A method for constructing an EBV-positive immunoreconstructed xenograft tumor model

Abstract

The application belongs to the field of medicine and particularly relates to a construction method of an EBV-positive immune-reconstructed xenograft tumor model; the method comprises the following steps: taking SPF female severely immune-deficient mice, and after adapting to the environment for 7-3 days, injecting human peripheral blood mononuclear cells into the caudal vein of the mice, and the next day, transplanting human EBV-positive gastric cancer cell line AGS / EBV under the skin of the right armpit of the mice. The EBV-positive immune-reconstructed xenograft tumor model provided by the application has significant advantages in aspects of simulating the human immune system, shortening the tumor formation time, revealing that EBV has a double function of 'immune reconstruction-promoting tumor', and supporting the development of treatment strategies, and provides an efficient and precise tool and method for the research and treatment of EBV-related gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a method for constructing an EBV-positive immune reconstitution xenograft tumor model. Background Technology

[0002] Animal models of EBV-related gastric cancer are mainly constructed by subcutaneously implanting EBV-positive gastric cancer cell lines into immunodeficient mice (such as NSG mice), as described in the paper "Gastric Cancer, 2021, 24(5):1076-1088". However, these models have significant limitations: First, they lack the involvement of the human immune system and cannot simulate the dual functions of EBV in immune reconstitution and tumor microenvironment regulation. Second, they are difficult to fully reveal the mechanism by which EBV promotes tumor progression by regulating the immune microenvironment. In addition, the existing models have limited application scope and cannot be used to evaluate treatment strategies that combine immune reconstitution and tumor microenvironment regulation. Furthermore, they cannot simulate the complex immune responses and changes in the tumor microenvironment in clinical practice, which limits their ability to predict clinical efficacy.

[0003] This invention effectively overcomes these shortcomings by constructing an EBV-positive immune reconstitution xenograft tumor model, providing more comprehensive tools and theoretical support for the research and treatment of EBV-related gastric cancer. Summary of the Invention

[0004] To address the shortcomings of existing methods, the purpose of this invention is to provide a method for constructing an EBV-positive immune-reconstructed xenograft tumor model.

[0005] The technical solution adopted by this invention to solve its technical problem is:

[0006] In a first aspect, the present invention protects a method for constructing an EBV-positive immune reconstitution xenograft tumor model, the method comprising the following steps: taking SPF-grade female severely immunodeficient mice, acclimating them to the environment for 7±3 days, injecting human peripheral blood mononuclear cells (PBMCs) into the tail vein, and then subcutaneously transplanting human EBV-positive gastric cancer cell line AGS / EBV into the right axilla of the mouse the next day.

[0007] In the specific implementation plan, the adaptation time to the environment is 7±1 days.

[0008] In the specific implementation plan, there are no specific restrictions on the injection volume of human peripheral blood mononuclear cells (PBMCs) and the transplantation volume of human EBV-positive gastric cancer cell line AGS / EBV; the selection can be made according to actual needs.

[0009] In a more specific implementation, the amount of human peripheral blood mononuclear cells (PBMCs) injected is 3 to 10 × 10^6, preferably 5 × 10^6.

[0010] In a more specific implementation plan, the number of human EBV-positive gastric cancer cell lines AGS / EBV transplanted is 3 to 10 × 10^6, preferably 5 × 10^6.

[0011] In the specific implementation plan, the SPF-grade female severely combined immunodeficient mice are 6-8 week old mice.

[0012] Secondly, the present invention also protects the EBV-positive immune-reconstructed xenograft tumor model constructed by the construction method described above.

[0013] Beneficial effects

[0014] This invention provides a method for constructing an EBV-positive immune-reconstituted xenograft model. By constructing an EBV-positive immune-reconstituted xenograft model (PBMC+AGS / EBV), significant advantages and effects have been demonstrated in the study of EBV-related gastric cancer.

[0015] First, this invention successfully reconstructs the human immune system in severely immunodeficient C-NKG mice by injecting human peripheral blood mononuclear cells into the tail vein. It can fully simulate the dual functions of EBV in immune reconstitution and tumor microenvironment regulation, overcoming the limitation of existing models that cannot simulate the human immune system.

[0016] Secondly, experimental data show that existing EBV-positive gastric cancer models typically require 30 to 40 days to form tumors, while the model of this invention only requires 10-14 days to form tumors, significantly shortening the experimental cycle and improving research efficiency.

[0017] Furthermore, this invention is the first to demonstrate that EBV has a dual function of "immune reconstitution-tumor promotion," not only enhancing PBMC-mediated immune reconstitution but also significantly accelerating gastric cancer progression by regulating the tumor microenvironment, providing a new perspective for the study of the mechanism of EBV-related gastric cancer.

[0018] Meanwhile, the model of this invention can simultaneously assess immune reconstitution and tumor progression, providing an experimental platform for developing novel therapeutic strategies that combine immune reconstitution and tumor microenvironment regulation. Experimental data show that the PBMC+AGS / EBV group successfully formed tumors within 10-14 days, with significantly larger tumor volumes than other groups (p<0.05), and significantly improved immune reconstitution levels (hCD45-positive cell proportion >40%).

[0019] Finally, by optimizing experimental procedures and reducing experimental cycles, this invention reduces the number of experimental animals used and the cost of experiments, making it more environmentally friendly and economical.

[0020] In summary, this invention has significant advantages in simulating the human immune system, shortening tumor formation time, revealing the dual function of EBV, and supporting the development of treatment strategies, providing efficient and precise tools and methods for the research and treatment of EBV-related gastric cancer. Attached Figure Description

[0021] Figure 1 Flowchart for constructing a humanized EBV-positive xenograft tumor model and its control group.

[0022] Figure 2 The flow cytometry plots represent the expression of hCD45 and mCD45 in the humanized EBV-positive xenograft model and its control group.

[0023] Figure 3 A statistical graph showing the hCD45 / (mCD45+hCD45) ratio in a humanized EBV-positive xenograft model and its control group.

[0024] Figure 4 Direct images of the spleen and spleen index statistics of the humanized EBV-positive xenograft model and its control group.

[0025] Figure 5 Line graph showing the body weight of the humanized EBV-positive xenograft model and its control group mice.

[0026] Figure 6 The tumor volume growth curves of the humanized EBV-positive xenograft model and its control group are shown.

[0027] Figure 7 A statistical graph showing the tumor weight of a humanized EBV-positive xenograft model and its control group.

[0028] Figure 8 Direct images of tumors in a humanized EBV-positive xenograft model and its control group. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the embodiments. Reagents or instruments used without a specified manufacturer are considered to be conventional products that can be purchased on the market.

[0030] Human EBV-positive gastric cancer cell line AGS / EBV was purchased from the Cell Biology Laboratory of Central South University (Hunan, China). Upon arrival, cells were expanded and immediately aliquoted and cryopreserved. All cell lines were identified by STR and used within 6 months, with passages controlled between 15 and 20. MycAway was used. TMThe Plus-Color One-Step Mycoplasma Detection Kit (40612ES, Yisheng Biotechnology) was used to exclude mycoplasma contamination. Cell lines were cultured in RPMI-1640 medium (KGL1501-500, Kaiji Biotechnology) containing 10% fetal bovine serum (C2910-0500, Viva Cell) and 1% penicillin-streptomycin. All cells were maintained in an incubator at 37°C and 5% CO2.

[0031] Human peripheral blood mononuclear cells (PBMCs), catalog number: FPB004F-C, were purchased from Shanghai Aoneng Biotechnology Co., Ltd.

[0032] Six- to eight-week-old SPF-grade female C-NKG mice with severe combined immunodeficiency (SICF) and a weight of 22 ± 2 g were purchased from Cyagen (Suzhou) Biotechnology Co., Ltd., and housed in the SPF-grade barrier facility of the Laboratory Animal Center of China Pharmaceutical University. A 12-hour light / dark cycle was maintained (light from 07:30 to 19:30, 5 mice per group, housed in individual ventilated cages; darkness from 19:30 to 07:30), with controlled room temperature (23 ± 2 °C) and relative humidity (40–50%). Mice were marked before grouping and then randomly assigned to different groups by an independent person. We used 6 mice per group. We ensured that the experimental groups were balanced in terms of animal age and weight. All animal handling complied with the standards of the Laboratory Animal Welfare and Ethics Review Committee of China Pharmaceutical University.

[0033] Example 1: Construction of a humanized EBV-positive xenograft tumor model

[0034] To establish a humanized EBV-positive xenograft model, C-NKG mice were injected with 5 × 10^6 PBMCs via the tail vein 7 days after acclimatization. The following day, 5 × 10^6 AGS / EBV cells (100 μL) were subcutaneously transplanted into the right axilla of the mice. Once the tumor was palpable, its size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W). 2 Two to three weeks after PBMC injection, blood samples were collected from the tail tip, and the expression levels of mCD45 and hCD45 were detected by flow cytometry to monitor the level of immune reconstitution in mice.

[0035] Example 2: Construction of a humanized EBV-negative xenograft model

[0036] To establish a humanized EBV-negative xenograft model, C-NKG mice were injected with 5 × 10^6 PBMCs via the tail vein 7 days after acclimatization. The following day, 5 × 10^6 AGS cells (100 μL) were subcutaneously transplanted into the right axilla of the mice. Once the tumor was palpable, its size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W). 2 Two to three weeks after PBMC injection, blood samples were collected from the tail tip, and the expression levels of mCD45 and hCD45 were detected by flow cytometry to monitor the level of immune reconstitution in mice.

[0037] Example 3: Construction of an EBV-positive xenograft tumor model

[0038] To establish an EBV-positive xenograft tumor model, C-NKG mice were subcutaneously transplanted with 5 × 10^6 AGS / EBV cells (100 μL) in the right axilla after 7 days of environmental acclimatization. Once the tumor was palpable, its size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W). 2 Two to three weeks after tumor implantation, blood samples were collected from the tail tip, and the expression levels of mCD45 and hCD45 were detected by flow cytometry to monitor the level of immune reconstitution in mice.

[0039] Example 4: Construction of an EBV-negative xenograft model

[0040] To establish an EBV-negative xenograft tumor model, C-NKG mice were subcutaneously transplanted with 5 × 10^6 AGS cells (100 μL) in the right axilla after 7 days of environmental acclimatization. Once the tumor was palpable, its size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W). 2 Two to three weeks after tumor implantation, blood samples were collected from the tail tip, and the expression levels of mCD45 and hCD45 were detected by flow cytometry to monitor the level of immune reconstitution in mice.

[0041] Figure 1 A schematic diagram of the experimental procedure of the present invention is provided, illustrating the model construction process; Figure 2 and Figure 3 The results of flow cytometry analysis according to the present invention are as follows: Figure 2 Representative flow cytometry plots of hCD45 and mCD45 in the peripheral blood of each group of mice. Figure 3Statistical analysis of the hCD45 / (hCD45+mCD45) ratio showed that the immune system was not significantly activated in the PBMC+AGS group compared to the AGS group, while the immune system was activated in the PBMC+AGS / EBV group compared to the PBMC+AGS group, with a significantly higher hCD45 ratio (>40%). This finding strongly suggests that EBV has a significant activating effect on the human immune system.

[0042] In addition, direct images of mouse spleens and bar charts of spleen index ( Figure 4 This further validated the results of the flow cytometry analysis. Both showed trends consistent with the flow cytometry data. Notably, during the experiment, there were no significant fluctuations or differences in the body weight of the mice in each group. Figure 5 This, to some extent, indicates that our experimental model has a relatively small impact on the overall physiological state of mice.

[0043] Figure 6 and Figure 7 The results showed that neither EBV nor the immune system significantly changed tumor volume or weight when either was present alone, indicating that EBV or the immune system alone does not affect tumors in the absence of either. This suggests that in this model, the immune system does not suppress tumor cells in the absence of EBV. Conversely, when EBV coexisted with the immune system, both tumor volume and weight increased significantly, indicating that EBV can directly regulate the immune system to promote tumor growth. This finding differs from the traditional understanding that EBV has an immune escape effect that promotes tumor growth.

[0044] Furthermore, direct imaging of the tumor tissue reveals that the tumor tissue in the two groups with EBV (AGS / EBV group and PBMC+AGS / EBV group) is redder. Figure 8 This may be due to EBV promoting tumor angiogenesis.

[0045] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A method for constructing an EBV-positive immune reconstitution xenograft tumor model, the method comprising the following steps: taking SPF-grade female severely immunodeficient mice, acclimating them to the environment for 7±3 days, injecting human peripheral blood mononuclear cells into the tail vein, and then subcutaneously transplanting human EBV-positive gastric cancer cell line AGS / EBV into the right axilla of the mouse the next day.

2. The construction method of claim 1, wherein, The adaptation time to the environment is 7±1 days.

3. The construction method according to claim 1, characterized in that, The injection dose of human peripheral blood mononuclear cells is 3 to 10 × 10^6.

4. The construction method according to claim 3, characterized in that, The injection dose of human peripheral blood mononuclear cells is 4~8×10^6.

5. The construction method according to claim 1, characterized in that, The number of human EBV-positive gastric cancer cell lines AGS / EBV transplanted is 3 to 10 × 10^6.

6. The construction method according to claim 5, characterized in that, The number of human EBV-positive gastric cancer cell lines AGS / EBV transplanted is 4~8×10^6.

7. The construction method according to claim 1, characterized in that, The SPF-grade female severely immunodeficient mice were 6-8 weeks old.

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