Construction method of EBV positive immune reconstruction xenotransplantation tumor model

Abstract

The invention belongs to the field of medicines, and particularly relates to a construction method of an EBV positive immune reconstruction xenotransplantation tumor model. The method comprises the following steps: taking an SPF-level female severe immunodeficiency mouse, adapting to the environment for 7 + / -3 days, then performing caudal vein injection of human peripheral blood mononuclear cells, and subcutaneously transplanting a human EBV positive gastric cancer cell line AGS / EBV to the right armpit of the mouse next day. The EBV positive immune reconstruction xenotransplantation tumor model provided by the invention has remarkable advantages in the aspects of simulating a human immune system, shortening tumor formation time, revealing that EBV has dual functions of'immune reconstruction-tumor promotion ', supporting treatment strategy development and the like, and an efficient and accurate tool and method are provided for research and treatment of EBV related gastric cancer.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and in particular relates to a method for constructing an EBV-positive immune reconstruction xenograft tumor model. Background Art

[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), such as the method described in the literature "Gastric Cancer, 2021, 24(5):1076-1088". However, these models have significant defects: first, the lack of involvement of the human immune system cannot simulate the dual functions of EBV in immune reconstitution and tumor microenvironment regulation; second, it is difficult to fully reveal the mechanism by which EBV promotes tumor progression by regulating the immune microenvironment; in addition, the existing models have a limited scope of application and cannot be used to evaluate treatment strategies that combine immune reconstitution and tumor microenvironment regulation, and cannot simulate the complex immune responses and tumor microenvironment changes in clinical practice, resulting in limited ability to predict clinical efficacy.

[0003] The present invention effectively compensates for these defects by constructing an EBV-positive immune reconstruction xenograft tumor model, and provides more comprehensive tools and theoretical support for the research and treatment of EBV-related gastric cancer. Summary of the invention

[0004] In view of the shortcomings of the existing problems, the purpose of the present invention is to provide a method for constructing an EBV-positive immune reconstruction xenograft tumor model.

[0005] The technical solution adopted by the present invention to solve the technical problem is:

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

[0007] In a specific embodiment, the acclimatization time is 7±1 days.

[0008] In a specific implementation scheme, the injection amount of human peripheral blood mononuclear cells (PBMC) and the transplantation amount of human EBV-positive gastric cancer cell line AGS / EBV are not specifically limited and can be selected according to actual needs.

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

[0010] In a more specific embodiment, the transplantation amount of human EBV-positive gastric cancer cell line AGS / EBV is 3 to 10×10^6, preferably 5×10^6.

[0011] In a specific embodiment, the SPF-grade female severe combined immunodeficiency mice are 6 to 8 weeks old.

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

[0013] Beneficial Effects

[0014] The present invention provides a method for constructing an EBV-positive immune-reconstructed xenograft tumor model, which demonstrates significant advantages and effects in the study of EBV-related gastric cancer by constructing an EBV-positive immune-reconstructed xenograft tumor model (PBMC+AGS / EBV):

[0015] First, the present invention successfully reconstructed the human immune system in severely immunodeficient C-NKG mice by injecting human peripheral blood mononuclear cells through the tail vein, and was able to fully simulate the dual functions of EBV in immune reconstitution and tumor microenvironment regulation, breaking through the limitation that existing models cannot simulate the human immune system.

[0016] Secondly, experimental data show that the existing EBV-positive gastric cancer model usually takes 30 days or even 40 days to form a tumor, while the model of the present invention only takes 10-14 days to form a tumor, which significantly shortens the experimental cycle and improves research efficiency.

[0017] In addition, the present invention confirms for the first time that EBV has the dual functions of "immune reconstitution-tumor promotion", which not only enhances PBMC-mediated immune reconstitution, but also significantly accelerates the progression of gastric cancer by regulating the tumor microenvironment, providing a new perspective for the study of the mechanism of EBV-related gastric cancer.

[0018] At the same time, the model of the present invention can simultaneously evaluate immune reconstruction and tumor progression, providing an experimental platform for the development of new treatment strategies that combine immune reconstruction and tumor microenvironment regulation. Experimental data showed that the PBMC+AGS / EBV group successfully formed tumors within 10-14 days, with tumor volumes significantly larger than those of other groups (p<0.05), and significantly improved immune reconstruction levels (hCD45 positive cell ratio>40%).

[0019] Finally, the present invention reduces the use of experimental animals and experimental costs by optimizing experimental steps and shortening experimental cycles, making it more environmentally friendly and economical.

[0020] In summary, the present invention has significant advantages in simulating the human immune system, shortening the time of tumor formation, revealing the dual functions of EBV, and supporting the development of treatment strategies, and provides efficient and accurate tools and methods for the research and treatment of EBV-related gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Flowchart for the construction of humanized EBV-positive xenograft tumor models and their control groups.

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

[0023] Figure 3 Statistical graph of hCD45 / (mCD45+hCD45) in the humanized EBV-positive xenograft tumor model and its control group.

[0024] Figure 4 This is a direct shot of the spleen and a statistical graph of the spleen index of the humanized EBV-positive xenograft tumor model and its control group.

[0025] Figure 5 This is a line graph of the body weight of mice in the humanized EBV-positive xenograft tumor model and its control group.

[0026] Figure 6 This is a graph showing the tumor volume growth curve of the humanized EBV-positive xenograft tumor model and its control group.

[0027] Figure 7 Statistical graph of tumor weight of humanized EBV-positive xenograft tumor model and its control group.

[0028] Figure 8 Direct images of tumors in the humanized EBV-positive xenograft tumor model and its control group. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below with reference to the examples. The reagents or instruments used without indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0030] The human EBV-positive gastric cancer cell line AGS / EBV was purchased from the Biological Cell Laboratory of Central South University (Hunan, China). Upon arrival, the cells were amplified and immediately aliquoted for cryopreservation. All cell lines were authenticated by STR and used within 6 months, with the number of passages controlled at 15 to 20. TMPlus-Color One-Step Mycoplasma Detection Kit (40612ES, Yisheng Bio) was used to exclude mycoplasma contamination. Cell lines were cultured in RPMI-1640 medium (KGL1501-500, Keygen Bio) 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 (PBMC), catalog number: FPB004F-C, were purchased from Shanghai Aoneng Biotechnology Co., Ltd.

[0032] SPF female severe combined immunodeficient mice C-NKG aged 6 to 8 weeks, weighing 22 ± 2 g, were purchased from Saiye (Suzhou) Biotechnology Co., Ltd. and raised in the SPF barrier facility of the Experimental Animal Center of China Pharmaceutical University. Mice were housed in a 12-h light / dark cycle (07:30-19:30 light, 5 mice per group, each individually ventilated cage; 19:30-7:30 dark), room temperature (23 ± 2 ° C) and relative humidity (40-50%) were controlled. Mice were marked before grouping and then randomly divided into different groups by an independent person. We used 6 mice in each group. We ensured that the experimental groups were balanced in terms of animal age and weight. The experimental animal operations were in accordance with the standards of the Ethical Review Committee of Laboratory Animal Welfare of China Pharmaceutical University.

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

[0034] To establish a humanized EBV-positive xenograft tumor model, 5×10^6 PBMCs were injected into the tail vein of C-NKG mice after 7 days of adaptation, and 5×10^6 AGS / EBV cells (100μL) were transplanted subcutaneously in the right armpit of the mice the next day. After the tumor became palpable, the tumor size was measured every two days, and the tumor volume (TV) = 1 / 2×maximum length (L)×width (W) 2 2 to 3 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 immune reconstitution level of mice.

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

[0036] To establish a humanized EBV-negative xenograft tumor model, 5×10^6 PBMCs were injected into the tail vein of C-NKG mice after 7 days of adaptation, and 5×10^6 AGS cells (100μL) were transplanted subcutaneously in the right armpit of the mice the next day. After the tumor became palpable, the tumor size was measured every two days, and the tumor volume (TV) = 1 / 2×maximum length (L)×width (W) 2 2 to 3 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 immune reconstitution level of mice.

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

[0038] To establish an EBV-positive xenograft tumor model, 5×10^6 AGS / EBV cells (100 μL) were subcutaneously transplanted in the right axilla of C-NKG mice after acclimation for 7 days. After the tumor became palpable, the tumor size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W) 2 2 to 3 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 immune reconstitution level of mice.

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

[0040] To establish an EBV-negative xenograft tumor model, 5×10^6 AGS cells (100 μL) were subcutaneously transplanted in the right axilla of C-NKG mice after acclimation for 7 days. After the tumor became palpable, the tumor size was measured every two days. Tumor volume (TV) = 1 / 2 × maximum length (L) × width (W) 2 2 to 3 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 immune reconstitution level of mice.

[0041] Figure 1 A schematic diagram of the experimental process of the present invention is given, showing the process of building the model; Figure 2 and Figure 3 is the flow cytometry analysis result of the present invention, wherein Figure 2 Representative flow cytometry images of hCD45 and mCD45 in the peripheral blood of each group of mice. Figure 3The statistical analysis of the ratio of hCD45 / (hCD45+mCD45) showed that the immune system of the PBMC+AGS group was not significantly activated compared with the AGS group, while the immune system of the PBMC+AGS / EBV group was activated compared with the PBMC+AGS group, that is, the ratio of hCD45 increased significantly (>40%). This finding strongly reveals that EB virus has a significant activating effect on the human immune system.

[0042] In addition, mouse spleen vertical photography and spleen index bar graph ( Figure 4 ) further verified the above flow cytometry analysis results. Both showed a trend consistent with the flow cytometry data. It is worth noting that during the experiment, there was no significant fluctuation or difference in the body weight of the mice in each group ( Figure 5 ), which to some extent illustrates that our experimental model has relatively little effect on the overall physiological state of mice.

[0043] Figure 6 and Figure 7 The results showed that when the EB virus or the immune system existed alone, the tumor volume and weight did not change significantly, indicating that the EB virus or the immune system did not affect the tumor when acting alone without the involvement of the other. This shows that in this model, if the EB virus is not present, the immune system will not inhibit tumor cells. On the contrary, when the EB virus and the immune system coexist, the tumor volume and weight both increased significantly, indicating that the EB virus can regulate the immune system to directly promote tumor growth. This finding is different from the traditional sense that the EB virus has an immune escape effect to promote tumors.

[0044] In addition, the direct-shot images of tumor tissues show that the tumor tissues of the two groups with EBV (AGS / EBV group and PBMC+AGS / EBV group) are more red ( Figure 8 ), which may be due to the role of EBV in promoting tumor angiogenesis.

[0045] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the attached claims.

Claims

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

2. The construction method according to claim 1, characterized in that: The adaptation time is 7±1 days.

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

4. The construction method according to claim 3, characterized in that: The injection amount 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 transplanted human EBV-positive gastric cancer cell line AGS / EBV was 3~10×10^6.

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

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

8. The EBV-positive immune-reconstructed xenograft tumor model constructed by the construction method according to any one of claims 1 to 7.

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