Method of measuring length of telomeres of umbilical blood leukoconcentrate cells

FIELD: medicine.

SUBSTANCE: method includes separation by centrifugation of heparinised mononuclear cells of umbilical blood (further UB) in gradient of density on Ficoll-Paque™PLUS. After that realised are thorough washing in PBS+0.1 % BSA medium and denaturation at temperature 82°C of suspension, consisting of UB cells and control cell culture 1301, supported in RPMI medium with addition of 10% bull serum of glutamine and penicillin with streptomycin. After that, analysed and control cells are distributed in two pairs of test tubes, adding to them up to 0.5 ml PBS 0.1% BSA solution, centrifuging with acceleration 500 g for 5 minutes. Then in two rest tubes with cells poured is hybridisation buffer, and into the second pair - poured is hybridisation buffer with peptide-nuclein probe. After that, they are incubated in darkness for 2-20 hours, washed, mixed, centrifuged, stained in solution of propidium-iodide and RNK A. After that, analysis of cell samples is carried out with determination of their relative length telomeres depending on length of telomeres of cell culture 1301, taking into account DNA index.

EFFECT: invention makes it possible to increase quality of determination of properties of transplant, samples of hemopoetic stem cells of umbilical blood for transplantation.

1 ex

 

A method of measuring telomere length of cells lacokanostra cord blood relates to the field of biotechnology and genetic engineering can be used in medicine as an additional informational material in the diagnosis and medical evaluation of severe disease, when determining the quality of the sample for transplantation.

Telomeres are non-coding terminal regions of the chromosomes, which consist of repetitive DNA sequences and protein complex. Shortening the length of telomeres with each cell division are limiting cell proliferative potential in cells, are able to share almost unlimited, is embryonic stem cells, its germinative cell lines, as well as most cancer cells with active telomerase is expressed, is able to finish the telomeres and/or actively activates the alternative pathway of telomere elongation. In clinical practice, is actively developing the use of the measure of telomere length. Standard methods for the quality characteristics of the sample of umbilical cord blood for transplantation include the number of hematopoietic stem cells (HSC) on body weight of the patient, their viability, as colony forming units (CFU), the absence of contamination. Additional characterization of the proliferative activity and stem cells as measured by telomere length, is also informative indicator of the quality of the graft.

In article M.Hultdin, E.Gronlund, K.-F.Norrback, E.Eriksson-Lindstrom, T.Justl and G.Roos "Telomere analysis by fluorescence in situ hybridization and flow cytometry" Nucleic Acids Research, 1998, Vol.26? No.16 3651-3656, the telomere analysis after hybridization and staining using flow cytometry. The cell suspension is washed in phosphate-buffered saline (PBS) by centrifugation 400g, 5 min and resuspended in 1 ml PBS. Cells stained Img/ml trypan blue and counted in a cell counter Burker chamber. Cells mixed 1:1 with cell 1301 and the total number of 2×106placed in 1.5 ml tubes. After adding PBS tubes centrifuged with acceleration 4900g 30 seconds. Sediment resuspended in 400 μl of Fix &Perm Reagent A and incubated for 15 minutes at room temperature. Next, add 1 ml PBS, centrifuged, the precipitate resuspended in 400 μl of Fix &Perm Reagent B and after a 15 minute incubation, the cells washed twice in PBS. In hybridization solution containing 70% formamide, 4 nm fluorescein-3-fluorescein PNA probe in 10 mm Tris, pH 7.2. After 10-minute incubation at room temperature, the tubes are mixed on a vortex and placed in a water bath at 87 deg 10 min with stirring. The tubes are placed in the dark at room temperature overnight (15-20 hours), the cells are then centrifuged and incubated twice for anatoy temperature for 10 minutes in 70% formamide, 0,1% BSA and 0.1% Tween in 10 mM Tris pH 7.2. Cells resuspended in 1 ml of 0.15 M NaCl, 0.1% BSA, 0,1% Twin 20 in 50 mM Tris, pH 7.5, and transferred into a 5 ml test tube, add 3-4 ml of 0.15 M NaCl, 0.1% BSA, 0,1% Tween 20 in 50 mM Tris, ph 7.5, and after 10 minutes at room temperature, the tubes centrifuged with acceleration 400g for 5 minutes. Cells resuspended in 0.5 ml PBS and add propidium iodide to a final concentration of 0.1 micrograms/ml, vortexing and kept in the dark for 30 minutes before analysis.

The drawbacks of such telomere analysis are: the increased complexity of technology, the lack of standardization of methods, lack of effective measurement of telomere length, the method is inefficient, not technological and takes a lot of time, research is and is not effective in industrial applications.

In the journal of Haematology, issue No. 3, 2002, s-269 specified article "analysis of the telomere length of cells in umbilical cord blood according to the method of flow cytometry" - Nora Regeczy, Sandor Valent, Luca Kormos, Melinda Hajdu, Laszio Gopcsa and Katalin Paloczi "Telomere length analysis on cord blood cells by the flow-FISH method", Hematologia? Vol.32, No.3, pp.265-269, 2002, containing a method of measuring telomere length of cord blood, including centrifugation of heparinized samples of umbilical cord blood in the density gradient on Ficoll-Hypaque, mononuclear cells hybridizing and paint using a set of DAKO. As cell control use kletocna the line 1301. Cell suspension comprising cells of the sample and the control culture, denature at 82°C for 10 minutes in microcentrifuge tubes in hybridization solution with either probe or without it. When hybridization tube containing solution is kept at room temperature in the dark during the night. After hybridization conduct two 10-minute washing with a wash solution at 40°C. Next, the samples resuspended in the buffer for staining using propidium iodide. The fluorescence signal of the telomere is defined as the average signal in channel FL1 in the field of G0/G1 cells after subtraction of autofluorescence (signal from tubes without probe). Relative telomere length was calculated as the ratio of telomeric signal sample to the signal from the control cell line 1301.

The disadvantages of this method of measuring telomere length of cells in umbilical cord blood, which is the closest solution are: the shape of the analyzed cell technology this method is not well saves, control and analyze cells are mixed in a single vial that in the analysis of telomere complicates the allocation of the necessary cell population, the method does not effectively carry out the selection of cells to the station GO/G1 cell cycle is a big waste of time.

The technical result of the invention is to improve the quality determine the population properties of the graft, samples of hematopoietic stem cells in umbilical cord blood for transplantation, improving efficiency, more information about the proliferative potential of the cells of the graft, and to diagnose and assess the severity of serious diseases through early detection rapid shortening of telomere length, which is the most common marker of irreversible processes in various diseases.

This result is achieved in that in the method of measuring telomere length of cells lacokanostra cord blood, including selection by centrifugation of heparinized mononuclear cells of umbilical cord blood in the density gradient on Ficoll, denaturation at 82°C cell suspension consisting of umbilical cells and control cell cultures 1301, the hybridization solution in the dark with the probe and without it at room temperature in wash buffer, laundering at 40°C, resuspension analyze and control cells in the buffer for dyeing solution propidium iodide, counting cells on a flow cytometer and the determination of the telomere length of the analyzed cells in comparison with the control cell line 1301, lacokanostra cord blood isolated in the density gradient on Ficoll-Paque™PLUS, in the mononuclear fraction of umbilical cord blood to determine the number of the number of hematopoietic stem cells CD34+CD45 dim, lymphocytes and leukocytes, cell culture line T-lymphoblastoid leukemia 1301 support in the medium of RPMI solution with addition of 10% bovine serum, glutamine and penicillin with streptomycin, washed and analyzed control cells from debris in a solution of phosphate-saline buffer solution of 0.1% bovine serum albumin, thus maintaining the shape of the cells, lining : polyester taffeta and counting the number of analyzed and control cells, placed on 2×106analyze and control cells through two pairs of tubes, adding to them all the tubes in 0.5 ml of phosphate-saline buffer with 0.1% bovine serum albumin, centrifuged obtained solutions with cells with acceleration 500g for 5 minutes, after merging it supernatant, the first pair of tubes to analyze and control cells pour in 300 μl of hybridization buffer containing 70% formamide, the second pair of tubes with cells pour in 300 μl of solution hybridization buffer with peptido-nucleic acid probe labeled with fluorochromes FITC complementary telomeric DNA sequence, after mixing all solutions in the tubes on a vortex spend denaturation of DNA cells from double-stranded to single-stranded form by incubating solutions of all tubes in solid-state thermostat at a temperature of 82°C for 10 minutes to mix the fluids in the vortex, incubated in the dark in the range from 2 to 20 hours at room temperature, through a process of hybridization of peptide-nucleic acid probe to a telomeric sequence in the DNA of cells, then twice washed cells, adding in test tubes with the cells in 1 ml wash buffer from the set of Telomere PNA Kit/FITC for Flow Cytometry, diluted in 10-fold volume of distilled water, mix them into the vortex and innumerous in the dark at 40°C for 10 minutes, after another mixing on vortex centrifuged solutions with acceleration 500g for 5 minutes, merging the supernatant, cells in test tubes add 500 ál of buffer for staining DNA, diluted in 10-fold volume of distilled water and containing propidium of iodized and ribonuclease A, mix the contents of the vortex, and incubated in the dark samples with cells at 37°C for 30 minutes, to determine the analyzed cells with diploid set of chromosomes and tetraploid control cell culture 1301, after analysis of cell samples consider the relative telomere length (OTD) of the sample cells in umbilical cord blood according to the formula based on the average telomere length of the control cell line 1301 given index DNA tetraploid control cells, while

OTD=(P-A)samples×2×100/(P-A)1301where

(P-A)samples- the difference between the intensities of fluorescence in the analyzed cells without probe, measured in relative units on the first channel flowing cytofluorimetry,

(P-A)1301- the difference between the intensities of fluorescence of control cells 1301 placed in them by the probe.

The essence of the invention as a technical solution is expressed in a set of key characteristics, sufficient to achieve the solution maintains a technical result.

Essential features of the invention, coinciding with the characteristics of the prototype are: - separation by centrifugation of heparinized mononuclear cells umbilical cord blood Ficoll density gradient; - the holding of denaturation at 82°C kletocny suspension consisting of umbilical cells and control cell cultures 1301; - implementation of the hybridization solution in the dark with the probe and without it at room temperature; G - laundering at 40°C, resuspension analyze and control cells in the buffer for dyeing solution propidium iodide, counting cells on a flow cytometer and the determination of the relative telomere length of the analyzed cells in comparison with the control cell line 1301.

Salient features of the solution are: D - lacokanostra Popovi the Noi blood allocate the density gradient on Ficoll-Paque™PLUS; E - in the mononuclear fraction of umbilical cord blood to determine the number of hematopoietic stem cells CD34+CD45dim, lymphocytes and leukocytes; W - a cell culture line T-lymphoblastoid leukemia 1301 support in the medium of RPMI solution with addition of 10% bovine serum, glutamine and penicillin with streptomycin; And - washed analyze and control cells from debris in a solution of phosphate-saline buffer solution of 0.1% bovine serum albumin, thus maintaining the shape of the cells, lining : polyester taffeta and counting the number of analyzed and control cells; It is placed on 2×106analyze and control cells through two pairs of tubes, adding to them all the tubes in 0.5 ml of phosphate-saline buffer with 0.1% bovine serum albumin, centrifuged obtained solutions with cells with acceleration 500g for 5 minutes, after merging it supernatant; L - in the first pair of tubes to analyze and control cells pour in 300 μl of hybridization buffer containing 70% formamide; M second pair of tubes with cells pour in 300 μl of solution hybridization buffer with peptido-nucleic acid probe labeled fluorochromes FITC complementary telomeric DNA sequence; N after mixing all of the solutions in test tubes on a vortex spend denaturation of the DNA of the cells, at the reduced temperature of the PLA is ing DNA transfer DNA from double-stranded to single-stranded form, by incubating solutions of all tubes in solid-state thermostat at a temperature of 82°C for 10 minutes; mix the solutions on the vortex, and incubated in the dark in the range from 2 to 20 hours at room temperature, through a process of hybridization of peptide-nucleic acid probe to a telomeric sequence in the DNA of cells; P - double-washed cells, add 1 ml of wash buffer from the set of Telomere PNA Kit/FITC for Flow Cytometry, diluted in 10-fold volume of distilled water, mix them into the vortex and innumerous in the dark at 40°C for 10 minutes, after another mixing on vortex centrifuged solutions with acceleration 500g for 5 minutes, merging supernatant; P - cells in test tubes add 500 ál of buffer for staining DNA, diluted in 10-fold volume of distilled water and containing propidium iodide and ribonuclease A, mix the contents of the vortex, and incubated in the dark samples with cells at 37°C for 30 minutes, to determine the analyzed cells with diploid set of chromosomes and tetraploid control cell culture 1301; - after analysis of cell samples considered relative telomere length (OTD) of the sample cells in umbilical cord blood according to the formula based on the average telomere length of the control cells is the offered line 1301 given index DNA tetraploid control cells, this OTD=(P-A)samples×2×100/(P-A)1301where (P-A)samples- the difference between the intensities of fluorescence in the analyzed cells without probe, measured in relative units on the first channel flowing cytofluorimetry, (P-A)1301- the difference between the intensities of fluorescence of control cells 1301, placed in them by the probe.

When the method of measuring telomere length of cells lacokanostra umbilical cord blood aconcentric from umbilical cord blood is obtained by separation using a cell separator made even more S100 (Switzerland), which gives the fraction of mononuclear cells by gradient centrifugation on Ficoll-Paque™PLUS (Ge Healthtare). In the mononuclear fraction of umbilical cord blood to determine the number of hematopoietic stem cells CD34+CD45dimin leukocytes , lymphocytes and monoacetal. For comparison and control select 1.5 ml of cell suspension control line 1301. Cell culture cell lines T-lymphoblastoid leukemia 1301 support in RPMI medium with addition of 10% bovine serum, glutamine and penicillin - streptomycin. Analyze and control cells were washed off debris in the solution mixture of phosphate saline buffer (PBS) and bovine serum albumin (BSA), thus maintaining the shape of cells and aligning the number of analyzed and control cells. Count to icesto cells on a flow cytometer FC500 (Beckman Coulter, USA). In two test tubes (Eppendorf), labeled with the sample number control and a number of sample-probe, placed on 2×106the analyzed cells. In the other two test tubes, labeled 1301 control and 1301 probe, placed on 2×106control cells. In all test tubes with the content added to 0.5 ml of the total volume of phosphate-saline buffer with 0.1% bovine serum albumin and centrifuged obtained solutions with cells with acceleration 500g for 5 minutes, then decant the supernatant. In tubes labeled control, i.e. containing the analyzed and reference cells, contribute 300 ál of solution hybridization buffer containing 70% formamide without probe. In tubes labeled probe make 300 ál of solution hybridization buffer containing 70% formamide, with peptido-nucleic acid probe labeled fluorochromes FITC complementary to the telomeric DNA sequence. After mixing all four test tubes with solutions to the vortex spend denaturation of DNA of cells with a reduced melting temperature of DNA transfer DNA from double-stranded to single-stranded form by incubating solutions of all test tubes in solid-state thermostat (Bio TDB-120, Biosan, Latvia) at a temperature of 82°C for 10 minutes, allowing you to maintain cell shape. Through hybridization, mix the solutions on Vortec the e and incubated in the dark for 2 to 20 hours at room temperature, during the process of hybridization of peptide-nucleic acid probe to telomeric DNA sequence of cells. After denaturation of the DNA is single-stranded state, which allows the probe to hybridisierung (connect) with the complementary sequence on the DNA of cells. Gradually DNA is returned in double-stranded form, which probes remain complementary to their areas. The solution with the cells washed with 1 ml wash buffer (type Wash Solution) from a set of Telomere PNA Kit/FITC for Flow Cytometry, pre-diluting it with distilled water in a 10-fold volume, and mix on vortex. After incubating the washed cells for 10 minutes at 40°C in thermostat mix them on the vortex and centrifuged solutions with acceleration 500g for 5 minutes. After draining the supernatant was repeated twice, the process of washing, mixing, incubation and centrifugation. At this stage, the washing reprecipitate probes.

Tween 20 comprising a wash solution contributes to the creation of perfusion (holes) in the cell membrane that contributes to the output reprecipitate probes from cells. For staining DNA using 500 ál of buffer, diluted in 10-fold volume of distilled water and containing propidium iodide and ribonuclease A. Stained cells is transferred into a standard 12×75 mm polystyrene probe the key. Mix the contents of the vortex, incubate the samples with cells in the dark at 37°C for 30 minutes to determine the analyzed cells with diploid set of chromosomes and tetraploid control cell culture 1301. At this stage, the staining of the entire cell's DNA. This allows us to estimate the amount of DNA in the cells in the analysis and to highlight the cells for analysis, which are under G0/G1 cell cycle. This is done to implement the normalization of the number of telomeres in the cell.

Analysis of samples carried out on a flow cytometer and consider the relative telomere length (OTD) of the sample cells in umbilical cord blood according to the formula based on the average telomere length of the control cell line 1301 given index DNA tetraploid control cells.

OTD=(P-A)samples×2×100/(P-A)1301where

(P-A)samples- the difference between the intensities of fluorescence in the analyzed cells without probe in relative units defined by the first channel of flowing cytofluorimetry.

(P-A)1301- the difference between the intensities of fluorescence of control cells 1301 placed in them by the probe.

When measuring telomere length of cells leukocyte fraction 14 samples of umbilical cord blood was determined in parallel the number of hemopoetic the ski stem cells CD34+CD45 dim, lymphocytes and monocytes, data hematological analyzer, performed enzyme-linked immunosorbent assay for antibodies to pathogens standard. According to the data obtained relative telomere length (OTD, % of 1301) were identified and absolute telomere length (ADT), using the known coefficients, the following empirical formula. ADT=OTD×0,77+2,02 (ten - thousand base pairs).

Of the 14 samples of umbilical cord blood relative average length of telomeres in cells aconcentrated cord blood was 20.4±4.9 percent relative to the control cell line T-lymphoblastoid leukemia 1301. And the absolute average telomere length in cells lacokanostra cord blood is 17.7±5.9 and TPN as a result of the research shows that the determination of telomere length indicates that the presence of chronic infectious diseases adversely affect telomere length of blood cells.

In the application the description of the proposed solution method is specified example telomere test, which was conducted on the proposed method of fluorescence in Situ hybridization and flow cytofluorimetry. In the analysis was determined telomere length in cells of the leukocyte fraction of umbilical cord blood. As an internal control was used a standard cell line T-lymphoblastic leukemia 1301, characterizational telomere length, regardless of the passage. The length of telomeres of cells leukocyte fraction of cord blood sample norm may indicate the presence of pathology. The main causes of accelerated shortening of telomeres is oxidative damage, chronic infections, lack of exercise, chronic psychological stress and genetic factors.

The use of technical solutions "Method for measuring telomere length of cells lacokanostra cord blood compared with the prototype allows to improve the quality of the defining properties of the graft, as correlated with the percentage of monocytes, as well as the presence or absence of antibodies against different pathogens, the determination of which is necessary in the standards when making preparations umbilical cord blood. The proposed method makes it easier to keep the shape of the analyzed cells, since at the first stage of sample preparation, the cells are washed in a solution of PBS with 0.1% BSA, control and analyze cells are not mixed in the same tube that the analysis allows us to more clearly allocate the necessary cell population. In the Protocol analysis made changes unrealized, in the method prototype. In the diagram FS (forward scattering) - SS (side scattering) allocate the required population (lymphocytes and/or monocytes), for them, i.e. hathiramani in this area, construct diagra the mu distribution of cellular events FL3 in the logarithmic scale, that allows you to more effectively carry out the selection of cells on the plant G0/G1 of the cell cycle, for which then, i.e. hathiramani on this selection, build a distribution diagram of cellular events FL1 in linear scale, the average value at which the fluorescence probes or autofluorescence in this region of the spectrum. In addition, changes in the conditions of incubation at the stage of staining DNA, instead of 2-3 hours at 2-8°C incubation is conducted for 30 minutes at 37°C, hybridization in the dark can be carried out within 2 hours, which significantly reduces the operating time. Measurement of telomere length of cells of a sample of umbilical cord blood allows to evaluate the proliferative activity of stem cells, which is an important indicator, as the success of transplantation often depends on the speed of recovery of hemopoiesis. Longer telomeres donor is associated with a more rapid recovery of granulocytes recipient. The proposed method of measuring telomere length of cells lacokanostra cord blood is industrially applicable and worked at LLC "Pokrovsky stem cell Bank" (LLC "Pokrovsky BSC") in St. Petersburg.

A method of measuring telomere length of cells lacokanostra cord blood, including selection by centrifugation of heparinized mononuclear cells in umbilical cord blood is the density gradient on Ficoll, denaturation at 82°C. cell suspension consisting of umbilical cells and control cell cultures 1301, the hybridization solution in the dark with the probe and without it at room temperature, laundering at 40°C, resuspension analyze and control cells in the buffer for dyeing solution propidium iodide, counting cells on a flow cytometer and the determination of the relative telomere length of the analyzed cells in comparison with the control cell line 1301, characterized in that lacokanostra cord blood isolated in the density gradient on Ficoll-Paque™PLUS, in the mononuclear fraction of umbilical cord blood to determine the number of hematopoietic stem cells CD34+CD45dim, lymphocytes and leukocytes, cell culture line T-lymphoblastoid leukemia 1301 support in the medium of RPMI solution with addition of 10%bovine serum, glutamine and penicillin with streptomycin, washed and analyzed control cells from debris in a solution of phosphate-saline buffer solution of 0.1%bovine serum albumin, thus maintaining the shape of the cells, lining : polyester taffeta and counting the number of analyzed and control cells, placed on 2·106analyze and control cells through two pairs of tubes, adding to them all the tubes in 0.5 ml of phosphate-saline buffer with 0.1%bovine, Suvereto the major albumin, centrifugate obtained solutions with cells with an acceleration of 500 g for 5 minutes, after merging it supernatant, the first pair of tubes to analyze and control cells pour in 300 μl of hybridization buffer containing 70% formamide, the second pair of tubes with cells pour in 300 μl of solution hybridization buffer with peptido-nucleic acid probe labeled fluorochromes FITC complementary to the telomeric DNA sequence, after mixing of all the solutions in test tubes on a vortex spend denaturation of DNA by cells and reduced the melting temperature of DNA transfer DNA from double-stranded to single-stranded form by incubating solutions of all test tubes in solid-state thermostat at a temperature of 82°C for 10 minutes, mix the solutions on the vortex, and incubated in the dark in the range from 2 to 20 h at room temperature, through a process of hybridization of peptide-nucleic acid probe to a telomeric sequence in the DNA of cells, then twice washed cells, adding in test tubes with the cells in 1 ml wash buffer from the set of Telomere PNA Kit/FITC for Flow Cytometry, diluted in 10-fold volume of distilled water, mix them into the vortex and innumerous in the dark at 40°C for 10 min, after another mixing on vortex centrifuged solution the acceleration of 500 g for 5 min, merging the supernatant, the cells in test tubes add 500 ál of buffer for staining DNA, diluted in 10-fold volume of distilled water and containing propidium iodide and ribonuclease A, mix the contents of the vortex, and incubated in the dark samples with cells at 37°C for 30 min, to determine the analyzed cells with diploid set of chromosomes and tetraploid control cell culture 1301, after analysis of cell samples consider the relative telomere length (OTD) of the sample cells in umbilical cord blood according to the formula based on the average telomere length of the control cell line 1301 given index DNA tetraploid control cells, while
OTD=(P-A)samples·2·100/(P-A)1301where
(P-A)samples- the difference between the intensities of fluorescence in the analyzed cells without probe, measured in relative units on the first channel flowing cytofluorimetry,
(P-A)1301- the difference between the intensities of fluorescence of control cells 1301 placed in them by the probes.



 

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19 cl, 4 dwg, 5 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method for Apo2L/TRAIL sensitivity prediction of a malignant tissue or cell sampled from a mammal, involving the stages as follows: sampling a malignant tissue or cell from a mammal; analysing the sample malignant tissue or cell for detecting expression of one or more biomarkers selected from a group of fucosyl transferase 3, fucosyl transferase 6, sialyl-Lewis A and/or X antigen (antigens) where expression of one or more specified biomarkers is an indicator of the fact that the specified sampled tissue or cell is sensitive to apoptosis-inducing activity Apo2L/TRAIL. Also, what is described is a method of apoptosis induction in the sampled malignant tissue or cell of a mammal. What is offered is a method of treating a malignant tumour in a mammal. The inventions enables using the detection of expression of one or more biomarkers as the indicator of the fact that a sample is sensitive to apoptosis-inducing agents, such as Apo2L/TRAIL and DR5 agonist antibodies. Specific biomarkers to be examined include fucosyl transferases, particularly fucosyl transferase 3 (FUT3) and/or fucosyl transferase 6 (FUT6), as well as sialyl-Lewis A and/or X antigens.

EFFECT: method improvement.

35 cl, 22 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: cell suspension under investigation is incubated with biochip, containing immobilised on biochip antibodies, which have specificity to superficial antigens of investigated cells. After incubation, biochip is washed from non-specifically bound cells. Cells, which remain bound with biochip, are subjected to processing with labelled polynucleotide probes of one or several types with further hybridisation. Reading and processing of results are performed by presence of cell binding in area of biochip sites, containing immobilised antibodies, presence in them of determined superficial antigens is detected, and presence and character of binding of labelled polynucleotide probes are used to determine genetic signs in the same cells.

EFFECT: method application makes it possible to increase quantity of simultaneously determined superficial antigens on different cells with application of non-conjugated with label antibodies, simultaneously reducing number of used antibodies.

9 cl, 2 dwg, 2 ex

Biomarkers // 2429297

FIELD: medicine.

SUBSTANCE: what is offered is applying an analysis of p53(TP53) gene status and/or expression level as a biomarker while evaluating sensitivity of an individual suffering a proliferative disease to treatment by an mTOR inhibitor combined with a cytotoxic agent or while selecting individuals sensitive to the specified combined therapy for the following treatment of the disease by this method. Thus sensitivity to treatment of the proliferative disease by the mTOR inhibitor combined with the cytotoxic agent is predicted if wild-type functionally active p53 gene is found in a sample taken from the patient.

EFFECT: higher analysis accuracy.

14 cl, 5 ex

FIELD: medicine.

SUBSTANCE: what is offered is a method of structure stabilisation of thrombin binding DNA-aptamers, and also DNA-aptamers stabilised in such a way. The presented method provides formation of an additional base-stacking system by means of heterocycles or their analogues by means of increasing a surface of an aromatic system of heterocycles or their analogues, owing to using methods of determining a tertiary structure or molecular simulation with stating the fact of contact formation of the aromatic system of heterocyclic bases or their analogues with a G-quadruplex quartet which is related to a lateral loop.

EFFECT: method allows more effective assembly of antithrombin DNA-aptamers and improved structural stability under physiological conditions.

7 cl, 7 dwg, 1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: method involves allele-specific Nested-PCR with primers which are matched with nucleotide sequences coding amino acids in positions 70-71 of the amino acid sequence. The allele-specific primers E70f1 - 5'-AGAAGGAGATCCTGGAGGATAG - 3' and R71r1 - 5'-CCTGTCCACCTCGGCCCGCCTATC - 3' are matched with a part of BoLA-DRB3 gene located on chromosome 23 (localisation 23q21). They interact only with the nucleotide sequences coding alleles *11, *23, *28 =*7A causing genetic stability to cattle leukaemia. Then sequencing primer Zond 70/71 5'-GCCCGGCTACACCTGT - 3' is used to identify homo- or heterozygosity of an individual by the given alleles. If observing the primers interacting with alleles *11, *23, *28 =*7A, animals are considered to be leukaemia stable, while the absence of interaction with the same alleles can enable to refer to leukaemia unstable, and to neutral.

EFFECT: invention can be used for mass genetic typing of BoLA-DRB3 leukaemia tolerable animals in livestock and commodity economies for animal selection in a nuclear stock.

2 dwg, 4 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: set contains species-specific oligonucleotide primer pairs and appropriate fluorescent-marked probes for conducting one-stage instant identification of several human-pathogenic Orthopoxviruses (VARV, MPXV, CPXV and VACV) by means of real-time multiplex PCR.

EFFECT: invention is intended for instant diagnostics of human and animal Orthopoxvirus infections by real-time multiplex PCR.

10 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: tissue is homogenised in a buffer and centrifuged at 105000 g for 60-90 min at 0-4°C to produce a cytoplasmic fraction which is then incubated with 10 mM of phosphocreatine and 10 mcg/ml of phosphocreatine kinase for 25-45 minutes at 35°C. Cytoplasmic fraction proteins are divided by ammonium sulphate at three stages, at the first stage ammonium sulphate is added to 38% of saturation and centrifuged to isolate a precipitate containing a 26S-proteasome pool, at the second stage, a supernatant is added with ammonium sulphate to 42 % of saturation and centrifuged to isolate a precipitate containing ballast proteins, at the third stage to the supernatant is added with ammonium sulphate to 70 % of saturation and centrifuged to isolate a precipitate containing a 20S-npoteasome pool. Ammonium sulphate is added in portions during 20 min on a magnetic stirrer and further mixed for 20 minutes.

EFFECT: invention allows dividing native 26S- and 20S-proteasomes and isolating them in those amounts they exist in living cells, with preserving at most an undamaged 26S-proteasome structure.

3 cl, 1 ex

FIELD: medicine.

SUBSTANCE: there are offered versions of antibodies and their antigen-binding IL-13, particularly human IL-13 specific fragments. There are described: a pharmaceutical composition, a pharmaceutical compound of the antibody, versions of coding and hybridising nucleic acids and expression vectors. There are offered versions of: cells and methods of producing the antibody, methods of treating IL-13 associated disorders. A method of IL-13 detection in a sample is described.

EFFECT: use of the invention provides new IL-13 antibodies with KD about 10-10 M which can be used for diagnosing, preventing or treating one or more IL-13 associated diseases.

87 cl, 37 dwg, 5 tbl, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed group of inventions relates to medicine, namely to neurosurgery and biotechnology, and can be used for cultivation of nerve stem cells of mammal, excluding embryonic human cells, and for treatment of spasticity, rigidity, spinal cord or amniotrophic state in subject, requiring such treatment. For cultivation of said cells preliminarily incubated in culture vessel is solution, which contains poly-D-lysine in concentration from 0.1 mkg/ml to 1 mg/ml during from 5 minutes to 3 hours. After that, culture vessel is rinsed and dried. Nerve stem cells are inoculated into said culture vessel without serum. After that, solution of fibronectin and, at least, one mitogen is added into culture vessel and nerve stem cells are cultivated without serum. Then, cultivated nerve stem cells are passed until fusion. For treatment of said states nerve stem cells, cultivated be claimed method, are concentrated. Therapeutically efficient amount of said concentrated nerve stem cells is introduced into region of patient's central nervous system tissue with decreased level of GABA-producing or glycin-producing neurons.

EFFECT: group of inventions ensures efficient method of cultivating mammalian nerve stem cells, excluding embryonic human cells, which makes it possible to achieve in vitro higher degree of neuronal differentiation to the level sufficient for treatment of said states and improvement of survival in vivo of such cells, as well as efficient treatment of spasticity, rigidity or amniotrophic state in subject due to introduction into their central nervous system tissues with lower level of GABA-producing or glycin-producing neurons of cells, able to differentiate into sufficient number of GABA-producing or glycin-producing neurons in said tissue.

10 dwg, 7 ex

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