WO2019113744A1 - Application of tipe2 gene-targeted tumor mdscs in preparation of drugs for tumor prevention or treatment - Google Patents

Abstract

Provided is application of TIPE2 gene-targeted tumor MDSCs in preparation of drugs for tumor prevention or treatment, wherein TIPE2 gene targeting comprises decreasing the expression of TIPE2 gene by using gene knockout, gene knock-down, or a chemical drug. TIPE2 gene-deleted tumor MDSCs, on the one hand, can prevent and inhibit tumorigenesis and tumor growth and inhibit a growth speed of an existing tumor, and on the other hand, can enhance the immune response against the tumor and be used for preparing new targeted drugs for tumor prevention or treatment.

Description

Application of tumor MDSC targeting TIPE2 gene in preparing drugs for preventing or treating tumors Technical field

The present application relates to the field of tumor targeted therapy, and in particular to the use of a tumor MDSC targeting the TIPE2 gene for the preparation of a medicament for preventing or treating a tumor.

Background technique

TIPE2, a tumor necrosis factor alpha-induced protein 8-2, is a member of the TNFAIP8 family. It has been reported that TIPE2 is an immunoregulatory factor, which is selectively expressed in the immune tissues and inflammatory tissues of the thymus, spleen, lymph nodes, etc., and plays a key role in inhibiting inflammation and maintaining immune homeostasis. TIPE2 negatively regulates NF-κB and MAPK signaling pathways, inhibits TCR-mediated T cell activation and TLR-mediated innate immunity of macrophages, and plays an anti-inflammatory role. The down-regulation of expression levels is often accompanied by the occurrence of inflammatory diseases such as hepatitis B, systemic lupus erythematosus, diabetic nephritis, experimental stroke, childhood immune asthma, and atherosclerosis.

Myeloid-derived suppressor cells (abbreviated MDSC) are a major component of the tumor microenvironment. The main feature of these cells is their strong immunosuppressive activity. In tumor-bearing hosts, MDSC is produced in the bone marrow and then migrates into peripheral lymphoid organs and tumors, helping to form the tumor microenvironment. Recent studies have shown that MDSCs have different functions and fates in tumors and peripheral lymphoid organs. Differentiation and dysfunction of myeloid cells is a hallmark of cancer. MDSC can accelerate tumor progression, mainly by increasing tumor cell survival, promoting angiogenesis, promoting tumor cell invasion to healthy tissues, and accelerating metastasis.

MDSC is differentiated from common myeloid progenitor cells in the bone marrow (abbreviated BM). The development of MDSC is regulated by two complex signaling networks. One type of signal promotes the aggregation of such immature myeloid cells, while the other provides a signal for the pathological activation of such cells. The composition of the myeloid myeloid cells in the tumor environment has been confirmed to have changed, as a large number of studies have shown that MDSCs are aggregated in the bone marrow of the tumor-bearing host. The pathological activation of MDSC is the result of sustained lower-intensity tumor signal stimulation of myeloid cells, characterized by relatively low phagocytic activity, sustained production of reactive oxygen species (ROS), nitric oxide (NO), and substantial resistance. Inflammatory cytokines. This is in stark contrast to the activation of myeloid cells in the absence of bacteria and viruses, characterized by rapid activation of phagocytosis, triggering of respiratory bursts, and release of pro-inflammatory cytokines. After the inflammation disappears, the normal myeloid system is restored.

MDSC has different biochemical and genomic features than neutrophils and monocytes. These features include the expression of large amounts of NADPH oxidase (Nox2), resulting in increased production of reactive oxygen species in the form of superoxide anion (O ₂ ^- ), hydrogen peroxide (H ₂ O ₂ ), and peroxynitrate (PNT, ONOO ^- ). Cluster (ROS); up-regulated expression of arginase 1 (arg1) and nitric oxide synthase 2 (nos2) genes, respectively, increased production of arginase 1 (Arg1) and nitric oxide (NO); up-regulation Activation of transcriptional regulators C/EBPβ and STAT3; down-regulation of transcription factor IRF8 activity and increased production of S100A8/9 protein. In addition, recent studies have also shown that miR-142-3p also has a specific regulatory effect on MDSC. These different features attenuate the ability of MDSCs to differentiate into mature myeloid cells including macrophages, dendritic cells, and mature neutrophils. Excessive production of ROS, NO, Arg1 and inhibitory cytokines determines the immunosuppressive activity of MDSC.

MDSC is derived from bone marrow hematopoietic progenitor cells and immature myeloid cells. Under normal physiological conditions, it can differentiate into mature neutrophils, dendritic cells and macrophages. Therefore, promoting differentiation and maturation of MDSC is targeted for MDSC tumor immunotherapy. a strategy. The rational elimination of the expanded and activated MDSC is a particularly important therapeutic strategy. Because MDSC has not yet differentiated from specific surface markers of IMC and mature neutrophils, it has brought difficulties to MDSC targeted rejection strategies. For example, Gr-1 is also expressed on the surface of mature neutrophils. While anti-Gr-1 antibody scavenges MDSC, functional neutrophils are also cleared accordingly, resulting in a decrease in immune defense functions such as anti-infection. Therefore, the search for new therapeutic targets that can effectively limit MDSC immunosuppressive function and minimize its side effects is the key to the success of clinical application of tumor immunotherapy targeting MDSC.

Summary of the invention

The purpose of the present application is to provide a target for the prevention or treatment of new tumors, and the use of the target in the preparation of a medicament for preventing or treating a tumor.

In order to achieve the above objectives, the present application adopts the following technical solutions:

One aspect of the present application discloses a use of a tumor MDSC targeting a TIPE2 gene for the preparation of a medicament for preventing or treating a tumor; wherein targeting the TIPE2 gene includes using a gene knockout, a gene knockdown or a chemical drug Decrease the expression of the TIPE2 gene.

It should be noted that the present study found that the tumor MDSC deficient in TIPE2 gene can inhibit the growth of newly implanted tumor cells, has the effect of preventing tumors, and can treat and inhibit existing tumor cells; therefore, the TIPE2 gene is deleted. The tumor MDSC can be used to prepare a medicament for preventing or treating a tumor. TIPE2 gene deletion can be achieved by gene knockout, gene knockdown or chemical drugs to reduce the expression of TIPE2 gene.

It should be noted that, in an implementation manner of the present application, the tumor MDSC of the mouse lung cancer with TIPE2 gene deletion, that is, the TIPE2 gene-positive lung cancer tumor MDSC, is confirmed to be effective for inhibiting the growth of lung cancer tumors. Anti-lung cancer effect. It is understood that the tumor MDSC targeting the TIPE2 gene can be used in the preparation of a medicament for preventing or treating tumors, and the corresponding tumor MDSC can be used. The preparation of a medicament for treating or preventing a corresponding tumor is not specifically limited herein.

Preferably, the application of the present application specifically refers to the prevention or treatment of tumors by cellular immunotherapy based on tumor MDSC.

Another aspect of the present application discloses a use of the TIPE2 gene as a target for the preparation of a medicament for preventing or treating a tumor; wherein targeting the TIPE2 gene comprises using a gene knockout, a gene knockdown or a chemical to reduce TIPE2 Gene expression.

It should be noted that the present application has found that the TIPE2 gene deletion itself has an inhibitory effect on tumors, and therefore, the TIPE2 gene can be used as a target for preparing a medicament for preventing or treating tumors.

Another aspect of the application discloses a TIPE2 gene knockout tumor MDSC.

Another aspect of the present application discloses a method for preparing a TIPE2 gene knockout tumor MDSC of the present application, comprising the following steps,

Tumor cell implantation was performed on mice with TIPE2 gene deletion by subcutaneous injection;

After the tumor cells to be implanted grow into tumor tissue blocks, the tumor tissue blocks are removed from the mice, and the tumor tissue blocks are digested with an enzyme mixture including collagenase I, collagenase IV, hyaluronidase and deoxygenation. Ribozyme; wherein the tumor cells grow into tumor tissue blocks generally within 2-3 weeks, that is, the tumor tissue blocks can be taken within 2-3 weeks after the tumor cells are implanted;

After the digestion is completed, the digestion is terminated by using a complete medium, and the tumor tissue block is placed on a cell sieve to be ground, and the tissue slurry passing through the cell sieve is collected; wherein, the complete medium is used to terminate the digestion, and the centrifuge tube is directly filled with the complete medium. The digestion can be terminated; in one implementation of the present application, the cell screen is a 70 μm sieve, and after the grinding, the tumor cells are all suspended in the tissue polishing liquid through the cell sieve;

The tissue slurry was subjected to low-speed centrifugation at 4 ° C to obtain a cell supernatant, and the precipitated impurities were discarded; the cell supernatant was centrifuged at 4 ° C at high speed to collect precipitated cells; wherein, 4 ° C low-speed centrifugation means lower at 4 ° C. Centrifugation at a rate at which the cells are still suspended in solution, while larger impurities are removed by centrifugation. In one implementation of the present application, the speed of low speed centrifugation is 60 g; 4 °C high speed centrifugation means 4 Centrifugation at a higher speed, at which the cells are pelleted by centrifugation for removal of the supernatant. In one implementation of the present application, the speed of high-speed centrifugation is 1260 g;

The collected precipitated cells were resuspended in RPMI 1640 complete medium, and the collected precipitated cells were isolated using mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit. Specifically, the cells were completely cultured with RPMI 1640. The base weight suspension is added to the liquid surface of the separation liquid prepared according to the kit, centrifuged at 1260 g, 25 ° C, and the two layers of annular milky white cell layers appearing in the centrifuge tube are aspirated and collected by a pipette; wherein the mouse tumor tissue lymph, The mononuclear macrophage and granulocyte separation solution kit, in one implementation manner of the present application, specifically adopts the LDS1090Z kit purchased from Tianjin Yuyang Biological Products Technology Co., Ltd., and the liquid level or gradient interface of the separation liquid is in accordance with Prepared by the conventional method disclosed in the specification, not here Specifically defined, 1260g, 25 ° C centrifugation is mainly to achieve better separation, the parameters can be adjusted within the tolerances allowed by the test;

The collected milky white cell layer was washed with PBS solution, 1260 g, centrifuged at 4 ° C, the supernatant was discarded, and resuspended in PBS to obtain a PBS cell suspension; in the present application, 1260 g, centrifuged at 4 ° C, wherein the centrifuge speed of 1260 g was The cells are precipitated without rupture; the centrifugation temperature at 4 °C is mainly to keep the cells at a lower temperature, slowing down their growth and metabolism, and making them more stable;

The PBS cell suspension was stained with the flow antibody CD45/CD11b/Gr-1 at 4 °C, and the CD45 ⁺ CD11b ⁺ Gr-1 ⁺ cells were sorted by flow cytometry to obtain the TIPE2 knockout tumor. MDSC. In one implementation of the present application, the obtained cells are resuspended by PBS. Among them, PBS is a phosphate buffer commonly used in laboratories.

Preferably, before the digestion of the tumor tissue block by the enzyme mixture, the tumor tissue block is further cut into small pieces and then digested. It can be understood that cutting the tumor tissue block into small pieces can better digest and improve the quality and efficiency of digestion.

Preferably, the enzyme mixture contains 50 mg/mL of collagenase I, 50 mg/mL of collagenase IV, 5 mg/mL of hyaluronidase, and 1 U/mL of deoxyribonuclease.

Preferably, in the tumor cell planting, the tumor cells used are LLC mouse lung cancer cells.

Preferably, in tumor cell planting, the amount of tumor cells implanted is 2 x 10 ⁶ tumor cells per mouse.

Preferably, the tumor tissue block is removed from the mouse, specifically, the mice that have been sacrificed by cervical dislocation are immersed in 75% alcohol, and then the mouse is placed on a sterile clean bench, and the tumor is removed with surgical scissors and forceps. Organization block.

Still another aspect of the present application discloses the use of the TIPE2 gene knockout tumor MDSC of the present application for the preparation of a medicament for preventing or treating a tumor.

It can be understood that the TIPE2 knockout tumor MDSC of the present application has a good tumor suppressing effect, and therefore, it can be completely prepared into a corresponding drug for preventing or treating a tumor. The drug may also include other pharmaceutically acceptable auxiliary ingredients or active ingredients, which are not specifically limited herein.

Due to the adoption of the above technical solutions, the beneficial effects of the present application are:

The present application relates to a tumor MDSC targeting TIPE2 gene in the preparation of a medicament for preventing or treating a tumor, wherein the tumor MDSC deficient in TIPE2 gene, on the one hand, can prevent and inhibit tumorigenesis and growth, and can inhibit existing The growth rate of tumors; on the other hand, it can enhance the anti-tumor immune response; provide a new targeted drug for the prevention or treatment of tumors.

DRAWINGS

1 is a diagram showing the results of a prophylactic test of a primary tumor of a wild type mouse in a TIPE2 knockout tumor MDSC in the examples of the present application;

2 is a diagram showing the results of a prophylactic test of a TIPE2 knockout tumor MDSC on a primary tumor of a Tipe2 ^−/− mouse in the examples of the present application;

Figure 3 is a diagram showing the results of treatment of an existing tumor with a TIPE2 knockout tumor MDSC in the examples of the present application;

Fig. 4 is a graph showing the results of enhancing the anti-tumor immune response of the TIPE2 knockout tumor MDSC in the examples of the present application.

Detailed ways

In the process of studying MDSC, this application found a new therapeutic target, TIPE2 gene. It was confirmed by research that knocking out the TIPE2 gene reduced the expression of TIPE2 gene, which can effectively inhibit tumor growth and enhance anti-tumor immunity. The reaction plays a role in preventing or treating a tumor. It can be understood that as long as the expression of the TIPE2 gene can be reduced, the tumor growth can be inhibited and the effect of preventing or treating the tumor can be achieved; therefore, in addition to gene knockout, gene knockdown or chemical drugs can also reduce the expression of the TIPE2 gene, as well as Inhibit tumor growth and achieve the effect of preventing or treating tumors.

In addition, it has been confirmed by the study of the present application that the TIPE2 knockout tumor MDSC can also inhibit tumor growth and enhance the anti-tumor immune response, thereby preventing or treating tumors.

The present application will be further described in detail below by way of specific embodiments and the accompanying drawings. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.

Example

In this case, TIPE2 gene-deficient mice were obtained by gene knockout, and LLC mouse lung cancer cells were implanted by subcutaneous injection. The same number of wild-type and TIPE2 gene-deficient tumor MDSCs and LLC tumors were taken within 2-3 weeks. The cells were mixed in a 1:1 ratio, and then subcutaneously implanted into the wild type and TIPE2 gene-deficient mice. The mouse lung cancer cell group was implanted as a control group by subcutaneous injection alone to observe TIPE2-deficient MDSC prevention or The effect of treating tumors. In addition, wild-type mice in which LLC mouse lung cancer cells had been cultured for 3 days and 6 days were treated with the same number of wild-type and TIPE2 gene-deficient tumor MDSCs, and the therapeutic effect of TIPE2-deficient MDSC on existing tumors was observed. Among them, TIPE2-deficient MDSC is the TIPE2 gene knockout MDSC. The details are as follows:

Test 1 TIPE2 knockout MDSC prevents or treats tumors

Tumor cell planting

The LLC mouse lung cancer cells purchased from ATCC were cultured in DMEM complete medium. To obtain a sufficient number of LLC cells, the specific required amount of LLC cells in this example was 2×10 ⁶ /mouse, using subcutaneous injection. They were planted in wild-type mice (abbreviated WT) and TIPE2 gene-deficient mice (Tipe2 ^-/- ), and the tumors were grown for 2-3 weeks. The same number of tumor tissue blocks of WT and Tipe2 ^-/- were used for Tumor MDSC was extracted.

Among them, DMEM complete medium includes: high glucose DMEM + 10% fetal bovine serum + 1% penicillin and streptomycin, all purchased from Hyclone.

2. Tumor MDSC extraction

The mice sacrificed by necking were immersed in 75% alcohol for 5 minutes, and then the mice were placed on a sterile clean bench, and the tumor tissue pieces were removed with surgical scissors and forceps and placed in a 10 cm culture dish. The tumor tissue was cut into small pieces with scissors, and 10 mL of the enzyme mixture was added and resuspended in a 50 mL centrifuge tube, and shaken in a shaking incubator at 200 rpm for 45-60 minutes, and the digestion was terminated by filling the centrifuge tube with complete medium.

The tissue block suspension was placed on a 70 μm cell sieve, and the cell screen was purchased from BD Falcon Co., Ltd., and repeatedly ground with a 5 mL syringe handle, and the cells were all dropped through a sieve into a new 50 mL centrifuge tube to obtain a tissue slurry. The sieve was discarded, and the polishing liquid was centrifuged at 60 g for 5 min at 4 ° C, and the precipitate was discarded. The supernatant of the cells was centrifuged at 1260 g, centrifuged at 4 ° C for 10 min, the supernatant was discarded, and the cell pellet was resuspended in RPMI 1640 complete medium. Take a 15 mL centrifuge tube and carefully add 3 mL of the separation solution, 1 mL of the separation solution 2, and 1 mL of the separation solution 3 according to the mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit instructions to prepare a volume ratio of 3 : 2:1, the total volume is equal to the gradient interface of the RPMI 1640 complete medium resuspended single cell suspension volume. The mouse tumor tissue lymph, mononuclear macrophage, granulocyte separation solution kit was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LDS1090Z.

A sample of the cell suspension was carefully pipetted with a pipette and applied to the liquid surface of the separation solution at the gradient interface, and centrifuged at 1260 g for 30 minutes at 25 ° C. At this time, two layers of ring-shaped milky white cells appeared in the centrifuge tube. Two layers of the circular target cell layer were pipetted into a new 15 mL centrifuge tube, and 10 mL of PBS was added to the obtained centrifuge tube to mix the cells. After centrifugation at 1260 g for 10 min at 4 ° C, the supernatant was discarded and the cell pellet was resuspended in PBS. The cells were stained with the flow antibody CD45/CD11b/Gr-1 from BioLegend for 30 minutes at 4 ° C, and then CD45 ⁺ CD11b ⁺ Gr-1 ⁺ cells were sorted by up-flow cytometry, that is, the obtained tumor MDSC. In this example, tumor MDSC obtained by resuspending PBS at a concentration of 1 × 10 ⁷ /mL was placed on ice for use.

The flow cytometer of this example was purchased from BD Bioscience. RPMI 1640 complete medium included RPMI 1640 + 10% fetal calf serum + 1% penicillin and streptomycin, all available from Hyclone. The enzyme mixture included 50 mg/mL collagenase I, 50 mg/mL collagenase IV, 5 mg/mL hyaluronidase, and 1 U/mL deoxyribonuclease, all of which were purchased from Sigma.

3. Tumor MDSC prevention test for primary tumors

1×10 ⁶ WT tumor MDSCs, ie tumor MDSC extracted from tumor tissue blocks of LLC mouse lung cancer cells without TIPE2 knockout; and 1×10 ⁶ Tipe2 ^−/− tumor MDSC, ie 1×10 ⁶ TIPE2 knockout mice were implanted with tumor MDSC extracted from tumor tissue blocks of LLC mouse lung cancer cells, which is the TIPE2 knockout tumor MDSC of this example. 1×10 ⁶ WT tumor MDSC and 1×10 ⁶ Tipe 2 ^−/− tumor MDSC were mixed with 1×10 ⁶ LLC tumor cells in a 1:1 ratio, respectively. Then, subcutaneous injection was used to implant a new group of WT and Tipe2 ^-/- mice subcutaneously, and 1×10 ⁶ LLC mouse lung cancer cell group was implanted as a control group by subcutaneous injection alone, using a ruler measurement method. The tumor long diameter and short diameter of the mouse were recorded every day, the tumor volume was calculated according to the tumor volume formula, and the tumor volume change was counted, and the data was analyzed by GraphPad 6.0 software.

Among them, the tumor volume (mm ³ ) = short diameter ² × long diameter × 1/2.

In this case, the tumor volume of each group of test mice was tested and counted on 0 days, 3 days, 6 days, 8 days, 11 days, 14 days and 16 days. The test results are shown in Figure 1 and Figure 2, among which 1 is a test result of three groups of tests on WT mice, and FIG. 2 is a test result of three sets of tests on Tipe2 ^−/− mice; in FIG. 1 and FIG. 2 , the abscissa is the number of days after planting, and the ordinate is Tumor volume. In Fig. 1, the "▲" curve is the tumor volume growth curve of the mixed cells of Tipe2 ^-/- tumor MDSC and LLC tumor cells implanted into WT mice, and the "■" curve is the mixed cell growth of WT tumor MDSC and LLC tumor cells. The tumor volume growth curve after WT mice, the "●" curve is the tumor volume growth curve after isolation of individual LLC tumor cells into WT mice. In Fig. 2, the "△" curve is the tumor volume growth curve after the mixed cells of the Tipe2 ^-/- tumor MDSC and LLC tumor cells are implanted into the Tipe2 ^-/- mice, and the "□" curve is the WT tumor MDSC and LLC tumor cells. The tumor volume growth curve after mixing the cells into Tipe2 ^−/− mice, the “○” curve is the tumor volume growth curve after the individual LLC tumor cells were implanted into the Tipe 2 ^−/− mice. The results in Figure 1 show that for wild-type mice, co-injection of LLC+Tipe2 ^-/- tumor MDSC has a significant inhibitory effect on tumor growth compared with injection of LLC alone, whereas co-injection of LLC+WT tumor MDSC has no inhibition compared with injection of LLC alone. Tumor growth effect. For TIPE2 gene-deficient mice, as shown in Figure 2, co-injection of LLC+Tipe2 ^-/- tumor MDSC has the same tumor growth inhibition effect as LLC injection alone, while co-injection of LLC+WT tumor MDSC is compared with injection of LLC alone. Instead, it has the effect of promoting tumor growth. The above results indicate that the TIPE2 knockout tumor MDSC has an anti-tumor effect and can be used to prevent or treat tumors. Second, TIPE2 gene knockout inhibits TIPE2 gene expression, in addition to the simultaneous implantation of LLC+WT tumor MDSC. In addition to the case, it is possible to suppress tumor growth, has an antitumor effect, and can be used for preventing or treating a tumor.

Test 2 Treatment of existing tumors with TIPE2 knockout MDSC

1. Tumor MDSC extraction

LLC mouse lung cancer cells were cultured in DMEM complete medium, and a sufficient number of LLC cells, ie, 2×10 ⁶ /mouse dose, were obtained by subcutaneous injection in wild type mice (abbreviated WT) and TIPE2 gene. Deletion mice (abbreviated Tipe2 ^-/- ), tumor growth was performed for 2-3 weeks, and the same number of tumor tissue blocks of WT and Tipe2 ^-/- were taken for tumor MDSC extraction.

The specific method of tumor MDSC extraction is the same as Experiment 1.

2. Tumor MDSC treatment of existing tumors

A new group of WT mice were implanted with a 2×10 ⁶ / mouse dose of LLC tumors by subcutaneous injection, and a tail vein injection was performed on days 3 and 6, and a group of 3×10 ⁶ WT tumor MDSCs were injected. The other group was injected with 3×10 ⁶ of Tipe2 ^−/− tumor MDSC. In addition, a group of WT mice bearing individual LLC tumors was set as a control group, and the diameter and short diameter of the tumor were recorded every day by means of ruler measurement. Diameter, the tumor volume was calculated and counted in the same manner as in Test 1.

In this case, tumor volumes of 0, 3, 6, 8, 11, 14 and 16 days of each group of test mice were tested and counted, and one group was injected on days 3 and 6, respectively. There were WT tumor MDSCs, and the other group was injected with Tipe2 ^-/- tumor MDSC on days 3 and 6, respectively, and a separate group of LLC tumors as controls; the results are shown in Figure 3. In Fig. 3, the abscissa is the number of days after planting, the ordinate is the tumor volume, and the "▲" curve is the tumor volume growth curve after injection of Tipe2 ^-/- tumor MDSC in WT mice implanted with LLC. The "■" curve is Tumor volume growth curve after injection of WT tumor MDSC in WT mice bearing LLC, the "●" curve is the tumor volume growth curve of control WT mice bearing LLC tumor alone. The results in Figure 3 show that tumor MDSCs injected with TIPE2 knockout have significantly inhibited tumor growth compared with LLC alone, whereas wild-type tumor MDSCs do not inhibit tumor growth compared with LLC alone. The above results indicate that TIPE2 knockout tumor MDSC has the effect of treating existing tumors.

Test 3 TIPE2 knockout MDSC enhances anti-tumor immune response test

1. Tumor MDSC extraction

LLC mouse lung cancer cells were cultured in DMEM complete medium, and a sufficient number of LLC cells, ie, 2×10 ⁶ /mouse dose, were obtained by subcutaneous injection in wild type mice (abbreviated WT) and TIPE2 gene. Deletion mice (abbreviated Tipe2 ^-/- ), after 17 days of tumor growth, took the same number of tumor tissue blocks of WT and Tipe2 ^-/- for tumor MDSC extraction.

The specific method for tumor MDSC extraction is referred to test 1, except that the cells obtained by flow cytometry sorting are resuspended in RPMI 1640 complete medium at a concentration of 1 × 10 ⁶ /mL, and placed on ice for use. Test 1 was resuspended in PBS; the rest was the same as Test 1.

2.CD8 ⁺ T cells

Add allogeneic C57BL/6 mouse spleen-derived CD8 ⁺ T cells to 1 μM CFSE, incubate at 37 ° C for 10-15 minutes, add 5 volumes of cold RPMI 1640 complete medium, and place on ice for 5 minutes to stop staining. Centrifuge at 2500 rpm for 5-10 minutes. The cells were washed twice with RPMI 1640 complete medium, and finally adjusted to a cell concentration of 2 × 10 ⁵ /well with RPMI 1640 complete medium, and plated in 96-well plates.

Among them, CD8 ⁺ T cells were obtained by aseptically sorting CD45 ⁺ CD3 ⁺ CD8 ⁺ cells. CFSE is a succinimide ester available from Invitrogen, Cat. No. C34570.

3. Enhance the test of anti-tumor immune response

CD8 ⁺ T cells stained with CFSE were pre-stimulated with anti-CD3/CD28 magnetic beads, and then one group was co-cultured with Tipe2 ^-/- tumor MDSC in the following ratio, and one group was co-cultured with WT tumor MDSC.

According to tumor MDSC: pre-stimulation staining CD8 ⁺ T cells were mixed and co-cultured in a ratio of 1:2, specifically, 1 × 10 ⁵ tumor MDSC: 2 × 10 ⁵ pre-stimulated stained CD8 ⁺ T cells;

According to tumor MDSC: pre-stimulated staining CD8 ⁺ T cells were mixed and co-cultured in a ratio of 1:4, specifically, 0.5×10 ⁵ tumor MDSC: 2×10 ⁵ pre-stimulated stained CD8 ⁺ T cells;

According to tumor MDSC: pre-stimulated stained CD8 ⁺ T cells were mixed and co-cultured in a ratio of 1:8, specifically, 0.25 × 10 ⁵ tumor MDSC: 2 × 10 ⁵ pre-stimulated stained CD8 ⁺ T cells.

In addition, unstimulated CD8 ⁺ T cells were used as a negative control, and CD8 ⁺ T cells pre-stimulated with anti-CD3/CD28 magnetic beads alone were used as positive controls. After 72 hours of culture, CD8 ⁺ T cell proliferation was measured by flow cytometry of BD Biosciences, and data was analyzed using BD Biosciences' FlowJo7.6 software.

The results are shown in Fig. 4. In Fig. 4, the ordinate is the amount of CD8 ⁺ T cell proliferation, the abscissa sequence negative control, the positive control, the tumor MDSC and the pre-stimulated CD8 ⁺ T cell 1:2 group, 1:4 The group and the 1:8 group, wherein the Unsti group was a negative control, the Sti group was a positive control, and the WT group was a culture result of blending WT tumor MDSC with pre-stimulated CD8 ⁺ T cells in different ratios, Tipe2 ^-/- The group was cultured after blending different ratios of Tipe2 ^-/- tumor MDSC and pre-stimulated CD8 ⁺ T cells. The results in Figure 4 show that WT tumor MDSC can significantly inhibit CD8 ⁺ T cell proliferation in 1:2 and 1:4 ratio co-culture conditions compared to the positive control group, while in the 1:8 ratio co-culture conditions not inhibit the proliferation of CD8 T cells ^+; this is different, TIPE2 knockout MDSC tumor in 1: 2, 1: 4 and 1: 8 ratio of co-culture conditions are not inhibit the proliferation of CD8 T cells ^+. In addition, TIPE2 knockout tumor MDSCs had higher levels of CD8 ⁺ T cell proliferation under 1:2:1:4 co-culture conditions compared to WT tumor MDSCs. This indicates that the TIPE2 knockout tumor MDSC has the ability to promote CD8 ⁺ T cell proliferation and enhance the anti-tumor immune response.

The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. For the ordinary person skilled in the art to which the present invention pertains, a number of simple deductions or substitutions may be made without departing from the spirit of the present application.

Claims (10)

The use of a tumor MDSC targeting the TIPE2 gene for the preparation of a medicament for preventing or treating a tumor; the targeting of the TIPE2 gene includes gene knockout, gene knockdown or chemical drugs to reduce the expression of the TIPE2 gene. The use according to claim 1, wherein the application comprises performing cellular immunotherapy based on the tumor MDSC to prevent or treat a tumor. The use of the TIPE2 gene as a target for the preparation of a medicament for preventing or treating tumors; the targeting of the TIPE2 gene includes gene knockout, gene knockdown or chemical drugs to reduce the expression of the TIPE2 gene. A TIPE2 gene knockout tumor MDSC. The method for preparing a TIPE2 gene knockout tumor MDSC according to claim 4, comprising the steps of: Tumor cell implantation was performed on mice with TIPE2 gene deletion by subcutaneous injection; After the tumor cells to be implanted grow into tumor tissue blocks, the tumor tissue pieces are removed from the mice, and the tumor tissue blocks are digested with an enzyme mixture including collagenase I, collagenase IV, and hyaluronidase. And deoxyribonuclease; After the digestion is completed, the digestion is terminated by using a complete medium, and the tumor tissue block is placed on a cell sieve to be ground, and the tissue slurry passing through the cell sieve is collected; The tissue slurry is subjected to low-speed centrifugation at 4 ° C to obtain a cell supernatant, and the precipitated impurities are discarded; the cell supernatant is subjected to high-speed centrifugation at 4 ° C to collect precipitated cells; The collected precipitated cells were resuspended in RPMI 1640 complete medium, and the collected precipitated cells were isolated using mouse tumor tissue lymph, mononuclear macrophage, and granulocyte separation solution kit. Specifically, the cells were completely cultured with RPMI 1640. The base weight suspension is added to the liquid surface of the separation liquid prepared according to the kit, centrifuged at 1260 g, 25 ° C, and the two layers of annular milky white cell layers appearing in the centrifuge tube are aspirated and collected by a pipette; The collected milky white cell layer was washed with PBS solution, 1260 g, centrifuged at 4 ° C, the supernatant was discarded, and resuspended in PBS to obtain a PBS cell suspension; The PBS cell suspension was stained with the flow antibody CD45/CD11b/Gr-1 at 4 °C, and the CD45 ⁺ CD11b ⁺ Gr-1 ⁺ cells were sorted by flow cytometry to obtain the TIPE2 knockout tumor. MDSC. The preparation method according to claim 5, wherein the enzyme mixture contains 50 mg/mL of collagenase I, 50 mg/mL of collagenase IV, 5 mg/mL of hyaluronidase, and 1 U/mL. Deoxyribonuclease. The preparation method according to claim 5 or 6, wherein the tumor cells are planted, The tumor cells used were LLC mouse lung cancer cells. The preparation method according to claim 5 or 6, wherein in the tumor cell planting, the amount of the tumor cells implanted is 2 × 10 ⁶ tumor cells per mouse. The preparation method according to claim 5 or 6, wherein the removing the tumor tissue block from the mouse comprises: immersing the neck-deprived mouse in 75% alcohol, and then placing the mouse on the mouse. On a sterile, clean bench, remove the tumor tissue block with surgical scissors and forceps. The use of the TIPE2 gene knockout tumor MDSC according to claim 4 for the preparation of a medicament for preventing or treating a tumor.

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