Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea
Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea
复制标题
DOI：
10.1016/j.heares.2008.03.003
复制
发表时间：
2008-06-01
期刊：
影响因子：
2.8
通讯作者：
Cotanche, Douglas A.
中科院分区：
文献类型：
--
作者：
Kaiser, Christina L.;Chapman, Brittany J.;Cotanche, Douglas A.
氨基糖苷类抗生素诱导耳蜗毛细胞caspase依赖性凋亡死亡。细胞凋亡是细胞死亡的一种调节形式，可由许多应激源诱导，激活信号通路，导致受影响细胞的受控分解。半胱氨酸天冬氨酸氨基转移酶家族在细胞凋亡信号通路中被激活，并负责细胞的破坏。氨基糖苷类药物暴露后，鸡耳蜗毛细胞中的启动子caspase-9和效应子caspase-3均被激活。我们比较了两种不同的caspase检测方法：caspase抗体和CaspaTag试剂盒，分析了庆大霉素诱导毛细胞死亡过程中caspase在鸟类耳蜗组织中的激活情况。Caspase抗体在固定组织中的特定位置与caspase-9或caspase-3的裂解激活形式结合。CaspaTag是一种荧光抑制剂，能结合固定组织中caspase异二聚体大亚基上的活性半胱氨酸残基。为了诱导耳蜗毛细胞丢失，1-2周龄的雏鸡一次注射庆大霉素(300 mg/kg)。使用caspase导向的抗体或CapaTag试剂盒对雏鸡进行caspase-9或caspase-3检测。耳朵被用鬼臼毒素或肌球蛋白VI共同标记，以显示毛细胞并确定耳蜗损伤的进展。两种检测方法的caspase激活时间相似；然而，caspase-9和caspase-3抗体只标记那些正在经历细胞凋亡死亡的细胞。相反，CaspaTag标记了所有经历了细胞凋亡并从感觉上皮细胞中排出的细胞，以及那些目前处于细胞死亡过程中的细胞。这使得CaspaTag非常适合显示一段时间内细胞死亡的总体模式或水平，而Caspase抗体则提供特定时间点的细胞死亡快照。(C)2008 Elesvier B.V.保留所有权利。
Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The intiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death of compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase anti-bodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in in fixed tissue.To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg.) Chicks were caspase-9 or caspase-3 using either caspase-directed anitbodies or CapaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; how-ever, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cells death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point. (C) 2008 Elesvier B.V. All rights reserved.