Poster Session 2
Poster Session 2
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海报展示2
DOI：
10.1177/2048872612461726e
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Marcel Leist
中科院分区：
文献类型：
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作者：
S. Schildknecht;Daniel M. Nagel;Marcel Leist
背景：帕金森病以黑质多巴胺能神经元的逐渐变性为特征。多巴胺能神经元持续暴露于升高的氧化应激条件，这是由于不稳定的神经递质多巴胺可以容易地经历氧化以形成超氧化物和能够与蛋白质中的半胱氨酸残基或谷胱甘肽反应以形成多巴胺缀合物的奎宁形式。为了研究在这些条件下发生的分子事件，以及验证潜在的药理学干预，需要一种与体内多巴胺能神经元特征非常相似的实验性人体体外模型。这种体外模型不仅可以显著减少体内动物试验的使用，而且还具有允许对人类系统中神经退行性疾病中发生的分子事件进行研究的优势。材料和方法：LUHMES是用v-myc逆转录病毒载体条件永生化的人中脑细胞。在这个系统中，四环素关闭v-myc表达，并允许分化为多巴胺能神经元。四环素与dbcAMP和GDNF（神经胶质细胞衍生的神经营养因子）组合导致在3-4天内分化成多巴胺能细胞，如通过特异性标志物如酪氨酸羟化酶或多巴胺转运蛋白（DAT）的表达所示。结果：LUHMES细胞对帕金森病毒素MPP +和甲基苯丙胺（METH）/Fe 2+的反应得到验证。在这两种情况下，观察到神经突的时间依赖性降解，伴随着细胞ATP和GSH的损失，以及自由基种类的形成增加。这些作用仅在完全分化的细胞中检测到，而未分化的LUHMES对相同的毒性损伤没有显着反应。通过与混合谱系激酶抑制剂CEP 1347共孵育或通过抑制聚ADP-核糖聚合酶（PARP），部分预防或延迟观察到的神经退行性作用。通过应用多巴胺转运蛋白或酪氨酸羟化酶抑制剂，进一步强调了多巴胺在神经退行性过程中的参与，这些抑制剂显著防止MPP +诱导的变性。讨论内容：帕金森病和其他神经退行性疾病，如阿尔茨海默病或多发性硬化症，是发达国家老龄化人口中最具挑战性的健康问题之一。因此，可以假设，制药工业在这一领域的努力将在未来几十年内急剧增加。这些研究项目将大大增加对可靠和有代表性的测试系统的需求。然而，最广泛使用的模型仍然是实验室动物。本文引入的人神经元细胞系密切反映了体内多巴胺能细胞的独特性质。该模型不仅可用于神经退行性疾病事件的基础研究，也可作为神经毒理学试验项目的筛选系统。
Background: Parkinsons disease is characterized by a gradual degeneration of dopaminergic neurons in the substantia nigra. Dopaminergic neurons are continuously exposed to elevated oxidative stress conditions due to the unstable neurotransmitter dopamine that can easily undergo oxidation to form superoxide and a quinine-form capable to react with cysteine residues in proteins or with glutathione to form dopamine-conjugates. For investigations on the molecular events occurring under these conditions, as well as for the validation of potential pharmacological interventions, an experimental human in vitro model that closely resembles the characteristics of dopaminergic neurons in vivo is desired. This in vitro model would not only allow to significantly reduce the use of in vivo animal testing, but concomitantly would have the advantages to allow studies on molecular events occurring in neurodegenerative disorders in a human system. Materials and Methods: LUHMES are human mesencephalic cells conditionally immortalized with a v-myc retroviral vector. In this system, tetracycline shuts down v-myc expression and allows differentiation into dopaminergic neurons. Tetracycline in combination with dbcAMP and GDNF (glial cell-derived neurotrophic factor) leads to a differentiation into dopaminergic cells within 3-4 days, as shown by the expression of specific markers such as tyrosine hydroxylase or dopamine transporter (DAT). Results: LUHMES cells were validated with respect to their response toward the parkinsonian toxins MPP + and methamphetamine (METH)/Fe2 + . In both cases, a time-dependent degradation of neurites, accompanied by a loss of cellular ATP and GSH, and increased formation of radical species was observed. These effects were only detected in fully differentiated cells, whereas undifferentiated LUHMES demonstrated no significant response to the same toxic insult. The neurodegenerative effects observed were partially prevented or delayed by co-incubation with the mixed lineage kinase inhibitor CEP1347, or by inhibition of poly-ADP-ribose polymerase (PARP). The involvement of dopamine in the neurodegenerative process was further underlined by application of dopamine transporter, or tyrosine hydroxylase inhibitors that significantly protected against MPP + -induced degeneration. Discussion: Parkinsons disease and other neurodegerative disorders such as Alzheimers disease or multiple sclerosis are one of the most challenging health issues in the aging populations of developed countries. It can therefore be assumed that efforts of the pharmaceutical industry within this field will increase dramatically within the next decades. These research projects will create a massive rise in the demand for reliable and representative test systems. The most widely used model for this purpose however is still the laboratory animal. The herein introduced human neuronal cell line closely reflects the unique properties of dopaminergic cells in vivo. This model can not only serve for basic research on the events occurring in neurodegenerative diseases, but can also be used as a screening system within neurotoxicological testing programs.