A型肉毒毒素裂解SNAP-25在被川楝素阻断后仍抑制乙酰胆碱诱发离体幽门肌收缩

发布时间：2018-03-19 06:55

本文选题：A型肉毒毒素(BTX-A)　切入点：川楝素　出处：《兰州大学》2007年硕士论文　论文类型：学位论文

【摘要】： 背景与目的：A型肉毒毒素(botulinum toxin type A.BTX-A)选择性地裂解突触相关蛋白—25(synaptosomal-associated protein of 25 kDa，SNAP-25)，阻断突触囊泡中神经递质与突触前膜的融合、胞吐和释放。川楝素(toosendanin，TSN)近期被证实通过阻断BTX-A裂解SNAP-25而具有抗肉毒毒素功能。本研究依离体大鼠幽门平滑肌为实验对象，应用TSN阻断BTX-A裂解SNAP-25后，观察BTX-A是否仍对乙酰胆碱(acetylcholine，ACh)诱发的平滑肌收缩起抑制作用，以期揭示BTX-A对平滑肌存在的作用途径。方法：三组Sprague-Dawley大鼠的幽门离体平滑肌条在Krebs液中1g的前负荷下孵育52分钟左右，待出现自发性收缩波且平稳后分别加入ACh，BTX-A，TSN和Atropine。第一组(n=10)：肌条对ACh 1001μM出现反应10分钟后，分别加入Atropine 1μM(n=5)或BTX-A 10 U/ml(n=5)记录4小时；第二组(n=10)：肌条对BTX-A 10U/ml(n=5)或AtropinelμM(n=5)出现反应30分钟后，加入ACh 100μM记录4小时；第三组(n=5)：肌条在含TSN 29.6μM溶液中孵育20分钟后，加BTX-A10U/ml，30分钟后再加ACh 100μM记录4小时。结果：第一组：ACh 100μM显著地增强了幽门收缩的张力(P＜0.001)和频率(P＜0.001)，但振幅无显著性差异；后续加入BTX-A 10 U/ml抑制了ACh诱发的收缩作用(张力，P＜0.001：频率，P＜0.001；振幅，P=0.014)。然而，Atropine 1μM却完全性抑制了ACh诱发的收缩反应。这一结果显示BTX-A10 U/ml直接抑制了ACh诱发的收缩反应。第二组：BTX-A 10 U/ml直接抑制了幽门自发性收缩(张力，P=0.006；频率，P＜0.001；振幅，P＜0.001)；AtropinelμM同样抑制了幽门肌的收缩。随后加入ACh100μM不能再激发幽门肌的收缩。第三组：TSN 29.6μM孵育20分钟，，幽门平滑肌的自发性收缩末发生变化；加入BTX-A 10 U/ml后，BTX-A降低幽门肌收缩反应(张力，P＜0.001；振幅，P＜0.001)。虽然这种抑制作用与未加TSN时BTX-A的效应不尽相似，但是BTX-A10 U/ml仍然抑制了幽门平滑肌收缩，即使ACh 100μM也同样不能激发它再收缩。结论：A型肉毒毒素不仅抑制幽门平滑肌的自发性收缩，而且抑制了乙酰胆碱诱发的收缩；即使它介导SNAP-25裂解被川楝素阻断后，也同样抑制乙酰胆碱诱发的幽门部平滑肌收缩。这提示A型肉毒毒素不仅抑制胆碱能神经中乙酰胆碱的释放，而且抑制外源性乙酰胆碱与胆碱能毒蕈碱受体结合后产生的肌肉收缩反应。
[Abstract]:Background & AIM: botulinum toxin type A.BTX-A selectively cleavage synaptosomal-associated protein of 25 kDaof SNAP-25 to block the fusion of neurotransmitters in synaptic vesicles and presynaptic membranes. Cytoemetic exocytosis and release. Toosendanin TSNs have recently been proved to have the function of anti-botulinum toxin by blocking BTX-A fragmentation of SNAP-25. In this study, the isolated rat pyloric smooth muscle was used as experimental object, and TSN was used to block SNAP-25 fragmentation by BTX-A. To observe whether BTX-A still inhibits the contraction of smooth muscle induced by acetylcholine acetylcholine acetylcholine in order to reveal the mechanism of BTX-A on smooth muscle. Methods: the isolated smooth muscle strips of three groups of Sprague-Dawley rats were incubated under the preload of 1 g in the Krebs solution for about 52 minutes, and after the spontaneous contraction wave appeared and stabilized, they were added into the ACh BTX-ACTN and Atropine respectively. The first group was treated with ACh 1001 渭 M for 10 minutes. The second group was incubated in the solution containing TSN 29.6 渭 M for 20 minutes. After 30 minutes of reaction to BTX-A 10 U / ml 5) or Atropinel 渭 MN 5), ACh 100 渭 M was added to the third group for 4 hours, and the third group was incubated in TSN 29.6 渭 M solution for 20 minutes, and the second group was incubated in the solution containing TSN 29.6 渭 M for 20 minutes, and the second group was incubated in the solution containing TSN 29.6 渭 M for 20 minutes after adding ACh 100 渭 M for 4 hours. BTX-A 10 U / ml for 30 minutes and ACh 100 渭 M for 4 hours. Results: in the first group, the tension and frequency of pyloric contraction were significantly enhanced by 1: ach 100 渭 M (P < 0.001), but there was no significant difference in amplitude. The subsequent addition of BTX-A10 U / ml inhibited the contraction induced by ACh (tension P < 0.001: frequency P < 0.001; amplitude P < 0.014). However, Atropine 1 渭 M completely inhibited the contraction induced by ACh, which indicated that BTX-A10 Ur / ml directly inhibited the contraction induced by ACh. The second group: BTX-A 10 U / ml directly inhibited the spontaneous contraction of pylorus (tension P0. 006; frequency P < 0. 001; amplitude P < 0. 001); Atropin 渭 M also inhibited the contraction of pyloric muscle. Then, the addition of ACh100 渭 M could not stimulate the contraction of pylorus. In the third group, the spontaneous contraction of pyloric smooth muscle changed after incubating with 29.6 渭 M for 20 minutes, and the contractile response of pyloric muscle was decreased after adding 10 Uml BTX-A (tension P < 0.001; amplitude P < 0.001). Although the inhibitory effect was not similar to that of BTX-A without TSN. However, BTX-A10 U / ml still inhibited the contraction of pyloric smooth muscle, and even ACh (100 渭 M) could not induce the contraction of pyloric smooth muscle. Conclusion botulinum toxin type A not only inhibits spontaneous contraction of pyloric smooth muscle, but also inhibits acetylcholine-induced contraction, even when SNAP-25 cleavage is blocked by azadirachtin. It also inhibited the contraction of pyloric smooth muscle induced by acetylcholine. This suggests that botulinum toxin A not only inhibits the release of acetylcholine from cholinergic nerves, It also inhibits the muscle contraction induced by the binding of exogenous acetylcholine to cholinergic muscarinic receptors.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R33

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