RESUMEN
Placenta is a highly specialized organ that is necessary for successful gestation. Several models of the placental barrier are used to study how it functions, including the transplacental transport of xenobiotics. One of these models, human choriocarcinoma cell line BeWo is widely used in vitro. Notably, cancerous BeWo cells form multilayer structures that normally are not found in the human placenta. Here, we aim to develop techniques suitable for monitoring BeWo b30 cells in culture. To assess the state of BeWo b30 cells growing on a membrane, we use impedance spectroscopy, which allows us to estimate the number of cell layers by the change in the electrical parameters of the biological system. In mature BeWo b30 cell cultures, we also note a significant increase in the expression of genes encoding metallothioneins (particularly, MT1B, MT1F, and MT2A) and syncytins (ERVW-1 and ERVFRD-1), which can be used as biomarkers reflecting the development of mature phenotypic characteristics, namely, trophoblastic invasion and formation of the syncytium.
Asunto(s)
Coriocarcinoma/genética , Coriocarcinoma/patología , Espectroscopía Dieléctrica , Perfilación de la Expresión Génica , Modelos Biológicos , Placenta/citología , Placenta/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Femenino , Humanos , EmbarazoRESUMEN
We compared available methods for monitoring the integrity of in vitro models of barrier tissues and studied the possibility of using impedance spectroscopy to solve this problem. It was demonstrated (theoretically and experimentally) that TEER measurements are not sufficiently sensitive to detect small defects in the cell barrier that significantly affect its permeability. For obtaining reliable results, it is necessary to set a sufficiently high threshold TEER, which leads to the loss of many intact samples. At the same time, impedance spectroscopy has all advantages of the classical method of measuring TEER (it is rapid and non-invasive method), while its application in combination with the methods of machine learning allows reliable detection of defects in the cell barrier.
Asunto(s)
Espectroscopía Dieléctrica/métodos , Mucosa Intestinal/metabolismo , Células CACO-2 , Impedancia Eléctrica , HumanosRESUMEN
Extracellular microRNA are one of the indicators of the functional state of cells. Culturing of Caco-2 cells under the conditions of microcirculation in a Homunculus microfluidic device allows better simulating natural environment of the body in comparison with static culturing. Impedance spectroscopy (BioClinicum Research Center) was used for non-invasive estimation of the monolayer quality and changes in the cell apical membrane due to the formation of microvilli. Under static conditions, Caco-2 cells release more microRNA from the apical membrane than under microcirculation conditions, while secretion of miR-320a, miR-24-3p, and miR-221-3p microRNA under static conditions can indicate stress of the cells and activation of inflammatory response. Under microcirculation conditions, the expression of laminin-α1 (LAMA1) was lower than under static conditions, which indicates deeper differentiation of cells.
Asunto(s)
Membrana Celular/metabolismo , Dispositivos Laboratorio en un Chip , MicroARNs/metabolismo , Microcirculación/fisiología , Células CACO-2 , Diferenciación Celular/fisiología , Espectroscopía Dieléctrica , HumanosRESUMEN
We studied the possibility of using viscumin lectin (MLI) for targeted delivery of antitumor drugs. Affinity of MLI for more than 600 oligosaccharide structures was determined and the glycosylation profiles of cell surface in various mouse tissues were analyzed. It was found that biodistribution of MLI was determined by not only expression of oligosaccharides specifically recognized by the lectin in tissue cells, but also by the structure of glycan in general.
Asunto(s)
Lectinas de Plantas/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 2/metabolismo , Toxinas Biológicas/metabolismo , Animales , Femenino , Glicosilación , Masculino , Ratones , Ratones Endogámicos BALB C , Oligosacáridos/metabolismo , Polisacáridos/metabolismoRESUMEN
External magnetic field is characterized by low toxicity and existence of magnetic properties, which contributes to an interest in the development of products from ferromagnetic nanoparticles (FNP) for antitumor therapy. Previously we synthesized a conjugate of ferromagnetic magnetite nanoparticles and viscumin (mistletoe lectin I, MLI), which exhibits the antitumor activity. Studying the pharmacological properties of this conjugate (FNP-MLI) was directed to the evaluation of FNP-MLI elimination after intratumor injection in mice. The elimination rate of FNP-MLI was much lower than that of native plant MLI. The presence of FNP-MLI was not accompanied by undesired changes in the tumor tissue. The use of a FNP-MLI conjugate allowed us to prolong the time of MLI presence in tissues without increasing the dose of exogenous lectin. These features contribute to the prolongation of an immunomodulatory effect of MLI.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antineoplásicos Fitogénicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Portadores de Fármacos/administración & dosificación , Nanopartículas de Magnetita/administración & dosificación , Proteínas Inactivadoras de Ribosomas Tipo 2/farmacocinética , Toxinas Biológicas/farmacocinética , Adenocarcinoma/diagnóstico por imagen , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Portadores de Fármacos/farmacocinética , Femenino , Humanos , Inyecciones Intralesiones , Inyecciones Subcutáneas , Imagen por Resonancia Magnética , Ratones , Ratones SCID , Proteínas Inactivadoras de Ribosomas Tipo 2/farmacología , Toxinas Biológicas/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Magnetite particles modified by polyethylene glycol with a molecular weight of 3 kDa and hydrodynamic diameter of ~60 nm were used. Plant lectin viscumin covalently immobilized on these nanoparticles retained its binding activity. Immunochemical characteristics of conjugated viscumin were evaluated using monoclonal antibodies. The resultant conjugate with a hydrodynamic diameter of 70 nm was used for studies of binding and internalization by target cells. Binding of viscumin and its conjugate was determined by receptors containing terminal galactose, while intracellular distribution varied. The model system presented in this study can be used for creation of drugs for target therapy.
Asunto(s)
Nanopartículas de Magnetita/química , Nanoconjugados/química , Proteínas Inactivadoras de Ribosomas Tipo 2/química , Toxinas Biológicas/química , Línea Celular Tumoral , Glioblastoma , Humanos , Tamaño de la Partícula , Proteínas Inactivadoras de Ribosomas Tipo 2/metabolismo , Toxinas Biológicas/metabolismoRESUMEN
We analyzed hepatotoxicity of three drugs: acetaminophen, metformin, and isoniazid. Spheroids of differentiated HepaRG cells cultured under microfluidic conditions were used as the model. Acute toxicity of substances was assessed by analyzing cell viability, while lactate concentration in the culture medium was used as the potential marker for evaluation of chronic exposure and non-lethal side effects of xenobiotics. The results were compared with mitochondrial activity and DNA fragmentation data. The efficiency and possibility of applying the integrated approach for assessment of drug hepatotoxicity are discussed.
Asunto(s)
Hepatocitos/efectos de los fármacos , Microfluídica/métodos , Xenobióticos/toxicidad , Línea Celular , Fragmentación del ADN/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Pruebas de Toxicidad/métodosRESUMEN
We studied the effects of DMSO and fibroblasts during HepaRG cell spheroid formation and conditions of their subsequent culturing on the levels of mRNA of the major cytochromes P450. A protocol of spheroid formation from differentiated HepaRG cells and their culturing in serum- and DMSO-free medium is developed.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Hepatocitos/enzimología , Esferoides Celulares/enzimología , Línea Celular , Medios de Cultivo/química , Sistema Enzimático del Citocromo P-450/metabolismo , Dimetilsulfóxido/química , Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
3D cell cultures are extensively used to study in vitro toxic effect of xenobiotics. When using multicellular spheroids, the question about their optimal size should be solved: small spheroids are difficult to manipulate, while large size of spheroids impairs the transport of nutrients and oxygen into the center. Mathematical models describing the distribution of substances in multicellular spheroids numerical procedure for solving differential equation system, which complicates their use in laboratory practice. We proposed and experimentally evaluated a new mathematical model describing oxygen distribution in HepaRG cell spheroids. Markers of functional activity were studied in spheroids of different size. The maximum size of spheroids that can be maintained in culture for 9 days without necrosis was determined.
Asunto(s)
Oxígeno/metabolismo , Esferoides Celulares/metabolismo , Línea Celular , Difusión , Hepatocitos/metabolismo , Humanos , Modelos Biológicos , Consumo de OxígenoRESUMEN
Acetaminophen in a concentration of 5 mM increased the expression of JNK, HIF1A (hypoxiainduced factor), and CASP3, which indicated development of oxidative stress and apoptotic cell death. Acetaminophen in a concentration of 10 mM did not induce expression of HIF1A and CASP3, but reduced expression of chaperone HSP90, which attested to activation of a caspase-3-independent mechanism of cell death. The methods of preventing acetaminophen intoxication are discussed.
Asunto(s)
Acetaminofén/farmacología , Analgésicos no Narcóticos/farmacología , Caspasa 3/metabolismo , Neuronas/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Estrés OxidativoRESUMEN
The behavior of magnetite nanoparticles was studied in the cell chip microcapillaries. No aggregation of magnetite nanoparticles under conditions of long-term circulation was noted. Biodistribution and toxicity of magnetite nanoparticles (14 nm) and aminated magnetite after their intragastric administration to mice were studied in vivo. According to mass spectrometry and microscopy data, accumulation of nanoparticles occurred mainly in the liver cells.
Asunto(s)
Nanopartículas de Magnetita/química , Animales , Animales no Consanguíneos , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Nanopartículas de Magnetita/toxicidad , Masculino , Ratones , Microcirculación , Técnicas Analíticas Microfluídicas , Modelos Biológicos , Soluciones , Distribución TisularRESUMEN
We developed a cytochrome P450 substrate-inhibitor panel for preclinical in vitro evaluation of drugs in a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). The concentrations of substrates and inhibitors were optimized to ensure reliable detection of the principal metabolites by HPLC-mass-spectroscopy. The selected specific substrate-inhibitor pairs, namely bupropion/2-phenyl-2-(1-piperidinyl)propane) for evaluation of CYP2B6B activity, tolbutamide/sulfaphenazole for CYP2C9, omeprazole/(+)-N-benzylnirvanol for CYP2C19, and testosterone/ketoconazole for CYP3A4, enable reliable evaluation of the drug metabolism pathway. In contrast to animal models characterized by species-specific expression profile and activity of cytochrome P450 isoforms, our in vitro model reflects the metabolism of human hepatocytes in vivo.
Asunto(s)
Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2C9/metabolismo , Citocromo P-450 CYP3A/metabolismo , Dispositivos Laboratorio en un Chip , Bupropión/metabolismo , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B6/análisis , Citocromo P-450 CYP2C19/análisis , Citocromo P-450 CYP2C9/análisis , Citocromo P-450 CYP3A/análisis , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Humanos , Cetoconazol/farmacología , Hígado/efectos de los fármacos , Hígado/enzimología , Espectrometría de Masas , Mefenitoína/análogos & derivados , Mefenitoína/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Omeprazol/metabolismo , Fenciclidina/análogos & derivados , Fenciclidina/farmacología , Especificidad por Sustrato , Sulfafenazol/farmacología , Testosterona/metabolismo , Tolbutamida/metabolismoRESUMEN
We studied the relationship between microcirculation parameters and functional status of HepaRG cells in spheroids and chose an optimal regimen within the physiologically permissible limits of mechanical impact for the cells that maintains the expression of functional genes of the liver.
Asunto(s)
Hígado/citología , Reactores Biológicos , Supervivencia Celular/fisiología , Hepatocitos/citología , Humanos , Microcirculación/fisiologíaRESUMEN
The central pattern generator (CPG) is defined as a set of neurons involved in joint production of patterned motor output. The roundtable discussion on the CPG was a part of the 5th All-Russian Conference on Animal Behavior (Moscow, Nov. 21, 2012). The discussion centred on three core themes: 1) the mechanisms of the organization and reconfiguration of pattern generating neuronal ensembles, 2) extrapolations that extend the CPG concept beyond the motor systems, and 3) evolutionary and developmental aspects of CPG.
Asunto(s)
Generadores de Patrones Centrales/fisiología , Animales , Evolución Biológica , Generadores de Patrones Centrales/crecimiento & desarrollo , HumanosRESUMEN
There is a growing recognition that Central Pattern Generators (CPGs) play the fundamental role in the production of motor commands. A CPG is defined as a set of neurons involved in joint production of patterned sequenced output. It is generally believed that the sequence arises from the pattern of synaptic connections among these neurons. Alternatively, it was suggested (Sakharov, 1985) that the orderly organization of pattern-generating units might be due to the chemical diversity of their constituent neurons representing different phenotypes. Recent researches demonstrate that a given CPG can be reorganized to produce a different pattern of output activity. This multifunctionality can hardly be explained in terms of the anatomical (= synaptic) organization. The heterochemical approach appears to be more flexible. We hypothesize that dynamic fluctuations of the local extracellular milieu determine the physiological properties and receptor profile of individual neurons, and thus the self-organisation of the latter into a CPG.
Asunto(s)
Encéfalo/fisiología , Generadores de Patrones Centrales/fisiología , Actividad Motora/fisiología , Neuronas Motoras/fisiología , Animales , HumanosRESUMEN
The analysis of the protein composition of exhaled breath to diagnose diseases of the respiratory system raises a problem of differentiation proteins of expressed in the tissues of the lungs and respiratory tract (endogenous) and got in the respiratory system from the ambient air in the process of respiration (exogenous). In this work an attempt was made to estimate a set of exhaled exogenic proteins by mass spectrometry coupled with nanoflow HPLC. Six-month isolation of healthy donors indoors with air cleaned of dust leads to removal from the spectrum of exhaled proteins of some keratins that are considered therefore to be exogenic. Non-keratin proteins may also circulate between the ambient air and human respiratory ways, but their concentration appears to be significantly lower the keratin concentrations (especially epidermis keratin). Among non-keratins dermcidin seems to be the most significant exogenic protein of exhaled air. The conclusion of the diagnostic value of exhaled proteins can be done only after careful comparison of the results of quantitative and qualitative analysis of their composition in norm and pathology for a statistically significant sample of donors.
Asunto(s)
Proteínas/análisis , Adulto , Contaminación del Aire Interior , Secuencia de Aminoácidos , Pruebas Respiratorias , Cromatografía Líquida de Alta Presión , Espiración , Humanos , Queratinas/análisis , Masculino , Espectrometría de Masas , Mapeo Peptídico , Factores de TiempoRESUMEN
Lactate (lactic acid) concentrations in sweat and venous and capillary blood of athletes were measured before and after exercise of the maximum aerobic power. Correlations between the increment of blood and sweat lactate concentrations were found. Lactate concentrations in the sweat can be used for evaluation of changes in blood lactate levels.
Asunto(s)
Ejercicio Físico/fisiología , Ácido Láctico/sangre , Ácido Láctico/metabolismo , Sudor/química , Adulto , Atletas , Humanos , Masculino , Adulto JovenRESUMEN
Extracellular concentration of heat shock protein (Hsp) with a molecular weight of 70 kDa (Hsp70) rapidly increases in the serum in response to stress and returns to the basal level during recovery. Further regulation of its blood concentration is unclear. A possible regulator is HspBP1, a protein binding Hsp70. Binding to ATPase domain of Hsp70, HspBP1 inactivates it, thus acting as a factor of nucleotide exchange. Blood sera from athletes were examined at the beginning and end of the last mesocycle of the training period by two-staged immunoaffinity test system. The concentration of HspBP1 increased with decreasing Hsp70 concentration under conditions of long-term training. Presumably, the dynamics of Hsp70 and HspBP1 concentrations can serve as the test for evaluating the adaptation potential.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/sangre , Proteínas HSP70 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Aptitud Física/fisiología , Adolescente , Prueba de Esfuerzo , HumanosRESUMEN
The mechanisms of early activation of natural killers were studied in volunteers of different athletic qualification. The initial levels of expression of KIR2DL2, PRF1, and GZMB genes regulating the functions of natural killer differ in athletes and untrained subjects. Moderate exercise stimulates transcription activities of these genes. In athletes, the expression increases more intensely than in controls. Stimulation of inhibitory (KIR2DL3) and activation (KIR2DS2) receptors was revealed. This indicated nonspecific stimulation of natural killers, probably mediated by an increase in serum concentration of heat shock protein with a molecular weight of 70 kDa.
Asunto(s)
Ejercicio Físico , Receptores KIR2DL3/genética , Receptores KIR/genética , Receptores de Células Asesinas Naturales/metabolismo , HumanosRESUMEN
Extracellular heat shock protein with molecular weight of 70 kDa is a signal molecule of the immune system. It is secreted by the peripheral blood, liver and muscle cells in response to physiological, thermal, and mental stresses. The main goal of our study was to compare the levels of expression of heat shock protein (70 kDa) matrix ribonucleic acid in leukocytes and serum concentrations of the protein before and after physiological stress. In order to solve this problem, we developed enzyme immunoassay of serum heat shock (70 kDa) protein concentration and a method for evaluating the expression of matrix ribonucleic acid of this protein in leukocytes by the real time PCR. The concentration of 70 kDa heat shock protein in the serum increased 1.7 times as a result of even a short-term highly intense physiological stress, while the expression of its matrix ribonucleic acid in leukocytes increased 1.5 times. The individual features determine the response to physiological stress. Probable sources of 70 kDa heat shock protein are discussed.