Method for detecting tumor killing activity of NK (Natural Killer) cells

Abstract

The invention discloses a method for detecting tumor killing activity of NK (Natural Killer) cells. The method for detecting the tumor killing activity of the NK cells comprises the following steps: recovering and culturing K562 cells; preparing a target cell K562; preparing effector cells NK; the K562 cell suspension and the NK cell suspension are mixed according to the effect-target ratio to serve as an experimental group, the K562 cell suspension serves as a control group, after the experimental group and the control group are incubated in an incubator for a period of time, the cells are transferred into a centrifugal tube and cleaned with PBS, then PI dye liquor is added for incubation, and the cell suspension is obtained. Detecting the apoptosis rate of the K562 cells in the experimental group and the control group by adopting a flow cytometer, and calculating the tumor killing rate of the NK cells; the killing activity of the NK cells to the K562 tumor cells is evaluated through a CFSE / PI labeling method, operation is relatively simple, consumed time is short, sensitivity is high, repeatability is good, and the method has the advantages of being safe, efficient, economical, stable and the like and has good application value.

Description

Technical Field

[0001] The present invention relates to the technical field of NK cell killing activity detection, and in particular to a method for detecting NK cell tumor killing activity. Background Art

[0002] Natural killer cells (NK) are cytotoxic lymphocytes in the innate lymphoid cell family. They are mainly found in the spleen, bone marrow and lymph nodes, accounting for about 10-15% of the total number of peripheral blood lymphocytes. Human peripheral blood NK cells are characterized by CD16 and CD56 as the main phenotypes, and their development mainly depends on cytokines and transcription factors. The anti-tumor effect of NK cells in clinical practice does not require antigen sensitization or antibody participation, and is not restricted by the major histocompatibility complex (MHC). It plays an important role in controlling the occurrence and development of tumors and is a new strategy for tumor immunotherapy.

[0003] The advantage of NK cells lies in their inherent natural killing ability, no need for activation and antigen recognition, and a wide range of anti-tumor effects. They are applicable to a variety of blood system tumors and solid tumors, and have broad clinical application prospects. Therefore, it is necessary to evaluate the tumor killing effect of the prepared NK cells before clinical use. At present, the methods for in vitro detection of NK cell tumor killing activity mainly include isotope release method, CCK-8 method, lactate dehydrogenase (LDH) release method, etc. However, the above methods have the disadvantages of high cost, cumbersome operation, low sensitivity, poor repeatability, and large errors. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting the tumor killing activity of NK cells. The detection method of the present invention has higher sensitivity and stability, can be used to detect the killing ability of NK cells on tumor cells, and the detection method is simple and easy to operate.

[0005] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0006] The first aspect of the present invention provides a method for detecting the tumor killing activity of NK cells, and the method for detecting the tumor killing activity of NK cells comprises the following steps:

[0007] (a) The frozen K562 cells were rapidly thawed and placed in RPMI1640 basal medium, pipetted and mixed, centrifuged, and the supernatant was discarded. The cells were then resuspended in IMDM complete medium, and the resuspended cells were inoculated into a culture flask for static culture. When the cell confluence reached 80% to 90%, the cells were subcultured into a culture flask for further culture.

[0008] (b) When the cell confluence is 80% to 90%, the cells in the culture flask are transferred to a centrifuge tube, mixed by pipetting, and samples are taken for counting, centrifuged and the supernatant is discarded, washed with PBS, the cell concentration is adjusted, CFSE dye solution is added and incubated in the dark, then pre-cooled IMDM complete medium is added and incubated on ice, washed with PBS, and resuspended in RPMI 1640 complete medium to obtain a K562 cell suspension;

[0009] (c) centrifuging the cultured NK cells, discarding the supernatant, and resuspending the cells in RPMI 1640 complete medium to obtain a NK cell suspension;

[0010] (d) K562 cell suspension and NK cell suspension were mixed according to the effector-target ratio as the experimental group, and K562 cell suspension was used as the control group. After the experimental group and the control group were incubated in an incubator for a period of time, the cells were transferred to a centrifuge tube and washed with PBS, and then PI dye was added for incubation. The apoptosis rate of K562 cells in the experimental group and the control group was detected by flow cytometry, and then the NK cell tumor killing rate was calculated according to NK cell tumor killing rate = (K562 cell apoptosis rate in the experimental group - K562 cell apoptosis rate in the control group) / (1 - K562 cell apoptosis rate in the control group) × 100%.

[0011] Preferably, in the step (d), compensatory adjustment is performed when the apoptosis rate of K562 cells in the experimental group and the control group is detected by flow cytometry.

[0012] Preferably, in step (d), the effector-target ratio is (5-20):1.

[0013] Preferably, in step (a), the resuspended cells are inoculated into a culture bottle for static culture at a concentration of (1-5)×10 5 / mL.

[0014] Preferably, in step (a), the volume ratio of the thawed K562 cell suspension to the RPMI1640 basal culture medium is 1:(8-12).

[0015] Preferably, in step (b), the final concentration of the added CFSE dye solution is 4-6 μmol / L.

[0016] Preferably, in step (b), the concentration of K562 cell suspension is 2×10 5 Pieces / mL.

[0017] Preferably, in step (c), the concentration of the NK cell suspension is (1-4)×10 6 Pieces / mL.

[0018] Preferably, in step (d), equal volumes of RPMI 1640 complete culture medium are added to the experimental group and the control group.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least:

[0020] The present invention evaluates the killing activity of NK cells against K562 tumor cells by the CFSE / PI labeling method, which has the advantages of relatively simple operation, short time consumption, high sensitivity, good repeatability, safety, high efficiency, economic stability, etc., and has good application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0022] Figure 1 This is a morphological characteristic diagram of the effector cell NK under a microscope in Example 1 of the present invention;

[0023] Figure 2 This is a morphological characteristic diagram of the target cell K562 under a microscope in Example 1 of the present invention;

[0024] Figure 3 This is a diagram showing the killing effect of NK on K562 cells at different effector-target ratios in Example 1 of the present invention;

[0025] Figure 4 This is a flow cytometric graph of the killing effect of NK on K562 cells at different effector-target ratios in Example 1 of the present invention. DETAILED DESCRIPTION

[0026] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the embodiments. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0027] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in this application should have the common meanings understood by those skilled in the art to which the present invention belongs.

[0028] The embodiment of the present invention provides a method for detecting the tumor killing activity of NK cells, and the method for detecting the tumor killing activity of NK cells comprises the following steps:

[0029] (a) The frozen K562 cells were rapidly thawed and placed in RPMI1640 basal medium, pipetted and mixed, centrifuged, and the supernatant was discarded. The cells were then resuspended in IMDM complete medium, and the resuspended cells were inoculated into a culture flask for static culture. When the cell confluence reached 80% to 90%, the cells were subcultured into a culture flask for further culture.

[0030] (b) When the cell confluence is 80% to 90%, the cells in the culture flask are transferred to a centrifuge tube, mixed by pipetting, and samples are taken for counting, centrifuged and the supernatant is discarded, washed with PBS, the cell concentration is adjusted, CFSE dye solution is added and incubated in the dark, then pre-cooled IMDM complete medium is added and incubated on ice, washed with PBS, and resuspended in RPMI 1640 complete medium to obtain a K562 cell suspension;

[0031] (c) centrifuging the cultured NK cells, discarding the supernatant, and resuspending the cells in RPMI 1640 complete medium to obtain a NK cell suspension;

[0032] (d) K562 cell suspension and NK cell suspension were mixed according to the effector-target ratio as the experimental group, and K562 cell suspension was used as the control group. After the experimental group and the control group were incubated in an incubator for a period of time, the cells were transferred to a centrifuge tube and washed with PBS, and then PI dye was added for incubation. The apoptosis rate of K562 cells in the experimental group and the control group was detected by flow cytometry, and then the NK cell tumor killing rate was calculated according to NK cell tumor killing rate = (K562 cell apoptosis rate in the experimental group - K562 cell apoptosis rate in the control group) / (1 - K562 cell apoptosis rate in the control group) × 100%.

[0033] The present invention evaluates the killing activity of NK cells against K562 tumor cells by the CFSE / PI labeling method, which has the advantages of relatively simple operation, short time consumption, high sensitivity, good repeatability, safety, high efficiency, economic stability, etc., and has good application value.

[0034] In some embodiments, in step (d), compensatory adjustment is performed when the apoptosis rate of K562 cells in the experimental group and the control group is detected by flow cytometry.

[0035] In some embodiments, in step (d), the effector-target ratio is (5-20):1.

[0036] In some embodiments, in step (a), the resuspended cells are inoculated into a culture flask for static culture at a concentration of (1-5)×10 5 / mL.

[0037] In some embodiments, in step (a), the volume ratio of the thawed K562 cell suspension to the RPMI1640 basal culture medium is 1:(8-12).

[0038] In some embodiments, in step (b), the final concentration of the added CFSE dye solution is 4-6 μmol / L.

[0039] In some embodiments, in step (b), the concentration of K562 cell suspension is 2×10 5 Pieces / mL.

[0040] In some embodiments, in step (c), the concentration of the NK cell suspension is (1-4)×10 6 Pieces / mL.

[0041] In some embodiments, in step (d), equal volumes of RPMI1640 complete culture medium are added to the experimental group and the control group.

[0042] The technical solution of the present invention is further described in detail below through specific embodiments.

[0043] Example 1

[0044] This embodiment is a method for detecting the tumor killing activity of NK cells, and the method for detecting the tumor killing activity of NK cells comprises the following steps:

[0045] (a) Recovery and culture of K562 cells:

[0046] Add 9 mL RPMI 1640 basal medium to the centrifuge tube at a ratio of 1:9 for later use; take out the cells to be revived, thaw quickly in a constant temperature water bath, disinfect the thawed cryopreservation tube with 75% alcohol, and place it in a biosafety cabinet or clean bench; transfer the cell suspension to the RPMI 1640 basal medium prepared in advance, mix well by pipetting, and centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with IMDM complete medium (containing 10% FBS), count and calculate the viability, prepare a T-25 culture bottle, mark the cell name, date and other information on the bottle, and press 3×10 5 / mL density was inoculated into a T-25 culture flask, and the culture medium was supplemented to 7mL after mixing, and the cells were placed in a 37°C, 5% CO2 incubator for static culture. When the cell confluence reached 80%-90%, the cells were subcultured into a T-75 culture flask (with the cell name and date marked) and continued to be cultured until the cell confluence reached 80%-90%. The cells can be used as target cells for subsequent experiments;

[0047] (b) Preparation of K562 cells:

[0048] Transfer the cells in the culture flask to a 50 mL centrifuge tube, mix thoroughly by pipetting, and then take samples for counting; centrifuge at 1000 rpm for 5 min, discard the supernatant, wash twice with PBS, and adjust the cell concentration to 5 × 10 6 / mL, add CFSE dye to a final concentration of 5μmol / L, incubate in a dark incubator for 15min, add 5 times the volume of pre-cooled IMDM complete medium, incubate on ice for 5min, wash twice with PBS, add RPMI 1640 complete medium to resuspend, and adjust the cell concentration to 2×10 5 / mL, and a K562 cell suspension was obtained for later use. The morphological characteristics of K562 cells under a microscope are shown in the figure below. Figure 2 As shown;

[0049] (c) Preparation of effector NK cells:

[0050] The NK cells cultured for 12 days were transferred to a 50 mL centrifuge tube, centrifuged at 500 g for 10 min, and resuspended in RPMI 1640 complete medium to adjust the cell concentration to 8 × 10 6 / mL, and NK cell suspension was obtained. The morphological characteristics of NK cells under the microscope are shown in the figure below. Figure 1 As shown;

[0051] (d) Plating and killing activity detection:

[0052] K562 cells and NK cells were inoculated into a 24-well plate (see Table 1), double negative wells (i.e., A1), PI (A2) and CFSE single staining wells (A3) were set for compensation adjustment, and three experimental groups (B1, B2, B3) with effector-target ratios of 5:1, 10:1, and 20:1, as well as a K562 cell control group (C) were set. After adding the cells, the well plate was placed in a 37°C, 5% CO2 incubator for incubation for 4 hours; the content distribution diagram of the 24-well plate is shown in Table 2; the killing effect diagram of NK cells on K562 cells at different effector-target ratios is shown in Figure 3 As shown;

[0053] Table 1 Types and volumes of contents added to the experimental wells, compensation wells and control wells

[0054]

[0055] Table 2 Distribution chart of contents in 24-well plate

[0056] A1 B1 B2 B3 C PBS A2 B1 B2 B3 C PBS A3 B1 B2 B3 C PBS PBS PBS PBS PBS PBS PBS

[0057] After the incubation, the cells were transferred to a 15 ml centrifuge tube, washed twice with PBS, and the remaining wells except the A1 well were added with PI dye solution and transferred to the sample tube, and incubated at room temperature in the dark for 15 minutes; the apoptosis rate of target cells K562 in each group was detected by flow cytometry, and then the NK cell tumor killing rate was calculated according to NK cell tumor killing rate = (experimental group K562 cell apoptosis rate - control group K562 cell apoptosis rate) / (1 - control group K562 cell apoptosis rate) × 100%. The flow cytometric graph of the killing effect of NK cells on K562 cells at different effector-target ratios is shown in the figure below. Figure 4 As shown;

[0058] The flow cytometry results are shown in Table 3 , and the NK cell tumor killing rate is shown in Table 4 ;

[0059] Table 3 Flow cytometry results (3 replicate wells)

[0060]

[0061] Table 4 Tumor killing rate

[0062] Grouping B1 B2 B3 Effect-target ratio 5:1 10:1 20:1 Kill rate (%) 75.3 97.4 98.8

[0063] The apoptosis rates of the three replicate wells in each group in Table 3 are relatively close, indicating that the experimental repeatability is good. The present invention achieves repeatable detection of NK cell tumor killing activity through a unified detection process, including preparation, staining, plating and flow cytometry of target cells and effector cells, thereby measuring the tumor killing ability of harvested NK cells.

[0064] Experimental example

[0065] The detection was repeated twice according to the detection method of Example 1. The flow cytometry detection results of the first time are shown in Table 5, and the cell killing calculation results are shown in Table 6; the flow cytometry detection results of the second time are shown in Table 7, and the cell killing calculation results are shown in Table 8;

[0066] Table 5 Flow cytometry results (3 replicates)

[0067]

[0068] Table 6 Tumor killing rate

[0069] Grouping B1 B2 B3 Effect-target ratio 5:1 10:1 20:1 Kill rate (%) 76.8 97.8 99.1

[0070] Table 7 Flow cytometry results (3 replicates)

[0071]

[0072]

[0073] Table 8 Tumor killing rate

[0074] Grouping B1 B2 B3 Effect-target ratio 5:1 10:1 20:1 Kill rate (%) 78.7 97.4 99.0

[0075] From Tables 4 to 8, we can see that:

[0076] After three repeated experiments, the following conclusions were drawn: the killing rate of NK cells increased with the increase of cell concentration. When the effector-target ratio was 20:1, the killing rate was 98.96%, which was the average of the three experiments. Moreover, NK showed good tumor cell killing activity when the effector-target ratio was 10:1. Under the same effector-target ratio, the killing rate results of the three groups were very close. It can be understood that when the initial cell culture process was determined, the experimental repeatability was good, and this method could stably measure the tumor killing ability of the harvested NK cells.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. A method for detecting NK cell tumor killing activity, characterized in that: The method for detecting the tumor killing activity of NK cells comprises the following steps: (a) The frozen K562 cells were rapidly thawed and placed in RPMI1640 basal medium, pipetted and mixed, centrifuged, and the supernatant was discarded. The cells were then resuspended in IMDM complete medium, and the resuspended cells were inoculated into a culture flask for static culture. When the cell confluence reached 80% to 90%, the cells were subcultured into a culture flask for further culture. (b) When the cell confluence is 80% to 90%, the cells in the culture flask are transferred to a centrifuge tube, mixed by pipetting, and samples are taken for counting, centrifuged and the supernatant is discarded, washed with PBS, the cell concentration is adjusted, CFSE dye solution is added and incubated in the dark, then pre-cooled IMDM complete medium is added and incubated on ice, washed with PBS, and resuspended in RPMI 1640 complete medium to obtain a K562 cell suspension; (c) centrifuging the cultured NK cells, discarding the supernatant, and resuspending the cells in RPMI 1640 complete medium to obtain a NK cell suspension; (d) K562 cell suspension and NK cell suspension were mixed according to the effector-target ratio as the experimental group, and K562 cell suspension was used as the control group. After the experimental group and the control group were incubated in an incubator for a period of time, the cells were transferred to a centrifuge tube and washed with PBS, and then PI dye was added for incubation. The apoptosis rate of K562 cells in the experimental group and the control group was detected by flow cytometry, and then the NK cell tumor killing rate was calculated according to NK cell tumor killing rate = (K562 cell apoptosis rate in the experimental group - K562 cell apoptosis rate in the control group) / (1 - K562 cell apoptosis rate in the control group) × 100%.

2. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (d), compensation adjustment is performed when the apoptosis rate of K562 cells in the experimental group and the control group is detected by flow cytometry.

3. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (d), the effector-target ratio is (5-20):

1.

4. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (a), the resuspended cells are inoculated into a culture bottle for static culture at a concentration of (1-5)×10 5 / mL.

5. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (a), the volume ratio of the thawed K562 cell suspension to the RPMI1640 basal culture medium is 1:(8-12).

6. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (b), the final concentration of the added CFSE dye solution is 4-6 μmol / L.

7. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In step (b), the concentration of K562 cell suspension is 2×10 5 Pieces / mL.

8. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (c), the concentration of the NK cell suspension is (1-4)×10 6 Pieces / mL.

9. The method for detecting the tumor killing activity of NK cells according to claim 1, characterized in that: In the step (d), equal volumes of RPMI 1640 complete culture medium were added to the experimental group and the control group.

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