Successful mouse hepatocyte culture with sandwich collagen gel formation / 대한외과학회지

PURPOSE: Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking. METHODS: Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied. RESULTS: After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture. CONCLUSION: The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.

Brain trptophan metabolism remained unaltered in restraint stressed rats following fluoxetine administration

Effects of two hours immobilization stress on tryptophan [Tp] metabolism in rats was studied following fluoxetine-HCl [20mg/kg] administration. After 2hr immobilization stress there was no effect on peripheral liver Tp metabolism but there was marked increase in brain Tp metabolism leading to increases in 5-HT [5-Hydroxy tryptamine] synthesis and turn over. Rats subjected to 2hrs immobilization stress immediately after fluoxetine-HCl [20mg/kg] administrations show significant increases in holo and total Tp pyrrolase enzyme activities leading to decreased serum total Tp concentrations. Brain Tp and 5-HT remain unchanged but 5-hydroxy indole acetic acid [5-HIAA] concentration was significantly increased when compared to fluoxetine injected non-immobilized rats. Administration of fluoxetine-HCl [20mg/kg] inhibited holo and total Tp pyrrolase enzyme activities when compared to saline injected rats and ultimately increases liver Tp concentrations. Although serum total Tp remained un- changed but brain Tp and 5-HT concentrations were significantly increased. It is therefore concluded that pre-treatment of rats with fluoxetine-HCl did not alter conditioned stress induced change in brain Tp metabolism. Future studies on chronic administration of the drug will be fruit full to investigate its effects on Tp metabolism in conditioned stress

Injected monosodium glutamate increases holotryptophan pyrrolase activity and plasma corticosterone levels more in male than female rats

Monosodium glutamate [MSG] injected intra - peritoneally at doses 4 g/kg increased holotryptophan pyrrolase activity significantly by 61.6% in male rats. The increases were smaller [24%] and non- significant in females. Apotryptophan was decreased by 61% and comparably in the two sexes. Cumulative effect on total enzyme activity was a reduction of 37.5% in female rats. A decrease of 20.6% in male rats was not significant. Plasma levels of corticosterone were increased significantly in male but not in female rats. The sex differences are explained in terms of female hormones being effected by MSG administration and higher circulating MSG - induced corticosterone levels being involved in the enhancement of holotryptophan pyrrolase activity in male rats. Plasma total tryptophan levels were increased due to an inhibition of total tryptophan pyrrolase activity in female but not in male. Plasma free tryptophan concentrations were decreased in both sexes

Lack of sodium salicylate-induced enhancement of hepatic tryptophan pyrrolase in 5 day salicylate injected rats

Single administration of sodium salicylate at doses of 400 mg/kg increased hepatic holo-tryptophan pyrrolase activity but did not produce any effect an apotryptophan pyrrolase activity in rats. Repeated administration of the drug at doses of 100 mg/kg did not alter holo-enzyme activity but decreased apo and total enzyme activity in rats killed 24 hours later. Administration of 400 mg/kg sodium salicylate to 5 day, 100 mg/kg sodium salicylate injected rats did not increase holo or apo-tryptophan pyrrolase activity. Possible role of tryptophan pyrrolase in the sodium- salicylate induced changes of brain serotonin metabolism is discussed

Effect of triiodothyronine in the regulation of haem biosynthetic pathway

La administración de triyodotironina a animales tiroidectomizados, disminuyó en un 50% el contenido de citocromo P-450. La actividad de hemo oxigenasa no se modificó por el tratamiento con triyodotironina, ya sea solo o con una dosis subóptima de Cl2Co. Bajo las mismas condiciones la actividad de la amino levulínico sintelasa no fue afectada. La triyodotironina produjo un incremento del 100% en la actividad de triptófano pirrolasa. Tanto la holo como la enzima total fueron aumentadas en el mismo grado. La actividad de la porfobilinógeno deaminasa-uroporfirinógeno co-sintetasa, fue inducida en los animales tratados con triyodotironina, en un 67% por sobre los valores de los animales tiroidectomizadosm y sólo 32% con respecto a los animales con operación simulada. Nuestros resultados sugieren que bajo estimulación por triyodotironina, la disminución en el contenido de citocromo P-450 no es debida a un aumento en la velocidad de degradación del hemo, sino a una disociación de éste para incrementar el "pool" celular del hemo, y saturar en parte a la nueva apotriptófano pirrolasa sintetizada