CN115353556A - Fusion protein-based anti-NK cell senescence method and culture medium containing fusion protein - Google Patents

Abstract

The invention discloses a fusion protein-based method for resisting NK cell senescence and a culture medium containing the fusion protein, and protects a fusion protein-recombinant human synapsis fusion protein 12 for resisting Natural Killer cell senescence. The cell test proves that the recombinant human synapsin 12 can effectively inhibit the senescence of the Natural Killer cells when the in-vitro intervention of the Natural Killer cell culture is carried out; animal experiments prove that the recombinant human syntaxin 12 intervenes in cultured Natural Killer cells and has stronger lethality to tumor cells in vivo. These experiments demonstrate that recombinant human syntaxin 12 not only can resist Natural Killer cell senescence, but also can improve its lethality to tumors.

Description

Fusion protein-based anti-NK cell senescence method and culture medium containing fusion protein

Technical Field

The invention belongs to the field of cellular immunotherapy, and particularly relates to a fusion protein-based method for resisting Natural Killer cell senescence and a culture medium containing the fusion protein.

Background

NK cells are the core components of human innate immunity and play an important role in anti-tumor immunity of organisms. NK cells are characterized by non-restricted killing by the Major Histocompatibility Complex (MHC). The phenotype of NK cells is CD3-CD56+. The killing of NK cells to tumor cells mainly comprises the following 4 ways: (1) perforin and granzyme mediated direct killing; (2) Membrane tumor necrosis factor family molecule-mediated tumor cell apoptosis; (3) Through secreting various cytokines, the tumor cells are synergistically inhibited or killed; (4) NK cells express on their surface the low affinity receptor CD16 molecule of the Fc fragment of immunoglobulins, which when bound to antibodies, induces antibody-dependent cellular cytotoxicity (ADCC) specifically killing tumor cells bound by the antibody IgG. This ensures the effect of NK cells for tumor immunotherapy.

At present, NK cells are mainly used for tumor immunotherapy, namely, NK cells of a patient are obtained firstly, then are cultured in vitro, and then are returned to the body of the patient so as to play a therapeutic role. However, some patients have low lethality due to their own body reasons, and their NK cells are very susceptible to aging in vitro, which leads to poor therapeutic effect after reinfusion.

The invention is especially provided for solving the problems that NK cells of partial patients are easy to age in vitro culture and have low lethality after reinfusion.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a fusion protein-based method for resisting Natural Killer cell senescence and a culture medium containing the fusion protein.

The purpose of the invention is realized by the following technical scheme:

a fusion protein for resisting Natural Killer cell senescence is a recombinant human syntaxin 12.

An in vitro culture method for resisting Natural Killer cell senescence based on fusion protein, which comprises the step of giving recombinant human synapsin 12 to intervene culture during in vitro culture of Natural Killer cells.

A culture medium for resisting Natural Killer cell senescence, which contains recombinant human synapsin 12.

The beneficial technical effects are as follows:

the cell test proves that the recombinant human synapsin 12 can effectively inhibit the senescence of the Natural Killer cells when the in-vitro intervention of the Natural Killer cell culture is carried out; animal experiments prove that the recombinant human syntaxin 12 intervenes in cultured Natural Killer cells and has stronger lethality to tumor cells in vivo. These experiments demonstrate that recombinant human syntaxin 12 not only can resist senescence of native Killer cells, but also can improve the lethality of native Killer cells to tumors. Accordingly, a corresponding native Killer cell culture medium can be developed.

Drawings

FIG. 1 is the results of SA- β -Gal staining of various groups of senescent NK cells.

Fig. 2 is a comparison of the sizes of the transplanted tumors in each group after they were exfoliated.

Detailed Description

1. Experimental materials

The human lymphocyte separation solution was purchased from Hangzhou Union Biotechnology GmbH. NK cell immunomagnetic bead sorting systems were purchased from pecan organisms. NK cell culture medium was purchased from Yaji Biotech, inc., shanghai. Recombinant human syntaxin 12 (STX 12) was purchased from shanghai high-invasive chemical technology, ltd. SA- β -Gal stabilizing Kit was purchased from Cell signalling. SGC-7901 cells were purchased from ATCC. BALB/C nude mice, male, 8 weeks old, with a body mass of 20-24 g, purchased from a racing organism.

2. Experimental method

1. Peripheral blood mononuclear cell isolation

A proper amount of peripheral blood of healthy volunteers is collected under the aseptic condition for preparing NK cells. Proper amount of human lymphocyte separating liquid is added into a centrifuge tube, and proper amount of peripheral blood is slowly added along the tube wall to avoid damaging the liquid level of the lymphocyte separating liquid. Centrifuge at 894 Xg for 20min at room temperature. And sucking the light yellow mononuclear cell layer out, putting the light yellow mononuclear cell layer into a centrifuge tube, centrifuging for 6min at room temperature of 572 Xg, and removing supernatant to obtain a precipitate, namely the peripheral blood mononuclear cells.

2. NK cell separation and purification

The peripheral blood mononuclear cells were washed with PBS, resuspended in PBS, counted, and 20. Mu.L (about 1X 10) of the total amount was collected according to the protocol of NK cell immunomagnetic bead sorting System ⁷ Respectively) cell suspension, adding anti-CD 3 immunomagnetic beads, incubating at 4 ℃ for 15min, adding 2ml LPBS, centrifuging to wash the cells, diluting to 500 μ L, slowly passing through a MACS chromatographic column, washing the column, collecting unbound cells (namely CD 3-cells), washing with PBS, repeating the above steps, adding anti-CD 56 immunomagnetic beads, passing through the column, collecting cells bound to the chromatographic column, namely CD3-CD56+ NK cells (determination of phenotype by flow cytometry), resuspending with PBS, centrifuging, washing with NK cell culture medium at 37 ℃ and 5% CO ₂ Culturing under the condition.

3. NK cell grouping, modeling and intervening culture

The NK cells are subjected to modeling (high-sugar induced aging) and intervening culture according to the following groups, and the culture medium is replaced every 2-3 d:

control group: by using NK cell culture medium at 37 deg.C, 5% CO ₂ Culturing under the condition;

and (3) an aging group: using NK cell medium containing 25mmol/L glucose at 37 ℃ 5% CO ₂ Culturing under the condition;

STX12-A group: using NK cell medium containing 25mmol/L glucose and 400ng/mL recombinant human synaptogusin 12, at 37 ℃ and 5% CO ₂ Culturing under the condition;

STX12-B group: using NK cell medium containing 25mmol/L glucose and 800ng/mL recombinant human synaptogusin 12 at 37 ℃ and 5% CO ₂ Culturing under the condition.

4. Cellular senescence level determination

Senescent cells were assayed using SA- β -Gal staining. After intervening culture for 12d in groups according to '3, NK cell grouping, modeling and intervening culture', each group was collected at 1X 10 ⁵ Fully and uniformly mixing NK cells with 1mL of Fixative working solution according to the instruction, and fixing for 10min; washing with PBS for 2 times, adding 1mL of working Solution containing Solution A, solution B and stabilizing Solution, mixing, and keeping at 37 deg.C without CO ₂ Incubate under conditions for 16h, mount glycerol for microscopic examination, and randomly select three fields under light microscope (200 ×) to determine the number of positive cells.

5. In vivo lethality assay

Tumor cell culture: the SGC-7901 cells were subjected to 5% CO at 37 ℃ in 10% FBS-containing RPMI-1640 medium ₂ Culturing under the condition, changing the culture solution every 2-3 days, subculturing, taking cells in logarithmic growth phase, digesting with 0.25% trypsin, collecting the cells, determining that the cell activity is not less than 95%, and adjusting the final cell density to 3 x 10 ⁶ 0.2mL of cell suspension was ready for use.

Conventional NK cells: the isolated and purified NK cell culture medium was adjusted to 37 ℃ and 5% CO ₂ Culturing under the condition of 12d.

STX12 intervention NK cells: isolated and purified NK cells were treated with NK cell medium containing 800ng/mL recombinant human synapsin 12 at 37 ℃ with 5% CO ₂ Culturing under the condition of 12d.

0.2mL of each of the above cell suspensions was inoculated subcutaneously into the right lower limb of nude mice, and the nude mice were kept in a SPF laminar flow laboratory under standard conditions, and were given a free diet of sterile feed and water. After inoculation, the presence of tumor formation and the presence of ulceration and red swelling at the injection site were observed every day, and the tumor size was measured on a sliding scale. When the tumor body grows to a proper size, the nude mice are randomly divided into 2 groups of 4 mice each. Conventional NK cell panel: intraperitoneal injection of regular NK cells at intervals of 2 days at a dose of 1X 10 ⁶ 0.2 mL/0.2 mL; STX12 intervention NK cell group: 2d intraperitoneal injection of STX12 at intervals to interfere 1X 10 NK cells ⁶ 0.2mL. NK cells were co-injected 5 times, and the day after the last 1 injection nude mice were sacrificed using chloral hydrate. Tumor tissue was stripped and weighed.

6. Statistical analysis

Statistical analysis was performed using SPSS 19.0 software, and the measurements were expressed as mean. + -. Standard deviation. The two groups were compared using independent sample t-test. * P <0.05 is statistically significant for the differences.

3. Results of the experiment

1. NK cell separation and purification

NK cell immunomagnetic bead sorting is the most efficient means for separating and purifying NK cells. Through NK cell immunomagnetic bead sorting, the purity of NK cells represented by CD3-CD56+ is improved to 99.3% from 9.4% before sorting, and NK cells are successfully separated and purified.

2. Cellular senescence level determination

Cells staining positive for SA- β -Gal represent senescent cells. The staining results of each group are shown in fig. 1, and compared with the control group, the number of positive cells in the aging group is obviously increased, which indicates that the high-glucose-induced aging model is successful; compared with the aging group, the STX12-A group and the STX12-B group have obviously reduced positive cells and the dose dependence is seen, which indicates that the STX12 intervention culture can effectively resist the NK cell aging.

3. In vivo lethality assay

As shown in FIG. 2, compared with conventional NK cells, the size and weight of STX12 intervention NK cell group transplantation tumor are obviously reduced, which indicates that recombinant human synapsin 12 intervention cultured NK cells have stronger lethality to tumors in vivo.

The above examples are intended to specifically describe the substance of the present invention. It will be appreciated by persons skilled in the art that the scope of the present invention should not be limited to the specific embodiments described above.

Claims (3)

1. A fusion protein for resisting Natural Killer cell senescence is a recombinant human syntaxin 12.

2. An in vitro culture method for resisting Natural Killer cell senescence based on fusion protein is characterized in that: recombinant human synapsin 12 was given to intervene in the culture when native Killer cells were cultured in vitro.

3. A culture medium for resisting Natural Killer cell senescence, which contains recombinant human synaptotagmin 12.

Images