Heterogeneity of Ductular Reactions in Adult Rat and Human Liver Revealed by Novel Expression of Deleted in Malignant Brain Tumor 1
2002; Elsevier BV; Volume: 161; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64395-7
ISSN1525-2191
AutoresHanne Cathrine Bisgaard, Uffe Holmskov, Eric Santoni‐Rugiu, Péter Nagy, Ole Nielsen, Peter Ott, Ester Hage, Kim Dalhoff, Lene Juel Rasmussen, Niels Tygstrup,
Tópico(s)Hepatitis B Virus Studies
ResumoThe regenerative capacity of mammalian adult liver reflects the ability of a number of cell populations within the hepatic lineage to take action. Limited information is available regarding factors and mechanisms that determine the specific lineage level at which liver cells contribute to liver repair as well as the fate of their progeny in the hostile environment created by liver injury. In the present study, we attempted to identify novel molecules preferentially involved in liver regeneration by recruitment of transit-amplifying, ductular (oval) cell populations. With a subtractive cDNA library screening approach, we identified 48 enriched, nonredundant gene products associated with liver injury and oval cell proliferation in the adult rat liver. Of these, only two, namely α-fetoprotein and a novel transcript with high homology to human DMBT1 (deleted in malignant brain tumor 1), were specifically associated with the emergence of ductular (oval) cell populations in injured liver. Subsequent cloning and characterization of the rat DMBT1 homologue revealed a highly inducible expression in ductular reactions composed of transit-amplifying ductular (oval) cells, but not in ductular reactions after ligation of the common bile duct. In human liver diseases, DMBT1 was expressed in ductular reactions after infection with hepatitis B and acetaminophen intoxication, but not in primary biliary cirrhosis, primary sclerosing cholangitis, and obstruction of the large bile duct. The expression heterogeneity in ductular reactions and multiple functions of DMBT1 homologues point to intriguing roles in regulating not only tissue repair but also fate decision and differentiation paths of specific cell populations in the hepatic lineage. The regenerative capacity of mammalian adult liver reflects the ability of a number of cell populations within the hepatic lineage to take action. Limited information is available regarding factors and mechanisms that determine the specific lineage level at which liver cells contribute to liver repair as well as the fate of their progeny in the hostile environment created by liver injury. In the present study, we attempted to identify novel molecules preferentially involved in liver regeneration by recruitment of transit-amplifying, ductular (oval) cell populations. With a subtractive cDNA library screening approach, we identified 48 enriched, nonredundant gene products associated with liver injury and oval cell proliferation in the adult rat liver. Of these, only two, namely α-fetoprotein and a novel transcript with high homology to human DMBT1 (deleted in malignant brain tumor 1), were specifically associated with the emergence of ductular (oval) cell populations in injured liver. Subsequent cloning and characterization of the rat DMBT1 homologue revealed a highly inducible expression in ductular reactions composed of transit-amplifying ductular (oval) cells, but not in ductular reactions after ligation of the common bile duct. In human liver diseases, DMBT1 was expressed in ductular reactions after infection with hepatitis B and acetaminophen intoxication, but not in primary biliary cirrhosis, primary sclerosing cholangitis, and obstruction of the large bile duct. The expression heterogeneity in ductular reactions and multiple functions of DMBT1 homologues point to intriguing roles in regulating not only tissue repair but also fate decision and differentiation paths of specific cell populations in the hepatic lineage. The proficiency of the adult mammalian liver to regenerate under pathophysiological conditions has long been recognized. However, only recently has it been firmly established that this regenerative capacity reflects the ability of several recognized populations of cells with stem-like characteristics to respond to damage of liver tissue. Because hepatic cells with stem-like properties are ideal therapeutic targets in patients suffering chronic liver failure, further knowledge of the mechanisms regulating the commitment of a particular cell type to participate in liver repair is important both for identification of therapeutic potentials and in understanding developmental processes and tissue homeostasis. The hallmarks of stem cells include competency to renew repeatedly, to renew the stem cell population, and to generate sufficient differentiated progeny to maintain or regenerate the functional capacity of a tissue.1Blau HM Brazelton TR Weimann JM The evolving concept of a stem cell: entity or function.Cell. 2001; 105: 829-841Abstract Full Text Full Text PDF PubMed Scopus (959) Google Scholar In rodent models, reconstruction of liver mass lost to surgical resection is achieved through proliferation of fully differentiated, normally quiescent hepatocytes and bile duct cells in the residual tissue. These mature cells in the hepatic lineage are numerous, can respond rapidly, and give rise to a large number of progeny while maintaining their differentiated phenotype.2Michalopoulos GK De Frances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2956) Google Scholar, 3Overturf K Al-Dhalimy M Ou C Grompe M Serial transplantation reveals the stem-cell-like regenerative potential of adult mouse hepatocytes.Am J Pathol. 1997; 51: 1273-1280Google Scholar However, in certain types of toxic hepatic injury impairing the replication of hepatocytes, a large population of ductular epithelial cells, possibly originating from endogenous stem cells in the canal of Hering, is produced. The resulting intricate network of ductular structures invade the parenchyma, where the ductular epithelial cells may differentiate to hepatocytes or bile duct cells to reconstitute the architecture and function of the damaged liver tissue.4Lemire JM Shiojiri N Fausto N Oval cell proliferation and the origin of small hepatocytes in liver injury induced by D-galactosamine.Am J Pathol. 1991; 139: 535-552PubMed Google Scholar, 5Dabeva MD Shafritz D Activation, proliferation, and differentiation of progenitor cells into hepatocytes in the D-galactosamine model of liver regeneration.Am J Pathol. 1993; 43: 1606-1620Google Scholar, 6Evarts RP Hu Z Omori N Omori M Marsden ER Thorgeirsson SS Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers.Carcinogenesis. 1996; 17: 2143-2151Crossref PubMed Scopus (67) Google Scholar The ductular epithelial cells are, therefore, at least bipotential progenitor cells and have, because of their cytological appearance with an oval-shaped nucleus and a high nuclear to cytoplasmic ratio, been named oval cells. Nevertheless, regeneration in response to other classes of hepatotoxins impairing hepatocyte replication seems to be accomplished by vigorously proliferating small hepatocyte-like progenitor cells expressing phenotypic characteristics of fetal hepatoblasts and adult mature hepatocytes.7Gordon GJ Coleman WB Hixson DC Grisham JW Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.Am J Pathol. 2000; 156: 607-619Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 8Gordon GJ Coleman WB Grisham JW Temporal analysis of hepatocyte differentiation by small hepatocyte-like progenitor cells during liver regeneration in retrorsine-exposed rats.Am J Pathol. 2000; 157: 771-786Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar It is not established if these small incompletely differentiated hepatocyte-like cells resemble the population of mature hepatocytes in adult liver giving rise only to new hepatocytes or the bipotential fetal hepatoblasts capable of differentiation into mature hepatocytes and bile duct cells during fetal liver development. Finally, there may be two origins of stem cells in the liver: endogenous stem cells located in the canal of Hering and exogenous stem cells derived from the bone marrow and capable of differentiation into hepatocytes and bile duct cells after homing to the injured liver.9Petersen BE Bowen WC Patrene KD Mars WM Sullivan AK Murase N Boggs SS Greenberger JS Goff JP Bone marrow as a potential source of hepatic oval cells.Science. 1999; 284: 1168-1170Crossref PubMed Scopus (2213) Google Scholar, 10Theise ND Badve S Saxena R Henegariu O Sell S Crawford JM Krause DS Derivation of hepatocytes from bone marrow cells in mice after radiation-induced myeloablation.Hepatology. 2000; 31: 235-240Crossref PubMed Scopus (913) Google Scholar The existence of hepatic cells with stem-like properties in humans is less well established. However, the ability of hepatocytes and bile epithelial cells to reconstitute liver mass and function after surgical resection is well known. Furthermore, ductular reactions recognized in a variety of genetic, acute, and chronic liver diseases display cells with phenotypic characteristics of the bipotential ductular (oval) cells in rodent models.11Hsia CC Evarts RP Nakatsukasa H Marsden ER Thorgeirsson SS Occurrence of oval-type cells in hepatitis B virus-associated human hepatocarcinogenesis.Hepatology. 1992; 16: 1327-1333Crossref PubMed Scopus (179) Google Scholar, 12De Vos R Desmet V Ultrastructural characteristics of novel epithelial cell types identified in human pathological liver specimens with chronic ductular reactions.Am J Pathol. 1992; 140: 1441-1450PubMed Google Scholar, 13Sell S Comparison of liver progenitor cells in human atypical ductular reactions with those seen in experimental models of liver injury.Hepatology. 1998; 27: 317-331Crossref PubMed Scopus (127) Google Scholar, 14Lowes KN Brennan BA Yeoh GC Olynyk JK Oval cell numbers in human chronic liver diseases are directly related to disease severity.Am J Pathol. 1999; 154: 537-541Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 15Crosby HA Hubscher S Fabris L Joplin R Sell S Kelly D Strain AJ Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.Am J Pathol. 1998; 152: 771-779PubMed Google Scholar As in rodent liver, the intrahepatic origin of these stem-like cells is likely to be the canals of Hering.16Theise ND Saxena R Portmann BC Thung SN Yee H Chiriboga L Kumar A Crawford JM The canals of Hering and hepatic stem cells in humans.Hepatology. 1999; 30: 1425-1433Crossref PubMed Scopus (639) Google Scholar Finally, it has recently been shown that in human transplant recipients, hepatocytes and bile duct cells can be derived from extrahepatic circulating multipotent stem cells, probably of bone marrow origin, and that these cells are involved in replenishment of hepatic epithelial cells.17Theise ND Nimmakayalu M Gardner R Illei PB Morgan G Teperman L Henegariu O Krause DS Liver from bone marrow in humans.Hepatology. 2000; 32: 11-16Crossref PubMed Scopus (1171) Google Scholar, 18Alison MR Poulsom R Jeffery R Dhillon AP Quaglia A Jacob J Novelli M Prentice G Williamson J Wright NA Hepatocytes from non-hepatic adult stem cells.Nature. 2000; 406: 257Crossref PubMed Scopus (965) Google Scholar Thus, the tremendous regenerative capacity of the mammalian liver seems to be reflected by the ability to call forth a cellular response at different levels in the hepatic lineage. This has led to the hypothesis that similar to other organ systems, the cellular lineage of the liver consists of true stem cells located in the canal of Hering, progenitor cells [transit-amplifying bipotential ductular (oval) cells], and mature hepatocytes and bile duct cells.19Sell S Heterogeneity and plasticity of hepatocyte lineage cells.Hepatology. 2001; 33: 738-750Crossref PubMed Scopus (386) Google Scholar Limited information is available regarding factors and mechanisms that determine the contribution of a liver cell population at a specific lineage level to liver repair as well as the fate of its progeny in the hostile environment created by liver injury. However, the ability of a particular cell population in the hepatic lineage to respond and participate in liver regeneration seems to be associated with the type of injury inflicted.2Michalopoulos GK De Frances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2956) Google Scholar, 7Gordon GJ Coleman WB Hixson DC Grisham JW Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.Am J Pathol. 2000; 156: 607-619Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 20Alison M Golding M Lalani E Sarraf C Wound healing in the liver with particular reference to stem cells.Phil Trans R Soc Lond B. 1998; 353: 877-894Crossref PubMed Scopus (73) Google Scholar These differences are reflected in the hepatic expression patterns of several growth regulatory molecules.21Nagy P Bisgaard HC Schnur J Thorgeirsson SS Studies on hepatic gene expression in different liver regenerative models.Biochem Biophys Res Commun. 2000; 272: 591-595Crossref PubMed Scopus (11) Google Scholar, 22Bisgaard HC Santoni-Rugiu E Nagy P Thorgeirsson SS Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells.Lab Invest. 1998; 78: 237-246PubMed Google Scholar, 23Bisgaard HC Müller S Nagy P Rasmussen LJ Thorgeirsson SS Modulation of the gene network connected to interferon-γ in liver regeneration from oval cells.Am J Pathol. 1999; 155: 1075-1085Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 24Tygstrup N Bangert K Ott P Bisgaard HC Messenger RNA profiles in liver injury and stress: a comparison of lethal and nonlethal rat models.Biochem Biophys Res Commun. 2002; 290: 518-525Crossref PubMed Scopus (17) Google Scholar Hence, it is tempting to hypothesize that the specific microenvironment created in response to a particular liver injury may determine fate decision and differentiation paths of the recruited cell populations, allowing the progeny to survive and proliferate in a hostile environment. Therefore, if a more targeted approach to decreasing tissue injury and enhancing repair/regeneration in humans is to be achieved a better understanding of the cellular and molecular mechanisms allowing stem cells to repopulate, proliferate, and differentiate in a hostile environment is required. In the present study, we have focused our efforts on identifying novel molecules and mechanisms defining the microenvironment necessary for proliferation of transit-amplifying ductular (oval) cells in injured liver. We have used a selective subtractive cDNA library screening approach applying a polymerase chain reaction (PCR)-based cDNA library construction and cDNA array analysis, extensive Northern blot analysis of identified molecules in representative models of rat liver regeneration, PCR-based cDNA cloning, and immunostaining of formalin-fixed tissue from experimental rat models as well as from a number of human liver diseases displaying ductular reactions. Here we report the identification and characterization of DMBT1 and its rat homologue dmbt1 4.7kb as novel molecules in liver regeneration. The molecules are rapidly induced after liver injury, and show strong heterogeneity of expression in ductular reactions of adult human and rat liver dependent on the injury induced. These findings point to intriguing roles of the molecules as factors in the microenvironment regulating not only tissue repair but also fate decision and differentiation paths of transit-amplifying ductular (oval) cells. Male Wistar rats, 8 weeks of age, were purchased from M&B A/S (Ry, Denmark) and kept under standardized conditions with access to food and water ad libitum. Liver regeneration through replication of mature hepatocytes and bile duct cells was achieved by surgical resection of the median and left lateral liver lobes removing ∼70% of the liver mass (PHx protocol).2Michalopoulos GK De Frances MC Liver regeneration.Science. 1997; 276: 60-66Crossref PubMed Scopus (2956) Google Scholar Liver regeneration by transit-amplifying ductular (oval) cells was achieved using the AAF/PHx protocol in which treatment with 2-acetylaminofluorene (2-AAF) (9 days, 20 mg/kg/day by gavage) was interrupted at day 5 to perform a 70% hepatectomy.6Evarts RP Hu Z Omori N Omori M Marsden ER Thorgeirsson SS Precursor-product relationship between oval cells and hepatocytes: comparison between tritiated thymidine and bromodeoxyuridine as tracers.Carcinogenesis. 1996; 17: 2143-2151Crossref PubMed Scopus (67) Google Scholar, 25Bisgaard HC Nagy P Santoni-Rugiu E Thorgeirsson SS Proliferation, apoptosis, and induction of hepatic transcription factors are characteristics of the early response of biliary epithelial (oval) cells to carcinogens.Hepatology. 1996; 23: 62-70Crossref PubMed Google Scholar Liver regeneration through proliferation of small hepatocyte-like progenitor cells was achieved by inhibiting replication of mature hepatocytes by two intraperitoneal injections of retrorsine (30 mg/kg) 2 weeks apart, followed by a 70% hepatectomy 5 weeks after the last treatment with retrorsine (retrorsine/PHx protocol).7Gordon GJ Coleman WB Hixson DC Grisham JW Liver regeneration in rats with retrorsine-induced hepatocellular injury proceeds through a novel cellular response.Am J Pathol. 2000; 156: 607-619Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar Control groups included: 1) a sham operation with laparotomy only; 2) treatment with 2-AAF (9 days, 20 mg/kg/day by gavage) combined with a sham operation at day 5; and 3) treatment with retrorsine (30 mg/kg i.p. 2 weeks apart) followed by a sham operation 5 weeks after the last treatment with toxin. Finally, proliferation of mature bile duct cells was induced by ligation of the common bile duct for 5 days.26Polimeno L Azzarone A Zeng QH Panella C Subbotin V Carr B Bouzahzah B Francavilla A Starzl TE Cell proliferation and oncogene expression after bile duct ligation in the rat: evidence of a specific growth effect on bile duct cells.Hepatology. 1995; 21: 1070-1078PubMed Google Scholar Groups of three animals were sacrificed by cervical dislocation at the time points indicated in the figures, and parts of the livers were snap-frozen in liquid nitrogen for RNA extraction, or fixed for histological examination. The Danish Council for Supervision with Experimental Animals had approved the studies. Normal liver tissue from liver transplant donors or taken for diagnostic purposes (n = 7) served as control samples. Diseased tissue samples were obtained from transplant recipients with primary biliary cirrhosis (n = 5, cirrhotic stage with active inflammation), primary sclerosing cholangitis (n = 3, cirrhotic stage with active inflammation), obstruction of the large bile duct (n = 2, cirrhotic stage), acetaminophen intoxication (n = 5, submassive liver cell necrosis), and hepatitis B infection (n = 3, submassive liver cell necrosis). A few tissue samples were received fresh allowing one part to be snap-frozen in liquid N2 for later extraction of total RNA and one part to be fixed in formalin. The remaining samples were only fixed in formalin. All fixed tissues were embedded in paraffin for routine histology and immunohistochemistry. The suppression subtractive hybridization cDNA library was constructed with the PCR-Select cDNA subtraction kit (Clontech Laboratories Inc., Palo Alto, CA) according to the manufacturer's instructions. The rat livers chosen for library construction were selected according to histological evaluations of liver morphology and cellular composition. A rat liver from the AAF/PHx protocol at day 10 after partial hepatectomy harboring a very large population of transit-amplifying ductular (oval) cells was chosen as the tester sample. As the driver sample a normal liver from an untreated rat was used. Polyadenylated RNA [poly(A)+ RNA] was prepared from snap-frozen liver tissue by ultracentrifugation through a cesium chloride cushion and enrichment by oligo(dT)-cellulose chromatography. Samples of 2 μg of poly(A)+ RNA were reverse-transcribed to cDNA. Subsequently, tester and driver cDNA were hybridized, the remaining unhybridized sequences amplified by PCR, and cloned into pCRII plasmid vectors (TA cloning kit; Invitrogen, Carlsbad, CA). Differentially expressed cDNA sequences were identified by cDNA array analysis as described in the PCR-Select cDNA subtraction kit protocol (Clontech). In the present study, 600 Escherichia coli transformants carrying the pCRII plasmid vector were analyzed for cDNA inserts by PCR. Of these, 196 transformants containing cDNA inserts larger than 250 bp were chosen for further array analysis. The cDNA products were amplified by PCR and arrayed on nylon membranes. Subsequently, the membranes were hybridized to cDNA probes produced by forward and reverse subtraction and labeled with [32P]dCTP (Rediprime; Amersham Pharmacia Biotech, Uppsala, Sweden). Clones containing cDNA inserts that exclusively hybridized to the forward-subtracted probe [representing cDNAs expressed highly in liver regenerating by recruitment of transit-amplifying ductular (oval) cells] were sequenced using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit with an automated sequencer (ABI373; PE Applied Biosystems, Foster City, CA). DNA sequences and conceptual translations were compared with known nucleotide and protein sequences using the BLAST algorithm (www.nci.nlm.nih.gov/BLAST/). Four publicly accessible databases were searched: SwissProt, GenBank nr protein, GenBank nr nucleotide, and dbEST-expressed sequence tags. The full-length cDNA for rat dmbt1 4.7kb was cloned by reverse transcribing 1 μg of total RNA (AAF/PHx protocol 10 days after PHx) into cDNA with the Smart Race cDNA amplification kit (Clontech). The resulting cDNA was then amplified in a 50-μl 5′ Race PCR reaction composed of 2.5 μl of cDNA template (diluted 1:100), 5 μl of Universal primer mix, 5 μl of Buffer A, and 5 μl of Buffer B (Elongase Enzyme Mix System; Life Technologies, Palo Alto, CA), 200 μmol/L each of dNTP, 1 μl Elongase Enzyme Mix, and 400 nmol/L gene-specific primer (Eb-3-UTR rev1, 5′-CTAGCTAGAGAAAGGATGGTGATGCCA-3′). The cDNA was amplified by an initial denaturing step for 2 minutes at 94°C followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 60°C, and 12 minutes at 68°C. The PCR products were cloned into the pCR-XL-TOPO plasmid vector (TOPO XL PCR cloning kit, Invitrogen) and sequenced. In experiments on liver regeneration, animals were sacrificed as indicated and total RNA extracted from ∼50 mg of snap-frozen liver tissue (RNeasy kit; Qiagen Inc., Santa Clarita, CA). Populations of viable nonparenchymal cells were isolated by perfusion of the liver in situ as described.22Bisgaard HC Santoni-Rugiu E Nagy P Thorgeirsson SS Modulation of the plasminogen activator/plasmin system in rat liver regenerating by recruitment of oval cells.Lab Invest. 1998; 78: 237-246PubMed Google Scholar The nonparenchymal cell populations were isolated 7 days after partial hepatectomy in the AAF/PHx protocol combining treatment with 2-AAF and a 70% hepatectomy, or 0 (untreated), 3, 6, 24, 48, and 96 hours after initiation of treatment with 2-AAF alone (20 mg/kg/day). Hepatocytes were isolated 0 (untreated), 3, 6, 24, 48, and 96 hours after treatment with 2-AAF alone (20 mg/kg/day) by a two-step in situ perfusion procedure.27Gant TW Silverman JA Bisgaard HC Burt RK Marino PA Thorgeirsson SS Regulation of 2-acetylaminofluorene- and 3-methylcholanthrene-mediated induction of multidrug resistance and cytochrome P450IA gene family expression in primary hepatocyte cultures and rat liver.Mol Carcinog. 1991; 4: 499-509Crossref PubMed Scopus (128) Google Scholar All cell preparations were snap-frozen in liquid nitrogen and stored at −70°C until total RNA was isolated (RNAstat Reagent; Tel-Test Inc., Friendswood, TX). Northern blot analysis was performed by electrophoresis of 10 μg of total RNA in 1% agarose/0.2 formaldehyde gels and transfer onto nylon membranes. A rat Multiple Tissue Northern (MTN) blot was purchased from Clontech. For quantification, a slot blot analysis was performed by immobilizing 10 μg of total RNA onto nylon membranes. Membranes were hybridized to cDNA probes labeled with [32P]dCTP. The probe for rat dmbt1 4.7kb encompassed nucleotides 4260 to 4623 of the 3′-untranslated sequence (GenBank accession number to be deposited), and for rat α-fetoprotein (AFP) nucleotides 101 to 329 (GenBank accession no. X02361). Both cDNA fragments were isolated in the subtraction library analysis. For semiquantitative reverse transcriptase-PCR analysis of DMBT1 in human liver, samples of 1 μg of total RNA were reverse-transcribed and amplified using the Advantage 2 PCR enzyme system (Clontech) and the following forward and reverse primers: hDMBT1-ZP-fwd3 (5′-CCTGCTCTGTCTGCCAAATCACATGCAAGCC-3′) and hDMBT-3-UTR rev1 (5′-GCATGGATTCTGGGACTGCAGGTCTATGGGCCA-3′). As internal standard of the RNA amount used in the PCR, a primer pair of human β-actin (5′-TCTGGCCGTACCACTGGCAT-3′) and (5′-cactgtgttggcgtacaggt-3′) were used for amplification. Amplification conditions were 35 cycles of 30 seconds at 94°C, 30 seconds at 65°C, and 2 minutes at 72°C. Tissue specimens were fixed in 4% neutral formalin or paraformaldehyde for 24 hours and processed for routine histology. Immunostaining of liver tissue was performed on 5-μm paraffin sections that were cleared with xylene and rehydrated through a graded series of alcohols (5 minutes each) ending with 10 minutes of incubation in phosphate-buffered saline. After quenching of endogenous peroxidase activity (15 minutes of incubation in methanol containing 2% H2O2), antigenic unmasking was accomplished by either heating in 10 mmol/L of sodium citrate (pH 6.0) for 15 minutes (DMBT1 and cytokeratin 7) or treatment with 0.1% trypsin for 15 minutes at 37°C (AFP). Nonspecific activity was blocked by a 60-minute incubation in blocking solution composed of 100 mmol/L Tris (pH 7.6), 550 mmol/L NaCl, 10 mmol/L KCl, 0.05% Triton X-100, 1% bovine serum albumin, and 1.5% serum of the secondary antibody species in a humidified chamber at room temperature. The sections were subsequently incubated overnight with the primary antibodies diluted in the above solution at 4°C. Specific binding of antibody was revealed by streptavidin/biotin immunoperoxidase techniques (Vectastain ABC Elite kit; Vector Laboratories, Burlingame, CA) with diaminobenzidine as substrate followed by light staining with Mayer's hematoxylin. The primary antibodies used on rat tissue were rabbit polyclonal against gp-340/DMBT1 (rabbit anti-human 421-gp-340,28Holmskov U Lawson P Teisner B Tornøe I Willis AC Morgan C Koch C Reid KBM Isolation and characterization of a new member of the scavenger receptor subfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule.J Biol Chem. 1997; 272: 13743-13749Crossref PubMed Scopus (192) Google Scholar, 29Holmskov U Mollenhauer J Madsen J Vitved L Grønlund J Tornøe I Kliem A Reid KBM Poustka A Skjødt K Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.Proc Natl Acad Sci USA. 1999; 96: 10794-10799Crossref PubMed Scopus (189) Google Scholar diluted 1:400), and AFP (rabbit anti-human AFP, diluted 1:800; DAKO, Glostrup, Denmark). Immunostaining of human tissues was achieved with mouse monoclonal antibodies against human gp-340/DMBT1 (mouse monoclonal Hyb213-6,28Holmskov U Lawson P Teisner B Tornøe I Willis AC Morgan C Koch C Reid KBM Isolation and characterization of a new member of the scavenger receptor subfamily, glycoprotein-340 (gp-340), as a lung surfactant protein-D binding molecule.J Biol Chem. 1997; 272: 13743-13749Crossref PubMed Scopus (192) Google Scholar, 29Holmskov U Mollenhauer J Madsen J Vitved L Grønlund J Tornøe I Kliem A Reid KBM Poustka A Skjødt K Cloning of gp-340, a putative opsonin receptor for lung surfactant protein D.Proc Natl Acad Sci USA. 1999; 96: 10794-10799Crossref PubMed Scopus (189) Google Scholar diluted 1:100) and human cytokeratin 7 (mouse monoclonal anti-human cytokeratin 7, diluted 1:100; BioGenex, San Ramon, CA). The specificities of the gp-340/DMBT1 antibodies were verified by Western blot analysis of DMBT1 protein purified from rat liver and human bronchi alveolar lung washings (data not shown). We approached the isolation of genes preferentially expressed in liver regenerating by recruitment of transit-amplifying ductular (oval) cells by identifying uniquely expressed transcripts in the AAF/PHx protocol as compared to the normal liver. Our overall screening approach, encompassing PCR-based construction of a subtractive cDNA library and cDNA array analysis combined with sequence acquisition and bioinformatics of resulting library clones, identified 48 enriched, nonredundant gene products (Table 1).Table 1Subtractive cDNA Library of Genes Highly Expressed in Rat Liver Regeneration by Transit-Amplifying Ductular (Oval) CellsNo.Insert identityAccession no.No.Insert identityAccession no.No.Insert identityAccession no.1Endomembrane protein emp70*cDNA inserts used in the comparative Northern blot analysis.AF160213176.2 kda, proteinNM_01905933MtN3-like proteinAF1517262α-Fetoprotein*cDNA inserts used in the comparative Northern blot analysis.V0125418Ebnerin/dmbt1*cDNA inserts used in the comparative Northern blot analysis.U3268134hnRNP HY141963Carbamyl phosphate synthetase 1*cDNA inserts used in the comparative Northern blot analysis.M123221914-kda, ubiquitin conjugating enzymeU0430835Lysosomal-associated transmembrane protein 4ANM_0086404α-1-acid glycoprotein*cDNA inserts used in the comparative Northern blot analysis.V0121620Receptor for activated kinase CAJ13286036Voltage-dependent anion channel 3 (Vdac3)*cDNA inserts used in the comparative Northern blot analysis.NM_0116965Na-, K-ATPase
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