ABSTRACT
Los hepatocitos son células epiteliales polarizadas que, al ser aisladas y cultivadas, pierden la polaridad y las propiedades de célula diferenciada. El cultivo de células hepáticas como esferoides permite obtener estructuras con organización de tipo tisular. En este trabajo se analizó estructural y funcionalmente la polaridad de esferoides porcinos. Para ello, las células hepáticas porcinas fueron aisladas y cultivadas en agitación constante. La actividad metabólica de los esferoides fue probada mediante el metabolismo de diazepam y de amonio, así como con síntesis de albúmina. Sus características estructurales mostraron la polaridad de las células. Fueron observados paquetes de fibras de colágeno distribuidas irregularmente y fibras reticulares en formahomogénea en todo el volumen del esferoide. Se hallaron células Ck19+ formando estructuras tipo ducto biliar, así como también _ y _-cateninas y pan-cadherinas en diferentes zonas, especialmente en las laminas externas, con características de epitelio cuboidal. Por microscopía electrónica de barrido se observaron estructuras muy compactas con superficie lisa, y por microscopía electrónica de transmisión, canalículos biliares con microvellosidades, uniones tight, zonula adherens y desmosomas. Las organelas celulares como mitocondrias, núcleos, nucleolos, peroxisomas, retículo endoplásmico estaban bien conservadas. Una compleja red de canalículos biliares fue observada mediante la incorporación y excreción de un análogo de sal biliar fluorescente. El análisis de los ácidos biliares excretados mostró un patrón normal. La morfología y funcionalidad de los esferoides puede aportar un modelo apropiado para aplicaciones en las que es primordial mantener las funciones específicas del hígado, como un dispositivo de hígado bioartificial.
Hepatocytes are epithelial cells that show a complex polarity in vivo. However, hepatocytes isolated and cultured in vitro normally lose both their differentiated properties and polarity. Culturing hepatocyte spheroids seems to be the accurate approach to maintain tissue level of organization. The structural and functional polaritiesof pig liver spheroids were analyzed in this work. Swine liver cells were isolated and cultured as spheroids. Their metabolic activity was proved through the metabolism of diazepam, ammonium and synthesis of albumin. Several structural features show the presence of polarity in the cells inside the spheroids. Reticular and collagen fibers, as well as Ck19(+) cells forming duct-like structures were found. _eta and _-catenins and pancadherins were positive in different regions of the spheroids, mainly in the outer cell layers, which have cuboidal epithelia features. The scanning electron microscopy showed a tightly compacted architecture, with smooth surface. The transmission electron microscopy analysis showed bile canaliculi with microvilli, tight junctions, zonula adherens and desmosome-like junctions. Wellmaintained cellular organelles, as mitochondria, nucleus,nucleolus, peroxisomes, endoplasmic reticulum, were seen in the spheroids. A complex inner bile canaliculinetwork was shown by using a fluorescent bile acid analogue incorporated and excreted by the spheroids. Furthermore, excretion of a normal pattern of bile acids was demonstrated. The morphology and functionality of the spheroids may provide an appropriate model for applications where the maintenance of liver-specific functions is crucial, as a bioartificial liver device.
Subject(s)
Animals , Cell Polarity/physiology , Hepatocytes/cytology , Hepatocytes/physiology , Spheroids, Cellular/cytology , Spheroids, Cellular/physiology , Albumins/metabolism , Diazepam/metabolism , Fluorescent Antibody Technique , Hepatocytes/metabolism , Immunohistochemistry , Microscopy, Electron , Spheroids, Cellular/metabolism , Swine , Urea/metabolismABSTRACT
Sedative effects and drug elimination characteristics after intravenous bolus administration of diazepam 0.15 mg/kg were investigated in 15 patients with liver disease [Group I], and in 15 normal patients [Group II] during diagnostic laparoscopy. Diazepam induced heavier sedation in patients with liver disease [p < 0.02]. Serum concentration of diazepam 30 minutes after administration was significantly lower in Group I [group with liver disease] when compared with Group II [control group] [210.68 +/- 112.65 ng/ml vs 451.57 +/- 239.87 ng/ml, p < 0.02]. The sedation scores during the laparoscopy procedures correlate negatively with serum benzodiazepine levels. The benzodiazepine concentration-time profiles of the groups vary significantly [p < 0.02]. Heavier sedative effect and lower benzodiazepine concentrations in patients with liver diseases suggests that the permeability of blood brain barrier increases and that higher affinity to benzodiazepine receptors exists. Differences in concentration-time courses of diazepam in patients with liver diseases is a warning indicating the accumulation of drug when infused or used frequently
Subject(s)
Humans , Diazepam/blood , Hypnotics and Sedatives , Laparoscopy/methods , Diazepam/metabolismABSTRACT
With a view to providing perfection to the benzodiazepine receptor model proposed earlier [S P Gupta, R N Saha & V Mulchandani (1992) J. Mol. Recog, 5, 75-80] a few more QSAR studies on a series of 9-benzylpurines and tetracyclic 1,4-benzodiazepine derivatives have been made. The models showing the interaction of these compounds with the receptor are proposed. It is found that the receptor model, unlike the one proposed earlier, requires the presence of a polar site along with all the other essential sites.
Subject(s)
Benzodiazepines/metabolism , Binding Sites , Binding, Competitive , Diazepam/metabolism , Kinetics , Molecular Structure , Protein Conformation , Receptors, GABA-A/chemistry , Structure-Activity RelationshipABSTRACT
Se analiza el efecto producido por diferentes dosis de diazepam sobre el EEG de ratas utilizando las Medidas Espectrales de Banda Ancha y el Espectro de Potencia. De estas medidas las que presentan diferencias significativas son: Poder Relativo, Frecuencia Dominante y la Coherencia sólo para la dosis más alta: observándose mayor energia en las frecuencias más rápidas alfa y beta y menor energía para las frecuencias más lentas delta y theta. Estos resultados brindan la posibilidad de utilizar este método en la caracterización electroencefalográfia de drogas psicotrópicas relacionadas con los ansiolíticos