• Molecules and Cells
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• 제42권1호
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• pp.45-55
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• 2019

The liver is involved in a wide range of activities in vertebrates and some other animals, including metabolism, protein synthesis, detoxification, and the immune system. Until now, various methods have been devised to study liver diseases; however, each method has its own limitations. In situ liver perfusion machinery, originally developed in rats, has been successfully adapted to mice, enabling the study of liver diseases. Here we describe the protocol, which is a simple but widely applicable method for investigating the liver diseases. The liver is perfused in situ by cannulation of the portal vein and suprahepatic inferior vena cava (IVC), with antegrade closed circuit circulation completed by clamping the infrahepatic IVC. In situ liver perfusion can be utilized to evaluate immune cell migration and function, hemodynamics and related cellular reactions in each type of hepatic cells, and the metabolism of toxic or other compounds by changing the composition of the circulating media. In situ liver perfusion method maintains liver function and cell viability for up to 2 h. This study also describes an optional protocol using density-gradient centrifugation for the separation of different types of hepatic cells, allowing the determination of changes in each cell type. In summary, this method of in situ liver perfusion will be useful for studying liver diseases as a complement to other established methods.

• Biomolecules & Therapeutics
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• 제13권3호
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• pp.127-132
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• 2005

We investigated whether ischemic preconditioning (IPC) protects liver against cold ischemic injury using isolated perfused rat liver. Rat livers were preconditioned by 5 minutes of ischemia and 5 minutes of reperfusion and preserved for 30 hours at $4^{\circ}C$ in University of Wisconsin solution. Livers were then reperfused for 120 minutes. Oxygen uptake and bile flow in ischemic livers markedly decreased during reperfusion. These decreases were prevented by IPC. Portal pressure was elevated in cold ischemic and reperfused livers and this elevation was prevented by IPC. Lactate dehydrogenase and purine nucleoside phosphorylase activities markedly increased during reperfusion. These increases were prevented by IPC. The ratio of reduced glutathione to glutathione disulfide was lower in ischemic livers. This decrease was prevented by IPe. Our findings suggest that IPC protects the liver against the deleterious effect of cold ischemia/reperfusion, and this protection is associated with the reduced oxidative stress.

• Archives of Pharmacal Research
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• 제23권6호
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• pp.620-625
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• 2000

The mechanisms of liver injury from cold storage and reperfusion are not completely under-stood. The aim of the present study was to investigate whether the inactivation of Kupffer cells (KCs) by gadolinium chloride ($GdCl_3$) modulates ischemia-reperfusion injury in the rat liver. Hepatic function was assessed using an isolated perfused rat liver model. In livers subjected to cold storage at $4^{\circ}C$ in University of Wisconsin solution for 24 hrs and to 20 min rewarm-ing ischemia, oxygen uptake was markedly decreased, Kupffer cell phagocytosis was stimulated, releases of purine nucleoside phosphorylase and lactate dehydrogenase were increased as compared with control livers. Pretreatment of rats with $GdCl_3$) , a selective KC toxicant, suppressed kupffer cell activity, and reduced the grade of hepatic injury induced by ischemia-reperfusion. While the initial mixed function oxidation of 7-ethoxycoumarin was not different from that found in the control livers, the subsequent conjugation of its meta-bolite to sulfate and glucuronide esters was suppressed by ischemia-reperfusion, CdCl$_3$restored sulfation and glucuronidation capacities to the level of the control liver. Our findings suggest that Kupffer cells could play an important role in cold/warm ischemia-reperfusion hepatic injury.

• 대한예방의학회:학술대회논문집
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• 대한예방의학회 1999년도 제51차 추계 학술대회 연제집
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• pp.193-194
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• 1999

• 대한자기공명의과학회:학술대회논문집
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• 대한자기공명의과학회 1997년도 제2차 추계학술대회 초록집
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• pp.15-15
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• 1997

• BMB Reports
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• 제33권3호
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• pp.213-220
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• 2000

A fulminant hepatitis is associated with massive liver cell necrosis and a high mortality rate. But survivors regenerate a normal liver and do not have chronic liver disease. This clinical course suggests that the acutely injured livers release a factor that allows a recovery from chronic hepatitis or cirrhosis. The objective of this study was to isolate and characterize an anti-fibrotic factor from acutely damaged rat livers. The liver cell necrosis was prepared from rat by warm ischemical perfusion and the perfusates were assessed against the growth inhibition of fat-storing cells (FSC). A liver-derived growth inhibitory factor (LDGIF) was purified from ischemically damaged rat livers by chromatographies on Sephacryl S-300, CM Sepharose, hydroxyapatite, and Superose 12. The LDGIF was isolated with an overall purification of 194-fold and 40% recovery. Although LDGIF was identified as the rat liver arginase by Nterminal sequence analysis, LDGIF exists as a monomer and the purified native arginase has a trimer form. Furthermore, LDGIF has a lower enzyme activity on the hydrolysis of L-arginine and a higher inhibitory effect on proliferation of FSC than the normal rat liver arginase. The catalytic activity of LDGIF is ascribed to the monomeric characteristics of the LDGIF. Therefore, the inhibitory action of LDGIF might not be due to the arginine depletion by the catalytic activity of arginase. In conclusion, the presence of the LDGIF could interpret the clinical course that serious fibrosis is not found in the liver of patients recovering from severe hepatic necrosis due to fulminant hepatitis, suggesting that this LDGIF may provide a novel target for the prevention and treatment of hepatic fibrosis.

• Biomolecules & Therapeutics
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• 제5권3호
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• pp.233-238
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• 1997

The distribution and excretion of radioactivity were examined after intraportal administration of sup 166/Ho-chitosan complex at a dose of 1 mcitg (10 mg chitosan/kg) to rats. Whole body macroautoluminographs showed that the radioactivity after an administration was concentrated in liver and perfused primarily to organs including kidney, spleen, and bone marrow, then to muscle and brain. Similar profiles were observed from 2 hr to 168 hr after the administration. The relative percentage of radioactivity in bone and spinal column increased with time, suggesting that free $^{166}$ Ho, released from chitosan complex deposited in the liver, selectively binds to these tissues. $^{166}$ Ho-chitosan complex administered intraportally was excreted less than 4% through urine (2.7$\pm$0.8%) and feces (0.65 $\pm$ 0.4%) up to seven days. These results demonstrate that the radio-activity of $_{166}$ Ho-chitusan complex when administered intraportally, mainly localizes in liver without affec-ting other tissues and organs. Considering the short half life of $^{166}$ Ho and the localization to the liver, $^{166}$ Ho-chitosan complex might be a useful agent in the treatment of hepatic carcinoma.

• 한국응용약물학회:학술대회논문집
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• 한국응용약물학회 1995년도 춘계학술대회
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• pp.128-128
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• 1995

To investigate the mechanism of the regulation of cytochrome P450IA1 gene expression, ethoxyresorufin deethylase(EROD) and benzo(a)pyrene hydroxylase in B6 mouse liver, in isolated perfused rat liver system. and in B6 mouse hepatocyte Hepa-I cells were examined. In C57BL/6N mouse, 3-methylcholan- throne( 3MC ) treatment have resulted in the stimulation of EROD activity based on fluorometry by 2.79 fold comparirng with that of control. Measurement of mRNA of cytochrome P450 was carried out by either nothern blot or dot blot analysis. Findings are similar to that of studies with enzymes. Furhtermore, when RTPCR method was applied to detect mRNA in Hepa I cell and liver tissues the results were more clear. Cytochrome P450IA1 upstream DNA containing CAT construct was transfected into Hepa-1 cells. After transfection of CAT construct, 3MC and flavonoids, such as, chrysin, hesperetin, kaempferol, morin, myricetin and aminoyrine were treated. 48 Hours after treatments, cells were harvested and assayed for CAT mRNA by RTPCR. 3MC treatment to hepa I cells transfected with trout P450IA1-CAT construct increased CAT mRNA by 2.81 fold when it was compared with that of control. This increase CAT mRNA was decreased by concomitantly treated flavonoids and aminopyrine. The level of CAT protein was 29.2-58.0% of 3MC stimulated CAT protein. Results of this study suggested that RTPCR seems to be a very good method to study regulation of gene expression in liver tissue or Hepa cells.

• 한국산업보건학회지
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• 제6권1호
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• pp.28-37
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• 1996

Benzidine, an aromatic amine used primarily in the manufacture of azo dyes, is recognized as a urinary bladder carcinogen in humans. In rats, mice, and hamsters, chronic exposure to benzidine resulted in tumors of the liver. The present study was undertaken to suggest analyzing the metabolites of benzidine with the optimal condition, identify the metabolites of benzidine, and observe time variance of the metabolites in the isolated perfusated rat liver. N-acetylbenzidine was synthesized by acetylation of benzidine with acetic anhydride and separated by thin layer chromatography(TLC) and high performance liquid chromatography(HPLC). To analysis benzidine and the metabolites of benzidine, HPLC operating condition has been optimized by means of preliminary experiment. The mobile phase consisted of acetonitrile(37%) in phosphate buffer, flow rate maintained at 1.0 ml/min. Optimal detective conditions were electrochemicaldetector(ECD) at 0.75 V for benzidine and N-acetylbenzidine and ultravioletdetector(UVD) at 287 nm for N,N'-diacetylbenzidine. The separation system was composed of a guard column and a separation column(Polymer C18, $4.6{\times}250cm$) at a temparature of $40^{\circ}C$. The perfusion system was equilibrated for 30 minutes before addition of benzidine to the perfusate. Samples of the perfusate were collected at time intervals(0, 10, 20, 30, 60, 90, 120 min) during the 2 hour perfusion. Before analyzing samples by HPLC/ECD/UVD, samples had been treated with sep-pak. Samples of perfusate analyzed by HPLC/ECD/UVD and the metabolites of benzidine in the isolated perfused rat liver were N-acetylbenzidine and N,N'-diacetylbenzidine. Benzidine metabolized over 60% during the initial 30 minutes of perfusion, extensively by 1 hour, and was undetectable in the perfusate. N-acetylbenzidine increased by 30 minutes of perfusion, declined. N,N'-diacetylbenzidine increased the 0-90 minutes period, remained constant during the 90-120 minutes period.

• Journal of Preventive Medicine and Public Health
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• 제33권1호
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• pp.117-124
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• 2000

목적 : 이 연구의 목적은 적출간 관류법을 통하여 염화니켈과 염화카드뮴의 간 독성을 비교하는 데에 두었다. 방법 : AST(aspartate aminotransferase ferase), ALT(alanine aminotransferase), LDH(lactate dehydrogenase) 등의 간기능을 나타내는 생화학적 지표와 관류속도를 간독성 지표로 이용하였으며 산소 소모율은 생존력을 나타내는 지표로 이용하였다. $300({\pm}50)g$ 정도의 흰쥐를 대조군과 각 노출군($50{\mu}M$ 및 $200{\mu}M$의 염화니켈과 염화카드뮴)에 5마리씩 총 25마리를 무작위로 할당하였다. Krebs-Ringer bicarbonate buffer solution을 관류액 용도로 제조하여 간문맥에 투입하고 간세포를 통과하여 vena cava로 배출되는 관류액을 일정한 시간 간격을 두고 계속적으로 채취하였으며 채취한 관류액은 간독성 지표를 측정하는 데에 이용하였다. 결과 : AST, ALT, LDH 모두 염하니켈 보다는 염화카드뮴 투여군에서 시간의 경과에 따라 큰 폭으로 증가하였으며 반복 측정된 2요인 분산분석을 실시한 결과, 통계적으로 유의한 차이를 나타냈다. 생존력은 두 물질 모두에서 시간의 경과에 따라 감소하는 경향을 나타냈다. 결론 : 결론적으로, 이 실험상에서는 염화니켈 보다 염화카드뮴의 간독성이 더욱 강한 것으로 드러났다. 적출간 더욱 강한 것으로 드러났다. 적출간 관류법의 특성상 급성 간독성 평가에서 적절한 기법으로 보이며 국내 연구에서도 이 방법이 널리 이용되기를 기대한다.