PDF(Pubmed)
Abstract：
Exposure to toxic molecules from food or oral medications induces toxicity in colon cells that cause various human diseases; however, in vitro monitoring systems for colon cell toxicity are not well established. Stress granules are nonmembranous foci that form in cells exposed to cellular stress. When cells sense toxic environments, they acutely and systemically promote stress granule formation, with Ras GTPase-activating protein-binding protein 1 (G3BP1) acting as a core component to protect their mRNA from abnormal degradation. Here, we knocked in green fluorescent protein (GFP)-coding sequences into the C-terminal region of the G3BP1 gene in a human colon cell line through CRISPR-Cas9-mediated homologous recombination and confirmed the formation of stress granules with the G3BP1-GFP protein in these cells under cellular stress exposure. We demonstrated the formation and dissociation of stress granules in G3BP1-GFP expressing colon cells through real-time monitoring using a fluorescence microscope. Furthermore, we validated the toxicity monitoring system in the established colon cell line by observing stress granule formation following exposure to dihydrocapsaicin, bisphenol A, and sorbitol. Taken together, we established a stress granule reporter system in a colon cell line, providing a novel assessment for the real-time monitoring of colon toxicity in response to various chemicals.
摘要：
暴露于食物或口服药物中的有毒分子会在结肠细胞中引起毒性，从而导致各种人类疾病；然而，结肠细胞毒性的体外监测系统尚未建立。应激颗粒是在暴露于细胞应激的细胞中形成的非膜性病灶。当细胞感觉到有毒环境时，它们急剧和系统地促进应力颗粒的形成，RasGTP酶激活蛋白结合蛋白1（G3BP1）作为核心成分，保护其mRNA免受异常降解。这里,我们通过CRISPR-Cas9介导的同源重组将绿色荧光蛋白（GFP）编码序列敲入人结肠细胞系G3BP1基因的C末端区域，并证实了在细胞应激暴露下这些细胞中G3BP1-GFP蛋白的应激颗粒的形成。我们通过使用荧光显微镜进行实时监测，证明了G3BP1-GFP表达结肠细胞中应激颗粒的形成和解离。此外，我们通过观察暴露于二氢辣椒素后应激颗粒的形成，验证了建立的结肠细胞系的毒性监测系统，双酚A,和山梨醇。一起来看,我们在结肠细胞系中建立了应激颗粒报告系统，为实时监测各种化学物质对结肠的毒性提供了一种新的评估。
