Method for delaying senescence of human natural killer cells

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly relates to a method for delaying senescence of human natural killer cells. The invention relates to a method for delaying senescence of human natural killer cells. The method comprises the following steps: S1, culturing and amplifying enriched human NK cell species bottles on an NK cell culture medium; s2, adjusting the NK cell concentration, adding nicotinamide mononucleotide and quercetin into the NK cell culture medium, and continuing to culture; s3, collecting the NK cells to prepare the NK cell preparation. The NK cell culture medium containing the NMN and the quercetin is used for treating the NK cells of human peripheral blood or umbilical cord blood, and the NMN and the quercetin are found to be capable of promoting relative increase of SIRT1 protein of the NK cells and have no influence on safety of the human NK cells, so that aging of the human NK cells can be delayed, support can be provided for development of anti-aging cell drugs, and the application of the NMN and the quercetin in the preparation of anti-aging drugs is promoted. Good prospects and application values are realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy in biopharmaceuticals, and particularly relates to a method for delaying the senescence of human natural killer cells. Background Art

[0002] Natural killer cells (NK cells), the third major type of lymphocytes besides T cells and B cells, are large granular lymphocytes with natural cytotoxicity against tumor cells. They are not only related to anti-tumor, anti-viral infection and immune regulation, playing an important role in immune surveillance and anti-tumor responses, but also involved in the occurrence of hypersensitivity reactions and autoimmune diseases. NK cells are derived from bone marrow hematopoietic stem cells and are a core component of the innate immune system. They are mainly distributed in the bone marrow, peripheral blood, lungs, liver and spleen, and there are also a small number of NK cells in secondary lymphoid tissues (SLT), mucosa-associated lymphoid tissues (MALT) and the thymus. In general, NK cells in human peripheral blood account for 5-15% of lymphocytes, and they are the first line of defense for the body to defend against infection and tumors. NK cells do not need to be pre-sensitized and have no major histocompatibility complex (MHC) restriction, and can kill tumor cells or infected cells, showing a broad-spectrum anti-tumor effect. NK cells have a wide range of tumor-killing pathways, do not cause graft-versus-host disease (GvHD), do not secrete inflammatory factors such as IL-1, and do not induce cytokine storms, with relatively high safety.

[0003] Cell senescence is a well-established cancer defense mechanism and is also thought to play a role in aging and age-related diseases. It can occur through the depletion of stem cells and progenitor cells and the adverse effects of the senescence-associated secretory phenotype, which includes many pro-inflammatory cytokines, chemokines, matrix metalloproteinases, and growth factors. Senescence is characterized by a gradual loss of physiological function and is a risk factor for several of the world's most common diseases. Aging is a multifactorial process, and the immune system plays a crucial role in the aging process. Chronic inflammation is closely related to aging. Cell senescence is the progressive decline of cell physiological function that occurs after an organism reaches sexual maturity. It is a cell state that is closely related to multiple physiological processes and aging-related diseases. Cell senescence and the senescence-associated secretory phenotype (SASP) have become major drivers of aging and many chronic diseases, including cancer, neurodegeneration, heart disease, and osteoarthritis. The SASP is a series of inflammatory cytokines, chemokines, growth factors, and proteases secreted after cell senescence and is closely related to multiple physiological processes and aging-related diseases. It is a characteristic of cell senescence. In recent years, studies have found that the SASP is related to stem cell senescence, fibroblast senescence, epithelial cell senescence, endothelial cell senescence, and smooth muscle cell senescence. As the core cells of innate immune cells, NK cells are the main force in clearing senescent cells. The clearance of senescent cells by immune cells mainly composed of NK cells can extend the lifespan of animals.

[0004] In the field of anti-aging, the accelerated aging of organs and functions caused by immune system imbalance is the most crucial link in this vicious cycle of aging, and it is also an important research direction in the exploration of scientists to delay aging and even block the aging process. Baker Darren J et al. (2016) found that eliminating senescent cells is a powerful method to delay tumor formation, maintain tissue and organ functions, and even extend healthy lifespan, without side effects. As the core cells of innate immune cells, NK cells are the main force in eliminating senescent cells. The elimination of senescent cells by immune cells mainly composed of NK cells can extend the lifespan of animals. Chelyapov Nickolas et al. (2022) used autologous proliferated and activated NK (aNK) cells, which can reduce the senescence markers p16 and β-galactosidase (β-gal) in peripheral blood. aNK cells can reduce senescent cells in vitro and human immune senescence. In addition, it can eliminate the effect of immune senescence to reduce senescence-related inflammatory responses, that is, NK cells can improve chronic inflammation and have the effect of delaying immune senescence. Therefore, NK cells can be an important way and choice to restore physical health and keep the body young. Xiaofeng T et al. (2022) from the Department of Blood Transfusion of Shanghai Changzheng Hospital, Institute of Immunology of Shanghai Jiao Tong University and other units cooperated to publish a paper in Frontiers in Immunology, which for the first time confirmed at the data level that autologous NK cell infusion can significantly relieve T cell senescence and exhaustion, and is an effective method to inhibit the key component SASP (senescence-associated secretory phenotype). Especially for the immune system, aging can develop in a younger direction through NK cell infusion, further corroborating the feasibility and effectiveness of the method of delaying aging through autologous NK cells.

[0005] In mammals, the longevity protein Sirtuins family consists of 7 proteins and is a key player in regulating cell functions. Increasing the content of Sirtuins in mammals can extend the lifespan of cells. Sirtuin 1 protein (SIRT1) is the most widely studied member of the Sirtuins family. It is involved in many biological processes, including metabolic regulation, DNA repair, cell proliferation, aging, inflammation, and apoptosis, etc. SIRT1 can enhance the defense mechanism of cells against stress responses, improve the overall state of cells, and can promote cell survival by regulating multiple signaling pathways and maintain the health of tissues.

[0006] As people age, the human body gradually ages, and the number of human NK cells also gradually decreases with age. While the number of NK cells decreases, their activity increases; the expression of the anti-aging protein SIRT1 in NK cells of the elderly increases, and these small amounts of NK cells have a stronger ability to resist the aging environment brought by age. There have been a large number of clinical studies on anti-aging by expanding NK cells in vitro and then reinfusing them. After NK cells are activated and expanded in large numbers, although the quantity and anti-tumor activity are guaranteed, the NK cells begin to age at this time, the expression of related anti-aging proteins is missing, and the ability of the aging NK cells to resist the body's aging becomes poor.

[0007] NMN, also known as nicotinamide mononucleotide, has the chemical formula C 11 H 15 N2O8P, is soluble in water and is widely used in drug, medical, and cosmetic raw materials and food additives. The industrial synthesis methods include chemical methods and biological methods.

[0008] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art already known to those of ordinary skill in the art. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for delaying the aging of human natural killer cells, which further processes highly purified NK cells that have been expanded in large numbers to delay the aging of human NK cells, so as to better play a role in the anti-aging treatment of NK cells.

[0010] To achieve the above purpose, the present invention provides the following technical solutions:

[0011] A method for delaying the aging of human natural killer cells, comprising the following steps:

[0012] S1. Seed the enriched human NK cells in a flask and culture and expand them on an NK cell medium.

[0013] S2. Adjust the NK cell concentration, add nicotinamide mononucleotide and quercetin to the NK cell medium, and continue culturing.

[0014] S3. Collect the cultured NK cells and prepare them into NK cell preparations, such as: preparing NK cell preparations containing 10% human serum albumin and 0.9% NaCl injection solution, with specifications of 2 billion NK / 100 mL / bag and 4 billion NK / 200 mL / bag for intravenous drip.

[0015] Further, the formula of the NK cell medium is: serum-free X-VIVO-15 medium, cytokine rhIL-2, cytokine IL-15, cytokine IL-18, and autologous plasma.

[0016] Furthermore, the formulation of the NK cell culture medium is as follows: serum-free X-VIVO-15 medium, 1000 IU / mL cytokine rhIL-2, 10 ng / mL cytokine IL-15, 30 ng / ml cytokine IL-18, and 10% autologous plasma.

[0017] Furthermore, in step S2, the final concentrations of nicotinamide mononucleotide and quercetin in the NK cell culture medium are 0.625 μM / mL and 12.5 nM / mL, respectively.

[0018] Furthermore, in step S1, the seeding concentration of the NK cells is 1.5×10 6 cells / mL, and the culture conditions are: 37 °C, 5% CO2, replenishing the culture medium every 3 days, and culturing for 10 - 20 days.

[0019] Furthermore, in step S2, after adjusting the NK cell concentration, the concentration of the NK cells is 1.0×10 6 cells / mL, and the culture conditions are: 37 °C, 5% CO2, and the continuous culture time is 72 h.

[0020] The second object of the present invention is to provide the use of nicotinamide mononucleotide and quercetin in delaying the senescence of NK cells, promoting the expression of SIRT1 protein in NK cells, and inhibiting the decrease of SIRT1 protein.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses an NK cell culture medium containing NMN and quercetin to treat human peripheral blood or cord blood NK cells, and finds that NMN and quercetin can promote the relative increase of SIRT1 protein in NK cells and have no safety impact on human NK cells, which helps to delay the senescence of human NK cells, can provide support for the development of anti-aging cell drugs, and has good prospects and application value. Description of the Drawings

[0023] Figure 1 For the preparation and identification of human peripheral blood NK cells;

[0024] Figure 2 For the effect of different concentrations of NMN on SIRT1 in human NK cells;

[0025] Figure 3 For the effect of different concentrations of quercetin on SIRT1 in human NK cells;

[0026] Figure 4 For the effect of different concentrations of NMN and quercetin on SIRT1 in human NK cells;

[0027] Figure 5 Effects of NMN and quercetin at different time points on SIRT1 in human NK cells

[0028] Figure 6 Effects of NMN and quercetin on SIRT1 in human NK cells with different densities

[0029] Figure 7 Effects of NMN and quercetin on the proliferation of human NK cells

[0030] Figure 8 Effects of NMN and quercetin treatments on the tumorigenicity of human NK cells in vitro

[0031] Figure 9 Effects of NMN and quercetin treatments on the karyotype of human NK cells Specific embodiments

[0032] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.

[0033] 1. Preparation of human peripheral blood NK cells

[0034] 1.1 Preparation of peripheral blood mononuclear cells

[0035] Collect 120 mL of human peripheral blood according to the "Standard Operating Procedure for Blood Collection". Wipe the anticoagulant blood collection tube or blood collection bag containing the whole blood sample with a dust-free cloth sprayed with 75% alcohol, and then place it in the transfer window (do not turn on the ultraviolet lamp).

[0036] Divide the peripheral blood equally into 50 mL centrifuge tubes, centrifuge at 800 g for 15 min at room temperature, transfer the upper plasma to a new 50 mL centrifuge tube, inactivate the plasma in a water bath at 56 °C for 30 min, centrifuge the inactivated plasma at 1000 g for 10 min, take the supernatant, place it at -20 °C for 15 min, centrifuge again at 1000 g for 10 min, and take the supernatant and store it at 4 °C.

[0037] The lower red blood cell layer after separating the plasma from the whole blood is used to separate peripheral blood mononuclear cells. First, dilute it 1:1 with physiological saline and mix well. Take another 2 50 mL centrifuge tubes, and slowly add the diluted blood to the upper layer of lymphocyte separation medium according to a 1:1 ratio based on the volume of the diluted blood, and centrifuge at 800 g for 30 min at room temperature. After centrifugation, gently aspirate the mononuclear cell layer, add an equal volume of physiological saline, and wash 2 times for later use.

[0038] 1.2 Enrichment of human peripheral blood NK cells

[0039] Use the human CD3+ cell sorting kit (ImunoSepTM Human CD3+ Cell Positive Selection Kit) and the human NK cell negative sorting kit (IPHASE Human NK Cells Isolation Kit) to highly enrich human peripheral blood NK cells.

[0040] 1. Three-flask culture

[0041] Prepare one T75 culture flask (TC), add 9 mL of PBS (20 mM pH 7.0) and anti-h CD16 mAb (final concentration: 15 μg / mL), spread evenly and mix well, and incubate overnight at 4°C.

[0042] Seed the flask at a density of 1.5×10 6 cells / mL in NK cell medium, replenish the medium every 3 days, and activate and amplify a large number of NK cells. Culture at 37°C and 5% CO2 for 10 - 20 days to make the purity ≥ 80%, and then treat with nicotinamide mononucleotide (NMN) and quercetin. It should be noted here that treatment with nicotinamide mononucleotide (NMN) and quercetin means adding nicotinamide mononucleotide (NMN) and quercetin to the NK cell medium after adjusting the NK cell concentration.

[0043] Among them, the NK cell medium is: serum-free X-VIVO-15 medium, 1000 IU / mL cytokine rhIL-2, 10 ng / mL cytokine IL-15, 30 ng / mL cytokine IL-18, and 10% autologous plasma.

[0044] 2. The anti-aging effect of NMN and quercetin on human NK cells

[0045] 2.1 Detection of SIRT1

[0046] Detect using a kit. The kit uses a double-antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). Add specimens, standards, and HRP-labeled detection antibodies to the pre-coated microwells with silent information regulator 1 (SIRT1) antibodies in sequence, incubate, and wash thoroughly. Color with the substrate TMB. TMB is converted into blue under the catalysis of peroxidase and into the final yellow under the action of acid. The color depth is positively correlated with silent information regulator 1 (SIRT1) in the sample. Measure the absorbance (OD value) with an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm and calculate the sample concentration.

[0047] 2.1.1 Sample treatment

[0048] a. Take 1 tube of NK cell suspension and centrifuge it. The centrifugation parameters are as follows: 4°C, 3000 rpm / min, 10 min, 0 for ascending speed and 0 for descending speed. After centrifugation, discard the supernatant.

[0049] b. Washing

[0050] Resuspend the NK cells collected above with 1 mL of pre-cooled PBS, transfer to a 2 mL EP tube, mix well, and centrifuge at 4°C, 3000 rpm / min, 5 min, 0% speed increase and 0% speed decrease. After centrifugation, discard the supernatant. Wash 2: Resuspend the cells in Wash 1 with 1 mL of pre-cooled PBS, mix well, take 20 μL of cell suspension for counting, and centrifuge the remaining suspension at 4°C, 3000 rpm / min, 5 min, 0% speed increase and 0% speed decrease. After centrifugation, discard the supernatant.

[0051] c. Preparation of lysis working solution

[0052] RIPA high-efficiency lysis buffer: PMSF = 100:1 ratio, add 100 μL lysis working solution (lysis concentration is 10 7 cells / mL);

[0053] d. Lyse cells

[0054] Add 100 μL of lysis working solution to the EP tube and place it on ice for 30 min. During this period, vortex vigorously for 10 s every 10 min to completely lyse the cells.

[0055] e. After the cells were lysed, centrifuged at 4°C, 12,000 rpm / min for 5 min, with an acceleration of 0 and a deceleration of 0. After centrifugation, the supernatant was collected into another labeled 2 mL EP tube for detection of SIRT1 content.

[0056] 2.1.2 Detection Operation

[0057] a. Take out the required strips of microwell ELISA plate from the aluminum foil bag, seal the remaining strips in a ziplock bag and store them at 4℃;

[0058] b. Set up 5 standard wells, 1 zero value well, 2 sample wells and 2 sample spike wells. Use a pipette to add 50 μL of standard to each of the 5 standard wells, with concentrations of 2.5, 5, 10, 20, and 40 ng / mL respectively; do not add anything to the zero value well; first add 10 μL of the sample to be tested to the sample well, then add 40 μL of sample diluent; add 10 μL of the sample to be tested to the sample spike well, then add 10 μL of standard (40 ng / mL) and 30 μL of sample diluent;

[0059] c. Except for the zero-value wells, add 100 μL of the detection antibody labeled with horseradish peroxidase (HRP) to each of the standard wells, sample wells, and sample spiked wells using a pipette. Seal the reaction wells with a sealing film, and place the microplate in an incubator at 37 ± 2 °C for incubation for 60 ± 5 min. Make sure the reaction wells do not dry out completely;

[0060] d. After incubation, take out the microplate, remove the sealing film, shake off the liquid in the reaction wells, lay absorbent paper on the laboratory bench, and invert the microplate on the absorbent paper to pat it dry. Add 350 μL of the diluted washing buffer to each well using an 8-channel pipette, let it stand for 1 min, shake off the washing solution, and invert the microplate on the absorbent paper to pat it dry. Repeat the washing step 5 times;

[0061] e. Pipette 600 μL of substrate A and 600 μL of substrate B into a 2.0 mL EP tube, mix well to obtain the mixed chromogenic solution. Add 100 μL of the mixed chromogenic solution to each well using a pipette, seal the reaction wells with a sealing film, gently shake the microplate to mix evenly, wrap the microplate with aluminum foil, and place it in a 37 °C microplate constant temperature oscillator for incubation in the dark for 15 min. After incubation, remove the aluminum foil and the sealing film, add 50 μL of the stop solution to each well using a pipette, and gently shake the microplate to mix evenly;

[0062] f. Within 15 min, use the Antu biological microplate reader to detect the OD values of the reaction wells. Make sure there are no bubbles in the wells before detection. If there are bubbles, eliminate them first to prevent excessive errors in the measured OD values. Calibrate with the zero-value wells using the Antu instrument control and analysis system (AUTOSOFT software), and measure the absorbance values (OD values) of each well at a wavelength of 450 nm.

[0063] g. Judgment of experimental results

[0064] Using the OD values of the measured standards as the abscissa and the concentration values of the standards as the ordinate, and using computer software, perform four-parameter Logistic curve fitting to create a standard curve;

[0065] When the R2 of the standard curve satisfies > 0.990 and the coefficient of variation of the replicate wells < 15%, this test is valid; otherwise, it is invalid and needs to be retested.

[0066] Substitute the average OD value of the sample into the equation to calculate the concentration of the sample to be measured. When the sample concentration is within the linear range and the spike recovery rate satisfies 80% - 120%, this test is valid; otherwise, it is invalid and needs to be retested.

[0067] Measure the SIRT1 protein concentrations (ng / mL) of the control group and the experimental group, and then, taking the SIRT1 protein content of the control group as 100%, calculate the SIRT1 protein content (relative content) of the experimental group. The calculation formula is as follows:

[0068]

[0069] 2.2 Effects of NMN and quercetin at different concentrations on the senescence of human NK cells

[0070] a. Five groups of experiments were designed, with 1 control group. Using the NMN concentration of 2.5 μM / ml as 1X, the cells were cultured for 72 hours (h), as shown in Table 1 below.

[0071] Table 1 Effects of NMN at different concentrations on SIRT1 in NK cells

[0072]

[0073] b. Five groups of experiments were designed, with 1 control group. Using the quercetin concentration of 50.0 nM / mL as 1X, the cells were cultured for 72 hours (h), as shown in Table 2 below.

[0074] Table 2 Effects of quercetin at different concentrations on SIRT1 in NK cells

[0075]

[0076] c. Five groups of experiments were designed, with 1 control group. Using the NMN concentration of 2.5 μM / mL and the quercetin concentration of 50.0 nM / mL as 1X, the cells were cultured for 72 hours (h), as shown in Table 3 below.

[0077] Table 3 Effects of NMN and quercetin at different concentrations on SIRT1 in NK cells

[0078]

[0079] Taking the SIRT1 protein content in the blank control group without adding NMN and quercetin as 100%, calculate the SIRT1 protein content (relative content) in the experimental groups:

[0080]

[0081] 2.3 Effects of NMN and quercetin on SIRT1 in human NK cells at different action times

[0082] Three groups of experiments were designed, with 3 control groups, as shown in Table 4 below. Taking the SIRT1 protein content in the blank control group as 100%, the cells were centrifuged to collect the cells, lysed to collect the supernatant, and stored in a refrigerator at 4°C for measuring the intracellular SIRT1 content.

[0083] Table 4 Effects of different action times on SIRT1 in NK cells

[0084]

[0085] Note: 0.25X in Table 4 represents: 0.625 μM / mL NMN and 12.5 nM / mL quercetin.

[0086] Effect of 2.4NMN and Quercetin at Different Concentrations on SIRT1 in Human NK Cells

[0087] Design 10 groups of experiments and 10 groups of controls. Taking the SIRT1 protein content of the blank control group as 100%, as shown in Table 5 below, centrifuge to collect cells, lyse to collect the supernatant, and store it in a 4°C refrigerator for measuring the intracellular SIRT1 content.

[0088] Table 5 Effect of Different NK Cell Densities on SIRT1 in NK Cells

[0089]

[0090] Note: 0.25X in Table 5 represents: 0.625 μM / mL NMN and 12.5 nM / mL quercetin.

[0091] 2.5 Determination of NK Cell Purity

[0092] Use flow cytometry to detect the purity of NK cells.

[0093] 3. Effect of NMN and Quercetin on the Toxicity of Human NK Cells

[0094] 3.1 Effect of NMN and Quercetin on the Proliferation of Human NK Cells

[0095] Design a blank control group and experimental groups supplemented with NMN and quercetin. The initial cell amount is 1 billion / L / bag. Detect the cell proliferation at time points of 1 day (1d), 3 days (3d), 5 days (5d), and 7 days (7d) respectively.

[0096] Use trypan blue staining to detect the cell viability.

[0097] 3.2 Effect of NMN and Quercetin on the Tumorigenicity of Human NK Cells

[0098] Use the soft agar cloning method to detect the tumorigenicity of human NK cells treated with NMN and quercetin.

[0099] 3.3 Effect of NMN and Quercetin on the Karyotype of Human NK Cells

[0100] Use G-banding chromosome karyotype analysis to detect the karyotype changes of human NK cells treated with NMN and quercetin.

[0101] 4. Results

[0102] From Figure 1 it can be seen that the prepared NK cells are CD3 - CD56 + / CD16 +≥80%, and the content of SIRT1 in NK cells was measured to be 2.10 - 15.08 ng / 10 7 NK cells at days 10 - 20 (d), and the number of NK cells was sufficient at this time. Subsequently, NMN and quercetin were added to the prepared high-purity human NK cells, and single-factor experiments were designed to investigate the effects of NMN and quercetin concentrations, action time, and cell density on SIRT1 in human NK cells.

[0103] It can be seen from Figures 4 - 6 that when the NK cell density was 1×10 6 / mL, NMN was added to a final concentration of 0.625 μM / mL, quercetin was added to a final concentration of 12.5 nM / ml, and the action time was 72 h, the increment of SIRT1 in NK cells was the highest, reaching 125%.

[0104] To further investigate the effects of NMN and quercetin treatments on the safety of NK cells, the effects of NMN and quercetin on the proliferation toxicity, tumorigenicity, and karyotype of NK cells were detected respectively. As Figures 7 - 9 shown, it can be seen that NMN and quercetin did not affect the proliferation of NK cells, did not affect the karyotype stability of NK cells, and no tumorigenicity was detected.

[0105] In summary, in the present invention, human peripheral blood or cord blood NK cells were treated with an NK cell culture medium containing NMN and quercetin, and it was found that NMN and quercetin can promote the relative increase of SIRT1 protein in NK cells and have no safety impact on human NK cells. This helps to delay the aging of human NK cells, provides support for the development of anti-aging cell drugs, and has good prospects and application value.

Claims

1. A method for delaying the aging of human natural killer cells, characterized in that: The following steps are involved: S1. Human NK cells enriched from human peripheral blood or umbilical cord blood are cultured and expanded in NK cell culture medium; S2. adjusting the NK cell concentration, adding nicotinamide mononucleotide and quercetin to the NK cell culture medium, and continuing the culture; S3. Collect NK cells and prepare NK cell preparations.

2. The method for delaying senescence of human natural killer cells according to claim 1, characterized in that: The formula of the NK cell culture medium is: wherein, the formula of the NK cell culture medium is: serum-free X-VIVO-15 culture medium, cytokine rhIL-2, cytokine IL-15, cytokine IL-18 and autologous plasma.

3. The method for delaying the aging of human natural killer cells according to claim 1, characterized in that: The formula of the NK cell culture medium is: serum-free X-VIVO-15 culture medium, 1000 IU / mL cytokine rhIL-2, 10 ng / mL cytokine IL-15, 30 ng / ml cytokine IL-18 and 10% autologous plasma.

4. The method for delaying the aging of human natural killer cells according to claim 1, characterized in that: In step S1, the NK cell seed bottle concentration is 1.5×10 6 / mL, culture conditions: 37℃, 5% CO2, culture medium supplemented every 3 days, cultured for 10-20 days.

5. The method for delaying the aging of human natural killer cells according to claim 1, characterized in that: In step S2, the final concentrations of nicotinamide mononucleotide and quercetin in the NK cell culture medium are 0.625 μM / mL and 12.5 nM / mL, respectively.

6. The method for delaying senescence of human natural killer cells according to claim 1, characterized in that: In step S2, the concentration of NK cells was adjusted to 1.0×10 6 / mL, the culture conditions were: 37℃, 5% CO2, and the culture time was continued for 72h.

7. Use of NMN and quercetin in delaying the aging of human natural killer cells.

8. The use according to claim 7, characterized in that Application of nicotinamide mononucleotide and quercetin in delaying NK cell aging, promoting NK cell SIRT1 protein expression, and inhibiting SIRT1 protein reduction.

Images