State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, National Medical Center for Infectious Diseases, Zhejiang University School of Medicine, 79 Qingchun Road, Hangzhou, 310003, Zhejiang Province, P. R. China.
Department of Dermatology, Affiliated Hangzhou Dermatology Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Sci Rep. 2024 Nov 3;14(1):26544. doi: 10.1038/s41598-024-78183-y.
Acute liver injury (ALI) presents a challenging problem worldwide, prompting extensive research efforts. Cellular senescence has been found to be induced following ALI, and targeting cellular senescence has shown therapeutic potential. Therefore, understanding the expression of senescence-related genes in ALI can help to explore pathogenesis and treatment of this common disease. Carbon tetrachloride (CCl) is commonly used to study ALI. Although polymerase chain reaction (PCR) is a convenient and economical molecular biology technique widely used in basic medicine, research on selecting suitable reference genes to obtain objective and reproducible PCR data is scarce. Moreover, evidence supporting the choice of reference genes for experimental studies of CCl4-induced ALI and hepatic senescence in mice is limited. In this study, we obtained murine livers at four time points (0, 12, 24, and 48 h) following CCl4 treatment. We used five algorithms (geNorm, BestKeeper, NormFinder, delta Ct, and RefFinder) to rank 12 candidate genes in real-time reverse-transcription quantitative PCR (RT-qPCR) experiments. Focusing on cellular senescence in this model, we adopted four senescence-associated secretory phenotype (SASP) genes (Il6, Il1b, Ccl2, and Ccl5) as target genes. Our results confirmed Gapdh and Tbp as suitable reference genes in murine CCl-induced ALI models. Furthermore, we provide a table of published studies recommending reference genes for various liver disease models. This study provides a valuable reference for enhancing the reliability and reproducibility of ALI molecular findings.
急性肝损伤（ALI）是一个全球性的挑战，促使了广泛的研究努力。研究发现，ALI 后会诱导细胞衰老，靶向细胞衰老具有治疗潜力。因此，了解 ALI 中与衰老相关基因的表达可以帮助探索这种常见疾病的发病机制和治疗方法。四氯化碳（CCl）常用于研究 ALI。聚合酶链反应（PCR）虽然是一种广泛应用于基础医学的方便、经济的分子生物学技术，但关于选择合适的参考基因以获得客观和可重复的 PCR 数据的研究却很少。此外，支持选择参考基因用于 CCl4 诱导的 ALI 实验研究和小鼠肝衰老的证据有限。在本研究中，我们在 CCl4 处理后 0、12、24 和 48 小时四个时间点获取了小鼠肝脏。我们使用了 5 种算法（geNorm、BestKeeper、NormFinder、delta Ct 和 RefFinder）对实时逆转录定量 PCR（RT-qPCR）实验中的 12 个候选基因进行排名。我们重点关注该模型中的细胞衰老，采用了 4 个衰老相关分泌表型（SASP）基因（Il6、Il1b、Ccl2 和 Ccl5）作为靶基因。结果证实 Gapdh 和 Tbp 是小鼠 CCl 诱导的 ALI 模型中合适的参考基因。此外，我们提供了一份发表研究的表格，推荐了各种肝脏疾病模型的参考基因。本研究为提高 ALI 分子研究的可靠性和可重复性提供了有价值的参考。
