Method for in-vivo detection of tumor cell killing effect of NK cells
Through in vivo detection methods, the survival rate is calculated using the proportion of labeled cells, which solves the problem of difficulty in evaluating NK cells to kill tumor cells in vivo in the prior art, and accurately evaluates the killing effect of NK cells, with high sensitivity and application prospects.
Patent Information
- Application Number
- CN202510054914.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-14
- Publication Date
- 2025-05-13
AI Technical Summary
The prior art is difficult to accurately and realistically evaluate the killing effect of NK cells on human tumor cells, especially in complex environments in the body.
By using male C57BL/6 mice as a model, the labeled tumor cells and Hela cells were injected into the mixture of labeled tumor cells and Hela cells, and the mice were subsequently sacrificed and the single-cell suspension was extracted from the lungs. The ratio of CFSE and PKH26 labeled cells was detected using flow cytometry to calculate the survival rate of tumor cells to be tested.
This method can effectively evaluate the killing of NK cells to human tumor cells in the body, has high sensitivity and effectiveness, and is suitable for basic research and treatment of tumor immunity.
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Figure CN119970797A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of tumor cell killing, and in particular to a method for detecting the in vivo effect of NK cells killing tumor cells. Background Art
[0002] Natural Killer (NK) cells are important executors of human immune defense and immune surveillance, and are the body's first line of defense against tumors. Due to the wide range of NK cells, the ability to quickly identify and kill target cells without prior sensitization, and the absence of risks such as graft-versus-host disease, cytokine release syndrome, and neurotoxicity, NK cell immunotherapy is currently considered to be a new type of tumor treatment with great potential. NK cell function is regulated by both inhibitory and activating signals. If the activation signal is stronger than the inhibitory signal, the NK cell is activated, and vice versa. Therefore, the activation of activating receptors is the first step in NK cell activation. The NKG2D (Natural-killer Group 2, Member D) receptor is the most important activating receptor for NK cells, and its binding to the corresponding ligand on the surface of target cells can directly cause NK cell activation. Human NKG2D ligands (NKG2DL) include MICA (Major-histocompatibility-complex Class I-related ChainA), MICB (Major-histocompatibility-complex Class I-related Chain B) and ULBP (UL16-binding protein) 1-6. These NKG2DLs are hardly expressed in normal cells, but their expression may increase when cells become malignant. After activation, NK cells kill target cells mainly by releasing perforin, granzymes and other cytokines or antibody-dependent cell-mediated cytotoxicity. Therefore, tumor cells expressing high levels of NKG2DL are conducive to NK cell immune clearance, while downregulation of expression can promote tumor cell immune escape. Research on NK cell function and application based on NKG2D receptors has been one of the hot topics in the field of tumor immunity research in recent years.
[0003] In the study of NK cell-related tumor immunity, it is inevitable to quantify the killing effect of NK cells. Due to the wide distribution range of NK cells in the body, the lack of intuitive and reliable evaluation indicators, and the susceptibility to interference from adaptive immunity, how to evaluate the killing of tumor cells by NK cells in vivo is a technical problem in NK cell research. At present, the NK cell in vitro killing detection method is commonly used. This method co-incubates tumor cells with NK cells in vitro and evaluates the killing situation by detecting the release level of radionuclides or lactate dehydrogenase. This method only simulates the killing process of NK cells on tumor cells in vitro, which is quite different from the real and complex killing environment and process in vivo. Therefore, in order to more accurately and truly evaluate the killing effect of NK cells on target cells, it is necessary to establish an effective in vivo killing system. Summary of the invention
[0004] The purpose of the present invention is to provide a method for in vivo detection of the killing effect of NK cells on tumor cells, which is used to solve the problem in the prior art that it is difficult to accurately and truly evaluate the killing effect of NK cells on human tumor cells.
[0005] To achieve the above object, the present invention provides the following technical solution: a method for detecting the effect of NK cells killing tumor cells in vivo, characterized in that it comprises the following steps: S1. Select a model for in vivo detection of the NK cell-killing tumor cell effect; NKG2D expressed by male C57BL / 6 mouse NK cells can effectively recognize human NKG2DL, which is a prerequisite for constructing mouse NK cells to kill human tumor cells in vivo. Human cervical cancer cell line Hela cells cannot be killed by NK cells, so they are used as internal reference cells. That is, this method uses the killing of Hela cells and other types of human tumor cells by male C57BL / 6 mouse NK cells under in vivo conditions as a model, in which Hela cells are used as a control; S2, grouping the selected mice; Mice were randomly divided into a detection group, a NK cell-depleted group, and a NKG2D antibody-blocked group; The mice in the detection group were intraperitoneally injected with isotype IgG, the mice in the NK cell-depleted group were intraperitoneally injected with anti-NK1.1 antibody, and the mice in the NKG2D antibody-blocked group were intraperitoneally injected with anti-mouse NKG2D monoclonal antibody; Among them, the male C57BL / 6 mice selected were 8 to 9 weeks old, the number of mice in each group was no less than three, the intraperitoneal injection dose of the antibody for each mouse was 300 μg, and the mice after injection were fed normally and observed for 24 hours before the experiment.
[0006] S3, preparing a cell mixture of tumor cells to be tested and Hela cells; Wherein, the tumor cells to be tested are human-derived tumor cells, and the tumor cells to be tested and Hela cells are cultured, labeled and mixed in sequence; Hela cells were cultured in DMEM medium, and the tumor cells to be tested were cultured in the corresponding culture medium. All complete culture media were added with 10% fetal bovine serum (FBS). The cells were cultured in a cell culture incubator at 37°C and 5% CO2. The culture medium was replaced every 2 to 3 days according to the cell growth, and the cells in the logarithmic growth phase were used for subsequent experiments.
[0007] 1) Take cells in logarithmic growth phase, when the cell confluence reaches 70%, discard the cell culture medium, wash with PBS, add 0.25% trypsin for digestion, centrifuge and resuspend in complete culture medium into a single cell suspension.
[0008] 2) Label the tumor cells to be tested with CFSE and accurately count the cells; label HeLa cells with PKH26 dye and accurately count the cells.
[0009] 3) Mix the above two cells in equal amounts so that each mL of cell mixture contains 5×10 6 tumor cells to be tested and 5×10 6 HeLa cells.
[0010] S4, injecting the tumor cell mixture into mice; The cell mixture was injected into mice by tail vein injection; 1) Fix the mouse in the holder, expose the tail, and grasp the middle of the tail of the experimental mouse with your left hand.
[0011] 2) Use 75% alcohol cotton balls to repeatedly wipe the tail to achieve disinfection, dilate the tail blood vessels, and soften the epidermal keratin. If the blood vessels are not dilated significantly, it is recommended to soak in warm water, the water temperature should not be too hot. After soaking in warm water for 2 to 3 minutes, take out the mouse tail and wipe it with a dry cotton ball. After the blood vessels are filled, wipe with alcohol cotton balls and prepare for needle insertion.
[0012] 3) Pinch the sides of the mouse's tail with the thumb and index finger of your left hand to fill the veins and point them upward. Use your middle finger to lift the tail from below and pinch the tip of the tail with your ring finger and little finger.
[0013] 4) Hold the needle syringe in your right hand, take 0.4mL of the cell mixture, make the needle parallel to the vein, and insert the needle from the lower 1 / 4 of the tail. With the bevel of the needle tip facing upward, gently pierce the skin and the needle is immediately parallel to the blood vessel. Fix the needle and tail, release the pressure on the base of the tail, and continue to inject until all the cell mixture is injected into the mouse vein.
[0014] 5) After the injection, press the injection site with your left thumb for 1 to 2 minutes to stop the bleeding.
[0015] S5, the mice in each group were killed, and the lungs were collected to prepare single cell suspensions; 1) Anesthetize the mice 5 hours after the injection of the cell mixture. After the mice enter the anesthetized state, kill them by cervical dislocation and carefully separate the lung tissue.
[0016] 2) Cut the lung tissue into a paste and place it in a collagenase solution.
[0017] 3) Place the container containing lung tissue and collagenase on a shaking platform at 37°C, 300 rpm, and digest for 40 to 60 minutes until the lung tissue is almost completely digested.
[0018] 4) Centrifuge (1200 rpm, 5 minutes) to remove collagenase, add 2 ml PBS, and mix by pipetting.
[0019] 5) Filter through a 200-mesh sieve, collect the filtrate, centrifuge again (1000 rpm, 5 minutes), and discard the supernatant.
[0020] 6) Add red blood cell lysis buffer, incubate on ice, centrifuge again (1000 rpm, 5 minutes), and remove the supernatant.
[0021] 7) Add 2 ml PBS and mix thoroughly. Centrifuge twice (1200 rpm, 5 minutes) and discard the supernatant.
[0022] 8) Add 0.5 ml of PBS and mix thoroughly by pipetting to obtain a mouse lung single cell suspension.
[0023] S6. Flow cytometry was used to detect the number of CFSE-positive cells and PKH26-positive cells in the mouse lung single cell suspension.
[0024] S7, calculating the survival rate of the tumor cells to be tested; Among them, the survival rate of tumor cells to be tested = the number of CFSE-positive cells / the number of PKH26-positive cells × 100%.
[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention relates to a method for detecting the in vivo effect of NK cells in killing tumor cells, which can effectively evaluate the killing of human tumor cells by NK cells under real and complex in vivo conditions.
[0026] The present invention relates to an in vivo method for detecting the effect of NK cells killing tumor cells, which has high sensitivity and effectiveness in evaluating the difference in NK cells killing tumor cells caused by different NKG2DL levels.
[0027] 3. The method for in vivo detection of the effect of NK cells killing tumor cells involved in the present invention has good application prospects in basic research on tumor immunity and tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a schematic diagram of the process of detecting the in vivo killing of human tumor cells by mouse NK cells in the present invention; Figure 2 This is a flow cytometer detection diagram of in vivo killing of breast cancer Bcap37 cells in the present invention; Figure 3 This is a schematic diagram of the in vivo killing effect of the present invention on breast cancer Bcap37 cells; Figure 4 The effect of ULBP2 small interfering RNA and overexpression plasmid on ULBP2 RNA expression and the schematic diagram of primer sequences in the present invention; Figure 5 Schematic diagram of the effect of ULBP2 small interfering RNA and overexpression plasmid on ULBP2 protein expression in the present invention; Figure 6 Figure 2 shows the in vivo killing flow cytometer detection of breast cancer MDA-MB-231 cells with different ULBP2 expression levels by mice injected with IgG antibodies in the present invention and a schematic diagram of the killing effect; Figure 7 The figure is a flow cytometric detection diagram of in vivo killing of breast cancer MDA-MB-231 cells with different ULBP2 expression levels by mice in the NKG2D antibody blocking group of the present invention and a schematic diagram of the killing effect; Figure 8 This is a flow cytometer detection diagram and a schematic diagram of the killing effect of the NK cell-depleted mice on breast cancer MDA-MB-231 cells with different ULBP2 expression levels in vivo. DETAILED DESCRIPTION
[0029] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Example 1, reference Figures 1 to 3 , detect the killing effect of NK cells on breast cancer.
[0031] 1. Experimental methods 1) Six male C57BL / 6 mice aged 8 to 9 weeks were selected and randomly divided into a detection group and a NK cell removal group, with three mice in each group.
[0032] Test group: each mouse was injected intraperitoneally with 300 μg of isotype IgG; NK cell-depleted group: each mouse was intraperitoneally injected with 300 μg of anti-mouse NK1.1 antibody; The animals were fed normally and observed for 24 hours.
[0033] 2) Breast cancer cell line Bcap37 and internal reference HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a cell culture incubator containing 5% CO2. The culture medium was changed every 2 to 3 days according to the cell growth.
[0034] 3) Take cells in logarithmic growth phase, when the cell confluence reaches 70%, discard the cell culture medium, wash with PBS, add 0.25% trypsin for digestion, centrifuge and resuspend in complete culture medium into a single cell suspension.
[0035] 4) Label Bcap37 cells with CFSE and accurately count the cells; label HeLa cells with PKH26 dye and accurately count the cells.
[0036] 5) Mix the above two cells in equal amounts so that each mL of the cell mixture contains 5×10 6 Bcap37 cells and 5×10 6 HeLa cells.
[0037] 6) Take 0.4 mL of the above cell mixture and inject it into the three groups of mice through the tail vein of mice.
[0038] 7) After 5 hours, the mice were anesthetized and killed by cervical dislocation. The lung tissues were carefully separated.
[0039] 8) Cut the lung tissue into a paste and place it in the collagenase solution. Place the container containing the lung tissue and collagenase on a shaking table at 37°C, 300 rpm, and digest for 40 to 60 minutes until the lung tissue is almost completely digested.
[0040] 9) Centrifuge (1200 rpm, 5 minutes) to remove collagenase, add 2 ml PBS, pipette and mix. Filter through a 200-mesh sieve, collect the filtrate, centrifuge again (1000 rpm, 5 minutes), and remove the supernatant.
[0041] 10) Add red blood cell lysis buffer, incubate on ice, centrifuge again (1000 rpm, 5 minutes), and remove the supernatant.
[0042] 11) Add 2 ml of PBS, mix well by pipetting, centrifuge twice (1200 rpm, 5 minutes), and remove the supernatant.
[0043] 12) Add 0.5 ml of PBS, mix by pipetting to obtain a mouse lung single cell suspension, transfer the suspension into a flow tube, and detect by flow cytometry.
[0044] 2. Experimental results Flow cytometry detected a group of cells emitting green fluorescence in the first channel, namely, CFSE-labeled BCap37 cells, and a group of cells emitting red fluorescence in the second channel, namely, PKH26-labeled HeLa cells. Figure 2 described.
[0045] After the NK cells were eliminated by anti-NK1.1 antibody, the survival rate of BCap37 cells was 71.33%; however, in the IgG antibody injection group, the survival rate of breast cancer BCap37 cells decreased to 44.93% due to the retention of NK cells (e.g. Figure 3 As shown, **p<0.01).
[0046] The decrease in BCap37 survival rate is due to the killing of breast cancer cells by NK cells. Therefore, this model can effectively reflect the killing of tumor cells by NK cells in vivo.
[0047] Example 2, see Figure 1 , Figures 4 to 8 , in vivo detection of the difference in NK cell killing of breast cancer cells expressing different levels of ULBP2.
[0048] 1. Experimental methods 1) Breast cancer cell line MDA-MB-231 and internal reference HeLa cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a cell culture incubator containing 5% CO2. The culture medium was changed every 2 to 3 days according to the cell growth.
[0049] 2) Construction of ULBP2 small interfering RNA (si-ULBP2) and ULBP2 overexpression plasmid (pcDNA-ULBP2). qRT-PCR was used to detect the expression of ULBP2 RNA after transfection with si-ULBP2 and pcDNA-ULBP2 and corresponding controls. The primer sequences and RNA expression levels were as follows: Figure 4 As shown; Western blot was used to detect ULBP2 protein expression, and the results were as follows Figure 5 As shown in the figure, the constructed si-ULBP2 and pcDNA-ULBP2 had obvious effects on the interference and overexpression of ULBP2 expression in tumor cells.
[0050] 3) Digest the MDA-MB-231 cells in the logarithmic growth phase with 0.25% trypsin, remove the culture medium by centrifugation, resuspend the cells in complete culture medium, gently pipette and evenly mix, and count the cells. 5 The amount of cells was inoculated into 6-well plates and cultured overnight.
[0051] 4) Cells were transfected with si-ULBP2 and control siRNA.
[0052] ① Dilute Lipofectamine 3000 with serum-free Opti-MEM to a concentration of 3%, mix gently, and incubate at room temperature for 5 minutes.
[0053] ② Dilute si-ULBP2 or control siRNA in serum-free Opti-MEM, mix gently, and incubate at room temperature for 5 minutes.
[0054] ③ Gently mix solutions ① and ②, incubate at room temperature for 15 minutes, then add the mixed reagent dropwise to the cells in the six-well plate, place in an incubator with saturated humidity, a constant temperature of 37°C, and 5% CO2, and change the solution 24 hours after transfection.
[0055] 5) Cells were transfected with pcDNA-ULBP2 and control pcDNA3.1.
[0056] ① Dilute Lipofectamine 3000 with serum-free Opti-MEM to a concentration of 3%, mix gently, and incubate at room temperature for 5 minutes.
[0057] ② Dilute pcDNA-ULBP2 or control pcDNA3.1 with serum-free Opti-MEM, mix gently, and incubate at room temperature for 5 minutes.
[0058] ③ Gently mix solutions ① and ②, incubate at room temperature for 15 minutes, then add the mixed reagent dropwise to the cells in the six-well plate, place in an incubator with saturated humidity, a constant temperature of 37°C, and 5% CO2, and change the solution 24 hours after transfection.
[0059] 6) Male C57BL / 6 mice aged 8 to 9 weeks were randomly divided into a detection group, a NK cell removal group, and a NKG2D antibody blocking group. In the detection group, each mouse was intraperitoneally injected with 300 μg of isotype IgG; in the NK cell removal group, each mouse was intraperitoneally injected with 300 μg of anti-mouse NK1.1 antibody; in the NKG2D antibody blocking group, each mouse was intraperitoneally injected with 300 μg of anti-mouse NKG2D monoclonal antibody; the mice were fed normally and observed for 24 hours.
[0060] 7) MDA-MB-231 cells transfected with si-ULBP2, control siRNA, pcDNA-ULBP2, and control pcDNA3.1 treated as above were labeled with CFSE and the cells were accurately counted; HeLa cells were labeled with PKH26 dye and the cells were accurately counted.
[0061] 8) Mix equal amounts of MDA-MB-231 and HeLa cells in each group so that each mL of cell mixture contains 5×10 6 MDA-MB-231 cells and 5×10 6 HeLa cells.
[0062] 9) Take 0.4 mL of the above cell mixture and inject it into the three groups of mice through the tail vein of mice.
[0063] 10) After 5 hours, the mice were anesthetized and killed by cervical dislocation. The lung tissues were carefully separated.
[0064] 11) Cut the lung tissue into a paste and place it in the collagenase solution. Place the container containing the lung tissue and collagenase on a shaking table at 37°C, 300 rpm, and digest for 40 to 60 minutes until the lung tissue is almost completely digested.
[0065] 12) Centrifuge (1200 rpm, 5 minutes) to remove collagenase, add 2 ml PBS, pipette and mix. Filter through a 200-mesh sieve, collect the filtrate, centrifuge again (1000 rpm, 5 minutes), and remove the supernatant.
[0066] 13) Add red blood cell lysis buffer, incubate on ice, centrifuge again (1000 rpm, 5 minutes), and remove the supernatant.
[0067] 14) Add 2 ml PBS and mix thoroughly. Centrifuge twice (1200 rpm, 5 minutes) and discard the supernatant.
[0068] 15) Add 0.5 ml of PBS and mix thoroughly by pipetting. The mouse lung single cell suspension is obtained and transferred into a flow cytometer for detection.
[0069] 2. Experimental results Flow cytometry detected a group of cells emitting green fluorescence in the first channel, namely, CFSE-labeled MDA-MB-231 cells, and a group of cells emitting red fluorescence in the second channel, namely, PKH26-labeled HeLa cells (e.g. Figure 6~Figure 8 In the IgG antibody injection group, after small interfering RNA inhibited the expression of ULBP2, the survival rate of tumor cells in the si-ULBP2 group was 1.46 times that of the negative control group ( Figure 6 , **p<0.01), while the tumor cell survival rate of the ULBP2 overexpression pcDNA-ULBP2 group was 60.66% of that of the control group ( Figure 6, ***p<0.001). After blocking NK cells with anti-NKG2D antibody and clearing NK cells with anti-NK1.1 antibody, there was no significant difference in tumor cell survival rate between the si-ULBP2 group and the pcDNA-ULBP2 group and the control group ( Figure 7 , Figure 8 ). It can be seen that this model can sensitively distinguish the clearance of tumor cells with different expression levels of NK cell activating ligand ULBP2 in mice, and prove that this difference in killing level is caused by NK cell activation mediated by NKG2D receptor.
[0070] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.
[0071] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A method for detecting the effect of NK cells killing tumor cells in vivo, characterized in that: The following steps are involved: S1. Select a model for in vivo detection of the NK cell-killing tumor cell effect; The killing of Hela cells and other types of human tumor cells by male C57BL / 6 mouse NK cells in vivo was used as a model, in which Hela cells were used as a control; S2, grouping the selected mice; Mice were randomly divided into a detection group, a NK cell-depleted group, and a NKG2D antibody-blocked group; Among them, mice in the detection group were intraperitoneally injected with isotype IgG, mice in the NK cell removal group were intraperitoneally injected with anti-NK1.1 antibody, and mice in the NKG2D antibody blocking group were intraperitoneally injected with anti-mouse NKG2D monoclonal antibody; S3, preparing a cell mixture of tumor cells to be tested and Hela cells; Wherein, the tumor cells to be tested are human-derived tumor cells, and the tumor cells to be tested and Hela cells are cultured, dye-labeled, counted and mixed in sequence; S4, injecting the tumor cell mixture into mice; The cell mixture was injected into mice by tail vein injection; S5, the mice in each group were killed, and the lungs were collected to prepare single cell suspensions; S6. Flow cytometry was used to detect the number of CFSE-positive cells and PKH26-positive cells in the single cell suspension of mouse lung; S7, calculating the survival rate of the tumor cells to be tested; Among them, the survival rate of tumor cells to be tested = the number of CFSE-positive cells / the number of PKH26-positive cells × 100%.
2. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: The male C57BL / 6 mice selected in step S2 were 8 to 9 weeks old, and the number of mice in each group was no less than three. The intraperitoneal injection volume of the antibody for each mouse was 300 μg. The injected mice were fed normally and observed for 24 hours before the experiment.
3. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: In step S3, the Hela cells are cultured in DMEM culture medium, and the tumor cells to be tested are cultured in the corresponding culture medium; Among them, 10% fetal bovine serum was added to all complete culture media, and the cells were cultured in a cell culture incubator at a constant temperature of 37°C and 5% CO2. According to the cell growth, the culture medium was replaced every 2 to 3 days, and the cells in the logarithmic growth phase were used for subsequent experiments.
4. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 3, characterized in that: When cells in logarithmic growth phase were taken, the cell culture medium was discarded, the cells were washed with PBS, 0.25% trypsin was added for digestion, and after centrifugation, they were resuspended in complete culture medium into a single cell suspension.
5. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: In step S3, the tumor cells to be tested are labeled with CFSE and the cells are accurately counted; HeLa cells are labeled with PKH26 and the cells are accurately counted; the above two cells are mixed in equal amounts so that each 1 mL of the cell mixture contains 5×10 6 tumor cells to be tested and 5×10 6 HeLa cells.
6. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: In step S4, the location for injecting the cell mixture into the mouse is the tail vein of the mouse.
7. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: In step S5, the mice in each group are killed by anesthetizing the mice 5 hours after the injection of the cell mixture, and then killing the mice by cervical dislocation after the mice enter an anesthetized state.
8. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 1, characterized in that: The steps for preparing mouse lung single cell suspension include: S5.
1. Take the mouse lungs, cut them into a paste, and place them in a collagenase solution; S5.
2. Place the container containing the lung tissue and collagenase mixture on a shaking table for digestion; S5.3, remove collagenase from the mixture by centrifugation, add 2 ml of PBS, and mix by pipetting; S5.4, filter the mixture through a 200-mesh sieve, collect the filtrate, centrifuge again, and remove the supernatant; S5.
5. Add red blood cell lysis buffer to the mixture, incubate on ice, centrifuge again and remove the supernatant; S5.
6. Add 2 ml of PBS to the mixture, pipette and mix well, centrifuge twice, and remove the supernatant; S5.
7. Add 0.5 ml of PBS to the mixture and mix well by pipetting to obtain a mouse lung single cell suspension.
9. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 8, characterized in that: The mixture was centrifuged at a speed of 1000-1200 rpm for 5 minutes.
10. The method for in vivo detection of the effect of NK cells killing tumor cells according to claim 8, characterized in that: In step S5.2, the shaking incubator temperature is 37°C, the rotation speed is 300 rpm, and the digestion is carried out for 40 to 60 minutes.