A culture method for improving the killing activity of NK cells

Abstract

The present invention belongs to the field of immunology, and discloses a culture method for improving the killing activity of NK cells. It comprises the following steps: (1) isolating and obtaining peripheral blood mononuclear cells, (2) coating a culture bottle with a coating solution, and inducing and culturing in a culture medium containing rice oligopeptides, docosahexaenoic acid, IL-15 and IL-12; (3) amplifying and culturing in a culture medium containing DMEM culture medium, pyrazinecarboxamide, scutellarin, glutamine, IL-2 and IL-21 to obtain NK cells. In the present invention, rice oligopeptides and docosahexaenoic acid can act synergistically to promote the induced differentiation of NK cells, and can cooperate with pyrazinecarboxamide and scutellarin in the amplification culture medium to effectively improve the in vitro tumor killing activity of NK cells, which plays an important role in tumor immunotherapy.

Description

Technical Field

[0001] The invention belongs to the field of immunology, and in particular relates to a culture method for improving the killing activity of NK cells. Background Art

[0002] At present, malignant tumors have always been the biggest threat to human health. People around the world have been constantly exploring better tumor treatment methods. Developing fast and effective treatment plans for different tumor types can effectively control the mortality rate of the disease. Tumor treatment includes surgery, radiotherapy, chemotherapy and immunotherapy.

[0003] In recent years, tumor immunotherapy has developed rapidly. It refers to the use of immunological principles to regulate the immune system, mobilize the body's anti-tumor specific immune response, and rely on the human immune system to inhibit the function of tumor cells to achieve the purpose of clearing tumor cells. Although with the advancement of science and the improvement of modern medical technology, the cure rate of some tumors has been improved to a certain extent in clinical practice, the overall incidence of tumors is still high. Therefore, in-depth research on the anti-tumor immune system and improving patients' response to tumor immunotherapy are of great social significance.

[0004] Natural killer cells (NK cells) are immune effector cells with certain killing capabilities. They are an important component of the anti-tumor innate immune response. They can secrete a large number of chemokines and cytokines, etc., and play a key role in the body's anti-tumor, anti-viral infection and immune regulation. Compared with T cells, NK cells have complex and diverse activation and inhibitory receptors on their surfaces. They can respond quickly and directly identify tumor cells without the need for pre-stimulation of antigens. Therefore, more and more researchers have begun to focus on NK cells as the focus of tumor immunotherapy.

[0005] Improving the number of cell proliferation and cell killing ability is the main factor to improve the effect of cell therapy, but in peripheral blood lymphocytes, the number of NK cells is relatively small, accounting for only 5-10%, and the existing methods of culturing NK cells in vitro have the problems of low induction efficiency and poor killing activity. Therefore, it is necessary to improve the culturing process of NK cells. Summary of the invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a method for cultivating NK cells to improve their killing activity, which can effectively increase the number of CD3 - CD56 + The proportion of cells can improve the tumor cell killing activity of NK cells.

[0007] The purpose of the present invention is achieved by the following technical solutions:

[0008] A method for improving the killing activity of NK cells comprises the following steps:

[0009] (1) Isolation and acquisition of peripheral blood mononuclear cells;

[0010] (2) Coating the culture flask with the coating solution, using the coated culture flask as an induction culture flask, resuspending the peripheral blood mononuclear cells in step (1) with the induction culture medium, inoculating the cells into the induction culture flask, and inducing the cells for 2-3 days to obtain a cell culture solution;

[0011] The induction medium comprises: AIM-V medium, rice oligopeptide 3.5-10 μg / mL, docosahexaenoic acid 1-5 μg / mL, IL-15 80-100 ng / mL, IL-12 200-300 IU / mL;

[0012] (3) Add expansion medium to the cell culture medium in step (2) and continue culturing for 8-10 days. Replenish expansion medium every 2-3 days to keep the cell density at 1-3×10 6 / mL, and NK cells were obtained;

[0013] The expansion culture medium is composed of: DMEM culture medium, pyrazinecarboxamide 10-20 ng / mL, scutellarin 1-5 μg / mL, glutamine 5-15 μg / mL, IL-2 800-1000 IU / mL, and IL-21 90-120 IU / mL.

[0014] Furthermore, in step (2), the rice oligopeptide is obtained by enzymatic hydrolysis and filtration of rice protein powder.

[0015] Furthermore, in step (2), the coating solution consists of AIM-V culture medium and anti-CD3 antibodies added thereto, and the concentration of the anti-CD3 antibodies in the coating solution is 0.5-5 μg / mL.

[0016] Furthermore, in step (1), the specific method for preparing the peripheral blood mononuclear cells is: collecting human peripheral blood under sterile conditions, diluting the peripheral blood with PBS buffer, and separating and obtaining peripheral blood mononuclear cells by dextran-diatrizoate density gradient centrifugation.

[0017] Furthermore, in step (2), the seeding density of the peripheral blood mononuclear cells is 1-5×10 6 Pieces / mL.

[0018] Furthermore, the induction culture and expansion culture are both carried out at 37° C. and 5% CO 2 .

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention has shown through research that rice oligopeptides and docosahexaenoic acid in the induction medium promote the induction and differentiation efficiency of NK cells, and can cooperate with pyrazinecarboxamide and breviscapine in the expansion medium to effectively increase the number of CD3 - CD56 + ratio, thereby enhancing the tumor cell killing activity of NK cells.

[0021] 2. The present invention provides a method for culturing the killing activity of NK cells, which has high safety and the prepared NK cells play an important role in tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow cytometry test result diagram of NK cells cultured in Example 1 of the present invention;

[0023] Figure 2 This is a flow cytometry test result diagram of NK cells cultured in Comparative Example 1 of the present invention;

[0024] Figure 3 This is a flow cytometry test result diagram of NK cells cultured in Comparative Example 2 of the present invention;

[0025] Figure 4 This is a flow cytometry test result diagram of NK cells cultured in Comparative Example 3 of the present invention;

[0026] Figure 5 This is a flow cytometry test result diagram of NK cells cultured in Comparative Example 4 of the present invention;

[0027] Figure 6 This is a flow cytometry test result diagram of NK cells cultured in Comparative Example 5 of the present invention;

[0028] Figure 7 This is a graph showing the killing rate of K562 cells by NK cells cultured in Example 1 of the present invention and Comparative Examples 1-5;

[0029] Figure 8 The figure is a graph showing the killing rate of A549 cells by NK cells cultured in Example 1 of the present invention and Comparative Examples 1-5. DETAILED DESCRIPTION

[0030] The following examples are only used to illustrate the present invention and should not be construed as limiting the present invention. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0031] Acquisition of experimental materials:

[0032] The specific preparation method of rice oligopeptide is as follows: take 200g rice protein powder, add water and stir evenly, then use a sodium hydroxide solution with a concentration of 1mol / L to adjust the pH to 9, then add 2g alkaline protease, and stir for 2 hours in a water bath at 45°C. Adjust the pH to 6.8, then add 2.1g neutral protease, stir for 2.5h, pay attention to control the pH value of the hydrolyzate to remain unchanged, after the enzymolysis is completed, kill the enzyme at 98°C for 10min, then centrifuge the above mixture at room temperature at 5000rpm for 20min, first filter the solution with a ceramic membrane with a pore size of 200nm, then filter with an ultrafiltration membrane with a cutoff of 1000, and obtain rice oligopeptide after drying.

[0033] Example 1

[0034] A method for improving the killing activity of NK cells comprises the following steps:

[0035] (1) Isolation and acquisition of peripheral blood mononuclear cells (PBMC): Collect 20 mL of peripheral blood from subjects without autoimmune diseases, tumors, viral and bacterial infections, etc., and add heparin for anticoagulation. Dilute the peripheral blood with PBS buffer at a ratio of 1:1, add an equal volume of Ficoll separation solution to a sterile centrifuge tube in advance, and then add the diluted peripheral blood to the Ficoll separation solution (be careful not to shake or blow), centrifuge at 500×g in a centrifuge for 15 minutes, carefully remove the upper plasma, aspirate the middle white film layer cells, and then wash twice with 3 times PBS buffer, and centrifuge again at 500×g for 15 minutes. The precipitate is PBMC.

[0036] (2) Prepare coating solution: It is composed of AIM-V medium and anti-CD3 antibody added thereto. The concentration of CD3 antibody in the coating solution is 3 μg / mL. Add it to T75 cell culture flask for coating. Culture it overnight at 4°C. Use the coated culture flask as the induction culture flask. Take the peripheral blood mononuclear cells obtained in step (1) and resuspend them in the induction medium (AIM-V medium, 7.5 μg / mL rice oligopeptide, 3 μg / mL docosahexaenoic acid, 90 ng / mL IL-15, 260 IU / mL IL-12). 6 The cells were inoculated into the coated induction culture flask at a density of 100 cells / mL and induced for 3 days under the following culture conditions: 37°C, 5% CO2 incubator to obtain cell culture fluid;

[0037] (3) Add expansion medium (DMEM medium, 15 ng / mL pyrazine carboxamide, 3 μg / mL scutellarin, 10 μg / mL glutamine, 900 IU / mL IL-2, 100 IU / mL IL-21) to the cell culture medium in step (2), and continue culturing at 37°C and 5% CO2 for 9 days. Replenish expansion medium every 2 days to keep the cell density at 2×10 6 / mL, and NK cells were obtained.

[0038] Example 2

[0039] A method for improving the killing activity of NK cells comprises the following steps:

[0040] (1) Isolation and acquisition of peripheral blood mononuclear cells: the steps are the same as those in Example 1.

[0041] (2) Prepare coating solution: It is composed of AIM-V medium and anti-CD3 antibody added thereto. The concentration of CD3 antibody in the coating solution is 0.5 μg / mL. Add it to T75 cell culture flask for coating. Culture it overnight at 4°C. Use the coated culture flask as the induction culture flask. Take the peripheral blood mononuclear cells obtained in step (1) and resuspend them in the induction medium (AIM-V medium, 3.5 μg / mL rice oligopeptide, 1 μg / mL docosahexaenoic acid, 80 ng / mL IL-15, 200 IU / mL IL-12). 6 The cells were inoculated into the coated induction culture flask at a density of 100 cells / mL and induced for 2 days under the following culture conditions: 37°C, 5% CO2 incubator to obtain cell culture fluid;

[0042] (3) Add expansion medium (DMEM medium, 20 ng / mL pyrazine carboxamide, 5 μg / mL scutellarin, 15 μg / mL glutamine, 1000 IU / mL IL-2, 120 IU / mL IL-21) to the cell culture medium in step (2), and continue culturing at 37°C and 5% CO2 for 8 days. Replenish expansion medium every 2 days to keep the cell density at 1×10 6 / mL, and NK cells were obtained.

[0043] Example 3

[0044] A method for improving the killing activity of NK cells comprises the following steps:

[0045] (1) Isolation and acquisition of peripheral blood mononuclear cells: the steps are the same as those in Example 1.

[0046] (2) Prepare coating solution: It is composed of AIM-V medium and anti-CD3 antibody added thereto. The concentration of CD3 antibody in the coating solution is 5 μg / mL. Add it to T75 cell culture flask for coating. Culture it overnight at 4°C. Use the coated culture flask as the induction culture flask. Take the peripheral blood mononuclear cells obtained in step (1) and resuspend them in the induction medium (AIM-V medium, 10 μg / mL rice oligopeptide, 5 μg / mL docosahexaenoic acid, 100 ng / mL IL-15, 300 IU / mL IL-12). 6 The cells were inoculated into the coated induction culture flask at a density of 100 cells / mL and induced for 2 days under the following culture conditions: 37°C, 5% CO2 incubator to obtain cell culture fluid;

[0047] (3) Add expansion medium (DMEM medium, 10 ng / mL pyrazine carboxamide, 1 μg / mL scutellarin, 5 μg / mL glutamine, 800 IU / mL IL-2, 90 IU / mL IL-21) to the cell culture medium of step (2), and continue culturing at 37°C and 5% CO2 for 10 days. Replenish expansion medium every 3 days to keep the cell density at 3×10 6 / mL, and NK cells were obtained.

[0048] Comparative Example 1

[0049] Comparative Example 1 provides a culture method for improving the killing activity of NK cells, which is different from Example 1 in that rice oligopeptide is omitted and the rest is the same as Example 1.

[0050] Comparative Example 2

[0051] Comparative Example 2 provides a culture method for improving the killing activity of NK cells, which is different from Example 1 in that docosahexaenoic acid is omitted, and the rest is the same as Example 1.

[0052] Comparative Example 3

[0053] Comparative Example 3 provides a culture method for improving the killing activity of NK cells, which differs from Example 1 in that the order of rice oligopeptides and docosahexaenoic acid in the induction medium of step (2) and pyrazinecarboxamide and scutellarin in the expansion medium of step (3) is swapped, and the rest is the same as Example 1.

[0054] Comparative Example 4

[0055] Comparative Example 4 provides a culture method for improving the killing activity of NK cells, which is different from Example 1 in that pyrazinecarboxamide is omitted and the rest is the same as Example 1.

[0056] Comparative Example 5

[0057] Comparative Example 5 provides a culture method for improving the killing activity of NK cells, which is different from Example 1 in that breviscapine is omitted and the rest is the same as Example 1.

[0058] Test Example 1

[0059] The NK cells obtained in Example 1 and Comparative Examples 1-5 were centrifuged at 500 rpm for 5 min, the supernatant was discarded, and then washed 3 times with PBS buffer, and the cells were resuspended to adjust the concentration to 1×10 6 / mL, and obtain the cell sample to be tested. Add 10μL of phycoerythrin conjugate-labeled CD56 monoclonal antibody (CD56-PE) and 10μL of fluorescein isothiocyanate-labeled CD3 monoclonal antibody (CD3-FITC) to the above cell sample to be tested (100μL), incubate at room temperature for 15min, wash with PBS three times to wash away the excess antibody. Finally, resuspend the cells with PBS again, load on flow cytometer, and analyze CD3 - CD56 + The expression of Figures 1 to 6 shown.

[0060] Depend on Figures 1 to 6 The test results show that CD3 - CD56 + The expression rate of the cells was significantly better than that of Comparative Examples 1 to 5. Comparative Examples 1 and 2 omitted rice oligopeptides and docosahexaenoic acid, respectively, and Comparative Example 3 adjusted the timing of adding soybean oligopeptides and docosahexaenoic acid. The results showed that CD3 - CD56 + The expression rates of the cells were significantly lower than those in Example 1, and the CD3 - CD56 + The cell expression rate was the lowest, indicating that the addition amount of the above rice oligopeptides and docosahexaenoic acid, as well as the addition timing of rice oligopeptides, docosahexaenoic acid, pyrazinecarboxamide, and scutellarin all had a significant effect on the expression of NK cells. Compared with Example 1, Comparative Examples 4 and 5 omitted pyrazinecarboxamide and scutellarin, respectively, and CD3 - CD56 + The cells showed different degrees of decrease. It can be seen that the addition of pyrazinecarboxamide and breviscapine also had an effect on CD3 - CD56 + The expression of cells is affected.

[0061] Experimental Example 2 CCK-8 method to measure the killing ability of NK cells on human chronic myeloid leukemia cells K562 cells

[0062] The NK cells obtained in Example 1 and Comparative Examples 1-5 were prepared to a density of 1×10 5 The cell suspension of 100 μL / mL was used as effector cells and inoculated into a 96-well plate. K562 tumor cells in the logarithmic growth phase were used as target cells. The effector-target ratio was controlled at 10:1, 20:1 and 40:1, respectively, and inoculated into a 96-well round-bottom plate. A blank control group, a control group with only effector cells and a control group with only target cells were also set up. Three replicate wells were set up in each group. The cells were cultured for 24 h, 10 μL of CCK-8 solution was added, and the cells were incubated at 37°C for 4 h. The absorbance value was measured at a wavelength of 450 nm using an ELISA reader. The killing rate was calculated according to the following formula. The experimental results of the killing rate of NK cells of Example 1 and Comparative Examples 1-5 on K562 tumor cells are shown in FIG. Figure 7 shown.

[0063] Killing rate (%) = [1-(OD value of experimental group-OD value of effector cell control group only) / (OD value of target cell control group only)] × 100%

[0064] Depend on Figure 7 From the statistical results of the killing experiment, it can be seen that the higher the ratio of effector cells to target cells, the greater the killing rate of NK cells. When the effector-target ratio is 40:1, the killing rate of Example 1 is maintained at more than 95%, which is significantly higher than that of Comparative Examples 1 to 5, among which the killing efficiency of Example 1 is as high as 95.79%, while the killing rate of Comparative Example 3 is only 44.89% when the effector-target ratio is 40:1. It can be seen that rice oligopeptides and docosenoic acid promote the induction efficiency of NK cells in the induction medium, and pyrazinecarboxamide and scutellarin promote the proliferation of NK cells in the amplification medium. The two interact with each other and significantly improve the tumor killing rate of NK cells against K562 cells.

[0065] Experimental Example 3 MTT method to measure the killing ability of NK cells on A549 lung cancer cells

[0066] A549 lung cancer cells with vigorous growth were selected as target cells, and NK cells obtained in Example 1 and Comparative Examples 1 to 5 were selected as effector cells. The above NK cells were prepared into 2×10 5 Then A549 lung cancer cells were mixed at the ratio of 10:1, 20:1, and 40:1, and three replicate wells were set for each group. Effector cells, target cells, and a blank control group were set up at the same time, and cultured in a 37°C, 5% CO2 incubator for 12 hours. Then 20 μL of MTT solution was added thereto, and the culture was continued for 4 hours. The original culture medium was discarded, and 200 μL of DMSO was added to each well. The mixture was shaken for 10 minutes. According to the instructions, the OD value at 450 nm was detected using an enzyme reader.

[0067] The formula for calculating the killing rate is the same as that of Experiment 2. The experimental results of the killing rate of NK cells on A549 lung cancer cells are as follows: Figure 8 shown.

[0068] By analyzing Figure 8 From the results of the killing activity of NK cells against A549 cells, it can be seen that with the increase of the effect-target ratio, the killing rate increases. The killing rate of NK cells prepared in Example 1 against A549 lung cancer cells is the highest, which is 84.81%, which is significantly better than the NK cells in each comparative example, while the killing rate of NK cells obtained in comparative example 3 against A549 is the lowest. This shows that the rice oligopeptides and docosenoic acid added in the induction culture process of the present application and the pyrazinecarboxamide and scutellarin added in the amplification culture process can cooperate with each other to improve the tumor killing activity of NK cells.

[0069] In combination with the above content, it can be seen that the present invention provides a culture method for improving the killing activity of NK cells. The method is simple and efficient. The present invention proves through a large amount of experimental data that rice oligopeptides and docosenoic acid in the induction medium can effectively promote the induced differentiation of NK cells, and the addition of pyrazinecarboxamide in the expansion medium can also improve the in vitro proliferation efficiency of NK cells and promote CD3 - CD56 + Therefore, the rice oligopeptides and docosenoic acid in the induction culture process and the pyrazinecarboxamide and breviscapine added in the expansion culture process cooperated with each other to significantly enhance the killing ability of NK cells against K562 cells and A549 lung cancer cells.

[0070] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for cultivating NK cells to improve their killing activity, characterized in that: The following steps are involved: (1) Isolation and acquisition of peripheral blood mononuclear cells; (2) Coating the culture flask with the coating solution, using the coated culture flask as an induction culture flask, resuspending the peripheral blood mononuclear cells in step (1) with the induction culture medium, inoculating the cells into the induction culture flask, and inducing the cells for 2-3 days to obtain a cell culture solution; The induction medium comprises: AIM-V medium, rice oligopeptide 3.5-10 μg / mL, docosahexaenoic acid 1-5 μg / mL, IL-15 80-100 ng / mL, IL-12 200-300 IU / mL; the coating solution is made of AIM-V medium and anti-CD3 antibody added thereto, and the concentration of anti-CD3 antibody in the coating solution is 0.5-5 μg / mL; (3) Add expansion medium to the cell culture medium in step (2) and continue culturing for 8-10 days. Replenish expansion medium every 2-3 days to keep the cell density at 1-3×10 6 / mL, and NK cells were obtained; The expansion culture medium is composed of: DMEM culture medium, pyrazinecarboxamide 10-20 ng / mL, scutellarin 1-5 μg / mL, glutamine 5-15 μg / mL, IL-2 800-1000 IU / mL, and IL-21 90-120 IU / mL.

2. The method for cultivating NK cells to improve their killing activity according to claim 1, characterized in that: In step (2), the rice oligopeptide is obtained by enzymatic hydrolysis and filtration of rice protein powder.

3. The method for cultivating NK cells to improve their killing activity according to claim 1, characterized in that: In step (1), the specific method for preparing the peripheral blood mononuclear cells is: collecting human peripheral blood under sterile conditions, diluting the peripheral blood with PBS buffer, and separating and obtaining peripheral blood mononuclear cells by dextran-diatrizoate density gradient centrifugation.

4. The method for cultivating NK cells to improve their killing activity according to claim 1, characterized in that: In step (2), the seeding density of the peripheral blood mononuclear cells is 1-5×10 6 Pieces / mL.

5. The method for cultivating NK cells to improve their killing activity according to claim 1, characterized in that: The induction culture and expansion culture were both carried out at 37° C. and 5% CO 2 .

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