Unknown,Transcriptomics,Genomics,Proteomics

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Analysis of gene expression in primary fibrotic hepatocytes (from animals treated for a month with CCl4) stimulated with the purinergic agonist, UTP

ABSTRACT: It is known that extracellular ATP is a Damage Associated Molecular Pattern, however the cellular actions of the nucleotide are not yet undestood. Primary hepatocytes from fibrotic or control C57black mice were stimulated by 24 h with 100 microM of UTP, to understand if P2Y2 receptor activation induce a differential gene expression. The analysis of regulated transcripts revealed the regulation of multiple pathways, between the most relevant we can mention the HIF-alpha pathway.

ORGANISM(S): Mus musculus

SUBMITTER: Erandi Velazquez-Miranda

PROVIDER: E-MTAB-8302 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Since xenobiotics enter the organism via the liver, hepatocytes must cope with numerous perturbations. In particular, exposure to hepatotoxic agents resulting in liver injury triggers a strong alteration in gene expression. The contribution of these transcriptional regulatory networks to the propagation of injury (i.e. by modulation of metabolic pathways, inflammation, proliferation) are not fully understood. Therefore, we conducted a time-resolved gene array study on mouse liver after exposure to a single intraperitoneal injection of the hepatotoxic compound carbon tetrachloride (CCl4). This model induces a transient injury which reaches its maximum between 2 to 3 days after injection, followed by a precise regenerative response. The experiment included early time points (i.e. 2 and 8h) and late time points (1 to 16 days) to cover the initial events induced by CCl4 metabolization and cell stress, and the regenerative phase between 2-6 days after intoxicaiton. C57BL/6N male mice (8-12 weeks old) were obtained from Charles Rivers (Sulzfeld, Germany). The local Ethics committee approved the experiments. 4-5 mice were used for each time point. Mice received a single intraperitoneal injection of CCl4 (1.6 g/kg body weight) dissolved in 0.5 ml olive oil. The liver tissue was collected after 2h, 8h, and 1, 2, 4, 6, 8 and 16 days following CCl4 administration. As control, mice received 0.5 ml of olive oil intraperitonealy for 1 or 8 days. Healthy liver was collected from untreated mice. At the indicated time points, mice were anesthetized and blood was collected from the heart with a 25 gauge needle, using EDTA as anticoagulant, for analysis of the liver transaminases GOT and GPT in serum. Afterwards, the liver was resected, washed in ice cold PBS and sectioned for further analysis. Liver tissue sections of about 0.5 cm2 were snap frozen in liquid nitrogen and stored at -80ﾰC for subsequent isolation of proteins or RNA. RNA was isolated from mouse liver tissue using the Phenol/Chloroform method (Trizolﾮ, Qiagen, Hilden, Germany) according to the manufacturerﾒs instructions. RNA concentration and integrity were determined spectrophotometrically in a Nanodropﾮ2000 (ThermoScientific, Waltham MA, USA) and in a Bioanalyzerﾮ (Agilent, Waldbronn, Germany), respectively.

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Analysis of gene expression in primary fibrotic hepatocytes (from animals treated for a month with CCl4) stimulated with the purinergic agonist, UTP

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