CN111826354B

CN111826354B - NK cell and application thereof in tumor treatment

Info

Publication number: CN111826354B
Application number: CN202010792695.7A
Authority: CN
Inventors: 不公告发明人
Original assignee: Shandong Hankang Biotechnology Co ltd
Current assignee: SHANDONG HANKANG BIOTECHNOLOGY Co.,Ltd.
Priority date: 2020-08-10
Filing date: 2020-08-10
Publication date: 2021-04-02
Anticipated expiration: 2040-08-10
Also published as: CN111826354A

Abstract

The invention discloses an NK cell and application thereof in tumor treatment. The acquisition of high killing activity NK cells is one of the keys for adoptive therapy of tumor cells by using NK cells. The invention discovers that the NK cells with high miR-124-3p expression have stronger killing activity and stronger killing power on tumor cells, so that the NK cells can be used for preparing an anti-tumor cell preparation.

Description

NK cell and application thereof in tumor treatment

Technical Field

The invention belongs to the field of adoptive cellular immunotherapy, and particularly relates to an NK cell and application thereof in tumor therapy.

Background

NK cells are main natural immune cells of an organism, antigen sensitization is not needed, abnormal cells in the body, such as tumor cells, bacteria or virus infected cells, can be directly identified and eliminated without antibodies, and play an extremely important role in early anti-tumor and anti-infection immune response of the organism. Besides directly killing tumor cells, NK cells can also promote the recruitment of effector T cells to a tumor microenvironment to play an anti-tumor role through the secretion of inflammatory cytokines and chemokines after being activated and the cooperation of the NK cells and dendritic cells, so that the subsequent specific anti-tumor immune response is effectively induced.

Adoptive cell immunotherapy is a biological therapy method for treating tumors by infusing lymphocytes to tumor patients after in vitro stimulation and culture, and directly killing tumor cells or exciting the immune reaction of the organism to kill tumor cells. In recent years, with the development of medical technology, adoptive cellular immunotherapy of tumors based on NK cells is becoming more and more widely used.

The acquisition of high killing activity NK cells is one of the keys for adoptive therapy of tumor cells by using NK cells.

Genomics is a cross-biology discipline that performs collective characterization, quantitative studies and comparative studies of different genomes on all genes of an organism. Genomics mainly studies the structure, function, evolution, localization, editing, etc. of genomes, and their influence on organisms. The genetic targets of disease or functional differences can be found by comparing genomics.

In a previous comparative genomics study of high and low killer active NK cells, a variety of differentially expressed genes were found, including a variety of miRNAs. At present, no disclosures are provided on which differentially expressed miRNAs can be used as targets to regulate the killing activity of NK cells on tumor cells.

Disclosure of Invention

The invention provides an NK cell and application thereof in tumor treatment in order to overcome the defects of the prior art.

The technical scheme is as follows:

an NK cell with high lethality, which is an NK cell with high miR-124-3p expression.

The high lethality NK cell is applied to the preparation of the medicine for treating tumors.

Has the advantages that:

the acquisition of high killing activity NK cells is one of the keys for adoptive therapy of tumor cells by using NK cells. The invention discovers that the NK cells with high miR-124-3p expression have stronger killing activity and stronger killing power on tumor cells, so that the NK cells can be used for preparing an anti-tumor cell preparation.

Drawings

FIG. 1 is a flow identification chart of NK cells cultured for 14d, from which it can be seen that the proportion of CD3-CD56+ phenotype cells (i.e., NK cells) reached 83.75%, and NK cell culture was successful.

FIG. 2 shows the results of RT-PCR measurement of the expression level of miR-124-3p in each group of NK cells, and it can be seen from the graph that miR-124-3p is highly expressed in the NK cells of the miR-124-3p group compared with the blank group, and the expression level of miR-124-3p in the transfection control group is substantially consistent with that of the blank group.

FIG. 3 shows that the killing power of NK cells of miR-124-3p group to HepG2 cells is significantly higher than that of NK cells of blank group and transfection control group, and the killing power of NK cells of miR-124-3p group to tumor cells is significantly better than that of NK cells of blank group and transfection control group.

Detailed Description

Example 1:

first, test materials

Lymphocyte isolates were purchased from Solarbio.

NK cell culture medium was purchased from CellGro SCGM and fetal bovine serum from Gibco.

CD3-PerCP-Cy5.5 and CD56-FITC were purchased from BD.

miR-124-3p mimics, miR-124-3p NC and RT-PCR primers are purchased from Ruibo, Guangzhou.

Lipofectamine^TMThe 2000 transfection kit was purchased from Invitrogen.

Second, test method

1. Culture and characterization of NK cells

Taking 100mL of peripheral anticoagulation blood of healthy blood donor according to a conventional method, separating mononuclear cells by using lymphocyte separation liquid, and then re-suspending the mononuclear cells by using NK cell culture medium containing 500U/mLrhIL-2 to prepare the mononuclear cells with the cell density of 2 multiplied by 10⁵The cell suspension/mL, inoculated in a culture flask, at 37 ℃, 5% CO₂Culturing under the condition that half amount of the culture solution is changed for 1 time every 2-3 d, and adjusting the cell number to 5 × 10 during each liquid change⁴mL, after 14 days of culture, cells were collected.

Flow identification: cells cultured for 14d were harvested, washed with PBS, and cell surface markers were detected by adding PerCP-Cy5.5 labeled CD3 and FITC labeled CD56 antibody according to conventional flow cytometry.

2. miR-124-3p transfection, RT-PCR identification and CD3-CD56+ phenotype determination

Cells cultured for 14 days were collected, washed with PBS, resuspended in NK cell medium containing 5% fetal bovine serum to give a cell suspension at 1X 10 per well⁵The density of each cell is inoculated on a 6-well plate, the cells are divided into a blank group (without any gene transfection), a miR-124-3p group (miR-124-3 p mimics transfection) and a transfection control group (miR-124-3 p NC transfection), and the cells are plated for 12h according to Lipofectamine^TM2000 transfection kit instruction for transfection operation, miR-124-3p mimics and miR-124-3p NC and liposome Lipofectamine to be transfected^TM2000 diluting with NK cell culture medium, mixing at a certain ratio, acting at room temperature for 30min, adding to-be-transfected cells, standing at 37 deg.C and 5% CO₂After 8h of culture, the solution was changed, and cells were collected after 48 h.

RT-PCR identification of transfection effects:

extracting total RNA of cells by a Trizol method, detecting the purity of the total RNA, carrying out reverse transcription on the RNA into cDNA by an RT-PCR kit after the purity meets the requirement, carrying out PCR amplification on the cDNA, and finally taking a proper amount of PCR products to carry out agarose gel electrophoresis detection on the amplification products.

miR-124-3p upstream primer (sequence NO. 1): 5'-CGGGTAGCAGGCTTCTGAGT-3', respectively;

miR-124-3p downstream primer (sequence NO. 2): 5'-AAACCCCTCTCTGTCGGTAGCT-3', respectively;

GAPDH upstream primer (sequence No. 3): 5'-AATGGGCAGCCGTTAGGAAA-3', respectively;

GAPDH downstream primer (sequence No. 4): 5'-TGAAGGGGTCATTGATGGCA-3' are provided.

CD3-CD56+ phenotypic assay:

the collected cells were washed with PBS and cell surface markers were detected by adding PerCP-Cy5.5 labeled CD3 and FITC labeled CD56 antibodies according to conventional flow cytometry.

3. Flow cytometry method for detecting lethality of miR-124-3p high-expression NK cells

Collecting NK cells transfected by a blank group (without any gene transfection), a miR-124-3p group (with miR-124-3p mimics) and a transfection control group (with miR-124-3p NC transfection) and continuously culturing for 48h, washing with PBS, adding APC-Anti-CD107a according to a conventional flow cytometry method, and detecting the expression level of CD107 a.

4. CCK-8 method for detecting lethality of miR-124-3p high-expression NK cells to tumor cells

Collecting NK cells transfected by a blank group (without any gene transfection), a miR-124-3p group (with miR-124-3p mimics transfection) and a transfection control group (with miR-124-3p NC transfection) and continuously culturing for 48h, washing with PBS, and preparing into 2 × 10 by using RPMI-1640 medium containing 10% fetal calf serum⁶A cell suspension in/mL as effector cells; taking HepG2 liver cancer cells in logarithmic growth phase, preparing into 2 × 10 with RPMI-1640 culture medium containing 10% fetal calf serum⁵A cell suspension in/mL as target cells. Respectively inoculating 100 μ L each of effector cell suspension and target cell suspension (effective target ratio 10:1) to 96-well culture plate, simultaneously setting independent effector cell well and independent target cell well, each group has 5 multiple wells, and culturing at 37 deg.C and 5% CO₂Culturing for 8h under the condition, adding 20 mu L CCK8 reagent into each hole, incubating for 4h, measuring the light absorption value (OD) of each hole on a 450nm wavelength microplate reader, calculating the average value, and calculating the killing rate of each group of NK cells on HepG2 liver cancer cells according to the following formula.

The killing rate (%) [1- (experimental OD value-OD value of effector cell alone)/OD value of target cell alone ]. times.100%

5. Statistical treatment

The data were processed using the SPSS17.0 statistical software package and expressed as mean. + -. standard deviation, and the comparisons between groups were by t-test, with P <0.05 representing a statistical significance for the differences.

Third, test results

1. Culture and characterization of NK cells

The flow assay results showed that the proportion of cells of the CD3-CD56+ phenotype reached 83.75% after 14d of culture, as shown in FIG. 1. Whereas the proportion of CD3-CD56+ phenotype cells before culture was only 6.62%. This indicates that NK cells have been successfully cultured.

2. miR-124-3p transfection, RT-PCR identification and CD3-CD56+ phenotype determination result

The RT-PCR result is shown in figure 2, compared with the blank group, the miR-124-3p group NK cells highly express miR-124-3p, and the miR-124-3p expression level in the transfection control group is basically consistent with that in the blank group.

The proportion of the groups of cells with the phenotype of CD3-CD56+ was shown in Table 1, with no significant difference.

TABLE 1 proportion of groups of cells of the CD3-CD56+ phenotype

3. NK cell lethality measurement result (flow cytometry)

The expression level of a killer activity marker molecule CD107a of each group of NK cells is shown in Table 2, and the result shows that the expression level of CD107a in the NK cells of the miR-124-3p group is remarkably higher than that of CD107a in the NK cells of the blank group and the transfection control group, and the killing power of the NK cells of the miR-124-3p group is remarkably better than that of the NK cells of the blank group and the transfection control group.

TABLE 2 expression levels of the NK cell killing activity marker molecule CD107a of each group

4. NK cell lethality to tumor cell assay result (CCK-8 method)

The killing power of each group of NK cells on HepG2 liver cancer cells is shown in Table 3 and figure 3, and the results show that the killing power of miR-124-3p group NK cells on HepG2 cells is obviously higher than that of blank group and transfection control group NK cells, and the killing power of miR-124-3p group NK cells on tumor cells is obviously better than that of the blank group and the transfection control group.

TABLE 3 lethality of various NK cells against HepG2 hepatoma cells

The experiment shows that the NK cell with high miR-124-3p expression has stronger killing activity and stronger killing power on tumor cells, so that the NK cell can be used for preparing an anti-tumor cell preparation.

The above embodiments are intended to specifically describe the substance of the present invention, but should not limit the scope of the present invention to the above specific embodiments.

Sequence listing

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Claims

1. An application of NK cell in preparing medicine for treating hepatocarcinoma; wherein the NK cell is a miR-124-3p high-expression NK cell and is constructed by transfecting miR-124-3p into the NK cell.