Project description:The objective of this study was to determine the gene expression profile of rat hepatocytes grown on decellularized rat liver matrices as a function of time.
Project description:We investigated the effects of the flutamide (FLU) -induced liver injury in primary rat hepatocytes using our liver microfluidic biochips. Flutamide is used a non-steroidal anti-androgenic drug. Two flutamide concentrations, 10µM and 100µM, were used to expose the hepatocytes for 24h under perfusion.
Project description:The transcriptional response to interferon alpha is cell type specific. To data, majority of investigations of interferon alpha induced gene expression have been made using immortalized or transformed cell lines underlining interferon's importance for treatment of cancer but omitting physiological relevance. Here in we have determined gene expression change in primary culture of hepatocytes after interferon alpha treatment. We used Affymetrix Rat Genome 230 2 microarrays to determine gene expression profiles in primary rat hepatocytes after interferon alpha treatment and untreated cells. Primary hepatocytes were chosen because they are most relevant to physiological state in intact liver. Hepatocytes was isolated from rat liver using collagenase treatment procedure and purified on percoll gradient. Next day after isolation primary hepatocytes culture was cultivated with (or without) 250u/ml rat interferon alpha for 3 and 6 hours. Experiment was performed in 3 biological replicates. cRNA preparation was performed according manufacturer's recommendation from 5ug of total RNA and gene expression profiles were determined.
Project description:We investigated the effects of the flutamide (FLU) -induced liver injury in primary rat hepatocytes using our liver microfluidic biochips. Flutamide is used a non-steroidal anti-androgenic drug. Two flutamide concentrations, 10M-BM-5M and 100M-BM-5M, were used to expose the hepatocytes for 24h under perfusion. Primary rat hepatocytes were cultivated in microfluidic biochips and treated with 10 and 100M-BM-5M of flutamide for 24h
Project description:The biological effects of the pesticide and complex I inhibitor tebufenpyrad (TEBU) on liver cells were investigated by proteomic approaches. Cellular contents and culture media were analyzed in dose-response experiments on primary rat hepatocytes (PRHs).
Project description:To investigate whether rat adult hepatocytes would exhibit different characteristics dependent on their ploidy statuses, we compared the transcriptome profile of 2c, 4c and 8c hepatocytes by mRNA microarray.
Project description:The effect of drugs, disease and other perturbations on mRNA levels are studied using gene expression microarrays or RNA-seq, with the goal of understanding molecular effects arising from the perturbation. Previous comparisons of reproducibility across laboratories have been limited in scale and focused on a single model. The use of model systems, such as cultured primary cells or cancer cell lines, assumes that mechanistic insights derived with would have been observed via in vivo studies. We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), primary human hepatocytes (HPH) from TG, and mouse liver / HepG2 results from the Gene Expression Omnibus (GEO) repository. Gene expression changes for treatments were normalized to controls and analyzed with three methods: 1) gene level for 9071 high expression genes in rat liver, 2) gene set analysis (GSA) using canonical pathways and gene ontology sets, 3) weighted gene co-expression network analysis (WGCNA). Co-expression networks performed better than genes or GSA on a quantitative metric when comparing treatment effects within rat liver and rat vs. mouse liver. Genes and modules performed similarly at Connectivity Map-style analyses, where success at identifying similar treatments among a collection of reference profiles is the goal. Comparisons between rat liver and RPH, and those between RPH, HPH and HepG2 cells reveal low concordance for all methods. We investigate differences in the baseline state of cultured cells in the context of drug-induced perturbations in rat liver and highlight the striking similarity between toxicant-exposed cells in vivo and untreated cells in vitro. Gene expression studies in model systems are widely used for understanding the mechanism of drugs and other perturbations in biological systems. Other researchers have examined the reproducibility of microarray studies between laboratories, or comparing microarrays and/or RNA sequencing. However, no large scale studies have compared results from protocols which differ in minor details, or results generated in vivo vs. in vitro culture system thought to serve as useful models. The rat liver is by far the most extensively studied model evaluating effects of drugs and other perturbations, and existing data allowed us to assess the level of concordance between rat liver and rat primary hepatocytes cultured in collagen-coated plates (i.e. “flat” culture) for hundreds of drugs. We found that the mouse liver serves as a better model of the rat liver than do rat primary hepatocytes, even after allowing for differences due to pharmacokinetics. The low concordance observed between rat liver and rat hepatocytes suggests that validating the utility of ‘omics data generated on emerging cell culture approaches (e.g. “organ-on-a-chip”, 3D-printed tissues) using rat cells and comparison to the rat liver may be necessary in order to gain confidence these approaches substantially improve on traditional culture models of human cells.
Project description:The effect of drugs, disease and other perturbations on mRNA levels are studied using gene expression microarrays or RNA-seq, with the goal of understanding molecular effects arising from the perturbation. Previous comparisons of reproducibility across laboratories have been limited in scale and focused on a single model. The use of model systems, such as cultured primary cells or cancer cell lines, assumes that mechanistic insights derived with would have been observed via in vivo studies. We examined the concordance of compound-induced transcriptional changes using data from several sources: rat liver and rat primary hepatocytes (RPH) from Drug Matrix (DM) and open TG-GATEs (TG), primary human hepatocytes (HPH) from TG, and mouse liver / HepG2 results from the Gene Expression Omnibus (GEO) repository. Gene expression changes for treatments were normalized to controls and analyzed with three methods: 1) gene level for 9071 high expression genes in rat liver, 2) gene set analysis (GSA) using canonical pathways and gene ontology sets, 3) weighted gene co-expression network analysis (WGCNA). Co-expression networks performed better than genes or GSA on a quantitative metric when comparing treatment effects within rat liver and rat vs. mouse liver. Genes and modules performed similarly at Connectivity Map-style analyses, where success at identifying similar treatments among a collection of reference profiles is the goal. Comparisons between rat liver and RPH, and those between RPH, HPH and HepG2 cells reveal low concordance for all methods. We investigate differences in the baseline state of cultured cells in the context of drug-induced perturbations in rat liver and highlight the striking similarity between toxicant-exposed cells in vivo and untreated cells in vitro. Gene expression studies in model systems are widely used for understanding the mechanism of drugs and other perturbations in biological systems. Other researchers have examined the reproducibility of microarray studies between laboratories, or comparing microarrays and/or RNA sequencing. However, no large scale studies have compared results from protocols which differ in minor details, or results generated in vivo vs. in vitro culture system thought to serve as useful models. The rat liver is by far the most extensively studied model evaluating effects of drugs and other perturbations, and existing data allowed us to assess the level of concordance between rat liver and rat primary hepatocytes cultured in collagen-coated plates (i.e. âflatâ culture) for hundreds of drugs. We found that the mouse liver serves as a better model of the rat liver than do rat primary hepatocytes, even after allowing for differences due to pharmacokinetics. The low concordance observed between rat liver and rat hepatocytes suggests that validating the utility of âomics data generated on emerging cell culture approaches (e.g. âorgan-on-a-chipâ, 3D-printed tissues) using rat cells and comparison to the rat liver may be necessary in order to gain confidence these approaches substantially improve on traditional culture models of human cells. To identify transcriptional changes in culture, rat primary hepatocytes (RPH) were isolated from three male Sprague Dawley rats. During the isolation and prior to perfusion, a lobe of liver was tied off to serve as the liver in situ reference sample. Cells were isolated and samples from the cell pellet (time zero) and cells cultured for 4, 24 and 48 hours. Three biological replicates were generated for each group (one from each rat). Each biological replicate was analyzed via 3 technical replicates, for a total of 9 array hybridizations per group.
Project description:The structural-functional organization of ammonia and glutamine metabolism in the liver acinus involves highly specialized hepatocyte subpopulations such as glutamine producing perivenous scavenger cells. However, it is still unclear whether this cell population is homogeneous and involves further subpopulations. This was investigated in the present study by proteome profiling of periportal glutamine synthetase-negative hepatocytes and perivenous glutamine synthetase (GS) expressing scavenger cells isolated from mouse and rat liver. Apart from established markers of GS+ hepatocytes such as glutamate transporter 1 (GLT1), ornithine aminotransferase (OAT) or ammonium transporter Rh type B (RHBG), we identified novel scavenger cell-specific proteins such as the basal transcription factor 3 (BTF3) and the heat-shock protein 25 (HSP25). Interestingly, BTF3 and HSP25 were heterogeneously distributed among GS+ hepatocytes as shown by immunofluorescence analyses in mouse, rat and human liver slices. Feeding experiments showed that RHBG but not GS protein levels were increased in the liver of mice fed with a high protein diet compared to standard chow. While the spatial distribution of GS and carbamoylphosphate synthetase-1 (CPS1) was not affected, periportal areas constituted by GLS2 positive hepatocytes were enlarged or reduced in response to high or low protein diet, respectively. The data suggest that the population of perivenous GS+ scavenger cells is heterogeneous and not uniform as previously suggested which may reflect a functional heterogeneity, possibly relevant for liver regeneration.