Comprehensive gene expression profile of extrahepatic bile ducts in mice with experimental biliary atresia
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ABSTRACT: Newborn Balb/c mice were injected intraperitoneally with 1.5x10^6 fluorescent-forming units (ffu) of type- A Rhesus Rotavirus (RRV) or 0.9% normal saline (NS; control) within 24 hours of birth to induce experimental model of biliary atresia. Extrahepatic bile ducts including gallbladder were microdissected en bloc at 3, 7 and 14 days after RRV or saline injections. GeneChipM-BM-. Mouse Gene 1.0 ST Array (Affymetrix, CA) were used to screen mRNAs whose expression was differently regulated after RRV challenge compared to normal saline controls. Gene expression profiling: Each experimental conditon contains 3 sets of samples. Each sample consists of two to six extrahepatic bile ducts pooled prior to isolation of total RNA to ensure generation of adequate mRNA to perform quantitative experiments.
Project description:Newborn Balb/c mice were injected with 1.5x10^6 fluorescent-forming units (ffu) of Rhesus rotavirus type-A or 0.9% NaCl (normal saline) intraperitoneally within 24 hours of birth to induce experimental model of biliary atresia. The extrahepatic bile ducts including gallbladder were microdissected en bloc at 3, 7 and 14 days after rhesus rotavirus or saline injection. GeneChipM-BM-. Mouse Gene 1.0 ST Array (Affymetrix, CA) were used to screen mRNAs whose expression was differently regulated after rhusus rotavirus injection compare to the normal saline controls. Gene expression profiling. Each experimental conditon has 3 sets of samples . Two to six extrahepatic bileducts were pooled prior to isolating total RNA depending on the size to ensure adequate RNA quantities to perform experiments quantifying mRNA expression.
Project description:Newborn Balb/c mice were injected with 1.5x10^6 fluorescent-forming units (ffu) of Rhesus rotavirus type-A or 0.9% NaCl (normal saline) intraperitoneally within 24 hours of birth to induce experimental model of biliary atresia. The extrahepatic bile ducts including gallbladder were microdissected en bloc at 3, 7 and 14 days after rhesus rotavirus or saline injection. TaqManM-BM-. Array Rodent MicroRNA Card v2.0 (A and B) were used to screen microRNAs whose expression was differently regulated after rhusus rotavirus injection compare to the normal saline controls. microRNA expression profiling. Each experimental conditon has 3 sets samples . Two to six extrahepatic bileducts were pooled prior to total RNA isolation depending on the size to ensure adequate RNA quantities to perform experiments quantifying microRNA expression.
Project description:RNA-Sequencing was performed on mechanically dissociated, epithelial-enriched samples, of human extrahepatic biliary tissue from Gallbladder, Common Bile Duct, and Pancreatic Duct tissues. Sequencing was also performed on in vitro cultures of Organoid cell lines at passage 5 that were derived from human Gallbladder, Common Bile Duct, Pancreatic Duct, or Intrahepatic Bile Ducts.
Project description:3D cell culture systems (organoids), cultured from extrahepatic bile duct biopsies, resemble biliary epthelium (cholangiocytes) upon culture according to the protocol previously established for culturing intrahepatic cholangiocyte organoids from liver biopsies (Huch et al. Cell 2015). These extrahepatic cholangiocyte organoids (ECOs) in Canonical-Wnt culture conditions maintain their genetic stability, and transcription profile and can be maintained and propagated during long periods of culturing while still maintaining their functional properties. The data presented in this ArrayExpress submission contained micro array results of 3 different ECO lines that were analyzed for their functional cholangiocyte ion-channels, involved in secreting a protective bicarbonate layer. It was shown that these data closely resemble the expression profile of intrahepatic cholangiocyte organoids (ICOs) as described by our group. Results of the ICOs are deposited in the EMBL-EBI ArrayExpress repository under number E-MTAB-9044.
Project description:The aim is to characterize rat liver fibrosis induced by bile duct ligation (BDL). To induce hepatic fibrosis, Male Sprague Dawley rats (9-12 weeks of age and 380-420 g of weight upon arrival, supplied by Beijing Vital River laboratory animal Co., Ltd.) underwent surgery of bile duct ligation (BDL). The bile ducts of Sprague-Dawley rats were ligated after 12 hours of fasting and water deprivation. Rat liver samples were collected from three groups of rats at week 1, 2 and 5 after BDL surgery. Three control groups of rats underwent sham operation, including bile duct mobilization, but without BDL. Three biological replicates were used for each group.
Project description:Extrahepatic cholestasis leads to complex injury and repair processes that result in bile infarct formation, neutrophil infiltration, cholangiocyte and hepatocyte proliferation, extracellular matrix remodeling, and fibrosis. To identify early molecular mechanisms of injury and repair after bile duct obstruction, microarray analysis was performed on liver tissue 24 hours after bile duct ligation (BDL) or sham surgery. The most upregulated gene identified encodes plasminogen activator inhibitor 1 (PAI-1, Serpine 1), a protease inhibitor that blocks urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Because PAI-1, uPA, and tPA influence growth factor and cytokine processing as well as extracellular matrix remodeling, we evaluated the role of PAI-1 in cholestatic liver injury by comparing the injury and repair processes in wild-type (WT) and PAI-1-deficient (PAI-1-/-) mice after BDL. PAI-1-/- mice had fewer and smaller bile infarcts, less neutrophil infiltration, and higher levels of cholangiocyte and hepatocyte proliferation than WT animals after BDL. Furthermore, PAI-1-/- mice had higher levels of tPA activation and mature hepatocyte growth factor (HGF) after BDL than WT mice, suggesting that PAI-1 effects on HGF activation critically influence cholestatic liver injury. This was further supported by elevated levels of c-Met and Akt phosphorylation in PAI-1-/- mice after BDL. In conclusion, PAI-1 deficiency reduces liver injury after BDL in mice. These data suggest that inhibiting PAI-1 might attenuate liver injury in cholestatic liver diseases. Total RNA isolated using TRI Reagent (Sigma, St. Louis, MO) was purified with an RNeasy mini kit (Qiagen, Valencia, CA). Twenty micrograms cRNA was hybridized to a mouse GeneChip (U74Av2, Affymetrix, Santa Clara, CA) at the Siteman Cancer Center GeneChip facility as described by the manufacturer. Analyses used one mouse per chip. Gene expression changes were analyzed using Affymetrix MicroArray Suite 4.0 and GeneChip 3.1 Expression Analysis and Statistical Algorithms (Affymetrix). The complete methodology and full data sets for all 6 analyzed chips are available at http://bioinformatics.wustl.edu.beckerproxy.wustl.edu This study compares the injury and repair processed in wild-type mice after BDL.
Project description:Despite the impact of bile duct disorders, treatment options remain very limited. Poor access to biliary tissue and restrictions in long-term culture or significant expansion of primary cholangiocytes have posed major challenges for research in the field. These limitations have so far precluded large scale experiments such as transcriptomic and genome-wide analyses which are urgently needed to better understand biliary physiology and pathophysiology. To address this issue, we have developed a novel system for the isolation and propagation of primary cholangiocytes from the extrahepatic bile ducts. The resulting Extrahepatic Cholangiocyte Organoids (ECOs) maintain their genetic stability, transcriptomic profile and function over long term culture and are compatible with regenerative medicine applications such as biliary reconstruction. We established a novel protocol for the isolation and propagation of primary cholangiocytes from the extrahepatic biliary tree in the form of extrahepatic cholangiocyte organoids (ECOs). The aim of this experiment was to provide in depth characterisation of the transcriptome of ECOs during long term culture. We compare the transcriptome of ECOs cultured for 1 passage (P1), 10 passages (P10) and 20 passages (P20) with freshly isolated primary cholangiocytes from the common bile duct. Embryonic Stem Cells (ES) cells are used as a negative control=