CN113913386B

CN113913386B - Trophoblast cell and application thereof in amplifying human NK cells

Info

Publication number: CN113913386B
Application number: CN202111183520.7A
Authority: CN
Inventors: 张欢; 于洋; 程铧
Original assignee: Beijing Yibo Puhui Biotechnology Development Co ltd
Current assignee: Beijing Yibo Puhui Biotechnology Development Co ltd
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2022-06-24
Anticipated expiration: 2041-10-11
Also published as: CN113913386A

Abstract

The application relates to the technical field of genetic engineering, and particularly discloses a trophoblast cell and application thereof in amplifying human NK cells. The trophoblast cell expressing cytokines including CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83 and MICA; the trophoblast cells are NIH3T3 cells; and a cytokine lentiviral vector for preparing the trophoblast cell and a cytokine lentivirus prepared by using the cytokine lentiviral vector; and an NK cell amplification medium prepared by using the trophoblast cells; and the application of the trophoblast in expanding NK cells. The NK cells prepared by the method have better safety and ideal killing data.

Description

Trophoblast cell and application thereof in amplifying human NK cells

Technical Field

The application relates to the technical field of genetic engineering, in particular to a trophoblast cell and application thereof in amplifying human NK cells.

Background

Natural killer cells (NK cells) are lymphocytes produced by CD34+ hematopoietic progenitors in the bone marrow. NK cells are mainly present in blood, liver, spleen, bone marrow, but factors such as inflammation, infection, etc. can trigger NK cell migration into almost any tissue. NK cells are important immune cells in the human immune system, can quickly react and kill cells infected by exogenous pathogens and having a malignant trend caused by pressure, and are in a key position in the processes of resisting viruses and tumors in the organism.

NK cells in the related art mediate cytotoxicity mainly through the following mechanism. Among these, the most studied route is degranulation, i.e. the release of cytotoxic particles by NK cells upon interaction with target cells. This pathway is triggered by signals that activate cell surface receptors, such as NKG2D, DNAM1 and the natural cytotoxicity receptors NKp30, NKp44 and NKp46, among others. Another pathway is the death receptor pathway, the Tumor Necrosis Factor (TNF) -related apoptosis-inducing ligand (TRAIL) -TRAIL receptor (TRAILR) pathway and the Fas-Fas ligand (FasL) pathway (also known as the CD 95-CD 95 ligand pathway). Rather than triggering the release of cytotoxic particles, the death receptor pathway induces apoptosis through caspase activation within the target cell, thereby inducing cytotoxicity.

Currently, NK cells are widely available from, for example, Peripheral Blood Mononuclear Cells (PBMC), cord blood, NK cell lines and human embryonic stem cells (hESC) and Induced Pluripotent Stem Cells (iPSC) as starting materials. The most predominant methods for expanding NK cells are two: the first is the co-stimulation of NK cell expansion by multiple cytokines; the second is the expansion of NK cells by trophoblast cell culture.

The methods for culturing NK cells by multi-cytokine co-stimulation can be divided into two methods: firstly, separating NK cells in PBMC, and then culturing and amplifying to obtain high-purity NK cells; second, PBMC cells were cultured directly to expand NK cells. In the method for amplifying NK cells by culturing trophoblast cells, commonly used trophoblast cells comprise genetically engineered K562 cells and other artificially established antigen presenting cells (aAPC), and researchers also use autologous PBMC as the trophoblast cells to amplify the NK cells.

Based on the above, although various methods for NK cell expansion are available to meet the dose required in clinical treatment. However, the killing data of NK cells and clinical manifestations of NK cells show that the activity of NK cells has a larger promotion space.

Disclosure of Invention

In order to obtain NK cells with higher activity, the application provides a trophoblast cell and application thereof in amplifying human NK cells.

In a first aspect, the present application provides a trophoblast cell, which adopts the following technical scheme:

a trophoblast cell expressing cytokines including CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83, and MICA; the trophoblast cells are NIH3T3 cells.

Preferably, the CD80 comprises the amino acid sequence shown as SEQ ID NO 1.

Preferably, the CD70 comprises the amino acid sequence set forth as SEQ ID NO 4.

Preferably, the CD86 comprises the amino acid sequence shown as SEQ ID NO 7.

Preferably, the 4-1BBL comprises an amino acid sequence as shown in SEQ ID NO 10.

Preferably, said CD40 comprises the amino acid sequence shown as SEQ ID NO 13.

Preferably, the CD58 comprises the amino acid sequence shown as SEQ ID NO 16.

Preferably, the CD83 comprises the amino acid sequence shown as SEQ ID NO 19.

Preferably, the MICA comprises the amino acid sequence shown as SEQ ID NO 22.

In a second aspect, the present application provides a cytokine lentiviral vector, which adopts the following technical scheme:

a cytokine lentiviral vector for preparing the above trophoblast cell.

Preferably, the lentiviral vector comprises any one of, or a combination of at least two of, a CD80 coding sequence, a CD70 coding sequence, a CD86 coding sequence, a 4-1BBL coding sequence, a CD40 coding sequence, a CD58 coding sequence, a CD83 coding sequence and a MICA coding sequence.

In a third aspect, the present application provides a cytokine lentivirus, which adopts the following technical scheme:

a cytokine lentivirus comprising the above cytokine lentivirus vector.

Preferably, the cytokine lentivirus is obtained by co-introducing the above-mentioned cytokine lentivirus vector and helper plasmid into mammalian cells.

Preferably, the mammalian cell comprises any one of 293 cells, 293T cells and 293FT cells.

In a specific embodiment, the mammalian cell is a 293 cell.

In a specific embodiment, the mammalian cell is a 293T cell.

In a specific embodiment, the mammalian cells are 293FT cells.

In a fourth aspect, the present application provides a method for preparing the above trophoblast cell, which adopts the following technical scheme:

the preparation method of the trophoblast cell specifically comprises the following steps:

constructing the cytokine lentivirus vector, and introducing the cytokine lentivirus vector and helper plasmid into mammalian cells together to obtain the cytokine lentivirus;

infecting the obtained cell factor lentivirus with host cell, and transducing to obtain trophoblast cell.

In a fifth aspect, the present application provides an NK cell amplification medium, which adopts the following technical scheme:

an NK cell expansion medium, which comprises the above trophoblast cells.

Preferably, the NK cell culture medium further comprises any one of or a combination of at least two of basal medium, serum, plasma or antibiotics.

In a sixth aspect, the present application provides a method for preparing the above trophoblast cell, cytokine lentiviral vector, cytokine lentiviral, trophoblast cell, or an NK cell expansion medium for expanding NK cells.

In summary, the present application has the following beneficial effects:

1. the NK cell activation and amplification is carried out on the basis of the trophoblast NIH3T3, and the amplified NK cell shows good anti-tumor activity and has better safety and ideal killing data.

2. The NIH3T3 cells as trophoblast cells are normal mouse fibroblasts, but not cancer cells which are similar to K562 and have potential safety problems, so that the potential safety hazard is reduced. And the feeder cells based on NIH3T3 do not contain replication viruses, such as EBV virus, and the like, so the feeder cells are safer than human-derived feeder cells.

3. In addition, NIH3T3, a cell of xenogeneic origin, which is a target cell of NK cells, is cleared by NK cells with high cytotoxicity, and has no safety problem caused by residue even without irradiation.

4. Meanwhile, NIH3T3 cells can be effectively engineered to express various NK cell activating molecules, so that the purity range of NK cells is controlled to be about 70-90%.

Drawings

FIG. 1 shows the expression of various cytokines in NIH3T3 cells.

FIG. 2 shows the result of measurement of NK cell induction after NIH3T3 cells were co-cultured with PBMC cells (derived from volunteers A) in example 1 (A: antibody isotype control group; B: antibody group).

FIG. 3 shows the results of detection of NK cells induced by the co-culture of NIH3T3 cells and PBMC cells (derived from volunteer B) in example 2 (A: antibody isotype control group; B: antibody group).

FIG. 4 shows the results of detection of NK cell expression inhibitory receptor KIR2DL1 in example 1 by expansion of NIH3T3 cells (A: antibody isotype control group; B: antibody group).

FIG. 5 shows the results of the detection of the expression of the inhibitory receptor KIR2DL1 by NK cells expanded from NIH3T3 cells in example 2 (A: antibody isotype control group; B: antibody group).

FIG. 6 shows the results of killing of pancreatic cancer cells PANC-1 by NK cells amplified in example 1 at different effective target ratios.

FIG. 7 shows the results of killing of pancreatic cancer cells PANC-1 by NK cells amplified in example 2 at different effective target ratios.

FIG. 8 shows the results of killing of pancreatic cancer cells BxPC-3 by NK cells amplified in example 1 at different effective target ratios.

FIG. 9 shows the results of killing of pancreatic cancer cells BxPC-3 by NK cells expanded in example 2 at different effective target ratios.

Detailed Description

The present application provides a trophoblast cell expressing cytokines including CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83, and MICA. Specifically, the trophoblast cells are NIH3T3 cells. Wherein the CD80 comprises an amino acid sequence shown as SEQ ID NO 1. The CD70 comprises the amino acid sequence set forth as SEQ ID NO 4. The CD86 comprises an amino acid sequence as shown in SEQ ID NO 7. The 4-1BBL comprises an amino acid sequence shown as SEQ ID NO 10. The CD40 comprises an amino acid sequence shown as SEQ ID NO 13. The CD58 comprises the amino acid sequence shown as SEQ ID NO 16. The CD83 comprises an amino acid sequence shown as SEQ ID NO 19. The MICA comprises an amino acid sequence as shown in SEQ ID NO 22.

The application also provides a cytokine lentiviral vector, which is used for preparing the trophoblast cell. Specifically, the lentiviral vector comprises any one or a combination of at least two of a CD80 coding sequence, a CD70 coding sequence, a CD86 coding sequence, a 4-1BBL coding sequence, a CD40 coding sequence, a CD58 coding sequence, a CD83 coding sequence and a MICA coding sequence.

In addition, the application also provides a cytokine lentivirus, and the cytokine lentivirus comprises the cytokine lentivirus vector. Specifically, the cytokine lentivirus is obtained by introducing the cytokine lentivirus vector and a helper plasmid into a mammalian cell together. In one step, the mammalian cell comprises any one of 293 cells, 293T cells and 293FT cells.

The application provides a preparation method of the trophoblast cell, which specifically comprises the following steps: constructing the cytokine lentivirus vector, and introducing the cytokine lentivirus vector and helper plasmid into mammalian cells together to obtain the cytokine lentivirus; infecting the obtained cell factor lentivirus with host cell, and transducing to obtain trophoblast cell.

In addition, the present application also provides an NK cell expansion medium comprising the above trophoblast cells. Further, the NK cell culture medium further comprises any one of or a combination of at least two of basal medium, serum, plasma or antibiotics.

The application also provides a preparation method of the trophoblast cell, the cytokine lentiviral vector, the cytokine lentivirus and the trophoblast cell or an application of the NK cell amplification culture medium in NK cell amplification.

The NK cell activation and amplification is carried out on the basis of the trophoblast NIH3T3, and the amplified NK cell shows good anti-tumor activity and good safety. In addition, NIH3T3, a cell of xenogeneic origin, which is a target cell of NK cells, is cleared by NK cells with high cytotoxicity, and has no safety problem caused by residue even without irradiation.

The following preparations 1 to 24, examples 1 to 2 and comparative examples 1 to 2 were combined. FIGS. 1-9 and the test assays illustrate the present application in further detail.

Preparation example

Preparation examples 1 to 8

Preparation examples 1-8 each provide a method for constructing a cytokine lentiviral vector. The difference is that the cytokine in the prepared cytokine lentiviral vector is different in type. The specific operation steps are as follows:

(1) obtaining coding sequence information of the cell factor, designing a primer and synthesizing the primer by a third party company;

(2) cloning a coding region of a cytokine by taking cDNA of a lymph node cell as a template, carrying out enzyme digestion, inserting a lentiviral vector EcoRI-XbaI or XhoI-XbaI site, and transforming to E.coli; after sequencing is correct, bacteria are shaken and extracted to obtain a cytokine lentiviral vector;

wherein, the acquisition way of the information of the cytokine coding sequence is NCBI database; lymph node cells are commercially available.

The cytokine types, coding sequence information, and primer information for the cytokines of preparation examples 1 to 8 are shown in Table 1.

TABLE 1 coding sequence information of each cytokine and corresponding primer information

Preparation examples 9 to 16

Preparation examples 9 to 16 each provide a method for producing a cytokine lentiviral vector. The method is characterized in that the types of the produced cytokine lentiviral vectors are different, and the specific operation steps are as follows:

(1) co-transfecting the cytokine lentiviral vectors obtained in preparation examples 1 to 8 and the mixed packaging plasmid into 293 cells (human kidney epithelial cell lines) by using a transfection reagent-calcium phosphate transfection reagent to obtain the 293 cells after co-transfection; co-transfected 293 cells at 37 ℃ in CO₂Culturing in an incubator, and taking the cultured supernatant when culturing for 24h, 48h and 72h respectively to obtain the supernatant of the 293 cells after co-transfection. Wherein, the mixed packaging plasmid comprises expression plasmids of VSV-G, Gag-Pol and Rev.

(2) Collecting the supernatant of the co-transfected 293 cells cultured in the step (1), and centrifuging the collected supernatant for 2h at 4 ℃ at 25000 r/min; discarding the centrifuged supernatant, and keeping the precipitate; resuspending the obtained precipitate with RPMI1640 culture medium to obtain lentivirus carrying cytokine gene, and freezing at-80 deg.C in refrigerator.

The cytokine lentiviral vectors corresponding to preparation examples 9 to 16 described above are shown in Table 2.

TABLE 2 cytokine Lentiviral vectors corresponding to preparation examples 9 to 16

Preparation example No	Lentiviral vector species	Post-production product
			9	Preparation example 1-CD80 Lentiviral vector	Lentivirus carrying CD80 gene
10	Preparation example 2-CD70 Lentiviral vector	Lentivirus carrying CD70 gene
			11	Preparation example 3-CD86 Lentiviral vector	Lentivirus carrying CD86 gene
12	Preparation example 4-4-1BBL Lentiviral vector	Lentiviruses carrying the 4-1BBL gene
			13	Preparation example 5-CD40 Lentiviral vector	Lentivirus carrying CD40 gene
14	Preparation example 6-CD58 Lentiviral vector	Lentivirus carrying CD58 gene
			15	Preparation example 7-CD83 Lentiviral vector	Lentivirus carrying CD83 gene
16	Preparation example 8-MICA Lentiviral vector	Lentivirus carrying MICA gene

Preparation example 17

This preparation provides a method for producing a cytokine lentiviral vector.

The present preparation differs from preparation 9 in that: the transfection reagent is a lipofectin.

Preparation example 18

This preparation provides a method for producing a cytokine lentiviral vector.

The present preparation differs from preparation 9 in that: the transfection reagent is a high molecular polymer transfection reagent.

Preparation example 19

The present preparation example provides a method for producing a cytokine lentiviral vector.

The present preparation differs from preparation 9 in that: the co-transfected cells were 293T cells (lymphocytes).

Preparation example 20

The present preparation differs from preparation 9 in that: the co-transfected cells were 293FT cells (human embryonic kidney cells).

Preparation example 21

The present preparation differs from preparation 9 in that: and (2) collecting supernatant of the co-transfected 293 cells cultured in the step (1), centrifuging at 3000r/min for 10min at room temperature, taking the supernatant, adding 4 XPEG 8000 concentrated solution (PEG-8000 concentration of 40% (W/V) and NaCl concentration of 1.2M), uniformly mixing, and standing at 4 ℃ overnight. Centrifuging at 4 deg.C for 30-60min the next day at 3000 r/min; discarding the centrifuged supernatant, and keeping the precipitate; the obtained precipitate was resuspended in RPMI1640 medium and frozen in a freezer at-80 ℃ for further use.

Preparation example 22

This preparation provides a method for producing a cytokine lentiviral vector.

The present preparation example differs from preparation example 21 in that: in the step (2), the rotation speed at 4 ℃ during centrifugation is 1600 g.

Preparation example 23

The present preparation example provides a method for preparing trophoblast cells (NIH 3T3 cells) comprising 8 cytokines. The 8 cytokines are CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83 and MICA respectively. The 8 cytokine lentiviral vectors used were the CD80 lentiviral vectors prepared in preparation examples 1 to 8 and produced in preparation examples 9 to 16, respectively. CD70 lentiviral vector. CD86 lentiviral vector. 4-1BBL lentiviral vector, CD40 lentiviral vector, CD58 lentiviral vector, CD83 lentiviral vector, MICA lentiviral vector. In the preparation example, the sequence of transferring 8 lentiviral vectors into NIH3T3 cells can be changed, and the induction effect of the NIH3T3 cells after transferring the lentiviral vectors on NK cells is not influenced.

The specific operation steps are as follows:

(1) resuscitating cultured NIH3T3 cells: reviving NIH3T3 cells purchased from ATCC to obtain revived NIH3T3 cells;

(2) recovering NIH3T3 cells obtained in step (1) according to 3 x 10⁵-5×10⁵Inoculating each well into a 6-well plate, and simultaneously adding the virus liquid carrying the CD80 gene obtained in the preparation example 9 and the virus liquid carrying the CD70 gene obtained in the preparation example 10 into the wells, wherein the MOI value of the virus is controlled to be 5-10; adding polybrene solution with final concentration of 10ug/ml, mixing, performing transduction culture at 37 deg.C, culturing for 6 hr, replacing culture medium containing serum, and culturing for 24 hr;

(3) absorbing the culture obtained in the step (2) and discarding the supernatant of the culture medium, adding the cell factor lentiviral vector and polybrene again according to the operation of the step (2), performing virus transduction culture again, replacing the culture medium containing serum after culturing for 6h, and continuing culturing for 24 h;

(4) repeating the above operation, digesting the cells on the third day after virus transfection, taking part of the cells for flow cytometry analysis, and determining the expression of CD80 cytokines and CD70 cytokines in NIH3T3 cells;

(5) after confirming that the CD80 cytokines and CD70 cytokines were expressed in the NTH3T3 cells, the procedures from step (1) to step (4) were continued, and the viral fluid carrying the CD86 gene, the viral fluid carrying the 4-1BBL gene, the viral fluid carrying the CD40 gene, the viral fluid carrying the CD58 gene, the viral fluid carrying the CD83 gene, and the viral fluid carrying the MICA gene obtained in preparation examples 11 to 16 were used to transfer the respective genes into the NTH3T3 cells in order to obtain the NTH3T3 cells expressing 8 cytokines, and the expression of the 8 cytokines in the NTH3T3 cells was confirmed by flow cytometry.

The expression of 8 cytokines in NIH3T3 cells infected with 8 lentiviruses obtained in this preparation is shown in FIG. 1.

FIG. 1 shows the expression of various cytokines in NIH3T3 cells.

As shown in FIG. 1, NIH3T3 cells efficiently expressed CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83 and MICA cytokines.

Examples

Example 1

This example provides a method for amplifying NK cells by co-culturing NIH3T3 cells with PBMC cells. Among them, NIH3T3 cells were NIH3T3 cells containing 8 cytokines prepared in preparation 23. PBMC cells were derived from healthy volunteer a for subsequent experiments. The specific operation steps are as follows:

(1) taking normally cultured NIH3T3 cells, inoculating into 6-well plate by trypsinization, when the confluence degree of NIH3T3 cells reaches about 80% -90%, replacing the liquid in the well with a culture system containing 5% human serum and serum-free culture medium (such as X-VIVO15), and simultaneously carrying out the steps of 2 × 10⁶-3×10⁶The PBMC cells were added at a cell concentration of/ml, and the recombinant interleukin 2(IL2) was co-cultured at 37 ℃ with 500-1000 units/ml.

(2) After 24 hours of co-culture, the state of NIH3T3 cells after co-culture with PBMC cells was observed, and it was found that the medium had a yellowish color, and the PBMC cells had a high cell density and had a morphology. According to the cell density of 1X 10 in the culture system⁶～1.5×10⁶Adding a culture medium containing 5% human serum and 500-1000unit/ml IL2 according to the proportion of each ml, gently blowing, uniformly mixing, and continuously culturing for 24 hours.

(3) After 3-5 days, the cells were counted according to a cell density of 1X 10 in the culture system⁶～1.5×10⁶Adding a culture medium containing 5% human serum and 500-1000unit/ml IL2 at a ratio of one to two to continue culturing.

(4) The growth rate of the immune cells is basically stable, and the immune cells are half-and-half supplemented with fresh X-VIVO15 containing 5% human serum every day from the 6 th dayThe cell density before fluid replacement on the next day was maintained at 2X 10 by adding 1000uni/mL IL2 based on the volume of the new fluid replacement⁶～3×10⁶And/ml. Replacing a large-capacity culture bottle or culture bag according to the total volume of the culture solution. At this stage, autologous serum is not used for culture, cells can maintain good growth, and NK cells which are co-cultured and amplified by NIH3T3 cells and PBMC cells are obtained.

Example 2

This example provides a method for amplifying NK cells by co-culturing NIH3T3 cells with PBMC cells. It differs from example 1 in the origin of the PBMC cells. In this example, PBMC cells were derived from healthy volunteer B for subsequent experiments.

Comparative example

Comparative example 1

This comparative example provides a NK cell obtained by amplification using a pure cytokine method. The specific amplification method refers to CN108676775A in the scheme of example 2. The NK cell takes A549 (human lung cancer cell) as a target cell, and the effective-target ratio is 5: 1 hour and overnight culture, the killing rate of the NK cells to the A549 cells is 26.17 percent.

Comparative example 2

This comparative example provides an NK cell expanded using K562 cells expressing various cytokines as trophoblast cells. The specific amplification method refers to the protocol of the test group in the specification of CN 105985931A. The NK cell takes A549 (human lung cancer cell) as a target cell, and the effective-target ratio is 10: when 1 hour, the killing rate of the NK cells cultured for 90 hours to A549 reaches 90.64 percent.

Detection test

Test for detection

NK cells induced by co-culture of NIH3T3 cells and PBMC cells and expression of the NK cell inhibitory receptor KIR2DL1 were examined by flow cytometry using the cells cultured up to day 14 in example 1-2 as the examination subjects.

The results of the tests are shown in FIGS. 2 to 5.

Among them, FIGS. 2 and 3 show the results of NK cell induction in the co-culture of NIH3T3 cells and PBMC cells.

FIG. 3 shows the result of measurement of NK cell induction after NIH3T3 cells were co-cultured with PBMC cells (derived from volunteer B) in example 2 (A: antibody isotype control group; B: antibody group).

Wherein, FIGS. 4 and 5 show the results of detecting the expression of inhibitory receptors by NK cells expanded from NIH3T3 cells.

FIG. 5 shows the results of detection of NK cell expression inhibitory receptor KIR2DL1 in example 2 by expansion of NIH3T3 cells (A: antibody isotype control group; B: antibody group).

As can be seen from FIGS. 2 and 3, the NK cells induced by the co-culture of NIH3T3 cells and PBMC cells according to the present invention are healthy and full in morphology. Wherein, the proportion of CD3-CD56+ cells amplified after the NIH3T3 cells and PBMC cells (from the volunteer A) are cultured together is 71.6%; the proportion of CD3-CD56+ cells expanded after the NIH3T3 cells were co-cultured with PBMC cells (from volunteer B) was 73.8%.

In addition, as can be seen from fig. 4 and 5, the present application utilizes the fact that the expression of inhibitory receptor KIR2DL1 recombinant protein (killer cell immunoglobulin-like receptor 2DL1 recombinant protein) in NK cells induced and generated after the co-culture of NIH3T3 cells and PBMC cells is low, which indicates that the NK cells prepared by the present application are in an activated state, and are favorable for exerting a potent killing effect.

Test No. 2

The NK cells obtained by amplification in example 1 and example 2 were used as detection targets, and detection tests were performed to examine the killing ability against pancreatic cancer cells.

Detection method

Preparation of NK cells for assay

(1) 10ml of cell suspension cultured to day 14 in example 1 and example 2 was taken and cultured at 1000r/minCentrifuging for 5min, and discarding the supernatant; resuspending and washing the cells by using 5ml of PBS, centrifuging for 5min under the condition of 1000r/min, and removing the supernatant; resuspending cells using 500ul serum-free X-VIVO15 medium, at 1X 10 as per the instructions⁷1X 10 for each cell⁷Adding CD3 magnetic beads according to the number of cells, and placing the mixture on a shaking table at room temperature for 15min at a speed of 50r/min to ensure that the magnetic beads are fully contacted with the cells to obtain a cell suspension with the magnetic beads;

(2) diluting the cell suspension with the magnetic beads obtained in the step (1) by using 5ml of PBS, reversing and uniformly mixing, and then placing on a magnetic frame for standing for 2 min; carefully transfer the supernatant into a clean centrifuge tube; the above washing operation was repeated using PBS; twice, transferring the washed supernatant into the same centrifugal tube to obtain cell suspension separated from the cell suspension with the magnetic beads;

(3) centrifuging the cell suspension obtained in the step (2) for 5min under the condition of 1000r/min, centrifuging and discarding the supernatant; carrying out heavy suspension negative screening by using an X-VIVO15 culture medium containing 5% human serum to obtain NK cells, transferring the NK cells into a culture dish, adding IL2 into the culture medium according to the final concentration of 500-;

(4) and culturing until the 5 th day, and taking the NK cells for a tumor cell killing test.

Secondly, preparing pancreatic cancer cells

On the first three days of the killing test, pancreatic cancer cells stably expressing luciferase were recovered and subcultured so as to be in an optimum growth state at the time of the killing test.

Third, killing test

(1) Pancreatic cancer cells were counted by digestion according to 5X 10 cell number⁴Pancreatic cancer cells were seeded in 24-well plates; after pancreatic cancer cells are fully adherent, the ratio of effective targets (E: T) is 1.25: 1. 2.5: 1. 5: 1. 10: 1, respectively adding NK cells into a 24-pore plate, arranging three parallel pores in each effective target ratio, uniformly mixing, placing the 24-pore plate into an incubator at 37 ℃, and standing and culturing for 4 hours.

(2) Taking the 24-well plate cultured in the step (1), and observing the pancreatic cancer cells under a microscope.

(3) Taking the 24-pore plate cultured in the step (1), sucking and removing culture medium supernatant, gently washing cells in the pores for 2 times by using PBS, and completely sucking liquid in the pores; cracking cells with 100ul of cell lysate special for luciferase detection, standing on ice for 10min, fully blowing the bottom cells of the pores, transferring the cell lysate into an EP tube, and centrifuging for 5min at 4 ℃ and 14000 r/min; then transferring the centrifuged supernatant into a clean EP tube to obtain a cell lysate;

(4) and (3) detection: adding 100ul of luciferase substrate into the enzyme label plate, rapidly sucking 20ul of cell lysate into substrate holes, and immediately detecting on a machine; selecting a luciferase reporter gene detection scheme on a multifunctional microplate reader, and detecting the fluorescence intensity in each hole; and calculating the killing capacity of the NK cells to the pancreatic cancer cells under each effect target ratio according to the ratio of the fluorescence intensity of the experimental wells to the fluorescence intensity of the control wells.

The result of the detection

By observing pancreatic cancer cells in the 24-well plate wells through a microscope in the step (2), the number of pancreatic cancer cells was significantly reduced.

The results of the killing assays are shown in FIGS. 6-9.

FIG. 9 shows the results of killing of pancreatic cancer cells BxPC-3 by NK cells amplified in example 2 at different effective target ratios.

As can be seen from fig. 6 and 7, the NK cells induced by the co-culture of NIH3T3 cells and PBMC cells in the present application have a significant killing effect on pancreatic cancer cells PANC-1, and only in the ratio of 1.25: at 1-effect target ratio, the PANC-1 can generate 40-60% killing rate on pancreatic cancer cells. In addition, as can be seen from fig. 8 and 9, the NK cells induced by the co-culture of NIH3T3 cells and PBMC cells according to the present application also have a significant killing effect on pancreatic cancer cells BxPC-3, and only in the range of 1.25: under the condition of 1-effect target ratio, the BxPC-3 can generate 45-75% killing rate on pancreatic cancer cells.

Compared with comparative examples 1 and 2, the NK cell activation and expansion is carried out based on the trophoblast cell NIH3T3, and the expanded NK cell can be cultured in vitro for a longer time and shows good antitumor activity. At the same time, the present application is safer than the K562 trophoblast cells used in comparative example 2.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.

Sequence listing

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<400> 4

atgccggagg agggttcggg ctgctcggtg cggcgcaggc cctatgggtg cgtcctgcgg 60

gctgctttgg tcccattggt cgcgggcttg gtgatctgcc tcgtggtgtg catccagcgc 120

ttcgcacagg ctcagcagca gctgccgctc gagtcacttg ggtgggacgt agctgagctg 180

cagctgaatc acacaggacc tcagcaggac cccaggctat actggcaggg gggcccagca 240

ctgggccgct ccttcctgca tggaccagag ctggacaagg ggcagctacg tatccatcgt 300

gatggcatct acatggtaca catccaggtg acgctggcca tctgttcctc cacgacggcc 360

tccaggcacc accccaccac cctggccgtg ggaatctgct ctcccgcctc ccgtagcatc 420

agcctgctgc gtctcagctt ccaccaaggt tgtaccattg cctcccagcg cctgacgccc 480

ctggcccgag gggacacact ctgcaccaac ctcactggga cacttttgcc ttcccgaaac 540

actgatgaga ccttctttgg agtgcagtgg gtgcgcccct ga 582

<210> 5

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gcgaattcgc caccatgccg gaggagggtt cgggc 35

<210> 6

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctctagatc aggggcgcac ccactgcact cc 32

<210> 7

<211> 990

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggatcccc agtgcactat gggactgagt aacattctct ttgtgatggc cttcctgctc 60

tctggtgctg ctcctctgaa gattcaagct tatttcaatg agactgcaga cctgccatgc 120

caatttgcaa actctcaaaa ccaaagcctg agtgagctag tagtattttg gcaggaccag 180

gaaaacttgg ttctgaatga ggtatactta ggcaaagaga aatttgacag tgttcattcc 240

aagtatatgg gccgcacaag ttttgattcg gacagttgga ccctgagact tcacaatctt 300

cagatcaagg acaagggctt gtatcaatgt atcatccatc acaaaaagcc cacaggaatg 360

attcgcatcc accagatgaa ttctgaactg tcagtgcttg ctaacttcag tcaacctgaa 420

atagtaccaa tttctaatat aacagaaaat gtgtacataa atttgacctg ctcatctata 480

cacggttacc cagaacctaa gaagatgagt gttttgctaa gaaccaagaa ttcaactatc 540

gagtatgatg gtattatgca gaaatctcaa gataatgtca cagaactgta cgacgtttcc 600

atcagcttgt ctgtttcatt ccctgatgtt acgagcaata tgaccatctt ctgtattctg 660

gaaactgaca agacgcggct tttatcttca cctttctcta tagagcttga ggaccctcag 720

cctcccccag accacattcc ttggattaca gctgtacttc caacagttat tatatgtgtg 780

atggttttct gtctaattct atggaaatgg aagaagaaga agcggcctcg caactcttat 840

aaatgtggaa ccaacacaat ggagagggaa gagagtgaac agaccaagaa aagagaaaaa 900

atccatatac ctgaaagatc tgatgaagcc cagcgtgttt ttaaaagttc gaagacatct 960

tcatgcgaca aaagtgatac atgtttttaa 990

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcctcgaggc caccatggat ccccagtgca ctatg 35

<210> 9

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gctctagatt aaaaacatgt atcacttttg tc 32

<210> 10

<211> 765

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atggaatacg cctctgacgc ttcactggac cccgaagccc cgtggcctcc cgcgccccgc 60

gctcgcgcct gccgcgtact gccttgggcc ctggtcgcgg ggctgctgct gctgctgctg 120

ctcgctgccg cctgcgccgt cttcctcgcc tgcccctggg ccgtgtccgg ggctcgcgcc 180

tcgcccggct ccgcggccag cccgagactc cgcgagggtc ccgagctttc gcccgacgat 240

cccgccggcc tcttggacct gcggcagggc atgtttgcgc agctggtggc ccaaaatgtt 300

ctgctgatcg atgggcccct gagctggtac agtgacccag gcctggcagg cgtgtccctg 360

acggggggcc tgagctacaa agaggacacg aaggagctgg tggtggccaa ggctggagtc 420

tactatgtct tctttcaact agagctgcgg cgcgtggtgg ccggcgaggg ctcaggctcc 480

gtttcacttg cgctgcacct gcagccactg cgctctgctg ctggggccgc cgccctggct 540

ttgaccgtgg acctgccacc cgcctcctcc gaggctcgga actcggcctt cggtttccag 600

ggccgcttgc tgcacctgag tgccggccag cgcctgggcg tccatcttca cactgaggcc 660

agggcacgcc atgcctggca gcttacccag ggcgccacag tcttgggact cttccgggtg 720

acccccgaaa tcccagccgg actcccttca ccgaggtcgg aataa 765

<210> 11

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gcgaattcgc caccatggaa tacgcctctg acgct 35

<210> 12

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctctagatt attccgacct cggtgaaggg ag 32

<210> 13

<211> 834

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atggttcgtc tgcctctgca gtgcgtcctc tggggctgct tgctgaccgc tgtccatcca 60

gaaccaccca ctgcatgcag agaaaaacag tacctaataa acagtcagtg ctgttctttg 120

tgccagccag gacagaaact ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt 180

ccttgcggtg aaagcgaatt cctagacacc tggaacagag agacacactg ccaccagcac 240

aaatactgcg accccaacct agggcttcgg gtccagcaga agggcacctc agaaacagac 300

accatctgca cctgtgaaga aggctggcac tgtacgagtg aggcctgtga gagctgtgtc 360

ctgcaccgct catgctcgcc cggctttggg gtcaagcaga ttgctacagg ggtttctgat 420

accatctgcg agccctgccc agtcggcttc ttctccaatg tgtcatctgc tttcgaaaaa 480

tgtcaccctt ggacaagctg tgagaccaaa gacctggttg tgcaacaggc aggcacaaac 540

aagactgatg ttgtctgtgg tccccaggat cggctgagag ccctggtggt gatccccatc 600

atcttcggga tcctgtttgc catcctcttg gtgctggtct ttatcaaaaa ggtggccaag 660

aagccaacca ataaggcccc ccaccccaag caggaacccc aggagatcaa ttttcccgac 720

gatcttcctg gctccaacac tgctgctcca gtgcaggaga ctttacatgg atgccaaccg 780

gtcacccagg aggatggcaa agagagtcgc atctcagtgc aggagagaca gtga 834

<210> 14

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gcctcgaggc caccatggtt cgtctgcctc tgcag 35

<210> 15

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctctagatc actgtctctc ctgcactgag at 32

<210> 16

<211> 723

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atggttgctg ggagcgacgc ggggcgggcc ctgggggtcc tcagcgtggt ctgcctgctg 60

cactgctttg gtttcatcag ctgtttttcc caacaaatat atggtgttgt gtatgggaat 120

gtaactttcc atgtaccaag caatgtgcct ttaaaagagg tcctatggaa aaaacaaaag 180

gataaagttg cagaactgga aaattctgaa ttcagagctt tctcatcttt taaaaatagg 240

gtttatttag acactgtgtc aggtagcctc actatctaca acttaacatc atcagatgaa 300

gatgagtatg aaatggaatc gccaaatatt actgatacca tgaagttctt tctttatgtg 360

cttgagtctc ttccatctcc cacactaact tgtgcattga ctaatggaag cattgaagtc 420

caatgcatga taccagagta ttacaacagc catcgaggac ttataatgta ctcatgggat 480

tgtcctatgg agcaatgtaa acgtaactca accagtatat attttaagat ggaaaatgat 540

cttccacaaa aaatacagtg tactcttagc aatccattat ttaatacaac atcatcaatc 600

attttgacaa cctgtatccc aagcagcggt cattcaagac acagatatgc acttataccc 660

ataccattag cagtaattac aacatgtatt gtgctgtata tgaatggtat gtatgctttt 720

taa 723

<210> 17

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gcgaattcgc caccatggtt gctgggagcg acgcg 35

<210> 18

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gctctagatt aaaaagcata cataccatt 29

<210> 19

<211> 618

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atgtcgcgcg gcctccagct tctgctcctg agctgcgcct acagcctggc tcccgcgacg 60

ccggaggtga aggtggcttg ctccgaagat gtggacttgc cctgcaccgc cccctgggat 120

ccgcaggttc cctacacggt ctcctgggtc aagttattgg agggtggtga agagaggatg 180

gagacacccc aggaagacca cctcagggga cagcactatc atcagaaggg gcaaaatggt 240

tctttcgacg cccccaatga aaggccctat tccctgaaga tccgaaacac taccagctgc 300

aactcgggga catacaggtg cactctgcag gacccggatg ggcagagaaa cctaagtggc 360

aaggtgatct tgagagtgac aggatgccct gcacagcgta aagaagagac ttttaagaaa 420

tacagagcgg agattgtcct gctgctggct ctggttattt tctacttaac actcatcatt 480

ttcacttgta agtttgcacg gctacagagt atcttcccag atttttctaa agctggcatg 540

gaacgagctt ttctcccagt tacctcccca aataagcatt tagggctagt gactcctcac 600

aagacagaac tggtatga 618

<210> 20

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gcgaattcgc caccatgtcg cgcggcctcc agctt 35

<210> 21

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gctctagatc ataccagttc tgtcttgtga gg 32

<210> 22

<211> 1152

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atggggctgg gcccggtctt cctgcttctg gctggcatct tcccttttgc acctccggga 60

gctgctgctg agccccacag tcttcgttat aacctcacgg tgctgtcctg ggatggatct 120

gtgcagtcag ggtttctcac tgaggtacat ctggatggtc agcccttcct gcgctgtgac 180

aggcagaaat gcagggcaaa gccccaggga cagtgggcag aagatgtcct gggaaataag 240

acatgggaca gagagaccag agacttgaca gggaacggaa aggacctcag gatgaccctg 300

gctcatatca aggaccagaa agaaggcttg cattccctcc aggagattag ggtctgtgag 360

atccatgaag acaacagcac caggagctcc cagcatttct actacgatgg ggagctcttc 420

ctctcccaaa acctggagac taaggaatgg acaatgcccc agtcctccag agctcagacc 480

ttggccatga acgtcaggaa tttcttgaag gaagatgcca tgaagaccaa gacacactat 540

cacgctatgc atgcagactg cctgcaggaa ctacggcgat atctaaaatc cggcgtagtc 600

ctgaggagaa cagtgccccc catggtgaat gtcacccgca gcgaggcctc agagggcaac 660

attaccgtga catgcagggc ttctggcttc tatccctgga atatcacact gagctggcgt 720

caggatgggg tatctttgag ccacgacacc cagcagtggg gggatgtcct gcctgatggg 780

aatggaacct accagacctg ggtggccacc aggatttgcc aaggagagga gcagaggttc 840

acctgctaca tggaacacag cgggaatcac agcactcacc ctgtgccctc tgggaaagtg 900

ctggtgcttc agagtcattg gcagacattc catgtttctg ctgttgctgc tgctgctatt 960

tttgttatta ttattttcta tgtccgttgt tgtaagaaga aaacatcagc tgcagagggt 1020

ccagagctcg tgagcctgca ggtcctggat caacacccag ttgggacgag tgaccacagg 1080

gatgccacac agctcggatt tcagcctctg atgtcagatc ttgggtccac tggctccact 1140

gagggcgcct ag 1152

<210> 23

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gcgaattcgc caccatgggg ctgggcccgg tcttc 35

<210> 24

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

gctctagact aggcgccctc agtggagcca gt 32

Claims

1. A method for amplifying NK cells by using NIH3T3 cells and PBMC cells for co-culture, which is characterized in that the method uses NIH3T3 cells as trophoblasts to amplify the NK cells; the NIH3T3 cells expressed cytokines CD80, CD70, CD86, 4-1BBL, CD40, CD58, CD83 and MICA 8.

2. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD80 is shown as SEQ ID NO 1.

3. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD70 is shown as SEQ ID NO 4.

4. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD86 is shown as SEQ ID NO 7.

5. The method for amplifying NK cells by using the co-culture of the NIH3T3 cells and the PBMC cells according to claim 1, wherein the amino acid sequence of 4-1BBL is shown as SEQ ID NO 10.

6. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD40 is shown as SEQ ID NO 13.

7. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD58 is shown as SEQ ID NO 16.

8. The method for amplifying NK cells by using the co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of CD83 is shown as SEQ ID NO 19.

9. The method for amplifying NK cells by co-culture of NIH3T3 cells and PBMC cells according to claim 1, wherein the amino acid sequence of MICA is represented by SEQ ID NO 22.

10. The method for amplifying NK cells by coculture of NIH3T3 cells and PBMC cells according to any one of claims 1 to 9, wherein the method for preparing trophoblast cells specifically comprises the following steps: constructing a cytokine lentivirus vector, and introducing the cytokine lentivirus vector and helper plasmid into mammalian cells together to obtain the cytokine lentivirus; infecting the obtained cell factor lentivirus with host cell, and transducing to obtain trophoblast cell.

11. An NK cell expansion medium comprising NIH3T3 cells according to claim 1.

12. The NK cell expansion medium of claim 11, further comprising any one or a combination of at least two of basal medium, serum, plasma, or antibiotics.

13. Use of the NK cell expansion medium of claim 11 or 12 for expanding NK cells.