CN112826921B - Application of VEGF165b protein in preparation of tumor inhibitor, tumor inhibitor and preparation method thereof - Google Patents
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Abstract
The invention relates to an application of VEGF165b protein in preparing a tumor inhibitor, the tumor inhibitor and a preparation method thereof, belonging to the technical field of tumor inhibition drugs. The VEGF165b protein disclosed by the invention is preferably used for treating NK92 cells by using the VEGF165b protein, and the treated NK92 cells are the tumor suppressor. VEGF165b can competitively bind with VEGFR1 receptors on NK92 cells with VEGF165, so that the levels of granzyme and perforin released by the NK92 cells are increased, and the killing effect on cancer cells is further improved. VEGF165b can promote the killing effect of NK92 cells, and has great application value in the aspects of immunotherapy based on NK 92-based immune cells and preparation of antitumor drugs.
Description
Technical Field
The invention relates to an application of VEGF165b protein in preparing a tumor inhibitor, the tumor inhibitor and a preparation method thereof, belonging to the technical field of tumor inhibition drugs.
Background
Natural killer cells NK (NK) are an important immune effector cell in the body, accounting for about 10% of healthy human peripheral blood lymphocytes, and are an essential role in immune surveillance and immune response in the development of early solid tumors as a first line of defense, and thus are widely used in clinical therapy. NK cells have unique immunity: (1) NK cells are non-specifically killed, the killing effect is not limited by HLA, cytotoxic receptors generated on the surfaces of the NK cells cause the secretion of perforin and granzyme B, IL-2 is not generated, but cytokines such as IFN-gamma and TNF-alpha are generated; (2) CD16 + on the surface of NK cells can generate ADCC killing effect; (3) The killing of NK cells is continuous, killing one to another, about 7-10 tumor cells killed; (4) NK cells are short-lived, disappear quickly after exerting efficacy, and are effective and safe for treating patients. NK92 is an NK cell line with the following properties: NK92 cells are highly IL-2 dependent; the immunophenotype is CD56 + CD 16-, thus no ADCC effect is generated; can be amplified in vitro and can be cultured for a long time; therefore, the NK92 host as a Chimeric Antigen Receptor (CAR) in vivo is expected to be applied to adoptive immunotherapy of tumors.
Among the studies of angiogenesis, VEGFA is the most widely studied. VEGFA not only promotes tumor formation but also induces immune tolerance by interacting with its receptors VEGFR1, VEGFR2 and Nrp 1. Vascular endothelial growth factor VEGF-A is selectively sheared by mRNA and can be divided into two main categories: a class of pro-angiogenic factors represented by the vegfxxxx family; the other is an anti-angiogenic factor based on VEGFxxxb family, of which VEGF165 and VEGF165b are two representatives. Various experiments show that VEGF165 can inhibit the killing effect of immune cells on cancer cells through ways of inhibiting maturation of DCs, proliferation of T cells, activation of NK cells and the like. At present, no better method for improving the killing capacity of NK cells to tumor cells exists.
Disclosure of Invention
The invention aims to provide application of VEGF165b protein in preparing a tumor suppressor, wherein the VEGF165b protein can enhance the capability of immune effector cells in killing tumor cells, so that the VEGF165b protein can be used for preparing the tumor suppressor.
The invention also provides a tumor inhibitor.
The invention also provides a preparation method of the tumor inhibitor.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the use of VEGF165b in the preparation of a tumour suppressor which enhances the ability of immune effector cells to kill tumour cells.
In particular, VEGF165b enhances the ability of immune effector cells to kill tumor cells by competitive inhibition with VEGF 165.
Preferably, the immune effector cells express VEGF165 themselves and do not express VEGF165b, and VEGF165b enhances the ability of the immune effector cells to kill tumor cells by competitively binding to VEGFR1 receptors on the immune effector cells with VEGF 165.
More preferably, the VEGF165b protein is used for treating the NK92 cells, and the treated NK92 cells are the tumor suppressor.
VEGF165b is a natural competitive inhibitor of VEGF165, and both VEGF165 and VEGFR1 can act. The NK92 cells express VEGF165 by themselves but not VEGF165b, and the VEGF165 inhibits the release of NK92 cell granzyme by binding with VEGFR1 receptors on the NK92 cells, so that the killing capacity of the NK92 cells on cancer cells is weakened. Therefore, the VEGF165b protein can be used for preparing tumor inhibitors.
According to the invention, the VEGF165b protein is used for treating the NK92 cells, and the VEGF165b and the VEGF165 compete to bind to VEGFR1 receptors on the NK92 cells, so that the levels of granzyme and perforin released by the NK92 cells are increased, and the killing effect on cancer cells is further improved. VEGF165b can promote the killing effect of NK92 cells, and has great application value in the aspects of immunotherapy based on NK 92-based immune cells and preparation of antitumor drugs.
Preferably, the treatment is incubation culture of VEGF165b protein and NK92 cells.
Preferably, the concentration of VEGF165b protein in the co-incubation culture is 40-100ng/mL. Further preferably, the concentration of VEGF165b protein in the co-incubation culture is 70-100ng/mL.
Still more preferably, the concentration of VEGF165b protein in the co-incubation culture is 80ng/mL.
Preferably, the co-incubation culture time is 18-30h.
A tumor suppressor which is an immune effector cell enhanced by VEGF165b by competing with VEGF165 for binding to VEGFR1 receptors on immune effector cells. Specifically, VEGF165b protein is used for treating NK92 cells, and the treated NK92 cells are the tumor suppressor.
NK92 cells with high killing activity obtained by VEGF165b protein treatment can be used as antitumor drugs for tumor treatment.
The preparation method of the tumor inhibitor comprises the following steps: and (3) treating the NK92 cells by using VEGF165b protein, wherein the treated NK92 cells are the tumor suppressor.
In the method provided by the invention, VEGF165b and VEGF165 can competitively bind with VEGFR1 receptors on NK92 cells, so that the levels of granzyme and perforin released by the NK92 cells are increased, and the killing effect on cancer cells is further improved.
Preferably, the treatment is incubation culture of VEGF165b protein and NK92 cells.
Preferably, the concentration of VEGF165b protein in the co-incubation culture is 40-100ng/mL. Further preferably, the concentration of VEGF165b protein in the co-incubation culture is 70-100ng/mL.
Still more preferably, the concentration of VEGF165b protein in the co-incubation culture is 80ng/mL.
Preferably, the time of the co-incubation culture is 18-30h.
Drawings
FIG. 1 is a graph showing a comparison of mRNA levels of NK92 cell Granzyme (Granzyme B) of each group in Experimental example 1 of the present invention;
FIG. 2 is a graph showing a comparison of mRNA levels of NK92 Perforin (Perforin) of each group in test example 1 of the present invention;
FIG. 3 is a graph showing a comparison of granzyme content in NK92 cells of each group in Experimental example 2 of the present invention;
FIG. 4 is a graph showing a comparison of the content of NK92 cell perforin in each group in test example 2 of the present invention;
FIG. 5 is a graph showing a comparison of the killing effect of NK92 cells on K562 cells in each group in Experimental example 3 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to specific examples. The equipment and reagents used in the examples and the experimental examples were commercially available except as specifically indicated.
Example 1
The application of the VEGF165b protein in preparing the tumor inhibitor for enhancing the capability of immune effector cells in killing tumor cells in the embodiment comprises the following steps: and (3) incubating and culturing the VEGF165b protein and the NK92 cells for 24h, wherein the final concentration of the VEGF165b protein is 40 ng/mL, and then separating the NK92 cells to obtain the tumor inhibitor.
Example 2
The application of the VEGF165b protein in preparing the tumor inhibitor for enhancing the capability of immune effector cells to kill tumor cells in the embodiment comprises the following steps: and (3) incubating VEGF165b protein and NK92 cells for 24h, wherein the final concentration of the VEGF165b protein is 80ng/mL, and then separating the NK92 cells to obtain the tumor inhibitor.
Example 3
The application of the VEGF165b protein in preparing the tumor inhibitor for enhancing the capability of immune effector cells in killing tumor cells in the embodiment comprises the following steps: and (3) incubating VEGF165b protein and NK92 cells for 24h, wherein the final concentration of VEGF165b protein is 100ng/mL, and then separating NK92 cells to obtain the tumor inhibitor.
Example 4
A tumor inhibitor is prepared by incubating VEGF165b protein and NK92 cell for 24h, and separating NK92 cell to obtain the final concentration of VEGF165b protein of 80ng/mL.
Example 5
The method of preparing the tumor suppressor of example 4, comprising: and (3) incubating VEGF165b protein and NK92 cells for 24h, wherein the final concentration of VEGF165b protein is 80ng/mL, and then separating NK92 cells to obtain the tumor inhibitor.
Test example 1 qPCR detection of granzyme and perforin mRNA level expression in NK92 cells with high killer Activity
1. Treatment of NK92 cells
1. Culture of NK92 cells
(1) Preparation of medium (500 mL): 368 mL of alpha-MEM medium, 64 mL of fetal bovine serum (12.5%), 64 mL of horse serum (12.5%), 500 μ L of IL-2 (100U), 5 mL of diabody (1%).
(2) Recovery of NK92 cells: taking out the frozen NK92 cells from the liquid nitrogen, and quickly thawing the cells in a water bath at 37 ℃; transferring the culture medium to a 15 mL centrifuge tube containing 10 mL culture medium on a clean bench, centrifuging the culture medium for 5 min at 300 g; discarding the supernatant, adding 3mL of culture medium into a centrifuge tube, resuspending the cell pellet, adding culture medium into a culture flask in advance, adding the cell suspension into the culture flask, 37 ℃, and 5% CO 2 Cultured in an incubator.
(3) Passage of NK92 cells: NK92 cells are suspension cells, and all cell suspensions are collected and centrifuged for 5 min at 300 g when passage is carried out; discarding the supernatant, adding 3mL of complete culture medium, resuspending the cell pellet, and uniformly distributing into 3 culture flasks; placing into an incubator for culturing.
2. Culture of K562 cells (cancer cells)
(1) Preparation of medium (550 mL): 500 DMEM medium (mL), fetal bovine serum (10%) 50 mL, and double antibody (%) 5.5 mL.
(2) Recovery of K562 cells: taking out the frozen K562 cells from the liquid nitrogen, and quickly thawing the cells in a water bath kettle at 37 ℃; in a clean bench, turn to contain 10 mCentrifuging the L culture medium in a centrifuge tube of 15 mL for 5 min at 300 g; discarding the supernatant, adding 3mL of the culture medium into a centrifuge tube, resuspending the cell pellet, adding the culture medium into the flask in advance, adding the cell suspension into the flask, 37 ℃, and 5% CO 2 Cultured in an incubator.
(3) Passage of K562 cells: k562 cells are suspension cells, and when passage is carried out, all cell suspensions are collected, 300 g of the cell suspensions are centrifuged for 5 min at room temperature; discarding the supernatant, adding 3mL of complete culture medium, resuspending the cell pellet, and uniformly distributing into 3 culture flasks; placing into an incubator for culturing.
3. NK92 cells incubated by VEGF165b protein
Collecting NK92 cells with logarithmic survival period, counting, spreading in 24-well plate, inoculating at cell density of 3 × 10 5 Individual cells/well; PBS or VEGF165b is added, the final concentration of VEGF165b is 80ng/mL, and the mixture is placed into an incubator to be incubated and cultured for 24 hours. After culturing, each group of NK92 cells is centrifuged, the supernatant is discarded, and then the culture solution is diluted for use.
2. qPCR detection of granzyme and perforin mRNA level expression
NK92 cells kill K562 cells: k562 cells in logarithmic growth phase are plated and K562 cells are used as target cells to be inoculated on three 24-well plates, and the density of the inoculated cells is 6 multiplied by 10 4 Each cell/well, provided with 3 secondary wells; effector cells were added according to the effective target ratio of 5.
Since K562 cells do not produce granzyme and perforin, the expression of mRNA level in cultured mixed cells is detected, and the final detection result is not influenced.
1. qPCR detection of granzyme mRNA level expression in NK92 cells treated with VEGF165b
(1) Extraction of RNA (the kit used was UNIQ-10 column type Total RNA extraction kit from Sangon Biotech, cat. No. B511361-0100)
(1) Suspension culture of cells: taking about 5X 10 of the above cultured 6 One cell, centrifugation at 3,000 rpmThoroughly removing supernatant after 5 min, adding RLT Solution of 350 μ L, mixing well, homogenizing for 30 sec with a homogenizer or sucking for 5 times with a 20-G (0.9 mm) needle;
(2) adding 1/2 volume of absolute ethyl alcohol into the cracking sample, and fully and uniformly mixing;
(3) putting the adsorption column into a collecting pipe, adding all the solution into the adsorption column by using a liquid transfer device, standing for 2 min, centrifuging at 8,000 rpm for 1 min, and pouring waste liquid in the collecting pipe;
(4) putting the adsorption column back into the collecting pipe, adding 500 mu L RW Solution, standing for 1 min, centrifuging at 10,000 rpm for 1 min, and pouring the waste liquid in the collecting pipe;
(5) putting the adsorption column back into the collection pipe, adding 500 muL of RPE Solution, standing for 2 min, centrifuging at 10,000 rpm for 1 min, and pouring the waste liquid in the collection pipe;
(6) repeating the step (4) once;
(7) placing the adsorption column back into the collection tube, and centrifuging at 10,000 rpm for 2 min;
(8) putting the adsorption column into a 1.5 mL centrifuge tube of RNase-free, and adding 30-50 mu L DEPC-treated ddH into the center of an adsorption film 2 O, standing for 5 min, and centrifuging at 12,000 rpm for 2 min;
(9) the resulting RNA solution was then assayed for concentration.
(2) RT-PCR (kit used is 5 x al-In-One RT MasterMix kit from abm company, cat number G490)
(1) The PCR reaction system is as follows: RNA template 10 μ L, RT MasterMix 4 μ L, nuclear-free H 2 O 6µL；
(2) The PCR reaction conditions were as follows: 10 min at 25 ℃,50 min at 42 ℃ and 5 min at 85 ℃;
(3) qPCR (kit used was EvaGreen 2 x qPCR MasterMix-No Dye kit from abm, cat # 1051844647001)
(1) The qPCR reaction system was as follows: qPCR MasterMix 5 muL, forward Primer 0.3 muL, reverse Primer 0.3 muL, template DNA 400ng, nuclear-free H 2 O 10µL；
The primers used for detection are shown below:
granular enzyme qPCR upstream primer: 5 'CGACAGTACATTGGAGTTGTGCG-3' (shown as SEQ ID NO. 1);
granular enzyme qPCR downstream primer: 5 'TTCGTCCATAGGAGACAATGCCC-3' (shown as SEQ ID NO. 2);
internal reference qPCR upstream primer: 5' and-;
internal reference qPCR downstream primer: 5-.
(2) qPCR reaction conditions were as follows: 1 cycle at 95 ℃ for 10 min; (95 ℃ 15 secs, 60 ℃ 60 secs) 40 cycles.
The results are shown in FIG. 1, PBS group represents granzyme mRNA level after PBS treatment, and VEGF165b group represents granzyme mRNA level after VEGF165b treatment. From FIG. 1 it can be seen that VEGF165b treatment increased NK92 cell granzyme mRNA levels.
2. qPCR detection of perforin mRNA level expression
The procedure was identical to that of qPCR for detection of granzymes.
The primers used for detection are shown below:
perforin qPCR upstream primer: 5 'ACTCACACAGGCAGCAGCCAACTTTGC-3' (shown as SEQ ID NO. 5);
perforin qPCR downstream primer: 5-.
Internal reference qPCR upstream primer: 5' and-;
internal reference qPCR downstream primer: 5-.
The results are shown in FIG. 2, in which the PBS group in FIG. 2 represents perforin mRNA levels after PBS treatment and the VEGF165b group represents perforin mRNA levels after VEGF165b treatment. From FIG. 2, it can be seen that VEGF165b treatment increased the mRNA level of NK92 cell perforin.
Test example 2 ELISA for measuring secretion levels of granzyme and perforin
1. ELISA method for detecting the amount of released Granzyme B (Kit used was LEGEND MAX ELISA Kit with Pre-coated Plates Human Granzyme B, cat # B267000, bioLegend)
(1) Preparation of samples
Plating K562 cells in logarithmic phase, taking K562 cells as target cells, inoculating the target cells into two 96-well plates, wherein the density of the inoculated cells is 1 × 10 4 4 secondary wells per cell/well; adding effector cells (PBS treated NK92 and 80ng/mL VEGF165b treated NK 92) according to the effective target ratio of 5; putting the 96-well plate into a multi-well plate centrifuge for centrifugation, and centrifuging for 5 min at room temperature at 300 g; taking the supernatant, wherein the supernatant is the sample to be detected.
(2) Preparation of standards
(1) Preparing 7 centrifuge tubes with the volume of 1.5 mL, marking the centrifuge tubes with the number of 1-7, sucking 200 mu L of standard samples with the concentration of 600 pg/mL, and adding the standard samples into a tube No. 1;
(2) firstly adding 100 muL of 1X standard substance diluent into a No. 2-7 tube;
(3) sucking 100 muL of standard sample from the No.1 tube, adding the standard sample into the No.2 tube, and fully and uniformly mixing; then, sucking 100 mu L from the No.2 tube and adding the 100 mu L into the No.3 tube; by analogy, the addition to tube 6 was stopped and tube 7 was used as a standard sample at a concentration of 0 pg/mL.
(3) The specific steps
(1) Taking out the particle/enzyme/kit from a refrigerator, recovering all reagents to room temperature, fully and uniformly mixing to avoid generating bubbles, and simultaneously preparing a sufficient amount of consumables required in operations such as a pore plate, a centrifuge tube and the like;
(2) adding 1 Xwashing liquor of 300 muL by using a discharge gun, washing for 5 min each time for 4 times, and adding 50 muL of Assay Buffer A;
(3) adding 100 muL of standard substances with 7 concentrations into the wells, wherein each concentration is provided with 2 auxiliary wells, and simultaneously, blank control wells are arranged; adding 50 muL of samples to be detected into corresponding holes, arranging 2 auxiliary holes for each sample, sealing the plate with a plate sealing film, and incubating at room temperature at 200 rpm for 2 h;
(4) discarding the liquid in the well plate, and repeating the washing process of (2);
(5) adding 200 muL of detection antibody of the granular enzyme B into each hole, sealing the plate with a plate film, and incubating for 1h at room temperature and 200 rpm;
(6) adding 1 Xwashing solution of 300 muL into a discharge gun, washing for 5 min each time, and washing for 4 times;
(7) adding 100 mu L of Avidin HRP E solution, sealing a plate and a membrane sealing plate, and incubating for 30 minutes at room temperature and 200 rpm;
(8) repeating the step (6);
(9) adding 100 muL of Substrate Solution F Solution into each hole, sealing a plate and a membrane sealing plate, and incubating for 30min at room temperature and 200 rpm in a dark place (the incubation time can be controlled according to color change);
and adding 100 muL of stop solution into the red, and immediately detecting the absorbance at the wavelength of 450 nm and 570 nm by using an enzyme labeling instrument.
The results are shown in FIG. 3, the PBS group represents the granzyme release level after PBS treatment, and the VEGF165b group represents the granzyme release level after VEGF165b treatment. From FIG. 3, it can be seen that VEGF165b significantly promoted the release of NK92 cell granzyme.
2. ELISA for the amount of Perforin released (the Kit used was Human Perforin ELISA Kit (PRF 1) from Abcam, cat # GR 3223536-8)
(1) Sample preparation
The sample preparation procedure was the same as for granzyme B.
(2) Preparation of standards
(1) Preparing 7 centrifuge tubes of 1.5 mL, marking as 1-7, and adding 200 mu L of the standard mother liquor with the concentration of 2000 pg/mL into a No.1 tube;
(2) adding 100 muL of 1 multiplied standard substance diluent into a 2-7 tube in advance;
(3) sucking 100 mu L of standard substance from the No.1 tube, adding the standard substance into the No.2 tube, mixing the standard substance and the No.2 tube completely, sucking 100 mu L of standard substance from the No.2 tube, adding the standard substance into the No.3 tube, and repeating the steps in sequence until the No.6 tube is stopped, wherein the No. 7 tube is the standard substance with the concentration of 0 pg/mL;
(3) Specific operation flow
(1) Taking the kit out of the refrigerator in advance, standing at room temperature, and fully and uniformly mixing all the reagents when all the reagents are recovered to the room temperature to avoid generating bubbles; meanwhile, preparing a sufficient amount of consumables required in the experimental process such as a pore plate, a centrifuge tube and the like;
(2) respectively sucking 100 mu L of standard substances with 7 concentrations, adding the standard substances into corresponding holes, setting 2 auxiliary holes for each concentration, and setting blank control holes at the same time; respectively sucking 100 mu L of samples and adding the samples into corresponding holes, and setting 2 auxiliary holes for each sample; sealing the plate with a sealing plate film, and then incubating at room temperature for 1h;
(3) discarding the liquid, washing the plate with 1 × 300 μ L of washing liquid for 5 min each time, and washing for 3 times;
(4) adding 1 multiplied biotinylated perforin detection antibody of 50 mu L into each well, sealing a plate with a plate film, and incubating for 1h at room temperature;
(5) washing the plate according to the process of the step (3);
(6) adding 100 muL of 1 XStreptavidin-HRP solution into each hole, sealing the plate, and incubating for 30min at room temperature;
(7) repeating the step (3);
(8) adding 100 muL of Chromogen TMB Substrate solution into each hole, and incubating for 10-20 min at room temperature in a dark place (the incubation time is controlled according to the change of color);
(9) 100 muL of stop solution is added into each well, and the absorbance at the wavelength of 450 nm and 620 nm is immediately detected by an enzyme-labeling instrument.
The results are shown in FIG. 4, in which the PBS group in FIG. 4 represents the perforin release level after PBS treatment, and the VEGF165b group represents the perforin release level after VEGF165b treatment. From FIG. 4, it can be seen that VEGF165b significantly promoted the release of NK92 cell perforin.
Test example 3 lactic acid dehydrogenase method for measuring toxicity of NK92 cells (kit used was CytoTox96 from Promega corporation) ® Non-Radioactive cytotoxin Assay, product number G1780, reaction solution and stop solution are all substances in the kit)
Collecting K562 cells in logarithmic growth phase, counting, inoculating to 96-well cell culture plate, inoculating cell density of 1 × 10 4 Each cell/well, provided with 3 secondary wells; according to the effective target ratio of 5VEGF165b (DevX) in 24h treated the resulting NK92 cells. Then centrifuging the 96-well plate added with the cells in a multi-well plate centrifuge at 300 g for 5 min at room temperature, taking out the 96-well plate, and putting the 96-well plate into an incubator for continuous culture for 5 h; taking out a 96-well plate 45 min before detection, adding 10 muL 10 xLDH into the single target cell hole and the blank culture medium hole, and putting the single target cell hole and the blank culture medium hole back to the incubator for continuous culture; taking out the 96-well plate at the co-culture time point, centrifuging the plate at room temperature for 4 min at 300 g, taking 50 muL of supernatant, adding the supernatant into a new 96-well plate, and adding 50 muL of reaction liquid prepared in advance; incubating for 30min at room temperature in dark; adding 50 mu L of reaction stop solution; absorbance was measured at 490 nm using a microplate reader, and the absorbance of the background blank well was subtracted from each of the values.
The cytotoxicity calculation formula is as follows:
toxicity of cells (%) = (OD value of experimental group-OD value of effector cell control well-OD value of target cell control well) ÷ (OD value of sample maximum enzyme activity control well-OD value of target cell control well) × 100%.
Toxicity of NK92 cells was calculated for each group, and the level of killing of K562 cells by NK92 cells after VEGF165b treatment is shown in fig. 5, where NS means "no statistical significance"; as can be seen from FIG. 5, when the concentration of VEGF165b is 80ng/mL, the toxicity of NK92 cells is strongest, and the killing capability of the NK92 cells on K562 cells after VEGF165b treatment is obviously improved.
The results of the above experiments show that the NK92 cells treated by VEGF165b of the invention can detect the upregulation of granzyme and perforin at both mRNA level and protein level when killing K562 cells. The toxicity experiment of NK92 cells detected by a lactic acid dehydrogenase method proves that the killing effect of NK92 on K562 cells after the action of VEGF165b is obviously enhanced. The experimental results all prove that VEGF165b can finally and remarkably enhance the killing effect of NK92 cells on cancer cells by promoting the NK92 cells to release granzyme and perforin, and the VEGF165b can be used for preparing the anti-tumor drugs with strong killing activity of effector cells.
<110> Xinxiang medical college
Application of VEGF165b protein in preparation of tumor inhibitor, tumor inhibitor and preparation method thereof
<160> 6
<170> SIPOSequenceListing 1.0
<211> 23
<212> DNA
<213> Artificial sequence
<221> granzyme qPCR upstream primer
<400> 1
cgacagtacc attgagttgt gcg 23
<211> 23
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<213> Artificial sequence
<221> granzyme qPCR downstream primer
<400> 2
ttcgtccata ggagacaatg ccc 23
<211> 23
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<213> Artificial sequence
<221> internal reference qPCR upstream primer
<400> 3
ggtggtctcc tctgacttca aca 23
<211> 23
<212> DNA
<213> Artificial sequence
<221> internal reference qPCR downstream primer
<400> 4
gttgctgtag ccaaattcgt tgt 23
<211> 22
<212> DNA
<213> Artificial sequence
<221> perforin qPCR upstream primer
<400> 5
actcacaggc agccaacttt gc 22
<211> 22
<212> DNA
<213> Artificial sequence
<221> perforin qPCR downstream primer
<400> 6
ctcttgaagt cagggtgcag cg 22
Claims (5)
- Application of NK92 cells treated by VEGF165b protein in preparation of tumor inhibitors is characterized in that: the treatment is to incubate and culture VEGF165b protein and NK92 cells; the concentration of VEGF165b protein in the co-incubation culture is 80-100ng/mL; the co-incubation time was 24h.
- 2. Use of NK92 cells treated with VEGF165b protein according to claim 1 in the preparation of a tumor suppressor, wherein: VEGF165b enhances NK92 cells' ability to kill tumor cells by competitive inhibition with VEGF 165.
- 3. Use of NK92 cells treated with VEGF165b protein according to claim 2 in the preparation of a tumor suppressor, wherein: the NK92 cells themselves express VEGF165 but do not express VEGF165b, VEGF165b enhances the ability of NK92 cells to kill tumor cells by competitively binding to VEGFR1 receptors on NK92 cells with VEGF 165.
- 4. A tumor suppressor agent, characterized by: treating NK92 cells by using VEGF165b protein, wherein the VEGF165b competitively binds with VEGFR1 receptors on the NK92 cells to obtain NK92 cells with enhanced immune effect by using VEGF165, and the NK92 cells are tumor inhibitors; the treatment is to incubate and culture VEGF165b protein and NK92 cells; the concentration of VEGF165b protein in the co-incubation culture is 80-100ng/mL; the co-incubation time was 24h.
- 5. The method for preparing a tumor suppressor according to claim 4, wherein: the method comprises the following steps: treating the NK92 cells by using VEGF165b protein, wherein the treated NK92 cells are the tumor suppressor; the treatment is to incubate and culture VEGF165b protein and NK92 cells; the concentration of VEGF165b protein in the co-incubation culture is 80-100ng/mL; the co-incubation time was 24h.
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| WO2018081652A2 (en) * | 2016-10-28 | 2018-05-03 | Nant Holdings Ip, Llc | Avatar dendritic cells: the neoantigen natural killer t-cell chemo immuno radiation composition inducing immunogenic cell death |
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| CN104628866A (en) * | 2015-01-21 | 2015-05-20 | 中国药科大学 | Antibody fusion protein targeting VEGFR2 as well as preparation and use thereof |
| WO2018081652A2 (en) * | 2016-10-28 | 2018-05-03 | Nant Holdings Ip, Llc | Avatar dendritic cells: the neoantigen natural killer t-cell chemo immuno radiation composition inducing immunogenic cell death |
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