RU2632109C1 - Method for nk-cells cytotoxic activity determination - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method comprises isolation of a mononuclear cells fraction containing NK-cells from the peripheral blood. Incubation is carried out in the culture medium of mononuclears in the presence of target cells, followed by estimation of the number of dead cells by flow cytometry. At that, peripheral blood mononuclear cells containing NK-cells are incubated in the DMEM culture medium for 4 hours in the presence of trophoblast cells of the Jeg-3 line. Some of the trophoblast cells are incubated in DMEM culture medium without mononuclears for 4 hours. The number of nonviable and viable trophoblast cells incubated with peripheral blood mononuclear cells and the base number of nonviable and viable trophoblast cells incubated in the culture medium without peripheral blood mononuclear cells are then determined. The cytotoxic effect of NK-cells on trophoblast cells is determined by the formula: CE=(X1/X2)×100-(X1_base/X2_base)×100, where CE is the cytotoxic activity of NK-cells, X1 is the number of non-viable trophoblast cells incubated with peripheral blood mononuclear cells, X2 is the number of viable trophoblast cells incubated with peripheral blood mononuclear cells, X1_bases is the base number of non-viable trophoblast cells incubated in the culture medium without addition of mononuclear cells; X2_base is the base number of viable trophoblast cells incubated in the culture medium without addition of mononuclear cells.

EFFECT: use of this method allows to obtain quantitative evaluation of the NK-cells cytotoxic activity, which makes it possible to monitor the course of pregnancy.

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Description

The invention relates to medicine and can be used to assess the cytotoxic activity of NK cells of a fraction of peripheral blood mononuclear cells.

A known method for determining the cytotoxic activity of NK cells by the intensity of the induction of death of target cells of the transplanted line K562 (V. Murugin. The complex of research methods of NK cells in normal and pathological conditions // Dissertation ... Candidate of Medical Sciences. Moscow, 2012, p. . 134). To label target cells of the K562 line, a fluorescent dye 5-, 6-carboxyfluorescein diacetate succinylmidyl ether (CPDE) is used, which forms a strong covalent bond with intracellular proteins and does not affect cell viability in the concentration used (Kovalchuk L.V., Ignatieva G.A. ., Gankovskaya L.V. Immunology. Workshop. Textbook // 2010. - R. 176). Fluorescence is counted using flow cytometry, which allows a large number of cells to be analyzed.

The disadvantage of this method: does not take into account the features of the functional interactions of NK cells in various physiological processes, for example during pregnancy.

Literature data (Hunt JS, Petroff MG, McIntire RH, Ober C. HLA-G and immune tolerance in pregnancy // FASEB J. - 2005. - Vol. 19, No. 7. P. 681-93) indicate the possibility of NK formation -decimal cells from peripheral blood NK cells. The direct interaction of trophoblast cells and NK cells is an integral process that occurs during physiological pregnancy. Currently, the question of the nature of the interaction of trophoblast cells and NK cells during pregnancy remains poorly understood. Using existing methods for assessing the cytotoxic activity of NK cells, it is not possible to fully simulate in vitro such interaction of cells during pregnancy.

The technical result of the invention is to provide a method for determining the cytotoxic activity of the NK cell fraction of peripheral blood mononuclear cells in relation to trophoblast cells, which can be used to monitor the cytotoxic activity of NK cells of patients with physiological pregnancy, as well as to study the functional activity of NK cells of patients with pathologies pregnancy.

The specified technical result is achieved by the fact that in the claimed method for determining the cytotoxic activity of NK cells, comprising isolating a fraction of mononuclear cells containing NK cells from peripheral blood, incubating mononuclear cells in the culture medium in the presence of target cells and then estimating the number of dead cells by flow cytofluometry according to the invention, peripheral blood mononuclear cells containing NK cells are incubated in DMEM culture medium for 4 hours in the presence of trophoble cells one hundred Jeg-3 lines, some trophoblast cells are incubated in DMEM culture medium without mononuclear cells for 4 hours, then the number of non-viable and viable trophoblast cells incubated with peripheral blood mononuclear cells is determined, and the base number of non-viable and viable trophoblast cells incubated in a culture medium without peripheral blood mononuclear cells, determine the cytotoxic effect of NK cells in relation to trophoblast cells according to the formula:

CE = (X1 / X2) × 100- (X1 _bases / X2 _bases ) × 100,

where CE is the cytotoxic activity of NK cells,

X1 is the number of non-viable trophoblast cells incubated with peripheral blood mononuclear cells,

X2 is the number of viable trophoblast cells incubated with peripheral blood mononuclear cells,

X1 _bases - the base number of non-viable trophoblast cells incubated in culture medium without the addition of mononuclear cells,

X2 _bases. - the base number of viable trophoblast cells incubated in culture medium without the addition of mononuclear cells.

The study on which the invention is based included 89 patients, among whom were 54 healthy non-pregnant women with no previous pregnancies (Group 1), 14 healthy men (Group 2) and 21 healthy pregnant women for 6-7 weeks.

For each patient, the cytotoxic effect of NK cells was evaluated in four repetitions.

The obtained data were statistically processed using the Statistica 10 program. To compare the obtained data, the Mann-Whitney U-test was used, which is a nonparametric analogue of the Student t-test. The differences were recognized statistically significant at p <0.05.

Table 1 shows the median values for each group of patients, and the values of the upper and lower quartiles are shown in braces.

It was found that group 1 (healthy non-pregnant women) is not statistically different from group 2 (healthy men). Group 3 (healthy pregnant women for a period of 6-7 weeks) is different from group 1 (healthy non-pregnant women).

It is likely that the reduced cytotoxic effect of NK cells with respect to trophoblast cells in the group of healthy pregnant women compared to the group of healthy non-pregnant women, detected by the proposed method, is determined by the in vivo contacts of NK cells of these patients with trophoblast cells and suppression of their cytotoxic function.

The method is illustrated in FIG. 1-6, where:

in FIG. 1 shows a two-dimensional histogram of the relative number of trophoblast cells of the Jeg-3 line with coordinates along the abscissa axis FSC, along the ordinate axis SSC. The Cells region contains trophoblast cells;

in FIG. Figure 2 shows a two-dimensional histogram of a sample of trophoblast cells of the Jeg-3 line, unpainted with KFDE dye (negative control), with coordinates along the abscissa axis FSC, along the ordinate axis FITC. Cells are projected from the Cells region;

in FIG. Figure 3 shows a two-dimensional histogram of the relative number of trophoblast cells of the Jeg-3 line with coordinates along the FSC abscissa axis and along the FITC ordinate axis. The Jeg-3 region contains trophoblast cells stained with KPDE. Cells are projected from the Cells region;

in FIG. Figure 4 shows a two-dimensional histogram of the relative number of trophoblast cells of the Jeg-3 line, unpainted with propidium iodide dye (negative control), with coordinates along the abscissa axis FSC, along the ordinate axis PE. Cells are projected from the Jeg-3 region;

in FIG. Figure 5 shows a two-dimensional histogram of the relative number of dead trophoblast cells of the Jeg-3 line stained with propidium iodide dye, with coordinates along the abscissa FSC, along the ordinate PE. The Dead cells region contains trophoblast cells stained with PE dye. Cells are projected from the Jeg-3 region;

in FIG. Figure 6 presents a two-dimensional histogram of the relative number of dead Jeg-3 trophoblast cells stained with propidium iodide dye after incubation in the presence of peripheral blood mononuclear cells, with coordinates along the FSC abscissa axis, along the PE ordinate axis. The Dead cells region contains trophoblast cells stained with PE dye. Cells are projected from the Jeg-3 region.

The method is carried out, for example, as follows.

Jeg-3 trophoblast cells are used to evaluate the cytotoxic activity of NK cells of the peripheral blood mononuclear fraction in relation to trophoblast cells.

I. The order of the study.

1. Preparation of working solutions.

1.1. DMEM culture medium supplemented with 10% inactivated fetal calf serum (ETS), 100 U / ml penicillin and 100 μg / ml streptomycin, 2 mM L-glutamine, 10 mM sodium pyruvate (complete culture medium).

1.2. Versen's solution is sterile.

1.3. Trypsin solution is sterile.

1.4. Ficoll-Verographin Density Gradient Solution.

1.5. Hanks solution is sterile.

2. Preparation of the necessary materials.

2.1. The tablet is 96-well, sterile, with a lid, transparent, round-bottom.

2.2. Adhesive culture bottle with a ventilated blue cap 75 cm ² .

2.3. Goryaev’s camera.

2.4. 5 ml sterile tips.

2.5. Disposable tips for dispensers of variable volume up to 10 μl, up to 300 μl, up to 1000 μl.

2.6. Conical sterile tubes with a volume of 13 ml, 15 ml, 50 ml.

2.9. A solution of propidium iodide 1 μg / ml, prepared on PBS.

3. Preparation of the necessary equipment.

3.1. Vertical laminar flow cabinet.

3.2. A set of mechanical dispensers of variable volume.

3.3. Centrifuge.

3.4. Thermostat.

3.5. Vortex.

3.6. Refrigerator 6 + 2 ° С.

3.7. CO ₂ incubator.

3.8. Flow cytometer equipped with a 488 nm laser, direct and indirect light scattering sensors, FITC fluorescence sensors (519 nm), PE (578 nm).

II. Cultivation of trophoblast cells of the Jeg-3 line.

Operating procedure

1. Before the start of the procedure, warm sterile containers with medium for culturing trophoblast cells, sterile Trypsin solution and sterile Versen solution at a temperature of 37 ° C.

2. All the necessary materials listed in paragraph 2 of Section I should be sterilized in a vertical laminar-flow cabinet for working with cell cultures.

3. Mix sterile Trypsin solution with Versen solution (TB), containing equal parts of Trypsin solution and Versen solution.

4. Carefully drain the old medium from a 75 cm ² vial for cultivation with trophoblast cells, add 5 ml of sterile TB solution to disintegrate the cell monolayer. Wait 30 seconds, gently shake the bottle and drain the TV solution. Repeat the operation 2 times, then add two drops of TV solution. Close the bottle cap. Shake the vial for 5 minutes so that the cells are washed all the time with the solution. Pat the bottle on its sides to remove cells from the walls of the bottle.

5. Add 5 ml of the previously prepared medium for trophoblast cells into a 75 cm ² vial.

6. Carefully resuspend. Transfer 700 μl of cell medium to a new 75 cm ² vial and add 10 ml of culture medium to it.

7. Cultivate the cell culture in a humid atmosphere with 7% CO ₂ at 37 ° C for 72 hours or until the cells form 2/3 of the monolayer.

III. Isolation of mononuclear cells from peripheral blood.

1. All the necessary materials listed in paragraph 2 of Section I should be sterilized in a vertical laminar-flow cabinet for working with cell cultures.

2. Heat Hanks and a solution of density gradient ficoll-verographin in a thermostat 37 ° C.

3. Sign and add to the laminar (based on the analysis of 1 patient): 1 test tube per 50 ml, 3 test tubes per 13 ml, 1 test tube per 15 ml.

4. Add 4 ml of gradient solution to 13 ml tubes. Add 50 ml of Hanks to 50 ml tubes; add 10 ml of Hanks to tubes for washing mononuclear cells.

5. Mix 6 ml of peripheral blood with a warm Hanks solution in a ratio of 1: 2.

6. Using a 1 ml pipette, carefully lay the diluted peripheral blood onto a gradient solution based on a 4 ml gradient of 6 ml diluted blood.

7. Centrifuge 400g for 30 min at 22 ° C.

8. Sterilize the tubes in the laminar-flow cabinet. Take 4-5 ml of plasma with a 1 ml pipette and drain it. Assemble the mononuclear ring.

9. Transfer the mononuclear cells into a test tube with 10 ml of Hanks solution.

10. Centrifuge 200g for 10 min at 22 ° C.

11. Carefully drain the supernatant, add 3 ml of Hanks solution to the mononuclear cells, resuspend.

12. Calculate the concentration of mononuclear cells in the Goryaev chamber. Add 10 ml of Hanks solution.

13. Centrifuge 200g for 10 min at 22 ° C.

14. Carefully drain the supernatant. Dilute cells in a DMEM-based culture medium to a concentration of 3 ppm.

15. Transfer 100 μl of cells to the wells of round-bottom plates.

16. Incubate for 96 hours at 37 ° C in a humid atmosphere and 5% CO ₂ .

IV. Conducting an experiment.

1. Heat the culture medium DMEM, the complete culture medium for trophoblast cells of the JEG-3 line, a solution of TB in a 37 ° C thermostat.

2. All the necessary materials listed in paragraph 2 of Section I should be sterilized in a vertical laminar-flow cabinet for working with cell cultures.

3. Dilute 4 μl of CPDE (stock solution: 4.48 mmol / L) in 5 ml of warm DMEM culture medium and resuspend.

4. Carefully drain the old medium from the culture vial with the trophoblast cells, add 5 ml of sterile solution, add a solution of dye KFDE.

5. Incubate for 10 min at 37 ° C, 7% CO ₂ .

6. Drain the dye solution, add 10 ml of cold culture medium for JEG-3 without ETS.

7. Repeat washing: Drain the cold culture medium solution and re-add 10 ml of cold culture medium for JEG-3.

8. Drain the culture medium solution.

9. Add 5 ml of sterile TB solution to the trophoblast cell culture vial to disintegrate the cell monolayer. Wait 30 seconds, gently shake the bottle and drain the TV solution. Repeat the operation 2 times, then add two drops of TV solution. Close the bottle cap. Shake the vial for 5 minutes so that the cells are washed all the time with the solution. Pat the bottle on its sides to remove cells from the walls of the bottle.

10. Dispense 2 ml of warm trophoblast cell medium.

11. Calculate the concentration of cells in the chamber of Goryaev. Dilute cells in JEG-3 cell medium to a concentration of 0.6 ppm.

12. Transfer 50 μl of cells to a plate with previously seeded peripheral blood mononuclear cells, resuspend. Dispense 150 μl of JEG-3 cells into 5 wells without mononuclear cells.

13. Centrifuge the plate at 100g for 3 min at 22 ° C.

14. Incubate samples for 4 hours at 37 ° C, 5% CO ₂ .

15. Stain samples with dye propidium iodide. The final concentration of propidium iodide should be 2 μg / ml. Leave unpainted 1 sample with JEG-3 cells.

16. Incubate samples for 20 minutes at 4 ° C.

17. Add 300 μl of standard phosphate buffered saline to the samples.

V. Analysis and recording of results.

Cell analysis is carried out on a flow cytometer equipped with a 488 nm laser according to four parameters: direct and side light scattering intensities, FITC fluorescence (absorption spectrum 488 nm, 519 nm emission spectrum), fluorescence through PE channel (absorption spectrum 488 nm, emission spectrum 578 nm). 10,000 events are analyzed. In the two-dimensional histogram with FSC-SSC coordinates, the Cells region containing trophoblast cells is isolated (Fig. 1). Cells from the Cells region project onto a two-dimensional histogram with FSC-FITC coordinates (FIG. 2). The border of the quadrants on the graph is set based on the previous measurement of the control sample, unpainted with a solution of KPDE (Fig. 2). When measuring a test sample stained with CPED solution, cells from the Cells region project onto a two-dimensional histogram with FSC-FITC coordinates. The number of events in the Jeg-3 quadrant is determined (Fig. 3), this number corresponds to the number of trophoblast cells. Then, events from the Jeg-3 quadrant are projected onto a two-dimensional histogram with FSC-PE coordinates. The border of the quadrants on the graph is set based on the previous measurement of the control sample, unpainted with propidium iodide solution (Fig. 4). When measuring a test sample stained with solutions of CPED and propidium iodide, the relative number of dead trophoblast cells of the Jeg-3 line (cells from the Jeg-4 quadrant stained with propidium iodide dye) is analyzed on a two-dimensional histogram with FSC-PE coordinates (Fig. 5). The Dead cells region contains trophoblast cells stained with propidium iodide solution. The number of events in the Dead cells quadrant corresponds to non-viable trophoblast cells (Fig. 5). Cell death in the control well (after incubation of trophoblast cells without peripheral blood mononuclear cells) should not exceed 30%.

Then, the number of events is recorded in the Dead cells quadrant after incubation of trophoblast cells with peripheral blood mononuclear cells (Fig. 6). The percentage of non-viable trophoblast cells is determined by the number of events in the quadrants of Dead cells.

Then calculate the cytotoxic effect of NK cells of the fraction of peripheral blood mononuclear cells according to the formula:

CE = (X1 / X2) × 100- (X1 _bases / X2 _bases ) × 100.

Parameter X1 corresponds to the number of events in the Dead cells gate after measuring a sample of trophoblast cells incubated with peripheral blood mononuclear cells (Fig. 6). Parameter X2 corresponds to the number of events in the Jeg-3 gate after measuring a sample of trophoblast cells incubated with peripheral blood mononuclear cells (Fig. 6). The parameter X1 of the _bases corresponds to the number of events in the Dead cells gate after measuring a sample of trophoblast cells incubated without peripheral blood mononuclear cells (Fig. 5). The parameter X2 _bases corresponds to the number of events in the Jeg-3 gate after measuring a sample of trophoblast cells incubated without peripheral blood mononuclear cells (Fig. 5).

Example 1. Patient A. Parameter X1 = 959 event. Parameter X2 = 2378 events. Parameter X1 _bases = 624 events. Parameter X2 _bases = 3003 events.

CE = (X1 / X2) × 100- (X1 _bases / X2 _bases ) × 100 = (959/2378) × 100- (624/3003) × 100 = 19.5%.

The method allows to obtain a quantitative assessment of the cytotoxic activity of NK cells against trophoblast cells during their interaction. Assessment of the cytotoxic effect of NK cells in relation to trophoblast cells can be used to monitor the physiological course of pregnancy, as well as to study the functional activity of NK cells in pregnancy pathology.

Claims (7)

A method for determining the cytotoxic activity of NK cells, comprising isolating a fraction of mononuclear cells containing NK cells from peripheral blood, incubating mononuclear cells in the culture medium in the presence of target cells and then estimating the number of dead cells by flow cytometry, characterized in that the peripheral blood mononuclear cells containing NK cells are incubated in DMEM culture medium for 4 hours in the presence of Jeg-3 trophoblast cells, a portion of trophoblast cells are incubated in culture DMEM without mononuclear cells for 4 hours, then determine the number of non-viable and viable trophoblast cells incubated with peripheral blood mononuclear cells, and the base number of non-viable and viable trophoblast cells incubated in a culture medium without peripheral blood mononuclear cells determine the cytotoxic effect of NK cells in relation to trophoblast cells according to the formula: CE = (X1 / X2) × 100- (X1 _bases / X2 _bases ) × 100, where CE is the cytotoxic activity of NK cells, X1 is the number of non-viable trophoblast cells incubated with peripheral blood mononuclear cells, X2 is the number of viable trophoblast cells incubated with peripheral blood mononuclear cells, X1 _bases - the base number of non-viable trophoblast cells incubated in culture medium without the addition of mononuclear cells; X2 _bases - the base number of viable trophoblast cells incubated in culture medium without the addition of mononuclear cells.

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