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- 저자 : Yurianna Shin (검색결과 3 건)
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Imran Ullah,옥선아,김영지,임맑음,오건봉,황성수,Yurianna Shin,김영임,임기선,허태영 한국통합생물학회 2017 Animal cells and systems Vol.21 No.5
Primary hepatocytes (PHs) are considered the ‘gold standard’ in drug screening owing to their ability to express many drug-metabolizing enzymes and transporters. Culturing hepatocytes and maintaining their fate in vitro is a major issue since last decade. The main problem with in vitro hepatocytes culture is that they rapidly lose their hepatic morphology and liver-specific functions in culture. Herein, we isolated rat PHs, and cultured them in monolayers (2-D) and spheroids (3-D). The 2-D-cultured PHs exhibited elongated morphology, whereas the 3-Dcultured PHs exhibited spheroid morphology with gradual diameter decrease until 7 days. After 7 days of in vitro culture, PHs were analyzed for the expression of hepatic (Alb, Tf, and Afp) and apoptotic markers (Bax and Bcl2), and co-expression of CYP3A1 and Abumin after 2 and 7 days. Furthermore, in both cultures, PHs were induced with 3-methylcholanthrene (3-MC, Cyp1a-specific inducer) and dexamethasone (Cyp3a-specific inducer) for 48 and 72 h, respectively. The mRNA levels of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PHs. After 7 days of in vitro culture, PHs exhibited dramatic downregulation of hepatic marker expression in both cultures. Furthermore, apoptotic marker expression was higher in the 2-D-cultured PHs than 3-D-cultured PHs. The mRNA levels of Cyp1a and Cyp3a indicated higher RNA content in the 2-D-cultured PHs after 48 h of induction. Therefore, we concluded that there was no significant difference between the culture systems, and further studies are required to identify the essential components for in vitro PH culture rather than culture systems.
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Small Molecules-Driven Pancreatic ß-Cells Differentiation of Bone Marrow Mesenchymal Stem Cells
Imran Ullah,Keon Bong Oh,Yurianna Shin,Youngim Kim,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 발생공학 국제심포지엄 및 학술대회 Vol.2017 No.10
Diabetes mellitus, a hyperglycemic condition, in which the patients either fail to secrete insulin because of ß-cells destruction (type I) or shows insulin resistance (type II), affecting more than 300 million people around the globe. Generation of effective ß-cells for the treatment of diabetes is a key area in modern translational medicines. Here we tried to generate porcine induced pancreatic ß-cells (piPan ß-cells) from Gal- TKO+MCP porcine bone marrow mesenchymal stem cell (pBM-MSCs) by overexpressing set of transcription factors included EGFP along with step-wise induction of small molecules. pBM-MSCs were cultured in media with 5-azacytidine for 24 hours and then proceeded with transfection using episomal transfection system. Transfected cells were replaced with basal media containing growth factors cocktail, Activin A and 2 uM of A83-01 followed by retinoic acid treatment. Finally, cells were replaced by maintenance media until maturation. After 3 days of transfection, EGFP expression was observed showing successful transfection which disappeared completely after 21 days. pBM-MSCs attained definitive endoderm morphology after Activin A treatment and formed exact pancreatic clusters after 4 weeks and 8 weeks in maintenance media, respectively. After 4 and 8 weeks, piPan ß-cells were analyzed for the expression of pancreatic markers by immunofluorescence (Insulin, PAX6, Somatostatin) and RT-qPCR (GCG, INS, NKX6.1, PDX1 and NEUROD1), which showed significantly higher expression as compared with pBM-MSCs. Morphological changes and expression of pancreatic markers revealed successfully differentiation of PiPan ß-cells; however, more in depth studies for its functional characteristic and in vivo maintenance are still in progress.
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In Vitro Culture of Rat Primary Hepatocytes Employing Monolayer and Spheroid Culture Environments
Imran Ullah,Yeongji Kim,Malgum Lim,Keon Bong Oh,Seongsoo Hwang,Yurianna Shin,Youngim Kim,Gi-Sun Im,Tai-Young Hur,Sun A Ock 한국동물생명공학회(구 한국동물번식학회) 2017 Reproductive & Developmental Biology(Supplement) Vol.41 No.2
Primary hepatocytes (PH) are considered as “gold standard” for drug screening because of its ability to express the entire set of drug metabolizing enzymes and transporters. Hepatocytes culturing and maintaining their hepatocyte fate in vitro is one of the major issue from last decade. The main problem in hepatocytes in vitro culturing is that, these cells rapidly loss their hepatic morphology and liver specific function in culture condition. In the present study, we isolated rat PH and cultured in monolayer (2D) as well as spheroid (3D) culture system. The 2D cultured PH hepatocyte showed an elongated hepatocyte morphology while, 3D cultured PH showed spheroid morphology with gradual decrease in diameter up to 7 days. After 7 days of in vitro culture, these cells were analyzed for the expression of hepatic markers (Alb, Tf, Afp) and apoptotic markers (Bax, Bcl2). Furthermore, PH in both culture systems was induced with two inducers i.e. 3-methylcholanthrene (3-MC, Cyp1a specific inducer) and dexamethasone (Cyp3a specific inducer) for 48 and 72 hours, respectively. The mRNA level of Cyp1a and Cyp3a were analyzed in induced (3-MC, dexamethasone) and non-induced PH, respectively. After 7 days in vitro culture, PH showed dramatic down regulation of hepatic markers in both culture systems. Furthermore, expression of apoptotic markers was higher in 2D cultured PH as compared to 3D. Cyp1a and Cyp3a mRNA level showed higher RNA content in 2D culture PH after 48 of induction. Therefore, we concluded that there was no significant difference found in two culture system and further studies are needed to find out the essential components for PH in vitro culture rather than culture system.
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