A method for improving the killing function of nk cells and application thereof
By adding heme oxygenase 1 antibody to the NK cell culture medium, the expression of heme oxygenase 1 in NK cells was blocked or its activity was inhibited, promoting the secretion of granzyme B and perforin, thus solving the problem of insufficient NK cell killing function and significantly improving the killing ability of NK cells against tumor cells.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SHUNDE HOSPITAL SOUTHERN MEDICAL UNIV (THE FIRST PEOPLES HOSPITAL OF SHUNDE FOSHAN)
- Filing Date
- 2023-02-20
- Publication Date
- 2026-06-02
AI Technical Summary
The cytotoxic function and antitumor activity of NK cells in clinical practice still need to be further improved.
By adding heme oxygenase 1 antibody to NK cell culture medium, the expression of heme oxygenase 1 in NK cells was blocked or its activity was inhibited, thereby promoting the secretion of granzyme B and perforin.
It significantly enhances the killing effect of NK cells on target cells Yac-1 and K562, and strengthens the anti-tumor function of NK cells.
Smart Images
Figure HDA0004087101370000011 
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Abstract
Description
Technical Field
[0001] This invention relates to the field of NK cell culture technology, specifically to a method and application for improving the cytotoxic function of NK cells. Background Technology
[0002] Natural killer cells (NK cells) are effector cells of the innate immune system, playing a crucial role in defending against pathogen invasion and in anti-tumor activity. NK cells have attracted attention due to their ability to kill cells without pre-stimulation. In addition to their powerful killing function, NK cells can also regulate immune function by secreting cytokines. NK cells can kill target cells directly or indirectly. Direct killing occurs through the Fas-FasL pathway and the TNF-α pathway, recruiting Caspase 8 to induce apoptosis in target cells. Indirect killing occurs through the secretion of granzymes, perforin, and interferon. NK cells primarily exert their function through cytotoxicity. Currently, the killing function of NK cells in the clinical field is still not strong enough, especially their anti-tumor activity in immunotherapy, which needs further improvement. Therefore, it is necessary to investigate a method to enhance the killing function of NK cells, thereby improving their anti-tumor function. Summary of the Invention
[0003] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a method and application for enhancing the cytotoxic function of NK cells, thereby improving the anti-tumor function of NK cells.
[0004] A first aspect of the present invention provides a method for improving the killing function of NK cells.
[0005] Specifically, the method includes the following steps:
[0006] (1) Add heme oxygenase 1 antibody to NK cell culture medium to prepare antibody culture medium;
[0007] (2) The antibody culture medium was subjected to aseptic filtration.
[0008] (3) Isolate NK cells and culture them in antibody culture medium.
[0009] Preferably, in step (1), the clone number of the heme oxygenase 1 antibody is EP1391Y, and the antibody was purchased from Abcam.
[0010] Preferably, in step (1), the final concentration of heme oxygenase 1 antibody in the antibody culture medium is 9.5 μg / ml-10.5 μg / ml.
[0011] More preferably, in step (1), the final concentration of heme oxygenase 1 antibody in the antibody culture medium is 10 μg / ml.
[0012] Preferably, in step (1), the NK cell culture medium is DMEM culture medium with 10% fetal bovine serum added and 1000 U / ml IL-2 added.
[0013] Preferably, in step (2), the aseptic filtration process uses a sterile vacuum filtration bottle or a needle filter.
[0014] Preferably, the pore size of the sterile filtration process is 0.22 μm.
[0015] Preferably, in step (3), the NK cells are mouse spleen NK cells.
[0016] Preferably, the mouse is an 8-week-old C57 / BL6 mouse.
[0017] Preferably, in step (3), the NK cells are treated in antibody culture medium for 48 hours.
[0018] Preferably, in step (3), the NK cells are cultured in a cell culture incubator.
[0019] A second aspect of the present invention provides a method for enhancing the cytotoxic function of NK cells and its application in NK cell culture.
[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0021] This invention treats NK cells with heme oxygenase 1 antibody, blocking the expression of heme oxygenase 1 in NK cells or inhibiting the effective substances of heme oxygenase 1 activity. Treatment with heme oxygenase 1 antibody promotes the secretion of granzyme B and perforin from NK cells and enhances the killing effect of NK cells on target cells Yac-1 and K562, thereby improving the anti-tumor function of NK cells. Attached Figure Description
[0022] Figure 1 The absorbance of NK cells in each group after adding different concentrations of heme oxygenase-1 antibody;
[0023] Figure 2 The granzyme B and perforin secretion of NK cells in each group after in vitro culture medium treatment;
[0024] Figure 3 The killing effect of NK cells on Yac-1 cells and K562 cells in each group;
[0025] Figure 4This shows the secretion of granzyme B and perforin in NK cells of different groups after treatment in mice. Detailed Implementation
[0026] To enable those skilled in the art to more clearly understand the technical solutions described in this invention, the following embodiments are provided for illustration. It should be noted that the following embodiments do not constitute a limitation on the scope of protection claimed by this invention.
[0027] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.
[0028] Example 1
[0029] A method to enhance the killing function of NK cells.
[0030] (1) Preparation of the drug solution: Prepare NK cell culture medium by adding 100 ml fetal bovine serum (FBS) and 1×10⁻⁶ ions to 1 L of DMEM medium. 6 UIL-2; Prepare NK cell culture medium containing heme oxygenase 1 antibody. Add the heme oxygenase 1 antibody (clone number: EP1391Y, purchased from Abcam) to the NK cell culture medium; the final concentration is 10 μg / ml. This solution can then be used to treat NK cells. Prepare a blank control solution and NK cell culture medium. Add 100 ml fetal bovine serum (FBS) and 1 × 10⁻⁶ ions to 1 L of DMEM medium. 6 U IL-2;
[0031] (2) Sorting of mouse spleen NK cells: Eight-week-old C57 / BL6 mice were euthanized by cervical dislocation, soaked in 75% ethanol for 5 min, and the spleen was removed in a biosafety cabinet. Single lymphocytes were obtained by grinding, and 6 ml of PBS was added. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and 2 ml of erythrocyte lysis buffer was added for resorting. The cells were lysed on ice for 5 min, and 4 ml of PBS was added to terminate the lysis. The cells were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and 1 ml of NK cell culture medium was added to resuspend the cells. The cells were then passed through a 100-mesh sieve. Flow cytometry was used to stain the cells, with each 1x10 cells... 6 1 μg of CD3-Percp-cy5.5 and NK1.1-APC antibody were added to the cells and incubated at 4°C in the dark for 1 h. After incubation, PBS was added to stop staining, and the cells were centrifuged at 1500 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in 1 ml of NK cell culture medium. NK cells were sorted using a BD Arial II flow cytometer with a 70 μm nozzle in Purity mode for CD3 cell sorting. - NK1.1 +NK cells were in a 96-well plate;
[0032] (3) 5000 mouse spleen NK cells were seeded into 24-well plates with 100 μl of culture medium per well. The cells were divided into two groups: 0 μg / ml group (blank group) and 10 μg / ml group, with 6 replicates in each group.
[0033] (4) After overnight culture, cells were collected and the culture medium was replaced. The 0 μg / ml group was replaced with new NK cell culture medium, and the 10 μg / ml group was replaced with NK cell culture medium containing a final concentration of 10 μg / ml HO-1 antibody.
[0034] (5) After 48 hours of treatment, 10 μl of CCK-8 reagent was added to each well and incubated in an incubator for 4 hours. The absorbance of each group at 450 nm was then measured.
[0035] The experimental results are as follows Figure 1 As shown, the OD value of the 0 μg / ml group (blank group) was 0.343±0.050, and the OD value of the 10 μg / ml group was 0.513±0.052.
[0036] Comparative Example 1
[0037] A method to enhance the killing function of NK cells.
[0038] The difference between Comparative Example 1 and Example 1 is that in step (1), the final concentration of the heme oxygenase 1 antibody added to the NK cell culture medium is 2 μg / ml.
[0039] Following the same experimental procedures as in Example 1 Figure 1 The results showed that the OD value of the 2 μg / ml group was 0.323 ± 0.077.
[0040] Comparative Example 2
[0041] A method to enhance the killing function of NK cells.
[0042] The difference between Comparative Example 2 and Example 1 is that in step (1), the final concentration of the heme oxygenase 1 antibody added to the NK cell culture medium is 5 μg / ml.
[0043] Following the same experimental procedures as in Example 1 Figure 1 The results showed that the OD value of the 5 μg / ml group was 0.370 ± 0.051.
[0044] Comparative Example 3
[0045] A method to enhance the killing function of NK cells.
[0046] The difference between Comparative Example 3 and Example 1 is that in step (1), the final concentration of the heme oxygenase 1 antibody added to the NK cell culture medium is 15 μg / ml.
[0047] Following the same experimental procedures as in Example 1 Figure 1 The results showed that the OD value of the 15 μg / ml group was 0.362 ± 0.041.
[0048] Based on the results of Example 1 and Comparative Examples 1-3, the differences in OD values between the 2 μg / ml group, the 5 μg / ml group, and the 15 μg / ml group and the 0 μg / ml group were not statistically significant. However, the OD value of the 10 μg / ml group was significantly higher than that of the 0 μg / ml group, being 1.5 times the OD value of the 0 μg / ml group. This indicates that the use of 10 μg / ml HO-1 antibody in Example 1 can promote the growth of NK cells.
[0049] To further illustrate the cytotoxic function of NK cells after treatment with the 10 μg / ml group in Example 1, the following is an identification of the NK cell cytotoxic function.
[0050] 1. Identification of NK cell granzyme B and perforin secretion:
[0051] (1) Experimental grouping and treatment:
[0052] The cells were divided into four groups, with three replicates per group. The rabbit IgG control group was treated with 10 μg / ml rabbit IgG for 12 h; the heme oxygenase-1 antibody treatment group was the same as the group treated with 10 μg / ml HO-1 antibody for 48 h as in Example 1; and the rabbit IgG control + RMA-S cell stimulation group was treated with 10 μg / ml rabbit IgG for 12 h, followed by 1x10... 6 Cells were stimulated for 6 hours; the group treated with heme oxygenase 1 antibody + RMA-S cell stimulation group, and the group treated with 10 μg / ml HO-1 antibody for 48 hours in Example 1, were further stimulated with 1x10 6 Cells were stimulated for 6 hours. Each group required the addition of a protein transport inhibitor to suppress the extracellular secretion of granzyme B and perforin.
[0053] (2) Detection of NK cell granzyme B and perforin secretion:
[0054] Collect cells into flow cytometry tubes by adding 2 ml of PBS, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 100 μl of staining solution containing 1 μg CD3-Percp-cy5.5 and NKp46-efluro450 antibody, and stain at 4°C in the dark for 1 h. After staining, add 2 ml of PBS to stop, centrifuge at 1500 rpm for 5 min, discard the supernatant, add 100 μl of fixation and transmembrane incubation solution, and incubate at 4°C in the dark for 1 h. Add 1 ml of fixation and transmembrane washing buffer, centrifuge at 3000 rpm for 5 min, discard the supernatant, add 100 μl of staining solution containing 2 μg GranzymeB-PE antibody and Perforin-FITC, and stain at 4°C in the dark for 8 h. Add 2 ml of PBS, centrifuge at 3000 rpm for 5 min, discard the supernatant, and resuspend the cells in 400 μl of PBS. Analyze the proportion of GranzymeB-positive and Perforin-positive NK cells using flow cytometry.
[0055] (3) Experimental results:
[0056] like Figure 2 As shown, regardless of whether in the RMA-S stimulation state or the non-stimulation state, refer to Figure 2 As shown in Part A, treatment with heme oxygenase 1 antibody significantly increased the secretion of NK cell granzyme B, indicating that heme oxygenase 1 antibody treatment enhanced the degranulation function of NK cells; regardless of whether RMA-S stimulation or non-stimulation was performed, refer to Figure 2 As shown in Part B, treatment with heme oxygenase 1 antibody significantly increased the secretion of perforin by NK cells, indicating that heme oxygenase 1 antibody has the effect of enhancing the cytotoxicity of NK cells.
[0057] 2. Identification of the cytotoxic effect of NK cells on target cells Yac-1 and K562:
[0058] (1) 51 Cr release assay was used to detect the killing effect of NK cells on Yac-1 and K562 tumor cells, using 100 μCi 51 Cr-labeled target cells were added to a 96-well cell culture plate (1 x 10⁻⁶ cells per well). 4NK cells were prepared in three replicates per group, with 100 μl per well. 100 μl of NK cells treated in Example 1 (i.e., HO-1Ab treatment group) or NK cells treated with rabbit IgG (control group) for 48 h were added to each well (target cells were Yac-1, effector-to-target ratio was 10:1, 5:1, 1:1; target cells were K562, effector-to-target ratio was 40:1, 20:1, 10:1). Cells were incubated at 37°C for 4 h, centrifuged at 1000 rpm for 10 min, and 100 μl of supernatant was transferred to a detection tube. The radioactivity per minute (cpm value) was measured using a liquid scintillation counter, and the killing rate of NK cells against target cells was calculated.
[0059] (2) Experimental results: such as Figure 3 As shown in Part A of Example 1, treatment with heme oxygenase 1 antibody significantly enhanced the killing effect of NK cells on Yac-1 tumor cells. Regardless of the effector-to-target ratio of 10:1 or 5:1, heme oxygenase 1 antibody significantly improved the killing function of NK cells against Yac-1. Figure 3 As shown in Part B of Example 1, the killing effect of NK cells on K562 tumor cells was significantly enhanced after treatment with heme oxygenase 1 antibody. Regardless of whether the effector-target ratio was 40:1 or 20:1, heme oxygenase 1 antibody could significantly enhance the killing function of NK cells on K562.
[0060] Comparative Example 4
[0061] A method to enhance the killing function of NK cells.
[0062] The difference between Comparative Example 4 and Example 1 is that the heme oxygenase 1 antibody was directly injected into mice, instead of treating NK cells in vitro with the heme oxygenase 1 antibody in Example 1.
[0063] Identification of NK cell granzyme B and perforin secretion:
[0064] (1) Experimental grouping and treatment:
[0065] The mice were divided into two groups, with 10 mice in each group. The rabbit IgG control group received an intraperitoneal injection of rabbit IgG (10 μg) every 2 days; the HO-1Ab treatment group received an intraperitoneal injection of HO-1 antibody (10 μg) every 2 days.
[0066] (2) Detection of NK cell granzyme B and perforin secretion:
[0067] After treatment, mice were injected intraperitoneally with 200 μg Poly I:C. Eighteen hours later, they were euthanized by cervical dislocation. The cells were soaked in 75% ethanol for 5 minutes. The spleen was harvested in a biosafety cabinet, and single lymphocytes were obtained by grinding. 6 ml of PBS was added, and the cells were centrifuged at 1500 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in 2 ml of erythrocyte lysis buffer and lysed on ice for 5 minutes. Lysis was terminated by adding 4 ml of PBS, centrifuged at 1500 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in 1 ml of NK cell culture medium and passed through a 100-mesh sieve. Cell counting was performed, with cells from each mouse divided into two wells, 2 x 10-1 cells per well. 6 One lymphocyte was seeded into a 24-well plate, one well was left unstimulated, and 2 x 10^6 lymphocytes were added to the other well. 6 RMA-S cells were stimulated. A protein transport inhibitor was added, and the cells were cultured in a cell culture incubator for 6 hours. 2 ml of PBS was added to collect the cells into a flow cytometry tube, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and 100 μl of staining solution containing 1 μg of CD3-Percp-cy5.5 and NKP46-efluro450 antibody was added. Staining was performed at 4°C in the dark for 1 hour. After staining, 2 ml of PBS was added to stop the incubation, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and 100 μl of fixation and transmembrane incubation solution was added. Incubation was performed at 4°C in the dark for 1 hour. 1 ml of fixation and transmembrane washing buffer was added, centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and 100 μl of staining solution containing 2 μg of Granzyme B-PE and Perforin-FITC antibody was added. Staining was performed at 4°C in the dark for 8 hours. 2 ml of PBS was added, centrifuged at 3000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in 400 μl of PBS. The proportion of GranzymeB-positive and Perforin-positive NK cells was detected by flow cytometry.
[0068] (3) Experimental results: such as Figure 4 As shown, regardless of whether in the RMA-S stimulation state or the non-stimulation state, refer to Figure 4 As shown in Part A, direct injection of heme oxygenase-1 antibody into mice did not significantly increase NK cell granzyme B secretion, indicating that direct injection of heme oxygenase-1 antibody into mice does not improve NK cell degranulation function; regardless of whether RMA-S stimulation or no stimulation was performed, refer to... Figure 4 As shown in Part B, direct injection of heme oxygenase 1 antibody into mice did not significantly increase the secretion of perforin by NK cells, indicating that direct injection of heme oxygenase 1 antibody into mice could not enhance the cytotoxic effect of NK cells.
[0069] In summary, in Example 1, in vitro treatment of NK cells with 10 μg / ml heme oxygenase 1 antibody promoted NK cell growth, enhanced NK cell degranulation and cytotoxicity, and improved the killing function of NK cells against Yac-1 and K562 tumor cells. However, changing the concentration of heme oxygenase 1 antibody or directly injecting heme oxygenase 1 antibody into mice did not improve the degranulation and cytotoxicity of NK cells.
[0070] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, any technical solutions obtained by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concept of the present invention and on the existing technology should be within the scope of protection defined by the claims.
Claims
1. A method for enhancing the cytotoxic function of NK cells, characterized in that, The method includes the following steps: (1) Add heme oxygenase 1 antibody to NK cell culture medium to prepare antibody culture medium, wherein the clone number of the heme oxygenase 1 antibody is EP1391Y, and the concentration of heme oxygenase 1 antibody in the antibody culture medium is 10 μg / ml; (2) Perform aseptic filtration on the antibody culture medium; (3) Isolate NK cells and culture them in antibody culture medium.
2. The method according to claim 1, characterized in that, In step (1), the NK cell culture medium is DMEM culture medium with 10% fetal bovine serum and 1000 U / ml IL-2 added.
3. The method according to claim 1, characterized in that, In step (2), the aseptic filtration process is performed using a sterile vacuum filtration bottle or a needle filter.
4. The method according to claim 3, characterized in that, The aseptic filtration process passes through a pore size of 0.22 μm.
5. The method according to claim 1, characterized in that, In step (3), the NK cells are mouse spleen NK cells.
6. The method according to claim 5, characterized in that, The mice were 8-week-old C57 / BL6 mice.
7. The method according to claim 1, characterized in that, In step (3), the NK cells are placed in antibody culture medium and treated for 48 hours.