• Animal Bioscience
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• v.35 no.11
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• pp.1787-1799
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• 2022

Objective: Choline deficiency, one main trigger for nonalcoholic fatty liver disease (NAFLD), is closely related to lipid metabolism disorder. Previous study in a choline-deficient model has largely focused on gene expression rather than gene structure, especially sparse are studies regarding to alternative splicing (AS). In modern life science research, primary hepatocytes culture technology facilitates such studies, which can accurately imitate liver activity in vitro and show unique superiority. Whereas limitations to traditional hepatocytes culture technology exist in terms of efficiency and operability. This study pursued an optimization culture method for duck primary hepatocytes to explore AS in choline-deficient model. Methods: We performed an optimization culture method for duck primary hepatocytes with multi-step digestion procedure from Pekin duck embryos. Subsequently a NAFLD model was constructed with choline-free medium. RNA-seq and further analysis by rMATS were performed to identify AS events alterations in choline-deficency duck primary hepatocytes. Results: The results showed E13 (embryonic day 13) to E15 is suitable to obtain hepatocytes, and the viability reached over 95% by trypan blue exclusion assay. Primary hepatocyte retained their biological function as well identified by Periodic Acid-Schiff staining method and Glucose-6-phosphate dehydrogenase activity assay, respectively. Meanwhile, genes of alb and afp and specific protein of albumin were detected to verify cultured hepatocytes. Immunofluorescence was used to evaluate purity of hepatocytes, presenting up to 90%. On this base, choline-deficient model was constructed and displayed significantly increase of intracellular triglyceride and cholesterol as reported previously. Intriguingly, our data suggested that AS events in choline-deficient model were implicated in pivotal biological processes as an aberrant transcriptional regulator, of which 16 genes were involved in lipid metabolism and highly enriched in glycerophospholipid metabolism. Conclusion: An effective and rapid protocol for obtaining duck primary hepatocytes was established, by which our findings manifested choline deficiency could induce the accumulation of lipid and result in aberrant AS events in hepatocytes, providing a novel insight into various AS in the metabolism role of choline.

• Korean Journal of Veterinary Service
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• v.31 no.4
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• pp.457-463
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• 2008

The sex steroid hormone estradiol-$17{\beta}(E_2)$ mediate their biological effects on development, differentiation and maintenance of reproductive tract and other target tissue through gene regulation by nuclear steroid receptors. Although the importance of $E_2$ in many physiological process has been reported, but little is known about the effects of $E_2$ on primary cultured chicken hepatocyte. therefore, in the present study, we have examined the effect of $E_2$ on cell proliferation and it's related signal cascades. $E_2$ increase $[^3H]$-thymidine incorporation in time-(${\leq}8hr$) and dose-($10^{-10}M$)dependent manner and treatment of $E_2$ increased the phosphorylation of p44/43 MAPKs(p44/42 mitogen-activated protein kinase) and JNK(c-Jun N-terminal kinase) in a time dependent manner. In addition, PD98059(p44/42 blocker, $10^{-5}M$), SP600125(JNK blocker, $10^{-6}M$) blocked the estrogen-induced increase in $[^3H]$-thymidine incorporation. In conclusion, $E_2$ stimulates the proliferation of primary cultured chicken hepatocytes and this action is mediated by p44/42 MAPKs and JNK signal transduction pathway.

• KSBB Journal
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• v.16 no.1
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• pp.24-29
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• 2001

Recently hepatocyte-based bioartificial liver (BAL) and hepatocyte transplantation have been actively investigated to treat acute hepatic failure. The BAL acts as a bridge to provide patients with more time until a donor organ becomes available for transplantation or until their own liver can be regenerated. In this study, we manufactured a polyurethane foam (PUF) using 15% NCO-prepolymer with a pore opening that allows it to be used as a hepatocyte immobilizing material. Cubes of PUF (3 mm dim.) were seeded with rat primary hepatocytes at a density of 5.5$\pm$1.1$\times$ $10^6$ cells/$cm^3$ PUF by centrifuging them together. The cell laden PUF cubes were packed into a prototype reactor and perfused with a hormonally defined medium for a week. Hepatocytes in the pores of the PUF formed spheroids that showed stable ammonia removal and urea synthesis activities. The albumin production level was comparable to other BAL systems. The PUF packed hepatocyte bioreactor has the potential to be used as a BAL.

• Korean Journal of Food Science and Technology
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• v.41 no.5
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• pp.597-601
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• 2009

Activation of hepatic stellate cells (HSCs) is known to be responsible for hepatic fibrosis and cirrhosis. When round-shape quiescent HSCs go to activation by liver injury, production of extracellular matrix is increased, and its shape becomes myofibroblast-like shape. The activated HSCs are characterized by the high rate of proliferation and the increased production of extracellular matrix. One way of the regeneration of activated HSCs is an apoptosis induction followed by removing the activated myofibroblast-like cells. The effect of extract of Terminalia chebula Retz. (TCE) on cytotoxicity was evaluated using the rat primary hepatocyte, HepG2 and T-HSC/Cl-6 by incubating these cells with TCE up to the dose of $1,000{\mu}g/mL$. At the maximum dose of TCE, no cytotoxicity was found on primary hepatocyte and HepG2, but cytotoxic effect of TCE was found on activated HSCs, and T-HSC/Cl-6 in a U-shaped dose-response manner with the highest effect at $500{\mu}g/mL$ of TCE. Finally, we confirmed the occurrence of apoptotic cell death by annexin-V/PI double staining. The population of annexin-V positive cells was increased in a dose dependent manner.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.6
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• pp.565-570
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• 1999

Intracellular accumulation of bile acids in the hepatocytes during cholestasis is thought to be pathogenic in cholestatic liver injury. Due to the detergent-like effect of the hydrophobic bile acids, hepatocellular injury has been attributed to direct membrane damage. However histological findings of cholestatic liver diseases suggest apoptosis can be a mechanism of cell death during cholestatic liver diseases instead of necrosis. To determine the pattern of hepatocellular toxicity induced by bile acid, we incubated primary cultured rat hepatocytes with a hydrophobic bile acid, Glycochenodeoxycholate (GCDC), up to 5 hours. After 5 hours incubation with $400\;{\mu}M$ GCDC, lactate dehydrogenase released significantly. Cell viability, quantitated in propidium iodide stained cells concomitant with fluoresceindiacetate was decreased time- and dose-dependently. Most nuclei with condensed chromatin and shrunk cytoplasm were heavily labelled time- and dose-dependently by a positive TUNEL reaction. These findings suggest that both apoptosis and necrosis are involved in hepatocytes injury caused by GCDC.

• Toxicological Research
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• v.8 no.2
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• pp.285-302
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• 1992

Human and rat hepatocytes were isolated by nonperfusion method and cultured for longer than 5 days. Human liver biopsy sample and rat liver were used as hepatocyte source. Several physical and chemical factors which were influencing on hepatocyte isolation procedure were examined and a batch isolation procedure was established for small size sample of rat liver. Isolated hepatocytes showed normal morphlologica characteristics in microscopy and electron microscopical examinations and a morphologica response to phalloidin. Isolated cells were cultured as a monolayer and proven to have intact morphological characteristics for longer than 15 days. Because human liver sample is harder and tighter compared with rat liver, a standard procedure for rat hepatocytes was slightly modified to reduce mechanical damage. Similarly with rat hepatocytes, isolated human hepatocytes showed a normal morphological characteristics and could be cultured for longer than 15days. Human and rat hepatocytes were examined on their functional integrities including cytochrome-P450 related enzyme activity and it's inducibility, hormonal inducibility of AIB uptake and TAT activity, albumin synthesis, DNA synthesis, cellular protein maintenance. In all parameters used in the present study, human and rat hepatocytes showed normal functional characteristics.

• Journal of Aquaculture
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• v.11 no.4
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• pp.557-564
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• 1998

Vitellogenin (VTG) induction in response estradiol-17${\beta}$ ($E_2$) were electrophoretically examined in primary hepatocyte cultures in rainbow trout. The hepatocytes were maintained as monolayers on positively charged dishes for up to 7 days. The viability of hepatocytes on Day 7 in cultures decreased about 20.7% and 23.6% with and without $E_2$, respecitively. The amount of DNA per dish also decreased to 13.7% and 14.0% with and without $E_2$, respectively. There were no differences in viability and DNA content between the control and $E_2$-treated culture. Moreover, the rate of VTG to total protein concentrations reached the maxium level at the addition of $10^{-6}$ M E2, to the incubation medium. However, the higher concentration of $10^{-5}$ M $E_2$ rater depressed the level.

• YAKHAK HOEJI
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• v.52 no.1
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• pp.56-61
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• 2008

The hepatoprotective effects of the water extracts of Hovenia dulcis Thunb (HD) were investigated in vitro. Following the induction of hepatotoxicity by ethanol in primary cultures of rat hepatocytes, the protective effects of four different water extracts of HD were determined through serial dose-response and time-dependent studies. The individual extracts used in these studies were prepared from fruits, seeds, leaves and tubes. Treatment of hepatocyte cultures with the water extracts of HD provided a significant protection from the increased lactate dehydrogenase activity induced by ethanol. Particularly, the fruits extract was the most effective against ethanol-induced hepatotoxicity in the primary cultures of rat hepatocytes. The results demonstrated that the extracts might have the protective effect against ethanol-induced toxicity in hepatocyte cultures.

• Archives of Pharmacal Research
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• v.13 no.4
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• pp.355-358
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• 1990

In order to investigate the cellular mechanisms of hypoglycemic of brazilin, hepatocyte monolayer culture was introduced and, glycogen synthesis rate and insulin binding were measured as parameters. Glycogen synthesis and insulin sensitivity were remarkably augmented by the treatment of brazilin. Brazilin slightely increased insulin binding. Scatchard analysis revealed that this increase in insulin binding was not due to increase in the binding capacity but in binding affinity. These results suggest that the augmentation of hepatic glycogenesis and insulin sensitivity by brazilin may play an important role in the improvement of hyperglycemia.

• Korean Journal of Environmental Agriculture
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• v.22 no.2
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• pp.94-99
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• 2003

The objective of present study was to investigate the antioxidative and hepatoprotective effects of selenium on cadmium-induced toxicity and lipid peroxidation in rat hepatocyte primary culture. To do this, two separate experiments were conducted. In Experiment 1, primary cultures of rat hepatocytes were incubated for 6 hr in the presence of various concentrations (1, 10, 50, 100, and $500\;{\mu}M$) of cadmium chloride. Cytotoxicity and lipid peroxidation were evaluated using the MTT assay and TBARS assay, respectively. Antioxidative and hepatoprotective effects were determined by measuring the activity of GOT and GSH-Px, respectively. Cell viability was reduced and lipid peroxidation was increased by cadmium in dose-dependent manners. There was significantly negative correlation (r=-0.943, p<0.01) between cell viability and lipid peroxidation GOT activity was increased and GSH-Px activity was decreased by cadmium at the concentration of $50\;{\mu}M$. In Experiment 2, primary cultures of rat hepatocytes were incubated for 6hr in the presence of 100\;{\mu}M$ of cadmium chloride and various concentrations (0.01, 0.1 and 1 ppm) of sodium selenite to assess the effect of selenium on cadmium-induced toxicity and lipid peroxidation. Cell viability and GSH-Px activity were increased by sodium selenite at the concentration of 1 ppm Whereas, lipid peroxidation and GOT activity were reduced by 0.1 ppm of sodium selenite. These results demonstrate that selenium has an antioxidative and hepatoprotective potentials against cadmium.