In-vitro amplification method and application of canine NK (Natural Killer) cells capable of efficiently killing tumor cells

Abstract

The invention belongs to the technical field of bioengineering, and particularly discloses an in-vitro amplification method of canine NK cells capable of efficiently killing tumor cells and application of the canine NK cells. 3 mL of dog peripheral blood is taken, and dog peripheral blood mononuclear cells PBMC are obtained through separation; activating, amplifying and culturing canine NK cells capable of efficiently killing tumor cells; and collecting the amplified canine NK cells capable of efficiently killing the tumor cells, and storing the canine NK cells in a cryopreservation solution according to the cell density of 1 * 10 < 8 > cells / mL. According to the method, the canine NK cell with high amplification multiple can be obtained by only needing 3mL of canine peripheral blood, and the amplification multiple is high; the number of the dog NK cells of the healthy volunteer dog sample is 3.02 * 108, the amplification multiple is 1008, the total number of the dog NK cells of the tumor volunteer affected dog sample reaches 6.84 * 107, and the amplification multiple is 340; and the canine NK cell obtained by the invention has the effect of efficiently killing tumor cells.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to an in vitro amplification method and application of canine NK cells that efficiently kill tumor cells. Background Art

[0002] Natural killer (NK) cells are important cells of innate immunity and are mainly distributed in peripheral blood, liver and spleen. NK cells are not restricted by major histocompatibility complex (MHC) and can recognize various tumor cells, senescent cells and viruses without relying on antibodies, and kill target cells. They play an important role in the body's anti-tumor, anti-viral, immune regulation and elimination of senescent cells.

[0003] In human medicine, NK cells can effectively treat malignant tumors of the blood system and also show their therapeutic potential in solid tumors. Some scholars have found that canine NK cells have anti-tumor potential, such as canine breast cancer, osteosarcoma, melanoma, adenocarcinoma and glioma.

[0004] Although the application of canine NK cell adoptive immunotherapy has great prospects, due to ethical and invasive issues, veterinary researchers have so far mostly used canine NK cells isolated from peripheral blood. Since NK cells only account for 2.5% to 15% of peripheral lymphocytes, in tumor treatment applications, NK cells need to be infused multiple times (7.5×10 6 ~1×10 8 / kg), so a large amount of NK cells is required, which limits its clinical application.

[0005] Since the research on canine NK cells is still in its preliminary stage and the biological characteristics of canine NK cells are not well understood, the current in vitro expansion methods of canine NK cells mainly draw on the research experience of human medicine. The current common in vitro expansion methods of NK cells mainly include the expansion method of antibody plus cytokine mixture and the expansion method of feeder layer cells plus cytokines. The former stimulates the proliferation of NK cells by adding a variety of antibodies and cytokines to produce activation signals. The purity of NK cells amplified by this method is low. When facing difficult samples, the failure rate is high due to the excessive cytokines that cause NK cells to be overstimulated and exhausted, and the production cost is high; the latter generates activation signals by adding NK cell recognition sensitive feeder layer cells and cytokines. This method is simple, avoids exogenous contamination, has high amplification efficiency and low cost. The memory generated by NK cells through contact with feeder layer cells can increase cytotoxicity, which is the best choice for in vitro expansion of canine NK cells with high tumor cytotoxicity.

[0006] Therefore, in order to meet the needs of research on anti-tumor therapeutic applications and future clinical applications, it is urgent to develop an in vitro expansion method for canine NK cells with high expansion efficiency, high cell purity and strong killing effect on tumor cells. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides an in vitro expansion method and application of canine NK cells that can effectively kill tumor cells.

[0008] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0009] One of the technical solutions of the present invention is to provide an in vitro amplification method of canine NK cells that can efficiently kill tumor cells, comprising the following steps:

[0010] S1, thawing frozen K562 engineered cells;

[0011] S2, taking 3 mL of dog peripheral blood, separating and obtaining dog peripheral blood mononuclear cells (PBMC);

[0012] S3, resuspending canine peripheral blood mononuclear cells PBMC in an activation composition and / or an expansion composition, culturing continuously for 11 days at 37°C and 5% CO2, and centrifuging at 700g for 5 minutes after the expansion culture on the 11th day to obtain canine NK cells; wherein the activation composition is composed of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C, the number of K562 engineered cells is 0.5 to 1 times that of canine peripheral blood mononuclear cells PBMC, the volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 1000 to 2500 IU / mL, the content of vitamin B6 is 20.5 to 30.8 ng / mL, and the content of vitamin C is 100 to 200 μg / mL; the expansion composition is composed of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin 2 is 1000-2500 IU / mL, the content of vitamin B6 is 20.5-30.8 ng / mL, and the content of vitamin C is 100-200 μg / mL.

[0013] S4. Collect the amplified canine NK cells that are highly efficient in killing tumor cells and use a cell density of 1×10 8 cells / mL stored in cryopreservation medium.

[0014] Furthermore, the step S1 specifically includes:

[0015] The frozen K562 engineered cells were taken out of the -80°C freezer and placed in a 37°C water bath for rapid shaking to thaw, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, resuspended in PBS, counted with trypan blue staining, divided into tubes according to the required number of cells, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and set aside.

[0016] Furthermore, the step S2 specifically includes:

[0017] S21, obtain 3 mL of fresh canine peripheral blood from the volunteer dog, add 3 mL of DPBS buffer to dilute, slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation solution, and centrifuge at 700 g for 30 min;

[0018] S22, after the centrifugation, extract the middle buffy coat layer into a centrifuge tube and wash it twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs);

[0019] S23, adding red blood cell lysis solution to the washed canine peripheral blood mononuclear cells PBMC, lysing at 4°C for 5 min, and after lysis, centrifuging at 300g for 10 min and discarding the supernatant to obtain lysed canine peripheral blood mononuclear cells PBMC;

[0020] S24. Wash the lysed canine peripheral blood mononuclear cells (PBMCs) twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells (PBMCs).

[0021] Furthermore, the step S3 specifically includes:

[0022] S31. On day 0, canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to a cell density of 1×10 6 cells / mL, cultured at 37°C, 5% CO2, final volume 6mL;

[0023] S32. On the third day, the cells were collected in a 15 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded, the cell pellet was resuspended with the expansion composition, and cultured at 37° C. with 5% CO2;

[0024] S33, day 5, replenishing with fresh amplification composition;

[0025] S34. On the 7th day, the cells were collected in a 50 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded, and the cell pellet was resuspended with DPBS buffer. The cells were counted to calculate the number of K562 engineered cells required for secondary stimulation; the cells were resuspended with the activation composition, and cultured at 37° C., 5% CO2;

[0026] S35. From the 8th to the 10th day, the cell status was observed, the medium was changed, and the cell density was adjusted. The expansion composition was replenished every 2 days to make the final cell density 0.05×10 5 / mL~1×10 6 / mL;

[0027] S36. On the 11th day, the expanded cells were collected and centrifuged at 700 g for 5 min. The supernatant was discarded to obtain canine NK cells that efficiently kill tumor cells.

[0028] The second technical solution of the present invention is to provide canine NK cells that are highly effective in killing tumor cells obtained by the above method.

[0029] The third technical solution of the present invention is to provide an application of canine NK cells that can effectively kill tumor cells in the preparation of drugs that kill tumor cells.

[0030] Preferably, the tumor cells include, but are not limited to, canine breast cancer cells, canine osteosarcoma cells, canine melanoma cells, canine adenocarcinoma cells and canine glioma cells.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) The present invention only requires 3 mL of canine peripheral blood. The small amount of blood drawn can meet the needs of NK cell culture for both small and large dogs. The expansion multiples can also meet the needs of autologous transfusion for healthy dogs and sick dogs, which is conducive to the clinical application of canine NK cell transfusion in the future.

[0033] (2) The canine NK cell culture composition of the present invention has simple ingredients and does not require excessive cytokines and antibodies; the culture method provided by the present invention is simple and convenient.

[0034] (3) The canine NK cells of the present invention have a high expansion multiple. Through the above technical scheme, the number of canine NK cells in the healthy volunteer dog sample was 3.02×10 8 The number of canine NK cells obtained from the tumor volunteer dog samples reached 6.84×10 7 The amplification factor was 340 times.

[0035] (4) The canine NK cells obtained by the present invention have a highly effective killing effect on tumor cells. The canine NK cells obtained by the present invention have a 92% killing effect on K562 tumor cells at an effector-target ratio of 40:1, and have a 98% killing effect on the solid tumor D17 cell line (canine osteosarcoma), and a 63% killing effect on the CMT1211 cell line (canine mammary tumor), showing the great potential of the canine NK cells obtained by the culture expansion composition and method in the treatment of canine tumor diseases.

[0036] (5) No K562 engineered cells remain, and the safety is high. The canine NK cells obtained by the present invention can be used for adoptive immunotherapy after 11 days of amplification culture, freezing and then thawing and culturing for 2 days, without any K562 engineered cells remaining. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The morphology of canine NK cells at different expansion and culture stages: A, day 0; B, day 7; C, day 9; D, day 11.

[0038] Figure 2 Figure 3 shows the growth curve of canine NK cells during the culture process of different samples.

[0039] Figure 3 This is the flow cytometry test result of canine NK cells after 11 days of expansion.

[0040] Figure 4 The killing effect of canine NK cells on K562, D17 and CMT1211 tumor cells at different effector-target ratios after 7 days of expansion and 2 days of recovery culture.

[0041] Figure 5 This is the target cell morphology after canine NK cells were revived for 2 days and co-cultured with K562, D17 and CMT 1211 tumor cell lines for 24 hours.

[0042] Figure 6 The results of STR residual detection of canine NK cells after 2 days of resuscitation culture. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0044] Unless otherwise specified, the reagents designed in the following examples can be purchased from commercial sources. For the sake of simplicity, some operations do not describe in detail the parameters, steps and instruments used, which are all well known and reproducible to those skilled in the art.

[0045] Source of experimental materials:

[0046] K562 engineered cells: purchased from Zhongke Saier Biotechnology (Heilongjiang) Co., Ltd.;

[0047] D17 cell line: purchased from ATCC;

[0048] CMT1211 cell line: kindly provided by China Agricultural University;

[0049] Advance RPMI 1640 medium is a commercial product from GIBCO with catalog number 12633012;

[0050] Canine interleukin-2: purchased from Shandong Aibei Runkang Biotechnology Co., Ltd.;

[0051] LDH detection kit: purchased from Promega (Beijing) Biotechnology Co., Ltd.

[0052] Example 1

[0053] 1) Thawing frozen K562 engineered cells

[0054] Take the frozen K562 engineered cells out of the -80℃ freezer and place them in a 37℃ water bath for rapid shaking and thawing. Centrifuge at 1200rpm for 5min, discard the supernatant, resuspend in PBS, count with trypan blue staining, divide according to the required number of cells, centrifuge at 1200rpm for 5min, discard the supernatant, and set aside.

[0055] 2) Isolating and obtaining canine peripheral blood mononuclear cells (PBMC);

[0056] ① Obtain 3 mL of fresh canine peripheral blood from the volunteer dog, add 3 mL of DPBS buffer to dilute, slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation solution, and centrifuge at 700 g for 30 min;

[0057] ②After centrifugation, extract the middle buffy coat layer into a centrifuge tube and wash it twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs);

[0058] ③ Add red blood cell lysis solution to the washed canine peripheral blood mononuclear cells PBMC, lyse at 4°C for 5 minutes, centrifuge at 300g for 10 minutes after lysis, and discard the supernatant to obtain the lysed canine peripheral blood mononuclear cells PBMC;

[0059] ④ Wash the lysed canine peripheral blood mononuclear cells PBMC twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells PBMC.

[0060] 3) Activation and expansion of canine NK cells that efficiently kill tumor cells

[0061] ① On day 0, canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to a cell density of 1×10 6 cells / mL, cultured at 37°C, 5% CO2, final volume 6mL;

[0062] ② On the third day, the cells were collected in a 15 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded, the cell pellet was resuspended with the amplification composition, and cultured at 37 ° C, 5% CO2; In this embodiment, the amplification composition is composed of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C, the volume ratio of Advance RPMI 1640 medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 1000 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 200 μg / mL;

[0063] ③ On the 5th day, fresh amplification composition was added;

[0064] ④ On the 7th day, the cells were collected in a 50 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded and the cell pellet was resuspended with DPBS buffer, and the number of K562 engineered cells required for secondary stimulation was calculated by cell counting; the cells were resuspended with the activation composition and cultured at 37° C., 5% CO2; in this embodiment, the activation composition consists of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The number of K562 engineered cells is 0.5 times that of canine peripheral blood mononuclear cells PBMC, the volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 1000 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 200 μg / mL;

[0065] ⑤ From the 8th to the 10th day, observe the cell status, change the medium and adjust the cell density. Replenish the expansion composition every 2 days to make the final cell density 0.05×10 5 / mL~1×10 6 / mL;

[0066] ⑥ On the 11th day, the expanded cells were collected and centrifuged at 700 g for 5 min. The supernatant was discarded to obtain canine NK cells that efficiently kill tumor cells. 8 cells / mL stored in cryopreservation medium.

[0067] Example 2

[0068] The difference from Example 1 is that the activation composition used is composed of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The number of K562 engineered cells is 0.5 times that of canine peripheral blood mononuclear cells PBMC. The volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 100 μg / mL. The expansion composition is composed of Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. Advance RPMI The volume ratio of 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 100 μg / mL.

[0069] Example 3

[0070] The difference from Example 1 is that the activation composition used is composed of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The number of K562 engineered cells is 0.5 times that of canine peripheral blood mononuclear cells PBMC. The volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 20.5 ng / mL, and the content of vitamin C is 100 μg / mL. The expansion composition is composed of Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. Advance RPMI The volume ratio of 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 20.5 ng / mL, and the content of vitamin C is 100 μg / mL.

[0071] Comparative Example 1

[0072] The difference from Example 1 is that the number of K562 engineered cells in the activation composition used is 1 times that of canine peripheral blood mononuclear cells PBMC.

[0073] Comparative Example 2

[0074] The difference from Example 1 is that the activation composition used is composed of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The number of K562 engineered cells is 1 times that of canine peripheral blood mononuclear cells PBMC. The volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum, and glutamine is 94:5:1. The content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 200 μg / mL. The expansion composition is composed of Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. Advance RPMI The volume ratio of 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 2500 IU / mL, the content of vitamin B6 is 30.8 ng / mL, and the content of vitamin C is 100 μg / mL.

[0075] The number of cells in different time periods of the activation compositions and expansion compositions of different formulations in Examples 1 to 3, Comparative Examples 1 and 2 were counted respectively, and the results are shown in Table 1; the morphology of canine NK cells at different expansion stages is shown in Table 1. Figure 1 shown.

[0076] Table 1

[0077]

[0078] From Table 1 and Figure 1 It can be seen that as the expansion process progresses, the volume of activated canine NK cells increases, taking on an irregular or comma shape, the cell density increases, and they grow in clusters. The cell clusters become larger and larger with the expansion time, and no feeder layer cells are observed on the 7th and 11th days of expansion culture.

[0079] Then, the expansion multiples and cell phenotypes of canine NK cells obtained in Examples 1 to 3, Comparative Example 1 and Comparative Example 2 were detected by flow cytometry:

[0080] 1) Canine NK cell expansion multiple

[0081] Trypan blue staining was used to count cells on the 7th, 9th, and 11th days of expansion culture and growth curves were drawn, as shown in Table 1 and Figure 2 As shown in Figure 2, the number of cells increased rapidly on the 7th day, and the total number of cells in healthy volunteer dogs reached 7.03×10 on the 11th day. 8 The number of cells in the volunteer tumor-bearing dogs reached 1.58×10 on day 11. 8 The amplification factor was 79 times.

[0082] 2) Flow cytometry detection of canine NK cell purity

[0083] Take 1×10 6 Cells were washed once with DPBS, and resuspended in 100 μl DPBS containing 2% FBS; flow cytometry antibodies of canine CD3, CD21, and CD94 were added to the cell suspension, and incubated in the dark for 30 min; cells were rinsed twice with 1 mL DPBS, and resuspended in 200 μl DPBS. Flow cytometry was used to detect the proportion of CD3-CD21-CD94+ canine NK cells in the total number of cells. The experimental results are shown in Tables 1 and Figure 3 As shown in the figure, CD3-CD21- cells accounted for 98.85%, and among the CD3-CD21- cell population, CD94+ NK cells representing mature development accounted for 43.03%. According to the percentage of CD3-CD21-CD94+ cells, the number of NK cells in healthy volunteer dogs was 3.02×10 8 The number of NK cells in the tumor-bearing dogs was 6.84×10 on day 11. 7 , amplified 340 times.

[0084] The above results indicate that the in vitro expanded culture composition of the present invention can efficiently and significantly stimulate the proliferation of NK cells and expand mature canine NK cells, and can obtain satisfactory expansion multiples even in poor samples of tumor-bearing dogs.

[0085] Application Example 1

[0086] In order to verify whether the method obtained by the present invention can be used to prepare drugs for killing tumor cells, this example takes K562 cells, D17 cells and CMT1211 cells as examples to detect the killing effect of amplified canine NK cells on tumor cells, as follows:

[0087] 5×10 3 K562 cells and D17 cells were added to each well, and then canine NK cells that had been amplified for 7 days and resuscitated for 2 days were added at E:T ratios of 40:1, 20:1, 10:1, 5:1, 2.5:1, and 1.25:1, respectively, and incubated for 4 hours. According to the LDH detection kit (CytoTox96 Non-Radioactive Cytotoxicity Assay), NK cell control group, experimental group, background group, volume correction group, target cell self-release group, and target cell maximum release group were set up, with 3 replicates in each group. After incubation, the value at 492 nm was detected by an ELISA instrument, and the killing rate was calculated according to the formula.

[0088] Test results such as Figure 4As shown, the killing rate of canine NK cells against tumor cells after 2 days of resuscitation and culture is higher than that after 7 days of expansion. At 40:1, the killing rate of canine NK cells is the highest. The killing rate of canine NK cells against K562 cells after 2 days of resuscitation and culture is 92%, the killing rate against D17 canine osteosarcoma cells is 98%, and the killing rate against CMT1211 canine breast tumor cell line is 63%.

[0089] After 2 days of recovery, canine NK cells were co-cultured with K562, D17 and CMT1211 for 24 hours. Figure 5 As shown, after being co-cultured with canine NK cells for 24 hours, the cell membranes of adherent D17 cells and CMT1211 cells were incompletely ruptured, the boundaries were unclear, the cell nuclei were lysed, and the fragments were sparse and granular. K562 cells were surrounded by activated canine NK cells, lost the luster of living cells, had incomplete cell membrane rupture, and the cytoplasm was vacuolated, and ruptured and died.

[0090] In addition, in this example, canine NK cells were cryopreserved and revived and then cultured for 2 days, and the residual feeder layer cells were detected by STR. The results were as follows: Figure 6 As shown. Figure 6 It can be seen that after 11 days of expansion, STR testing using the cryopreservation and resuscitation technology did not reveal any residual K562 engineered cells used as feeder cells, indicating high safety.

[0091] The above results show that the canine NK cells obtained by the present invention have strong cytotoxicity to tumor cells, have great potential for future application in the adjuvant treatment of tumor diseases, and can be used to prepare drugs that kill tumor cells.

[0092] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A method for in vitro expansion of canine NK cells that efficiently kill tumor cells, characterized in that: The following steps are involved: S1, thawing frozen K562 engineered cells; S2, taking 3 mL of dog peripheral blood, separating and obtaining dog peripheral blood mononuclear cells (PBMC); S3, resuspending canine peripheral blood mononuclear cells PBMC in an activation composition and / or an expansion composition, culturing continuously for 11 days at 37°C and 5% CO2, and centrifuging at 700g for 5 minutes after the expansion culture on the 11th day to obtain canine NK cells; wherein the activation composition is composed of K562 engineered cells, Advance RPMI 1640 culture medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C, the number of K562 engineered cells is 0.5 to 1 times that of canine peripheral blood mononuclear cells PBMC, the volume ratio of Advance RPMI 1640 culture medium, fetal bovine serum and glutamine is 94:5:1, the content of canine interleukin 2 is 1000 to 2500 IU / mL, the content of vitamin B6 is 20.5 to 30.8 ng / mL, and the content of vitamin C is 100 to 200 μg / mL; the expansion composition is composed of Advance RPMI 1640 medium, fetal bovine serum, glutamine, canine interleukin 2, vitamin B6, and vitamin C. The volume ratio of Advance RPMI 1640 medium, fetal bovine serum, and glutamine is 94:5:

1. The content of canine interleukin 2 is 1000-2500 IU / mL, the content of vitamin B6 is 20.5-30.8 ng / mL, and the content of vitamin C is 100-200 μg / mL. S4. Collect the amplified canine NK cells that are highly efficient in killing tumor cells and use a cell density of 1×10 8 cells / mL stored in cryopreservation medium.

2. The in vitro amplification method of canine NK cells that efficiently kill tumor cells according to claim 1, characterized in that: The step S1 specifically includes: The frozen K562 engineered cells were taken out of the -80°C freezer and placed in a 37°C water bath for rapid shaking to thaw, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, resuspended in PBS, counted with trypan blue staining, divided into tubes according to the required number of cells, centrifuged at 1200 rpm for 5 min, the supernatant was discarded, and set aside.

3. The in vitro expansion method of canine NK cells that efficiently kill tumor cells according to claim 2, characterized in that: The step S2 specifically includes: S21, obtain 3 mL of fresh canine peripheral blood from the volunteer dog, add 3 mL of DPBS buffer to dilute, slowly add the diluted canine peripheral blood to the upper layer of 6 mL of lymphocyte separation solution, and centrifuge at 700 g for 30 min; S22, after the centrifugation, extract the middle buffy coat layer into a centrifuge tube and wash it twice with DPBS buffer to obtain washed canine peripheral blood mononuclear cells (PBMCs); S23, adding red blood cell lysis solution to the washed canine peripheral blood mononuclear cells PBMC, lysing at 4°C for 5 min, and after lysis, centrifuging at 300g for 10 min and discarding the supernatant to obtain lysed canine peripheral blood mononuclear cells PBMC; S24. Wash the lysed canine peripheral blood mononuclear cells (PBMCs) twice with DPBS buffer, centrifuge at 1200 rpm for 5 min, discard the supernatant, and obtain canine peripheral blood mononuclear cells (PBMCs).

4. The in vitro expansion method of canine NK cells that efficiently kill tumor cells according to claim 3, characterized in that: The step S3 specifically includes: S31. On day 0, canine peripheral blood mononuclear cells (PBMCs) were resuspended with the activation composition to a cell density of 1×10 6 cells / mL, incubated at 37°C, 5% CO2, final volume 6mL; S32. On the third day, the cells were collected in a 15 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded, the cell pellet was resuspended with the expansion composition, and cultured at 37° C. with 5% CO 2 ; S33, day 5, replenishing with fresh amplification composition; S34. On the 7th day, the cells were collected in a 50 mL centrifuge tube, centrifuged at 700 g for 5 min, the supernatant was discarded, and the cell pellet was resuspended with DPBS buffer. The cells were counted to calculate the number of K562 engineered cells required for secondary stimulation; the cells were resuspended with the activation composition, and cultured at 37° C., 5% CO2; S35. From the 8th to the 10th day, the cell status was observed, the medium was changed, and the cell density was adjusted. The expansion composition was replenished every 2 days to make the final cell density 0.05×10 5 / mL~1×10 6 / mL; S36. On the 11th day, the expanded cells were collected and centrifuged at 700 g for 5 min. The supernatant was discarded to obtain canine NK cells that efficiently kill tumor cells.

5. A canine NK cell that is highly efficient in killing tumor cells obtained by the method according to any one of claims 1 to 4.

6. Use of the canine NK cells that efficiently kill tumor cells as claimed in claim 5 in the preparation of drugs that kill tumor cells.

7. Use of the canine NK cells that efficiently kill tumor cells according to claim 6 in the preparation of drugs that kill tumor cells, characterized in that: The tumor cells include canine breast cancer cells, canine osteosarcoma cells, canine melanoma cells, canine adenocarcinoma cells and canine glioma cells.

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