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BACKGROUND: There is no standard therapy for acute liver failure. Hepatocyte transplantation has been proposed for temporary liver function support, while the injured liver regenerates or while waiting for transplantation. We have previously shown such efficacy for microencapsulated porcine hepatocytes in mice with fulminant liver failure. We aimed to establish a large animal model for fulminant liver failure to assess the efficacy of microencapsulated porcine hepatocytes in temporary liver function support. METHODS: The model was developed in baboons; for testing microencapsulated hepatocytes, the best condition was 75% hepatectomy and 60 min warm ischemia time. Fulminant liver failure was characterized by steep increases in liver biochemical parameters, severe steatosis, and massive hepatocyte necrosis during the first 10 days. Hepatocytes from miniature swine were microencapsulated in alginate-poly-l-lysine microspheres, and transplanted intraperitoneally immediately after hepatectomy and warm ischemia (80-120 mL packed hepatocytes in 200-350 mL microspheres, about 30%-50% of the baboon's native liver volume). RESULTS: In the control group, three of five animals were sacrificed after 6-10 days because of fulminant liver failure, and two of five animals recovered normal liver function and survived until elective euthanasia (28 days). In the treatment group of four animals, one animal developed liver failure but survived to 21 days, and three animals recovered completely with normal liver function. CONCLUSIONS: The results indicate that microencapsulated porcine hepatocytes provide temporary liver function support in baboons with fulminant liver failure. These data support development of this cell therapy product toward clinical trials in patients with acute liver failure.
Assuntos
Transplante de Células/métodos , Hepatócitos/transplante , Falência Hepática Aguda/terapia , Transplante Heterólogo/métodos , Animais , Separação Celular/métodos , Modelos Animais de Doenças , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Antígeno Ki-67/metabolismo , Falência Hepática Aguda/patologia , Falência Hepática Aguda/fisiopatologia , Masculino , Camundongos , Microesferas , Papio hamadryas , Suínos , Porco MiniaturaRESUMO
Statins act by reducing hepatic cholesterol synthesis, thus stimulating uptake of serum cholesterol. Statin therapy modulates a number of genes involved in hepatic cholesterol homeostasis. These have rarely been analyzed simultaneously in the same experimental setting, with virtually no studies of primary human hepatocytes. This study analyzed the efficacy of rosuvastatin in the coordinated regulation of a number of genes implicated in cholesterol metabolism in primary human hepatocytes. Expression of five cholesterol-related genes were significantly upregulated, notably the Niemann-Pick C1 like 1 protein, for whom functional studies have been essentially limited to the intestine. Two genes were significantly downregulated, including sterol recognition element binding protein-1 gene that is implicated in control of hepatic lipogenesis. The results show the coordinated regulation of several genes implicated in hepatic cholesterol homeostasis and suggest therapeutic targets that could complement that clinical action of statins.
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Fluorbenzenos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas de Membrana/genética , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Células Cultivadas , Humanos , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Rosuvastatina CálcicaRESUMO
UNLABELLED: Phosphatase and tensin homolog (PTEN) is a regulator of phosphoinositide 3-kinase signaling and an important tumor suppressor mutated/deleted in human cancers. PTEN deletion in the liver leads to insulin resistance, steatosis, inflammation, and cancer. We recently demonstrated that unsaturated fatty acids trigger steatosis by down-regulating PTEN expression in hepatocytes via activation of a mammalian target of rapamycin (mTOR)/nuclear factor kappa B (NF-kappaB) complex, but the molecular mechanisms implicated in this process are still unknown. Here, we investigated potential genetic and epigenetic mechanisms activated by fatty acids leading to PTEN down-regulation. Our results indicate that unsaturated fatty acids down-regulate PTEN messenger RNA expression in hepatocytes through mechanisms unrelated to methylation of the PTEN promoter, histone deacetylase activities, or repression of the PTEN promoter activity. In contrast, unsaturated fatty acids up-regulate the expression of microRNA-21, which binds to PTEN messenger RNA 3'-untranslated region and induces its degradation. The promoter activity of microRNA-21 was increased by mTOR/NF-kappaB activation. Consistent with these data, microRNA-21 expression was increased in the livers of rats fed high-fat diets and in human liver biopsies of obese patients having diminished PTEN expression and steatosis. CONCLUSION: Unsaturated fatty acids inhibit PTEN expression in hepatocytes by up-regulating microRNA-21 synthesis via an mTOR/NF-kappaB-dependent mechanism. Aberrant up-regulation of microRNA-21 expression by excessive circulating levels of fatty acids exemplify a novel regulatory mechanism by which fatty acids affect PTEN expression and trigger liver disorders.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Hepatócitos/metabolismo , MicroRNAs/metabolismo , Ácido Oleico/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Serina-Treonina Quinases TOR , Fator de Transcrição RelA/metabolismo , Regulação para CimaRESUMO
Embryonic stem cells (ESC) can differentiate into all cell lineages, and ESC-like cells were shown to induce hematopoietic chimerism and tolerance in allogeneic models. The aim of our study was to test the capacity of mouse ESC (mESC) to engraft in rats in a xenotransplantation setting. Forty-six rats were transplanted intravenously with 1 million mESC, without immunosuppression (group 1, n = 23) or with cyclosporine (group 2, n = 23). Three months after mESC transplantation, skin grafts were performed from allogeneic, xenogeneic identical to mESC, or xenogeneic third party donors. At day 27 post-transplant, we detected circulating mouse cells in the blood of 4/23 and 5/23 animals of group 1 and group 2, respectively. Chimerism was confirmed by PCR. We also identified long-term surviving murine cells within livers of chimeric animals. Skin grafts showed no difference in survival between allogeneic and xenogeneic donors. Transplantation of xenogeneic mouse ESC induced short-term chimerism in the blood and persistent tissue chimerism in the liver of recipient rats, but did not induce tolerance to skin grafts. Improved immunosuppressive protocols should be tested to prolong chimerism and allow tolerance.
Assuntos
Quimerismo , Células-Tronco Embrionárias/fisiologia , Transplante de Células-Tronco , Transplante Heterólogo , Animais , Ciclosporina/farmacologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/efeitos dos fármacos , Sobrevivência de Enxerto , Sistema Hematopoético , Imunossupressores/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Transplante de Pele , Tolerância ao TransplanteRESUMO
UNLABELLED: For many years, adipose tissue has been mainly considered as an inert reservoir for storing triglycerides. Since the discovery that adipocytes may secrete a variety of bioactive molecules (hormones, chemokines, and cytokines), an endocrine and paracrine role for white adipose tissue (WAT) in the regulation of energy balance and other physiological processes has been established, particularly with regard to brain and muscle. In contrast, little is known about the interactions of WAT with liver. Hence, we examined the effect of the secretory products of WAT on hepatocytes. Conditioned medium of human WAT explants induced significant steatosis in hepatocyte cell lines. Factor(s) responsible for the conditioned medium-induced steatosis were screened by a battery of blocking antibodies against different cytokines/chemokines shown to be secreted by WAT. In contrast to interleukin-8 and interleukin-6, the monocyte chemoattractant protein-1 was capable of inducing steatosis in hepatocytes in a time-dependent manner at concentrations similar to those found in conditioned medium. Incubation of conditioned medium with antimonocyte chemoattractant protein-1 antibodies prevented triglyceride accumulation. Investigation of the mechanism leading to the triglyceride accumulation showed that both a diminution of apolipoprotein B secretion and an increase in phosphoenolpyruvate carboxykinase messenger RNA may be involved. CONCLUSION: The monocyte chemoattractant protein-1 secreted by adipose tissue may induce steatosis not only recruiting macrophages but also acting directly on hepatocytes.
Assuntos
Tecido Adiposo Branco/metabolismo , Quimiocina CCL2/metabolismo , Hepatócitos/metabolismo , Metabolismo dos Lipídeos/fisiologia , Animais , Apolipoproteínas B/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Células Cultivadas , Quimiocina CCL2/farmacologia , Meios de Cultivo Condicionados/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Hepatócitos/efeitos dos fármacos , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismoRESUMO
The aim of this study was to establish hepatocyte isolation in pigs, and to evaluate function of isolated hepatocytes after encapsulation, cryopreservation, and transplantation (Tx) in a mouse model of fulminant liver failure (FLF). After isolation, porcine hepatocytes were microencapsulated with alginate-poly-L-Lysine-alginate membranes and cryopreserved. In vitro, albumin production of free and encapsulated hepatocytes were measured by enzyme linked-immunoadsorbent assay. In vivo, encapsulated hepatocytes were transplanted into different groups of mice with FLF and the following experimental groups were performed: group 1, Tx of empty capsules; group 2, Tx of free primary porcine hepatocytes; group 3, Tx of fresh encapsulated porcine hepatocytes; group 4, Tx of cryopreserved encapsulated porcine hepatocytes. In vitro, fresh or cryopreserved encapsulated porcine hepatocytes showed a continuous decreasing metabolic function over 1 week (albumin and urea synthesis, drug catabolism). In vivo, groups 1 and 2 showed similar survival (18% and 25%, respectively, p > 0.05). In groups 3 and 4, Tx of fresh or cryopreserved encapsulated porcine hepatocytes significantly increased survival rate to 75% and 68%, respectively (p < 0.05). Primary porcine hepatocytes maintained metabolic functions after encapsulation and cryopreservation. In mice with FLF, Tx of encapsulated xenogeneic hepatocytes significantly improved survival. These results indicate that porcine hepatocytes can successfully be isolated, encapsulated, stored using cryopreservation, and transplanted into xenogeneic recipients with liver failure and sustain liver metabolic functions.
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Criopreservação/métodos , Hepatócitos/transplante , Falência Hepática Aguda/terapia , Transplante Heterólogo/métodos , Animais , Células Cultivadas , Composição de Medicamentos , Sobrevivência de Enxerto , Hepatócitos/metabolismo , Falência Hepática Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , SuínosRESUMO
Background: Dermoid cysts are benign tumors rarely arising from the cecum. Also, including this report, 10 cases have been described in the literature, to our knowledge. Dermoid cysts are benign tumors rarely found in the cecum. A literature research has found only 9 other cases. Case: We describe the case of a healthy 44-year-old man with no surgical history, in whom a well-defined cystic-mass was incidentally found on abdominal computed tomography, presenting as an appendicular mucocele. An hemicolectomy was performed and pathological analysis revealed a dermoid cyst of the cecum. This paper describes the case of a healthy 44-year-old male with no prior surgical history, with a well-defined cystic mass found incidentally on abdominal computed tomography, thought to be an appendicular mucocele. After hemicolectomy and pathological analysis, this was revealed to be a dermoid cyst of the cecum. Conclusion: Although dermoid cysts are a rare tumor of the cecum, they should be considered in the differential diagnosis of a nontender palpate mass in right lower quadrant (RLQ) or cecal mass on imaging. Although rarely found in the cecum, a dermoid cyst should be included in the differential diagnosis of nontender palpable masses of the RLQ, or if revealed by imagery.
RESUMO
Organ transplantation has encountered great development during the 80's. However, the number of organ donations and transplantations performed stabilized during the 90ies, with a concomitant increase of patients on the waiting list. Xenotransplantation, i.e. the use of animal organs for transplantation to humans, is one among various alternatives to human organ donation. Xenotransplantation offers several advantages, e.g. it would be possible to transplant all patients at an early stage of their disease. The main barriers to xenotransplantation are the strong immunological responses that human develop against animal antigens and zoonoses. To overcome these hurdles, genetically modified pigs have been engineered by cloning and could allow the initiation of new clinical trials in a near future.
Assuntos
Transplante de Órgãos/métodos , Transplante Heterólogo/métodos , Animais , Clonagem de Organismos , Humanos , Suínos , Obtenção de Tecidos e Órgãos , Imunologia de Transplantes , Zoonoses/transmissãoRESUMO
BACKGROUND: Cytokine administration is a potential therapy for acute liver failure by reducing inflammatory responses and favour hepatocyte regeneration. The aim of this study was to evaluate the role of interleukin-1 receptor antagonist (IL-1ra) during liver regeneration and to study the effect of a recombinant human IL-1ra on liver regeneration. METHODS: We performed 70%-hepatectomy in wild type (WT) mice, IL-1ra knock-out (KO) mice and in WT mice treated by anakinra. We analyzed liver regeneration at regular intervals by measuring the blood levels of cytokines, the hepatocyte proliferation by bromodeoxyuridin (BrdU) incorporation, proliferating cell nuclear antigen (PCNA) and Cyclin D1 expression. The effect of anakinra on hepatocyte proliferation was also tested in vitro using human hepatocytes. RESULTS: At 24h and at 48 h after hepatectomy, IL-1ra KO mice had significantly higher levels of pro-inflammatory cytokines (IL-6, IL-1ß and MCP-1) and a reduced and delayed hepatocyte proliferation measured by BrdU incorporation, PCNA and Cyclin D1 protein levels, when compared to WT mice. IGFBP-1 and C/EBPß expression was significantly decreased in IL-1ra KO compared to WT mice. WT mice treated with anakinra showed significantly decreased levels of IL-6 and significantly higher hepatocyte proliferation at 24h compared to untreated WT mice. In vitro, primary human hepatocytes treated with anakinra showed significantly higher proliferation at 24h compared to hepatocytes without treatment. CONCLUSION: IL1ra modulates the early phase of liver regeneration by decreasing the inflammatory stress and accelerating the entry of hepatocytes in proliferation. IL1ra might be a therapeutic target to improve hepatocyte proliferation.
Assuntos
Hepatectomia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Regeneração Hepática/efeitos dos fármacos , Alanina Transaminase/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1/deficiência , Proteína Antagonista do Receptor de Interleucina 1/genética , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Regeneração Hepática/genética , Masculino , Camundongos , Fatores de TempoRESUMO
The aim of this study was to evaluate the effects of intraperitoneal transplantation of encapsulated human hepatocytes on liver metabolism and regeneration of mice with acute liver failure. Primary human hepatocytes were immortalized using lentiviral vectors coding for antiapoptotic genes and microencapsulated using alginate-polylysine polymers. In vitro, immortalized human hepatocytes showed low, but stable, synthetic and catabolitic functions over time, when compared to primary hepatocytes. In vivo, mice with acute liver failure and transplanted with encapsulated immortalized human hepatocytes had a significantly improved survival and biochemical profile, compared to mice transplanted with empty capsules. Serum levels of cytokines implicated in liver regeneration were lower in mice transplanted with hepatocytes compared to mice receiving empty capsules. This decrease was significant for IL-6 and HGF at 3 h. Measurement of liver regeneration showed no significant difference between mice transplanted with hepatocytes compared to control groups. Intraperitoneal transplantation of encapsulated immortalized hepatocytes significantly improved survival of mice with acute liver failure by providing metabolic support and without modifying liver regeneration. The lower levels of cytokines implicated in liver regeneration suggest that the metabolic support provided by the encapsulated hepatocytes reduced the inflammatory stress on the liver and herein decreased the regenerative trigger on residual hepatocytes. These data emphasize that metabolic function and regeneration of hepatocytes are two distinct aspects that need to be studied and approached separately during acute liver failure.
Assuntos
Hepatócitos/citologia , Falência Hepática Aguda/terapia , Regeneração Hepática , Animais , Aspartato Aminotransferases/sangue , Bilirrubina/sangue , Cápsulas/química , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citocinas/sangue , Fator de Crescimento de Hepatócito/sangue , Hepatócitos/transplante , Humanos , Interleucina-6/sangue , Fígado/fisiologia , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/mortalidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Albumina Sérica/metabolismo , Taxa de Sobrevida , Transplante HeterólogoRESUMO
BACKGROUND: Xenotransplantation carries inherent risks of infectious disease transmission to the recipient and even to society at large, and it should only be carried out with strict regulation and oversight. In collaboration with the International Xenotransplantation Association, the University Hospital Geneva, and the World Health Organization, an international inventory has been established (www.humanxenotransplant.org) aiming to collect basic data on all types of currently ongoing or recently performed xenotransplantation procedures in humans. METHODS: We collected information from publications in scientific journals, presentations at international congresses, the internet, and declarations of International Xenotransplantation Association members on xenotransplantation procedures in humans performed during the past 15 years. RESULTS: We identified a total of 29 human applications of xenotransplantation, including 7 that were currently ongoing. Procedures involved transplantation of xenogeneic cells, i.e., islets of Langerhans, kidney cells, chromaffin cells, embryonic stem cells, fetal and adult cells from various organs or extracorporeal perfusion using hepatocytes, liver, spleen, or kidney. The treatments were performed in 12 different countries, 9 of them having no national regulation on xenotransplantation. CONCLUSION: Several clinical applications of cell xenotransplantation are ongoing around the world, often without any clear governmental regulation. This information should be used to inform national health authorities, healthcare staff, and the public, with the objective of encouraging good practices, with internationally harmonized guidelines and regulation of xenotransplantation.
Assuntos
Transplante Heterólogo/estatística & dados numéricos , Adulto , Animais , Cricetinae , Humanos , Infecções/epidemiologia , Infecções/etiologia , Infecções/transmissão , Transplante das Ilhotas Pancreáticas/estatística & dados numéricos , Transplante de Rim/estatística & dados numéricos , Coelhos , Ovinos , Suínos , Transplante/estatística & dados numéricos , Transplante Heterólogo/efeitos adversos , Transplante Heterólogo/legislação & jurisprudência , Organização Mundial da SaúdeRESUMO
OBJECTIVE: It is generally admitted that the endocrine cell organization in human islets is different from that of rodent islets. However, a clear description of human islet architecture has not yet been reported. The aim of this work was to describe our observations on the arrangement of human islet cells. RESEARCH DESIGN AND METHODS: Human pancreas specimens and isolated islets were processed for histology. Sections were analyzed by fluorescence microscopy after immunostaining for islet hormones and endothelial cells. RESULTS: In small human islets (40-60 mum in diameter), beta-cells had a core position, alpha-cells had a mantle position, and vessels laid at their periphery. In bigger islets, alpha-cells had a similar mantle position but were found also along vessels that penetrate and branch inside the islets. As a consequence of this organization, the ratio of beta-cells to alpha-cells was constantly higher in the core than in the mantle part of the islets, and decreased with increasing islet diameter. This core-mantle segregation of islet cells was also observed in type 2 diabetic donors but not in cultured isolated islets. Three-dimensional analysis revealed that islet cells were in fact organized into trilaminar epithelial plates, folded with different degrees of complexity and bordered by vessels on both sides. In epithelial plates, most beta-cells were located in a central position but frequently showed cytoplasmic extensions between outlying non-beta-cells. CONCLUSIONS: Human islets have a unique architecture allowing all endocrine cells to be adjacent to blood vessels and favoring heterologous contacts between beta- and alpha-cells, while permitting homologous contacts between beta-cells.
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Células Secretoras de Glucagon/citologia , Células Secretoras de Insulina/citologia , Ilhotas Pancreáticas/citologia , Adolescente , Adulto , Idoso , Células Cultivadas , Humanos , Técnicas In Vitro , Microscopia de Fluorescência , Pessoa de Meia-Idade , Adulto JovemRESUMO
Shortage of organ donors limits the number of possible liver transplantations. Alternative therapies for treatment of liver failure are currently being developed: (i) extracorporeal artificial liver devices; (ii) bioartificial liver devices using hepatocytes; and (iii) hepatocyte transplantation. The objective of these strategies is to bridge patients with liver failure until a suitable liver allograft is obtained for transplantation or the patient's own liver regenerates sufficiently to resume normal function. In this review, we discuss these strategies and summarize the current status of clinical experience.
Assuntos
Hepatócitos/transplante , Transplante de Fígado , Fígado Artificial , Ensaios Clínicos como Assunto , Desenho de Equipamento , Hepatócitos/fisiologia , Humanos , Fígado Artificial/estatística & dados numéricos , Fígado Artificial/tendências , Doadores de Tecidos/provisão & distribuiçãoRESUMO
Multipotent mesenchymal stromal cells (MSC) are currently investigated clinically as cellular therapy for a variety of diseases. Differentiation of MSC toward endodermal lineages, including hepatocytes and their therapeutic effect on fibrosis has been described but remains controversial. Recent evidence attributed a fibrotic potential to MSC. As differentiation potential might be dependent of donor age, we studied MSC derived from adult and pediatric human bone marrow and their potential to differentiate into hepatocytes or myofibroblasts in vitro and in vivo. Following characterization, expanded adult and pediatric MSC were co-cultured with a human hepatoma cell line, Huh-7, in a hepatogenic differentiation medium containing Hepatocyte growth factor, Fibroblast growth factor 4 and oncostatin M. In vivo, MSC were transplanted into spleen or liver of NOD/SCID mice undergoing partial hepatectomy and retrorsine treatment. Expression of mesenchymal and hepatic markers was analyzed by RT-PCR, Western blot and immunohistochemistry. In vitro, adult and pediatric MSC expressed characteristic surface antigens of MSC. Expansion capacity of pediatric MSC was significantly higher when compared to adult MSC. In co-culture with Huh-7 cells in hepatogenic differentiation medium, albumin expression was more frequently detected in pediatric MSC (5/8 experiments) when compared to adult MSC (2/10 experiments). However, in such condition pediatric MSC expressed alpha smooth muscle more strongly than adult MSC. Stable engraftment in the liver was not achieved after intrasplenic injection of pediatric or adult MSC. After intrahepatic injection, MSC permanently remained in liver tissue, kept a mesenchymal morphology and expressed vimentin and alpha smooth muscle actin, but no hepatic markers. Further, MSC localization merges with collagen deposition in transplanted liver and no difference was observed using adult or pediatric MSC. In conclusion, when transplanted into an injured or regenerating liver, MSC differentiated into myofibroblasts with development of fibrous tissue, regardless of donor age. These results indicate that MSC in certain circumstances might be harmful due to their fibrogenic potential and this should be considered before potential use of MSC for cell therapy.
Assuntos
Cirrose Hepática/patologia , Fígado/patologia , Mesoderma/citologia , Células Estromais/citologia , Actinas/metabolismo , Animais , Western Blotting , Diferenciação Celular , Colágeno/metabolismo , Humanos , Imuno-Histoquímica , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: Accurate determination of islet purity and mass before transplantation is an essential part of quality control. The standard method is based on manual evaluation of these parameters and thus subjective and prone to errors. Therefore, we developed a computerized approach aimed at evaluating more objectively the number and purity of isolated human islets. METHODS: Islets were isolated and purified from human pancreata according to a standard method. For each preparation, two samples were dithizone stained. One sample was analyzed manually by microscopy, following the standard procedure, and the other was digitally photographed for both digital manual and computerized analyses. Computerized analysis was performed using the MetaMorph and ImageJ softwares to automatically quantify purity and size of islets. Islet equivalent (IEQ) number was calculated using the Ricordi algorithm or considering the individual volume of each islet. Computerized analysis was validated using calibrated red glass microspheres. RESULTS: When digital manual and computerized analyses were compared, mean values of total islet number, IEQ number calculated using the Ricordi algorithm, and purity were similar. Comparisons of individual values showed good correlations (r>or=0.89). By standard manual analysis, total islet number and purity were higher and IEQ number similar compared with digital manual and computerized analyses. IEQ number was 10% lower (P<0.0001) when calculated using individual sphere volumes compared with the Ricordi algorithm. Measurement of red glass microspheres showed identical values comparing standard manual and computerized analyses. CONCLUSIONS: Computer-assisted digital image analysis is an objective and a reliable method for analyzing pancreatic islets before transplantation.
Assuntos
Transplante das Ilhotas Pancreáticas/instrumentação , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Algoritmos , Separação Celular/métodos , Vidro , Humanos , Processamento de Imagem Assistida por Computador/métodos , Ilhotas Pancreáticas/metabolismo , Microscopia , Microesferas , Pâncreas/patologia , Controle de Qualidade , Reprodutibilidade dos Testes , Software , Obtenção de Tecidos e Órgãos/métodosRESUMO
Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH represent a new valuable model of steatosis, not associated with insulin resistance, to study at both the genetic and functional level factors involved in the process of lipid accumulation and steatosis-associated liver injury.