RESUMEN
Primary human hepatocytes (PHHs) are the gold standard for drug development procedures; however, maintaining functional PHHs in vitro is challenging in conventional collagencoated cultures. In the present study, we developed a new scaffold comprising high amounts (≥1 mg/cm2) of atelocollagen exposed to ultraviolet radiation to induce crosslinking and improve stability. Scanning and transmission electron microscopy revealed a microdimpled surface (MDS) scaffold composed of randomly arranged atelocollagen fibrils. The scaffold was therefore designated as MDS atelocollagen. PHHs cultured on MDS atelocollagen were round with a compact cytoplasm and exhibited enhanced levels of albumin (ALB) secretion and cytochrome P450 (CYP) 3A4 activity. The expression of hepatocyterelated genes, such as serum proteins, drug metabolismrelated CYPs, and nuclear receptors, was enhanced in cells cultured on MDS atelocollagen, but not in those cultured on conventional atelocollagen. Moreover, the abnormal gene expression of cell adhesion molecules observed in conventional atelocollagen culture was suppressed when the cells were grown on MDS atelocollagen, thereby suggesting a cell behavior similar to that of in vivo hepatocytes. These results suggest that MDS atelocollagen functionally preserves PHHs while conserving the simplicity of conventional PHH atelocollagencoated cultures.
Asunto(s)
Colágeno/farmacología , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Biomarcadores , Técnicas de Cultivo de Célula , Células Cultivadas , Colágeno/química , Colágeno/metabolismo , Perfilación de la Expresión Génica , Hepatocitos/citología , HumanosRESUMEN
OBJECTIVES/HYPOTHESIS: Artificial tracheas prepared using a collagen sponge and polypropylene mesh have been implanted in patients who received tracheal resections, but epithelialization in the reconstructed area is slow. We determined the optimal bovine atelocollagen concentration necessary for the rapid and complete tracheal epithelial coverage of collagen sponge implants. STUDY DESIGN: Preliminary animal experiment. METHODS: Collagen sponges were prepared using lyophilizing 0.5%, 0.7%, and 1.0% atelocollagen solutions (0.5%, 0.7%, and 1.0% sponges) and were analyzed using scanning electron microscopy. Partial tracheal defects were prepared in rabbits and reconstructed using sponges. Epithelial regeneration in the reconstructed area was evaluated by endoscopic, histological, and scanning electron microscope analyses. RESULTS: All sponges had a membranous structural framework, and numerous fibrous structures filled the spaces within the framework in the 0.5% sponges. The membranous structure in the 0.7% sponges branched at many points, and intermembrane spaces were frequently observed. Conversely, the membranous structure in the 1.0% sponges was relatively continuous, thick, and closely arranged. Two weeks after implantation, tracheal defects were entirely covered with epithelium in two of the four and three of the four of the 0.5% and 0.7% sponge-implanted rabbits, respectively. The collagen sponges remained exposed to the tracheal lumen in four of the four rabbits in the 1.0% sponge group. Ciliogenesis in the center of the epithelialized region was detected only in the 0.7% sponge group. CONCLUSION: Collagen sponges prepared from various concentrations of bovine atelocollagen have different structures. Complete epithelial coverage was achieved in more rabbits implanted with sponges prepared using the 0.7% bovine atelocollagen solution than in those implanted with sponges prepared from the 0.5% and 1.0% solutions. LEVEL OF EVIDENCE: NA Laryngoscope, 126:E396-E403, 2016.
Asunto(s)
Órganos Artificiales , Colágeno , Mucosa Respiratoria/cirugía , Andamios del Tejido , Tráquea/cirugía , Animales , Bovinos , Regeneración Tisular Dirigida/métodos , Masculino , Microscopía Electrónica de Rastreo , Modelos Animales , Polipropilenos , Poríferos , Conejos , Regeneración , Ingeniería de TejidosRESUMEN
BACKGROUND: Helicobacter pylori survival in a hostile acidic environment is known to be caused by its production of urease, which is not released by known secretion pathways. It has been proposed that H. pylori cells undergo spontaneous autolysis during cultivation and that urease becomes surface-associated only concomitant with bacterial autolysis. The aim of this study was to elucidate mechanisms by which H. pylori cells undergo autolysis during cultivation. MATERIALS AND METHODS: Autolysis of H. pylori KZ109 cells was estimated by measuring the turbidity of the culture, by detection of cytoplasmic protein release into the culture supernatant and by scanning electron microscopic observation of H. pylori cells during cultivation. An autolysis-inducing factor (AIF) was partially purified from the culture supernatant by a partition method using ethyl acetate. RESULTS: Bacterial turbidity of KZ109 cells was drastically decreased after late-log phase accompanying release of urease and HspB into the extracellular space. Concomitantly, cell lytic activity was detected in the culture supernatant. Scanning electron microscopic observation suggested that partially purified AIF induced cell lysis. It was also shown that the AIF is different from other autolytic enzymes or substances so far reported. CONCLUSIONS: This study demonstrated the presence of the peptidergic autolytic substances in the culture supernatant of H. pylori KZ109 cells. The results of this study should be useful for further studies aimed at elucidation of the strategy of survival of H. pylori in the gastric environment and elucidation of the mechanisms of pathogenesis induced by H. pylori.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bacteriólisis/fisiología , Helicobacter pylori/fisiología , Péptidos/metabolismo , Proteínas Bacterianas/farmacología , Bacteriólisis/efectos de los fármacos , Campylobacter jejuni/efectos de los fármacos , Campylobacter jejuni/fisiología , Medios de Cultivo Condicionados/química , Citoplasma/metabolismo , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/crecimiento & desarrollo , Helicobacter pylori/ultraestructura , Humanos , Microscopía Electrónica de Rastreo , Péptidos/farmacología , Esferoplastos/fisiología , Esferoplastos/ultraestructura , Ureasa/metabolismoRESUMEN
The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.