX-A is lower than the clinical dose, it can reach the contralateral non-injected muscle through blood circulation and hydrolyze SNAP-25 in the neuromuscular junction. 2) even after the application of BTX-A, cl.SNAP-25 could not be detected in the central nervous system, indicating that the reverse axonal transport was very weak or non-existent. 3) after BTX-A injection, the nerve and junction of the injection side and the SNAP-25 in the nerve innervated the muscle were compensated.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R964

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本文編號：1531872