Enhanced NK cell as well as preparation method and application thereof

Abstract

The invention provides an enhanced NK cell as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Quercetin, paclitaxel and resveratrol are compounded to serve as a culture medium induction additive to pretreat the NK cells, the killing toxicity of the NK cells on human lung cancer cells is successfully improved, tumor growth is effectively inhibited, a new way is opened up for lung cancer treatment, and the method has a wide application prospect. The invention has important research value and potential clinical application prospect in the field of tumor immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an enhanced NK cell and a preparation method and application thereof. Background Art

[0002] Cancer is one of the major challenges facing the global medical and health field. Its morbidity and mortality rates have remained high among all kinds of diseases, seriously threatening human life and health. Although traditional cancer treatments such as surgical resection, radiotherapy and chemotherapy can control the growth and spread of tumors to a certain extent, they each have obvious limitations. For some patients with advanced cancer, surgical treatment is often unable to completely eliminate the lesions due to tumor metastasis or special location; while radiotherapy kills cancer cells, it will cause varying degrees of damage to surrounding normal tissues, causing a series of adverse reactions; the toxic side effects of chemotherapy drugs will not only affect the patient's physical functions, but may also cause damage to the immune system and reduce the patient's quality of life. These problems have prompted the scientific community to continuously seek more effective new strategies for cancer treatment.

[0003] Natural killer (NK) cells play a vital role in the human immune system's fight against cancer. They are the body's first line of defense against tumors and can quickly identify and kill tumor cells without prior sensitization. Their unique immune function makes them one of the core research directions of cancer immunotherapy. However, factors such as the complexity of the tumor microenvironment and the immune escape mechanism of cancer cells often lead to severe inhibition of NK cell activity and tumor killing ability, greatly limiting the actual application of NK cells in clinical cancer 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, Chinese medicine has shown unique advantages and potential. A large number of studies have shown that many Chinese medicine monomers and compounds have the effect of significantly enhancing the activity of NK cells. For example, ginsenosides, as an important Chinese medicine monomer, can promote the activation and proliferation of NK cells by regulating cell signaling pathways, enhance their cytotoxic effects, and thus effectively improve the killing ability of NK cells against tumor cells. For another example, the Sijunzi Decoction compound composed of multiple Chinese medicines such as Astragalus, Atractylodes, and Poria can regulate the body's overall immune function, improve the immune microenvironment, and promote the maturation and function of NK cells, playing a positive role in the adjuvant treatment of various cancers. These research results fully demonstrate the great value of Chinese medicine in cancer immunotherapy and provide 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, successfully screened and obtained three effective compound monomer active ingredients of traditional Chinese medicine, and fully combined the advantages of traditional Chinese medicine in regulating immunity with the anti-tumor properties of natural compounds. Summary of the invention

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

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a NK cell killing activity enhancer, comprising the following components: Quercetin, paclitaxel, and resveratrol.

[0008] In the present invention, quercetin refers to a natural flavonoid compound widely found in plants, and its chemical formula is C 15 H 10 O 7 , has rich biological activity and medicinal value.

[0009] In the present invention, the paclitaxel refers to a diterpenoid compound extracted from Taxus plants with significant anti-cancer activity, with a chemical name of 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], which is an important clinical chemotherapy drug.

[0010] In the present invention, the resveratrol is a natural polyphenol compound, the chemical name of which is 3,5,4'-trihydroxystilbene, which is mainly found in plants such as grapes, peanuts, and knotweed, and has a wide range of biological activities.

[0011] 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.

[0012] The present invention also provides a NK cell pretreatment culture medium, wherein the NK cell pretreatment culture medium contains the above-mentioned NK cell killing activity enhancer.

[0013] Preferably, 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.

[0014] Preferably, the NK cell pretreatment medium uses RPMI1640 medium as the basal medium.

[0015] Preferably, the NK cell pretreatment culture medium also contains FBS and penicillin-streptomycin dual antibody solution.

[0016] The present invention also provides a method for preparing enhanced NK cells, comprising the following steps: The NK cells were inoculated into the above-mentioned NK cell pretreatment medium and cultured for 24 to 72 hours to obtain enhanced NK cells.

[0017] Preferably, the seeding density of the NK cells is 10 5 ~10 7 cells / ml.

[0018] The present invention also provides enhanced NK cells prepared by the above preparation method.

[0019] The present invention also provides the use of the enhanced NK cells in the preparation of cancer therapeutic drugs.

[0020] Beneficial effects of the present invention: The present invention utilizes a combination of quercetin, paclitaxel and resveratrol as a culture medium induction additive to pretreat NK cells, successfully enhancing the cytotoxicity of NK cells to human lung cancer cells, effectively inhibiting tumor growth, and opening up a new approach for lung cancer treatment. It has important research value and potential clinical application prospects in the field of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is a graph of cytotoxicity results; Figure 2 The figure shows the tumor volume results of nude mice. DETAILED DESCRIPTION

[0022] The present invention provides a 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.

[0023] The present invention also provides a NK cell pretreatment culture medium, wherein the NK cell pretreatment culture medium contains the above-mentioned NK cell killing activity enhancer.

[0024] Preferably, 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. Further preferably, in the NK cell pretreatment medium, the final concentration of quercetin is 0.45-0.55 μmol / L; in the NK cell pretreatment medium, the final concentration of paclitaxel is 0.23-0.27 μmol / L; in the NK cell pretreatment medium, the final concentration of resveratrol is 0.23-0.27 μmol / L.

[0025] Preferably, the NK cell pretreatment medium uses RPMI1640 medium as the basal medium; in the present invention, the basal medium can also be selected from common cell culture media such as DMEM medium and MEM medium, which have the ability to provide cells with necessary nutrients and a suitable growth environment, and can be reasonably selected according to actual experimental requirements and cell culture characteristics. DMEM medium is rich in nutrients such as various amino acids, vitamins, glucose, and its high sugar form can better support cell metabolism and proliferation in certain cell cultures; MEM medium components are relatively simple, but for some cell cultures that are not particularly demanding in terms of nutritional requirements, they can also meet basic requirements, and the cost is relatively low. When using different basal culture media, the added 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 a good growth state and activity during the culture process, and then give full play to the enhancement of quercetin, paclitaxel and resveratrol on the killing activity of NK cells, and provide a reliable cell source and experimental basis for subsequent cancer treatment research or application.

[0026] In the present invention, preferably, the NK cell pretreatment medium also contains FBS and penicillin-streptomycin double antibody solution, which is used to provide NK cells with nutrients required for growth and inhibit microbial contamination, and ensure 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 activity states of cells. Penicillin-streptomycin double antibody solution can effectively prevent bacterial and fungal contamination, avoid adverse effects on cell culture due to microbial growth, and ensure the reliability and stability of experimental results. It can also contain L-glutamine, non-essential amino acids, sodium pyruvate and other ingredients. L-glutamine is an important precursor substance for cell synthesis of proteins and nucleic acids, plays a key role in cell metabolism, can enhance cell vitality and stability, and help maintain the normal growth and function of NK cells. Non-essential amino acids can supplement the additional demand of cells for amino acids during culture, promote the synthesis and renewal of intracellular proteins, and further optimize the growth environment of cells. Sodium pyruvate can be used as a supplementary substrate for cellular energy metabolism, improve the energy production efficiency of cells, enhance the metabolic activity of NK cells, and enable them to maintain a good physiological state during the culture process, thereby better responding to the effects of quercetin, paclitaxel and resveratrol and enhancing their killing activity.

[0027] 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, culturing for 24 to 72 hours, and obtaining enhanced NK cells. Preferably, the inoculation density of the NK cells is 10 5 ~10 7 cells / ml.

[0028] The present invention also provides enhanced NK cells prepared by the above preparation method.

[0029] The present invention also provides the use of the above-mentioned enhanced NK cells in the preparation of cancer therapeutic drugs. The enhanced NK cells of the present invention can exert their anti-cancer effects in the following ways: for example, the pretreated enhanced NK cells are prepared into cell preparations and returned to the patient 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, and thus inhibit tumor growth and metastasis. For example, enhanced NK cells can be combined with tumor-targeting nanocarriers. By utilizing the specific targeting ability of nanocarriers, NK cells can be delivered to the tumor microenvironment more accurately. Nanocarriers can carry some auxiliary substances that can enhance NK cell function or improve the tumor microenvironment, such as immunostimulatory factors, etc., to further synergistically enhance the killing effect of NK cells on cancer cells while reducing nonspecific damage to normal tissues. For example, when used together with immune checkpoint inhibitors, immune checkpoint inhibitors can relieve the inhibitory effect of tumor cells on the immune system, while enhanced NK cells can directly kill tumor cells. The two complement each other, which can greatly improve the effect of cancer treatment and bring new hope to cancer patients. It is expected to become an important part of future comprehensive cancer treatment plans.

[0030] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0031] Example Preparation of NK cell pretreatment medium containing the drug composition: Under sterile conditions, 0.5 μmol / L quercetin, 0.25 μmol / L paclitaxel and 0.25 μmol / L resveratrol were added to the RPMI1640 medium, and 10% FBS and 1% penicillin-streptomycin double antibody solution were added and mixed thoroughly.

[0032] Experimental example 1. Experimental materials: SPF grade 5-week-old healthy male BALB / c nude mice. Before the experiment, the nude mice were placed in a temperature of 22±2℃, relative humidity of 50%-60%, 12-hour light / dark alternation environment for 3-5 days for adaptive breeding, and free access to food and water; Human lung cancer cell line A549; 1.2 Reagents Mouse NK cell isolation kit, quercetin, paclitaxel, resveratrol, RPMI1640 medium (containing L-glutamine), fetal bovine serum (FBS), penicillin-streptomycin double antibody solution (100×), phosphate buffered saline (PBS, pH7.4), LDH detection kit, Trypsin-EDTA digestion solution (0.25% Trypsin, containing 0.02% EDTA), and trypan blue dye.

[0033] 1.3 Instruments Centrifuge, clean bench, CO 2 Incubator, microplate reader, electronic balance, vernier caliper, blood cell counting chamber, inverted microscope.

[0034] 2. Isolation of Mouse NK Cells Healthy mice were killed by cervical dislocation and quickly immersed in 75% alcohol for 5-10 minutes for disinfection. In the clean bench, the spleen of the mouse was carefully removed using forceps and scissors, placed in a sterile culture dish containing pre-cooled PBS, and the attached fat and connective tissue were carefully removed; Use forceps and scissors to mince the spleen into 1-2 mm pieces. 3 The tissue blocks were then transferred to a sterile centrifuge tube containing 5 ml of RPMI1640 medium and gently blown with a pipette to further disperse the tissue blocks. The cell suspension was then filtered through a 200-400 mesh cell sieve into a new sterile centrifuge tube to remove undispersed tissue debris to obtain a single cell suspension. The single cell suspension was centrifuged at 1500 rpm for 5-8 minutes at 4°C and the supernatant was discarded; Add an appropriate amount of the 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 instructions of the kit, mix gently and incubate at 4°C for 15-20 minutes to allow the magnetic beads to specifically bind to the NK cells. Transfer the incubated cell suspension to a sterile centrifuge tube placed on a magnetic rack and let it stand for 2-3 minutes until the NK cells bound to the magnetic beads are adsorbed on the tube wall. Carefully aspirate and discard the cell supernatant that is 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 rack for 2-3 minutes after each wash, and then discard the supernatant. Finally, resuspend the separated NK cells with an appropriate amount of RPMI1640 culture medium containing 10% FBS and transfer them to a sterile culture bottle.

[0035] Take a small amount of the separated NK cell suspension, mix it with trypan blue dye at a ratio of 1:1, and incubate it at room temperature for 2-3 minutes. On a blood cell counting plate, count the live cells (colorless) and dead cells (blue) with the help of an inverted microscope to calculate the cell viability, and the results show that the cell viability is 93.35%.

[0036] Take an appropriate amount of cell suspension, centrifuge at 1500rpm for 5 minutes, and discard the supernatant. Add an appropriate amount of fluorescently labeled anti-mouse NK cell specific surface marker (CD 3- NK 1.1+ ) and incubate at 4°C in the dark for 30 minutes. After incubation, wash the cells twice with PBS, centrifuge at 1500rpm 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. Flow cytometry was used to analyze the proportion of NK cells in the total cells. The results showed that the purity of the obtained NK cells was as high as 96.55%, which was relatively pure.

[0037] 3. NK Cell Pretreatment Culture 3.1 Set up the following groups: Experimental group culture medium: under sterile conditions, add 0.5 μmol / L quercetin, 0.25 μmol / L paclitaxel and 0.25 μmol / L resveratrol to RPMI1640 culture medium, add 10% FBS and 1% penicillin-streptomycin double antibody solution, and mix thoroughly; Control group culture medium 1: add quercetin and paclitaxel at a final concentration of 0.5 μmol / L to RPMI1640 culture medium, add 10% FBS and 1% double antibody solution, and mix thoroughly; Control group culture medium 2: Add quercetin and resveratrol at a final concentration of 0.5 μmol / L to RPMI1640 culture medium, and add 10% FBS and 1% double antibody solution, and mix thoroughly; 3.2 NK cells were cultured at 1×10 6 The cells / ml were inoculated into culture flasks containing the above-mentioned different culture media at a density of 1.5 cells / ml and placed at 37°C and 5% CO 2 incubator for 48 hours.

[0038] 4. Cytotoxicity assay (LDH method) 4.1 Resuscitation of human lung cancer cell line A549 was carried out in RPMI1640 medium containing 10% FBS and 1% penicillin-streptomycin solution at 37°C and 5% CO 2 The A549 cells were cultured in an incubator until the logarithmic growth phase. A549 cells were digested with Trypsin-EDTA digestion solution to prepare a single cell suspension and the cell concentration was adjusted to 1×10 5 cells / ml.

[0039] 4.2 Experimental Grouping: Experimental group: NK cells and A549 cells pretreated with the experimental culture medium were inoculated into 96-well plates at effector-target ratios (E:T) of 50:1, 25:1, and 12.5:1, respectively, with 100 μl per well, and 3 replicate wells were set for each ratio.

[0040] Control group 1: NK cells and A549 cells pretreated with the culture medium of control group 1 were inoculated into 96-well plates at the same effector-target ratio as above, 100 μl per well, and 3 replicate wells were set for each ratio.

[0041] Control group 2: NK cells and A549 cells pretreated with the culture medium of control group 2 were inoculated into 96-well plates at the same effector-target ratio as above, 100 μl per well, and 3 replicate wells were set for each ratio.

[0042] Target cell spontaneous release group: only 100 μl A549 cell suspension was added.

[0043] Target cell maximum release group: add 100 μl A549 cell suspension and then add 10 μl 1% TritonX-100 solution.

[0044] NK cell spontaneous release group: only 100 μl of pre-cultured NK cell suspension was added.

[0045] 4.3 Place the 96-well plate in a 37°C, 5% CO 2 Incubate in the incubator for 4 hours. After incubation, centrifuge the 96-well plate at 1500 rpm for 5 minutes. Pipette 100 μl of supernatant from each well and transfer it to a new 96-well plate. Add the corresponding reagents according to the instructions of the LDH detection kit and react at room temperature in the dark for 15 to 30 minutes. Use an enzyme-labeled instrument to measure the absorbance (OD value) of each well at a wavelength of 490 nm.

[0046] 4.4 Calculate the cytotoxicity results according to the following formula: Cytotoxicity (%) = [(OD value of experimental group - OD value of NK cell spontaneous release group - OD value of target cell spontaneous release group) / (OD value of target cell maximum release group - OD value of target cell spontaneous release group)] × 100% 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.

[0047] The results are shown in Table 1 and Figure 1 As shown: Table 1 Cytotoxicity test results (unit: %) Effector-target ratio (E:T) Cytotoxicity in experimental groups Control group 1 cytotoxicity Control group 2 cytotoxicity 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 In the table, * indicates P < 0.05 compared with other treatment groups.

[0048] 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.

[0049] 5. In vivo experiments in nude mice 5.1 Experimental Grouping: Blank control group: 10 nude mice, each injected subcutaneously with 0.2 ml PBS.

[0050] Negative control group: 10 nude mice, each subcutaneously injected with 1×10 6 A549 cells.

[0051] 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).

[0052] 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.

[0053] Control group 2: 10 nude mice, each of which was subcutaneously injected with 1×10 6 A549 cells were cultured and 7 days after tumor cell inoculation, 1×106 NK cells pretreated with 0.5 μmol / L quercetin + 0.5 μmol / L resveratrol were injected into the tail vein.

[0054] 5.2 Tumor inoculation and observation: Under sterile conditions, the A549 cell suspension was adjusted to an appropriate concentration, the cell suspension was drawn up with a 1 ml syringe, and 0.2 ml was slowly injected subcutaneously into the right armpit of the nude mouse. Starting from the day of NK cell inoculation, the long diameter (a) and short diameter (b) of the nude mouse tumor were measured with a vernier caliper every 3 days, according to the formula V = 1 / 2 × a × b 2 The tumor volume was calculated and recorded.

[0055] 5.3 End of experiment and data analysis: The experiment was terminated 21 days after NK cell inoculation.

[0056] The tumor volume data of each group of nude mice were statistically analyzed. SPSS 16.0 software was used for statistical analysis in the present invention, and the experimental data were expressed as "mean ± standard deviation". The data differences between the experimental group and the control group and between the experimental groups were compared by one-way analysis of variance, with P < 0.05 as the judgment standard. If this condition was met, the difference was considered to be statistically significant.

[0057] The nude mice in the blank control group were not inoculated with tumor cells, and the tumor volume was always 0 during the entire experimental period, which is not repeated in the table. In addition, there was no death of nude mice during the experiment.

[0058] The results are shown in Table 2 and Figure 2 As shown: Table 2 Nude mouse tumor volume results (mm 3 ) 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* In the table, * indicates P < 0.05 compared with the negative control group, and ** indicates P < 0.01 compared with the negative control group.

[0059] The experimental results show that the specific drug composition used in the experimental group of the present invention has the best effect on NK cell pretreatment in inhibiting tumor growth, which is significantly better than 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 tumor volume.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A NK cell killing activity enhancer, characterized in that: Includes the following components: Quercetin, paclitaxel, and resveratrol.

2. The NK cell killing activity enhancer according to claim 1, characterized in that 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.

3. A NK cell pretreatment medium, characterized in that The NK cell pretreatment culture medium contains the NK cell killing activity enhancer according to claim 1 or claim 2.

4. The NK cell pretreatment medium according to claim 3, characterized in that 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.

5. The NK cell pretreatment medium according to claim 4, characterized in that The NK cell pretreatment medium uses RPMI1640 medium as a basic medium.

6. The NK cell pretreatment medium according to claim 5, characterized in that The NK cell pretreatment culture medium also contains FBS and penicillin-streptomycin double antibody solution.

7. 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 3 to 6, and cultured for 24 to 72 hours to obtain enhanced NK cells.

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

9. Enhanced NK cells obtained by the preparation method according to claim 7 or 8.

10. Use of the enhanced NK cells according to claim 9 in the preparation of cancer therapeutic drugs.

Images