Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures
Rapid simultaneous determination of apoptosis, necrosis, and viability in sulfur mustard exposed HaCaT cell cultures
复制标题
DOI：
10.1016/j.toxlet.2009.09.008
复制
发表时间：
2009-12-15
影响因子：
3.5
通讯作者：
Kehe, K.
中科院分区：
文献类型：
--
作者：
Heinrich, A.;Balszuweit, F.;Kehe, K.
硫芥（SM；双（2-氯乙基）硫醚；HD）是一种水疱诱导剂，会导致 DNA 损伤，随后导致细胞死亡，主要是通过基底角质形成细胞的凋亡。尽管对细胞机制进行了深入研究，但到目前为止，还没有任何治疗方法可以预防或拮抗 SM 相关的细胞和组织损伤。为了制定针对水泡的治疗策略，区分 SM 处理的人角质形成细胞中的凋亡和坏死非常重要。 DNA 片段化是细胞凋亡的标志，并受到一系列酶（核酸内切酶、DNase1、NUC 18）的调节，最终将染色质切割成 180-200 个碱基对的特定形式，即核小体。监测细胞凋亡的一种可行方法是通过细胞死亡检测 ELISA(plus)(R) (CDDE) 检测核小体。相反，在坏死过程中，DNA 片段化是随机的，并产生较大的片段，因此数量明显较少，并且主要出现在细胞培养物上清液中。为了监测坏死，我们通过 ToxiLight (R) 生物发光测定法测量了细胞内腺苷酸激酶 (AK) 向细胞培养物上清液中的释放情况（TL）。通过结合细胞死亡检测 ELISA(plus)(R) 和 ToxiLight (R) 生物发光测定，我们在 SM 暴露后获得了有关细胞存活和细胞死亡机制的更全面的信息。为了验证该测定，我们测试了常见的细胞凋亡和坏死诱导剂，例如 SM 300 muM 30 分钟、Lewisite (L) 60 muM 5 分钟和 Triton X-100 0.1%。结果表明，可以区分两种细胞死亡模式并量化其程度。该测定在量化细胞毒性剂引起的细胞凋亡和坏死以及评估潜在活性药物成分的保护作用方面非常有效。 (C) 2009 Elsevier Ireland Ltd. 保留所有权利。
Sulfur mustard (SM; bis(2-chloroethyl)sulphide; HD) is a blister inducing agent causing DNA damage and subsequently, cell death, mostly by apoptosis in basal keratinocytes. Despite intensive investigations on the cellular mechanism, there are, as of now, no causal therapeutics to prevent or antagonize SM-related damage to cells and tissues. In order to develop treatment strategies against vesication, it is important to distinguish apoptosis from necrosis in SM treated human keratinocytes. DNA fragmentation is a hallmark of apoptosis and regulated by a cascade of enzymes(endonucleases, DNase1, NUC 18), which finally cut the chromatin into specific formations of 180-200 base pairs, the nucleosomes. A feasible way to monitor apoptosis is the detection of nucleosomes by means of the Cell Death Detection ELISA(plus)(R) (CDDE) In contrast, during necrosis DNA fragmentation is at random and delivers larger fragments, which therefore are significantly less in number and predominantly occur in cell culture supernatant.To monitor necrosis, we measured the release of intracellular adenylate kinase (AK) into cell culture supernatant by means of the ToxiLight (R) Bioluminescence Assay (TL). With combination of the Cell Death Detection ELISA(plus)(R) and the ToxiLight (R) Bioluminescence Assay, we acquired more comprehensive information on cell survival and mechanisms of cell death, following an SM exposure. To validate the assay we tested common apoptosis- and necrosis-inducing agents like SM 300 mu M for 30 min, Lewisite (L) 60 mu M for 5 min and Triton X-100 0.1%. The results show that it is possible to differentiate between the two modes of cell death and to quantify their extent.This assay is highly effective in quantifying apoptosis and necrosis caused by cytotoxic agents and in estimating protective effects of potential active pharmaceutical ingredients. (C) 2009 Elsevier Ireland Ltd. All rights reserved.