• Journal of Veterinary Clinics
• /
• v.14 no.1
• /
• pp.24-29
• /
• 1997

흰쥐 간정맥 및 간문맥에 PE10 튜브를 마취하에 이식시킨 후 5일째부터 고형사료와 10% creatine을 함유한 고형사료를 5일 동안 섭식 시켜 급속 냉동 시켰다. 냉동시킨 흰쥐 간장, 뇌 및 근육에서 creatine을 포함한 간장 metabolites을 조사하여 다음과 같은 결과를 얻었다. 또한 간장세포내의 water compartment의 변화를 동시에 실시 하였다. Creatine을 섭식시킨 실험군은 정상 대조군과 비교하여 섭식에 의한 간세포내의 water compartment 에 벼노하를 주지 않았고 간장 세포내의 creatine농도에 있어서는 약 43배 이상 세포내 증가를 유도하였으며, 이는 세포외 및 세포내의 creatine의 gradient에 의해 영향을 받고 있음을 알 수가 있었고, 또한 동시에 뇌, 근육 등에서는 섭식에 의해 유의성 있는 농도 변화는 보이지 않았다. 이는 세포 외액속에 함유된 creatine 이 여러 수송기전에 의하여 간세포내로 흡착이 이루어진 것을 알 수 있었으나, 간세포 분리 시험법에로 Creatine Kinase 효소 활성도를 측정한 결과 non-parenchymal hepatocytes에서만 거의 100% 효소 활성도가 있음을 증명 하였다.

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

• Proceedings of the KSMRM Conference
• /
• 1997.10a
• /
• pp.15-15
• /
• 1997

• The Korean Journal of Physiology and Pharmacology
• /
• v.3 no.6
• /
• pp.571-578
• /
• 1999

This study evaluated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) and PKC inhibition using the isoquinoline sulfomide derivative H-7 on hemodynamics and glucoregulation in the isolated perfused rat liver. Livers were isolated from fed male Holtzman rats and perfused with Krebs Ringer bicarbonate solution under a constant flow of 50 ml/min at $35^{\circ}C.$ Portal vein pressure, glucose and lactate concentrations in the medium and oxygen consumption rates were continuously monitored by a Grass polygraph, YSI glucose and lactate monitors, and a YSI oxygen monitor, respectively. PMA at concentration of 2 to 200 nM increased the portal vein pressure, glucose and lactate production, but decreased oxygen consumption rate in a dose-dependent fashion. H-7 $(200\;{\mu}M)$ attenuated PMA (50 nM)-induced vasoconstriction $(15.1{\pm}1.36\;vs\;10.56{\pm}1.17\;mmHg),$ glucose production rate $(91.3{\pm}6.15\;vs\;71.8{\pm}2.50\;{\mu}moles/g/hr),$ lactate production rate $(72.4{\pm}6.82\;vs\;53.6{\pm}4.82\;{\mu}moles/g/hr)$ and oxygen consumption rate $(33.7{\pm}1.41\;vs\;27.9{\pm}1.75\;{\mu}l/g/min).$ The effects of PMA were blocked either by addition of verapamil $(9\;{\mu}M)$ or perfusion with $Ca^{2+}-free$ KRB. These results suggest that the hemodynamic and glucoregulatory changes in the perfused rat liver are mediated by protein kinase C activation and require $Ca^{2+}$ influx from the extracellular fluid.

• Proceedings of the Korean Society of Applied Pharmacology
• /
• 1995.04a
• /
• pp.128-128
• /
• 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.

• Applied Microscopy
• /
• v.23 no.2
• /
• pp.53-77
• /
• 1993

In this study, an attempt was made to investigate the probable organelles participating in the secretion of biligrafin. The animals (ICR male mice, 25-30gm) were divided into normal control and 6 biligrafin injected groups to which 30% biligrafin (0.006ml/gm b.w.) were injected at 10, 20, 40, 80, 160 and 320 min prior to the sampling. The mice of each group were perfused through the heart with ice-cold 2.5% glutaraldehyde buffered with 0.1M Na-cacodylate (pH. 7.4) under the Na-pentobarbital (Nembtal 0.0015mg/gm b.w.) anesthesia and liver tissues were taken from each group. Some specimens were immersed 1 hr in the same solution used in the perfusion. After an overnight rinse in 0.1M Na-cacodylate buffer containing 10% DMSO and 7.6% sucrose, $75{\mu}m$ fronzen sections were made for cytochemical study. The sections were incubated in thiamin pyrophosphatase (TPPase) and inosine diphosphatase (ID Pase) media for 70 min at $37^{\circ}C$ respectively and acid phosphatase (AcPase) medium for 40 min at $37^{\circ}C$. They were postfixed in 1 % $OsO_4$ for 1 hr. The other specimens were immersed for 8 hrs in the fixative consisting of 2.5% glutaraldehyde and 3.0% paraformaldehyde buffered with Na-cacodylate (pH. 7.4). All of the osmificated specimens were processed for electron microscopy. In both normal and biligrafin injected groups, endoplasmic reticulum (ER), vacuoles, Golgi apparatus and lysosomes were seen in the vicinity of bile canaliculus. In the biligrafin injected groups, however, the Golgi apparatus appeared to be decreased and ER and vacuoles were dilated and increased. The rough endoplasmic reticulum (RER) having a few attached ribosomes appeared to be the round saccule, especially at 20 min after biligrafin injection. Smooth endoplasmic reticulum (SER) seemed to be formed by the detachment of ribosomes at the cisternal end of RER. The cistern of SER showed saccules which probably budded off to form the vacuole. The vacuoles were devoid of visible centents. This finding seemed to be in agreement with the biochemical property of the bile constituents. The fusion between the vacuoles and bile canaliculus were frequently seen in the groups injected with biligrafin. The lysosome did not show any changes in the biligrafin injected groups. Accumulation of some material and lipid droplets were seen at the 40 and 80 min after biligrafin injection, especially at the latter. At 160 and 320 min after biligrafin injections, however, they were decreased successively while the RER stack, free ribosomes and polysomes were increased. Although the reactive products of TPPase and IDPase were observed in the ER saccules and vesicles of the normal control and biligrafin injected groups, the fusion between the bile canaliculus and saccules or vesicles could easily be seen in the latter. The AcPase activity, however, was observed in the cistern at the maturing face of Golgi apparatus and lysosomes in both normal and biligrafin groups. The results suggest that the biligrafin is excreted via the vesicles, vacuoles or sacoules probably derived from the SER without the participation of Golgi apparatus and lysosomes, and the excess amount of material is stored as inclusions during the repairing of the organelles being overactive.