Method for prediction of early stage of apoptosis

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for the prediction of the early stage of lymphocyte apoptosis. That is ensured by isolating cells, incubating for 48 hours at temperature 37°C with 5% CO2 with adding apoptosis inductor, dexamethasone in the concentration of 10-4 mole/ml. A lymphocyte viability is quantified by trypan blue inclusion, the recovered and oxidised glutathione concentrations are measured in lymphocyte lysate after the 30-minute pre-incubation with 2-vinylpyridine 10 mM. The early stage of lymphocyte apoptosis is stated, if observing an integrated decrease of the recovered glutathione concentration by 17% and more and an increase of the oxidised glutathione concentration by 19% and more as compared to the reference.

EFFECT: using the presented method in medical practice enables predicting the antioxidant state of the patient's body accompanying various diseases as shown by the early stage of lymphocyte apoptosis evaluated.

1 tbl

 

Known methods of forecasting apoptosis and the development of ways to protect cells from apoptosis by activation of antioxidant activity by determining the growth of the total content of SH-groups of proteins [Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes / B. R. Burchill, J. M. Oliver, S. C. Pearson et al. // J. of Cell Biology. - 1978. - Vol.76, No. 2. - P. 439-447], but this technique does not allow to assess the level of reduced glutathione, and consequently, to evaluate the effectiveness of forecasting apoptosis. There is also known a method of indirect determination of the content of reduced glutathione on glutathione peroxidase activity [Medical laboratory technology: In 2 volumes. / Ed. by A. I. Karpischenko - So 2 / A. I. Karpischenko. - SPb.: Intermedica. - 1999. - 656 S.], but the activity of this enzyme depends on the conformation of the active center of the enzyme. When changing the conformation of the active center of the enzyme's activity is changing that it is not possible to accurately assess the level of reduced glutathione, and thus to predict apoptosis. Also known as the indirect method of determining the concentration of reduced glutathione in the activity of glutathione reductase [Medical laboratory technology: In 2 volumes. / Ed. by A. I. Karpischenko - So 2 / A. I. Karpischenko. - SPb.: Intermedica. - 1999. - 656 S.], but the activity of this enzyme depends on the conformation of outdoor activities the center of the enzyme. When changing the conformation of the active center of the enzyme activity changes that will not reliably in any situation to estimate with high accuracy the level of reduced glutathione, and thus to predict apoptosis.

There is also known a method of evaluating the efficiency of antioxidant activity in forecasting apoptosis on the content of reduced glutathione proposed by M. E. Anderson (1985) modified by S. Kojima et al. (2004) [Low dose gamma-rays activate immune functions via induction of glutathione and delay tumor growth / S. Kojima, K. Nakayama, H. Ishida // J. Radiat. Res. - 2004. - Vol.45, No. 1. - P. 33-39], based on the interaction of reduced glutathione (GSH) with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB). This forms the oxidized glutathione (GSSG), which is then restored and re-interacts with DTNB. This method is closest to the proposed to the technical essence and the achieved result, and selected as a prototype.

The aim of the invention is to improve the accuracy of predicting an early stage of apoptosis of lymphocytes.

This goal is achieved by the fact that it includes the stages of cell isolation, incubation of the cells for 48 hours at 37°C and 5% content of CO2with the addition of the inducer of apoptosis dexamethasone at a concentration of 10-4mol/ml, quantitative determination of the viability of the lymphocytes p is the inclusion Trypanosoma blue and biochemical determining the concentrations of reduced and oxidized glutathione in the lysate of lymphocytes after pre-incubation for 30 minutes with 10 mm 2-vinylpyridine) - derivatives while the comprehensive decrease in the concentration of reduced glutathione by 17% or more and the increase in the concentration of oxidized glutathione by 19% or more, compared with the control predict early stage of lymphocyte apoptosis".

New in the proposed as a method of the invention is a complex determination of reduced and oxidized glutathione after incubation of lymphocytes, which is essential for the prediction of early lymphocyte apoptosis.

Antioxidant system is aimed at the effective neutralization of hydroxyradicals and reducing toxic to the body of gidroperekisi. Hydroxyl radical (.OH) is involved in microbicide and cytotoxic action of neutrophils, monocytes and T-lymphocytes and other cells [Oxidative stress. Prooxidants and antioxidants / E. B. Menshikov, V. H. Lankin, N. To. Zenkov et al. - M.: The Word. - 2006. - S. 556]. There are two main mechanisms of synthesis.OH neutrophils: the first is the formation of hydrogen peroxide in the presence of metals of variable valence in the so-called "Fenton reaction": Fe2++H2O2→Fe3++.OH+OH-[Luwak C. I. Oxidative stress and mechanisms of protection against him in bacteria / Century Imperial Luwak // Biochemistry. - 2001. - T. 66, vol.5. - S. 592-609], the second in a series of reactions involving hypohalide:HO Cl+O2.-O.H+Cl-+O2; or the interaction of hypochlorite ions Fe2+[Spin trapping evidence for myeloperoxidase-dependent hydroxyl radical formation by human neutrophils and monocytes // C. L. Ramos, S. Pou, B. E. Britigan et al. // J. Biol. Chem. - 1992. - Vol.267. - P. 8307-8312; Candeias, L. P. Formation of hydroxyl radicals on reaction of hypochlorous acid with ferrocyanide, a model iron (II) complex / L. P. Candeias, M. R. L. Stratford, P. Wardman // Free Radical Res. - 1994. - Vol.20. - P. 241-249]. Therefore, if a cell has no variable valence metal in a free state, the Fenton reaction and this reaction will not be dangerous.

In the cells there are a number of protective reactions by blocking the free ions of Fe2+and Cu+. For example, activated cells synthesize lactoferrin binding free iron and convert it into a catalytically inactive form, and produce high concentrations of taurine, conjugating with hypochlorite and protects cells from its toxic effects [Taurine chloramines, a product of activated netrophils, inhibits in vitro the genetation of nitric oxide and other macrofage inflammatory mediators / J. Marcinkiewiez, A. Grabowska, J. Bereta et al. // J. Leukocyte Biol. - 1995. - Vol.58. - P. 667-674].

Education·HE shows during microsomal oxidation, oxidation of arachidonic acid in reactions with flavins what armentani, the ubiquinone, peroxynitrite. Several studies in vitro have been certified products 'IT is these cells [Hydroxylation of salicylate by activated neutrophils / W. B. Davis, B. S. Mohammed, D. C. Mays et al. // Biochem Pharmacol. - 1989. - Vol.38. - P. 4013-4019]. However, the study of these reactions are often based on the use of inhibitors and measure the level of secondary products. Therefore, reactions attributed.IT could be caused by other oxidants, in particular O2.-or hypochlorous acid [Do human neutrophils form hydroxyl radical. Evaluation of an unresolved controversy / M. S. Cohen, B. E. Britigan, D. J. Hassett et al. // Free It Biol Med. - 1988. - Vol.5. - P. 81-90]. Some authors consider that in vitro by the reaction of Fenton cells produce a small quantity of.HE [Rosen, G. M. Free radicals and phagocytic cells / G. M. Rosen, S. Pou, C. L. Ramos // FASEB J. - 1995. - Vol.9. - P. 200-211].

Generation.HE stimulated cells in inflammation may be significantly limited by the absence in the environment of iron ions study of the Fenton reaction in the cells revealed that blocking ions of iron by lactoferrin directly inhibits the reaction [Rosen, G. M. Free radicals and phagocytic cells / G. M. Rosen, S. Pou, C. L. Ramos // FASEB J. - 1995. - Vol.9. - P. 200-211], and disposal of H2O2the myeloperoxidase limits the reaction, even if iron is available [Winterbourn, C. C. Myeloperoxidase as an effective inhibitor of hydroxyl radical production: Implications for the oxidative reactions of neutrophils / C. C. Winterbourn // J. Clin. Invest. - 1986. - Vol.78. - P. 545-557]. Although Bo is isinto biological forms of iron catalytically inactive, the ability of cells to the production.HE's in the presence of transferrin, susceptible to proteolytic degradation [Phagocyte-derived free radicals stimulated by ingestion of ironrich Staphylococcus aureus: Aspin-trapping study / M. S. Cohen, B. E. Britigan, Y. S. Chai et al. // J. Infect Dis. - 1991. - Vol.163. - 819-826], or iron in the composition of Pseudomonas aeruginosa containing siderophore pyochelin [Possible role of bacterial siderophores in inflammation-Iron bound to the pseudomonas siderophore pyochelin can function as a hydroxyl radical catalyst / T. J. Coffrnan, C. D. Cox, B. L. Edeker et al. / J. Clin. Invest. - 1990. - Vol.86. - P. 1030-1038]. However, M. S. Cohen and co-authors found that intracellular iron is not always available in their experiments increased formation of radical.HE was not observed, even if cell absorbed Staphylococcus aureus, which was preincubated with Fe2+[Phagocyte-derived free radicals stimulated by ingestion of ironrich Staphylococcus aureus: Aspin-trapping study / M. S. Cohen, B. E. Britigan, Y. S. Chai et al. // J. Infect Dis. - 1991. - Vol.163. - 819-826].

Using sensitive spin labels detected the accumulation of hydroxyl-radical cells in vitro in response Cl and O2.-and transformation in.HE has been a very small portion of the used cells of oxygen [Free hydroxyl radicals are formed on reaction between the neutrophilderived species superoxide and hypochlorous acid / L. P. Candeias, K. B. Patel, M. R. L. Stratford et al. // FEBS Lett. - 1993. -Vol.333. - P. 151-159]. The question of whether or not such amount.HE is to play a significant role in cytotoxicity, still remains open. Here is necessary in order to consider what is much more bactericidal capacity.HE has in the presence of Cl [Radiation induced generation of chlorine derivatives in NaO-saturated phosphate buffered saline: Toxic effects on Escherichia coli cells / G. Czapski, S. Goldstein, N. Andorn et al. // Free It. Biol. Med. - 1992. - Vol.12. - P. 353-361], probably due to a reaction between them to form hypochlorite [Bactericidal potency of hydroxyl radical in physiological environments / R. G. Wolcott, B. S. Franks, D. M. Hannum et al. // J. Biol. Chem. - 1994. - Vol.269. - P. 9721-9734].

Hydroxyl radical is one of the most reactive oxidants and can interact with almost any molecule of the cell. He modifies the deoxyribose and the nitrogenous bases of DNA, oxidize molecules of proteins, carbohydrates and lipids. Particularly active.HE in the course of the reactions of lipid peroxidation attacks phospholipids containing fatty acid radicals of unsaturated communication, which leads to the formation of hydroperoxides [Dubinin E. E. Products of the metabolism of oxygen in the functional activity of cells (life and death, creation and destruction). Physiological and biochemical aspects / E. E. Dubinin. - SPb.: Medical press, 2006. - 400 C.; Oxidative stress. Prooxidants and antioxidants / E. B. Menshikov, V. H. Pankin, N. To.Zenkov et al. - M.: The Word. - 2006. - S. 556]. The main component of the antioxidant system is the reduced form of glutathione.

Glutathione is a Tripeptide (L-γ-glutamyl-L-sister licin) with molecular weight of 307 Da occupies a special place among the SH-containing compounds. The presence of γ-glutamine connection protects the Tripeptide from enzymatic degradation. In the body, glutathione is present in two forms: oxidized - GSSG and restored GSH and the GSH content in cells by several orders of magnitude higher than GSSG [Kolesnichenko h.p., 1989; G. Wu et al., 2004; Smirnova, C., October O. N., 2005; Murray, R. et al., 2009]. According to P. Pietarinen-Runtti et al. (2000) the concentration of GSH in the cells is about 5 nmol/mg protein. Glutathione content in blood serum of healthy people slightly, so cells are a basic need for GSH provide by amatrices synthesis [G. Wu et al., 2004] during two sequential reactions catalyzed by γ-glutamylcysteine-synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3) [kulinskaya C. I., 1990; Smirnova, C., October O. N., 2005; Murray, R. et al., 2009]. The limiting level synthesis is the formation of γ-glutamylcysteine dependent on the presence of L-cysteine and its ability to be oxidized to L-cystine [Zenkov N. To. et al., 2001]. At the same time, the failure glutathione synthetase promotes oxidative damage in neutrophils [Spielberg, S. P. et al., 1979].

Glutathione at physiological pH values has two anionic carboxy group, a positively charged amino group and an SH group of the cysteine residue, which gives GSH properties reductant and the ability to neutralize free the f radicals and ROS [Day R. M., 2005; Zhu Y., 2007; Circu C. L. et al, 2009]. Glutamin is a typical thiol and participating in one-electron reduction reactions, becomes GS', which dimerizes to GSSG, easily reacting with free SH-groups. The second type of redox transformations involving GSH is the reaction thioldisulfide exchange, which is known as the main way of formation of the mixed disulfide of glutathione protein (protein-SSG) and play a role in the regulation of biological processes [Chai Y. C. et al., 1994]. In the reactions of the third type is a two-electron oxidation of glutathione with the formation of the intermediate, which reacts with a second molecule of GSH (getting GSSG) or another molecule (the synthesis of the mixed disulfide) [Smirnova, C., October O. N., 2005].

GSH is the stabilizer of membranes [Bilenko, M., 1989; Udupi V., 1992; S. Trudel et al., 2009]. It protects cellular structures from highly toxic OCI- [A. C. Carr, C. Winterbourn C., 1997], while GSH is converted into glutathione-sulfonamide and dehydrogenation [Harwood D. T. et al., 2006]. Linking NO, glutathione forms toxic to cells nitrosyl complexes. Mono-nitrosoglutathione can activate apoptosis [Turpaev K. T. et al., 1997].

Not always the regenerative potential of GSH sufficient for complete neutralization of prooxidants. There is an opinion that the interaction of GSH with organic radicals effectively only in terms of what the pressure O 2.-so glutathione forms of superoxide dismutase kind of antioxidant system, because otherwise develop the reaction of formation of H2About2and GS.[Lankin C. H. et al., 1997; Menshikova, E. B. et al., 2006]. In combination with vitamin b12glutathione and N-acetylcysteine, can potentiate prooxidant and cytotoxic effect on the cell [Soloviev, M. E. et al., 2007].

The main antioxidant effect of GSH implements through participation in the work of enzymes. Glutathione acts as a donor of hydrogen at the restoration of the H2About2and lipid peroxides glutathione-peroxidase and glutathione-S-transferring enzyme (GT) [Hirayama K., 1989; Sies H. et al., 1997; Kulinsky C. I., 1990; Hayes, J. D. et al., 2005; Zenkov H. K. et al., 2009; Liu G. et al., 2010]. The high activity of glutathione reductase and the accumulation of GSH has protective effect on alveolar macrophages, inkubiruemykh with prooxidants in vitro [Pietarinen R. K. 1995] and other cells.

With changes in redox balance involves a large number of reactions, so maintain optimal redox state of cytosol is an important condition for the normal functioning of the cells. A high concentration of glutathione in the cytoplasm, its redox activity and the ability to maintain the restored condition make the system GSH/GSSG important the first intracellular redox buffer [Reed M. S. et al., 2008]. The concentration of GSH in the cell in 500-1000 times higher than the level of NADPH and other intracellular redox systems, so changes in the ratio of GSH/GSSG directly reflect changes in the redox status of the cells [kulinskaya C. I., 2007; Asian Feet, Canatan d, 2008; Reed, M. S., 2008]. Consider that the buffer capacity of the glutathione system protects replicative cell system, and a deficiency of GSH leads to reduced synthesis of DNA and proteins [Poot M, 1991; Lankin C. Z., 1997; Day, R. M., Suzuki Y. J., 2005; Liu G. et al., 2010], and then to apoptosis.

The natural antioxidants also include ascorbic acid, which plays an important role in the development of oxidative stress in the body.

Ascorbic acid implements its antioxidant activity in plasma, interstitial fluid and intracellular level. In humans, ascorbic acid predominantly in the L-form. Stressful situations increase the number of metabolites of vitamin C in the form of dehydroascorbic acid.

Ascorbic acid and dehydroascorbic acid play an active role in several processes, including protection from infection, increase the immunity in the healing process, as well as taking part in the formation of anti-stress hormones. Ascorbate is a cofactor dopamine-β-hydroxylase, which catalyzes the synthesis of norepinephrine and other catecholamines. Ascorbic acid is in what stenopetalum for L-prolylhydroxylase, which is necessary for synthesis of collagen and connective tissue in General. In the body with ascorbic acid is the regeneration of α-tocopherol from tocopheroxyl radical. Oxidative stress correlates with impairment of insulin secretion and therapy ascorbic acid interrupt the damaging effect of free radicals, reduces the degree of manifestation of insulin resistance [M I. Balabolkin et al., 2003]. Ions ascorbate is one of the active elements of the antioxidant defense system, protecting lipids from oxidation them peroxy radicals. Anti-oxidant effect of ascorbate is manifested when a sufficient number of other antioxidants, such as α-tocopherol and glutathione. Glutathione restores dehydroascorbic acid direct and non-enzymatic way to ascorbic acid. This reaction is one of the main mechanisms of the antioxidant system, often described as restorative cycles - glutathione/putationally and ascorbic/dehydroascorbic acid. The cells of peripheral tissues absorb exogenous dehydroascorbic acid and in the presence of glutathione convert it into the cytoplasm in ascorbic acid. Restoring glutathionylated in glutathione catalyzed by glutathione reductase and requires NADPH as the e of the cofactor. Deficiency of glutathione reduces the content of ascorbic acid in the tissues and at the same time increases the concentration of dehydroascorbic acid.

The lack of α-tocopherol and glutathione may prevail Pro-oxidant effect of ascorbate and its metabolites. Prooxidant effect of ascorbic acid can be observed not only at the lack of α-tocopherol and glutathione, but also the use of high doses of ascorbic acid. To avoid prooxidant effect of ascorbic acid in the case of creation of adequate intracellular levels of reduced glutathione.

Each newly introduced in the claims the sign performs the function of increasing the accuracy and efficiency of the method: a comprehensive determination of reduced and oxidized glutathione after incubation of cells for subsequent forecasting an early stage of apoptosis.

The role of the antioxidant system of the cell is to reduce the toxic effects of free radicals, including lipid hydroperoxides.

Antioxidant protection provides a wide range of substances of different origin, physical-chemical nature and mechanisms of action. Total property, by definition, J. M. Gutteridge (1992), is the ability to sit in on low in comparison with the oxidizable substrate concentrations, significantly zaderjyvat the or to inhibit its oxidation. Constant education of prooxidants must be balanced by their inactivation, therefore, to maintain homeostasis adequate situation of continuous regeneration of the antioxidant capacity of cells [Zenkov N. To. et al, 2001; O. Blokhina et al, 2003].

Standard nomenclature of antioxidants does not currently exist, although a number of authors [P. Dimascio, 1990; Kalra V et al, 2001; Zaitsev C., et al., 2003] distinguishes two classes: proactive, reduce the rate of initiating chain reactions of oxidation and quenching (interrupting circuit), preventing the development of a chain reaction. To include preventive catalase and peroxidase, destroying ROOH, as well as agents which form chelate complexes with metal ions of variable valence, to a terminating circuit - phenols, aromatic amines. In vivo the main quenching antioxidants are: vitamin E, neutralizing ROO.the lipid phase of membranes [Jore D. et al, 1990; J. H. Hong et al, 2004], the enzyme SOD, catching O2.-in the aqueous phase cells [Fridovich I., 1989; P. Dimascio, 1990; Ciurea D., 1992], and ceruloplasmin protein acute phase, performing radical scavenging function in blood [S. L. Marklund, 1987; Atanasiu RL. et al, 1998].

Better known division of antioxidant enzymes and nonenzymatic compounds of nature. The latter in certain concentrations are always present in the lipid phase of membranes and water environments of the body and R is shobuda first eliminating the symptoms of oxidative stress [Droge W, 2002; Blokhina O, 2003]. The enzymes most actively join antioxidant defense (AOD) after turning on the mechanisms of induction [Luwak C. I., 2001]. Upon the occurrence of oxidative stress (OS) consumption of antioxidants increases and changes the expression of genes encoding protein components of AOD [Dubinin E. E., 2006]. Between enzymes and non-enzymatic elements AOD there is an equilibrium, the latter in a number of pathological conditions can act as prooxidants [Zenkov N. To. and others, 2001].

The main role among the non-enzymatic antioxidant defense systems attached to glutathione.

The functioning of cells is associated with the level of protein-bound glutathione. Determination of protein-bound glutathione is based on the ability of bergerat sodium (NaBH4) release from binding proteins with glutathione, which when interacting with DTNB forms a colored compound, namely thio-2-nitrobenzoic acid, an aqueous solution which has a maximum absorption at a wavelength of 412 nm [Burchill, B. R. Microtubule dynamics and glutathione metabolism in phagocytizing human polymorphonuclear leukocytes [Text] / B. R. Burchill, J. M. Oliver, C. B. Pearson et al. // J. of Cell Biology. - 1978. - Vol.76, No. 2. - P. 439-447].

Currently, it is extremely important to determine the level of protein-bound glutathione to assess the effectiveness of the protection of cells from apoptosis.

In laboratory practice, the most most is n way to protect cells from apoptosis and pereokislenie by determining the concentration of protein-bound and reduced glutathione.

The content of reduced glutathione determine the method proposed by M. E. Anderson (1985) modified by S. Kojima et al. (2004) [Kojima, S. Low dose gamma-rays activate immune functions via induction of glutathione and delay tumor growth / S. Kojima, K. Nakayama, H. Ishida // J. Radiat. Res. - 2004. - Vol.45, No. 1. - P. 33-39]. The principle of the method is based on the interaction of GSH with 5,5'-dithio-bis(2-nitrobenzoic) acid (DTNB) with the formation of thio-2-nitrobenzoic acid, an aqueous solution which has a maximum absorption at a wavelength of 412 nm. This forms GSSG, which is to glutathion reductase GSH and again interacts with DTNB. The rate of formation of the colored product is proportional to the content of total glutathione. For the determination of GSSG sample preincubated with blocker SH-groups 2-vinylpyridine ("Wako, Japan), which irreversibly binds GSH and, consequently, the rate of formation of the colored product is proportional to the content of GSSG.

The cell lysate is prepared with 5% sulfosalicylic acid, which is beset by proteins, but did not inhibit the activity of glutathione reductase.

The protein concentration in the cells is determined by the method [A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding / M. M. Bradford // Analyt. Biochem. - 1976. - Vol.7, No. 1, 2. - P. 248-254], based on the interaction of Kumasi blue G-250 with residues of arginine and lysine in proteins. Free red dye (absorption maximum -495 nm) in the formation of the complex with the protein enters the blue form (maximum absorption - 595 nm).

To 0.1 ml of cell lysate add 1.0 ml of a solution of Kumasi blue (100 mg dye, 50 ml of 96° ethanol, 100 ml of 85% H3RHO4N2O to 1.0 l), mixed, incubated for 3 min at room temperature and measure the optical density of the samples (wavelength 595 nm against a control containing 0.1 ml of water and 1.0 ml of Kumasi blue. The protein content calculated using a calibration curve constructed by dilutions of a standard solution of albumin (1.0 mg/ml) and expressed in mg/ml

Currently, it is extremely important to predict the early stage of apoptosis to evaluate the concentration of different forms of glutathione.

All of the above indicates the extreme importance of developing a predictor of early stage apoptosis of lymphocytes.

The popularity of the above method proved its high sensitivity, simplicity of implementation and adequate adequacy of the obtained results, the underlying determine the concentration of glutathione in the incubation medium lymphocytes.

The essential features characterizing the invention, shown in a certain aggregate of new properties that are not explicitly derived from the prior art in this field, and are not obvious to the expert.

Identical set of features not found in the study of patent and scientific medical literature is.

This invention can be used in medical practice to improve the accuracy and efficiency of the prediction of early-stage apoptosis in various diseases. This method is one of the fundamental when assessing the viability of the cells during transplantation of organs and tissues. Thus, it should be considered the present invention with the relevant conditions of patentability: novelty", "inventive step", "industrial applicability".

The method is based on determining the concentration of protein-bound and reduced glutathione.

The method is carried out in stages as follows:

1. Selection of culture cell line Jurkat.

The cultivation and study of cells in apoptosis was carried out in hole tablets (2,0×104cells per well) in culture medium RPMI-1640 for the incubation of 48 hours, 37°C and 5% content of CO2with the addition of the minimum concentration of the inducer of apoptosis is a synthetic glucocorticoid dexamethasone ("KRCA", Slovenia), comprising 10-4mol/ml were Then evaluated the number of apoptotic cells using FITC - labeled annexin V and propidium iodide (PI) method running laser cytometry, and was conducted to determine the viability of cells to enable Trypanosoma blue. To control the amount of glue is OK in apoptosis was 7,06 (6,00-8,71)%, and after incubation of 28.2 (25,1-of 31.4)% (four times)

2. Quantitative determination of the number of viable cells after their incubation with staining Trifanova blue ("Serva", CUlA) microscopic method.

Cells resuspended 1 ml of cell suspension. Select 100 ál resuspending cell suspension and add 100 μl of 0.1% solution Trypanosoma blue in physiological solution, mix well and fill the camera Goryaeva. Previously to the camera grind in cover glass so that appeared promising, nuconomy ring (only under these conditions, a proper volume of the chamber). A drop of cell suspension with dye contribute pressed under cover glass. Counting cells produced in 5 large squares diagonally in the camera Goryaeva. The calculation of viable cells on the content of "dead" cells are colored blue, produced by the formula:

And×106=(number of cells)/4,

where A is the cellularity of blood lymphocytes.

3. Biochemical analysis of protein-bound, reduced and oxidized glutathione.

The lymphocyte lysate prepared with 5% sulfosalicylic acid, which is beset by proteins, but did not inhibit the activity of glutathione reductase. The amount of total glutathione (GSH and GSSG) is determined in a sample containing 0.1 M Na-phosphate buffer (pH=7,5) with 1 mm EDTA, 0.4 mm NDFN, 0.3 mm DTNB and 1 U/is l glutathione reductase ("Wako", Japan). Oxidized glutathione define in a similar way in the cell lysate after pre-incubation of the sample for 30 min with 10 mm 2-vinylpyridine. Calculate the content of total and oxidized glutathione is produced using the calibration graphs for plotting using the solutions of GSH and GSSG ("Mr", USA) at a concentration of from 3 to 100 μm, treated like experimental samples. The concentration of GSH is calculated as the difference between the concentration of total glutathione and GSSG, expressing the result in nmol/mg protein.

Determination of protein-bound glutathione.

After incubation in experimental conditions (5% and 20% oxygen) in the presence or absence of NEM, DTE, NAC cells are centrifuged for 5 minutes at 4°C and 1500 rpm for their deposition. Remove the supernatant. Add 1 ml of chilled PBS (pH 7,4). Resuspending on the vortex. Centrifuged 5 minutes at 4°C and 1500 rpm Remove supernatant. Sediment cells resuspended in 1 ml of 5% sulfosalicylic acid to obtain a cell lysate. Centrifuged at 3000 rpm for 10 min 1.0 ml protein precipitate is incubated for 1 h at 50°C with 1.0 ml of 1% NaBH4. Next, the remaining protein is precipitated by the addition of 0.4 ml of 30% THU. The sample is incubated for 15 min at 50°C. Then, the sample is cooled to 5 min (0°C). Centrifuged 10 min at 3000 rpm, the Supernatant is mixed with 2.5 ml PBS (pH 7,4) and add 2.0 ml of acetone for complete oxidation of NaBH . Mix. Zentrifugenbau 10 min at 3000 rpm to Remove the upper phase. The lower phase add an equal volume of diethyl ether (to remove THU). Mix. Centrifuged 10 min at 3000 rpm Then remove the upper phase. Next, the procedure of washing samples from THU with diethyl ether to produce 4-fold. Then select 0.1 ml liquid (lower phase) and mixed with 0.4 ml of 0.01 M phosphate buffer (pH=7.0). In the sample add 0.1 ml 0.4 mg/ml DTNB. Sample spectrophotometery at 412 nm against a control containing water instead of the solution of precipitated protein.

Calculated taking into account the molar extinction coefficient of 13·103M-1cm-1. The results of determining the concentration of protein-bound glutathione is expressed as nmol/mg protein.

Evaluation method for predicting an early stage of apoptosis of lymphocytes by the prototype method and the proposed method was carried out 40 times. Examination results were processed statistically using the software package (Stat Soft Statistica 6.0.

When conducting research on the prototype method the level of reduced glutathione in the incubation medium of the cells in the rate was 2.05±0.19 nmol/mg protein and oxidized glutathione 0.18±0.02 nmol/mg protein, and in the evaluation of the proposed method in case of an effective forecasting an early stage of apoptosis, the level of vosstanovleniya was 1.7±0.13 nmol/mg protein and oxidized glutathione 0.26±0.02 nmol/mg protein. In the case of inefficient forecasting an early stage of apoptosis of lymphocytes, the level of reduced glutathione was 2.0±0.2 nmol/mg protein and oxidized glutathione 0.21±0.01 nmol/mg protein (table.1).

That is, when the effective prediction of early stages of apoptosis of lymphocytes, the level of reduced glutathione was decreased by 17% or more, and oxidized glutathione were increased by 19% or more. Poor prediction of early stages of apoptosis of lymphocytes, the level of reduced glutathione was increased by 5% or less, and oxidized glutathione were increased by 6% or more.

The results of glutathione levels in the incubation medium lymphocytes correspond to the literature [Smirnova, C., October O. N., 2005].

So, when using the prototype method was obtained insufficiently accurate result, is not allowed to establish effective prediction of early lymphocyte apoptosis, which is associated with the lack of a comprehensive biochemical analysis of different forms of glutathione, and the most efficient and accurate was the proposed method.

Thus the proposed method is simple in execution and interpretation of results.

A method for predicting an early stage of apoptosis of lymphocytes, comprising the steps of cell isolation, incubation of the cells for 48 hours at the temperature 37°C and 5% content of CO 2with the addition of the inducer of apoptosis dexamethasone at a concentration of 10-4mol/ml, quantitative determination of the viability of the lymphocytes to enable Trypanosoma blue and biochemical determination of the concentration of reduced and oxidized glutathione in the lysate of lymphocytes after pre-incubation for 30 minutes with 10 mm 2-vinylpyridine) - derivatives while the comprehensive decrease in the concentration of reduced glutathione by 17% or more and the increase in the concentration of oxidized glutathione by 19% or more, compared with the control predict early stage of apoptosis of lymphocytes.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, specifically to oncology, and concerns the prediction of the clinical outcome of musculo-invasive bladder cancer. The method involves measuring the activity of 26S proteasomes and NF-kB and p50 amounts in the tumour tissue. If the 26S proteasome activity is more than 18-1,000 units/mg of protein, and if the NF-kB p65/p50 coefficient is more than 1.0, a low probability of the recurrence is predicted; if the 26S proteasome activity is less than 18-1,000 units/mg of protein, and if the NF-kB p65/p50 coefficient is less than 1.0, a high probability of the tumour recurrence is predicted. The invention can be used for detecting the progression of the disease in the form of the tumour recurrence following the transurethral resection of the bladder tumour and M-VAC polychemotherapy.

EFFECT: using the invention provides the higher accuracy and information value for predicting the clinical outcome of the bladder cancer.

2 ex, 2 dwg

FIELD: medicine.

SUBSTANCE: chemical elements are measured in human blood by X-ray fluorescence using synchrotron emission; a ratio of the total chemical elements with the prooxidant properties to the total chemical elements with the antioxidant properties is calculated by formula: АОP=i=1nxii=1nyi, wherein xi is the total chemical elements in the human blood with the prooxidant properties; yi is the total chemical elements in the human blood with the antioxidant properties; n is a quantity of the measured chemical elements in the human blood measured to the similar ratio of the chemical elements for a 'nominally healthy person'; if the derived antioxidant protection is more than that for the 'nominally healthy person', the antioxidant protection of the human body is stated to reduce.

EFFECT: higher objectivity and reliability of assessing the antioxidant protection of the human body.

3 ex

FIELD: medicine.

SUBSTANCE: photometric measurement of whole blood is taken in the visible and near IR wavelength region; spectral transmission factors of a whole blood cell are measured, while the primary haemoglobin derivative concentration values are determined by minimising a disparity The invention can be used to control the blood gas in resuscitation, toxicology, in intensive care to determine an effect on the haemoglobin composition of physiological, pathological and ecological factors, as well as in maternity hospitals, obstetric and child care hospitals to control haematological factors of newborn children and to monitor hyperbilirubinaemia.

EFFECT: more informative blood count.

5 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and concerns a method for carrying out an immunochromatographic assay with a dissociating fluorescent tag, wherein complexes comprising molecules of an antigen or antigens, specific antibodies and tag molecules are formed on a membrane test strip. After the assay is completed, the tag is extracted from the working membrane of the test strip, and a fluorescent signal is measured in a liquid volume.

EFFECT: method according to the invention provides creating the optimum conditions for tag fluorescence, as well as eliminating the harmful influence of the membrane which is impermeable to decrease an intensity of both exciting and emitted light.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method involves: peripheral blood is examined for cytomegalovirus titre, blood pH and oxyhaemoglobin on a biochemical analyser; if observing an increase of the cytomegalovirus titre to 1:1600, a decrease of blood pH to 7.25±0.03, and oxyhaemoglobin - to 90.15±0.35% with the reference of 95.20%, a threatening formation of hemic hypoxia is stated.

EFFECT: higher assessment accuracy.

FIELD: chemistry.

SUBSTANCE: carried out are: determination of the serum concentration of a factor of a tumour necrosis alpha (TNF-α), evaluation of a relative content of Th17-lymphocytes (%CD3+CD4+CD161+ ot CD3+CD4+) and an activity of succinatedehydrogenase in a population of regulatory T-cells (CD3+CD4+CD127low) (SDH-Treg) before and on the following day after infliximab introduction. A prognostic factor of the medicine efficiency K is calculated by formula. If the factor value is K<0.5, a minimal therapeutic effect of infliximab is predicted. When K=0.5-1.5, a moderate therapeutic effect, which demands correction of the medicine introduction plan, is predicted. If K is from over 1.5 to 2.5, a positive effect from introduction of infliximab is predicted.

EFFECT: possibility to predict long-term, not less than 1 year, effect from the infliximab therapy at any stage of therapy.

4 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry, in particular to aspects, related to methods of the nonenzymatic determination of the presence or a quantity of carbohydrates in a sample, and describes a method, device and set for the sample analysis to determine the presence or a quantity of an analysed substance, in particular a carbohydrate, in particular, sugar, in the sample with the application of fabric. The method includes the application of the sample on a synthetic fabric; chemical modification of the said carbohydrate, present in the sample, with an oxidising agent with a sufficient oxidising potential in order to cleave the carbohydrate between two hydroxyl groups; inactivation of the said oxidising agent, which would prevent the identification of the chemically modified carbohydrate; identification of the presence or the quantity of the said chemically modified carbohydrate by means of a copper-containing compound to obtain the visible change of colour.

EFFECT: invention can be used for the determination of the presence of a carbohydrate on the surface to indicate the surface contamination, in particular contamination with a substance, promoting the microbial growth.

20 cl, 2 dwg, 7 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to method of determining expression of inflammatory process in case of osteoarthrosis. Essence of method consists in the following: carried out is luminol-dependent iron-induced chemi-luminescence of model system, which has the following composition: 2.72 g of KH2PO4, 7.82 g of KCl, 1.5 g of sodium citrate C6H8O7Na3*5,5H2O per 1 liter of distilled water, pH 7.45 with 0.2 ml of 10-5 M luminol solution, after that, intensity of model system luminescence is determined in presence of synovial fluid before and after addition of synovial fluid. Degree of suppression of intensity of model system chemi-luminescence is calculated by formula. If its value is from 1.71 to 6.48%, high activity of inflammatory process is determined, if its value is from 6.49 to 21.55%, medium activity is determined, from 21.56 to 55.46 - small activity.

EFFECT: application of the method makes it possible to reduce time of determination and increases accuracy of assessment of inflammatory process degree in case of osteoarthrosis.

1 tbl, 1 dwg, 3 ex

FIELD: chemistry.

SUBSTANCE: diagnostic test element (110) for the detection of an analyte in a sample (126) of a body fluid, in particular whole blood with the volume not less than 2 microlitres, contains a test field (116) with a reagent-indicator, where the reagent-indicator is capable of feeling a detected change, in particular an optic change, in case of the analyte presence. The test field (116) includes a detector layer (118), containing the reagent-indicator, where the detector layer contains particles (137). 90% of all particles (137) of the indicator layer (118) have the actual size smaller than 10 microns. The diagnostic test element (110) contains a bearing element (112), which has a transparent region (114), where the test field (116) with its detection side (120) is partially applied on the transparent region (114).

EFFECT: detected change is an optically detectable change, where an optic detector with spatial resolution is applied for the detection of the detected change.

9 cl, 8 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to immunology and medical diagnostics and represents method of carrying out immunochromatographic analysis for serodiagnostics. Claimed invention is intended for immunochromatographic determination of antibodies to causative agents of infectious diseases or other antigens, for instance, allergens, in liquid samples. Claimed method of identifying antibodies is characterised by the following: solution for sample dilution contains specific antibodies against test strip of antigen (or antigens) immobilised in analytic zone. Used concentration of antibodies in diluting solution is lower than lower limit of determination of antibodies by said method with application of diluting solution, which does not contain specific antibodies, which results coloration of analytic zone of test strip is not observed if specific antibodies are absent in sample. If sample contains specific antibodies, presence of additional quantity of specific antibodies in diluting solution results in enhancement of intensity of coloration of test strip analytic zone.

EFFECT: application of invention makes it possible to register total concentration of specific antibodies in sample and in diluting solution, which results in reduction of limit of analysis identification due to displacement of working range of concentrations, determined by test, into the area of lower values.

3 dwg, 1 ex

FIELD: chemistry.

SUBSTANCE: method includes the selection and preparation of samples to be analysed, selection of specified volumes of solutions of a test system components, placement of the samples to be analysed and the test system components into a cuvette, registration of chemiluminiscence with further quantitative estimation of its value with taking into account the background signal of chemiluminiscence. The weight of porting of the sample of the material to be analysed is taken such that corresponds to the value of a specific surface 0.20±0.05 m2/g, and in case when it is not possible to determine the value of the specific surface of the sample to be tested, the weight of the taken portion is 0.010±0.005 g. The portion of the sample of the material to be analysed is placed into a cuvette with the further successive addition of the test system components: 0.01M solution of luminal in 0.5 NaOH solution and a solution of hydrogen peroxide of a 20-30% concentration to fill the working space in the cuvette, keeping the ratio luminal:hydrogen peroxide equal to 2:5. After that, values of chemiluminiscence are registered for 125 minutes and the total value of chemiluminiscence is calculated.

EFFECT: identification of the free-radical activity of solid materials by the method of the chemiluminiscence registration by means of the system of chemical reagents without the application of biological substrates in the test system.

3 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to method of estimating antioxidant activity of vegetable raw material from swamp cinquefoil (Comarum palustre L.). Method of estimating antioxidant activity of vegetable raw material from swamp cinquefoil (Comarum palustre L.) consists in determination of antioxidant activity in water tinctures of swamp cinquefoil by reduction of the level of free-radical oxidation, which is determined by the level of malonic dialdehyde (MDA) by method of interaction with thiobarbituric acid in model system of lipid peroxidation, represented by liposomes obtained from lecithin.

EFFECT: method reduces labour intensiveness of antioxidant activity determination and simplifies processing of obtained results, reduces cost of carrying out analysis, and increases determination accuracy.

1 tbl

FIELD: medicine.

SUBSTANCE: invention relates to the field of radiobiology and experimental medicine. A method of estimating pharmacological and toxicological properties of substances consist in the following: a substance to be analysed is introduced into a nutritional medium of larvae and flies of Drosophila melanogaster, combining in their genome hypomorphic mutations of ss- and CG5017-genes. The larvae and flies are irradiated with ionising rays with a dose of 1-10 roentgen. Viability, structures of extremities and a level of transcription of CG 1681, CYP6G1 and ss-genes are estimated. The obtained characteristics of the flies, grown on a medium, containing the analysed substance, and the flies, grown on a medium, which does not contain the analysed substance, irradiated and non-irradiated are compared, and pharmacological properties of the substance are determined by the results of comparison of viability, quantity of tarsal segments of extremity and the level of transcription of CG 1681, CYP6G1 and ss-genes in the flies of all formed groups.

EFFECT: method makes it possible to realise effective fast targeted selection and determine the properties of substances with toxicoperotective, radioprotective, toxicosensibilising and radiosensibilising properties.

7 dwg, 2 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: method includes the selection of a totality of substances, for which the following procedures will be performed: monitoring of the region around a point source, determination of a route of sampling by the seasonal wind direction and drawing a map of isolines of pollutions on the basis of obtained data. A vector of the prevailing seasonal wind direction is selected. On the said vector sampling for each pollutant is carried out in two points r1 and r2, spaced from the point source at distances in the interval of 5 heights of a source (h) to 15 heights of the source. Coefficients B=ln(q1/q2·exp(C·((1/r2)-(1/r1))))/ln(r1/r2) and A=q1/(r1B)·exp(-C/r1), where q1 and q2 are concentrations of a pollutant in the points of sampling r1 and r2, C=30 h, are calculated. A single-dimensional profile of the pollutant concentration is calculated by the direction of the prevailing wind by formula F(R,A,B)=A·RB·exp(-C/R), where R is the current distance from the source, with transition to an area image of the pollutant distribution in the region being performed by multiplication of the specific concentration F(R,A,B) by a transposed function of the wind rose G(φ+180°), known from meteorological observations for the said region in the selected season.

EFFECT: method makes it possible to estimate a degree of pollution of the environment from the technogenic point source in a quick and accurate way.

3 dwg, 1 tbl, 1 ex

FIELD: measurement equipment.

SUBSTANCE: invention relates to the field of surface events and may be used in different fields, also for characterisation of disperse materials or crushed materials, sand, cement, etc. The method is characterised by the fact that the studied disperse material is placed into a template made in the form of a plate having free space in its centre, which is arranged in the centre of the limiting circumference applied on an easily replaced surface, or in a cuvette with available internal area, covered with a layer of water, which is exposed to the surfactant, they fix appearance of moving objects and calculate speed of their motion with subsequent calculation of speed of water movement along the surface of the disperse material.

EFFECT: method improvement.

6 ex, 1 tbl, 6 dwg

FIELD: agriculture.

SUBSTANCE: energy value is determined based on the calculation of the activation energies of chemical components as the sum of the activation energies of the grain cover and core of white lupine, multiplied by the mass fraction of the cover and core in the grain, respectively. To calculate the activation energy the data of thermogravimetric and differential-thermal analysis are used, obtained in continuous heating of samples at a rate of 20 deg/min to thermal decomposition of the components of the grain cover and core.

EFFECT: invention enables to estimate fast and accurately the energy value of feed white lupine grains for feeding farm animals.

3 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to veterinary science and can be used for oestrus and ovulation monitoring in animals and planning of the preferential time of fertilisation. That is ensured by presenting a standing sensor placed in relation to an animal so that to detect the animal's standing position. That is followed by data collection from the standing position sensor, including the overall standing time data. The oestrus and ovulation time is determined by calculating the initial time of measurement and a peak on the basis of variations of the relation of the standing time. The preferential time of the animal's fertilisation is planned with using specifying the preferential time of fertilisation presented on a data detection device. The data detection device provides a convenient time zone of fertilisation and the preferential time of fertilisation within a time zone of ovulation. That is followed by presenting the data specifying the actual time of fertilisation. If observing a fertilisation failure, it is stated if the actual time of fertilisation that occurred in the above preferential time of fertilisation falls within the time zone of ovulation.

EFFECT: invention enables specifying the preferential time of the animal's fertilisation within the convenient time zone, as well as stating the animal's disease or weakness.

6 cl, 11 dwg

FIELD: physics.

SUBSTANCE: to visually establish movement and determine the trajectory of formed objects in form of particles, the method of rendering self-organisation and movement of objects of dispersed particles employs a preparation object having a limiting closed line with a marked centre. A pattern, in which a dispersed material is placed, is then placed in the marked centre of the limiting circle. The liquid under analysis is then placed within the limiting circle in an amount which provides a layer of liquid over the material under analysis. A capillary containing a surfactant is then guided to the centre. A video camera is turned on to record surface changes. The capillary is lowered until it touches the surface. The video camera is turned off once the process of movement of self-organising objects is complete.

EFFECT: visual establishment of movement and determining the trajectory of formed objects in form of particles.

8 dwg

FIELD: electricity.

SUBSTANCE: system comprises an assembly of capacitance-type primary converters to measure electric capacity (dielectric capacitivity) and electric resistance (electrical conductivity) and temperature, which is placed at mobile equipment inside a settling tank, an assembly of secondary converters coupled to the primary ones and supplying the primary converters with action signals of the preset frequency and amplitude and the instantaneous response values of voltage and current at the primary converters for further processing, a programmable device or an automated workspace for control coupled to the secondary converters through a wired or wireless communications line with functions of data collection, processing and storage, including control over changes in the measured values of dielectric capacitivity and electrical conductivity with time or in regard to a structure of the settling tank, as well as issuing of the final forecast for a level or properties of sediment or sludge.

EFFECT: improving efficiency of automated control for waste water settling tanks.

12 cl, 1 dwg

FIELD: biotechnologies.

SUBSTANCE: phospholipide fluorescent probe characterised by the following name 1-[13-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)tridecanoyl]-2-(10-{[(2-hydroxynaphthyl-1)azophenyl-4]azophenyl-4}decanoyl)-sn-glycero-3-phosphocholine, is used in the test system to determine activity of A2 phospholipase, group IIA (secFLA2(IIA)) in blood serum, which also contains vesicular and phospholipide matrix for inclusion of a probe, containing phosphatidyl choline, lisophosphatidyl choline and phosphatidyl glycerine, a buffer solution and A2 phospholipase of apitoxin as standard.

EFFECT: invention makes it possible to reliably detect activity of secFLA2 in human blood serum in clinical conditions.

2 cl, 5 dwg, 5 ex

FIELD: technologies for testing properties of materials.

SUBSTANCE: method for determining composition of bi-nonary condensed type systems, in case of core sizes in fractions d1>d2>d3>…>d9>d10 and in case of core dimensions relations d2/d1,d3/d2,…,d10/d9 greater than 0.155 fractions volumes are determined from formulas for binary systems V1=1m3, Y1=1-d2/d1, V2=1m3·Y1·Ve1,m3, for ternary systems Vsm2=1m3, Y2=1-d3/d2av, V3=1m3·Y2·Vemp,m3, for quaternary systems Vsm3=1m3, Y3=1-d4/d3av, V4=1m3·Y3·Vemp3,m3, for quinary systems Vsm4=1m3, Y4=1-d5/d4av, V5=1m3·Y4·Vemp4,m3, for senary systems Vsm5=1m3, Y5=1-d6/d5av, V6=1m3·Y5·Vemp5,m, for septenary systems Vsm6=1m3, Y6=1-d7/d6av, V7=1m3·Y6·Vemp6,m3, for octuple systems Vsm7=1m3, Y7=1-d8/d7av, V8=1m3·Y7·Vemp7,m3, for nonary systems Vsm8=1m3, Y8=1-d9/d8av, V9=1m3·Y8·Vemp8,m3, where V1,V2…,V9 - piled up fraction volume with core sizes respectively d1,d2,…,d9,m3,Vsm2,Vsm3,…,Vsm8 - piled up volume of binary, ternary,…,octuple friable condensed type system, m3, Y1,Y2,…,Y8 - coefficient of condensation level of fraction with core size d1 by fraction with core size d2, condensation of binary friable system with average core size d2av by fraction with core size d3,…, condensations of octuple friable systems with average core size d8av by fraction with core size d9, Ve1 - emptiness of fraction with core size d1, Vemt2, Vemt3,…,Vemt8 - emptiness value for binary, ternary,…, octuple friable condensed type system.

EFFECT: lower laboriousness, higher effectiveness, possible optimization by utilizing computer means.

11 ex, 1 tbl

Up!