Development of cell based assays as replacement assays for Botulinum toxins and antitoxins

肉毒杆菌毒素，一些最有毒的自然存在的物质是由肉毒杆菌产生的蛋白质。它们具有神经毒性，对神经细胞有毒性，并导致肉毒杆菌中毒，最常与食用含有毒素的食物或吸毒者有关。这些毒素通过阻断神经功能，导致呼吸和肌肉瘫痪，甚至死亡。只要及时给病人服用抗毒素药物，治疗是可能的。虽然毒素毒性极高，但极少量的毒素可以安全地用于治疗疼痛的肌肉痉挛和非随意肌肉收缩。它越来越多地应用于许多新的医疗条件和美容目的。肉毒杆菌中毒的诊断方法是向动物注射患者的体液和可疑的食物样本，以确认是否存在毒素。在制药行业，治疗性毒素和抗毒素的制造商要求许多动物在严重致死试验中确认安全性、效力和稳定性。迄今为止开发的替代方法存在局限性，要么仍然依赖动物，要么提供组织，要么只反映导致动物体内毒素作用模式的几个因素中的一个。避免这些限制的一种方法是开发体外功能测试，由于涉及肉毒杆菌中毒的所有关键步骤，因此提供了整个动物替代的可能性。由于肉毒杆菌毒素通过阻止神经肌肉神经末梢的化学物质(如乙酰胆碱等神经递质)的释放而导致瘫痪，因此相关的体外检测必须专注于这一活性。已建立的人类神经细胞系和人类干细胞还没有提供所需的敏感性，到目前为止的研究已经证实，在这些细胞可以与神经毒素一起使用之前，需要分化成神经样结构。在这项工作中，我们将分化的细胞应用到微电极阵列(MEA)上，以便进行细胞功能的原位测量。我们还将通过对参与神经递质释放的成分进行染色来观察细胞循环活动，并选择性地检测细胞内受到毒素攻击并与神经递质释放相关的关键蛋白质，以支持这些研究。这种新方法反映了毒素的全部功能，但它完全不以动物为导向，不会依赖于在其他应用和策略中使用的任何动物实验。

Botulinum toxins, some of the most poisonous naturally occurring substances are proteins produced by the bacterinum Clostridium botulinum. They are neurotoxic, that is toxic to the nerve cells and responsible for causing botulism, most often associated with eating food containing toxin or in hard drug users. The toxins cause respiratory and muscle paralysis, and even death, by blocking the nerve function. Treatment is possible with antitoxins, provided they are given to patients on time. Although highly toxic in extremely small quantities toxin can be administered safety to treat painful muscle spasms and involuntary muscle contractions. It is increasingly applied to many new medical conditions and for cosmetic purposes. Botulism is diagnosed by injecting animals with body fluids from patients and in suspected food samples to confirm presence of toxins. In pharmaceutical industries manufacturers of therapeutic toxins and antitoxins require many animals in severe lethality assay to confirm safety, potency and stability. Alternative methods developed to date have limitations by either still relying on animal or for provision of tissues or for reflecting only one of several factors that contribute to toxin mode of action in animals. One approach to avoid these limitations is to develop in vitro functional assay which offers the potential for entire animal replacement because of involving all key steps of botulinum intoxication. Because botulinum toxin induces paralysis by blocking the release of chemicals (neurotransmitters such as acetylcholine) at the neuromuscular nerve endings, relevant in vitro assays must focus on this activity. Established human neuronal cell lines and human stem cells have not offered the required sensitivity and studies to date have confirmed need for differentiation into neuronal like structures before these cells can be used with neurotoxins. In this work we will apply differentiated cells onto micro-electrode arrays (MEAs) in order to perform measurement of cell function in situ. We will support these studied by also looking at cell recycling activity by staining of the components involved in neurotransmitter release and by selectively detecting key proteins within the cells which are attacked by toxins and are also associated with neurotransmitter release. This new approach reflects the full function of the toxin yet it is entirely non animal orientated and will not rely on any animal experimentation used in other applications and strategies.