Uno Katsuhiro, Murotomi Kazutoshi, Kazuki Yasuhiro, Oshimura Mitsuo, Nakajima Yoshihiro
Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan.
Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Kagawa, Japan.
Luminescence. 2018 May;33(3):616-624. doi: 10.1002/bio.3454. Epub 2018 Feb 8.
We have developed a bioluminescence-based non-destructive cytotoxicity assay in which cell viability and membrane damage are simultaneously evaluated using Emerald luciferase (ELuc) and endoplasmic reticulum (ER)-targeted copepod luciferase (GLuc-KDEL), respectively, by using multi-integrase mouse artificial chromosome (MI-MAC) vector. We have demonstrated that the time-dependent concentration response curves of ELuc luminescence intensity and WST-1 assay, and GLuc-KDEL luminescence intensity and lactate dehydrogenase (LDH) activity in the culture medium accompanied by cytotoxicity show good agreement in toxicant-treated ELuc- and GLuc-KDEL-expressing HepG2 stable cell lines. We have clarified that the increase of GLuc-KDEL luminescence intensity in the culture medium reflects the type of cell death, including necrosis and late apoptosis, but not early apoptosis. We have also uncovered a strong correlation between GLuc-KDEL luminescence intensity in the culture medium and the extracellular release of high mobility group box 1 (HMGB1), a representative damage-associated molecular pattern (DAMP) molecule. The bioluminescence measurement assay using ELuc and GLuc-KDEL developed in this study can simultaneously monitor cell viability and membrane damage, respectively, and the increase of GLuc-KDEL luminescence intensity in the culture medium accompanied by the increase of cytotoxicity is an index of necrosis and late apoptosis associated with the extracellular release of DAMP molecules.
我们开发了一种基于生物发光的非破坏性细胞毒性检测方法，该方法使用多整合酶小鼠人工染色体（MI-MAC）载体，分别通过Emerald荧光素酶（ELuc）和内质网（ER）靶向的桡足类荧光素酶（GLuc-KDEL）同时评估细胞活力和膜损伤。我们已经证明，在经毒物处理的表达ELuc和GLuc-KDEL的HepG2稳定细胞系中，ELuc发光强度与WST-1检测的时间依赖性浓度响应曲线，以及GLuc-KDEL发光强度与培养基中乳酸脱氢酶（LDH）活性伴随细胞毒性的变化显示出良好的一致性。我们已经阐明，培养基中GLuc-KDEL发光强度的增加反映了细胞死亡的类型，包括坏死和晚期凋亡，但不包括早期凋亡。我们还发现培养基中GLuc-KDEL发光强度与高迁移率族蛋白B1（HMGB1）的细胞外释放之间存在很强的相关性，HMGB1是一种典型的损伤相关分子模式（DAMP）分子。本研究中使用ELuc和GLuc-KDEL开发的生物发光测量检测方法可以分别同时监测细胞活力和膜损伤，并且培养基中GLuc-KDEL发光强度随细胞毒性增加而增加是与DAMP分子细胞外释放相关的坏死和晚期凋亡的指标。
