[Phenotypical properties and ability to multilineage differentiation of adipose tissue stromal cells during subculturing].

Morphological and immunophenotypical properties of human adult adipose tissue stromal cells (ATSC) at cultivation passage 0 and 4 as well as their ability to induced in vitro differentiation into adipogenic and osteogenic directions were studied in this work. It was shown that primary cultures of ATSC were characterized by the presence of the lower number of cells expressing mesenchymal markers (CD73, CD105) than the cells of the 4th passage, but contained endothelial progenitor cells expressing CD34 and capable to form capillary-like structures within extracellular matrix. Both cell populations could equally differentiate into adipogenic and osteogenic lineages.

[Changes of ultrastructure of the capillary endotheliocytes of ischemized and nonaffected muscular tissue after transplantation of human hemopoietic stem cells of fetal liver in experiment in vivo].

In experiment was investigated ultrastructure of the capillaries endothelial cells and histological peculiarities of muscular tissue on various stages after transplantation of hemopoietic stem cells of fetal liver (HSCFL). There was proved, that in ischemic environment HSCFL stimulate processes of angiogenesis, and in the case of transplantation into intact muscular tissue they are differentiating into the tissue macrophages, not interfering with muscular tissue structure.

[Osteogenic and adipogenic capacity of fibroblast-like progenitor cells derived frow human fetal liver].

The study of differentiation potential of multiponent stromal progenitor cells (PCs) in embryogenesis is a crucial issue for understanding their biology and role in tissue regeneration of an adult organism. In this study in monolayer culture there were investigated osteogenic and adipogenic capacities of fibroblast-like PCs derived from human fetal liver of 8-11 gestation weeks before and after exposure to cryoprotectant dimethyl sulphoxide (DMSO). It was shown that the primary suspension of human fetal liver cells included immature stromal fibroblast-like PCs which were able to be induced into osteogenic and adipogenic differentiation. A short-time exposure of freshly isolated human fetal liver cells to cryoprotectant DMSO led to altering properties of the fibroblast-like PCs. Under subculture conditions, it was found an increase in the number of fibroblast-like PCs which were able to be induced to osteogenic differentiation in vitro. The established fact of DMSO influence on the differentiation capacity of fetal fibroblast-like PCs is necessary to take into consideration while developing cryopreservation methods for stem cells.

[Effect of human fetal liver cells on the rate of DNA and nRNA synthesis in regenerating rat liver]. / Vliianie émbrional'nykh kletok pecheni cheloveka na sintez DNK i iaRNK v regeneriruiushchei pecheni krys.

The model of partial hepatectomy was utilized to investigate the influence of human fetal liver cells (FLCs) and of human embryo tissue (PCF) postnuclear cytoplasm fraction injection on the rate of DNA and nRNA synthesis in regenerating rat liver cells. Single infusion of FLCs and PCF into the spleen pulp of rats has been shown to increase the DNA synthesis 24 hours after the operation in 2.5 +/- 0.4 and 3.2 +/- 0.5 times respectively. A group of rats has been identified with no influence of the infusion on the DNA synthesis 24 hours after partial hepatectomy, this process having been even retarding in 48 hours after the operation. Meanwhile the FLCs and PCF infusion enhanced the intensity of nRNA synthesis in 72 hours after the operation in all the animal groups. The effect demonstrated is probably caused by the biologically active substances contained in the fetal tissues.

[Comparative study of the effect of paracetamol, phenacetin and amidopyrine on mitochondrial function in isolated rat hepatocytes]. / Sravnitel'noe izuchenie deistviia paratsetamola, fenatsetina i amidopurina na funktsiiu mitokhondrii v izolirovannykh gepatotsitakh krys.

Effects of amidopyrine, phenacetin and paracetamol on the viability and energetic state of isolated rat hepatocytes were compared. During incubation in minimal salt solution all drugs in concentrations above 1 mM in dose-dependent manner decreased the viability of hepatocyte suspension assessed by trypan blue dye inclusion and inhibited the ATP synthesis and the respiratory activity. Cytotoxic effect of chemicals decrease in the order: amidopyrine-->phenacetin-->paracetamol. The inhibition of the rate of endogenous respiration were accompanied by stimulation of oxygen consumption in nonmitochondrial systems. An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and disruption of plasma membrane by digitonin followed by substrate succinate addition did not restore the respiratory activity of hepatocytes up to the level of control cells. These data show that cytotoxicity of the chemicals is determined by their interaction with enzymes of mitochondrial respiratory chain.

[Effect of starvation on the metabolism of the xenobiotic p-nitroanisole in isolated hepatocytes of rats of both sexes]. / Vliianie goloda na metabolizm ksenobiotika p-nitroanizola v izolirovannykh gepatotsitakh krys raznogo pola.

Hepatocytes isolated from fed female rats are characterized by the lower contents of cytochrome P-450 and more slowly metabolize the xenobiotic p-nitroanisole (p-NA) than hepatocytes from males. The starvation does not change the cytochrome P-450 contents, but depresses the p-NA biotransformation. The inhibition of p-NA O-demethylation capacity is a result of insufficiently reduced equivalent supply, because the addition of exogenous NADPH to the cells with digitonin-permeabilized plasma membrane increases p-nitrophenol (p-NPh) formation. In the presence of exogenous NADPH hepatocytes from fasting male and female rats produce more p-NPh, than those from fed ones. That suggests the induction of xenobiotic biotransformation system during starvation. Sex differences in response of xenobiotic biotransformation system to starvation manifest themselves in different ratio in contents of free and conjugated p-NPh.

[Effect of the composition of liver perfusion media on functional properties of isolated hepatocytes and effectiveness of the isolation method]. / Vliianie sostava sredy perfuzii pechnei na funktsional'nye svoistva izolirovannykh gepatotsitov i éffektivnost' metoda ikh vydeleniia.

Yield, viability and respiratory activity of hepatocytes isolated from rat after liver perfusion by media with various composition have been studied. Collagenase, EDTA or citrate were used as disaggregative agents, as well as sucrose or NaCl as a main osmotic component. The yield of intact cells after perfusion by different media decreased in the following order, collagenase -NaCl-->EDTA-sucrose-->citrate-sucrose-->EDTA-NaCl. The rate of endogenous respiration in hepatocytes isolated by non-enzymatic methods with EDTA did not depend on the nature of main osmotic component and was by 40% lower than in the cells isolated by the enzymatic method, however uncoupler-stimulated respiration changed significantly less. Replacement of EDTA by citrate increased the rate of endogenous respiration and particularly the uncoupler-stimulated respiration. The results show that the nature of disaggregative agent and main osmotic component has the influence on efficiency of cell isolation method as well as metabolic state of isolated hepatocytes. Sucrose leads to the cell shrinkage and have additional to Ca(2+)-chelator desaggregative effect, citrate plays two functions both as Ca(2+)-chelator and as mitochondrial substrate.

[The effect of paracetamol and its combinations with acetylsalicylic and ascorbic acids on lipid peroxidation processes in the rat liver]. / Vliianie paratsetamola, ego kombinatsii s atsetilsalitsilovoi i askorbinovoi kislotami na protsessy perekisnogo okisleniia lipidov v pecheni krys.

Paracetamolum (PC) after i.p. administration to rats at the therapeutic (100.0 mg/kg) and subtoxic (500.0 mg/kg) doses was shown to induce significant and dose-dependent elevation of the level of malone dialdehyde (MDA) as well as the increase in a certain extent of chemiluminescence (CL) being injected at the higher dose. Acetylsalicylic acid (ASA) at the same doses being injected at the lower one, induced the tendency toward the reduction of MDA level, while at the higher dose the rise of MDA content and CL intensity compared to PC have been observed. PC combination with ASA at therapeutic doses range (50.0 + 50.0 mg/kg) significantly diminished MDA formation, while at subtoxic doses range (250.0 + 250.0 mg/kg) such effect was not detected, although subsequent tendency toward CL intensity reduction was observed. Ascorbinic acid being administered in combination with PC and ASA had no influence on MDA formation but significantly weakened CL intensity when subtoxic doses of indicated drugs were used.

[Respiratory and detoxication activity of two fractions of hepatocytes isolated by a non-enzymatic method]. / Dykhatel'naia i detoksikatsionnaia aktivnost' dvukh fraktsii gepatotsitov, vydelennykh nefermentativnym metodom.

Slow centrifugation of isotonic sucrose medium containing the suspension of hepatocytes, obtained after disaggregation of liver, which has been preliminarily perfused by EDTA solution, results in the formation of two fractions of cells of different colour. Fraction 1 contains intact and metabolically active hepatocytes. Cells of fraction 2 have a damaged membrane, but they are capable of realizing biotransformation of xenobiotics in the presence of exogenous NADPH as well as maintaining efficiency of oxidative phosphorylation of mitochondria under the transfer to the medium simulating cytoplasm.

[Exogenous NADPH restores the detoxication function of isolated hepatocytes lost in the process of freezing-thawing]. / Ekzogennyi NADPH vosstanavlivaet detoksikatsionnuiu funktsiiu izolirovannykh gepatotsitov, utrachennuiu v protsesse zamorazhivaniia-otogreva.

Freezing and thawing have been studied for their effect on the rat hepatocyte detoxication system. Freezing-thawing of hepatocytes in the medium without cryoprotectors is to impair functioning of the biotransformation system of biphenyl, a hydrophobic xenobiotic. It is found that inhibition of the first stage of xenobiotics biotransformation is a result of the loss of pyridine nucleotides and substrates necessary for NADP+ reduction.

[Role of albumin in the biotransformation of biphenyl by isolated rat hepatocytes]. / Rol' al'bumina v biotransformatsii bifenila izolirovannymi gepatotsitami krys.

The role of albumin in biotransformation of biphenyl, a lipophilic xenobiotic, by isolated rat hepatocytes has been studied. It is shown that in the absence of albumin biphenyl is quickly and almost completely bound by cells. The rate of formation of 4-hydroxybiphenyl, the reaction product, depends on the substrate concentration (in the range 8.5-70 microM) and conforms with the Mikhaelis-Menten equation. An increase in the biphenyl concentration in the incubating medium to 140 microM induces no changes in the rate of its biotransformation. Serum albumin, while binding biphenyl, also reduces its effective concentration in a cell, which prevents the cytochrome-P-450-dependent monooxygenase system of hepatocytes from the inhibiting effect of high concentrations of the xenobiotic.

[Isolation of rat hepatocytes by a nonenzymatic method: detoxifying and respiratory activity]. / Vydelenie gepatotsitov krys nefermentativnym metodom: detoksikatsionnaia i dykhatel'naia aktivnosti.

A procedure for isolation of rat hepatocytes involving perfusion by EDTA, mechanical disaggregation of the liver and separation of intact cells from damaged ones by low-velocity centrifugation in isotonic sucrose media is described. A layer of a darker colour formed after separation contains hepatocytes with native plasma membrane characterized by respiratory activity and rate of biotransformation being close to the values obtained with cells isolated with the application of collagenase.

[Respiratory activity of isolated hepatocytes]. / Dykhatel'naia aktivnost' izolirovannykh gepatotsitov.

Oligomycin and uncoupler of oxidative phosphorylation have been studied for their effect on the respiration activity of hepatocytes in rats. The respiration rate in the presence of oligomycin and uncoupler is higher than it is with the respiration uncoupled in the absence of oligomycin. Exogenic succinate makes endogenic respiration of hepatocytes in the presence of digitonin 5 times more intensive. The obtained results evidence for the fact that the uncoupled respiration is limited by the concentration of substrates able to be oxidized in the respiration chain of mitochondria. Oligomycin induces accumulation of substrates and following addition of the disconnector evokes their fast oxidation.

[Energy state of hepatocytes of fed rats isolated by the means of EDTA and vibration]. / Energeticheskoe sostoianie gepatotsitov sytykh krys, vydelennykh s pomoshch'iu EDTA i vibratsii.

The energy state of mitochondria in fed rat hepatocytes isolated by the use of non-enzymatic method including liver perfusion with an EDTA-containing solution with a further mild mechanical effect of tissue fragments by vibration has been studied. The isolation procedure used permits to obtain significant amounts of hepatocytes whose viability is not less than 80%. The endogenous respiration rate (10-15 nm O2/min.mln cells) is slightly stimulated by succinate. In the course of incubation in a balanced salt medium, the cells accumulate ATP and the lipophilic cation, tetraphenylphosphonium. Data from the inhibitory analysis testify to the fact that tetraphenylphosphonium accumulation reflects the membrane potential of intact cell mitochondria, which are in a metabolic state similar to state 3.

[Isolation of intact mitochondria from the rat liver using vibration]. / Vydelenie intaktnykh mitokhondrii iz pecheni krys s ispol'zovaniem vibratsii.

A method for isolating mitochondria from the rat liver is described. In this method the homogenization step is replaced by vibration with the frequency of 50 Hz realized by a simple device. Mitochondria isolated by vibration demonstrate higher indices of the oxidative phosphorylation with succinate and glutamate + malate used as substrates than those isolated by homogenization do. The method described permits decreasing considerably the isolation medium expenditure remaining the mitochondria yield per gram of the liver unchanged.

[A non-enzymatic method of preparing isolated hepatocytes: assessment of their morpho-functional activity]. / Nefermentativnyi metod polucheniia izolirovannykh gepatotsitov: otsenka ikh morfo-funktsional'noi aktivnosti.

A method for isolating hepatocytes using the two-step liver perfusion by chelate-containing solutions is described. The hepatocytes obtained are highly inactive for the trypan blue staining test, (not less than 90%), ATP content, the rate of endogenic respiration and its stimulation by succinate, a degree of xenobiotic oxidation.

[Functional characteristics of rat hepatocytes isolated by a nonenzymatic method and incubated in vitro]. / Nekotorye kharakteristiki sostoianiia gepatotsitov krys, izolirovannykh nefermentativnym sposobom i inkubiruemykh in vitro.

A study was made of the effect of normothermic incubation on the metabolic state of parenchymal liver cells, isolated by the nonenzymatic method. In the process of a 2 hour incubation in the nutritious sodium-containing medium, a high level of the energy supply system is maintained, determined by the contents of adenine nucleotides, the rate of endogenous respiration and by its stimulation by succinate.

[The role of lipolysis and lipid peroxidation in the induction of ion transport in mitochondria after freezing and thawing]. / Rol' lipoliza i perekisnogo okisleniia lipidov v induktsii ionnogo transporta mitokhondrii posle zamorazhivaniia-ottaivaniia.

The reasons of ion transport induction observed after freezing-thawing of the rat liver mitochondria were investigated. It is found that a fall in the membrane potential value, an increase in the respiration rate and K+ ion release from mitochondria to the incubation medium with phosphate are averted when succinate is replaced by the Kreb's cycle substrates and when ionol (an antioxidant) or nupercain (an inhibitor of mitochondrial phospholipase A2) are incorporated into the incubation medium. The induction of ion transport is caused by the activation of peroxide processes and of lipolysis associated with them. It is supposed that under experimental conditions after freezing-thawing a degree of pyridine nucleotides and glutathione reduction lowers, that in its turn leads to the inhibition of the glutathione-peroxidase activity and development of peroxide processes.

[Ion transport in rat liver mitochondria after freezing and thawing]. / Ionnyi transport v mitokhondriiakh pecheni krys posle zamorazhivaniia-ottaivaniia.

Induction of ion transport in the rat liver mitochondria arising after their deep freezing is inhibited effectively by adenosine diphosphate, Mg ions, Ca2+ complexons (EDTA, EGTA) as well as by inhibitors of ATPase (oligomycin and dicyclohexylcarbodiimide) and of electrogenic transport of Ca2+ (ruthenium red). The value of ion flows is determined by adenine nucleotide concentration in the matrix. It is supposed that specific inhibitors of ion electrogenic transport promoting an increase in the transmembrane potential prevent from releasing adenine nucleotides from the mitochondrial matrix.

[Ca2+ release from rat liver mitochondria subjected to deep freezing]. / Vysvobozhdenie Ca2+ iz mitokhondrii pecheni krys, podvergnutykh glubokomu zamorazhivaniiu.

Freezing-thawing of rat liver mitochondria evokes a release of Ca2+ into the incubation medium containing oxidation substrates: succinate and phosphate. The release is accompanied by a decrease in the membrane potential value. ATPase and NADH-dehydrogenase inhibitors when introduced into the medium simultaneously with mitochondria prevent the both processes, but only till Ca2+ release. ADP added during Ca2+ release, initiates its uptake. When succinate is replaced by NAD-dependent substrates (malate in combination with glutamate) and in the presence of the antioxidant (ionol) Ca2+ is not released from mitochondria. It is supposed that Ca2+ release from mitochondria due to freezing-thawing is evoked by activation of lipid peroxidation and is controlled by the degree of pyridine nucleotide reduction.