Method for improving killing activity of NK cells

Abstract

The invention discloses a method for improving killing activity of NK cells, and belongs to the technical field of cell culture. The method comprises the following steps: (1) separating peripheral blood mononuclear cells; (2) sequentially carrying out induction culture and multiplication culture on the mononuclear cells; an induction culture medium used in the induction culture is an RPMI 1640 basic culture medium, fetal calf serum, IL-15, IL-12 and TGF-beta; a proliferation culture medium used in the proliferation culture is an RPMI 1640 basic culture medium, fetal calf serum, IL-13, IL-18, valine, leucine, isoleucine and glycyrrhizin. The culture medium obtained by scientific compatibility of cell factors, amino acids and plant active ingredients is used for proliferation culture of the NK cells, the killing activity of the NK cells to various tumor cells can be improved, and the culture medium has great significance in improving the clinical treatment effect of the NK cells.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, in particular to a method for improving the killing activity of NK cells. Background Art

[0002] NK cells (natural killer cells), as a key component of the human body's nonspecific immunity, play a vital role in the body's immune defense system. They are mainly distributed in the peripheral blood and spleen, and can directly attack tumor cells and virus-infected cells without the participation of antibodies and complement. They play a core role in immune surveillance and resistance to pathogenic microorganisms. Related studies have shown that NK cells not only participate in the innate immune response, but also play an important regulatory role in adaptive immune regulation. They play a major role in various pathological processes such as pathogenic microorganism infections, autoimmune diseases and tumors.

[0003] Currently, the number of NK cells in peripheral blood lymphocytes is relatively low, failing to meet clinical demands for both quantity and function. To address this issue, cytokine stimulation is currently the primary method used to expand NK cells. However, the quantity and quality of NK cells expanded by these methods are insufficient for clinical application. Furthermore, tumor cells often release a series of inhibitory factors, creating a suppressive tumor microenvironment that inhibits NK cell function and significantly reduces their cytotoxic activity, making it difficult for them to effectively kill tumor cells, thereby providing an opportunity for tumor cell survival and proliferation. Furthermore, in some viral infections, NK cell activity can be reduced by the virus itself or by the immune response triggered by the infection, impairing the body's ability to clear virus-infected cells and making it difficult to effectively control the disease process. Therefore, in-depth research and identification of methods to effectively enhance NK cell cytotoxicity is crucial for fully realizing the potential of NK cells in disease treatment, particularly in the treatment of autoimmune diseases and tumors. This will not only help improve clinical treatment outcomes but also provide patients with greater hope and better prognosis. Summary of the Invention

[0004] The present invention aims to provide a method for enhancing the cytotoxic activity of NK cells to address the problems of the prior art. The present invention utilizes a culture medium obtained by scientifically combining cytokines, amino acids, and plant active ingredients to proliferate and culture NK cells. This method can enhance the cytotoxic activity of NK cells against various tumor cells, which is of great significance for improving the clinical therapeutic effects of NK cells.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for improving the killing activity of NK cells, comprising the following steps:

[0007] (1) Isolation of peripheral blood mononuclear cells;

[0008] (2) sequentially performing induction culture and proliferation culture on the mononuclear cells;

[0009] The induction culture medium is: RPMI 1640 basal medium + fetal bovine serum + IL-15 + IL-12 + TGF-β;

[0010] The proliferation culture medium is RPMI 1640 basal medium + fetal bovine serum + IL-13 + IL-18 + valine + leucine + isoleucine + glycyrrhizin.

[0011] Furthermore, the induction culture medium is: RPMI 1640 basal culture medium + 10% fetal bovine serum + 40-55ng / ml IL-15 + 10-20ng / ml IL-12 + 20-40ng / ml TGF-β.

[0012] Furthermore, the proliferation culture medium is: RPMI 1640 basal culture medium + 10% fetal bovine serum + 15-30ng / ml IL-13 + 20-30ng / ml IL-18 + 0.5-1.0g / L valine + 0.5-1.0g / L leucine + 0.5-1.0g / L isoleucine + 0.2-0.4g / L glycyrrhizin.

[0013] Furthermore, the induction culture conditions are: culture at 37° C. and 5% CO 2 for 2-4 days.

[0014] Furthermore, the proliferation culture conditions are: culture at 37° C. and 5% CO 2 for 6-10 days.

[0015] Furthermore, during the proliferation culture process, IL-18 solution, valine solution and leucine solution are added after culturing for 2-3 days.

[0016] Furthermore, the amount of the IL-18 solution, valine solution and leucine solution added is 1.5%-3% of the volume of the initial proliferation culture medium.

[0017] Furthermore, the concentration of the IL-18 solution is 40-60 ng / ml; the concentration of the valine solution is 1.0-1.5 g / L; and the concentration of the leucine solution is 1.0-1.5 g / L.

[0018] The present invention also provides a proliferation culture medium for improving the killing activity of NK cells, wherein the proliferation culture medium comprises: RPMI1640 basal culture medium + 10% fetal bovine serum + 15-30ng / ml IL-13 + 20-30ng / ml IL-18 + 0.5-1.0g / L valine + 0.5-1.0g / L leucine + 0.5-1.0g / L isoleucine + 0.2-0.4g / L glycyrrhizin.

[0019] The present invention also provides use of the above proliferation culture medium in culturing NK cells with high killing activity.

[0020] The present invention discloses the following technical effects:

[0021] The present invention utilizes a scientific combination of cytokines, amino acids, and plant-based active ingredients to create a NK cell proliferation medium. This medium promotes NK cell growth, reduces functional aging and apoptosis of NK cells during prolonged culture, and significantly enhances the cytotoxic activity of NK cells against various tumor cells. This invention provides a novel technical solution for enhancing NK cell cytotoxicity and lays the foundation for improving the clinical therapeutic efficacy of NK cells. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] The present invention provides a method for improving the killing activity of NK cells, comprising the following steps:

[0028] (1) Isolation of peripheral blood mononuclear cells;

[0029] (2) performing induction culture and proliferation culture on the mononuclear cells in sequence;

[0030] The induction culture medium during induction culture was: RPMI 1640 basal medium + fetal bovine serum + IL-15 + IL-12 + TGF-β;

[0031] The proliferation culture medium is RPMI 1640 basal medium + fetal bovine serum + IL-13 + IL-18 + valine + leucine + isoleucine + glycyrrhizin.

[0032] IL-15 and IL-12 can promote the differentiation of mononuclear cells into NK cells. Proliferation and culture of NK cells differentiated from mononuclear cells are beneficial to enhancing the activity of NK cells.

[0033] IL13 can promote NK cell growth, maintain cell activity, and synergize with IL-18 to enhance the killing activity of NK cells; valine, leucine, and isoleucine can provide essential nutrients for cell body function and cell metabolism; glycyrrhizin is a natural ingredient extracted from plants with high safety, which has the effect of slowing down cell apoptosis and can reduce the functional aging and apoptosis of NK cells during culture.

[0034] In some embodiments of the present invention, the induction medium is: RPMI 1640 basal medium + 10% fetal bovine serum + 40-55ng / ml IL-15 + 10-20ng / ml IL-12 + 20-40ng / ml TGF-β; in some specific embodiments of the present invention, the IL-15 concentration in the induction medium may be 40ng / ml, 50ng / ml, or 55ng / ml, the IL-12 concentration may be 10ng / ml, 15ng / ml, or 20ng / ml, and the TGF-β concentration may be 20ng / ml, 30ng / ml, or 40ng / ml.

[0035] In some embodiments of the present invention, the proliferation medium is: RPMI 1640 basal medium + 10% fetal bovine serum + 15-30ng / ml IL-13 + 20-30ng / ml IL-18+0.5-1.0g / L valine+0.5-1.0g / L leucine+0.5-1.0g / L isoleucine+0.2-0.4g / L glycyrrhizin; in some specific embodiments of the present invention, the concentration of IL-13 in the proliferation medium may be 15ng / ml, 20ng / ml, or 30ng / ml, the concentration of IL-18 may be 20ng / ml, 25ng / ml, or 30ng / ml, the concentration of valine may be 0.5g / L, 0.8g / L, or 1.0g / L, the concentration of leucine may be 0.5g / L, 0.8g / L, or 1.0g / L, the concentration of isoleucine may be 0.5g / L, 0.8g / L, or 1.0g / L, and the concentration of glycyrrhizin may be 0.2g / L, 0.3g / L, or 0.4g / L.

[0036] In some embodiments of the present invention, the induction culture conditions are: culturing at 37° C. and 5% CO 2 for 2-4 days.

[0037] In some embodiments of the present invention, the proliferation culture conditions are: culturing at 37° C. and 5% CO 2 for 6-10 days.

[0038] In some embodiments of the present invention, IL-18 solution, valine solution, and leucine solution are added after 2-3 days of culture during the proliferation culture process. Supplementation of IL-18 and amino acids in the later stages of NK cell culture provides sustained growth momentum for NK cells. The amount of IL-18 solution (40-60 ng / ml), valine solution (1.0-1.5 g / L), and leucine solution (1.0-1.5 g / L) added is 1.5%-3% of the initial proliferation culture volume.

[0039] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0040] Example 1

[0041] This embodiment provides a method for culturing NK cells, and the specific process is as follows:

[0042] 1. Isolation of Mononuclear Cells

[0043] Peripheral blood was collected from healthy subjects and placed in anticoagulation tubes. Fresh anticoagulated umbilical cord blood was diluted with PBS solution at a 1:1 volume ratio. Lymphocyte separation solution was then added. The mixture was slowly added dropwise to the centrifuge tube, allowing the peripheral blood to spread evenly on top of the separation solution, maintaining a clear interface between the two liquid surfaces. The tube was then centrifuged at 500g for 20 minutes, the upper plasma layer was discarded, and the buffy coat layer was carefully aspirated and transferred to a new centrifuge tube. PBS solution was added to the new centrifuge tube to wash the cells, and the tube was centrifuged at 300g for 10 minutes. The supernatant was discarded. This step was repeated twice to obtain isolated mononuclear cells.

[0044] 2. Prepare culture medium

[0045] The induction medium was prepared by using RPMI 1640 medium as the basal medium and supplemented with 10% fetal bovine serum, 50 ng / ml IL-15, 10 ng / ml IL-12, and 30 ng / ml TGF-β.

[0046] The proliferation medium was prepared by using RPMI 1640 medium as the basal medium and supplemented with 10% fetal bovine serum, 15 ng / ml IL-13, 20 ng / ml IL-18, 0.5 g / L valine, 0.5 g / L leucine, 0.5 g / L isoleucine, and 0.2 g / L glycyrrhizin.

[0047] 3. NK cell induction culture and proliferation culture

[0048] Mononuclear cells were collected at a rate of 1×10 5 The cells were inoculated into the induction medium at a density of 10 cells / ml and cultured at 37°C in a 5% CO2 environment for 3 days. 5 NK cells were seeded at a density of 100 cells / ml in proliferation medium and cultured at 37°C in a 5% CO2 environment for 3 days. IL-18 solution (50 ng / ml), valine solution (1 g / L), and leucine solution (1 g / L) were then added daily. 15 μl of each of IL-18 solution, leucine solution, and isoleucine solution were added per mL of proliferation medium (original volume). After another 4 days of culture, NK cells were harvested by centrifugation at 500 g for 10 minutes.

[0049] Example 2

[0050] This embodiment provides a method for culturing NK cells, and the specific process is as follows:

[0051] 1. Isolation of Mononuclear Cells

[0052] Peripheral blood was collected from healthy subjects and placed in anticoagulation tubes. Fresh anticoagulated umbilical cord blood was diluted with PBS solution at a 1:1 volume ratio. Lymphocyte separation solution was then added. The mixture was slowly added dropwise to the centrifuge tube, allowing the peripheral blood to spread evenly on top of the separation solution, maintaining a clear interface between the two liquid surfaces. The tube was then centrifuged at 500g for 20 minutes, the upper plasma layer was discarded, and the buffy coat layer was carefully aspirated and transferred to a new centrifuge tube. PBS solution was added to the new centrifuge tube to wash the cells, and the tube was centrifuged at 300g for 10 minutes. The supernatant was discarded. This step was repeated twice to obtain isolated mononuclear cells.

[0053] 2. Prepare culture medium

[0054] The induction medium was prepared by using RPMI 1640 medium as the basal medium and adding 10% fetal bovine serum, 40 ng / ml IL-15, 15 ng / ml IL-12, and 40 ng / ml TGF-β.

[0055] The proliferation medium was prepared by using RPMI 1640 medium as the basal medium and supplemented with 10% fetal bovine serum, 20 ng / ml IL-13, 25 ng / ml IL-18, 0.8 g / L valine, 0.8 g / L leucine, 0.8 g / L isoleucine, and 0.3 g / L glycyrrhizin.

[0056] 3. NK cell induction culture and proliferation culture

[0057] Mononuclear cells were collected at a rate of 1×10 5 The cells were inoculated into the induction medium at a density of 10 cells / ml and cultured at 37°C in a 5% CO2 environment for 3 days. 5 NK cells were seeded at a density of 100 cells / ml in proliferation medium and cultured at 37°C in a 5% CO2 environment for 3 days. IL-18 solution (50 ng / ml), valine solution (1 g / L), and leucine solution (1 g / L) were then added daily. 20 μl of each of IL-18 solution, leucine solution, and isoleucine solution were added per mL of proliferation medium (original volume). After another 4 days of culture, NK cells were harvested by centrifugation at 500 g for 10 minutes.

[0058] Example 3

[0059] This embodiment provides a method for culturing NK cells, and the specific process is as follows:

[0060] 1. Isolation of Mononuclear Cells

[0061] Peripheral blood was collected from healthy subjects and placed in anticoagulation tubes. Fresh anticoagulated umbilical cord blood was diluted with PBS solution at a 1:1 volume ratio. Lymphocyte separation solution was then added. The mixture was slowly added dropwise to the centrifuge tube, allowing the peripheral blood to spread evenly on top of the separation solution, maintaining a clear interface between the two liquid surfaces. The tube was then centrifuged at 500g for 20 minutes, the upper plasma layer was discarded, and the buffy coat layer was carefully aspirated and transferred to a new centrifuge tube. PBS solution was added to the new centrifuge tube to wash the cells, and the tube was centrifuged at 300g for 10 minutes. The supernatant was discarded. This step was repeated twice to obtain isolated mononuclear cells.

[0062] 2. Prepare culture medium

[0063] The induction medium was prepared by using RPMI 1640 medium as the basal medium and adding 10% fetal bovine serum, 55 ng / ml IL-15, 20 ng / ml IL-12, and 20 ng / ml TGF-β.

[0064] The proliferation medium was prepared by using RPMI 1640 medium as the basal medium and supplemented with 10% fetal bovine serum, 30 ng / ml IL-13, 30 ng / ml IL-18, 1.0 g / L valine, 1.0 g / L leucine, 1.0 g / L isoleucine, and 0.4 g / L glycyrrhizin.

[0065] 3. NK cell induction culture and proliferation culture

[0066] Mononuclear cells were collected at a rate of 1×10 5 The cells were inoculated into the induction medium at a density of 10 cells / ml and cultured at 37°C in a 5% CO2 environment for 3 days. 5 NK cells were seeded at a density of 100 cells / ml in proliferation medium and cultured at 37°C in a 5% CO2 environment for 3 days. IL-18 solution (50 ng / ml), valine solution (1 g / L), and leucine solution (1 g / L) were then added daily. 30 μl of each of IL-18 solution, leucine solution, and isoleucine solution were added per mL of proliferation medium (original volume). After another 4 days of culture, NK cells were harvested by centrifugation at 500 g for 10 minutes.

[0067] Comparative Example 1

[0068] The only difference from Example 2 is that 20 ng / ml of IL-10 was additionally added to the original proliferation medium.

[0069] Comparative Example 2

[0070] The only difference from Example 2 is that valine in the original proliferation medium and the replenishment of valine solution during the culture process are omitted.

[0071] Comparative Example 3

[0072] The only difference from Example 2 is that leucine in the original proliferation medium and the replenishment of leucine solution during the culture process are omitted.

[0073] Comparative Example 4

[0074] The only difference from Example 2 is that valine in the original proliferation medium is replaced by lysine.

[0075] Comparative Example 5

[0076] The only difference from Example 2 is that glycyrrhizin in the original proliferation culture medium is omitted.

[0077] Comparative Example 6

[0078] The only difference from Example 2 is that valine and leucine are omitted from the original proliferation medium, and the valine solution and leucine solution are replenished during the culture process.

[0079] Comparative Example 7

[0080] The only difference from Example 2 is that the replenishment of the IL-18 solution, the valine solution, and the leucine solution during the culture process is omitted.

[0081] Test Example 1

[0082] The NK cells cultured in Examples 1-3 and Comparative Examples 1-7 were used to detect the killing activity of the NK cells. The specific detection process is as follows:

[0083] 1. Cytotoxicity of NK cells against K562 cells

[0084] K562 cells were used as target cells, and cells after proliferation and culture in each group were used as effector cells. The LDH assay was used to detect the cytotoxicity of NK cells against K562 cells. The effector-target ratio was set at 10:1, 20:1, and 40:1, respectively. A natural release well and a maximum release well were set for target cells, each containing 200 μl. Each well was incubated in a 37°C, 5% CO2 incubator for 3 h, centrifuged at 1500 rpm for 5 min, and 100 μl of the supernatant was aspirated from each well and placed in a flat-bottom 96-well culture plate. 100 μl of LDH matrix solution was added and reacted for 3 min. 30 μl of 1 mol / L HCl was added to each well, and the optical density (OD) was measured at 492 nm on a microplate reader. The cytotoxicity of NK cells against K562 cells was calculated according to the following formula:

[0085] NK cell killing activity against K562 cells = (OD of reaction well - OD of natural release well) / (OD of maximum release well - OD of natural release well) × 100%

[0086] The test results are shown in Table 1.

[0087] Table 1 Cytotoxicity of NK cells in each group against K562 cells (%)

[0088] Group 10:1 20:1 40:1 Example 1 63.47 81.03 92.11 Example 2 67.11 84.62 96.57 Example 3 66.52 80.92 94.50 Comparative Example 1 56.78 76.48 83.62 Comparative Example 2 49.67 77.95 72.44 Comparative Example 3 46.96 75.41 77.37 Comparative Example 4 60.49 79.84 83.30 Comparative Example 5 52.97 76.90 76.81 Comparative Example 6 41.65 70.11 62.75 Comparative Example 7 52.36 72.83 80.37

[0089] As can be seen from Table 1, the NK cells obtained by the methods of Examples 1-3 have a strong killing activity against K562 cells, and the NK cell killing activity of Example 2 is the strongest. Compared with Example 2, Comparative Example 1 adds IL-10 to the original proliferation medium, and the NK cell killing activity decreases. It is speculated that there may be a stable synergistic effect between the components of the original proliferation medium. The addition of IL-10 will break this effect, resulting in a decrease in the effect of certain components on NK cells. Comparative Examples 2, 3, and 6 omit valine and / or leucine in the original proliferation medium, and the NK cell killing activity decreases. When valine and leucine are omitted at the same time, the degree of decrease in NK cell killing activity is more significant, indicating that valine and leucine have a synergistic effect in the original proliferation medium. Comparative Example 4 replaces valine in the original proliferation medium with lysine, and the NK cell activity decreases, but is higher than Comparative Examples 2, 3, and 6, indicating that amino acids have a certain effect on NK cell killing activity, but the effects of different amino acids are different. When using the proliferation medium or culture method of the present invention, it is necessary to pay attention to the type of amino acids. In Comparative Example 5, glycyrrhizin was omitted from the original proliferation medium, and NK cell activity decreased, indicating that glycyrrhizin is an active ingredient that enhances NK cell killing activity. In Comparative Example 7, fluid replenishment during the culture process was omitted, and NK cell activity decreased, indicating that appropriate fluid replenishment of cytokines and amino acids during the culture process helps promote the growth of NK cells and promotes the continued increase of NK cell activity.

[0090] The above results indicate that the proliferation culture medium of the present invention produces a synergistic effect among the components through the scientific combination of specific components, thereby greatly improving the killing activity of NK cells.

[0091] 2. Cytotoxicity of NK cells against HepG2 cells

[0092] HepG2 cells were used as target cells, and cells after proliferation and culture in each group were used as effector cells. The LDH assay was used to detect the cytotoxicity of NK cells against HepG2 cells. The effector-target ratio was set at 10:1, 20:1, and 40:1, respectively. A natural release well and a maximum release well were set for target cells, each containing 200 μl. Each well was incubated in a 37°C, 5% CO2 incubator for 3 h, centrifuged at 1500 rpm for 5 min, and 100 μl of the supernatant was aspirated from each well and placed in a flat-bottom 96-well culture plate. 100 μL of LDH matrix solution was added and reacted for 3 min. 30 μL of 1 mol / L HCl was added to each well, and the optical density (OD) was measured at 492 nm on a microplate reader. The cytotoxicity of NK cells against HepG2 cells was calculated according to the following formula:

[0093] NK cell killing activity against HepG2 cells = (OD of reaction well - OD of natural release well) / (OD of maximum release well - OD of natural release well) × 100%

[0094] The test results are shown in Table 2.

[0095] Table 2 Cytotoxicity of NK cells in each group against HepG2 cells (%)

[0096]

[0097]

[0098] As can be seen from Table 2, the NK cells obtained by the methods of Examples 1-3 have strong killing activity against HepG2 cells, and the NK cell killing activity of Example 2 is the strongest. Compared with Example 2, the NK cell activity of Comparative Examples 1-7 is reduced, and the trend is consistent with the experimental results of NK cell killing activity against K562 cells.

[0099] 3. Cytotoxicity of NK cells against A549 cells

[0100] A549 cells were used as target cells, and cells after proliferation and culture in each group were used as effector cells. The LDH assay was used to detect the cytotoxicity of NK cells against A549 cells. The effector-target ratio was set at 10:1, 20:1, and 40:1, respectively. A natural release well and a maximum release well were set for target cells, each containing 200 μl. Each well was incubated in a 37°C, 5% CO2 incubator for 3 h, centrifuged at 1500 rpm for 5 min, and 100 μl of the supernatant was aspirated from each well and placed in a flat-bottom 96-well culture plate. 100 μL of LDH matrix solution was added and the reaction was continued for 3 min. 30 μL of 1 mol / L HCl was added to each well, and the optical density (OD) was measured at 492 nm on a microplate reader. The cytotoxicity of NK cells against A549 cells was calculated according to the following formula:

[0101] NK cell killing activity against A549 cells = (OD of reaction well - OD of natural release well) / (OD of maximum release well - OD of natural release well) × 100%

[0102] The test results are shown in Table 3.

[0103] Table 3 Cytotoxicity of NK cells in each group against A549 cells (%)

[0104]

[0105]

[0106] As can be seen from Table 3, the NK cells obtained by the methods of Examples 1-3 had strong killing activity against A549 cells, and the NK cells of Example 2 had the strongest killing activity. Compared with Example 2, the NK cell activity of Comparative Examples 1-7 decreased, and the trend was consistent with the experimental results of NK cell killing activity against K562 cells.

[0107] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for improving the killing activity of NK cells, characterized in that: The following steps are involved: (1) Isolation of peripheral blood mononuclear cells; (2) sequentially performing induction culture and proliferation culture on the mononuclear cells; The induction culture medium is: RPMI 1640 basal medium + fetal bovine serum + IL-15 + IL-12 + TGF-β; The proliferation culture medium is RPMI 1640 basal medium + fetal bovine serum + IL-13 + IL-18 + valine + leucine + isoleucine + glycyrrhizin.

2. The method according to claim 1, characterized in that The induction medium is: RPMI 1640 basic medium + 10% fetal bovine serum + 40-55 ng / ml IL-15 + 10-20 ng / ml IL-12 + 20-40 ng / ml TGF-β.

3. The method according to claim 1, characterized in that The proliferation culture medium is: RPMI 1640 basal culture medium + 10% fetal bovine serum + 15-30 ng / ml IL-13 + 20-30 ng / ml IL-18 + 0.5-1.0 g / L valine + 0.5-1.0 g / L leucine + 0.5-1.0 g / L isoleucine + 0.2-0.4 g / L glycyrrhizin.

4. The method according to claim 1, wherein The induction culture conditions are: culturing at 37° C. and 5% CO 2 for 2-4 days.

5. The method according to claim 1, wherein The proliferation culture conditions are: culturing at 37° C. and 5% CO 2 for 6-10 days.

6. The method according to claim 1, characterized in that During the proliferation culture process, IL-18 solution, valine solution and leucine solution are added after culturing for 2-3 days.

7. The method according to claim 6, characterized in that The amount of the IL-18 solution, valine solution and leucine solution added is 1.5%-3% of the volume of the initial proliferation culture medium.

8. The method according to claim 6, characterized in that The concentration of the IL-18 solution is 40-60 ng / ml; the concentration of the valine solution is 1.0-1.5 g / L; and the concentration of the leucine solution is 1.0-1.5 g / L.

9. A proliferation culture medium for improving the killing activity of NK cells, characterized in that: The proliferation culture medium comprises: RPMI1640 basal culture medium + 10% fetal bovine serum + 15-30 ng / ml IL-13 + 20-30 ng / ml IL-18 + 0.5-1.0 g / L valine + 0.5-1.0 g / L leucine + 0.5-1.0 g / L isoleucine + 0.2-0.4 g / L glycyrrhizin.

10. Use of the proliferation culture medium according to claim 9 in culturing NK cells with high cytotoxic activity.

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