Neuronal apoptosis in an in vitro model of photochemically induced oxidative stress.
Neuronal apoptosis in an in vitro model of photochemically induced oxidative stress.
复制标题
光化学诱导的氧化应激体外模型中的神经元凋亡。
DOI：
10.1006/exnr.1995.1022
复制
发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Grayson,DR
中科院分区：
文献类型：
--
作者：
Manev,H;Cagnoli,CM;Atabay,C;Kharlamov,E;Ikonomovic,MD;Grayson,DR
在神经元中，氧化应激可由神经递质相关机制触发，并可能导致细胞凋亡。一个简单的和可重复的模型，诱导氧化应激是必要的，以阐明氧化应激和神经细胞凋亡的联系机制。我们在这里报告了一种诱导细胞凋亡的方法，通过加载光敏染料，玫瑰红，并将培养物暴露于光，一个程序，产生活性单线态氧。我们用这个模型在大鼠小脑颗粒神经元的原代培养，并在非神经元的人胚肾293细胞系。我们已经测量了以下：（a）通过用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四唑溴化物（MTT）定量染色的线粒体代谢活性，（B）通过定量原位末端脱氧核苷酸转移酶测定的DNA片段化，和（c）通过台盼蓝排除试验的细胞活力。氧化应激导致线粒体功能的早期损害（MTT法）。随后是DNA片段化，最终是细胞死亡。使用大分子合成抑制剂茴香霉素和抗氧化剂维生素E可获得保护。该模型可用于研究氧化应激诱导神经元凋亡的机制，并有助于发现神经保护药物的新靶点。
In neurons, oxidative stress can be triggered by neurotransmitter-linked mechanisms and may lead to apoptotic cell death. A simple and reproducible model of inducing oxidative stress is needed to elucidate mechanisms which link oxidative stress and neuronal apoptosis. We report here a method of inducing apoptosis in cell cultures by loading them with a photosensitive dye, rose bengal, and exposing the cultures to light, a procedure which generates reactive singlet oxygen. We used this model in primary culture of rat cerebellar granule neurons, and in a nonneuronal human embryonic kidney 293 cell line. We have measured the following: (a) metabolic activity of the mitochondria by quantitative staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), (b) DNA fragmentation by quantitative in situ terminal deoxynucleotidyl transferase assay, and (c) cell viability by a trypan blue exclusion test. The oxidative stress caused an early impairment of mitochondrial function (MTT assay). This was followed by DNA fragmentation and ultimately by cell death. Protection was obtained with an inhibitor of macromolecular synthesis, anisomycin, and with antioxidant, vitamin E. This model can be used to study the mechanism of oxidative stress-triggered neuronal apoptosis, and it may help in discovering new targets for neuroprotective drugs.