局部肌肉注射A型肉毒毒素播散情况的研究

发布时间：2018-02-24 22:03

本文关键词： A型肉毒毒素突触体相关蛋白SNAP-25 逆向轴浆运输中枢效应　出处：《浙江大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景：A型肉毒毒素(Botulinum toxin type A,BTX-A)是革兰阳性厌氧产孢肉毒杆菌产生的一种神经外毒素,可特异性裂解运动终板中运动神经元突触前膜内突触体相关蛋白SNAP-25(synaptosomal-associated protein of25-kDa)使得递质囊泡无法与突触前膜融合,从而抑制神经末梢乙酰胆碱的释放而松弛肌纤维,因而一直被用于眼肌痉挛、面肌痉挛等肌张力障碍性疾病的治疗。临床应用也日渐增多。既往普遍认为临床局部肌肉注射BTX-A,其作用只局限于注射点以及附近的突触,但近年来有越来越多的证据提示BTX-A的局部注射后,不仅在注射部位发挥作用,并且对远隔的神经肌肉接头也有作用,甚至还能通过逆向轴浆运输的方式进入高一级神经系统。这一现象打破了人们一直以来教条式的认识。现今BTX-A的中枢效应已被逐步揭示,但对其机制的研究仍然十分匮乏。研究目的：外周肌肉注射BTX-A并检测BTX-A局部注射后逆向轴浆运输到中枢的情况,以及远隔对侧神经肌肉接头的播散情况,阐明BTX-A产生中枢效应的途径,将为应用BTX-A后出现的中枢效应给出合理解释,从而能为临床注射BTX-A后可能出现的中枢效应提供更多有用的信息。研究方法： 1)对成年小鼠的单侧腓肠肌注射,低于临床应用剂量(1U/kg)、临床应用剂量(5U/kg)和小鼠耐受的最大安全剂量(30U/kg)三种不同剂量的BTX-A后,不同时间点通过免疫印迹检测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响的范围。BTX-A在动物体内的播散和作用特点。 2)检测支配注射肌肉的神经和相应的高一级运动神经元内的cl.SNAP-25,从而了解是否真的存在BTX-A逆向轴浆运输到中枢的现象。结果： 1)单侧注射三个不同梯度的剂量后,均能在对侧肌肉中发现cl.SNAP-25的存在。 2)单侧注射最大耐受安全剂量后,均不能在支配该肌肉的神经以及脊髓运动区发现cl.SNAP-25的存在。 3)不同注射剂量下,机体对BTX-A削减功能性的SNAP-25进行不同程度的代偿。结论： 1)即使是低于临床应用剂量的BTX-A可通过血液循环达到对侧非注射肌肉,并水解神经肌肉接头内的SNAP-25。 2)即使是应用了小鼠最大耐受安全剂量的BTX-A后,在中枢神经系统内,仍然不能检测到cl.SNAP-25,说明其逆向轴浆运输的现象极其微弱或并不存在。 3)机体在接受BTX-A注射后,对注射侧肌肉的神经及接头和支配该肌肉的神经内的SNAP-25进行代偿补充。
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R964

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