1. A culture method for amplifying NK cells from umbilical cord blood is characterized in that inactivated LCL cells are used as trophoblasts and combined with cytokines for specific amplification; the LCL cell is an LCL-mbiL-15 cell or an EBV-LCL cell.

2. The method according to claim 1, wherein the NK cells are obtained by culturing CBMC cells using the inactivated LCL cells as feeder cells, wherein the ratio of the LCL cells to the CBMC cells is 1: 1-5.

3. The method of claim 2, wherein the cytokine comprises one or more of IL-2, IL-15, and IL-21.

4. The culture method according to claim 3, wherein the amount of IL-2 is 0-200U/mL, the amount of IL-15 is 0-200U/mL, and the amount of IL-21 is 0-3000U/mL.

5. The culture method according to claim 4, comprising the steps of:

(1) separating CBMC from umbilical cord blood;

(2) preparing EBV supernatant;

(3) EBV supernatant culture PBMC is used for preparing EBV-LCL cells;

(4) transfecting the EBV-LCL cell with the plasmid to obtain an LCL-mbiL-15 cell;

(5) irradiating LCL-mbiL-15 cells with ultraviolet rays, and culturing the CBMC obtained in the step (1).

6. The culture method according to claim 5, wherein the ultraviolet irradiation in the step (5) is carried out under conditions of 0.120J/cm²。

7. An umbilical cord blood-derived NK cell prepared by the culture method according to any one of claims 1 to 6.

8. Use of the culture method according to any one of claims 1 to 6 and/or the cord blood-derived NK cell according to claim 7 for the preparation of a medicament for immunotherapy.

9. An immunotherapeutic agent comprising the cord blood-derived NK cell according to claim 7.

10. Use of the culture method according to any one of claims 1 to 6 for the preparation of NK cells from other sources, including peripheral blood, lymph nodes, bone marrow.

Background

Natural Killer (NK) cells have the natural ability to kill tumor cells as lymphocytes for active immunization of the body. Studies show that the lymphocytes recovered first after hematopoietic stem cell transplantation are NK cells, and the NK cells from infusion donors in the initial stage of hematopoietic stem cell transplantation can reduce the occurrence of graft-versus-host disease (GVHD). The cord blood contains high ratio of CD34⁺Cells, therefore, are considered to be a safe and effective source of stem cells for clinical use. The research shows that the content of the umbilical cord blood is higherA proportion of NK cells, but the NK cells in the cord blood are immature NK cells, and the killing activity is low.

The existing culture methods of NK cells are mostly directed to NK cells derived from peripheral blood mononuclear cells.

Chinese patent 201380028253.3 discloses a method for inducing and amplifying natural killer cells derived from peripheral blood mononuclear cells, which comprises: irradiated Jurkat cells as feeder cells and irradiated continuous lymphocyte cell line cells transformed by EB virus were co-cultured with peripheral blood mononuclear cells in the presence of cytokines. The culture method provided by the invention patent can induce and proliferate a large amount of NK cells from a small amount of peripheral blood mononuclear cells, and can remarkably improve the efficiency and efficacy of preventing and treating cancers by using the NK cells.

Chinese patent 201610482853.2 discloses a method for amplifying NK cells, which uses inactivated LCL feeder cells, peripheral blood mononuclear cells, cytokines IL-2 and IL-15 to perform cell culture in a cell culture solution, and selectively amplifies the NK cells in the peripheral blood mononuclear cells, the invention obviously improves the amplification efficiency of the NK cells, improves the NK killing function, can amplify the NK cells by 800 times of 600-fold, improves the killing power of leukemia cells K562 by 10 times compared with freshly separated NK cells, and improves the killing power of leukemia cells K562 by more than 20% compared with the killing power of the leukemia cells K562 by pure NK cells cultured by using IL-2.

However, when the above method is applied to culture of cord blood-derived NK cells, the obtained NK cells do not show superior clinical application characteristics, and therefore, it is necessary to establish a culture method more suitable for cord blood-derived NK cells.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for specifically amplifying umbilical cord blood-derived NK cells using inactivated LCL cells as feeder cells in combination with cytokines, thereby enhancing the anti-tumor activity of NK cells to make them more suitable for clinical treatment.

LCL: lymphoblastoid cell lines, lymphoblast cell line.

CBMC: cord blood mononuclear cells.

PBMC: peripheral blood mononuclear cells.

EBV: Epstein-Barr virus, EB virus.

In one aspect, the invention provides a culture method for efficiently amplifying cord blood-derived NK cells.

The culture method comprises the steps of using the inactivated LCL cells as feeder cells and specifically amplifying the NK cells derived from the umbilical cord blood by combining with cytokines.

The NK cells are obtained by culturing CBMC.

The dosage ratio of the LCL cells to the CBMC is 1: 1-5, preferably 1: 1.

the LCL cell is an LCL-mbiL-15 cell and/or an EBV-LCL cell.

The EBV-LCL cells are obtained by culturing PBMC with a culture medium containing EBV supernatant.

The LCL-mbiL-15 cells were obtained by culturing PBMC using EBV supernatant-containing medium and then further transfecting mbiL-15.

The cytokines include but are not limited to IL-2, IL-15, IL-21.

Preferably, the IL-2 is applied in an amount of 0-200U/mL, the IL-15 is applied in an amount of 0-200U/mL, and the IL-21 is applied in an amount of 0-3000U/mL.

Preferably, the IL-2 is applied in an amount of 100U/mL, the IL-15 is applied in an amount of 100U/mL, and the IL-21 is applied in an amount of 2500U/mL.

Preferably, the basic culture medium used in the culture method is RPMI l640 culture solution containing 10% fetal bovine serum.

Preferably, the culture method comprises the following steps:

(1) separating CBMC from umbilical cord blood;

(2) preparing EBV supernatant;

(3) EBV supernatant culture PBMC is used for preparing EBV-LCL cells;

(4) transfecting LCL cells with plasmids to obtain LCL-mbiL-15 cells;

(5) irradiating LCL-mbiL-15 cells with ultraviolet rays, and culturing the CBMC obtained in the step (1).

Further preferably, the ultraviolet irradiation is performed under the condition of 0.120J/cm²。

In another aspect, the present invention provides an umbilical cord blood-derived NK cell.

The NK cells were prepared by the aforementioned culture method.

In still another aspect, the present invention provides the use of the aforementioned cord blood-derived NK cells for the preparation of an immunotherapeutic agent.

In yet another aspect, the invention is an immunotherapeutic agent.

The immunotherapy drug comprises umbilical cord blood-derived NK cells prepared by the culture method.

In still another aspect, the present invention provides the use of the aforementioned culture method for the preparation of an immunotherapeutic drug.

The immunotherapy medicine comprises the NK cells prepared by the culture method.

In yet another aspect, the invention provides the use of the aforementioned culture method for the preparation of NK cells of other origin.

Such other sources include, but are not limited to, peripheral blood, lymph nodes, bone marrow.

The invention has the beneficial effects that:

1. the purity of NK cells amplified by CBMC cells cultured by the method provided by the application is as high as 85%, the amplification multiple is 700 +/-50.5, the inhibitory receptors CD158a, CD158b and NKG2A have no obvious change (p is greater than 0.05), and the ratios of the activating receptors including NCKs (NKp30, NKp44, NKp46) and NKG2D are obviously increased (p is less than 0.05).

2. The NK cell obtained by the method obviously increases the killing activity on malignant tumor cells. Especially, the killing performance of freshly isolated and uncultured CB-NK cells on Raji cells expressing high HLA class I molecules is low, but the killing performance of amplified CB-NK cells on the Raji cells is obviously improved.

Drawings

FIG. 1 is a graph showing the proliferation of CB-NK cells after 21 days of culturing LCL-mbiL-5 as a feeder cell by irradiating the CB-NK cells with ultraviolet rays under the condition that LCL-mbiL-15 cells are used as the feeder cells.

Fig. 2 shows the expression levels of CB-NK cell surface activating and inhibitory receptors (n ═ 11) by flow cytometry under the condition of LCL-mbIL-15 cells as feeder cells, indicating that p is >0.05 and p is < 0.05.

FIG. 3 shows the killing effect of isolated and purified CB-NK cells before culture and CB-NK cells after culture on leukemia cells K562 which do not express HLA class I molecules under the condition that LCL-mbIL-15 cells are used as feeder cells.

FIG. 4 shows the killing effect of isolated and purified CB-NK cells before culture and CB-NK cells after culture on high HLA class I-expressing lymphoma cells Raji under the condition that LCL-mbIL-15 cells are used as feeder cells.

FIG. 5 shows the purity changes of isolated and purified CB-NK cells before culture and CB-NK cells after culture under conditions where LCL-mbiL-15 cells are used as feeder cells.

FIG. 6 shows the expression of antibodies in CB-NK cells after culturing LCL-mbiL-15 cells as feeder cells.

FIG. 7 shows the expression of antibodies in CB-NK cells after culturing LCL-mbiL-15 cells as feeder cells.

Detailed Description

The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

Example 1 culture method for efficiently amplifying cord blood-derived NK cells

Step 1, CBMC separation

5mL of cord blood is extracted, and the cord blood is mixed with PBS according to the mixing ratio of 1: 1. another 15mL centrifuge tube was used and 5mL of lymphocyte separation medium (available from CEDARLANE, cat # CL5020) was added. The mixed solution of the cord blood and the PBS is slowly dripped to the liquid level of the lymphocyte separation solution along the tube wall by using a dropper, and the layered liquid level is not damaged. Centrifuge horizontally 800g, 20 min. After centrifugation, the tubes were divided into 3 layers. The white cloud layer between the upper and middle layer interfaces is sucked by a suction apparatus into a new centrifuge tube, and other layer cells are not carried into the centrifuge tube as much as possible. PBS (5 times more than PBS) is added into the centrifuge tube, 300g is centrifugated for 5min, and the supernatant is discarded. Repeat 2 times to wash away lymphocyte separation liquid and cell debris. After the last centrifugation, the supernatant was discarded. All the steps described above are completed in as short a time as possible. The separated CBMC cells were stored in liquid nitrogen with cell freezing medium for later use.

Step 2, preparation of EBV supernatant

B95-8 cells (purchased from cell Bank, Cat. GNO3, China academy of sciences) were placed in PRMI 1640 medium (purchased from Saimer Feishale Sci technology (China) Ltd., Cat. 11875093) containing 10% fetal bovine serum (FBS, purchased from Saimer Feishale technology (China) Ltd., Cat. 10099141C), 50U/mL streptomycin (purchased from Saimer Feishale technology (China) Ltd., Cat. 15070063) at 37 ℃ with 5% CO₂And (5) normally culturing and subculturing under the condition. After starvation lysis of the cultured B958 cells, the supernatant was collected by centrifugation and filtration, and the supernatant was enriched with EBV. This EBV supernatant was stored in aliquots at-80 ℃. When in use, the solution is first re-warmed at 37 ℃ and then filtered by a 0.22 mu m filter membrane (completed within 30-60min as much as possible).

Step 3, preparation of EBV-LCL cells

9mL of PRMI 1640 culture medium was added to a T25 flask, and 2X 10 cells were added⁶PBMC; then adding 9mL of EBV supernatant prepared in the step 2; then 80. mu.L of cyclosporin A (purchased from Sigma, cat # 239835) was added; the culture was carried out at 37 ℃. After 7d, the flask was half-exchanged and 40. mu.L of cyclosporin A was added. This process was repeated 1 time every 7d until 28d of culture. And culturing for 28 days to obtain the EBV-LCL cell.

Step 4, culture of NK cells

EBV-LCL cells were irradiated with UV light at 0.120J/cm before culture experiments²。

The separated and frozen CBMC obtained in the step 1 is processed at 39 DEG CThawing in water bath, washing with RPMI1640 culture solution for 2 times, and suspending in RPMI l640 culture solution containing 10% fetal calf serum to 2 × 10⁶one/mL of cell suspension, then 1mL of cell suspension per well in a 24-well plate. To the cell suspension, IL-2 (final concentration 100U/mL) + IL-15 (final concentration 100U/mL) + IL-21 (final concentration 2500U/mL), EBV-LCL (1X 10 LCL) were added⁶One). At 37 deg.C, 5% CO₂The culture in the incubator is carried out, the liquid in each hole is half-changed and supplemented with corresponding cell factors every 3d, and the culture is carried out for 14-21 d.

Wherein IL-2 is available from PeproTech (PeproTech) of America, and has a product number of 200-02; IL-15 was purchased from PeproTech (PeproTech, Papt. Thaks) USA under a product number of 200-15; IL-21 was purchased from PeproTech (PeproTech) USA under a product number of 200-21.

Step 5, detection of NK cell immunophenotype

Flow cytometry analysis was performed on CBMC before and after amplification, and cells before and after amplification culture were classified and identified using CD45, CD56, CD16, and CD3 antibodies, respectively, to confirm the proportion of NK cells. Meanwhile, the NK cell surface receptors before and after amplification were analyzed by flow cytometry using CD158a, CDl58b, NKG2D, NK046, NKp30, and NKp44 antibodies.

Wherein CD45 is available from BD biosciences under the product number 555484; CD56 was purchased from BD biosciences under the accession number 555518; CD16 was purchased from BD biosciences under the accession number 555407; CD3 was purchased from BD biosciences under the accession number 555339; CD158a was purchased from BD biosciences under the accession number 556063; CDl58b was purchased from BD biosciences under the accession number 559785; NKG2D available from BD biosciences under the accession number 558071; NKp46 available from BD biosciences under the accession number 557991; NKp30 available from BD biosciences under the accession number 563385; NKp44 was purchased from BD biosciences under the accession number 558563.

The specific operation method comprises the following steps: NK cells before and after amplification were washed 2 times with PBS, respectively, and then the cells were resuspended. Each 1 × 10⁵Adding corresponding antibody 4 μ L into 100 μ L, keeping away from light at 4 deg.C for 30min, washing with PBS for 2 times, resuspending the cells with 300 μ L PBS, and detecting with flow cytometerFor measurement, flowjo762 software was used for analysis of the results.

Step 6, NK cell killing capacity detection

And (3) detecting the killing effect of the NK cells by using a flow cytometer. The isolated and purified NK cells were cultured as effector cells, respectively. The myelogenous leukemia cell line K562 (purchased from cell bank of Chinese academy of sciences, cat # SCSP-5054), lymphoma cell Raji (purchased from cell bank of Chinese academy of sciences, cat # TCHU 44), which had been incubated with a fluorescent dye CFSE (purchased from Saimer Feishell science and technology (China) Co., Ltd., cat # C34554) for 10min, washed 2 times with RPMI-1640 containing 10% FBS, resuspended, and resuspended in 2X 10 FBS-containing RPMI-1640⁵Each well was placed in a round-bottomed U-tube as a target cell, according to effector cell: adding corresponding cultured NK cells into different ratios of Target cells (Effector: Target, E: T), culturing in an incubator for 12h, washing with PBS for 1 time, adding a blank control group (without Effector cells), washing the effective Target cell mixed solution with PBS for 2 times, adding PI (dye), incubating in dark for 30min, and detecting with a flow cytometer.

Wherein the cells with double positive of CFSE and PI are the killed target cells. Specific killing calculation percentage equation (%): [ dead target cell (%) of experimental group-dead target cell (%) of nature/100-dead target cell (%) ] × 100.

The experimental results are as follows:

1. flow cytometry for detecting changes of NK cell surface receptors of umbilical cord blood before and after culture

21 days after CBMC cells were cultured and NK cells were expanded using EBV-LCL as feeder cells, CD3 was detected by flow cytometry^-CD56⁺And changes in their cell surface receptors. More than 33% of the cells after 21 days of culture were CD3^-CD56⁺(NK) cells, the absolute number of NK cells is 200 + -12.5 times before culture. The inhibitory receptors CD158a, CD158b and NKG2A were not significantly changed compared to pre-culture cells (p)>0.05), whereas the ratio of activating receptors including NCKs (NKp30, NKp44, NKp46) and NKG2D were all significantly increased (p)<0.05)。

2. Killing activity of cord blood NK cells on leukemia cells

The CB-NK cells are used as effector cells to detect the cytotoxicity effect on malignant tumor cells K562 and Raji. Compared with the prior culture, the killing activity of the CB-NK cell is obviously improved. Especially, the killing performance of freshly isolated and uncultured CB-NK cells on Raji cells expressing high HLA class I molecules is low, but the killing performance of amplified CB-NK cells on the Raji cells is obviously improved.

Example 2 culture method for efficiently amplifying NK cells derived from cord blood

Referring to the culture method of example 1, except that the feeder cells were replaced with LCL-mbiL-15 from EVB-LCL.

The preparation method of LCL-mbiL-15 is as follows:

(1) plasmid construction

The vector IS modified by taking a lentivirus vector pLenti6V5-GW-LaZ produced by Invitrogen company as a framework, an insulator (IS2) IS inserted into the 3LTR (Francisco Martin, Karim Benalbdelah, et al. A molecular HS4-SAR insulator (IS2) which present and enhance expression of viral vectors in ploripotent cells [ J ]. PLoS ONE,2014,9(1): e84268.), a human EF-1 promoter IS adopted, and a reporter gene EGFP IS connected by IRES.

mbIL-15PCR products: extracting RNA of human kidney cancer cells, and performing reverse transcription to form cDNA. The cDNA is used as a template to design a membrane-bound IL5 gene primer, a BamHI restriction enzyme is added to the 5 'end of an upstream primer, and an XbaI restriction enzyme is added to the 5' end of a downstream primer. The sequence of the upstream primer is shown in SEQ ID NO.1, and the sequence of the downstream primer is shown in SEQ ID NO. 2. The amplification reaction procedure was: denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, 35 cycles, and gel electrophoresis identification after amplification. The sequence of the mbiL-15 is shown in SEQ ID NO. 3. The PCR product is precipitated by alcohol and centrifugation for later use.

And (3) carrying out enzyme digestion on the vector: the digestion system consisted of 5. mu.L of 10 XBuffer, 5. mu.L of DNA, 1. mu.L each of BamHI and XbaI restriction enzymes, 4. mu.L of water in a water bath at 37 ℃ for 3 h. Then separated by gel electrophoresis and further purified by using a gel DNA extraction kit (purchased from Tiangen Biochemical technology (Beijing) Ltd., product number DP 214).

Connecting: the purified 2 DNA fragments were ligated by T4 DNA ligase. The linker was 10. mu.L, comprising 10 XBuffer 1. mu.L of T4 DNA ligase, 2. mu.L of the vector fragment, 4. mu.L of the desired fragment, 1. mu.L of T4 DNA ligase (available from NEB under the accession number M0202V), and ligated overnight at 14 ℃.

And (3) transformation: the ligation product was taken out and put into a 1.5mL EP tube containing a mixture of competent bacteria (purchased from Tiangen Biochemical technology (Beijing) Ltd., product No. CB101) and ice water, ice-washed for 30min, water-washed at 42 ℃ for 40s, ice-washed for 3min, added into 800. mu.L of LB liquid medium at 30 ℃ for shaking culture and centrifugation, the supernatant was discarded, immediately blown and mixed well, added onto a solid medium with ampicillin resistance, coated with a triangular glass rod, and the plate was placed into a 37 ℃ incubator for overnight culture. The colonies that were filled were selected in 1:1000 ampicillin liquid medium and cultured overnight at 30 ℃ with shaking.

Extracting an mbIL15 expression plasmid: the plasmid extraction kit is from Nanjing NuoZan biology company, and a sample is taken out for enzyme digestion identification after completion.

(2) Virus production: and (4) adopting a specific packaging system for virus packaging. Plasmid mixture was prepared from 9. mu.g of packaging plasmid (including the plasmids expressing respectively gap/pol, Rev, VSV-G, all from Invitrogen) and 3. mu.g of mbIL15 expression plasmid, and 50. mu.L of Buffer B, purified water to 500. mu.L; the mixture was dropped into an equal amount of Buffer A, and then allowed to stand at room temperature for 30 minutes. Adding the solution into a culture dish of a kit paved with 293T cells, gently mixing uniformly, and culturing in an incubator at 37 ℃. Transfection reagent (CPT Transfection Kit) was purchased from Viraltherapy, Wuhan. After 12-24 hours of transfection, serum-containing DMEM medium was replaced, culture was continued for 48-72 hours, and the medium was collected, centrifuged at 3000g for 15 minutes, cell debris was removed, and the virus supernatant was filtered using a 0.45 μm filter. The virus was precipitated with PEG-it and concentrated by centrifugation (1500g, 15 min). Dissolving the virus precipitate in 10-100 times volume of DMEM medium, subpackaging, and storing at-80 deg.C for use.

(3) Cell transfection: transfection of LCL cells was performed in 96-well plates treated with recombinant human fibronectin (50. mu.g/mL). The cells were suspended in the corresponding cell culture medium and then mixed with the corresponding volume of concentrated virus, supplemented with cytokines, added to a 96-well plate, and Polybrene (Polybrene) was added at a concentration of 4. mu.g/mL. After centrifugation (700-800g) of the 96-well plate at room temperature for 2 hours, the plate was cultured overnight in an incubator. Positive cells infected with virus were screened by flow cytometry. Obtaining LCL-mbiL-15 cells.

NK cells were cultured according to steps 4-6 of the method of example 1, except that step 4 was replaced as follows:

step 4, culture of NK cells

LCL-mbiL-15 cells were irradiated with UV light at 0.120J/cm before culture experiments²。

The separated and frozen CBMC obtained in the step 1 is thawed and recovered in a 39 ℃ water bath box, washed for 2 times by RPMI1640 culture solution and suspended to 2 multiplied by 10 by RPMI l640 culture solution containing 10 percent fetal calf serum⁶one/mL of cell suspension, then 1mL of cell suspension per well in a 24-well plate. To the cell suspension, IL-2 (final concentration 100U/mL) + IL-15 (final concentration 100U/mL) + IL-21 (final concentration 2500U/mL), LCL-mbiL-15 (1X 10)⁶One). At 37 deg.C, 5% CO₂The culture was performed in the incubator, and the liquid in each well was half-changed and supplemented with the corresponding cytokine every 3d for 21 days.

The experimental results are as follows:

1. flow cytometry for detecting changes of NK cell surface receptors of umbilical cord blood before and after culture

The CBMC cells were cultured and NK cells were expanded 21 days using LCL-mbiL-15 as feeder cells, and then CD3 was detected by flow cytometry^-CD56⁺And changes in their cell surface receptors. More than 85% of the cells after 21 days of culture were CD3^-CD56⁺(NK) cells, the absolute number of NK cells was 700. + -. 10.5 times that before culture (FIG. 1). Compared with the CB-NK cells before culture, the inhibitory receptors CD158a, CD158b and NKG2A have no obvious change (p)>0.05), whereas the ratio of activating receptors including NCKs (NKp30, NKp44, NKp46) and NKG2D were all significantly increased (p)<0.05) (FIG. 2), the purity and the amplification efficiency of NK cells were significantly improved as compared with those of NK cells cultured in example 1, and the purity of NK cells was varied as shown in FIG. 5. The expression of NK cell antibodies after culture is shown in FIGS. 6 and 7.

2. Killing activity of cord blood NK cells on leukemia cells

The CB-NK cells are used as effector cells to detect the cytotoxicity effect on malignant tumor cells K562 and Raji. Compared with the prior culture, the killing activity of the CB-NK cells to malignant tumor cells K562 and Raji is obviously improved (figure 3 and figure 4). Especially, freshly isolated uncultured CB-NK cells had very low killing of Raji cells expressing high HLA class I molecules, but the killing of Raji cells by amplified CB-NK cells was significantly increased (FIG. 4). The killing activity of K562 is basically consistent with that of NK cells cultured in example 1.

Comparative example

NK cells were cultured according to the method of example 1, except that step 4 was as follows:

and 4, step 4: the separated and frozen CBMC obtained in the step 1 is thawed and recovered in a 39 ℃ water bath box, washed for 2 times by RPMI1640 culture solution and suspended to 2 multiplied by 10 by RPMI l640 culture solution containing 10 percent fetal calf serum⁶one/mL of cell suspension, then 1mL of cell suspension per well in a 24-well plate. To the cell suspension, IL-2 (final concentration of 100U/mL) + IL-15 (final concentration of 100U/mL) + IL-21 (final concentration of 2500U/mL) was added. At 37 deg.C, 5% CO₂The culture in the incubator is carried out, the liquid in each hole is half-changed and supplemented with corresponding cell factors every 3d, and the culture is carried out for 14-21 d.

Compared with the NK cells obtained by culturing in the examples 1 and 2, the NK cells obtained by culturing in the comparative example have obviously reduced killing performance on malignant tumor cells K562 and obviously reduced killing performance on Raji cells. And the ratio of activating receptors including NCKs (NKp30, NKp44, NKp46) and NKG2D is significantly reduced.

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