Application of sulfhydryl compound GSHEE in enhancing killing activity of NK cells

Abstract

The invention discloses an application of a sulfhydryl compound GSHEE in enhancing the killing activity of NK cells, and discloses an action path of the GSHEE in enhancing the functions of the NK cells through a metabolism recovery mechanism for the first time, the level of glutathione (GSH) in the NK cells treated by the GSHEE is remarkably improved, and the accumulation of reactive oxygen species (ROS) can be reduced, so that the expression of functional molecules (such as CD107a and IFN-gamma) in the NK cells is improved, and the killing activity of the NK cells is enhanced. The killing activity of the NK cells on tumor cells is enhanced, and a new thought and scheme are provided for NK cell immunotherapy of tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a thiol compound GSHEE in enhancing the killing activity of NK cells. Background Art

[0002] Natural killer (NK) cells are a key immune cell in the human immune system and an important component of innate immunity. NK cells have a non-specific and direct killing mechanism, and can recognize and kill certain tumor cells, virus-infected cells, and abnormal cells of the body without prior sensitization. Therefore, NK cells play a vital role in anti-tumor immunity and have become a hot topic in tumor immunotherapy research. For example, NK cells play an important role in the immune defense of hematological malignancies such as acute leukemia (AL). Studies have shown that the NK cell function of AL patients is significantly impaired, and their cytotoxic activity is significantly weakened, which seriously weakens the effect of anti-leukemia immune response. Although NK cell-related immunotherapy has made certain progress, how to effectively restore or enhance NK cell function to enhance its anti-leukemia ability is still a difficult and hot issue in current research.

[0003] Intracellular redox balance plays an important role in regulating immune cell function, and this balance is closely related to glutathione (GSH) metabolism. As one of the most important antioxidants in cells, GSH supports the activity and function of immune cells by maintaining redox homeostasis. GSH has been widely recognized as an important regulator of immune cell function, especially in T cells, where GSH promotes cell activation, proliferation and differentiation by maintaining redox balance. Previous studies have shown that GSH plays a specific role in different immune cell subsets. However, current research on GSH in NK cells is still relatively limited, especially research on GSH metabolism in NK cells of tumor patients is even more scarce. Existing evidence shows that GSH metabolism in NK cells of AL patients is generally disordered, which may be an important reason for their impaired function.

[0004] Therefore, intervention based on GSH metabolism is expected to become an important strategy to restore NK cell function, which has important theoretical significance and clinical application potential. Summary of the invention

[0005] In view of this, the primary purpose of the present invention is to provide an application of a thiol compound, glutathione ethyl ester (GSHEE), in enhancing the killing activity of NK cells. The present invention has found that pretreatment of NK cells with GSHEE can significantly increase the level of glutathione (GSH) in NK cells and improve their redox metabolism, thereby increasing the killing activity of NK cells, enhancing their ability to kill tumor cells, and improving the anti-tumor effect of NK cells.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] One aspect of the present invention provides the use of the thiol compound GSHEE in enhancing the killing activity of NK cells.

[0008] Another aspect of the present invention provides the use of the thiol compound GSHEE in the preparation of a drug or a pharmaceutical composition, wherein the drug or the pharmaceutical composition has at least any one of the following functions:

[0009] Enhance the killing activity of NK cells;

[0010] and / or, increasing GSH levels in NK cells;

[0011] and / or, increasing the expression of CD107a in NK cells;

[0012] and / or, promoting the secretion of IFN-γ in NK cells;

[0013] and / or, anti-tumor.

[0014] Another aspect of the present invention provides a method for enhancing the killing activity of NK cells for non-therapeutic purposes, comprising the following steps:

[0015] Obtain and purify NK cells:

[0016] The purified NK cells were placed in an activation medium containing the thiol compound GSHEE for culture and activation.

[0017] Another aspect of the present invention provides a NK cell with enhanced killing activity, which is obtained by treating with the method described above.

[0018] Another aspect of the present invention provides the use of the NK cells described above in the preparation of anti-tumor drugs or pharmaceutical compositions.

[0019] Another aspect of the present invention provides an anti-tumor drug comprising the NK cells described above.

[0020] Beneficial effects of the present invention:

[0021] The present invention proposes for the first time the regulatory effect of the thiol compound GSHEE on NK cell metabolism and function. The NK cells treated with GSHEE have significantly enhanced cell killing activity by increasing the intracellular glutathione (GSH) level and improving redox metabolism. The expression of functional molecules in NK cells (such as CD107a and IFN-γ) is significantly increased. Therefore, the thiol compound GSHEE can enhance the anti-tumor function of NK cells of tumor patients by improving the GSH metabolism of NK cells, thereby providing a new idea for tumor treatment using NK cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The survival of NK cells of patients with acute leukemia pretreated with GSHEE in Example 1, Figure 1 a is a representative flow cytometry diagram of NK cell apoptosis. Figure 1 b is a bar chart showing the statistical data of NK cell apoptosis.

[0023] Figure 2 The results of intracellular GSH level detection after GSHEE pretreatment of NK cells from acute leukemia patients in Example 2 are as follows: Figure 2 a is the flow cytometry histogram of GSH levels in AL-NK cells. Figure 2 b is a bar chart showing the level of GSH in AL-NK cells.

[0024] Figure 3 The results of the cell killing ability of NK cells from acute leukemia patients after pretreatment with reduced GSH in Example 3 are as follows: Figure 3 a is a representative flow cytometry diagram of AL-NK cells killing THP-1 cells. Figure 3 b is a bar chart showing the killing of THP-1 cells by AL-NK cells.

[0025] Figure 4 The cell killing ability results of NK cells of acute leukemia patients after GSHEE pretreatment in Example 3 are as follows: Figure 4 a is a representative flow cytometry diagram of AL-NK cells killing KG-1a cells. Figure 4 b is a bar chart showing the killing of KG-1a cells by AL-NK cells.

[0026] Figure 5 The expression results of the killing function molecule CD107a of NK cells of acute leukemia patients pretreated with GSHEE in Example 4 are as follows: Figure 5 a is a representative flow cytometry image of CD107a on the surface of AL-NK cells, Figure 5 b is a bar chart showing the CD107a expression on the surface of AL-NK cells.

[0027] Figure 6The results of the IFN-γ test on NK cells of patients with acute leukemia treated with GSHEE in Example 5 are as follows: Figure 6 a is a representative flow cytometry image of intracellular IFN-γ in AL-NK cells. Figure 6 b is a bar chart showing the intracellular IFN-γ in AL-NK cells. DETAILED DESCRIPTION

[0028] Embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0029] The first aspect of the present invention discloses the use of the thiol compound GSHEE in enhancing the killing activity of NK cells. The present invention verifies through experiments that the killing activity of NK cells treated with GSHEE is significantly enhanced.

[0030] Those skilled in the art know that GSH is a key antioxidant that participates in the maintenance of intracellular redox balance, can remove excess reactive oxygen species (ROS), and protect cells from oxidative stress damage. ROS levels are usually elevated, and GSH levels may be impaired, resulting in a decrease in NK cell function. The present invention experimentally verified that the GSH level in GSHEE-treated NK cells was significantly increased, and the expression of CD107a was increased, and the secretion of IFN-γ was promoted, which suggests that GSHEE can improve the killing activity of NK cells by increasing the level of intracellular glutathione (GSH), improving redox metabolism.

[0031] The second aspect of the present invention discloses the use of the thiol compound GSHEE in the preparation of a drug or a pharmaceutical composition, wherein the drug or the pharmaceutical composition has at least any one of the following functions:

[0032] Enhance the killing activity of NK cells;

[0033] and / or, increasing GSH levels in NK cells;

[0034] and / or, increasing the expression of CD107a in NK cells;

[0035] and / or, promoting the secretion of IFN-γ in NK cells;

[0036] and / or, anti-tumor.

[0037] In the present invention, the thiol compound is reduced glutathione ethyl ester (GSHEE), and its molecular structure is as follows:

[0038]

[0039] GSHEE is a derivative of GSH, so it has good biocompatibility and low toxicity. The research in this invention provides a new drug option for enhancing NK cell activity and even anti-tumor treatment, which can avoid the severe immune activation syndrome (such as cytokine storm) that may be caused by traditional immune enhancement drugs. In addition, it can be used in combination with existing immunotherapy (such as CAR-NK or immune checkpoint inhibitors) to further improve the effectiveness of anti-tumor treatment.

[0040] In the present invention, the tumor can be any common tumor disease in the art that can be treated with NK cell immunotherapy, preferably a tumor with immune dysfunction, such as leukemia, preferably acute leukemia.

[0041] The third aspect of the present invention discloses a method for enhancing the killing activity of NK cells for non-therapeutic purposes, comprising the following steps:

[0042] Obtain and purify NK cells:

[0043] The purified NK cells were placed in an activation medium containing the thiol compound GSHEE for culture and activation.

[0044] In the present invention, NK cells are not particularly limited and can be common sources in the art, such as peripheral blood, umbilical cord blood, iPSC or NK cell lines (such as NK-92, NKG, etc.); preferably, the NK cells are from peripheral blood; in some specific embodiments of the present invention, the NK cells are from peripheral blood mononuclear cells of acute leukemia patients.

[0045] Furthermore, the separation and purification of NK cells can be carried out by conventional means in the art, including but not limited to density gradient centrifugation, flow cytometry or magnetic bead sorting, etc. The technicians in this field have such ability, so there is no special limitation.

[0046] In the present invention, the activation culture medium refers to the culture medium in which NK cells can receive specific signals and stimulations when cultured, thereby enabling NK cells to obtain higher killing activity and immune function.

[0047] Specifically, the composition of the activation medium is well known to those skilled in the art. In the present invention, the composition of the activation medium includes a basal medium, a cytokine, a fetal bovine serum and an antibiotic. In some specific embodiments of the present invention, the basal medium is an IMDM medium, but is not limited thereto. Any medium that can be used for NK cell culture in the art can be used, and a suitable medium and culture conditions can be selected according to the type of specific cells. In the present invention, the cytokine includes but is not limited to at least one of IL-12, IL-15, IL-2 and IL-18, etc., preferably IL-12 and IL-15.

[0048] It is understood that the dosage of each component in the activation medium can be adjusted or optimized as needed by those skilled in the art without special restrictions. In some specific embodiments of the present invention, the final concentration of IL-12 is preferably 5-15 ng / mL, preferably 10 ng / mL; the final concentration of IL-15 is preferably 30-70 ng / mL, preferably 50 ng / mL; the volume percentage of antibiotics is preferably 0.5-3%, preferably 1%; the volume percentage of fetal bovine serum is 8-12%, preferably 10%.

[0049] The activation medium of the present invention contains a certain concentration of the thiol compound GSHEE, thereby enhancing the killing activity of NK cells. The concentration of the thiol compound GSHEE is not particularly limited, and can be determined by a person skilled in the art through an experimental method familiar to the art. In some specific embodiments of the present invention, 1×10 5 ~1×10 6 NK cells, the added concentration of the thiol compound GSHEE is 30-70 μM, for example, it can be any value among 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM or 70 μM, preferably 40-60 μM, and more preferably 50 μM.

[0050] Further, the culture conditions can be selected and adjusted according to the specific cell type selection and the effect of the sulfhydryl compound GSHEE, without special restrictions. In some specific embodiments of the present invention, the culture conditions are: 37°C, 5% CO2 for 12 to 24 hours, and the specific culture time can be adjusted according to the concentration of the sulfhydryl compound GSHEE, but is not limited thereto.

[0051] The fourth aspect of the present invention discloses a NK cell with enhanced killing activity, which is obtained by treating with the method described above. After being treated with GSHEE, the cytotoxic activity of the NK cell is significantly enhanced.

[0052] The fifth aspect of the present invention discloses the use of the above-mentioned NK cells in the preparation of anti-tumor drugs or pharmaceutical compositions.

[0053] The sixth aspect of the present invention discloses an anti-tumor drug, which contains the NK cells described above.

[0054] By preparing corresponding anti-tumor drugs from NK cells with significantly enhanced cell activity, excellent anti-tumor effects can be obtained.

[0055] It is understandable that the drug also includes at least one pharmaceutically acceptable excipient and / or carrier. These excipients and / or carriers are conventional choices in the art, and their main function is to improve the absorption effect or therapeutic effect of the drug within a safe range, or to minimize side effects, etc. Those skilled in the art can make corresponding choices based on the specific dosage form, etc., and they have such ability, which will not be elaborated in detail here.

[0056] The present invention is described below by specific examples, it should be noted that the following specific examples are only for illustrative purposes, and do not limit the scope of the present invention in any way, unless otherwise defined, all technical and scientific terms used herein are identical with the meanings generally understood by the technicians belonging to the technical field of the present invention. The term used in the specification of the present invention herein is only for the purpose of describing specific embodiments, and is not intended to limit the present invention. In addition, if not otherwise specified, the method for not specifically recording conditions or steps is a conventional method, and the reagents and materials adopted can be obtained from commercial sources.

[0057] Example 1 Preparation of NK cells

[0058] 1. Isolation of peripheral blood mononuclear cells

[0059] Under sterile conditions, collect an appropriate amount of peripheral blood samples from patients with acute leukemia (the samples are all from the First Affiliated Hospital of the University of Science and Technology of China, and the patients' written informed consent is obtained) for the preparation of NK cells. Follow the steps below:

[0060] (1) Add sterile 1× PBS to the anticoagulant blood collection tube, dilute the peripheral blood sample at a 1:1 volume ratio and mix well.

[0061] (2) Add an appropriate amount of human lymphocyte separation solution (LST1077, TBDscience) into the centrifuge tube, and carefully add the diluted peripheral blood slowly along the wall of the centrifuge tube onto the separation solution to ensure a clear interface between the separation solution and the blood.

[0062] (3) After balancing, centrifuge at 450 g for 30 min at 20°C (both the acceleration and deceleration speeds are set to 0).

[0063] (4) After centrifugation, carefully aspirate the formed buffy coat layer (lymphocyte layer) with a Pasteur pipette and transfer it to a new centrifuge tube.

[0064] (5) Add sterile 1× PBS to wash the cells and centrifuge at 1800 rpm and 4°C for 10 minutes. Discard the supernatant and gently blow away the precipitated cell clusters with a pipette.

[0065] (6) Add sterile 1× PBS again, mix well, centrifuge at 1200 rpm and 4°C for 10 minutes, and discard the supernatant. The precipitate is the peripheral blood mononuclear cells, which can be used for subsequent experiments.

[0066] 2. Isolation and purification of NK cells

[0067] Flow cytometry was used to separate and purify NK cells from patients with acute leukemia. The specific steps are as follows:

[0068] (1) Mononuclear cells were isolated from peripheral blood samples of patients with acute leukemia and stained with flow cytometry antibodies CD45-APC / Cy7 (304014, Biolegend), CD3-FITC (300306, Biolegend), CD56-PE (318306, Biolegend), and CD33-APC (303408, Biolegend) and incubated at 4°C in the dark for 30 min.

[0069] (2) After incubation, add 1× PBS to wash away excess antibodies, centrifuge at 450 g for 10 minutes, and discard the supernatant.

[0070] (3) The cells were resuspended in 1× PBS, filtered, transferred to flow cytometry tubes, and sorted using a CytoFLEX SRT flow cytometer. The gating strategy for NK cells was CD45 high 、CD3 - 、CD56 + , sorting purity is greater than 95%.

[0071] 3. Cultivation and activation of NK cells

[0072] The purified NK cells were resuspended in IMDM medium (BL312A, Biosharp), and 10% fetal bovine serum (164210, Pricella), 1% antibiotics (BL142A, Biosharp), and IL-12 (10 ng / mL, CI58, Novoprotein) and IL-15 (50 ng / mL, C016, Novoprotein) were added to activate NK cells. At the same time, GSHEE (50 μM) (G1404, Sigma) was added to the culture medium and cultured at 37 ° C, 5% CO2. After 12 h of culture treatment, the survival of NK cells was detected by flow cytometry.

[0073] The experimental results are as follows Figure 1 As shown, where ns indicates no significant difference, the results indicate that the addition of the thiol compound GSHEE does not affect NK cell survival.

[0074] Example 2 Detection of GSH levels in NK cells of acute leukemia patients treated with GSHEE

[0075] GSH is a key antioxidant that participates in the maintenance of intracellular redox balance, can remove excessive reactive oxygen species (ROS), and protect cells from oxidative stress. In the tumor microenvironment, ROS levels are usually elevated, and GSH levels may be impaired, resulting in a decline in NK cell function. By detecting the level of GSH in NK cells, its antioxidant capacity and its potential in tumor immune surveillance can be evaluated. In this embodiment, the effect of GSHEE on improving NK cell GSH levels is evaluated by detecting the level of GSH in NK cells.

[0076] The specific experimental steps are as follows:

[0077] (1) Human peripheral blood mononuclear cells were isolated from fresh peripheral blood samples using the Ficoll method. AL-NK cells were separated and purified by flow cytometry, the NK cells were resuspended in IMDM complete medium, and the cell suspension was transferred to a cell culture plate. IL-12 (10 ng / mL) and IL-15 (50 ng / mL) were added to the culture plate to activate NK cells, and the thiol compound GSHEE (50 μM) of the present invention was added at the same time. The culture plate was placed in an incubator at 37°C and 5% CO2 for 12 hours.

[0078] (2) After 12 hours of GSHEE treatment, the cells were harvested and resuspended in 1× PBS. The cell wells were gently blown and collected into 1.5 mL EP tubes. The cells were centrifuged at 4°C and 450 g for 5 minutes and the supernatant was discarded.

[0079] (3) To measure the intracellular GSH content, ThiolTracker Violet (Invitrogen, Cat# T10095) dye stock solution was diluted to a concentration of 5 mM using 1× PBS, and 100 μL of the solution was taken to resuspend the cells and incubated at 37°C in the dark for 30 min.

[0080] (4) Add 1 mL of 1× PBS to wash away excess dye and centrifuge at 450 g for 5 min at 4°C. After the cells are washed, perform flow cytometry analysis.

[0081] The experimental results can be found in Figure 2 , where P<0.05 is *, indicating statistically significant difference.

[0082] Figure 2 The results showed that the GSH level in NK cells of acute leukemia patients pretreated with GSHEE increased significantly, indicating that NK cells pretreated with GSHEE improved GSH metabolism in NK cells and optimized the oxidative balance state.

[0083] Example 3 Detection of NK cell killing function in acute leukemia patients treated with GSHEE

[0084] 1. NK cell preparation

[0085] (1) AL-NK cells pretreated with thiol compound GSHEE (50 μM) were collected and placed in a 1.5 mL Eppendorf tube, centrifuged at 450 g for 5 minutes, and the supernatant was discarded. At the same time, AL-NK cells treated with reduced GSH (1 mM, HY-D0187, MCE) were collected as a comparative example, and the pretreatment steps were the same as those in Example 1.

[0086] (2) Wash the cells once with 1× PBS, then resuspend the cells in IMDM basal medium and count the cells.

[0087] 2. Labeling of target cells

[0088] (1) Collect KG-1a cells (ATCC, catalog number: CCL-246.1) in a 15 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes, discard the supernatant, and wash the cells once with 1× PBS. In the comparative example, the target cells used for the reduced GSH treatment were THP-1 cells (ATCC, catalog number: TIB-202).

[0089] (2) Resuspend the cells in 1 mL of 1× PBS, add 1 μL of CFSE (5 mM, HY-D0056, MCE), mix quickly, and stain at 37°C in the dark for 20 minutes.

[0090] (3) After staining, add IMDM complete medium containing 10% FBS to terminate the reaction, let stand for 5 minutes, centrifuge at 800 rpm for 5 minutes, and discard the supernatant.

[0091] (4) Resuspend and wash the excess dye with 1× PBS, repeat the centrifugation step, and finally resuspend the cells with IMDM basal medium and count them. Inoculate 2.5×10 cells per well. 4 target cells.

[0092] 3. Co-culture of effector and target cells

[0093] (1) According to the effector-target ratio (2.5:1), the NK cell suspension and the target cell suspension were added to a 96-well plate, and IMDM medium was supplemented to a final volume of 200 μL per well. After mixing, the plate was placed in a cell culture incubator (37° C., 5% CO 2 ) and incubated for 6 hours (the reduced GSH treatment was incubated for 4 hours in the comparative example).

[0094] (2) The experimental groups are as follows, with 3 replicate wells in each group:

[0095] Target cell only group: blank wells with only target cells were used as the target cell basic apoptosis group.

[0096] Untreated NK cell group: target cells and NK cells not treated with drugs were inoculated according to the effect-target ratio.

[0097] GSHEE-treated NK cell group: target cells and NK cells pre-treated with GSHEE were inoculated at the corresponding effector-target ratio.

[0098] The experimental grouping in the comparative example is the same as above.

[0099] 4. Target cell apoptosis detection

[0100] After incubation, the cells were centrifuged at 450 g for 5 min and the supernatant was discarded.

[0101] The apoptosis of target cells KG-1a (or THP-1 cells in the control example) was determined using an apoptosis kit (E-CK-A217, Elabscience). 100 μL of 1× binding buffer was added to each well, followed by 1 μL of Annexin V-APC and 1 μL of PPI. The cells were stained at room temperature in the dark for 10 min, and then analyzed by flow cytometry.

[0102] 5. Calculation of experimental results

[0103] Killing rate of untreated group = Annexin V positive rate of untreated group - positive rate of simple target cell group

[0104] Killing rate of GSHEE treatment group = Annexin V positive rate of GSHEE treatment group - positive rate of simple target cell group

[0105] Killing rate of reduced GSH treatment group = Annexin V positive rate of reduced GSH treatment group - positive rate of simple target cell group. Figure 3 and Figure 4 , where ns means no significant difference; P<0.01 means **, indicating statistical difference.

[0106] in, Figure 3 The experimental results of the comparative example of reduced GSH treatment are shown in Figure 2. Figure 4 These are the experimental results of GSHEE treatment in Example 3.

[0107] The results showed that GSHEE pretreatment enhanced the killing ability of NK cells from patients with acute leukemia, while reduced GSH pretreatment had no effect on the killing ability of NK cells from patients with acute leukemia.

[0108] Example 4 Detection of expression of killer function molecule CD107a

[0109] The surface expression of CD107a is a sign of NK cell degranulation and activation, and its expression level is significantly related to the killing activity of NK cells. When NK cells are activated, CD107a will appear on the cell surface, so the activation state of NK cells can be assessed by detecting its expression by flow cytometry. In this embodiment, the flow cytometry detection method marked by effector cell CD107a is used to assess the activity of natural killer cells. Since the NK cell cytoplasm contains a large number of granules with cell lysis, after activation, NK cells will release these granules into target cells through cell immune synapses. These cell granules include cell lytic proteins perforin and granzymes, wherein granzymes include lysosome-associated membrane protein 1 (LAMP1) expressed on the membrane, i.e. CD107a, which is a marker of NK cell degranulation. When NK cells kill target cells, as degranulation occurs, CD107a molecules are transported to the cell membrane surface, and the upregulation of CD107a molecules is consistent with the secretion of perforin, and its expression level is significantly correlated with the killing activity of NK cells. Therefore, NK cells with positive expression of CD107a molecules can represent NK cells with killing activity. In this embodiment, the expression of NK cell killing functional molecule CD107a is detected to evaluate the effect of GSHEE on NK cell activity.

[0110] The specific experimental steps are as follows:

[0111] (1) Human peripheral blood mononuclear cells were isolated from fresh peripheral blood samples using the Ficoll method. The isolated cells were resuspended in IMDM complete medium and the cell suspension was transferred to a cell culture plate. IL-12 (10 ng / mL) and IL-15 (50 ng / mL) were added to the culture plate to activate NK cells, and the thiol compound GSHEE (50 μM) of the present invention was added at the same time. The culture plate was placed in an incubator at 37°C and 5% CO2 for 12 hours.

[0112] (2) When the culture time reached 10 hours, the flow cytometry antibody CD107a-PE (328608, Biolegend) was added to the PBMCs cell suspension in the cell culture plate, and monensin (M5273, Sigma) was added at the same time to block the process of Golgi apparatus secreting cytokines outside the cells. 0.5 μL of antibody and monensin were added for every 100 μL, and the cells were cultured at 37°C for 2 hours.

[0113] (3) After 12 hours of GSHEE treatment, the cells were harvested and resuspended in 1× PBS. The cell wells were gently blown and collected into 1.5 mL EP tubes. The cells were centrifuged at 4°C and 450 g for 5 minutes and the supernatant was discarded.

[0114] (4) Add 0.5 μL of flow cytometry antibodies CD45-BV510 (304036, Biolegend), CD3-APC / CY7 (344818, Biolegend), and CD56-PE / CY7 (362510, Biolegend) to each 100 μL of cell suspension, and incubate the cells in a dark place at 4°C with fluorescently labeled antibodies for 30 min.

[0115] (5) After the incubation, add 1 mL of 1× PBS to elute the excess antibody and centrifuge at 4°C and 450 g for 5 minutes. After the cells are washed, perform flow cytometry.

[0116] The experimental results can be found in Figure 5 , where P<0.05 is *, indicating statistically significant difference.

[0117] Figure 5 The results showed that the expression of the killing function molecule CD107a of NK cells from acute leukemia patients pretreated with GSHEE was significantly increased, indicating that the killing ability of NK cells pretreated with GSHEE was significantly increased.

[0118] Example 5 Detection of cytokine IFN-γ

[0119] NK cells can secrete interferon-γ (IFN-γ), and the production of IFN-γ is closely related to the activation and function of NK cells. When NK cells recognize and attack pathogens or tumor cells, they release IFN-γ, thereby enhancing the immune response. Therefore, detecting the level of cytokine IFN-γ can measure NK cell activity. In this example, the effect of GSHEE on NK cell activity was evaluated by detecting the cytokine IFN-γ in NK cells.

[0120] The specific experimental steps are as follows:

[0121] 1. Isolate human peripheral blood mononuclear cells from fresh peripheral blood samples using the Ficoll method, and resuspend the cells in IMDM complete medium. Transfer the cell suspension to a cell culture plate. Add IL-12 (10 ng / mL) and IL-15 (50 ng / mL) to the cell culture plate to activate NK cells, and add the thiol compound GSHEE (50 μM) of the present invention. Place the culture plate in an incubator at 37°C and 5% CO2 for 12 hours.

[0122] 2. When the culture time reaches 10 hours, monensin (M5273, Sigma) is added to block the process of Golgi apparatus secreting cytokines to the extracellular space, and 0.5 μL of monensin is added for every 100 μL.

[0123] 3. After 12 hours of GSHEE treatment, collect the cells, add 1×PBS to resuspend, gently blow the cell plate, collect the cells into a 1.5mL EP tube, centrifuge at 4°C, 450g for 5 minutes, and discard the supernatant.

[0124] 4. Add 0.5 μL of flow cytometry antibodies CD45-BV510 (304036, Biolegend), CD3-APC / CY7 (344818, Biolegend), and CD56-PE / CY7 (362510, Biolegend) to each 100 μL of cell suspension and incubate in a 4°C refrigerator in the dark for 30 minutes. After incubation, add 1 mL of 1× PBS to elute excess antibodies and centrifuge at 4°C, 450 g for 5 minutes.

[0125] 5. After discarding the supernatant, prepare the fixative solution with eBioscience flow cytometry fixation permeabilization concentrate (00-5123-43, Invitrogen) and diluent (00-5223-56, Invitrogen), resuspend the cells with 200 μL of fixative solution, and fix at 4°C for 2 h or overnight.

[0126] 6. Dilute eBioscience flow cytometry permeabilization buffer (10×) (00-8333-56, Invitrogen) with pure water, add 1 mL of 1× permeabilization buffer to wash the fixative, centrifuge at 900g for 10 min, and discard the supernatant. Prepare blocking buffer (1× permeabilization buffer + 10% mouse serum), add 100 μL of blocking buffer to each tube, and block at 4°C in the dark for 30 minutes.

[0127] 7. Add 0.5 μL of flow cytometry antibody IFN-γ-PerCP / Cy5.5 (506528, Biolegend) to each 100 μL of cell suspension and incubate at 4°C in the dark for 1-2 hours. Add 1 mL of permeabilization buffer to the cells to wash away excess antibodies and centrifuge at 4°C, 900 g for 10 minutes.

[0128] 8. Use 200 μL 1× PBS solution to resuspend the centrifuged cells, filter impurities through a 200-mesh nylon mesh, transfer to a 2 mL EP tube, and finally perform flow cytometry detection.

[0129] The experimental results are shown in Figure 6 , where P<0.01 is **, indicating statistically significant difference.

[0130] Figure 6 The results in the study showed that the cytokine IFN-γ of NK cells from patients with acute leukemia pretreated with GSHEE increased significantly, further indicating that the killing ability of NK cells pretreated with GSHEE was significantly increased.

[0131] In the present invention, NK cells separated from peripheral blood mononuclear cells of acute leukemia patients are used as examples to verify the action path of GSHEE in enhancing NK cell function through the metabolic recovery mechanism, which significantly enhances the killing ability of NK cells against leukemia cells. GSHEE significantly enhances the cytotoxic activity of NK cells and shows good anti-leukemia efficacy. However, it is understandable that the mechanism of action in the present invention is also applicable to other types of NK cells and other similar immune dysfunction tumors, and its effect is predictable, and is not limited to the acute leukemia in the present invention.

[0132] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0133] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of thiol compound GSHEE in enhancing NK cell killing activity; Preferably, the use further comprises increasing the level of GSH in NK cells, and / or increasing the expression of CD107a in NK cells, and / or promoting the secretion of IFN-γ.

2. The use of the thiol compound GSHEE in the preparation of a drug or a pharmaceutical composition, characterized in that: The drug or pharmaceutical composition has at least one of the following functions: Enhance the killing activity of NK cells; and / or, increasing GSH levels in NK cells; and / or, increasing the expression of CD107a in NK cells; and / or, promoting the secretion of IFN-γ in NK cells; and / or, anti-tumor; Preferably, the tumor is acute leukemia.

3. A method for enhancing the killing activity of NK cells for non-therapeutic purposes, characterized in that: The following steps are involved: Obtain and purify NK cells: The purified NK cells were placed in an activation medium containing the thiol compound GSHEE for culture and activation.

4. The method according to claim 3, characterized in that The NK cells are derived from peripheral blood, umbilical cord blood, iPSC or NK cell lines; Preferably, the NK cells are from peripheral blood; Preferably, the NK cells are derived from peripheral blood mononuclear cells of patients with acute leukemia; 5. The method according to claim 3, characterized in that The activation culture medium comprises a basal culture medium, cytokines, fetal bovine serum and antibiotics; Preferably, the cytokines are IL-12 and IL-15; Preferably, in the activation medium, the final concentration of IL-12 is 10 ng / mL, the final concentration of IL-15 is 50 ng / mL, the volume percentage of antibiotics is 1%, and the volume percentage of fetal bovine serum is 10%.

6. The method according to claim 3, characterized in that The culture conditions are: 37° C., 5% CO 2 for 12 to 24 hours.

7. The method according to claim 3, characterized in that Each 1×10 5 ~1×10 6 NK cells, the added concentration of the thiol compound GSHEE is 30-70 μM; Preferably, every 1×10 5 ~1×10 6 NK cells, the added concentration of the thiol compound GSHEE is 40-60 μM; Preferably, every 1×10 5 ~1×10 6 NK cells, the added concentration of the thiol compound GSHEE is 50 μM.

8. A NK cell with enhanced killing activity, characterized in that: The method is used to obtain the product according to any one of claims 3 to 7.

9. Use of the NK cells according to claim 8 in the preparation of anti-tumor drugs or pharmaceutical compositions; Preferably, the tumor is acute leukemia.

10. An anti-tumor drug, characterized in that: Containing the NK cell according to claim 8.

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