An enhanced NK cell, its preparation method and application

Abstract

The present invention provides an enhanced NK cell, its preparation method and application, belonging to the field of biomedical technology. The present invention uses a compound of quercetin, paclitaxel and resveratrol as a culture medium induction additive to pre-treat NK cells, successfully enhancing the killing toxicity of NK cells against human lung cancer cells and effectively inhibiting tumor growth, opening up a new way for the treatment of lung cancer and having important research value and potential clinical application prospects in the field of tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to an enhanced NK cell and its preparation method and application. Background Art

[0002] Cancer, as one of the major challenges faced by the global healthcare field, has consistently ranked high in terms of incidence and mortality among various diseases, seriously threatening human life and health. Traditional cancer treatment methods such as surgical resection, radiotherapy, and chemotherapy, although to a certain extent can control the growth and spread of tumors, each has obvious limitations. Surgical treatment for some patients with advanced cancer often cannot completely remove the lesions due to tumor metastasis or special location; radiotherapy, while killing cancer cells, will cause varying degrees of damage to surrounding normal tissues, triggering a series of adverse reactions; the toxic and side effects of chemotherapy drugs not only affect the patient's physical functions but may also lead to immune system damage and reduce the patient's quality of life. These problems have prompted the scientific community to continuously seek more effective new cancer treatment strategies.

[0003] Natural killer (NK) cells play a crucial role in the process of the human immune system's defense against cancer. It is the first line of defense in the body's anti-tumor immunity, capable of quickly identifying and killing tumor cells without prior sensitization. Its unique immune function makes it one of the core directions in cancer immunotherapy research. However, factors such as the complexity of the tumor microenvironment and the immune escape mechanism of cancer cells often severely inhibit the activity and tumor-killing ability of NK cells, greatly limiting the actual application effect of NK cells in cancer clinical treatment. Therefore, finding effective methods to enhance the function of NK cells has become a key topic in the field of cancer research.

[0004] In the exploration process of cancer treatment, traditional Chinese medicine has shown unique advantages and potential. A large number of studies have shown that many traditional Chinese medicine monomers and compounds have a significant effect on enhancing the activity of NK cells. For example, ginsenoside, as an important traditional Chinese medicine monomer, can promote the activation and proliferation of NK cells by regulating cell signaling pathways, enhance its cytotoxic effect, and thus effectively improve the tumor-killing ability of NK cells. Another example is the Sijunzi decoction compound composed of several traditional Chinese medicines such as Astragalus membranaceus, Atractylodes macrocephala, and Poria cocos, which can regulate the overall immune function of the body, improve the immune microenvironment, and promote the maturation and function of NK cells, playing an active role in the adjuvant treatment of various cancers. These research results fully demonstrate the great value of traditional Chinese medicine in cancer immunotherapy, providing new ideas and directions for cancer treatment.

[0005] In view of the above background, the present invention proposes a new NK cell activity enhancement scheme, and successfully screens out three active ingredients of traditional Chinese medicine monomers with effective compounding, fully combining the advantages of traditional Chinese medicine in regulating immunity and the anti-tumor characteristics of natural compounds. Summary of the Invention

[0006] The purpose of the present invention is to provide an enhanced NK cell and its preparation method and application, providing a new effective means for cancer treatment.

[0007] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0008] The present invention provides an NK cell killing activity enhancer, comprising the following components:

[0009] Quercetin, paclitaxel and resveratrol.

[0010] In the present invention, the quercetin refers to a natural flavonoid compound widely present in plants, with the chemical formula C 15 H 10 O7, having rich biological activities and medicinal values.

[0011] In the present invention, the paclitaxel refers to a diterpenoid compound with significant anti-cancer activity extracted from plants of the genus Taxus, with the chemical name 5β,20-epoxy-1,2α,4,7β,10β,13α-hexahydroxytaxane-11-ene-9-one-4,10-diacetate-2-benzoate-13-[(2’R,3’S)-N-benzoyl-3-phenylisoserine ester], and is an important clinical chemotherapy drug.

[0012] In the present invention, the resveratrol is a natural polyphenolic compound, with the chemical name 3,5,4'-trihydroxystilbene, mainly present in plants such as grapes, peanuts, and polygonum cuspidatum, and having a wide range of biological activities.

[0013] Preferably, the molar ratio of quercetin, paclitaxel and resveratrol in the NK cell killing activity enhancer is 1:0.3 - 0.8:0.3 - 0.8.

[0014] The present invention also provides an NK cell pretreatment culture medium, and the NK cell pretreatment culture medium contains the above-mentioned NK cell killing activity enhancer.

[0015] Preferably, in the NK cell pretreatment culture medium, the final concentration of quercetin is 0.4 - 0.6 μmol / L;

[0016] in the NK cell pretreatment culture medium, the final concentration of paclitaxel is 0.2 - 0.3 μmol / L;

[0017] In the NK cell pretreatment medium, the final concentration of resveratrol is 0.2 - 0.3 μmol / L.

[0018] Preferably, the NK cell pretreatment medium is based on RPMI1640 medium.

[0019] Preferably, the NK cell pretreatment medium also contains FBS and penicillin - streptomycin double - antibody solution.

[0020] The present invention also provides a method for preparing enhanced NK cells, comprising the following steps:

[0021] Inoculate NK cells into the above - mentioned NK cell pretreatment medium and culture for 24 - 72 h to obtain enhanced NK cells.

[0022] Preferably, the seeding density of the NK cells is 10 5 ~10 7 cells / ml.

[0023] The present invention also provides enhanced NK cells prepared by the above - mentioned preparation method.

[0024] The present invention also provides the application of the above - mentioned enhanced NK cells in the preparation of cancer treatment drugs.

[0025] Advantages of the present invention:

[0026] The present invention uses the compound of quercetin, paclitaxel and resveratrol as a culture medium induction additive to pretreat NK cells, successfully enhancing the killing toxicity of NK cells against human lung cancer cells, effectively inhibiting tumor growth, opening up a new way for lung cancer treatment, and having important research value and potential clinical application prospects in the field of tumor immunotherapy. Description of the Drawings

[0027] Figure 1 It is a graph of cytotoxicity results;

[0028] Figure 2 It is a graph of the tumor volume results of nude mice. Detailed Embodiments

[0029] The present invention provides an NK cell killing activity enhancer, comprising the following components: quercetin, paclitaxel and resveratrol. Preferably, the molar ratio of quercetin, paclitaxel and resveratrol in the NK cell killing activity enhancer is 1:0.3 - 0.8:0.3 - 0.8.

[0030] The present invention also provides an NK cell pretreatment medium, which contains the above - mentioned NK cell killing activity enhancer.

[0031] Preferably, in the NK cell pre-treatment medium, the final concentration of quercetin is 0.4 - 0.6 μmol / L; in the NK cell pre-treatment medium, the final concentration of paclitaxel is 0.2 - 0.3 μmol / L; in the NK cell pre-treatment medium, the final concentration of resveratrol is 0.2 - 0.3 μmol / L. Further preferably, in the NK cell pre-treatment medium, the final concentration of quercetin is 0.45 - 0.55 μmol / L; in the NK cell pre-treatment medium, the final concentration of paclitaxel is 0.23 - 0.27 μmol / L; in the NK cell pre-treatment medium, the final concentration of resveratrol is 0.23 - 0.27 μmol / L.

[0032] Preferably, the NK cell pre-treatment medium is based on RPMI1640 medium; in the present invention, the basal medium can also be selected from common cell media such as DMEM medium and MEM medium. These media all have the ability to provide necessary nutrients and a suitable growth environment for cells, and can be reasonably selected according to actual experimental needs and cell culture characteristics. DMEM medium is rich in various amino acids, vitamins, glucose and other nutrients, and its high-glucose type can better support cell metabolism and proliferation in certain cell cultures; MEM medium has relatively simple components, but it can also meet the basic requirements for cell cultures with less demanding nutritional needs, and the cost is relatively low. When using different basal media, the addition concentration and culture conditions of the NK cell killing activity enhancer need to be appropriately optimized and adjusted to ensure that NK cells can maintain good growth status and activity during the culture process, and then fully exert the enhancing effect of quercetin, paclitaxel and resveratrol on the killing activity of NK cells, providing a reliable cell source and experimental basis for subsequent cancer treatment research or application.

[0033] In the present invention, preferably, the NK cell pretreatment medium further contains FBS and penicillin-streptomycin double antibody solution, whose function is to provide the nutrients required for the growth of NK cells and inhibit microbial contamination, ensuring the stability of the cell culture environment and the normal growth and proliferation of cells. FBS is rich in various growth factors, proteins and other nutrients, which can promote the survival, proliferation and metabolic activities of NK cells, and help maintain the physiological functions and active states of cells. The penicillin-streptomycin double antibody solution can effectively prevent the contamination of bacteria and fungi, avoid the adverse effects on cell culture caused by the growth of microorganisms, and ensure the reliability and stability of experimental results. It may also contain components such as L-glutamine, non-essential amino acids, sodium pyruvate, etc. L-glutamine is an important precursor for cells to synthesize proteins and nucleic acids, plays a key role in the cell metabolism process, can enhance the vitality and stability of cells, and helps maintain the normal growth and function of NK cells. Non-essential amino acids can supplement the additional demand for amino acids during cell culture, promote the synthesis and renewal of intracellular proteins, and further optimize the cell growth environment. Sodium pyruvate can be used as a supplementary substrate for cell energy metabolism, improve the energy production efficiency of cells, enhance the metabolic activity of NK cells, enable them to maintain a good physiological state during culture, and thus better respond to the effects of quercetin, paclitaxel and resveratrol, and enhance their killing activity.

[0034] The present invention also provides a method for preparing enhanced NK cells, comprising the following steps: inoculating NK cells into the above-mentioned NK cell pretreatment medium and culturing for 24 - 72 h to obtain enhanced NK cells. Preferably, the inoculation density of the NK cells is 10 5 ~10 7 cells / ml.

[0035] The present invention also provides enhanced NK cells prepared by the above preparation method.

[0036] The present invention also provides the use of the above-mentioned enhanced NK cells in the preparation of cancer treatment drugs. The enhanced NK cells of the present invention can exert their anti-cancer effects in the following ways: for example, the pre-treated enhanced NK cells are prepared into cell preparations and infused back into the patient's body by intravenous infusion. Under the action of the blood circulation system, NK cells can quickly migrate to the tumor tissue site, recognize and directly attack cancer cells with their enhanced killing activity, induce apoptosis or necrosis of cancer cells, thereby inhibiting the growth and metastasis of tumors. Another example is that the enhanced NK cells can be combined with tumor-targeted nanocarriers. Utilizing the specific targeting ability of the nanocarriers, NK cells can be more precisely delivered to the tumor microenvironment. The nanocarriers can carry some auxiliary substances that can enhance the function of NK cells or improve the tumor microenvironment, such as immune stimulatory factors, etc., further synergistically enhancing the killing effect of NK cells on cancer cells while reducing non-specific damage to normal tissues. Another example is to use it in combination with immune checkpoint inhibitors. Immune checkpoint inhibitors can relieve the inhibition of tumor cells on the immune system, while enhanced NK cells can directly exert the function of killing tumor cells. The two complement each other, can greatly improve the effect of cancer treatment, bring new hope to cancer patients, and are expected to become an important part of future cancer comprehensive treatment programs.

[0037] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0038] Embodiment

[0039] Prepare an NK cell pre-treatment culture medium containing a drug composition:

[0040] Under sterile conditions, add quercetin with a final concentration of 0.5 μmol / L, paclitaxel with a final concentration of 0.25 μmol / L, and resveratrol with a final concentration of 0.25 μmol / L to RPMI1640 medium, and add 10% FBS and 1% penicillin-streptomycin double antibody solution, and mix well.

[0041] Experimental example

[0042] 1. Experimental materials: SPF-grade 5-week-old healthy male BALB / c nude mice. Before the experiment, the nude mice were placed in an environment with a temperature of 22 ± 2 °C, a relative humidity of 50%-60%, and a 12-hour light / dark cycle for adaptive feeding for 3-5 days, with free access to food and water;

[0043] Human lung cancer cell line A549;

[0044] 1.2 Reagents

[0045] Mouse NK Cell Isolation Kit, quercetin, paclitaxel, resveratrol, RPMI 1640 medium (containing L-glutamine), fetal bovine serum (FBS), penicillin-streptomycin double antibody solution (100×), phosphate buffer (PBS, pH 7.4), LDH detection kit, Trypsin-EDTA digestive solution (0.25% Trypsin, containing 0.02% EDTA), trypan blue stain solution.

[0046] 1.3 Instruments

[0047] Centrifuge, laminar flow hood, CO2 incubator, microplate reader, electronic balance, vernier caliper, hemocytometer, inverted microscope.

[0048] 2. Isolation of Mouse NK Cells

[0049] Healthy mice were sacrificed by cervical dislocation and quickly immersed in 75% alcohol for 5 - 10 minutes for disinfection. In the laminar flow hood, use forceps and scissors to carefully remove the spleen of the mouse and place it in a sterile petri dish containing pre-cooled PBS, and carefully remove the attached fat and connective tissue;

[0050] Use forceps and scissors to cut the spleen into tissue pieces of 1 - 2 mm 3 in size, and then transfer them to a sterile centrifuge tube containing 5 ml of RPMI 1640 medium. Gently pipette to further disperse the tissue pieces. Then filter the cell suspension through a 200 - 400 mesh cell sieve into a new sterile centrifuge tube to remove undispersed tissue residues, thus obtaining a single cell suspension. Centrifuge the single cell suspension at 1500 rpm for 5 - 8 minutes at 4°C and discard the supernatant;

[0051] Add an appropriate amount of buffer in the Mouse NK Cell Isolation Kit to the cell pellet, resuspend the cells and adjust the cell concentration to 2×10 7 cells / ml. Add the corresponding magnetic beads according to the kit instructions, gently mix and incubate at 4°C for 15 - 20 minutes to specifically bind the magnetic beads to NK cells. Transfer the incubated cell suspension to a sterile centrifuge tube placed on a magnetic stand, let it stand for 2 - 3 minutes, and wait for the NK cells bound to the magnetic beads to adsorb on the tube wall. Carefully aspirate and discard the cell supernatant that has not bound to the magnetic beads. Wash the NK cells adsorbed on the tube wall 2 - 3 times with pre-cooled PBS buffer, let it stand on the magnetic stand for 2 - 3 minutes after each wash, and then discard the supernatant. Finally, resuspend the isolated NK cells with an appropriate amount of RPMI 1640 medium containing 10% FBS and transfer them to a sterile culture flask.

[0052] Take a small amount of the separated NK cell suspension, mix it with trypan blue stain in a 1:1 ratio, and incubate at room temperature for 2 - 3 minutes. On a hemocytometer, count the viable cells (colorless) and dead cells (blue) under an inverted microscope to calculate cell viability. The result shows that the cell viability reaches 93.35%.

[0053] Take an appropriate amount of cell suspension, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant. Add an appropriate amount of antibody against the specific surface marker (CD 3- 、NK 1.1+ ) of mouse NK cells labeled with fluorescence, and incubate in the dark at 4°C for 30 minutes. After the incubation, wash the cells twice with PBS, centrifuge at 1500 rpm for 5 minutes each time, and discard the supernatant. Add an appropriate amount of PBS containing 1% paraformaldehyde to resuspend the cells and fix the cells. Use a flow cytometer to detect and analyze the proportion of NK cells in the total cells. The result shows that the purity of the obtained NK cells is as high as 96.55%, which is relatively pure.

[0054] 3. Pretreatment and culture of NK cells

[0055] 3.1 Set the following groups:

[0056] Experimental group medium: Under sterile conditions, add quercetin with a final concentration of 0.5 μmol / L, paclitaxel with a final concentration of 0.25 μmol / L, and resveratrol with a final concentration of 0.25 μmol / L to RPMI1640 medium. At the same time, add 10% FBS and 1% penicillin - streptomycin double - antibody solution, and mix well;

[0057] Control group medium 1: Add quercetin with a final concentration of 0.5 μmol / L and paclitaxel with a final concentration of 0.5 μmol / L to RPMI1640 medium. At the same time, add 10% FBS and 1% double - antibody solution, and mix well;

[0058] Control group medium 2: Add quercetin with a final concentration of 0.5 μmol / L and resveratrol with a final concentration of 0.5 μmol / L to RPMI1640 medium. At the same time, add 10% FBS and 1% double - antibody solution, and mix well;

[0059] 3.2 Inoculate NK cells at a density of 1×10 6 cells / ml into culture flasks containing the above different media respectively, and culture in an incubator at 37°C and 5% CO2 for 48 hours.

[0060] 4. Cytotoxicity detection (LDH method)

[0061] 4.1 Resuscitate the human lung cancer cell line A549 and culture it in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin double antibody solution in an incubator at 37°C and 5% CO2 until the logarithmic growth phase. Digest the A549 cells with Trypsin-EDTA digestive solution to make a single-cell suspension, and adjust the cell concentration to 1×10 5 cells / ml.

[0062] 4.2 Experimental grouping:

[0063] Experimental group: After culturing NK cells pretreated with the experimental group medium and A549 cells, inoculate them into 96-well plates at effector-to-target ratios (E:T) of 50:1, 25:1, and 12.5:1 respectively, 100 μl per well, and set 3 replicates for each ratio.

[0064] Control group 1: After culturing NK cells pretreated with the control group 1 medium and A549 cells, inoculate them into 96-well plates at the same effector-to-target ratios as above, 100 μl per well, and set 3 replicates for each ratio.

[0065] Control group 2: After culturing NK cells pretreated with the control group 2 medium and A549 cells, inoculate them into 96-well plates at the same effector-to-target ratios as above, 100 μl per well, and set 3 replicates for each ratio.

[0066] Target cell spontaneous release group: Only add 100 μl of A549 cell suspension.

[0067] Target cell maximum release group: Add 100 μl of A549 cell suspension, and then add 10 μl of 1% TritonX-100 solution.

[0068] NK cell spontaneous release group: Only add 100 μl of the pretreated and cultured NK cell suspension.

[0069] 4.3 Incubate the 96-well plates in an incubator at 37°C and 5% CO2 for 4 hours. After incubation, centrifuge the 96-well plates at 1500 rpm for 5 min in a centrifuge. Pipette 100 μl of the supernatant from each well and transfer it to a new 96-well plate. Add the corresponding reagents according to the LDH detection kit instructions and react for 15 - 30 minutes at room temperature in the dark. Measure the absorbance (OD value) of each well at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0070] 4.4 Calculate the cytotoxicity of the results according to the following formula:

[0071] Cytotoxicity (%) = [(OD value of the experimental group - OD value of the NK cell spontaneous release group - OD value of the target cell spontaneous release group) / (OD value of the target cell maximum release group - OD value of the target cell spontaneous release group)] × 100%

[0072] In the formula, the OD value of the experimental group can be replaced by the OD value of the control group 1 and the OD value of the control group 2 to obtain the corresponding results.

[0073] The results are shown in Table 1 and Figure 1 As shown:

[0074] Table 1 Cytotoxicity test results (unit: %)

[0075] Effector-to-target ratio (E:T) Cytotoxicity of experimental group Cytotoxicity of control group 1 Cytotoxicity of control group 2 50:1 76.31 ± 0.31* 44.82 ± 0.37 49.25 ± 0.38 25:1 68.39 ± 0.40* 35.56 ± 0.38 39.84 ± 0.39 12.5:1 59.43 ± 0.44* 26.25 ± 0.38 30.42 ± 0.39

[0076] In the table, * indicates P < 0.05 compared with other treatment groups.

[0077] It can be seen from the above experimental data that at each effect-target ratio, the mean cytotoxicity of the experimental group was significantly higher than that of the control group 1 and the control group 2, which indicates that the NK cells pretreated with the specific drug combination provided by the present invention have a stronger killing ability against A549 cells than the control group. It can be seen that the drug combination pretreatment method adopted by the experimental group more effectively enhances the cytotoxic effect of NK cells on lung cancer cells, and shows a better effect in killing lung cancer cells.

[0078] 5. In vivo experiments in nude mice

[0079] 5.1 Experimental Grouping:

[0080] Blank control group: 10 nude mice, each injected subcutaneously with 0.2 ml PBS.

[0081] Negative control group: 10 nude mice, each subcutaneously injected with 1×10 6 A549 cells.

[0082] Experimental group: 10 nude mice, each of which was subcutaneously injected with 1×10 6 A549 cells were injected into the tail vein 7 days after tumor cell inoculation with 1×10 6 The NK cells cultured were pretreated with the culture medium of the above experimental groups (centrifuge and discard the supernatant after pre-culture, resuspend the cells with PBS, and then centrifuge again and discard the supernatant, repeat 3-4 times to remove as much residual culture medium as possible).

[0083] Control group 1: 10 nude mice, each of which was subcutaneously injected with 1×10 6 A549 cells were injected into the tail vein 7 days after tumor cell inoculation with 1×10 6 NK cells were cultured after pretreatment with 0.5μmol / L quercetin + 0.5μmol / L paclitaxel.

[0084] Control group 2: 10 nude mice, each of which was subcutaneously injected with 1×10 6For A549 cells, 7 days after tumor cell inoculation, 1×106 NK cells pretreated with 0.5 μmol / L quercetin + 0.5 μmol / L resveratrol were injected via the tail vein.

[0085] 5.2 Tumor inoculation and observation: Under aseptic conditions, the A549 cell suspension was adjusted to an appropriate concentration. A 1 ml syringe was used to aspirate the cell suspension, and 0.2 ml was slowly injected subcutaneously into the right axilla of nude mice. Starting from the day of NK cell inoculation, the long diameter (a) and short diameter (b) of the tumors in nude mice were measured every 3 days using a vernier caliper. According to the formula V = 1 / 2 × a × b 2 calculate the tumor volume and record it.

[0086] 5.3 Experiment end and data analysis:

[0087] The experiment ended 21 days after NK cell inoculation.

[0088] Statistical analysis was performed on the tumor volume data of nude mice in each group. In this invention, SPSS 16.0 software was used for statistical analysis, and the experimental data was expressed as "mean ± standard deviation". One-way ANOVA was used to compare the data differences between the experimental group and the control group and between each experimental group. Taking P < 0.05 as the judgment standard, if this condition is met, the difference is considered statistically significant.

[0089] The nude mice in the blank control group were not inoculated with tumor cells, and the tumor volume was always 0 throughout the experimental period, which will not be elaborated in the table. In addition, there was no phenomenon of nude mouse death during the experiment.

[0090] The results are as shown in Table 2 and Figure 2 as follows:

[0091] Table 2 Results of Tumor Volume in Nude Mice Unit (mm 3 )

[0092] Group Negative control group Experimental group Control group 1 Control group 2 Day 0 27.34±2.15 26.89±2.08 27.11±2.10 27.05±2.09 Day 3 42.56±3.22 32.56±2.56* 38.78±3.01 38.21±2.98 Day 6 62.12±4.56 40.12±3.12** 52.34±4.01 51.11±3.98 Day 9 87.67±6.23 50.34±4.01** 70.56±5.34* 69.23±5.21* Day 12 122.56±8.45 65.67±5.23** 95.67±7.01* 93.45±6.89* Day 15 170.34±10.78 85.43±6.56** 130.45±9.11* 128.78±9.01* Day 18 235.67±13.56 115.67±8.45** 175.67±11.56* 172.34±11.23* Day 21 315.43±17.89 158.90±11.23** 230.12±14.56* 225.67±14.12*

[0093] In the table, * indicates P < 0.05 compared with the negative control group, and ** indicates P < 0.01 compared with the negative control group.

[0094] The experimental results show that the method of pre-treating NK cells with the specific drug composition used in the experimental group of this invention has the best effect in inhibiting tumor growth, significantly superior to Control Group 1 and Control Group 2, indicating that the compound combination of quercetin, paclitaxel, and resveratrol can effectively enhance the toxicity of NK cells to human lung cancer cells and reduce the tumor volume.

[0095] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A NK cell pretreatment medium, characterized in that The NK cell pretreatment culture medium contains an NK cell killing activity enhancer; The NK cell killing activity enhancer consists of quercetin, paclitaxel and resveratrol; In the NK cell pretreatment medium, the final concentration of quercetin is 0.4-0.6 μmol / L; In the NK cell pretreatment medium, the final concentration of paclitaxel is 0.2-0.3 μmol / L; In the NK cell pretreatment medium, the final concentration of resveratrol is 0.2-0.3 μmol / L.

2. The NK cell pretreatment medium according to claim 1, characterized in that The NK cell pretreatment medium uses RPMI1640 medium as a basic medium.

3. The NK cell pretreatment medium according to claim 2, characterized in that The NK cell pretreatment culture medium also contains FBS and penicillin-streptomycin double antibody solution.

4. A method for preparing enhanced NK cells, characterized in that: The following steps are involved: The NK cells are inoculated into the NK cell pretreatment medium according to any one of claims 1 to 3, and cultured for 24 to 72 hours to obtain enhanced NK cells.

5. The preparation method according to claim 4, characterized in that: The seeding density of the NK cells was 10 5 ~10 7 cells / ml.

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