Hwang S Y, Cho S H, Cho D Y, Lee M, Choo J, Jung K H, Maeng J H, Chai Y G, Yoon W J, Lee E K
Department of Chemical Engineering, Hanyang University, Ansan, Korea.
Biotech Histochem. 2011 Jun;86(3):181-7. doi: 10.3109/10520291003648367. Epub 2010 Mar 5.
Apoptosis is an important phenomenon for investigating the efficacy of anti-cancer drug candidates. The conventional assays for cellular apoptosis, such as enzyme-linked immunosorbent assay, absorbance monitoring for the activity of caspase, and flow cytometric assay, have focused only on biochemical events. We investigated the staurosporine (STS)-induced apoptosis of the murine macrophage RAW-264.7 cell using a cell based bioimaging technique. Using time-lapse confocal microscopy, we monitored caspase-3 activation during apoptosis by imaging the translocation of green fluorescent protein from the cytosol to the nuclei. Five hours after 1 μM STS treatment, caspase-3 was observed to be activated and membrane blebbing was observed simultaneously. Also, the loss of phosphatidylserine (PS) asymmetry in the phospholipid bilayer of plasma membrane during early apoptosis was monitored by imaging annexin-V labeled with fluorescein isocyanate binding to the externalized PS at various concentrations of STS. Moreover, disintegration of the plasma membrane during late apoptosis was confirmed using a nuclear dye, propidium iodide. The single cell based bioimaging data agreed well with those of the biochemical assays for caspase activation and morphological observation for membrane integrity.
细胞凋亡是研究抗癌候选药物疗效的一个重要现象。传统的细胞凋亡检测方法，如酶联免疫吸附测定、半胱天冬酶活性的吸光度监测和流式细胞术检测，都只关注生化事件。我们使用基于细胞的生物成像技术研究了星形孢菌素（STS）诱导的小鼠巨噬细胞RAW-264.7细胞凋亡。通过延时共聚焦显微镜，我们通过对绿色荧光蛋白从细胞质向细胞核的转位进行成像，监测了细胞凋亡过程中半胱天冬酶-3的激活。用1 μM STS处理5小时后，观察到半胱天冬酶-3被激活，同时观察到细胞膜起泡。此外，通过对不同浓度STS下异硫氰酸荧光素标记的膜联蛋白-V与外化的磷脂酰丝氨酸（PS）结合进行成像，监测了早期细胞凋亡过程中质膜磷脂双分子层中PS不对称性的丧失。此外，使用核染料碘化丙啶证实了晚期细胞凋亡过程中质膜的解体。基于单细胞的生物成像数据与半胱天冬酶激活的生化检测以及膜完整性的形态学观察数据吻合良好。
