- Email: [email protected]
505 INDUCTION OF PANCREATIC BETA-CELL PHENOTYPE IN LIVER OF DIABETES MICE
04D. MOLECULAR AND CELLULAR BIOLOGY – D) LIVER REGENERATION AND EXPERIMENTAL ONCOLOGY detect cell cycle phase. Apoptosis was examined by Annexin-V/PI doublelabelled FCM, gel electrophoresis and transmission electron microscope. Additionally, the expression of cell cycle related protein (Cyclin D1 and Cyclin-Dependent Kinase 4) in HSCs was detected by Western blot and RT-PCR. Results: The FRNK plasmids were transfected into HSCs successfully, and the overexpression of FRNK decreased FAK phosphorylation on Tyr397 without changing total FAK level. After FRNK transfection, the proliferation inhibition rates were 20.07%, 26.16% and 29.77% at 12 h, 24 h, and 48 h, respectively. Meanwhile, the apoptotic rate in HSCs exposed to FRNK plasmid for 48 h was higher than that in the blank plasmid group (25.37%±1.92% vs 9.28%±1.05%), P < 0.01. The morphology changes of HSCs observed by transmission electron microscopy also revealed that the apoptosis of HSCs was increased by FRNK. Apoptotic DNA Ladder was detected in HSCs exposed to FRNK. The percentage of cells at G0/G1 phase was significantly higher in FRNK plasmid group than that in blank plasmid group (71.4±2.81 vs 48.9±1.66), P < 0.01. Finally, it was also demonstrated that FRNK reduced the expression of Cyclin D1, CDK4 at the level of both mRNA and protein. Conclusion: FRNK transfection mediated by liposomes induced the overexpression of exogenous FRNK in HSCs, inhibited the phosphorylation of FAK, arrested HSCs at G0/G1 phase, inhibited the proliferation and induced the apoptosis of HSCs. The inhibition of FAK phosphorylation may be a potential therapeutic strategy for preventing liver fibrogenesis.
04D. MOLECULAR AND CELLULAR BIOLOGY – D) LIVER REGENERATION AND EXPERIMENTAL ONCOLOGY
505 INDUCTION OF PANCREATIC BETA-CELL PHENOTYPE IN LIVER OF DIABETES MICE M. Vorobeychik1 , L. Bachmetov2 , K. Bloch1 , P. Vardi1 , R. Zemel2 , R. Tur-Kaspa2,3 . 1 Diabetes Research Lab, 2 Molecular Hepatology Lab. Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Rabin Medical Center, Petach Tikva, 3 Departments of Medicine D & Liver Institute, Rabin Medical Center, Petach Tikva, Israel E-mail: [email protected] The presence of insulin expressing cells in livers of mice with streptozotocin (STZ) diabetes was recently described. Nevertheless, the type of cell populations in the liver possessing the ability to undergo differentiation towards pancreatic beta cell phenotype is not yet determined. The aim of our study was to determine the liver cells population, which acquires pancreatic beta cell specific markers upon induction of diabetes in mice. Diabetes was induced by a single injection of streptozotocin (150 mg/kg body mass). After 3 weeks of persistent hyperglycemia, livers were collected for immunohistohemical analysis and oval cell isolation. Expression of oval cell antigen (A-6) and proinsulin was identified by doubleimmunofluorescence staining with anti-A-6 and anti-proinsulin antibodies on paraffin sections. RT and Real-time PCR analysis was performed to evaluated expression of pancreatic gene specific expression. We have demonstrated an increased periportal number and density of A-6 positive oval cells in diabetic mouse liver in comparison to the intact controls. Diabetic liver-derived non mature cell population was characterized by increased expression of oval cell transcripts CK-19 and CD-34. In contrast to intact mice, intensive staining for proinsulin was found in periportal cell population of livers isolated from diabetic mice. Immunohistochemical co-localization of oval cell marker (A-6) and proinsulin in the same population of liver cells, reflects a possible role for
S191
activated oval cells in pancreatic genes expression in diabetic mice. RT and Real-time PCR analysis confirmed the presence of pancreatic betacell transcripts PDX-1 and Insulin1 in freshly-isolated liver oval cells in contrast to oval cells isolated from intact mice. In conclusion our preliminary results suggest that hepatic oval cells derived from diabetic animals have a potential to express beta-cell like phenotype. We speculate that induction of Diabetes activates liver oval cells towards pancreatic phenotype. 506 HEPATOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO S.H. Bae1 , J.Y. Choi1 , S.K. Yoon1 , K.H. Yoon1 , B.S. Kim1 , I.H. Oh2 . 1 Department of Internal Medicine, Kangnam St. Mary Hospital, Seoul, 2 Catholic High Performance Cell Therapy Center, The Catholic University of Korea, South Korea E-mail: [email protected] The bone marrow contains stem cells that have the potential to differentiate into a variety of organ specific mature cells including for example the liver. Recently, the origin of hepatic progenitors and hepatocytes was identified to be the bone marrow. However, evidence that describes which cells, among all bone marrow cells, differentiate into hepatocytes, has not yet been presented. To effectively induce hepatogenic differentiation, we designed a 2-step protocol with the use of hepatocyte growth factor, oncostatin M, and dexamethasone. After 3 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion. Rat F344 DPPIV(−) with liver injury induced by bile duct ligation were injected with 1 x 106 mesenchymal stem cells DPPIV(+) via direct liver injection. PCR analysis for the Y chromosome was done on DNA extracted from transplanted female liver with primer for sry gene of the Y chromosome. Comparing to PBS group, significant improvements in serum level of albumin, AST/ALT, r-GTP, total bilirubin and direct bilirubin were observed at 3 weeks in the group of hepatocyte-like cells from MSCs. Co-expression of peptidase dipeptidyl peptidase IV (CD26) and albumin in recipeint liver tissue was confirmed by double immunostaining. Mesenchymal stem cells can be differentiated to functional hepatocyte-like cells with 2-step protocol in vitro, and the differentiated MSCs restore the liver function in the liver injury model induced by bile duct ligation in vivo. In conclusion, Bone marrow mesenchymal stem cells have the potential to differentiate toward hepatocyte of endodermal origin in vitro and in vivo liver injury model. 507 ROLE OF MIR-21 IN REGENERATING RAT LIVER R.E. Castro1,2 , D.M. Santos1 , Y. Zeng3 , C.J. Steer2,4 , C.M. Rodrigues1 , B.T. Kren2 . 1 iMed, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal, 2 Department of Medicine, 3 Department of Pharmacology, 4 Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, USA E-mail: [email protected] Background and Aims: Much progress has been made in understanding the remarkable ability of the liver to regenerate; however, significant gaps still exist. New pathways of gene regulation coordinated via microRNAs (miRNAs), may help to establish how regeneration factors stimulate liver cells so rapidly after surgical resection, and what mechanisms are involved in establishing the final mass-to-liver ratio. This study was designed to determine if miRNAs were modulated during liver regeneration (LR) in rat following 70% partial hepatectomy (PH), and elucidate their potential role in that process.
04D. MOLECULAR AND CELLULAR BIOLOGY – D) LIVER REGENERATION AND EXPERIMENTAL ONCOLOGY
505 INDUCTION OF PANCREATIC BETA-CELL PHENOTYPE IN LIVER OF DIABETES MICE M. Vorobeychik1 , L. Bachmetov2 , K. Bloch1 , P. Vardi1 , R. Zemel2 , R. Tur-Kaspa2,3 . 1 Diabetes Research Lab, 2 Molecular Hepatology Lab. Felsenstein Medical Research Center, Sackler School of Medicine, Tel-Aviv University, Rabin Medical Center, Petach Tikva, 3 Departments of Medicine D & Liver Institute, Rabin Medical Center, Petach Tikva, Israel E-mail: [email protected] The presence of insulin expressing cells in livers of mice with streptozotocin (STZ) diabetes was recently described. Nevertheless, the type of cell populations in the liver possessing the ability to undergo differentiation towards pancreatic beta cell phenotype is not yet determined. The aim of our study was to determine the liver cells population, which acquires pancreatic beta cell specific markers upon induction of diabetes in mice. Diabetes was induced by a single injection of streptozotocin (150 mg/kg body mass). After 3 weeks of persistent hyperglycemia, livers were collected for immunohistohemical analysis and oval cell isolation. Expression of oval cell antigen (A-6) and proinsulin was identified by doubleimmunofluorescence staining with anti-A-6 and anti-proinsulin antibodies on paraffin sections. RT and Real-time PCR analysis was performed to evaluated expression of pancreatic gene specific expression. We have demonstrated an increased periportal number and density of A-6 positive oval cells in diabetic mouse liver in comparison to the intact controls. Diabetic liver-derived non mature cell population was characterized by increased expression of oval cell transcripts CK-19 and CD-34. In contrast to intact mice, intensive staining for proinsulin was found in periportal cell population of livers isolated from diabetic mice. Immunohistochemical co-localization of oval cell marker (A-6) and proinsulin in the same population of liver cells, reflects a possible role for
S191
activated oval cells in pancreatic genes expression in diabetic mice. RT and Real-time PCR analysis confirmed the presence of pancreatic betacell transcripts PDX-1 and Insulin1 in freshly-isolated liver oval cells in contrast to oval cells isolated from intact mice. In conclusion our preliminary results suggest that hepatic oval cells derived from diabetic animals have a potential to express beta-cell like phenotype. We speculate that induction of Diabetes activates liver oval cells towards pancreatic phenotype. 506 HEPATOGENIC DIFFERENTIATION OF RAT BONE MARROW MESENCHYMAL STEM CELLS IN VITRO AND IN VIVO S.H. Bae1 , J.Y. Choi1 , S.K. Yoon1 , K.H. Yoon1 , B.S. Kim1 , I.H. Oh2 . 1 Department of Internal Medicine, Kangnam St. Mary Hospital, Seoul, 2 Catholic High Performance Cell Therapy Center, The Catholic University of Korea, South Korea E-mail: [email protected] The bone marrow contains stem cells that have the potential to differentiate into a variety of organ specific mature cells including for example the liver. Recently, the origin of hepatic progenitors and hepatocytes was identified to be the bone marrow. However, evidence that describes which cells, among all bone marrow cells, differentiate into hepatocytes, has not yet been presented. To effectively induce hepatogenic differentiation, we designed a 2-step protocol with the use of hepatocyte growth factor, oncostatin M, and dexamethasone. After 3 weeks of induction, cuboidal morphology, which is characteristic of hepatocytes, was observed, and cells also expressed marker genes specific of liver cells in a time-dependent manner. Differentiated cells further demonstrated in vitro functions characteristic of liver cells, including albumin production, glycogen storage, urea secretion. Rat F344 DPPIV(−) with liver injury induced by bile duct ligation were injected with 1 x 106 mesenchymal stem cells DPPIV(+) via direct liver injection. PCR analysis for the Y chromosome was done on DNA extracted from transplanted female liver with primer for sry gene of the Y chromosome. Comparing to PBS group, significant improvements in serum level of albumin, AST/ALT, r-GTP, total bilirubin and direct bilirubin were observed at 3 weeks in the group of hepatocyte-like cells from MSCs. Co-expression of peptidase dipeptidyl peptidase IV (CD26) and albumin in recipeint liver tissue was confirmed by double immunostaining. Mesenchymal stem cells can be differentiated to functional hepatocyte-like cells with 2-step protocol in vitro, and the differentiated MSCs restore the liver function in the liver injury model induced by bile duct ligation in vivo. In conclusion, Bone marrow mesenchymal stem cells have the potential to differentiate toward hepatocyte of endodermal origin in vitro and in vivo liver injury model. 507 ROLE OF MIR-21 IN REGENERATING RAT LIVER R.E. Castro1,2 , D.M. Santos1 , Y. Zeng3 , C.J. Steer2,4 , C.M. Rodrigues1 , B.T. Kren2 . 1 iMed, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal, 2 Department of Medicine, 3 Department of Pharmacology, 4 Department of Genetics, Cell Biology, and Development, University of Minnesota Medical School, Minneapolis, MN, USA E-mail: [email protected] Background and Aims: Much progress has been made in understanding the remarkable ability of the liver to regenerate; however, significant gaps still exist. New pathways of gene regulation coordinated via microRNAs (miRNAs), may help to establish how regeneration factors stimulate liver cells so rapidly after surgical resection, and what mechanisms are involved in establishing the final mass-to-liver ratio. This study was designed to determine if miRNAs were modulated during liver regeneration (LR) in rat following 70% partial hepatectomy (PH), and elucidate their potential role in that process.