利用实时成像和荧光泛素化细胞周期指示剂胚胎干细胞来区分全氟辛酸和高渗山梨醇等一般应激源导致的 G1 细胞周期延迟,以及苯并(a)芘等致突变应激源导致的 G2 细胞周期延迟。

IF 2.5 3区 医学 Q3 CELL & TISSUE ENGINEERING
Mohammed Abdulhasan, Ximena Ruden, Teya Marben, Sean Harris, Douglas M Ruden, Awoniyi O Awonuga, Elizabeth E Puscheck, Daniel A Rappolee
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引用次数: 0

摘要

最低可观察到的不良效应水平(LOAEL)是毒理学中的标准起始剂量。不过,最近有报告称,首次可观察到的不良效应水平(FOAEL)在本研究中被用作检测活体成像仪中诱变刺激的一个标准。荧光泛素化细胞周期指示剂(FUCCI)胚胎干细胞(ESC)在细胞周期的 S-G2-M 期为绿色,在 G1 期不是绿色。这里的标准培养基变化是一种轻微的应激,会延迟G1期,培养基变化会使绿色增加2.5-5倍。由于应激温和,培养基更换会迅速增加绿色细胞数量,但环境毒物和阳性对照高渗应激会抑制培养基更换后绿色细胞数量的增加。此前,全氟辛酸(PFOA)和邻苯二甲酸二乙酯(DEP)抑制了非绿色细胞周期向绿色细胞周期的进展。在这里,双酚 A(BPA)、皮质醇和阳性对照高渗山梨醇也抑制了绿色荧光,但高剂量(10 μM)的苯并(a)芘(BaP)会在整个 74 小时的暴露过程中增加绿色荧光。由于任何应激都会影响许多细胞周期阶段,因此信使 RNA(mRNA)标记物最好以比率来解释,即剂量依赖性诱变剂会增加 G2/G1,而非诱变剂会增加 G1/G2。暴露 74 小时后,RNAseq 检测 G1 和 G2 标记,BaP 剂量增加会增加 G2/G1 比率,但高渗山梨醇和 PFOA 剂量增加会增加 G1/G2 标记比率。BaP 在刺激 2 小时后会导致 FOAEL 快速绿色增加,而维甲酸仅在培养后期才会导致绿色荧光显著增加。使用活体成像仪确定 FUCCI ESC 的 FOAEL 和 G2 延迟是一种新方法,可使商业和基础发育生物学家检测具有致突变性的药物和环境刺激。此外,它还可用于测试防止突变的化合物。在纵向研究中,通过这种独特的活体报告和活体成像仪方案,可以进行跟踪研究,以检测预防性化合物本身是否会造成危害。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Using Live Imaging and Fluorescence Ubiquitinated Cell Cycle Indicator Embryonic Stem Cells to Distinguish G1 Cell Cycle Delays for General Stressors like Perfluoro-Octanoic Acid and Hyperosmotic Sorbitol or G2 Cell Cycle Delay for Mutagenic Stressors like Benzo(a)pyrene.

Lowest observable adverse effects level (LOAEL) is a standard point-of-departure dose in toxicology. However, first observable adverse effects level (FOAEL) was recently reported and is used, in this study, as one criterion to detect a mutagenic stimulus in a live imager. Fluorescence ubiquitinated cell cycle indicator (FUCCI) embryonic stem cells (ESC) are green in the S-G2-M phase of the cell cycle and not green in G1-phase. Standard media change here is a mild stress that delays G1-phase and media change increases green 2.5- to 5-fold. Since stress is mild, media change rapidly increases green cell number, but higher stresses of environmental toxicants and positive control hyperosmotic stress suppress increased green after media change. Perfluoro-octanoic acid (PFOA) and diethyl phthalate (DEP) previously suppressed progression of nongreen to green cell cycle progression. Here, bisphenol A (BPA), cortisol, and positive control hyperosmotic sorbitol also suppress green fluorescence, but benzo(a)pyrene (BaP) at high doses (10 μM) increases green fluorescence throughout the 74-h exposure. Since any stress can affect many cell cycle phases, messenger RNA (mRNA) markers are best interpreted in ratios as dose-dependent mutagens increase in G2/G1 and nonmutagens increase G1/G2. After 74-h exposure, RNAseq detects G1 and G2 markers and increasing BaP doses increase G2/G1 ratios but increasing hyperosmotic sorbitol and PFOA doses increase G1/G2 marker ratios. BaP causes rapid green increase in FOAEL at 2 h of stimulus, whereas retinoic acid caused significant green fluorescence increases only late in culture. Using a live imager to establish FOAEL and G2 delay with FUCCI ESC is a new method to allow commercial and basic developmental biologists to detect drugs and environmental stimuli that are mutagenic. Furthermore, it can be used to test compounds that prevent mutations. In longitudinal studies, uniquely provided by this viable reporter and live imager protocol, follow-up can be done to test whether the preventative compound itself causes harm.

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来源期刊
Stem cells and development
Stem cells and development 医学-细胞与组织工程
CiteScore
7.80
自引率
2.50%
发文量
69
审稿时长
3 months
期刊介绍: Stem Cells and Development is globally recognized as the trusted source for critical, even controversial coverage of emerging hypotheses and novel findings. With a focus on stem cells of all tissue types and their potential therapeutic applications, the Journal provides clinical, basic, and translational scientists with cutting-edge research and findings. Stem Cells and Development coverage includes: Embryogenesis and adult counterparts of this process Physical processes linking stem cells, primary cell function, and structural development Hypotheses exploring the relationship between genotype and phenotype Development of vasculature, CNS, and other germ layer development and defects Pluripotentiality of embryonic and somatic stem cells The role of genetic and epigenetic factors in development
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