A quantitative real-time approach for discriminating apoptosis and necrosis.
A quantitative real-time approach for discriminating apoptosis and necrosis.
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一种定量的实时方法，用于区分凋亡和坏死。
DOI：
10.1038/cddiscovery.2016.101
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发表时间：
2017
影响因子：
7
通讯作者：
Santhoshkumar TR
中科院分区：
文献类型：
--
作者：
Lekshmi A;Varadarajan SN;Lupitha SS;Indira D;Mathew KA;Chandrasekharan Nair A;Nair M;Prasad T;Sekar H;Gopalakrishnan AK;Murali A;Santhoshkumar TR
凋亡和坏死是细胞死亡机制的两种主要形式。这两种细胞死亡形式都涉及多种生理和病理状况，并且在化疗成功后癌细胞的清除过程中也会出现。为了定性和定量的目的，已经发展出大量的细胞和生化检测方法来确定凋亡或坏死。对这些检测方法及其性能的进一步分析表明，由于凋亡细胞中会二次诱导坏死，因此很难将这些方法中的任何一种用作确证性手段。这凸显了一种具有实时分析能力的方法对于区分这两种细胞死亡形式的基本需求。本文描述了一种基于活细胞的灵敏方法，可在单细胞水平上区分凋亡和坏死。该方法使用稳定表达基于基因编码的FRET（荧光共振能量转移）的活性半胱天冬酶检测探针以及靶向线粒体的DsRed荧光蛋白的癌细胞。半胱天冬酶激活通过FRET探针切割时FRET的丧失来可视化，而线粒体荧光的保留以及FRET探针在切割前的丧失则证实为坏死。没有切割且线粒体荧光保留则表明是活细胞。这里描述的方法是一种极其灵敏的工具，可用于可视化和定量凋亡和坏死，适用于多种显微镜技术、流式细胞术以及高通量成像平台，在细胞生物学和肿瘤学药物筛选的多个领域具有潜在应用。
Apoptosis and necrosis are the two major forms of cell death mechanisms. Both forms of cell death are involved in several physiological and pathological conditions and also in the elimination of cancer cells following successful chemotherapy. Large number of cellular and biochemical assays have evolved to determine apoptosis or necrosis for qualitative and quantitative purposes. A closer analysis of the assays and their performance reveal the difficulty in using any of these methods as a confirmatory approach, owing to the secondary induction of necrosis in apoptotic cells. This highlights the essential requirement of an approach with a real-time analysis capability for discriminating the two forms of cell death. This paper describes a sensitive live cell-based method for distinguishing apoptosis and necrosis at single-cell level. The method uses cancer cells stably expressing genetically encoded FRET-based active caspase detection probe and DsRed fluorescent protein targeted to mitochondria. Caspase activation is visualized by loss of FRET upon cleavage of the FRET probe, while retention of mitochondrial fluorescence and loss of FRET probe before its cleavage confirms necrosis. The absence of cleavage as well as the retention of mitochondrial fluorescence indicates live cells. The method described here forms an extremely sensitive tool to visualize and quantify apoptosis and necrosis, which is adaptable for diverse microscopic, flow cytometric techniques and high-throughput imaging platforms with potential application in diverse areas of cell biology and oncology drug screening.