Drug combination composition for removing senescent cells and application thereof

Abstract

The invention relates to a drug combination composition for removing senescent cells and application of the drug combination composition. The drug combination composition comprises NK cells and calcium folinate. According to the invention, the NK cells and the calcium folinate are creatively combined. Tests show that compared with a use scheme of combining other medicines with the NK cells, the calcium folinate can remarkably improve the killing effect of the NK cells on senescent cells.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular immunology, and in particular to a combined drug composition for eliminating senescent cells and an application thereof. Background Art

[0002] For a long time, aging was considered inevitable, but with the progress of anti-aging research, anti-aging treatments are increasingly considered feasible. In anti-aging treatments, "senescent cells" are undoubtedly the most eye-catching. Although these senescent cells have stopped dividing and appear to be in a dormant state of growth stagnation, they persist and continue to secrete many pro-inflammatory and tissue remodeling molecules that affect other normal cells around them. Therefore, clearing senescent cells may be an effective means to delay or even reverse aging.

[0003] NK cells, or natural killer cells, are the core cells of the human body's innate immunity. NK cells have dual functions. They can not only play a killing role as immune cells, but also participate in immune responses as immune regulatory cells. Compared with adaptive immune cells, NK cells are more sensitive in anticipating "enemy" invasion and have a broader spectrum of "killing enemy" capabilities. Not only can they kill tumor cells and resist viruses, NK cells can also clear senescent cells, playing an important role in anti-aging. Studies have shown that NK cells can kill and clear senescent immune cells in vivo and in vitro, enhance the body's immunity, and improve the body's sub-health state. Although NK cells have a certain role in anti-aging treatment, the current clinical effect still has room for improvement, and a method to further improve the anti-aging effect of NK cells is urgently needed. Among them, the combined use of NK cells and drugs may be one of the solutions.

[0004] Calcium folinate is a formylated derivative of folic acid, which has similar effects to folic acid and is the activated form of folic acid in the body. After entering the human body, it can effectively inhibit the toxic reactions caused by methotrexate, is beneficial to the synthesis of thymine nucleotides, DNA, RNA and proteins, and can stimulate the growth and maturation of immune cells. In recent years, a large number of experiments and clinical studies at home and abroad have fully proved that calcium folinate can be used as a highly efficient biochemical regulator, which can significantly increase the anti-tumor activity of some drugs (such as strong fluorouracil). However, whether calcium folinate has anti-aging function or whether it can enhance the effect of anti-aging drugs has not been reported. Whether calcium folinate can further improve the ability of NK cells to eliminate senescent cells is still unknown. Therefore, how to prepare a drug combination composition that can effectively kill senescent cells has also become a problem that needs to be solved urgently. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a combined drug composition for removing senescent cells and its application. The present invention creatively combines NK cells and calcium folinate. According to experiments, compared with other drugs combined with NK cells, calcium folinate can significantly enhance the killing effect of NK cells on senescent cells.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a combination pharmaceutical composition for eliminating senescent cells, wherein the combination pharmaceutical composition comprises NK cells and calcium folinate.

[0008] In the present invention, after NK cells and calcium folinate are combined, the killing effect on senescent cells is significantly increased. The two complement each other and can synergistically enhance the effect, which significantly improves the killing effect on senescent cells. It is used in the field of scientific research, such as for the study of the mechanism of cell senescence or for screening more drugs that kill senescent cells.

[0009] Preferably, the concentration of calcium folinate is 25-100 μM, for example, it can be 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM or 100 μM, etc.

[0010] Preferably, the density of the NK cells is (1-4)×10 2 / μL, and the (1-4) can be, for example, 1, 1.5, 2, 2.5, 3, 3.5 or 4, etc.

[0011] In the present invention, the concentrations of NK cells and calcium folinate have a great influence on the killing effect. When the concentrations of NK cells and calcium folinate are outside the above range, the killing effect on senescent cells is low and no significant difference can be obtained.

[0012] Preferably, the NK cells are derived from allogeneic or autologous cells.

[0013] Preferably, the NK cells are derived from primary cultured cells or NK cell lines.

[0014] Preferably, the NK cells are derived from peripheral blood, umbilical cord blood, bone marrow or induced pluripotent stem cells.

[0015] Preferably, the method for obtaining senescent cells includes natural aging, drug induction, ultraviolet induction, ionizing radiation induction, ultrasonic treatment, mechanical damage, overexpression of aging genes, protease inhibitor induction, inhibition of cell metabolic state or induction of cell metabolic stress.

[0016] Preferably, the senescent cells include cells that overexpress senescence genes or naturally senescent cells.

[0017] Preferably, any one or a combination of at least two of HRAS, ATP2C2, BCAS3, CREG1, DYNC1I1, OAS2, OPRL1, PDE11A, PPFIBP2, RAI2, SYT17, TNFSF13B, RRM2, ACSS1, DMC1, HEY1, HTR1B, SLC25A10, ZNF214, C1ORF147, IL1B, SERPINB4, TNFSF8, WTAPP1, FPGT, GRIK2, GUCY1B1, ID2, KIAA0895, NAT1, PBX1, PLA2G12A, ROBO2, PRSS35, SLIT2, P21, P16, P53 or KRAS.

[0018] Preferably, the cell types of the senescent cells include any one of immune cells, red blood cells, nerve cells, muscle cells, epithelial cells, connective tissue cells, germ cells, stem cells, liver cells, kidney cells, lung cells, cardiomyocytes, lymphocytes, intestinal cells, adipocytes, ovarian cells, osteoblasts or fibroblasts, or a combination of at least two of them.

[0019] Preferably, the senescence detection index of the senescent cells includes any one of CD28, CD57, KLRG1, P21, and SA-β-Gal, or a combination of at least two or more thereof.

[0020] In the present invention, a senescent cell model constructed by IMR-90 cells overexpressing the HRAS gene is used for research. After detecting that the senescence-related β-galactosidase is significantly increased during senescence, it can be determined that the senescence cell model is successfully constructed. When cells are senescent, the cell proliferation and differentiation ability and physiological function gradually decline, and the activity of senescence-associated β-galactosidase (SA-βgal) will increase significantly, so the activity of SA-βgal is often detected to distinguish normal cells from senescent cells. SA-βgal can hydrolyze 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (X-gal) at pH 6.0 to produce a water-insoluble blue substance. This blue product is quite stable and can be stored for several months in the dark after being fixed and dried. The amount of blue product of each cell can be detected on a flow cytometer, and the average fluorescence intensity of blue cells in the total population can be calculated to evaluate the degree of senescence of the cell population.

[0021] Preferably, the combined pharmaceutical composition is a single compound preparation, or a combination of two separate preparations of calcium folinate and NK cells.

[0022] Preferably, the calcium folinate and NK cells are administered simultaneously.

[0023] Preferably, the calcium folinate and NK cells are administered sequentially.

[0024] In a second aspect, the present invention provides a drug for eliminating senescent cells, wherein the drug comprises the combined pharmaceutical composition for eliminating senescent cells described in the first aspect.

[0025] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0026] Preferably, the pharmaceutically acceptable excipients include any one or a combination of at least two of a carrier, a diluent, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

[0027] In a third aspect, the present invention provides a method for preparing the combined pharmaceutical composition for eliminating senescent cells according to the first aspect, the preparation method comprising: mixing NK cells with calcium folinate to prepare the composition.

[0028] Preferably, when the NK cells are in the culture stage, calcium folinate is added at any one or more time points and the cells are cultured until the purity of the NK cells reaches more than 90%.

[0029] Preferably, when the purity of the NK cells reaches 90% or more, calcium folinate is added at any one time point or a combination of at least two time points, and cultured until the harvest stage.

[0030] The harvesting stage is when the purity of NK cells reaches 95% or above, for example, it can be 95%, 96%, 97%, 98%, 99% or 100%.

[0031] Preferably, when the NK cells reach the harvesting stage, calcium folinate is added at any one time point or a combination of at least two time points for clearing senescent cells, and cultured until the end of the experiment.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects:

[0033] In the present invention, NK cells and calcium folinate are used alone, both of which have a certain killing effect on senescent cells. After the two are combined, the killing effect is significantly increased. The two complement each other and can synergistically enhance the effect, which significantly improves the killing effect on senescent cells. The concentrations of NK cells and calcium folinate have a certain influence on the killing effect. Too high or too low concentrations will reduce the killing effect on senescent cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The graph shows the results of SA-β-Gal activity levels in cells overexpressing the G12V mutant of HRAS.

[0035] Figure 2 These are graphs showing the killing effects of different treatments on IMR-90-HRAS-GFP cells, where Figure A shows the results of treatment with eight drugs, and Figure B shows the results of NK cell combined drug treatment.

[0036] Figure 3 This is a graph showing the killing effect of NK cells with different effector-target ratios and different concentrations of calcium folinate on IMR-90-HRAS-GFP cells.

[0037] Figure 4 This is an analysis chart of the real-time quantitative living cell imaging results of IMR-90-HRAS-GFP cells treated with NK cells and calcium folinate, where Figure A is a graph showing the change in relative fluorescence area; Figure B is the relative fluorescence area at the last time point in Figure A.

[0038] Figure 5 This is a graph analyzing the results of real-time quantitative live cell imaging of tumor cells SK-OV-3-mcherry treated with NK cells and calcium folinate. Figure A is a graph showing the change in relative fluorescence area; Figure B is the relative fluorescence area at the last time point in Figure A. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0040] Sources of materials used in the following examples:

[0041] IMR-90 cells: purchased from ATCC (American Type Culture Collection, CCL-186)

[0042] HaCaT cells: purchased from ATCC (American Type Culture Collection, PCS-200-011)

[0043] 293T cells: purchased from ATCC (American Type Culture Collection, CRL-3216)

[0044] NK cells: NK cell expansion kit (Cat. No. NE000-N022)

[0045] SK-OV-3 cells: purchased from ATCC (American Type Culture Collection, HTB-77)

[0046] Example 1

[0047] Construction and validation of senescent cell models

[0048] There are many ways to establish senescent cell models, such as radiation, H2O2 stimulation, high sugar stimulation or drug stimulation, but because the state of the cell itself has a great influence on the experimental results, the cell models treated with the same method show different treatment results, and the results vary greatly. The senescent cell model obtained by overexpressing senescent genes can avoid the above problems.

[0049] Among the three human Ras genes (HRAS, KRAS, and NRAS), mutations (such as the G12V mutation) or abnormal activation of the HRAS gene will lead to the continuous activation of the Ras protein, thereby affecting cell proliferation, differentiation, and survival, playing a key role in promoting cell aging, and can be used as a cell model for aging research. Therefore, this study first overexpressed the G12V mutant of HRAS (abbreviated as HRAS) in human embryonic lung fibroblasts IMR-90, human skin keratinocytes HaCaT, and human embryonic kidney cells 293T. G12V ) to obtain a senescent cell model.

[0050] IMR-90, HaCaT and 293T cells were cultured at 1×10 6 The cells were resuspended at a density of 100 / mL and then inoculated in a 6-well plate at a volume of 1 mL per well. G12V The above cells were transduced with a lentiviral vector expressing the green fluorescent protein (GFP) reporter gene to obtain cells overexpressing HRAS G12V and GFP-expressing IMR-90-HRAS-GFP cells, HaCaT-HRAS-GFP cells, and 293T-HRAS-GFP cells.

[0051] Fix the cells with 2% paraformaldehyde for 10 minutes, centrifuge at 600g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Stain the cells with senescence evaluation reagent X-Gal at 37°C for 2 hours to detect changes in senescence marker β-galactosidase (senescence-associated β-galactosidase, SA-β-Gal). Centrifuge at 600g for 5 minutes at room temperature, discard the supernatant, and wash the cells three times with PBS. Resuspend the cells with 200μL PBS solution for flow analysis.

[0052] Compared with normal cells, the activity level of SA-β-Gal in senescent cells is upregulated. Figure 1 As shown, compared with the non-overexpressed HRAS G12V Compared with the control group, the SA-β-Gal activity levels of IMR-90-HRAS-GFP cells, HaCaT-HRAS-GFP cells and 293T-HRAS-GFP cells were significantly upregulated, indicating that IMR-90-HRAS-GFP cells, HaCaT-HRAS-GFP cells and 293T-HRAS-GFP cells were induced to age to a certain extent. Among them, the SA-β-Gal activity level of IMR-90-HRAS-GFP cells was the highest (MFI was 65470), indicating that the cell had a higher level of senescence and could be used as a model for subsequent drug screening.

[0053] Example 2

[0054] Screening for drugs with anti-aging properties

[0055] (1) Detection of the direct killing effects of 8 drugs on senescent cells

[0056] Use culture medium at 1×10 5 IMR-90-HRAS-GFP cells were resuspended at a density of 100 μL / mL and inoculated into 96-well plates at a volume of 100 μL per well for overnight culture. In order to explore the direct killing effects of bumetanide, cyclophosphamide, folinic acid, nepafenac, cetirizine, alvimopan, melatonin and colchicine on senescent cells, the above drugs were added to a concentration of 50 μM. The culture plates were then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0057] The mortality rate of IMR-90-HRAS-GFP cells was detected by the lactate dehydrogenase method. In the maximum release well control group without drug treatment, 20 μL of cell lysate was added, and the mixture was mixed by repeated pipetting several times, and the culture was continued to be incubated at 37°C and 5% CO2 for 30 minutes. The cell culture plate was removed and centrifuged at 500g for 5 minutes using a multi-well plate centrifuge. 50 μL of the supernatant from each well was taken and transferred to a new 96-well plate. 50 μL of lactate dehydrogenase detection working solution was added to each well and mixed thoroughly, and incubated at 25°C in the dark for 30 minutes. Then, the absorbance was measured at 490 nm using a multifunctional microplate reader. Calculate the cell mortality rate (%) = (sample group - culture medium control group) / (maximum release well control group - culture medium control group) × 100%. The results are as follows: Figure 2 As shown in A, there was no statistical difference in the killing rate of the eight drugs compared with the control group without drug addition (P>0.05), indicating that the eight drugs had no direct killing effect on senescent cells.

[0058] (2) Detection of 8 drugs to enhance the effect of NK cells in killing senescent cells

[0059] Use culture medium at 1×10 5 IMR-90-HRAS-GFP cells were resuspended at a density of 100 μL / mL and inoculated into a 96-well plate at a volume of 100 μL per well for overnight culture. Harvest-stage NK cells, i.e., purity of 95% or more, were added to each well at an effector-target ratio of 2:1, and the above drugs were added to a concentration of 50 μM. The culture plate was then placed in a 37°C, 5% CO2 incubator for overnight culture.

[0060] The mortality rate of IMR-90-HRAS-GFP cells was detected by lactate dehydrogenase method. Figure 2 As shown in B. Compared with the group treated with only NK cells, the target cell death rate in the calcium folinate and NK cell combination group was significantly increased (P<0.05). In the other 7 drug combined with NK cell groups, the target cell death rate did not increase significantly (P>0.05). This result shows that calcium folinate can enhance the killing effect of NK cells on senescent cells, and this combination drug regimen has a better anti-aging effect than the NK cell regimen alone.

[0061] Example 3

[0062] Screening of NK cells and effective concentration of calcium folinate

[0063] Use culture medium at 1×10 5 IMR-90-HRAS cells were resuspended at a density of 1×10 / mL and inoculated in a 96-well plate at a volume of 100 μL per well. NK cells were inoculated at different effector-target ratios (2:1, 1:1, and 0:1) (i.e., the density of IMR-90-HRAS cells was 1×104 The concentration of NK cells was 2×10 4 pcs / hole, 1×10 4 100 μM, 50 μM, 25 μM, 5 μM and 0 μM were added to each effector-target ratio group. A maximum enzyme activity control group and a culture medium control group were set up at the same time. Finally, the culture plate was placed in a 37°C, 5% CO2 incubator for overnight culture.

[0064] Then the cell mortality rate was detected by lactate dehydrogenase method. 30 minutes before the scheduled detection time, the cell culture plate was taken out from the cell culture incubator. Figure 3 As shown in the results, in the group with an effect-target ratio of 0:1, the addition of different concentrations of calcium folinate had no effect on the survival of senescent cells (P>0.05). In the group with an effect-target ratio of 1:1, the addition of different concentrations of calcium folinate could not enhance the ability of NK cells to kill senescent cells (P>0.05). In the group with an effect-target ratio of 2:1, when the concentration of calcium folinate was lower than 50 μM, the killing effect of NK cells on senescent cells increased with the increase of calcium folinate concentration; when the concentration of calcium folinate was higher than 50 μM, the killing effect of NK cells decreased significantly; when the concentration of calcium folinate was 50 μM, the killing effect of NK cells on senescent cells was the best (P<0.05). This result shows that in the combination of NK cells and calcium folinate, the optimal effect-target ratio of NK cells is 2:1, and the optimal concentration of calcium folinate is 50 μM.

[0065] Example 4

[0066] NK cells and calcium folinate kill IMR-90-HRAS-GFP cells

[0067] Use culture medium at 1×10 5IMR-90-HRAS-GFP cells were resuspended at a density of 100 μL / mL and inoculated in a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, three groups were set up: Group ① added 100 μL of culture medium as a negative control group; Group ② added calcium folinate with a final concentration of 50 μM and NK cells with an effector-target ratio of 2:1; Group ③ only added NK cells with an effector-target ratio of 2:1. The green fluorescence signal and proliferation of IMR-90-HRAS-GFP cells were recorded in real time using the IncuCyte SX5 live cell imaging analysis system. When the IMR-90-HRAS-GFP cells added for the first time were completely eliminated, an equal amount of IMR-90-HRAS-GFP cells were added to evaluate the persistence of NK cell killing function in each group. After co-culture, the changes in GFP fluorescence signals of IMR-90-HRAS-GFP cells in each group were calculated using IncuCyte SX5 software. The lower the signal value, the fewer the number of IMR-90-HRAS-GFP cells in the group, and the better the effect of NK cells in killing IMR-90-HRAS-GFP cells.

[0068] The relative fluorescence area changes of IMR-90-HRAS-GFP during the killing process are shown in Figure 2. Figure 4 As shown in Figure A, compared with group ①, the relative fluorescence area of ​​target cells in groups ② and ③ decreased significantly after the addition of NK cells, indicating that NK cells in both groups had a significant killing effect on target cells. Figure 4 As shown in Figure B, by comparing the relative fluorescence area of ​​target cells at the last time point, it was found that the killing effect of NK cells on target cells in Group ② was stronger than that in Group ③, indicating that calcium folinate can improve the persistence of NK cells in killing senescent cells.

[0069] Example 5

[0070] Specific verification of the synergistic enhancement effect of NK cells combined with calcium folinate

[0071] Aging and tumors have some similar characteristics (such as genomic instability, epigenetic changes, chronic inflammation and intestinal flora imbalance), but some characteristics of the aging process (such as mitochondrial dysfunction, changes in intercellular communication, and imbalance in protein homeostasis) have not been found to have clear corresponding phenomena in tumors. In addition, phenomena such as cell telomere shortening and stem cell failure associated with the aging process can also inhibit tumor occurrence, indicating that there are essential differences between senescent cells and tumor cells. So is the synergistic enhancement effect of calcium folinate combined with NK cells in killing senescent cells specific?

[0072] Preparation of SK-OV-3-mCherry cells: SK-OV-3 cells were cultured at a concentration of 1 × 10 6The cells were resuspended at a density of 100 μL / mL and then inoculated in a 6-well plate at a volume of 100 μL per well. SK-OV-3 cells were transduced with a lentiviral vector containing a red fluorescent protein mCherry reporter gene at an MOI of 5 IU / ml to obtain SK-OV-3-mCherry cells.

[0073] Use culture medium at 1×10 5 SK-OV-3-mCherry cells were resuspended at a density of 100 μL / mL and inoculated in a 96-well plate at a volume of 100 μL per well. After being cultured overnight in the IncuCyte SX5 live cell imaging analysis system, three groups were set up: Group ① added 100 μL of culture medium as a negative control group; Group ② added calcium folinate with a final concentration of 50 μM and NK cells with an effector-target ratio of 2:1; Group ③ only added NK cells with an effector-target ratio of 2:1. The fluorescence signal and proliferation of SK-OV-3-mCherry cells were recorded in real time using the IncuCyte SX5 live cell imaging analysis system. After the co-culture, the changes in the mCherry fluorescence signal of SK-OV-3-mCherry cells in each group were calculated using the IncuCyte SX5 software. The lower the signal value, the fewer cells in the group, and the better the effect of killing SK-OV-3-mCherry cells.

[0074] The results of the relative fluorescence area changes of SK-OV-3-mCherry cells during the killing process are shown in the figure. Figure 5 As shown in Figure A, compared with group ①, the relative fluorescence area of ​​target cells in groups ② and ③ decreased significantly after the addition of NK cells, indicating that NK cells in both groups had a significant killing effect on target cells. Figure 5 Figure B compares the relative fluorescence area of ​​target cells at the last time point and finds that the killing effect of NK cells on target cells in group ② is not stronger than that in group ③, indicating that calcium folinate cannot enhance the ability of NK cells to kill tumor cells.

[0075] In summary, the present invention found that although calcium folinate has no direct killing effect on senescent cells, after the combined use of NK cells and calcium folinate, the killing effect of NK cells on senescent cells can be significantly improved by calcium folinate, which has a synergistic effect. The combined use of NK cells and calcium folinate did not enhance the killing effect on tumor cells. This shows that the synergistic enhancement effect of NK cells combined with calcium folinate in killing senescent cells is specific.

[0076] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A combined pharmaceutical composition for removing senescent cells, characterized in that: The combined drug composition comprises NK cells and calcium folinate.

2. The combined pharmaceutical composition according to claim 1, characterized in that: The concentration of calcium folinate is 25-100 μM; Preferably, the density of the NK cells is (1-4)×10 2 Pieces / μL.

3. The combined pharmaceutical composition according to claim 1 or 2, characterized in that: The NK cells are derived from allogeneic or autologous cells; Preferably, the NK cells are derived from primary cultured cells or NK cell lines; Preferably, the NK cells are derived from peripheral blood, umbilical cord blood, bone marrow or induced pluripotent stem cells.

4. The combined pharmaceutical composition according to any one of claims 1 to 3, characterized in that: The method for obtaining senescent cells includes natural aging, drug induction, ultraviolet induction, ionizing radiation induction, ultrasonic treatment, mechanical damage, overexpression of aging genes, protease inhibitor induction, inhibition of cell metabolic state or induction of cell metabolic stress; Preferably, the senescent cells include cells that overexpress senescence genes or naturally senescent cells; Preferably, the cell senescence gene includes any one of HRAS, ATP2C2, BCAS3, CREG1, DYNC1I1, OAS2, OPRL1, PDE11A, PPFIBP2, RAI2, SYT17, TNFSF13B, RRM2, ACSS1, DMC1, HEY1, HTR1B, SLC25A10, ZNF214, C1ORF147, IL1B, SERPINB4, TNFSF8, WTAPP1, FPGT, GRIK2, GUCY1B1, ID2, KIAA0895, NAT1, PBX1, PLA2G12A, ROBO2, PRSS35, SLIT2, P21, P16, P53 or KRAS, or a combination of at least two thereof; Preferably, the cell types of the senescent cells include any one or a combination of at least two of immune cells, red blood cells, nerve cells, muscle cells, epithelial cells, connective tissue cells, germ cells, stem cells, liver cells, kidney cells, lung cells, cardiomyocytes, lymphocytes, intestinal cells, adipocytes, ovarian cells, osteoblasts or fibroblasts; Preferably, the senescence detection index of the senescent cells includes any one of CD28, CD57, KLRG1, P21, and SA-β-Gal, or a combination of at least two or more thereof.

5. The combined pharmaceutical composition according to any one of claims 1 to 4, characterized in that: The combined pharmaceutical composition is a single compound preparation, or a combination of two separate preparations of calcium folinate and NK cells; Preferably, the calcium folinate and NK cells are administered simultaneously; Preferably, the calcium folinate and NK cells are administered sequentially.

6. A drug for removing senescent cells, characterized in that: The medicine comprises the combined pharmaceutical composition for eliminating senescent cells according to any one of claims 1 to 5.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. The drug according to claim 7, characterized in that The pharmaceutically acceptable excipients include any one or a combination of at least two of carriers, diluents, fillers, binders, wetting agents, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, coating materials, colorants, pH regulators, antioxidants, antibacterial agents or buffers.

9. A method for preparing a combined pharmaceutical composition for eliminating senescent cells according to any one of claims 1 to 6, characterized in that: The preparation method comprises: mixing NK cells with calcium folinate to obtain the product.

10. The preparation method according to claim 9, characterized in that: When the NK cells are in the culture stage, calcium folinate is added at any one or more time points and then cultured until the purity of the NK cells reaches more than 90%; Preferably, when the purity of the NK cells reaches 90% or more, calcium folinate is added at any one time point or a combination of at least two time points, and cultured until the harvest stage; Preferably, when the NK cells reach the harvesting stage, calcium folinate is added at any one time point or a combination of at least two time points for clearing senescent cells, and cultured until the end of the experiment.

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