CN115305238B - Application of component for improving natural killer cell killing power in natural killer cell culture - Google Patents
Application of component for improving natural killer cell killing power in natural killer cell culture Download PDFInfo
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- 210000000822 natural killer cell Anatomy 0.000 title claims abstract description 75
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- 238000004113 cell culture Methods 0.000 title abstract description 5
- 230000002147 killing effect Effects 0.000 claims abstract description 36
- 239000004480 active ingredient Substances 0.000 claims abstract description 10
- 230000002708 enhancing effect Effects 0.000 claims abstract description 9
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- 108090000765 processed proteins & peptides Proteins 0.000 claims description 18
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- KHGNFPUMBJSZSM-UHFFFAOYSA-N Perforine Natural products COC1=C2CCC(O)C(CCC(C)(C)O)(OC)C2=NC2=C1C=CO2 KHGNFPUMBJSZSM-UHFFFAOYSA-N 0.000 description 2
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- C12N5/0646—Natural killers cells [NK], NKT cells
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Abstract
The invention discloses an application of a component for improving natural killer cell killing power in natural killer cell culture, and an application of protecting AMARApeptide as an active component for improving human natural killer cell killing power in-vitro culture of human natural killer cells to improve the killing power. Experimental data shows that: AMARApeptide promotes the expression of killer core proteins CD107a, performin and Granzyme B in human natural killer cells from a molecular level; AMARApeptide enhances the killing power of human natural killer cells on tumor cells from the cell level. The research result proves that AMARApeptide is an effective active ingredient capable of enhancing the killing power of human natural killer cells, and has good development and application prospects in preparing a culture medium for enhancing the killing power of human natural killer cells.
Description
Technical Field
The invention belongs to the field of cellular immunotherapy, and in particular relates to an application of a component for improving natural killer cell killing power in natural killer cell culture.
Background
Natural killer cells (NK cells) are a very important class of cytotoxic lymphocytes in the natural immune system, which is the first line of defense in the immune system. NK cells are a group of specialized cells that differ from the need for antibodies and Major Histocompatibility Complex (MHC) when acquired immunity is functional. NK cells react very rapidly when mammals are infected with viruses or when they are affected by tumors. Clinical experiments show that: after obtaining immune cells of a tumor patient, the immune cells are stimulated in vitro, amplified and cultured and then returned to the patient, so that the immune function of the patient can be directly killed, restored or enhanced, and the effect of inhibiting the growth of the tumor can be achieved. Therefore, the killing power of NK cells has great influence on the strength of the mammal in resisting virus, tumor and other abnormal invasion capacity.
At present, NK cells are mainly used for tumor immunotherapy by firstly obtaining NK cells of a patient, then culturing the NK cells in vitro and then infusing the NK cells back into the patient to play a role in therapy. However, some patients have poor NK cell activity and low killing power due to the body itself, resulting in unexpected therapeutic effects after feedback.
In order to improve the therapeutic effect after reinfusion, it is important and necessary to improve the killing power of NK cells in the in vitro culture link. Currently, various active ingredients capable of stimulating NK cells to enhance killing power are usually added into an in vitro culture medium, and the active ingredients comprise polysaccharide, monoclonal antibody, polypeptide and small molecule compound.
The active ingredients for stimulating NK cells to enhance the killing power are expanded, and more choices are provided for NK cell immunotherapy.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of a component for improving natural killer cell killing power in natural killer cell culture.
The invention aims at realizing the following technical scheme:
the application of AMARA peptide as an active ingredient for enhancing the killing power of human natural killer cells in-vitro culture of the human natural killer cells to improve the killing power of the human natural killer cells. The activity research shows that the in vitro stimulation of the human natural killer cells by the AMARA peptide can effectively improve the killing power of the human natural killer cells, in particular: from the molecular level, AMARA peptide promotes the expression of the killing core proteins CD107a, performin, granzyme B in human natural killer cells; AMARA peptides enhance the killing power of human natural killer cells on tumor cells at the cell level.
The application of AMARA peptide as an active ingredient for enhancing the killing power of human natural killer cells in preparing a human natural killer cell culture medium. The active ingredient AMARA peptide is added into the culture medium of the human natural killer cells for culturing the human natural killer cells, so that the killing power of the human natural killer cells can be improved.
The beneficial technical effects are as follows:
from the molecular level, AMARA peptide promotes the expression of the killing core proteins CD107a, performin, granzyme B in human natural killer cells; AMARA peptides enhance the killing power of human natural killer cells on tumor cells at the cell level. The research result proves that the AMARA peptide is an effective active ingredient capable of enhancing the killing power of the natural killer cells, and has good development and application prospects in preparing a culture medium for enhancing the killing power of the natural killer cells.
Drawings
FIG. 1 shows cell phenotypes before and after NK cell immunomagnetic bead sorting as measured by flow cytometry.
FIG. 2 shows the result of western blotting.
FIG. 3 shows the killing power of each group of NK cells against tumor SGC-7901 cells.
Detailed Description
1. Experimental materials
Human lymphocyte isolates were purchased from Hangzhou Union Biotechnology Co. NK cell immunomagnetic bead sorting systems were purchased from Biyun Tian organisms. NK cell medium was purchased from Shanghai Gei Biotech Co. AMARA peptides are purchased from MCE and have purity of more than or equal to 98 percent. CCK-8 reagent was purchased from Biyunshan organisms. Fluorescein isothiocyanate-labeled perforin mab, granzyme B mab, CD107a mab was purchased from beijing-bai-oibo technologies. SGC-7901 cells were purchased from ATCC.
2. Experimental method
1. Peripheral blood mononuclear cell isolation
A suitable amount of peripheral blood from healthy volunteers was collected under sterile conditions for NK cell preparation. Adding proper amount of human lymphocyte separating liquid into the centrifuge tube, and slowly adding proper amount of peripheral blood to the tube wall to avoid damaging the liquid level of lymphocyte separating liquid. Centrifuge at 894 Xg for 20min at room temperature. Sucking out the pale yellow mononuclear cell layer, placing into a centrifuge tube, centrifuging for 6min at 572 Xg at room temperature, discarding the supernatant, and precipitating to obtain peripheral blood mononuclear cells.
2. NK cell separation and purification
The peripheral blood mononuclear cells were washed with PBS, resuspended in PBS, counted, and 20. Mu.L (about 1X 10) 7 Adding anti-CD 3 immunomagnetic beads, incubating at 4deg.C for 15min, adding 2mL PBS, centrifuging to wash cells, fixing volume to 500 μL, slowly passing through MACS chromatographic column, washing the column, collecting unbound cells (i.e. CD 3-cells), PBS washing, repeating the above steps, adding anti-CD 56 immunomagnetic beads, passing through the column, collecting cells bound to the chromatographic column, i.e. CD3-CD56+ NK cells (phenotype measured by flow cytometry), PBS resuspension, centrifuging to wash, washing withNK cell culture medium at 37℃with 5% CO 2 Culturing under the condition.
3. NK cell grouping and intervention culture
NK cells were cultured by the following group intervention, with medium change every 2-3 d:
control group: NK cell culture medium at 37deg.C, 5% CO 2 Culturing under the condition;
low group: NK cell culture medium containing 5 μg/mL AMARA peptide was used at 37℃with 5% CO 2 Culturing under the condition;
high group: NK cell culture medium containing 10 μg/mL AMARA peptide was used at 37deg.C with 5% CO 2 Culturing under the condition.
4. Determination of killing force (molecular level)
NK cells cultured for 9d in a grouping interference way are taken, the cells are collected, total proteins are extracted, and protein quantification is carried out by a BCA method. 40 mug total protein is taken from each group, the total protein is loaded on sodium dodecyl sulfate polyacrylamide gel electrophoresis, film transfer is carried out, 5% of skimmed milk powder by mass fraction is added, and a shaking table is closed for 1h at room temperature and low speed. Diluted CD107a, performin, granzyme B, beta-actin primary antibodies were incubated overnight in a shaker at 4 ℃. TBST washes the membrane 4 times, 10min each. Diluted IgG secondary antibody was added and incubated for 2h at room temperature. And (3) adding ECL luminescent liquid for developing, exposing the film, developing the film, and photographing.
5. Determination of killing force (cell layer)
Target cells: SGC-7901 cells were cultured in RPMI-1640 medium containing 10% FBS at 37℃with 5% CO 2 Culturing under the condition, taking cells in logarithmic growth phase for testing.
Effector cells: the above grouping interfered with NK cells cultured for 9 d.
The target cells and effector cells were prepared into 5X 10 cells using RPMI-1640 medium containing 10% FBS 4 Individual/mL and 2.5X10 5 100 mu L of each cell suspension is inoculated into a 96-well plate (the effective target ratio is 5:1), a single-effect cell group, a single-target cell group and a blank group are simultaneously arranged, 5 compound holes are arranged in each group, and the temperature is 37 ℃ and the concentration of CO is 5 percent 2 After 48h incubation, 10. Mu. LCCK-8 reagent was added to each well, incubation was continued in the incubator for 4h, OD at 450nm was detected, and tumor cells were counted as followsKilling activity of (2): killing force (%) = [1- (effective target group OD value-single effect cell group OD value)/(single target cell group OD value-blank group OD value)]×100％。
6. Statistical analysis
Statistical analysis was performed using SPSS 19.0 software and the metrology data were expressed as mean.+ -. Standard deviation. The comparison between the two groups uses independent sample t-test. P <0.05 is statistically significant for the differences.
3. Experimental results
1. NK cell separation and purification
FIG. 1 shows the cell phenotype of NK cells before and after immunomagnetic bead sorting as measured by a flow cytometer, the purity of NK cells represented by CD3-CD56+ after sorting is remarkably improved (8.7% before sorting, 99.8% after sorting), and NK cells are successfully separated and purified.
2. Determination of killing force (molecular level)
NK cell-secreted performin, granzyme B plays an important role in promoting target cell death. CD107a is a marker of degranulation when NK exerts a killing effect. Therefore, the skilled artisan usually detects the expression levels of these three proteins at the molecular level to evaluate NK cell killing. FIG. 2 shows the western blotting results, which clearly show that the expression of CD107a, perforin and Granzyme B in NK cells of the Low group and the High group is obviously improved compared with that of the control group.
3. Determination of killing force (cell layer)
The killing power of each group of NK cells against tumor SGC-7901 cells is shown in Table 1 and FIG. 3. The results clearly show that the killing power of the NK cells of the Low group and the High group on SGC-7901 cells is obviously improved compared with the control group (P < 0.05).
TABLE 1 killing of NK cells of groups on tumor SGC-7901 cells
| Group of | Control group | Low group | High group |
| Killing power (%) | 40.73±3.29 | 55.18±2.77* | 79.25±3.41* |
The above experiments prove that AMARA peptide has the effect of enhancing killing in human natural killer cells. Specifically: the molecular level AMARA peptide promotes the expression of killing power core proteins CD107a, performin and Granzyme B in human natural killer cells; at the cell level, AMARA peptides enhance the killing of human natural killer cells against tumor cells. The test result proves that AMARApeptide is an effective active ingredient capable of enhancing the killing power of human natural killer cells.
The above embodiments are intended to specifically describe the essential aspects of the present invention. It will be appreciated by persons skilled in the art that the scope of the invention should not be limited to the specific embodiments described above.
Claims (1)
- Application of AMARA peptide as an active ingredient for enhancing killing power of human natural killer cells in preparation of human natural killer cell culture medium, wherein the concentration of the AMARA peptide in the culture medium is 5 mug/mL or 10 mug/mL.
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