两种肝毒性化学物诱导的肝纤维化小鼠模型转录组学的比较研究
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1.上海中医药大学附属曙光医院肝病研究所,上海 201203;2.上海市中医临床重点实验室,上海 201203;3.肝肾疾病病证教育部重点实验室,上海 201203

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45

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R-33

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Comparative study of transcriptomics in two murine liver fibrosis models induced by hepatotoxic chemicals
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1. Institute of Liver Diseases, Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203, China. 2. Shanghai Key Laboratory of Traditional Chinese Clinical Medicine, Shanghai 201203.3. Key Laboratory of Liver and Kidney Diseases, Ministry of Education, Shanghai 201203

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    摘要:

    目的 比较四氯化碳(CCl4 )和 3,5-二乙氧基羰基-1,4-二氢可力丁(DDC)两种肝毒性化学物诱导的肝纤维化小鼠模型的转录组学差异,为使用肝纤维化小鼠模型的研究提供参考依据。 方法 分别采用 10% CCl4(2 mL/ kg)腹腔注射和 0. 1% DDC 饮食喂养诱导小鼠肝纤维化模型。 造模 4 周后,检测血清 ALT、AST、TBil 水平;HE 染色观察肝内炎症浸润情况;天狼猩红染色观察肝胶原沉积情况;Jamall’s 法测定肝组织羟脯氨酸(Hyp)含量;ELISA 检测肝组织上清 TNF-α、IL-6 和 IL-1β 含量。 提取小鼠总 RNA 进行测序(RNA-Seq),使用 R 软件分析 2 种肝纤维化模型的差异基因,并进行 GO 和 KEGG 富集分析,并验证差异显著的基因。 结果 与正常小鼠相比,CCl4 染毒小鼠和 DDC 饮食小鼠血清 ALT、AST、TBil 水平和肝组织 TNF-α、IL-6 和 IL-1β 含量显著升高,血清 Alb 水平下降。 病理染色提示 CCl4 染毒小鼠肝组织结构破坏,中央静脉周围大量肝细胞玻璃样变及坏死;DDC 饮食小鼠肝内卟啉沉积,大量炎症细胞在汇管区和胆管周围浸润;两种模型小鼠肝内均有不同程度胶原沉积。通过筛选条件(|logFC |>2 倍且 P<0. 05)获得 CCl4 染毒模型、DDC 饮食模型的差异基因分别为 1820、2373 个,其中上调基因分别为 1302、1978 个,下调基因分别为 518、395 个。 GO 注释发现 2 种模型在分子功能(MF)、生物学过程(BP)、细胞成分(CC)等方面具有重要功能,KEGG 分析发现 CCl4 染毒模型、DDC 饮食模型分别激活 22、29 条信号通路,其中细胞外基质受体的相互作用、细胞周期、蛋白的消化与吸收、焦点粘附、PI3K-Akt 等 16 条信号通路在 2 种模型中均显著富集(P<0. 05)。 聚类分析发现在 2 种模型中均明显下调基因包括 Mup11、Mup15、Mup17、Mup1 等,采用 RTqPCR 得以证实(P<0. 05)。 结论 本研究报道了 CCl4 染毒和 DDC 饮食两种肝纤维化模型的 RNA-Seq 转录组学特点并进行比较,观察基因表达的场所、基因表达所调节的通路等方面,为后续肝纤维化的发病机制和治疗研究的动物模型的选择提供参考依据。

    Abstract:

    Objective To assess transcriptomic differences between carbon tetrachloride ( CCl4 )-induced and diethyl 1,4-dihydro-2, 4, 6-trimethyl-3, 5-pyridinedicarboxylate ( DDC) diet-induced mouse models of liver fibrosis to provide a framework for future research using mouse liver fibrosis models. Methods Mouse models of liver fibrosis were induced by a 10% CCl4(2 mL/ kg) injection or a 0. 1% DDC diet. After 4 weeks of induction, serum levels of ALT, AST,and TBil were measured. HE and Sirius red staining were used to observe hepatic inflammation and collagen deposition.Jamall’ s method was used to evaluate hydroxyproline ( Hyp) content in liver tissues. Hepatic tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β were measured by ELISA. Total RNA was extracted from murine liver tissues for RNA-sequencing (RNA-seq). Differentially expressed genes of the two models were analyzed by R software and then GO and KEGG enrichment was performed. Then, genes with significant differences were verified. Results Compared with normal mice, serum levels of ALT, AST, and TBil and hepatic expression of TNF-α, IL-6, and IL-1β were significantly increased in mice that received CCl4 and DDC, while the Alb serum level was decreased. Pathological staining showed that the structures of liver tissues were destroyed and a large number of hepatocytes around the central vein were hyalinized and necrotic in CCl4 -treated mice. In DDC diet-treated mice, a large amount of porphyrins had been deposited in the liver and a large number of inflammatory cells had infiltrated into the portal area and bile duct. Different degrees of collagen deposition were observed in the liver tissues of the two model mice. Different genes (DEGs) of CCl4- and DDC diet-treated mice were screened using a filter (| logFC | > 2-fold and P<0. 05). As a result , 1820 and 2373 DEGs in CCl4- and DDC diet-treated mice were analyzed, including 1302 and 1978 upregulated genes, and 518 and 395 downregulated genes,respectively. GO annotation showed that the two models had important functions in molecular function, biological process,and cell component. KEGG analysis showed that 22 and 29 signaling pathways were activated in CCl4- and DDC dietinduced models, respectively. Among them, 16 signaling pathways, such as extracellular matrix receptor interaction, cell cycle, protein digestion and absorption, focal adhesion, and PI3K-Akt, were significantly enriched in the two models (P<0. 05). Cluster analysis showed that Mup11, Mup15, Mup17, and Mup1 were significantly down-regulated in both models,which were identified by RT-qPCR (P<0. 05). Conclusions This study conducted a comparative analysis of the RNASeq transcriptomic features of liver fibrosis models induced by exposure to CCl4 and a DDC diet. It examined the gene expression patterns and the pathways influenced by gene expression. The findings serve as a valuable resource for selecting appropriate animal models for future research on the pathogenesis and treatment of liver fibrosis.

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闫阮玉,吴洪雨,黄 恺,孙 鑫,薛静波,陶艳艳,刘成海,彭 渊.两种肝毒性化学物诱导的肝纤维化小鼠模型转录组学的比较研究[J].中国比较医学杂志,2024,34(5):~32.

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  • 收稿日期:2023-11-03
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  • 在线发布日期: 2024-06-26
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