RESUMO
AIMS/BACKGROUND: We report in this study that, 10 mM nicotinamide can stimulate the proliferation of primary rat hepatocytes in serum-free Dulbecco's modified Eagle's medium supplemented with 10 ng/ml epidermal growth factor and that small hepatocyte colonies appear from 4 to 5 days after plating. We examined the effects of nicotinamide-related agents on the growth and differentiation of primary rat hepatocytes and on the appearance of small hepatocyte colonies. METHODS: As nicotinamide is an aqueous vitamin named niacin and known to act as an inhibitor of poly (ADP-ribose) polymerase (PARP), we therefore chose to examine the effects on hepatocytes of three nicotinamide-related agents, nicotinic acid (NA) which is also a niacin, 3-aminobenzamide (3-AB) which is a strong inhibitor of PARP but is not a niacin, and 3-acetylpyridine (3-AP) which is a weak inhibitor of PARP and also not a niacin. To examine their effects on the growth of the cells and on the formation of the colony, immunocytochemistry for BrdU was carried out. Expression of albumin, tryptophan 2,3-dioxygenase (TO), and connexin 32 (Cx32) mRNAs were used as marks of hepatic differentiation. Intracellular NAD+ content was also measured. RESULTS: At concentration of 10 mM, NA could not enhance the proliferation of mature hepatocytes but induced the appearance of small hepatocyte colonies. At concentration of 5 mM, 3-AB enhanced the proliferation of the hepatocytes but did not induce small hepatocyte colonies. On the other hand, although 10 mM 3-AP remarkably inhibited the DNA synthesis of the cells, the expression not only of albumin but also of TO and Cx32 mRNAs in the cells was well maintained for more than one week. The intracellular NAD+ concentration was correlated with the proliferation of the hepatocytes. CONCLUSION: These results suggest that the intracellular NAD+ content may be correlated with the proliferation of primary hepatocytes and that the supplementation of niacin in the medium may be important for the appearance of small hepatocyte colonies.
Assuntos
Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Niacina/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Piridinas/farmacologia , Albuminas/metabolismo , Animais , Northern Blotting , Bromodesoxiuridina/metabolismo , Contagem de Células , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Conexinas/metabolismo , Fígado/metabolismo , Masculino , NAD/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Triptofano Oxigenase/metabolismo , Proteína beta-1 de Junções ComunicantesRESUMO
Hepatic cells isolated from an adult rat liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)-positive cells, which are thought to be markers for hepatic immature cells, were rarely observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P. LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies. The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type IV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MHs by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.
Assuntos
Fígado/ultraestrutura , Organoides/ultraestrutura , Animais , Bile/metabolismo , Canalículos Biliares/citologia , Northern Blotting , Western Blotting , Diferenciação Celular , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Matriz Extracelular/ultraestrutura , Fígado/citologia , Fígado/metabolismo , Masculino , Microscopia de Fluorescência , Organoides/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Proliferation of adult rat hepatocytes is observed in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less-differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well-developed bile canaliculus structures are often observed in the cell-cell borders. Thus, we suggest that small hepatocytes may be considered to be 'committed progenitor cells' that can further differentiate into mature hepatocytes.
Assuntos
Fígado/citologia , Animais , Diferenciação Celular , Células Cultivadas , Células Clonais , Fígado/ultraestrutura , Niacinamida/farmacologia , RatosRESUMO
To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo, but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentitate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Fígado/citologia , Niacinamida/farmacologia , Animais , Sangue , Bovinos , Adesão Celular , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Colágeno , Meios de Cultura Livres de Soro , Técnicas de Cultura/métodos , Feto , Cinética , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Masculino , Ratos , Ratos Sprague-Dawley , Albumina Sérica/biossínteseRESUMO
Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear. It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals. In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA). DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not. The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU. Furthermore, extensive GJIC was also observed with DMSO and DMTU. These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression.
Assuntos
Conexinas/metabolismo , Dimetil Sulfóxido/farmacologia , Sequestradores de Radicais Livres/farmacologia , Junções Comunicantes/efeitos dos fármacos , Fígado/metabolismo , Tioureia/análogos & derivados , Vitamina E/farmacologia , Acetamidas/farmacologia , Animais , Comunicação Celular/efeitos dos fármacos , Conexina 26 , Indicadores e Reagentes/farmacologia , Fígado/citologia , Fígado/efeitos dos fármacos , Masculino , Nitroazul de Tetrazólio/farmacologia , Estresse Oxidativo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Tioureia/farmacologia , Fatores de Tempo , Proteína beta-1 de Junções ComunicantesRESUMO
Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion. The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor. One small cell finally proliferated to several hundred cells. In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei. The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes. Thus, the small hepatocytes may be "committed progenitor cells" which can further differentiate into mature hepatocytes.
Assuntos
Fígado/citologia , Animais , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/farmacologia , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Colagenases , Fator de Crescimento Epidérmico/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Niacinamida/farmacologia , Ratos , Ratos Sprague-Dawley , Albumina Sérica/biossínteseRESUMO
The ability to swallow may be affected by administration of a small dose of muscle relaxant. To test the hypothesis that a subparalyzing dose of a muscle relaxant can impair swallowing, effects of partial paralysis produced by pancuronium on the swallowing reflex were investigated in eight conscious subjects. The swallowing reflex was induced by a bolus injection or a continuous infusion of distilled water into the mesopharynx. The swallowing function was assessed by electromyogram of suprahyoid muscles (EMGSH), mesopharyngeal pressure (Pmeso), and hypopharyngeal pressure (Phypo). Peripheral muscle activity was simultaneously determined by train of four ratio (TOFR) of hypothenar muscles to electrical stimulation of ulnar nerve and by hand grip strength (HGS). Following control measurements, measurements during partial paralysis and after recovery from partial paralysis were performed after intravenous administration of pancuronium 0.02 mg/kg. Partial paralysis significantly depressed EMGSH (bolus injection 44.1 +/- 10.0%, continuous infusion 55.9 +/- 10.2% of control value, P less than 0.01). Pmeso also significantly decreased (bolus injection 64.9 +/- 6.7 to 47.8 +/- 5.8 mmHg, P less than 0.01; continuous infusion 63.4 +/- 7.7 to 52.5 +/- 5.8 mmHg, P less than 0.05). The TOFR of peripheral muscles decreased to 81.4 +/- 6.7% of control value (P less than 0.01), and HGS was reduced from 44.6 +/- 1.9 to 39.4 +/- 2.0 kg (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)