RESUMO
Macrovesicular steatosis in greater than 30% of hepatocytes is a significant risk factor for primary graft nonfunction due to increased sensitivity to ischemia reperfusion (I/R) injury. The growing prevalence of hepatic steatosis due to the obesity epidemic, in conjunction with an aging population, may negatively impact the availability of suitable deceased liver donors. Some have suggested that metabolic interventions could decrease the fat content of liver grafts prior to transplantation. This concept has been successfully tested through nutritional supplementation in a few living donors. Utilization of deceased donor livers, however, requires defatting of explanted organs. Animal studies suggest that this can be accomplished by ex vivo warm perfusion in a time scale of a few hours. We estimate that this approach could significantly boost the size of the donor pool by increasing the utilization of steatotic livers. Here we review current knowledge on the mechanisms whereby excessive lipid storage and macrosteatosis exacerbate hepatic I/R injury, and possible approaches to address this problem, including ex vivo perfusion methods as well as metabolically induced defatting. We also discuss the challenges ahead that need to be addressed for clinical implementation.
Assuntos
Fígado Gorduroso/cirurgia , Transplante de Fígado , Traumatismo por Reperfusão , Animais , Fígado Gorduroso/patologia , Sobrevivência de Enxerto , Humanos , Fatores de RiscoRESUMO
In addition to its maladaptive effects on psychiatric function, psychosocial deprivation impairs recovery from physical illness. Previously, we found that psychosocial deprivation, modeled by isolation rearing, depressed immediate early gene (IEG) expression in the medial prefrontal cortex (mPFC) and increased locomotion in the open field test [Levine JB, Youngs RM, et al. (2007) Isolation rearing and hyperlocomotion are associated with reduced immediate early gene expression levels in the medial prefrontal cortex. Neuroscience 145(1):42-55]. In the present study, we examined whether similar changes in behavior and gene expression are associated with the maladaptive effects of psychosocial deprivation on physical injury healing. After weaning, anesthetized rats were subjected to a 20% total body surface area third degree burn injury and were subsequently either group or isolation reared. After 4 weeks of either isolation or group rearing (a period that encompasses post-wearing and early adolescence), rats were killed, and their healing and gene expression in the mPFC were assessed. Locomotion in the open field test was examined at 3 weeks post-burn injury. We found that: 1) gross wound healing was significantly impaired in isolation-reared rats compared with group-reared rats, 2) locomotion was increased and IEG expression was suppressed for isolation-reared rats during burn injury healing, 3) the decreased activity in the open field and increased IEG expression was greater for burn injury healing group-reared rats than for uninjured group-reared rats, 4) the degree of hyperactivity and IEG suppression was relatively similar between isolation-reared rats during burn injury compared with uninjured isolation-reared rats. Thus, in the mPFC, behavioral hyperactivity to novelty (the open field test) along with IEG suppression may constitute a detectable biomarker of isolation rearing during traumatic physical injury. Implications of the findings for understanding, assessing, and treating the maladaptive effects of psychosocial deprivation on physical healing during childhood are discussed.
Assuntos
Regulação da Expressão Gênica/fisiologia , Genes Precoces/fisiologia , Atividade Motora/fisiologia , Córtex Pré-Frontal/fisiologia , Isolamento Social , Cicatrização/fisiologia , Envelhecimento/fisiologia , Animais , Biomarcadores , Química Encefálica/genética , Química Encefálica/fisiologia , Queimaduras/patologia , Interpretação Estatística de Dados , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Córtex Pré-Frontal/metabolismo , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Meio SocialRESUMO
This work describes the selective targeting of pigmented retinal pigment epithelial (RPE) cells by a single pulsed laser irradiation. We observed: (1) single pulsed laser irradiation caused cellular damages on pigmented, and not on non-pigmented RPE cells at laser radiant exposure up to 2550 mJ/cm(2); (2) in the mixture of pigmented and non-pigmented RPE cells, single pulsed laser-induced damage was confined to pigmented RPE cells. This study demonstrates that the pigmented RPE cells can be selectively damaged, using a single pulsed laser irradiation, without thermal coagulation to adjacent non-pigmented RPE cells.
Assuntos
Óptica e Fotônica , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos da radiação , Biotecnologia/métodos , Membrana Celular/metabolismo , Sobrevivência Celular , Células Cultivadas , Desenho de Equipamento , Humanos , Técnicas In Vitro , Lasers , Melaninas/metabolismo , Modelos Biológicos , Fagocitose , Retina/efeitos da radiação , Espectrofotometria/métodosRESUMO
Extending transplant criteria to include livers obtained from donors after cardiac death (DCD) could increase the liver donor pool, but conventional simple cold storage of these ischemic organs can lead to poor graft function after transplantation. Experimental normothermic machine perfusion has previously proven to be useful for the recovery and preservation of DCD livers, but it is more complicated than conventional cold storage, and, therefore, is perhaps not practical during the entire preservation period. In clinical situations, the combined use of simple cold storage and normothermic perfusion preservation of DCD livers might be more realistic, but even a brief period of cold storage prior to normothermic preservation has been suggested to have a negative impact on graft viability. In this study we show that rat livers subjected to 45 minutes of ex vivo warm ischemia followed by 2 hours of simple cold storage can be reclaimed by 4 hours of normothermic machine perfusion. These livers could be orthotopically transplanted into syngeneic recipients with 100% survival after 4 weeks (N = 10), similar to the survival of animals that received fresh livers that were stored on ice in University of Wisconsin (UW) solution for 6 hours (N = 6). On the other hand, rats that received ischemic livers preserved on ice in UW solution for 6 hours (N = 6) all died within 12 hours after transplantation. These results suggest that normothermic perfusion can be used to reclaim DCD livers subjected to an additional period of cold ischemia during hypothermic storage.
Assuntos
Isquemia/fisiopatologia , Circulação Hepática , Reperfusão/métodos , Alanina Transaminase/sangue , Animais , Bilirrubina/sangue , Temperatura Baixa , Morte , Humanos , Modelos Animais , Seleção de Pacientes , Ratos , Traumatismo por Reperfusão/fisiopatologia , TemperaturaRESUMO
Environmental deprivation contributes in important ways to the development of a wide range of psychiatric disorders. Isolation rearing of rodents, a model for environmental deprivation in humans, consistently produces hyperlocomotion, which provides a measurable parameter to study the underlying mechanisms of early adverse psychosocial stressors. Male Sprague-Dawley rat pups were separated from dams at postnatal (PN) day 20 and reared either in groups of three or in isolation. On PN 38, locomotion was assessed in the open field. On PN 46, rats were killed and gene expression patterns examined in the medial prefrontal cortex (mPFC). Isolation-reared rats displayed increased locomotor activity and decreased resting time in the open field. Specific gene expression patterns in the mPFC were associated with both isolation rearing and hyperlocomotive behavior in the open field. Genes involved in these expression patterns included immediate early genes (IEGs) and genes that regulate cell differentiation and apoptosis. The study of these genes could provide important insights into how abnormal early psychosocial events affect brain function and behavior.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genes Precoces/fisiologia , Locomoção/fisiologia , Córtex Pré-Frontal/metabolismo , Isolamento Social , Animais , Animais Recém-Nascidos , Comportamento Animal , Análise por Conglomerados , Perfilação da Expressão Gênica/métodos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ratos , Ratos Sprague-Dawley , Tempo de ReaçãoRESUMO
Fluid shear stress is a ubiquitous stimulus of mammalian cell metabolism; however, its signal transduction pathway is unknown. We hypothesized that shear stress may alter some physical properties of the cell membrane. Using primary human umbilical vein endothelial cells (HUVECs), we investigated the effects of shear on the cell membrane by monitoring flow-induced changes in the uptake of the amphipath merocyanine 540 (MC540). Under static conditions, MC540 was rapidly internalized by HUVECs at 37 degrees C, and so was the membrane impermeant dye lucifer yellow, suggesting that the MC540 uptake was partly due to endocytosis. However, exposure to steady flow for 5 min at 37 degrees C induced an increase in MC540 uptake while that of lucifer yellow was unchanged, suggesting that the flow-induced increase in MC540 uptake was not endocytosis-related. The increase in MC540 uptake was significant for levels of steady shear of 6 dyne/cm2 and above. Pulsatile flow was more stimulatory than steady flow at 2 dyne/cm2, but no significant difference between the two was seen at higher shear stress levels. We conclude that fluid shear stress enhanced the uptake of MC540 by a mechanism other than endocytosis, suggesting an increase in plasma membrane permeability during exposure of the cells to shear stress.
Assuntos
Permeabilidade da Membrana Celular , Endotélio Vascular/metabolismo , Corantes Fluorescentes/farmacocinética , Pirimidinonas/farmacocinética , Membrana Celular/metabolismo , Células Cultivadas , Endotélio Vascular/citologia , Citometria de Fluxo , Humanos , Reologia , Estresse MecânicoRESUMO
Numerous waterborne pathogens are difficult to detect and enumerate with accuracy due to methodological limitations and high costs of direct culturing. The purity of DNA extracted from wastewater samples is an important issue in the sensitivity and the usefulness of molecular methods such as polymerase chain reaction (PCR) and hybridizations on DNA microarrays. Ten different DNA extraction procedures, including physical and chemical extraction and purification steps, were examined to ascertain their relative effectiveness for extracting bacterial DNA from wastewater samples. The quality of the differentially extracted DNAs was subsequently assessed by PCR amplification and microarray hybridization. Our results showed that great differences existed among the ten procedures and only a few of the methods gave satisfactory results when applied to bacterial pathogens. This observation suggested that the extraction method needed to be carefully selected to produce significant and confident results in the detection of pathogens from environmental samples.
Assuntos
Bactérias/isolamento & purificação , DNA Bacteriano/isolamento & purificação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Eliminação de Resíduos Líquidos/métodos , Microbiologia da Água , Bactérias/genética , Bactérias/patogenicidade , Técnicas Bacteriológicas , DNA Bacteriano/análise , DNA Bacteriano/genéticaRESUMO
Recent in vivo studies suggest that tumor necrosis factor-alpha (TNF-alpha) is involved in the development of the thymus. We postulated that this inflammatory mediator could regulate the influx of progenitor T cells into the thymus. Using an in vitro static adhesion system, we found that TNF-alpha increases the adhesion of a murine progenitor T cell line (FTF1) to a bovine aortic endothelial cell line (1F8), human umbilical vein endothelial (HUVE) cells, and a murine arterial endothelial (MAE) cell line. TNF-alpha treatment of the 1F8 cells resulted in a time- and dose-dependent increase in the adherence of FTF1 cells. Adherence increased during the first 6 hr of treatment with TNF-alpha concentrations ranging from 10(-11) to 10(-9) M. Maximal adherence (6 hr treatment with 10(-10) M of TNF-alpha) was approximately 4.5-fold larger than that of untreated monolayers. A slow decrease in adherence, down to approximately 2-fold at 48 hr, was observed beyond 12 hr of TNF-alpha treatment; in contrast, removal of TNF-alpha after 6 hr of continued stimulation caused the adherence to return to pre-stimulation levels within 24-30 hr. Adhesion of FTF1 cells to TNF-alpha treated 1F8 cells was almost completely blocked by a monoclonal antibody against murine CD49d (very late antigen-4) expressed on FTF1 cells. TNF-alpha-induced adhesion of FTF1 cells to MAE cells was also blocked by monoclonal antibodies against murine CD49d and CD106 (vascular cell adhesion molecule-1). These results support the notion that local secretion of TNF-alpha could modulate the dynamics of adhesion of progenitor T cells to the thymic endothelium.
Assuntos
Endotélio Vascular/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Ligação Competitiva/imunologia , Bovinos , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular , Relação Dose-Resposta Imunológica , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Cinética , Camundongos , Linfócitos T/fisiologiaRESUMO
Burn injury initiates an inflammatory response as part of the healing process that is associated with extensive metabolic adjustments. While most studies have focused on understanding these changes from a biochemical perspective, not much work has been done to characterize these processes at the gene expression level. As a first step, we have comprehensively analyzed changes in gene expression in rat livers during the first 24 h after burn injury using Affymetrix GeneChips, which showed 339 genes to be differentially expressed at a statistical significance of P < 0.05 and changed at least twofold. Functional classification based on gene ontology terms indicated that two categories, metabolism (28%) and inflammation (14%), accounted for nearly 42%. Detailed analysis of the metabolism group of genes indicated that fatty acid (FA) and triglyceride (TG) biosynthesis in the liver were unchanged, whereas TG utilization, FA import, and beta-oxidation increased after burn injury. The increased FA pools after burn injury appear to serve as substrates for ATP production. Following burn injury, the cholesterol biosynthetic pathway was suppressed while cholesterol was increasingly imported and converted into bile acids. The inflammatory genes that were altered included several classic acute phase response markers, as well as genes involved in the complement, kinin, clotting, and fibrinolytic protein systems. These temporally coordinated changes in gene expression were also corroborated by biochemical measurements for FA, TG, cholesterol, and ATP. Together, these data indicate that FA are increasingly imported and oxidized in the liver to meet the enhanced energy demands arising from an inflammatory response during the first 24 h after burn injury.
Assuntos
Queimaduras/genética , Metabolismo Energético/genética , Perfilação da Expressão Gênica , Fígado/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Ácidos e Sais Biliares/metabolismo , Queimaduras/complicações , Queimaduras/metabolismo , Queimaduras/patologia , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Inflamação/etiologia , Inflamação/genética , Inflamação/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Sprague-Dawley , Triglicerídeos/metabolismoRESUMO
Fluid flow and several other agonists induce prostacyclin (PGI2) production in endothelial cells. G proteins mediate the response of a large number of hormones such as histamine, but the transduction pathway of the flow signal is unclear. We found that GDP beta S and pertussis toxin inhibited flow-induced prostacyclin production in human umbilical vein endothelial cells. In addition, flow potentiated the histamine-induced production of PGI2. This suggests that flow stimulates prostacyclin production via a pertussis toxin-sensitive G protein and modulates the stimulus-response coupling of other agonists.
Assuntos
Endotélio Vascular/metabolismo , Epoprostenol/biossíntese , Proteínas de Ligação ao GTP/fisiologia , Fenômenos Biomecânicos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Proteínas de Ligação ao GTP/antagonistas & inibidores , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Humanos , Toxina Pertussis , Tionucleotídeos/farmacologia , Veias Umbilicais , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Current fluorescence-based adhesion assays that use a 96-well plate format rely on the assumption that the fluorescent label does not significantly leak from the cells. Thus, we evaluated a calcein-based, in vitro adhesion assay in 96-well plates using five different types of leukocytes (HL60 cells, human neutrophils, rat neutrophils, mouse progenitor T cells and EL4 cells). Each cell type leaked calcein at a different rate, with the highest rates found for rat neutrophils and progenitor T cells, which lost as much as 20%-40% of the label within 90 min, the time required to complete the assay. Thus, we developed a procedure to measure the dye leakage rate during the assay in order to obtain a correction factor, which was then used to calculate the "true" number of adherent cells. Data for the adhesion of FTF1 cells to endothelial monolayers, after correction for calcein leakage, deviated less than 10% of adhesion data obtained with a well-established 51Cr-based assay.
Assuntos
Técnicas de Cultura de Células/métodos , Corantes Fluorescentes/metabolismo , Neutrófilos/fisiologia , Linfócitos T/fisiologia , Animais , Adesão Celular , Fluoresceínas/metabolismo , Células HL-60 , Humanos , Camundongos , Ratos , Espectrometria de FluorescênciaRESUMO
Several extracellular matrix (ECM) configurations involving type I collagen and Matrigel were examined for their ability to support differentiated function and polarity of cultured adult rat hepatocytes. Collagen sandwich- and Matrigel-based cultures yielded superior and comparable albumin secretion for at least 2 weeks. In collagen sandwich, hepatocytes were polygonal, and formed multicellular arrays. Collagen sandwich was also found to promote in vivo-like polarization of F-actin, cell adhesion molecules (E-cadherin), and lateral (Na+, K(+)-ATPase, glucose transporter) and apical (dipeptidyl peptidase, aminopeptidase) membrane polarity markers, but not the expression of the gap junction protein connexin 32 and the epidermal growth factor (EGF) receptor. In contrast, hepatocytes cultured in or on Matrigel were more rounded and formed aggregates. Matrigel-based cultures also elicited detectable levels of connexin and EGF receptor and an altered distribution of F-actin, E-cadherin, and apical and lateral membrane proteins. Composite sandwich configurations containing collagen I and Matrigel restored markers lacking in the collagen sandwich, and showed a variable morphology and membrane polarity. Hepatocyte polarity could thus be manipulated by the overall ECM composition. Furthermore, in composite sandwich cultures, these manipulations can be effected largely independent of changes in hepatocyte morphology and albumin secretion.
Assuntos
Matriz Extracelular , Fígado/citologia , Fígado/fisiologia , Actinas/análise , Animais , Materiais Biocompatíveis , Biomarcadores , Caderinas/análise , Membrana Celular/ultraestrutura , Células Cultivadas , Colágeno , Conexinas/análise , Técnicas de Cultura/instrumentação , Técnicas de Cultura/métodos , Dipeptidil Peptidase 4/análise , Combinação de Medicamentos , Feminino , Laminina , Microscopia Confocal , Proteoglicanas , Ratos , Ratos Endogâmicos Lew , ATPase Trocadora de Sódio-Potássio/análise , Proteína beta-1 de Junções ComunicantesRESUMO
In recent years, metabolic flux analysis has been widely used in bioprocess engineering to monitor cell viability and improve strain activity. Metabolic flux analysis refers to a methodology for investigating cellular metabolism whereby intracellular fluxes are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of reaction networks. By considering the stoichiometry of biochemical reactions, it is possible to estimate the degree of engagement of each pathway participating in overall cellular activity, and hence obtain a comprehensive view of a cell s metabolic state. Given the potential impact of cellular energy metabolism on the function of engineered tissues, such comprehensive analysis of metabolic activity can be an extremely useful tool for tissue engineers. Estimates of intracellular fluxes under various environmental conditions could be used to optimize function in vivo as well as culture conditions in vitro. In this review, we provide a brief theoretical background of metabolic flux analysis and summarize the most widely used experimental approaches to obtain flux data. This review is intended as an overview of the field and as a starting point for tissue engineers wishing to learn about and eventually employ this methodology.
Assuntos
Engenharia Biomédica , Fenômenos Fisiológicos Celulares , Células/metabolismo , Metabolismo , Modelos Biológicos , Animais , Sobrevivência Celular , Células/citologia , Humanos , CinéticaRESUMO
The thymus is the site of production of mature T lymphocytes and thus is indispensable for the development and maintenance of the T cell-mediated arm of the immune system. Thymic production of mature T cells is critically dependent on an influx of bone marrow-derived progenitor T cells that undergo replication and selection within the thymus. Thymus cellularity and thymic hormone secretion reach a peak during the first year of life and then decline gradually until the age of 50-60 years, a process known as "thymic involution." A rapid reduction of thymus cellularity occurs in young patients following injuries, chemotherapy, and other forms of stress. The mechanisms underlying the involution process appear to be dependent on factors intrinsic to the thymic tissue, such as the local production of cytokines and chemoattractants, promoting the recruitment, growth, and differentiation of bone marrow-derived T cell progenitors in the thymus, as well as extrinsic factors, such as systemic levels of endocrine hormones and mediators released by intrathymic nerves of the autonomic nervous system. Knowledge of these factors provides a rational basis for the development of an approach based on tissue engineering that could be used to provide either temporary or permanent reconstitution of thymic function.
Assuntos
Envelhecimento/imunologia , Síndromes de Imunodeficiência/terapia , Linfócitos T/imunologia , Timo/crescimento & desenvolvimento , Hormônios do Timo/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos T/análise , Sistema Nervoso Autônomo/fisiologia , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Citocinas/fisiologia , Hormônios/fisiologia , Humanos , Síndromes de Imunodeficiência/etiologia , Contagem de Linfócitos , Camundongos , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Estresse Fisiológico/imunologia , Linfócitos T/efeitos dos fármacos , Timo/embriologia , Timo/imunologia , Timo/metabolismo , Hormônios do Timo/uso terapêuticoRESUMO
Little information on the effect of plasma on hepatocyte cytochrome P450 (CYP) activities is currently available. We characterized the effect of plasma on CYPs of hepatocyte-mesenchymal cell co-cultures, which exhibit stable liver specific functions and may be potentially useful for bioartificial liver design. Rat hepatocyte-mouse 3T3-J2 cell co-cultures were maintained for 6 days in medium, and then switched to heparinized human plasma containing 3-methylcholanthrene (3MC; 2 microM), phenobarbital (PB; 1 mM), or no inducer for up to 7 days. CYP activities were measured in situ based on the o-dealkylation of ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD), or benzyloxy- (BROD) resorufin. Plasma alone increased PROD/BROD but not EROD/MROD. The endogenous inducer was in the high molecular weight fraction (>5 kD) of plasma and inhibited by >5 nM okadaic acid and >10 microM dibutyryl cyclic AMP, two inhibitors of PB-inducible CYPs. Furthermore, plasma increased CYP1A1 and CYP2B1/2 mRNA levels. In plasma, 3MC induced EROD/MROD to about 60% of the level induced in culture medium while PB induced PROD/BROD that were three- to 10-fold above levels induced in medium. CYP activities decreased between days 2 and 7 of plasma exposure, but were enhanced by plasma supplementation with amino acids, insulin, glucagon, and hydrocortisone.
Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Fibroblastos/enzimologia , Hepatócitos/enzimologia , Células 3T3 , Animais , Técnicas de Cocultura/métodos , Meios de Cultura , Sistema Enzimático do Citocromo P-450/análise , Fibroblastos/citologia , Heparina , Hepatócitos/citologia , Humanos , Metilcolantreno , Camundongos , Fenobarbital , RatosRESUMO
Maintaining hepatocyte function during plasma exposure is critical for the successful development of hepatocyte-based bioartificial liver assist systems. Past attempts to culture hepatocytes in plasma yielded discouraging results. Using a stable culture model based on sandwiching hepatocytes between two layers of collagen gel, we investigated the effect of hormone and amino acid supplementation during exposure of rat hepatocytes to heparin-treated human plasma for 1 week. Morphology and hepatocyte-specific functions were evaluated for hepatocytes cultured in Dulbecco's Modified Eagle medium (DMEM), nonsupplemented plasma, plasma supplemented with hormones, or with hormones plus amino acids. Amino acids were supplemented at four-fold concentration of Basal Medium Eagle with 4 mM glutamine, whereas hormones included 7.5 microg/mL of hydrocortisone and 50 microU/mL of insulin. Cuboidal structure and bile canaliculi formation were observed throughout the 1-week exposure period for control hepatocytes in DMEM and for hepatocytes cultured in hormone supplemented plasma. Albumin and urea synthesis rates of hepatocytes in hormone plus amino acid supplemented plasma during the last day of plasma exposure were 60.4 +/- 13.7 and 75.6 +/- 6.5 (microg/day per 1 x 10(6) cells, mean +/- SD), respectively, comparable to cultures in standard culture medium. On the other hand, hepatocytes exposed to nonsupplemented plasma suffered significant morphological and functional damage. The results of this study indicate that hormone plus amino acid supplementation help to restore function in hepatocytes exposed to plasma.
Assuntos
Aminoácidos/farmacologia , Fígado/metabolismo , Albuminas/biossíntese , Animais , Células Cultivadas , Meios de Cultura/farmacologia , Feminino , Glucagon/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/farmacologia , Insulina/farmacologia , Fígado/citologia , Ratos , Ratos Endogâmicos Lew , Ureia/metabolismoRESUMO
Techniques of liver replacement would benefit patients awaiting donor livers and may be a substitute for transplantation in patients whose livers can regenerate. Poly(lactic-co-glycolic acid) (PLGA) copolymers are biodegradable and have been shown to be useful as scaffolds for seeding and culturing various types of cells. In this study, foam disks were prepared from PLGA (lactic-to-glycolic mole ratio of 85:15) by lyophilization of benzene (5% w/v) solutions. These disks were then used as scaffolds for rat hepatocyte culture. Foams were coated with either a type I collagen gel (0.1% w/v), coated with gelatin (5% w/v), or treated with oxygen plasma (25 W, 90 s) to modify their surface chemistry and wettability. The disks were then seeded with rat hepatocytes (10(6)/mL) and cultured for a period of 2 weeks. All surface treatments resulted in increased hydrophilicity, the greatest being obtained by collagen treatment (contact angle < 10 degrees ), and a minimal decrease in void fraction (5%). DNA content after a 2-week culture period increased proportionally with the wettability of the treated foam surface. Urea synthesis in untreated foams averaged 15.3 +/- 2.3 microg/h/microg DNA, which was significantly higher than that for controls, whereas gelatin and collagen treated foams exhibited urea synthetic rates below the control levels at all times. The DNA content decreased significantly by about 50% between days 1 and 12. PLGA foams, treated and untreated, represent a promising scaffold for scaling up hepatocyte cultures.
Assuntos
Materiais Biocompatíveis , Ácido Láctico , Fígado/fisiologia , Ácido Poliglicólico , Polímeros , Engenharia Tecidual , Animais , Bioprótese , Adesão Celular/fisiologia , Células Cultivadas , Fígado/citologia , Transplante de Fígado , Masculino , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , Ratos Endogâmicos F344RESUMO
BACKGROUND: The direct impact of burn injury on liver metabolism was studied in a rat liver perfusion system to remove the influence of systemic factors that modulate liver metabolism. METHODS: Seven animals received a burn injury covering 20% of the total body surface area, and seven were sham burned. The in situ liver perfusion studies were carried out in these animals after 3 days of isonitrogenous-isocaloric enteral feeding. In each study oxygen consumption and the rates of uptake and release of glucose, urea, and various amino acids were measured. RESULTS: Burn injury significantly increased urea production (18.5 +/- 0.4 versus 12.2 +/- 0.6 micromol/gm liver/hr and oxygen consumption (3.23 +/- 0.17 versus 1.21 +/- 0.03 micromol/gm liver/min) in the liver but did not alter the rate of gluconeogenesis. The change in amino acid concentrations in the perfusion medium implies an increased net protein breakdown. CONCLUSIONS: Our study indicates that (1) burn injury induces a hypermetabolic state in the liver, (2) the observed enhancement of gluconeogenesis in vivo after burn in probably regulated by factors outside the liver, and (3) the liver itself plays an active role in up-regulating urea production in burn injury. Identifying intrahepatic factors that up-regulate urea production may provide an "intrahepatic approach" to ameliorate the severe nitrogen loss after burn injury.
Assuntos
Queimaduras/fisiopatologia , Glucose/metabolismo , Fígado/metabolismo , Nitrogênio/metabolismo , Aminoácidos/metabolismo , Animais , Queimaduras/metabolismo , Gluconeogênese/fisiologia , Glucose/biossíntese , Técnicas In Vitro , Masculino , Mitocôndrias/metabolismo , Oxirredução , Consumo de Oxigênio/fisiologia , Perfusão , Projetos Piloto , Ratos , Ratos Sprague-Dawley , Ureia/metabolismoRESUMO
Because the cell mass in a bioartificial tissue exceeds relatively small numbers, there is a requirement to incorporate a convective transport system that provides nutrients and removes waste products. This function is provided by the vascular tree in vivo, and it may be desirable that endothelial cells be used in the design of the vascular tree in the bioartificial tissue, because endothelial cells provide a nonthrombogenic surface inside normal blood vessels. Furthermore, there is an ongoing effort in the area of vascular graft development, and the development of these applications requires a reliable source of endothelial cells.
RESUMO
Long-term and stable hepatocyte culture systems have a wide variety of uses, both in basic science and in the development of hepatocyte-based applications. In most cases, long-term cultures of hepatocytes are superior to traditional cultures in collagen-coated dishes, which only transiently express a low level of liver-specific function during the first wk in culture (1,2). The collagen sandwich provides a system capable of maintaining long term and stable function of hepatocytes with which to study liver physiology (3,4). This system is now used along with several other long-term hepatocyte culture techniques that have been developed since the mid 1970s. These other methods include the use of special extracellular matrix (ECM) materials, such as an extract from the Engelbreth-Holm-Swarm sarcoma grown in mice [5] (under the commercial appellations of Matrigel and Biomatrix, Biomedical Technologies, Stoughton, MA), co-culture with mesenchymal, endothelial, or epithelial cells (6-8), special culture media (e.g., dimethyl sulfoxide supplementation or arginine-free formulas), and culture at high seeding densities. In the context of studying liver physiology and morphogenesis of the liver plate, the sandwich culture system appears to be particularly well-suited, since it exhibits in vivo-like ECM geometry, has relatively flexible medium requirements, and individual cell morphology and structure can be easily visualized. One disadvantage, however, is that, in current practice, the ECM layer on top of the cells may present a transport barrier that can slow down the exchange of nutrients, products, and chemical signals with the bulk of the medium.