Culture solution for culturing NK (Natural Killer) cells and technology for amplifying NK cells in vitro

Abstract

The invention provides a culture solution for culturing NK (Natural Killer) cells and a technology for amplifying the NK cells in vitro, and belongs to the technical field of immunobiology. The culture solution comprises an NK cell culture solution, namely an RPMI-1640 culture solution, fetal calf serum, L-glutamine, IL-2 and a polypeptide composition, namely polypeptide CT-K3K7NK cell promoting peptide. When the culture solution is used for culturing the NK cells obtained from the elderly individuals, the multiplication capacity, the cell killing activity and the cell factor IFN-gamma secretion capacity of the NK cells can be remarkably improved. Therefore, the invention provides a new scheme and thought for solving the problem of insufficient proliferation and killing activity of the NK cells of the elderly individuals in the autoculture process, and has a wide application prospect in the field of senescence-related immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of immunobiological technology, and in particular to a culture medium for culturing NK cells and a technology for amplifying NK cells in vitro. Background Art

[0002] Natural killer cells (NK cells) are important innate immune cells that have the ability to recognize and kill tumor cells, virus-infected cells, and other abnormal cells. NK cells recognize target cells through their surface receptors and release cytotoxic molecules (such as perforin) and cytokines (such as interferon-γ) to perform immune clearance functions. Unlike adaptive immune cells (such as T cells), NK cells do not rely on antigen presentation, can respond quickly and perform immune surveillance functions, and therefore play a vital role in antiviral, anti-tumor and immune responses.

[0003] Due to its immune effect and tumor immune surveillance function, NK cells have also become a research hotspot in immunotherapy, especially in autologous cell therapy. As part of the treatment method, NK cells are gradually being used in clinical practice. Autologous NK cell therapy separates NK cells from the patient's peripheral blood, expands and activates them in vitro, and then returns them to the patient's body. The expanded high-function NK cells are used to enhance the patient's immune defense ability and treatment effect.

[0004] The core of autologous NK cell therapy lies in the in vitro expansion and activation of a sufficient number of highly functional NK cells. However, in actual operation, especially for elderly patients, this treatment method faces many challenges: First, the proliferation capacity of NK cells isolated from the peripheral blood of elderly individuals is significantly lower than that of young patients when cultured in vitro. This makes it difficult to expand a sufficient number of NK cells within a limited time. Second, the functionality of NK cells in elderly individuals is significantly reduced, including the reduction of cytokine secretion capacity and cytotoxicity. This means that even if the number of expanded cells reaches the requirement, the therapeutic effect is often not as expected.

[0005] Therefore, in order to address the problems of insufficient NK cell proliferation and decreased function in elderly individuals, designing a specially adapted proliferation culture medium has become a key technical problem that needs to be solved urgently. Summary of the invention

[0006] The purpose of the present invention is to provide a culture medium for culturing NK cells and a technology for in vitro amplification of NK cells, so as to achieve in vitro rapid amplification and enhancement of the cytotoxic activity of NK cells obtained from elderly individuals.

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

[0008] First, the present invention provides a culture solution for promoting the proliferation and cell killing activity of senescent NK cells, wherein the culture solution comprises a common NK cell culture solution and a polypeptide composition;

[0009] The polypeptide composition consists of 10-40 μg / mL polypeptide CT-K3K7 and 10-40 μg / mL NK cell promoting peptide;

[0010] The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD;

[0011] The senescent NK cells are naturally senescent NK cells obtained from elderly individuals or induced senescent NK cells obtained by induced aging.

[0012] Preferably, the common NK cell culture medium consists of RPMI-1640 culture medium, 10% fetal bovine serum, 2mM L-glutamine and 200IU / mL IL-2.

[0013] Preferably, the polypeptide composition consists of 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide.

[0014] Preferably, the cell killing activity is the ability to kill human chronic myeloid leukemia cells, and the improvement of the cell killing activity is achieved by increasing the secretion of IFN-γ.

[0015] Secondly, the present invention provides an application of a polypeptide composition in preparing a culture fluid for promoting proliferation and cell killing activity of senescent NK cells, wherein the polypeptide composition is composed of 10-40 μg / mL polypeptide CT-K3K7 and 10-40 μg / mL NK cell promoting peptide;

[0016] The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD.

[0017] Preferably, the polypeptide composition consists of 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide.

[0018] Preferably, the senescent NK cells are naturally senescent NK cells obtained from elderly individuals or induced senescent NK cells obtained by induced senescence;

[0019] The cell killing activity is the ability to kill human chronic myeloid leukemia cells.

[0020] Preferably, the elderly individual is an individual older than 60 years old.

[0021] Secondly, the present invention provides a culture technology for in vitro expansion of senescent NK cells, characterized in that the culture technology comprises the following steps:

[0022] (1) Anticoagulated blood was collected from the peripheral veins of elderly individuals, and after dilution, centrifugation, separation of the buffy coat layer, washing, and lysis of red blood cells, NK cells were isolated using magnetic bead sorting;

[0023] (2) The NK cells were cultured in senescent NK cell culture medium at 37°C and 5% CO2 with an initial culture density of 1×10 6 cells / mL;

[0024] (3) After 5-14 days of culture, the cells are collected for subsequent applications;

[0025] The senescent NK cell culture medium comprises RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2200IU / mL, 10-40μg / mL polypeptide CT-K3K7 and 10-40μg / mL NK cell promoting peptide;

[0026] The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD.

[0027] Preferably, the senescent NK cells are naturally senescent NK cells obtained from elderly individuals.

[0028] Preferably, the senescent NK cell culture medium comprises RPMI-1640 culture medium, 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide.

[0029] Secondly, the present invention provides an application of a polypeptide composition in the preparation of a culture fluid for promoting the IFN-γ secretion ability of senescent NK cells, wherein the polypeptide composition is composed of 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide;

[0030] The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD;

[0031] The senescent NK cells are naturally senescent NK cells obtained from elderly individuals.

[0032] The beneficial effects of the present invention are:

[0033] 1. Significantly improve the proliferation capacity of NK cells in elderly individuals: The culture fluid of the present invention can significantly improve the proliferation capacity of NK cells in elderly individuals by adding polypeptide CT-K3K7 and NK cell promoting peptide in a specific ratio compared with ordinary NK cell culture fluid. This means that more NK cells can be expanded within a limited time, providing a sufficient cell source for autologous NK cell therapy, and solving the problem of insufficient NK cell proliferation capacity in the peripheral blood of elderly patients.

[0034] 2. Effectively enhance the cytotoxic activity of NK cells in elderly individuals: The basal cytotoxic activity of NK cells obtained from elderly individuals is relatively weak. The culture medium provided by the present invention greatly enhances the cytotoxic activity of elderly NK cells against abnormal cells such as tumor cells through the synergistic effect of CT-K3K7 and NK cell-promoting peptide, thereby enhancing the efficacy of autologous NK cell therapy in anti-tumor and other aspects.

[0035] 3. Promote the secretion of cytokines by NK cells in elderly individuals: The culture medium of the present invention can effectively promote the secretion of IFN-γ by NK cells, thereby enhancing the role of NK cells in antiviral and immune response, and improving the overall immunotherapy effect.

[0036] 4. Restoration effect on senescent NK cells: In the senescent NK cell model experiment, the culture fluid of the present invention also showed good effects. Compared with the senescent model group, the proliferation ability and killing activity of senescent NK cells cultured with the culture fluid were significantly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The difference in absorbance of NK cells treated for different intervention groups;

[0038] Figure 2 The difference in killing rate of NK cells treated for different intervention groups;

[0039] Figure 3 The difference in IFN-γ concentration in the supernatant of co-culture of effector and target cells;

[0040] Figure 4 Effects of the NK cell culture fluid prepared by the present invention on the proliferation ability (a) and killing activity (b) of senescent NK cells. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0042] The polypeptide CT-K3K7 involved in the present invention is an optimized scorpion venom polypeptide, and its amino acid sequence is FLKFLKKLF-NH2; the NK cell promoting peptide is a polypeptide for promoting the growth of NK cells involved in the invention patent application number: 202010624330.3, and its amino acid sequence is KGNPDTDHQVFWMWMLGEAAYD, which is named NK cell promoting peptide in the present invention. The above-mentioned polypeptide is synthesized by Shanghai Jier Polypeptide Co., Ltd.

[0043] Example 1

[0044] Obtaining NK cells from elderly individuals

[0045] (1) 20 mL of anticoagulated blood was collected from the peripheral vein of elderly individuals (average age 63 years old), diluted once with PBS, and stored in a 4°C refrigerator;

[0046] (2) equilibrate the lymphocyte separation solution to room temperature for later use;

[0047] (3) Mix the diluted peripheral blood and lymphocyte separation solution in a ratio of 2:1 and transfer them into a 50 mL centrifuge tube;

[0048] (4) Place a 50 mL centrifuge tube in a low-temperature high-speed centrifuge and centrifuge at 400 g, 20°C for 20 min, using a centrifugation mode with an increase speed of 1 and a decrease speed of 1;

[0049] (5) After centrifugation, gently take out the sample, discard the upper plasma under sterile conditions, gently aspirate the buffy coat layer and place it in PBS containing 2% fetal bovine serum, and centrifuge at 1200 rpm for 5 min;

[0050] (6) After centrifugation, discard the supernatant and wash twice with PBS containing 2% fetal bovine serum;

[0051] (7) After centrifugation, the supernatant was discarded, and red blood cell lysis buffer was added to lyse the mixture on ice for 8 minutes. After lysis, PBS was added to dilute the red blood cell lysis buffer, and the mixture was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded;

[0052] (8) PBMCs were resuspended in culture medium and NK cells were isolated using magnetic bead sorting for subsequent experiments.

[0053] Example 2

[0054] Prepare different types of NK cell culture medium

[0055] NK cell culture medium 1: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL;

[0056] NK cell culture medium 2: RPMI-1640 culture medium, 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, 50 μg / mL peptide CT-K3K7;

[0057] NK cell culture medium 3: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 50μg / mL NK cell promoting peptide;

[0058] NK cell culture medium 4: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 40μg / mL peptide CT-K3K7, 10μg / mL NK cell promoting peptide;

[0059] NK cell culture medium 5: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 30μg / mL peptide CT-K3K7, 20μg / mL NK cell promoting peptide;

[0060] NK cell culture medium 6: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 25μg / mL peptide CT-K3K7, 25μg / mL NK cell promoting peptide;

[0061] NK cell culture medium 7: RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 20μg / mL peptide CT-K3K7, 30μg / mL NK cell promoting peptide;

[0062] NK cell culture medium 8: RPMI-1640 culture medium, 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, 10 μg / mL polypeptide CT-K3K7, 40 μg / mL NK cell promoting peptide.

[0063] Example 3

[0064] Detection of the effect of the culture medium prepared in Example 2 on the proliferation ability of NK cells in elderly individuals

[0065] (1) After culturing the NK cells obtained in Example 1 for 5 days, the cells were equally divided into 8 groups (intervention groups 1-8), and the NK cell culture medium prepared in Example 2 was used for culturing:

[0066] The initial culture density is: 1×10 6 cells / mL, culture conditions were 37°C, 5% CO2;

[0067] (2) After 5 days of culture, cells were collected by centrifugation. Cell suspensions were prepared using NK cell culture medium 1 in intervention groups 1-8 and added to 96-well cell culture plates. 100 μL (1×10 4 cells), with 3 replicate wells in each group;

[0068] (3) After culturing in a 37°C, 5% CO2 incubator for 3 days, remove the culture plate, add 10 μL of CCK-8 reagent to each well, and mix gently;

[0069] (4) Incubate the cells in a cell culture incubator for 2 h, and then read the absorbance of each well at 450 nm using an ELISA reader.

[0070] Table 1 Differences in absorbance of NK cells treated with different intervention groups

[0071]

[0072]

[0073] From the results in Table 1, it can be seen that the absorbance values ​​of intervention groups 2 to 8 were significantly higher than those of intervention group 1, indicating that compared with the ordinary NK cell culture medium (intervention group 1), the intervention groups (intervention groups 2 to 8) with the addition of peptide CT-K3K7 and NK cell promoting peptide in the culture medium can significantly improve the proliferation capacity of NK cells. These results show that the culture medium with the addition of these two factors has a significant promoting effect on promoting NK cell proliferation.

[0074] Further analysis showed that compared with the addition of peptide CT-K3K7 alone (intervention group 2) or NK cell-promoting peptide alone (intervention group 3), the effect of combined use of peptide CT-K3K7 and NK cell-promoting peptide was improved in intervention groups 4 to 8. This result shows that the combined use of the two can promote the proliferation of NK cells more effectively than when used alone, suggesting that the combined effect of peptide CT-K3K7 and NK cell-promoting peptide has a stronger synergistic effect on enhancing the proliferation capacity of NK cells in elderly individuals.

[0075] Further observation of the data showed that the absorbance value of intervention group 7 was significantly higher than that of all other intervention groups, especially compared with intervention groups 2 and 3. The proliferation effect of intervention group 7 was particularly obvious. The absorbance value of intervention group 7 reached 0.864±0.045, which was significantly higher than that of other intervention groups. This shows that the combination ratio of peptide CT-K3K7 and NK cell promoting peptide in intervention group 7 may have reached the optimal point, and the synergistic effect of the two played the best effect in this group, greatly promoting the proliferation of NK cells in elderly individuals.

[0076] In summary, intervention group 7 not only showed that the combined use of peptide CT-K3K7 and NK cell-promoting peptide had a significant promoting effect on the proliferation of elderly NK cells, but also the synergistic effect was better than that when used alone, and it may be the best combination to promote the proliferation of elderly NK cells.

[0077] Example 4

[0078] Detection of the difference in killing activity of NK cells cultured in different NK cell culture media

[0079] (1) After culturing the NK cells obtained in Example 1 for 5 days, the cells were evenly divided into 8 groups (intervention groups 1-8), and cultured using the NK cell culture fluids 1-8 prepared in Example 2, respectively:

[0080] The initial culture density is: 1×10 6 cells / mL, culture conditions were 37°C, 5% CO2;

[0081] (2) After 5 days of culture, cells were collected by centrifugation. All intervention groups 1-8 used NK cell culture medium 1 to prepare 1×10 6 cells / mL cell suspension;

[0082] (3) Human chronic myeloid leukemia K562 cells were prepared into 1×10 5 / ml cell suspension, NK cells cultured in different intervention groups were mixed with K562 cells at an effector-target ratio of 1:10;

[0083] (4) The mixed cells were inoculated into a 96-well cell culture plate, and a control target cell natural group (LDH release was 0) and a target cell maximum release group (LHD release was 100%) were set up at the same time;

[0084] (5) Place the cell culture plate in a cell culture incubator at 37°C and 5% CO2 and continue incubating for 4 h;

[0085] (6) After the incubation, the supernatant was collected by centrifugation, and the LDH release amount after treatment in different intervention groups was detected using an LDH detection kit, and the difference in the killing activity of NK cells cultured in each intervention group was calculated.

[0086] Table 2 Differences in killing rate of NK cells treated by different intervention groups

[0087] Experimental Grouping Kill rate / % Intervention Group 1 19.23±1.73 Intervention Group 2 24.59±2.56 Intervention Group 3 26.03±2.91 Intervention Group 4 31.68±2.78 Intervention Group 5 34.43±2.61 Intervention Group 6 35.42±3.01 Intervention Group 7 42.41±4.10 Intervention Group 8 32.34±3.16

[0088] From the results in Table 2, it can be seen that the cell killing rate of intervention group 1 was 19.23±1.73, showing a lower killing activity, indicating that the NK cells obtained from elderly individuals themselves have weak killing activity, which is consistent with the characteristics of the functional decline of elderly NK cells. Therefore, the basic killing activity of NK cells in elderly individuals is subject to certain restrictions, and may play a more limited role in the immune response.

[0089] Compared with intervention group 1, the killing rates of intervention groups 2 and 3 were both improved, 24.59±2.56 and 26.03±2.91, respectively, indicating that the culture medium with the addition of CT-K3K7 or NK cell-promoting peptide can enhance the killing activity of NK cells to a certain extent. This shows that when these two components are used alone, they can have a positive effect on the function of elderly NK cells and enhance their anti-tumor ability, but the overall improvement is very limited and the improvement effect is poor.

[0090] Further analysis of the results of intervention groups 4 to 8 showed that, similar to the results of the cell proliferation experiment, when the total amount of addition was the same, the effect of the combination of CT-K3K7 and NK cell-promoting peptide was significantly better than that of either alone, indicating that the synergistic effect of these two factors played a key role in enhancing the killing activity of NK cells.

[0091] It is worth noting that the killing rate of intervention group 7 was 42.41±4.10, which was significantly better than all other intervention groups, further proving that the combination ratio of CT-K3K7 and NK cell-promoting peptide in intervention group 7 reached the optimal point. At this ratio, the synergistic effect of the two was maximized, greatly promoting the killing activity of elderly NK cells.

[0092] Example 5

[0093] Detection of the difference in IFN-γ concentration in the supernatant of co-culture of effector and target cells

[0094] (1) The cells were treated according to the steps of Example 4. After incubation for 4 hours, the culture medium was collected and centrifuged to remove NK cells and dead cells to obtain the effector-target cell co-culture supernatant;

[0095] (2) According to the instructions of the ELISA kit, the content of IFN-γ in the supernatant of the co-culture of effector and target cells was detected.

[0096] Table 3 Differences in IFN-γ concentrations in the supernatant of co-culture of effector and target cells

[0097]

[0098]

[0099] From the results in Table 3, it can be seen that the results of IFN-γ concentration are basically the same as the trend of the results of NK cell killing rate detection. This result further illustrates that the combination of CT-K3K7 and NK cell promoting peptide can effectively promote NK cells to secrete IFN-γ, thereby improving the killing activity of NK cells obtained in elderly individuals.

[0100] Example 6

[0101] Detecting the effect of the NK cell culture medium prepared by the present invention on the proliferation ability of senescent NK cells

[0102] (1) The NK92 cells at generation P3 were divided into different centrifuge tubes, centrifuged to obtain cell pellets, and the cells were treated according to the following groups:

[0103] Control group: cell suspension was prepared using RPMI-1640 culture medium containing 10% FBS, 2 mM L-glutamine and IL-2 200 IU / mL;

[0104] Aging model group: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, and 30 mmol / L glucose;

[0105] Intervention group 1: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 30mmol / L glucose, and 20μg / mL peptide CT-K3K7;

[0106] Intervention group 2: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, 30 mmol / L glucose, and 30 μg / mL NK cell-promoting peptide;

[0107] Intervention group 3: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 30mmol / L glucose, 20μg / mL peptide CT-K3K7, and 30μg / mL NK cell-promoting peptide;

[0108] (2) The cell suspension was inoculated into a 96-well cell culture plate and 100 μL (1×10 4 cells), with 3 replicate wells in each group;

[0109] (3) After culturing in a 37°C, 5% CO2 incubator for 3 days, remove the culture plate, add 10 μL of CCK-8 reagent to each well, and mix gently;

[0110] (4) Incubate the cells in a cell culture incubator for 2 h, and then read the absorbance of each well at 450 nm using an ELISA reader.

[0111] Table 4 Effect of the NK cell culture medium prepared by the present invention on the proliferation ability of senescent NK cells

[0112] Experimental Grouping Absorbance value Control group 0.808±0.031 Aging model group 0.430±0.039 Intervention Group 1 0.519±0.039 Intervention Group 2 0.557±0.043 Intervention Group 3 0.752±0.041

[0113] From the results in Table 4, it can be seen that compared with the control group, the absorbance of the aging model group induced by high sugar was significantly decreased, indicating that the model induced aging successfully.

[0114] Compared with the aging model group, the absorbance of intervention group 1 increased by 20.70%, the absorbance of intervention group 2 increased by 29.53%, and the absorbance of intervention group 3 was 74.88%;

[0115] The q value of the combined drug effect was calculated using King's formula: q = E (A + B) / (EA + EB - EA EB):

[0116] q value = 0.7488 / 0.2953+0.2070-(0.2953×0.2070)≈1.70.

[0117] It can be seen that the q value when the polypeptide CT-K3K7 and the NK cell promoting peptide are used in combination is significantly higher than 1.15, indicating that the combination of polypeptide CT-K3K7 and the NK cell promoting peptide for the proliferation of aging NK cells can synergistically promote the proliferation of aging NK cells.

[0118] Example 7

[0119] (1) The NK92 cells at generation P3 were divided into different centrifuge tubes, centrifuged to obtain cell pellets, and the cells were treated according to the following groups:

[0120] Control group: cell suspension was prepared using RPMI-1640 culture medium containing 10% FBS, 2 mM L-glutamine and IL-2 200 IU / mL;

[0121] Aging model group: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, and 30 mmol / L glucose;

[0122] Intervention group 1: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 30mmol / L glucose, and 20μg / mL peptide CT-K3K7;

[0123] Intervention group 2: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2 mM L-glutamine, IL-2 200 IU / mL, 30 mmol / L glucose, and 30 μg / mL NK cell-promoting peptide;

[0124] Intervention group 3: Cell suspension was prepared using RPMI-1640 containing 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 30mmol / L glucose, 20μg / mL peptide CT-K3K7, and 30μg / mL NK cell-promoting peptide;

[0125] (2) Human chronic myeloid leukemia K562 cells were prepared into 1×10 5 / ml cell suspension, and the NK cell suspensions of different treatment groups were mixed with K562 cells at an effector-target ratio of 1:10;

[0126] (3) The mixed cells were inoculated into a 6-well cell culture plate, and a control target cell natural group (LDH release was 0) and a target cell maximum release group (LHD release was 100%) were set up at the same time;

[0127] (4) Place the cell culture plate in a cell culture incubator at 37°C and 5% CO2 and continue incubating for 4 hours;

[0128] (5) After the incubation, the supernatant was collected by centrifugation, and the LDH release amount after treatment in different intervention groups was detected using an LDH detection kit, and the difference in the killing activity of NK cells cultured in each intervention group was calculated.

[0129] Table 5 Effect of the NK cell culture medium prepared by the present invention on the killing activity of senescent NK cells

[0130] Experimental Grouping Kill rate / % Control group 45.04±2.71 Aging model group 20.34±1.98 Intervention Group 1 25.31±2.09 Intervention Group 2 26.83±2.83 Intervention Group 3 39.71±3.26

[0131] According to the results in Table 5, we can observe that the senescent NK cells induced in a high-glucose environment showed a significant decrease in the killing activity against tumor cells. At the same time, it can be seen that compared with the aging model group, the NK cell killing rate of intervention group 1 increased by 24.41%, intervention group 2 increased by 31.91%, and the killing rate of intervention group 3 increased significantly by 95.24%.

[0132] To further analyze these intervention effects, we used King's formula to calculate the q value of combined medication: q = E(A+B) / (EA+EB-EA·EB)

[0133] According to the calculation, the q value is: q value = 0.9524 / 0.2441 + 0.3191-(0.2441 × 0.3191) ≈ 1.96. The calculation results show that the q value of the combined use of peptide CT-K3K7 and NK cell promoting peptide is significantly higher than 1.15, suggesting that these two components have a significant synergistic effect in improving the killing activity of aging NK cells.

[0134] Comprehensive analysis shows that the test results of the aging model are highly consistent with the test results of NK cells in elderly individuals, which further verifies that the addition of peptide CT-K3K7 and NK cell-promoting peptide in the NK cell culture medium in an appropriate ratio can significantly restore the proliferation ability and killing activity of aged NK cells. This discovery provides new ideas and potential treatment options for solving the problem of insufficient proliferation and killing activity of NK cells in elderly individuals during autologous culture.

Claims

1. A culture medium for promoting the proliferation and cell killing activity of senescent NK cells, characterized in that: The culture fluid comprises common NK cell culture fluid and a polypeptide composition; The polypeptide composition consists of 10-40 μg / mL polypeptide CT-K3K7 and 10-40 μg / mL NK cell promoting peptide; The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD; The senescent NK cells are naturally senescent NK cells obtained from elderly individuals or induced senescent NK cells obtained by induced aging.

2. The culture solution according to claim 1, characterized in that The common NK cell culture medium consists of RPMI-1640 culture medium, 10% fetal bovine serum, 2mM L-glutamine and 200IU / mL IL-2.

3. The culture solution according to claim 2, characterized in that The polypeptide composition consists of 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide.

4. The culture solution according to claim 3, characterized in that The cell killing activity is the ability to kill human chronic myeloid leukemia cells; The improvement of the cell killing activity is achieved by increasing the secretion of IFN-γ.

5. Use of a polypeptide composition in preparing a culture medium for promoting proliferation and cell killing activity of senescent NK cells, characterized in that: The polypeptide composition consists of 10-40 μg / mL polypeptide CT-K3K7 and 10-40 μg / mL NK cell promoting peptide; The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD.

6. The use according to claim 5, characterized in that: The polypeptide composition consists of 20 μg / mL polypeptide CT-K3K7 and 30 μg / mL NK cell promoting peptide.

7. The use according to claim 6, characterized in that: The senescent NK cells are naturally senescent NK cells obtained from elderly individuals or induced senescent NK cells obtained by induced senescence; The cell killing activity is the ability to kill human chronic myeloid leukemia cells.

8. A culture technique for in vitro expansion of senescent NK cells, characterized in that: The culture technique comprises the following steps: (1) Anticoagulated blood was collected from the peripheral veins of elderly individuals, and after dilution, centrifugation, separation of the buffy coat layer, washing, and lysis of red blood cells, NK cells were isolated using magnetic bead sorting; (2) The NK cells were cultured using senescent NK cell culture medium at 37°C and 5% CO2, with an initial culture density of 1×10 6 cells / mL; (3) After 5-14 days of culture, the cells are collected for subsequent applications; The senescent NK cell culture medium comprises RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2200IU / mL, 10-40μg / mL polypeptide CT-K3K7 and 10-40μg / mL NK cell promoting peptide; The amino acid sequence of the polypeptide CT-K3K7 is FLKFLKKLF-NH2, and the amino acid sequence of the NK cell promoting peptide is KGNPDTDHQVFWMWMLGEAAYD.

9. The culture technique according to claim 8, characterized in that: The senescent NK cells are naturally senescent NK cells obtained from elderly individuals.

10. The culture technique according to claim 9, characterized in that: The aging NK cell culture medium comprises RPMI-1640 culture medium, 10% FBS, 2mM L-glutamine, IL-2 200IU / mL, 20μg / mL polypeptide CT-K3K7 and 30μg / mL NK cell promoting peptide.

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