CN115161278A - Application of small peptide derivative in improving killing power and migration activity of natural killer cells - Google Patents
Application of small peptide derivative in improving killing power and migration activity of natural killer cells Download PDFInfo
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- 230000005012 migration Effects 0.000 title claims abstract description 19
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Abstract
The invention discloses an application of a small peptide derivative in improving killing power and migration activity of natural killer cells, belongs to the field of biology, and relates to an application of a small peptide derivative in natural killer cell culture. The invention provides application of a small peptide derivative in improving killing power and migration activity of natural killer cells in vitro, wherein the small peptide derivative is N-Formyl-MLFK. Experiments show that the small peptide derivative N-Formyl-MLFK can improve the killing power of natural killer cells and the migration activity of the natural killer cells, and is beneficial to the natural killer cells to exert the killing effect of the natural killer cells.
Description
Technical Field
The invention relates to natural killer cells, in particular to application of a small peptide derivative in improving killing power and migration activity of natural killer cells.
Background
Natural Killer (NK) cells are important innate immune cells that the body resists infection by pathogenic microorganisms and clears the cancerous cells of the body. The function of NK cells is regulated by the balance between the signal intensity mediated by inhibitory receptors and activated receptors, and the activated NK cells can directly kill target cells and can secrete cytokines and inflammatory factors to participate in immune regulation. In recent years, anti-tumor immunotherapy based on NK cell immune function has become an emerging hot field of tumor therapy.
The killing power and the migration activity of the NK cells have important influence on the NK cells to play the immune function, and the NK cells with strong killing power and strong migration activity have strong killing and clearing capacity on target cells.
Therefore, improving the killing power and migration activity of NK cells is an important direction for NK cell research.
Disclosure of Invention
The invention aims to provide application of a small peptide derivative in improving killing power and migration activity of natural killer cells, wherein the small peptide derivative is N-Formyl-MLFK.
In order to achieve the purpose, the invention provides the following technical scheme:
an application of a small peptide derivative in improving killing power and migration activity of natural killer cells in vitro is disclosed, wherein the small peptide derivative is N-Formyl-MLFK.
The application of a small peptide derivative in preparing a culture solution for improving killing power and migration activity of natural killer cells is disclosed, wherein the small peptide derivative is N-Formyl-MLFK.
The invention discovers that: the small peptide derivative N-Formyl-MLFK can not only improve the killing power of natural killer cells, but also improve the migration activity of the natural killer cells, and is beneficial to the natural killer cells to exert the killing effect of the natural killer cells.
Drawings
FIG. 1 shows the results of measurement of the expression levels of the Granzyme B and Perforin proteins in each NK cell group, (B) is a photograph showing the migration of each NK cell group, wherein Blank represents a control, high represents 50. Mu.g/mLN-Formyl-MLFK intervention, and Low represents 25. Mu.g/mLN-Formyl-MLFK intervention.
Detailed Description
1. Experimental materials
Human Peripheral Blood Mononuclear Cells (PBMCs) were purchased from wuhansaioo biotechnology limited. RPMI 1640 medium and FBS were purchased from Gibco. Flow antibody was purchased from BD. The brand of the human NK cell complete medium is Procell, and is purchased from Wuhan Punuo Sai Life technologies, inc.
N-Formyl-MLFK, namely N-Formyl-Met-Leu-Phe-Lys, is purchased from Nanjing Kingsler Biotech limited, and has the product number RP12958 and the purity of more than or equal to 95 percent.
2. Experimental methods
1. Resuscitation, passage and cryopreservation of PBMC
1.1 Resuscitation
1) Preheating water in a constant-temperature water bath to 37 ℃;
2) Preparing a 15mL centrifuge tube, adding 5mL RPMI 1640 complete medium containing 10% FBS, and preheating in a 37 ℃ water bath;
3) After wearing goggles and thick woolen gloves, taking out cells to be revived from a liquid nitrogen tank, transferring the cells into a constant-temperature water bath kettle at 37 ℃ as soon as possible, and rewarming and shaking the cryopreservation tube to improve the rewarming speed;
4) Sucking the thawed cells in the cryopreservation tube into a prepared centrifuge tube, uniformly mixing, and centrifuging at 1000rpm for 5min;
5) Preparing a T-25 culture bottle, writing the cell name and date, and adding 4mL of complete culture medium;
6) After centrifugation, the supernatant was discarded, the cells were resuspended in 1mL of complete medium, transferred to T-25 cell culture, mixed well and transferred to CO 2 And (5) culturing and standing in an incubator.
Note that: when taking out cell cryopreserving pipe from the liquid nitrogen, if there is liquid nitrogen to get into in the cryopreserving pipe, need unscrew cryopreserving pipe, discharge inside remaining liquid nitrogen, later screw up cryopreserving pipe, place on the dry ice, then put into 37 ℃ water bath, avoid the difference in temperature too big to cause liquid nitrogen rapid gasification and explode.
Passage 1.2 (one transmission two)
1) When the confluence degree of the cells reaches more than 85 percent, passage can be carried out;
2) Opening the mouth of a culture bottle in the biological safety cabinet, and collecting the culture medium in the bottle;
3) Adding 3mL of sterile 1 XPBS into the culture bottle, horizontally placing the culture bottle to enable the PBS to be capable of infiltrating all areas on the bottom surface of the culture bottle, and removing the PBS;
4) Adding 1mL of digestive juice into the bottle, soaking the bottom surface, and adding CO at 37 DEG C 2 Incubating in an incubator for 1-2 min;
5) Observing whether the cells become round and float under an inverted microscope after the incubation is finished, if the cells are completely digested, directly adding 2mL of complete culture medium containing 10% FBS into the culture flask, and sucking the suspension into a 15mL centrifuge tube;
6) Centrifuging at 1000rpm for 5min;
7) Two new T-25 flasks were prepared and 4mL of complete medium was added to each flask.
8) After centrifugation, the supernatant was discarded, the centrifuged cells were suspended in 2mL of complete medium, and the resuspended cells were transferred to 2T-25 flasks, 1mL each;
9) Placing the flask horizontally, shaking for mixing, placing the flask at 37 deg.C and 5% CO 2 And (5) standing and culturing in an incubator.
1.3 frozen storage (one frozen per bottle T-25)
1) -6) reference passage step
7) After centrifugation, discarding the supernatant, resuspending the cell pellet with 1mL of cryopreservation solution, and transferring into a 1.8mL cryopreservation tube;
8) Transferring the frozen tube into a programmed cooling box filled with isopropanol, and then transferring into a refrigerator at minus 80 ℃ for cooling overnight;
9) And taking out the freezing tube in the sequence cooling box after cooling, and transferring into a liquid nitrogen tank for storage as soon as possible.
2. PBMC cell culture and NK cell induced differentiation
Culturing PBMC cells in 10-percent FBS-containing RPMI 1640 at 37 ℃ in 5% CO 2 Culturing under saturated humidity condition, and culturing the cells until the density reaches about 80 percent for passage. Taking PBMC cells with good growth state, placing at 37 deg.C, 5% 2 An incubator, using complete culture medium containing 500U/mL IL-2 and 20ng/mL IL-15 for human NK cells, inducing differentiation according to the medium application instruction, supplementing liquid once every 3d, and collecting cells after 14 d. The cell density was controlled to about 2.0X 10 during the culture 6 /mL。
Labeling cells at 0d and 14d with PE-CD3 and APC-CD56, respectively, and flow-measuring CD3 - CD56 + The proportion of cells to total cells is the purity of NK cells.
3. NK cell lethality assay
NK cells cultured for 14 days were collected, trypsinized, washed with PBS, and resuspended in human NK cell complete medium (density 5X 10) 4 /mL), inoculating in 24-well plate, each well for 1mL, after adaptive culture for 24h, replacing with human NK cell complete medium containing 25. Mu.g/mL, 50. Mu.g/mL N-Formyl-MLFK to continue culture, while setting control without N-Formyl-MLFK. After 48h, the cells were harvested and washed 3 times with PBS, lysed, centrifuged to remove the supernatant, and the protein concentration was determined by BCA. Preparing gel according to the instruction of the kit prepared by SDS-PAGE gel, taking equal amount of protein from each group, performing electrophoresis, transferring to membrane,blocking, adding Granzyme B, perforin and beta-actin primary antibody, and incubating overnight at 4 ℃; and (3) adding a secondary antibody after washing the membrane, standing for 2 hours at room temperature, washing the membrane for 3 times, placing the membrane in an ECL luminous agent, developing, fixing, stopping washing with distilled water, and photographing and analyzing by a gel image processing system.
4. NK cell migration Activity assay
NK cells cultured for 14 days were collected, trypsinized, washed with PBS, and resuspended in human NK cell complete medium (density 5X 10) 4 and/mL) were inoculated into 6-well plates at 2.5mL per well, and after acclimation for 24h, the plates were replaced with complete medium of human NK cells containing 25. Mu.g/mL and 50. Mu.g/mL of N-Formyl-MLFK, while a sterile 10. Mu.L tip was used to uniformly line the bottom of the plate to form a "naked" area in the center of the well, and a control containing no N-Formyl-MLFK was set. After scratching, the cells are continuously cultured for 12h for photomicrography analysis, and the cell migration rate is calculated according to the formula:
mobility (%) = (1-12 h scratch distance/0 h scratch distance) × 100%.
5. Statistical analysis
Analysis was performed using SPSS 19.0 statistical software, expressed as mean. + -. Standard deviation, and comparisons between groups were by t-test, with P <0.05 representing a statistical significance of the difference.
3. Results of the experiment
1. PBMC cell culture and NK cell induced culture
The detection results of the cell phenotype by the flow cytometer are shown in Table 1, and the proportion of the CD3-CD56+ phenotype cells is increased from 0d (9.48 +/-2.93)% to 14d (80.67 +/-3.15)%, which indicates the success of NK cell induction.
TABLE 1 CD3-CD56+ phenotypic ratio
| Ratio of CD3-CD56+ phenotype | |
| 0d | (9.48±2.93)％ |
| 14d | (80.67±3.15)％ |
2. NK cell killing power
The result of measuring the expression levels of the Granzyme B and the Perforin protein in each group of NK cells is shown in A in figure 1, and compared with the control, the expression levels of the Granzyme B and the Perforin protein in the NK cells interfered by 25 mu g/mL and 50 mu g/mLN-Formyl-MLFK are obviously improved on average. Granzyme B and Perforin are important factors released by NK cells and acting on target cells to exert killing activity on the target cells, and the expression level of the factor has a decisive influence on the killing power of the NK cells. The results show that the intervention culture of N-Formyl-MLFK can effectively improve the lethality of NK cells.
3. NK cell migration Activity
Migration rates of NK cells of each group are shown in Table 2 and B in FIG. 1, and compared with the control, the 25. Mu.g/mL and 50. Mu.g/mL N-Formyl-MLFK interfered NK cells have obviously stronger migration activity.
TABLE 2 NK cell mobility
| Mobility ratio | |
| 25μg/mLN-Formyl-MLFK | (65.22±3.61)％ |
| 50μg/mLN-Formyl-MLFK | 100％ |
| Control | (26.09±2.35)％ |
The above experimental results can show that: the small peptide derivative N-Formyl-MLFK can not only improve the killing power of natural killer cells, but also improve the migration activity of the natural killer cells, and is beneficial to the natural killer cells to exert the killing effect of the natural killer cells.
Claims (2)
1. An application of a small peptide derivative in improving killing power and migration activity of natural killer cells in vitro is disclosed, wherein the small peptide derivative is N-Formyl-MLFK.
2. An application of a small peptide derivative in preparing a culture solution for improving killing power and migration activity of natural killer cells is disclosed, wherein the small peptide derivative is N-Formyl-MLFK.
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