A mouse immunosuppression model for long-term maintenance of immunosuppression state and a construction method and application thereof

Abstract

This invention belongs to the field of animal model construction technology, specifically relating to a mouse immunosuppressive model that maintains a long-term immunosuppressive state, its construction method, and its application. This invention establishes a stable immunosuppressive state model with reduced neutrophil count and no significant decrease in erythrocyte count by administering different doses of immunosuppressants (10-50 mg / kg body weight) to Balb / c mice via a combination of "reduced total dose + multiple gavage administration + satellite monitoring." The method for constructing this mouse immunosuppressive model reduces drug usage, controls neutrophil count rebound, establishes a stable model of reduced neutrophil count, and avoids deep bone marrow suppression (i.e., does not cause an uncontrollable decrease in erythrocyte or platelet count), which is beneficial for long-term experimental conduct and drug evaluation.

Description

Technical Field

[0001] This invention belongs to the field of animal model construction technology, specifically relating to a mouse immunosuppressive model that maintains an immunosuppressive state for a long time, its construction method and application. Background Technology

[0002] Invasive fungal infections are a rapidly growing cause of death in recent years, facing serious challenges such as drug scarcity, drug resistance, difficulty in early diagnosis, and a lack of vaccines. The body's defense mechanism against invasive fungi differs significantly from its efficient humoral immunity against viral and bacterial infections. It relies on Th1-induced innate immune enhancement to resist invasion, with neutrophils playing a crucial role in this process. A large proportion of patients with invasive fungal infections are immunosuppressed with neutropenia, and neutropenia has been established as a high-risk factor for the disease. Among numerous immunosuppressants, cyclophosphamide can significantly reduce neutrophil count, making it suitable for establishing animal models for preventing invasive fungal infections. Vaccination cycles are typically lengthy; for mice, at least three vaccinations are required over a month. Therefore, it is necessary to construct long-term, stable immunosuppressive models to assess whether vaccines can elicit a sufficient immune response in immunosuppressed individuals with neutropenia, thereby guiding vaccine design and optimization.

[0003] However, current cyclophosphamide-induced immunosuppression models have the following problems: 1. The number of peripheral blood neutrophils is easily affected by environmental changes, physiological stress, and inflammation, exhibiting significant fluctuations. These fluctuations include at least two scenarios: first, natural fluctuations without immunosuppressant intervention; and second, overdose rebound caused by compensatory recovery after immunosuppressant administration. 2. While high-dose cyclophosphamide can stably reduce neutrophil levels, it simultaneously inhibits non-immune cells such as erythrocytes and platelets, and its toxic effects may interfere with the scientific evaluation of experimental results. 3. A modeling method close to the commonly used clinical oral administration route needs to be established. For patient groups requiring long-term immunosuppression (such as organ transplant recipients), oral immunosuppressants have better medication adherence. If the traditional intraperitoneal injection modeling method is used when developing related vaccines, the significant differences in drug metabolites and pharmacokinetic characteristics compared to oral administration will make it difficult for the model to accurately simulate the real immunosuppressive state in humans.

[0004] Therefore, it is necessary to establish a mouse immunosuppressive model that maintains a long-term immunosuppressive state by continuously and stably reducing neutrophils without affecting the number of non-immune cells such as erythrocytes and platelets, in order to overcome the shortcomings of existing animal models. Summary of the Invention

[0005] To address the shortcomings of the existing technology, the purpose of this invention is to provide a mouse immunosuppressive model that maintains an immunosuppressive state for a long period of time, as well as its construction method and application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides a method for constructing a mouse immunosuppressive model that maintains a prolonged state of immunosuppression, comprising the following steps: S1. Select 4-5 week old Balb / c mice; S2. Mice were treated with immunosuppressants. In week 1, mice were given immunosuppressants 2-4 times, with a dose of 10-50 mg / kg each time; in week 2, mice were given immunosuppressants 1-3 times, with a dose of 10-50 mg / kg each time; in week 3, mice were given immunosuppressants 2-4 times, with a dose of 1-10 mg / kg each time; in week 4, mice were given immunosuppressants 1-2 times, with a dose of 1-10 mg / kg each time. Each group of mice underwent a routine blood test once a week. The blood test indicators included neutrophils, lymphocytes, white blood cells, and red blood cells. The degree of immunosuppression was determined by comparing the differences in these indicators.

[0007] In some embodiments, the immunosuppressant is cyclophosphamide.

[0008] In some embodiments, in step S2, the immunosuppressant treatment of mice is administered by gavage or intraperitoneal injection, preferably by gavage.

[0009] In some embodiments, in step S2, mice are given immunosuppressants four times in week 1, with each treatment dose being 10-50 mg / kg; mice are given immunosuppressants twice in week 2, with each treatment dose being 10-50 mg / kg; mice are given immunosuppressants three times in week 3, with each treatment dose being 1-10 mg / kg; and mice are given immunosuppressants once in week 4, with each treatment dose being 1-10 mg / kg.

[0010] In some embodiments, in step S2, the mice are given four doses of immunosuppressant on days 1, 3, 5, and 7 of week 1; two doses of immunosuppressant on days 2, 4, and 6 of week 2; three doses of immunosuppressant on days 2 and 5 of week 3; and one dose of immunosuppressant on days 3 and 6 of week 4.

[0011] In some implementations, in step S2, the dose of immunosuppressant treated with mice during weeks 1-2 is 30 mg / mL each time.

[0012] In some implementations, in step S2, the dose of immunosuppressant treated with mice during weeks 3-4 is 3.3 mg / mL each time.

[0013] In some implementations, the routine blood tests on mice are performed weekly by selecting the same batch of mice for blood collection and monitoring in weeks 1 and 3, and selecting different mice for blood collection and monitoring in weeks 2 and 4.

[0014] In some implementations, the indicators are statistically analyzed using a linear mixture model.

[0015] In a second aspect, the present invention provides a mouse immunosuppressive model that maintains an immunosuppressive state for a long period of time, which is constructed using the method described above.

[0016] A third aspect of the present invention provides the application of the above-described mouse immunosuppression model in immunotherapy-related diseases.

[0017] A fourth aspect of the present invention provides the application of the above-described mouse immunosuppression model in the screening of immune response-related vaccines.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: (1) This invention establishes a stable immunosuppressive state model with reduced neutrophil count and a basically stable and controllable recovery of erythrocyte count. This invention administers different doses of immunosuppressants (10-50 mg / kg body weight) to Balb / c mice via a method of "reduced total dose + multiple gavage administration + satellite group monitoring," administering the drugs 2-4 times every 7 days. From day 8 onwards, each dose group gradually enters a stable neutrophil immunosuppressive state, allowing for related vaccination and efficacy evaluation studies. The reduction in the number of administrations and the reduction in the total drug dose aim to decrease the rebound rate by reducing the degree of immunosuppression and shorten the maintenance time fluctuation by controlling the administration frequency. By setting up a satellite group, blood is collected from the submandibular region of the mice in rotation to monitor neutrophil counts and other related indicators. Any unexpected fluctuations are promptly adjusted to maintain model stability. This provides a reliable experimental basis for research on new drugs intended for use in immunosuppressive animals to assess immune response.

[0019] (2) The method for constructing the mouse immune model of the present invention reduces the amount of drugs used, controls the rebound of the number of neutrophils, establishes a stable model of reduced neutrophil count, and does not cause deep inhibition of bone marrow, that is, it does not cause an uncontrollable decrease in the number of red blood cells or platelets, which is conducive to long-term experimental development and drug evaluation. Attached Figure Description

[0020] Figure 1This is a comparison of the number of neutrophils in the grasping group and the blank group in this embodiment of the invention; Figure 2 This invention relates to the effects of cyclophosphamide on the levels of neutrophils, lymphocytes, white blood cells, and red blood cells in Balb / c mice. Data are expressed as mean ± standard error, and statistical analysis employed a linear mixed-effects model. p < 0.05 indicates a significant difference compared to the control group at the same time point (significant interaction effect of time × treatment group). Figure 3 This study compares the fluctuations of various indicators in Balb / c mice after gavage administration of 10 mg / kg cyclophosphamide on days 1, 3, 5, 7, 9, 11, and 13, and 1.1 mg / kg cyclophosphamide on days 16, 19, 24, and 27 in this embodiment of the invention. Figure 4 This study compares the fluctuations of various indicators in Balb / c mice after gavage administration of 30 mg / kg cyclophosphamide on days 1, 3, 5, 7, 9, 11, and 13, and 3.3 mg / kg cyclophosphamide on days 16, 19, 24, and 27 in this embodiment of the invention. Figure 5 This study compares the fluctuations of various indicators in Balb / c mice after gavage administration of 50 mg / kg cyclophosphamide on days 1, 3, 5, 7, 9, 11, and 13, and 10 mg / kg cyclophosphamide on days 16, 19, 24, and 27, as described in this embodiment of the invention. Figure 6 This invention provides a comparison of the fluctuations of various indicators in Balb / c mice after intraperitoneal injection of 200, 100, and 150 mg / kg cyclophosphamide on days 1, 8, and 18, respectively, in this embodiment of the invention. Figure 7 This invention provides a comparison of the fluctuations of various indicators in Balb / c mice after intraperitoneal injection of 200, 200, and 150 mg / kg cyclophosphamide on days 1, 8, and 18, respectively. Figure 8 This invention provides a comparison of the fluctuations of various indicators in Kunming mice after intraperitoneal injection of 200, 100, and 150 mg / kg cyclophosphamide on days 1, 8, and 18, respectively, in this embodiment of the invention. Figure 9 This invention provides a comparison of the fluctuations of various indicators in Kunming mice after intraperitoneal injection of 200, 200, and 150 mg / kg cyclophosphamide on days 1, 8, and 18, respectively. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0022] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0024] Example 1: Effect of grasping treatment on neutrophil fluctuations in mice Four-week-old Balb / c mice weighing 18.0–22.0 g were randomly divided into two groups (n=12 per group) after 3 days of acclimatization: a grasping treatment group and a control group. The grasping treatment lasted for four weeks, 2–3 times per week. Specifically, on days 1, 3, 5, 8, 12, 15, 19, 22, 24, and 26, mice were grasped and released from their cages by gavage for approximately 5 seconds around 9:00 AM, but no further gavage was administered. The mice had free access to food and water at other times. The control group received pure water via gavage (using the grasping posture) and had free access to food and water. Peripheral blood was collected periodically for analysis. Neutrophil levels were measured on days 1, 8, 15, 21, and 29, with three mice from each group sampled each time. The results are shown in Table 1 and [Table data would be inserted here]. Figure 1 As shown in the data, even if mice are not given cyclophosphamide by gavage, but only pure water by gavage, or even only local stress (such as grasping operation), the number of neutrophils in the mice fluctuates. Therefore, a method for constructing a mouse model that can maintain stable neutrophil levels over a long period of time is needed.

[0025] Table 1. Number of neutrophils in mice Grouping Week 1 Week 2 Week 3 Week 4 Week 5 gripping group 0.85±0.31 0.44±0.16 0.47±0.24 0.98±0.15 0.94±0.09 Blank group 0.88±0.19 0.82±0.09 0.8±0.09 0.55±0.15 0.9±0.26 Example 2 Four-week-old Balb / c mice weighing 18.0–22.0 g were randomly divided into four groups according to body weight after a 3-day acclimatization period: control group 1, experimental group 2, experimental group 3, and experimental group 4. Six animals were housed in each group in separate cages with free access to food and water. Administered medication every other day for weeks 1–2, and twice weekly at a reduced dose for weeks 3–4. Experimental group 1 received cyclophosphamide solution at approximately 9:00 AM on days 1, 3, 5, 7, 9, 11, and 13, administered at 10 mg / kg body weight seven times, and at 1.1 mg / kg body weight four times on days 16, 19, 24, and 27. Experimental group 2 received cyclophosphamide solution at approximately 9:00 AM on days 1, 3, 5, 7, 9, 11, and 13, administered at 30 mg / kg body weight seven times, and at 3.3 mg / kg body weight four times on days 16, 19, 24, and 27. Experimental group 3 was administered cyclophosphamide solution via gastric gavage at approximately 9:00 AM on days 1, 3, 5, 7, 9, 11, and 13, seven times at a dose of 50 mg / kg body weight. On days 16, 19, 24, and 27, it was administered cyclophosphamide solution at a dose of 10 mg / kg body weight four times. Note that needles from different dosage groups should not be mixed to avoid dosage contamination.

[0026] Observe the animals' condition daily (including skin and fur condition, eyes and mucous membranes, respiratory system, circulatory system, spontaneous movement, central nervous system, and behavioral characteristics), and record in detail the characteristics of adverse reactions, the time of occurrence, severity, and any animal mortality. Administer 0.2 mL of medication per 10 g body weight via gavage. Weigh the animals weekly to adjust the gavage volume and record their weight at the end of the experiment. After drug administration, collect 200 μL of blood from 3 mice in each group via the mandible in weeks 1 and 3, and collect 200 μL of blood from the other 3 mice in each group in weeks 2 and 4. Perform complete blood tests (including neutrophils, lymphocytes, white blood cells, and red blood cells). The degree of immunosuppression is determined by comparing the difference in neutrophil count and other indicators with the blank control.

[0027] To assess the time-dependent effects of different doses and timings of cyclophosphamide on complete blood count (CBC) parameters such as neutrophil count, this study employed a linear mixed-effects model (IBM SPSS Statistics, version 26.0) to analyze the data. Neutrophil count was the dependent variable. The model used time (as a continuous variable), treatment groups (control group, different drug administration groups), and the interaction between time and treatment groups as fixed effects. To control for correlations among multiple measurements from the same mouse, a random intercept with mouse individual ID as the random factor was included in the model. The model employed a first-order autoregressive (AR1) covariance structure, was fitted using restricted maximum likelihood (REML), and effectively handled randomly missing data. Statistical significance was set at p < 0.05. The linear mixed-effects model was used for data analysis, which effectively handles randomly missing data due to individual animal dropout or sample testing failures.

[0028] Given that this study employed a longitudinal repeated measures design and implemented a staggered blood collection strategy to ensure animal welfare (i.e., observations at adjacent time points did not completely overlap), systematic data gaps occurred. Traditional methods such as repeated measures ANOVA (RM-ANOVA) are not directly applicable. Therefore, this study used a linear mixed model (LMM) for statistical analysis. The core advantage of LMM lies in its ability to effectively handle unbalanced longitudinal data with missing values, incorporating all available observations for each individual into the model, thereby maximizing statistical power. More importantly, LMM can simultaneously estimate fixed effects and random effects. Fixed effects reflect the average impact of experimental treatments or time on the population and are the core focus of this study; while random effects capture inter-individual differences in the data (such as differences in initial levels and rates of change). This modeling approach, which clearly distinguishes between overall trends and individual variations, makes the assessment of treatment effects more accurate and robust. In the results graph, the bold solid line represents the population mean trend of the fixed effects estimate, while the semi-transparent thin lines in the background show the individual estimated trajectories based on random effects, intuitively presenting the consistency of the effects and the heterogeneity among individuals.

[0029] Table 2 Changes in mouse body weight Grouping Week 0 Week 1 Week 2 Blank control group 1 19.79±1.12 19.93±1.23 19.61±1.30 Experimental group 2 19.6±0.81 19.41±1.24 18.54±1.37 Experimental group 3 19.63±0.76 19.94±0.61 18.62±0.63 Experimental group 4 19.87±1.01 19.81±0.97 17.94±0.62 Note: Mouse body weight data are expressed as mean ± standard error, and analyzed by one-way ANOVA. The same applies below.

[0030] As shown in Table 2, the modeling process had no significant impact on mouse body weight. No obvious abnormalities in mouse condition or behavior were observed during the experiment.

[0031] Table 3 Blood routine tests of mice with immunosuppression-related conditions in week 1

[0032] Note: #Estimation of Immunosuppression Degree: Immunosuppression degree = (1 - baseline percentage) × 100%, where "baseline percentage" is the average value of relevant indicators in the dosage group divided by the average value of the blank control group. m indicates comparison with the blank control group, P < 0.05; n indicates comparison with the low-dose group, P < 0.05; o indicates comparison with the low-dose group, P < 0.05. Because the number of neutrophils in mice is easily affected by the peripheral environment and local stress (such as grasping operations), the blank data fluctuated significantly (as in Example 1). For ease of comparison, the blood routine data of the blank control group in week 1 were selected as the reference.

[0033] Experimental results showed that cyclophosphamide dose-dependently reduced the number of neutrophils in the peripheral blood of mice. Notably, in the untreated state, the number of neutrophils in mice was easily affected by the peripheral environment and local stress (such as grasping), leading to significant data fluctuations, manifested as a high standard deviation within the mean ± standard deviation. With increasing cyclophosphamide dosage, in addition to a decrease in the absolute number of neutrophils, the standard deviation also decreased in a dose-dependent manner, suggesting that drug treatment can reduce the sensitivity of neutrophils to external stimuli, thus stabilizing the data. However, in the initial low-dose phase, neutrophil counts still showed some volatility, with a relatively large range of mean ± standard deviation. But as the dosage further increased, the standard deviation gradually decreased, eventually becoming significantly lower than that of the blank control group.

[0034] Table 4. Blood routine tests of mice with immunosuppression-related conditions in week 2

[0035] Table 5. Blood routine tests of mice with immunosuppression-related conditions in week 3.

[0036] Table 6. Blood routine tests of mice with immunosuppression-related conditions in week 4.

[0037] Figure 2 To statistically analyze the effects of different cyclophosphamide doses on neutrophil levels in Balb / c mice using a linear mixed-effects model, [the study aimed to]... Figure 2As shown in Tables 3-6, the immunosuppressive model established via gavage administration exhibited significant stability. Statistical analysis revealed that the time-group interaction effects of neutrophils (Neu), white blood cells (WBC), and red blood cells (RBC) were all significant (p<0.05): Neu (F(9, 20.390)=2.784, p=0.026), WBC (F(9, 22.994)=2.772, p=0.023), and RBC (F(9, 32.000)=2.238, p =0.045), confirming the differentiated time-series action pattern produced by different doses.

[0038] The data above show that experimental groups 3-4 achieved significant inhibition of Neu and WBC levels within the first week after drug administration, and subsequently maintained a stable low level throughout weeks 2-4. Intra-group time trend analysis showed no significant fluctuations (p>0.05). Neu levels in experimental groups 3-4 remained consistently low below the reference value throughout the entire period, with fluctuations controlled within 0.2 × 10⁻⁶. 9 Within a range of / L, a stable immunosuppressive window of up to 21 days was successfully established; experimental group 2 entered steady state after week 2, maintaining the inhibitory effect for 14 days. Lymphocyte (Lym) analysis showed a highly significant main effect of grouping (p<0.001), while the time-group interaction effect was not significant (p=0.328), indicating that the inhibitory effect of the drug on lymphocytes remained stable and consistent throughout the observation period, without any drastic rebound or uncontrolled fluctuations. RBCs, as an indicator of toxicity monitoring, showed controllable inhibition during administration, and after dose adjustment in week 4, they demonstrated good hematopoietic compensatory recovery ability, confirming the safety of the dosing regimen.

[0039] Therefore, this stable period of immunosuppression provides an ideal experimental window for vaccine immunogenicity evaluation studies, supporting a 14-21 day vaccination regimen of 2-3 doses every 7 days, which meets the experimental requirements for evaluating vaccine efficacy under immunosuppressive conditions.

[0040] Comparative Example 1: Effects of cyclophosphamide on Balb / c levels and the number of neutrophils in Kunming mice. Four-week-old female Balb / c mice and Kunming mice weighing 18-22 grams were selected and divided into three groups: control group 1, control group 2, and control group 3, with six mice in each group. The mice were acclimatized for three days. Cyclophosphamide at a concentration of 20 g / L was prepared using physiological saline. The control groups were induced according to the protocol shown in Table 7, while the control groups were injected with the corresponding dose of physiological saline.

[0041] Table 7 Injection Protocol

[0042] Approximately two mice were used for blood collection during each rotation. Blood samples were collected from the lower jaw on days 3 and 7 for routine blood tests. The neutrophil count was <500 cells / mm². 3 The experiment can continue. Cyclophosphamide was injected once on day 8, and the mice's blood count was checked on days 11 and 14. Cyclophosphamide was injected once on day 18, and the mice's blood count was checked on days 18, 21, and 28.

[0043] Table 8. Changes in mouse body weight

[0044] p indicates that compared with the Balb / c blank control group, P<0.05; q indicates that compared with Balb / c control group 1, P<0.05; r indicates that compared with the Kunming blank control group, P<0.05.

[0045] As shown in Table 8, cyclophosphamide significantly reduced the body weight of mice in the second week of modeling. About 1 to 3 days after the injection of cyclophosphamide, mice showed hair frizz and lethargy, which gradually recovered.

[0046] Figures 3 to 9 Scatter plots showing the effects of different doses and timings of cyclophosphamide administration on immune-related Neu, Lym, and WBC counts in mice of the examples and comparative examples, using the same statistical methods, are presented. The plots show that the frequency and amplitude of fluctuations in the comparative examples are greater than those in the examples. Taking Neu count as an example, Balb / c mice and Kunming mice, administered intraperitoneally at two slightly different doses, exhibited fluctuations of 2-3 times, with the average values ​​distributed around the reference blank level, and the fluctuation amplitude reaching approximately 1.5 × 10^9 / L. In the examples, the neutrophil levels in each dose group consistently remained below the normal reference value, with the fluctuation amplitude strictly controlled within 0.2 × 10^9 / L. 9 Within / L, it is reduced by 87% compared to traditional methods. More importantly, experimental groups 3 and 4 were able to rapidly establish a stable immunosuppressive state in the first week after administration and maintain it for 21 days, while experimental group 2 could maintain it for 14 days. This can create testing opportunities for 2 to 3 vaccinations for vaccine studies that are administered once every 7 days, thus forming a predictable and controllable long-term inhibitory window.

[0047] Comparative Example 2: Effect of cyclophosphamide at a dose of 30 mg / kg on the number of Balb / c neutrophils, etc. Three female Balb / c mice weighing 8-22 grams were selected and injected intraperitoneally with cyclophosphamide at a dose of 30 mg / kg body weight on days 0 and 7. Blood samples were collected from the mandible of the mice on days 3, 7, and 10 to detect complete blood count.

[0048] Table 9. Effect of 30 mg / kg cyclophosphamide on Balb / c neutrophil count

[0049] As shown in Table 9, the number of neutrophils in the peripheral blood of mice at this dose did not reach the reported target of <500 cells / mm². 3 .

[0050] In summary, compared with the typical uncontrollable "oversuppression-rebound-re-suppression" pattern exhibited by the conventional intraperitoneal injection method (a representative example), the technology of this invention demonstrates superior stability control. The high-frequency fluctuations of traditional methods lead to uncertainty in immune status, limiting the experimental design for evaluating vaccine immunogenicity. This invention, through precise dose gradient design and optimized administration routes, solves the stability problem of immunosuppression models, providing a standardized experimental platform for vaccine inoculation studies under immunosuppressive conditions. This technological innovation not only improves the reproducibility and reliability of experimental results but also establishes a new technical standard for preclinical evaluation in the field of vaccine development.

[0051] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of prolonged immunosuppression, characterized in that, Includes the following steps: S1. Select 4-5 week old Balb / c mice; S2. Mice were treated with immunosuppressants. In week 1, mice were given immunosuppressants 2-4 times, with a dose of 10-50 mg / kg each time; in week 2, mice were given immunosuppressants 1-3 times, with a dose of 10-50 mg / kg each time; in week 3, mice were given immunosuppressants 2-4 times, with a dose of 1-10 mg / kg each time; in week 4, mice were given immunosuppressants 1-2 times, with a dose of 1-10 mg / kg each time. Each group of mice underwent a routine blood test once a week. The blood test indicators included neutrophils, lymphocytes, white blood cells, and red blood cells. The degree of immunosuppression was determined by comparing the differences in these indicators.

2. The method as described in claim 1, characterized in that, In step S2, the immunosuppressant is cyclophosphamide.

3. The method as described in claim 1, characterized in that, In step S2, the immunosuppressants were administered to the mice by gavage or intraperitoneal injection.

4. The method as described in claim 1, characterized in that, In step S2, mice were given immunosuppressants 4 times in week 1, with each treatment dose being 10-50 mg / kg; mice were given immunosuppressants 2 times in week 2, with each treatment dose being 10-50 mg / kg; mice were given immunosuppressants 3 times in week 3, with each treatment dose being 1-10 mg / kg; and mice were given immunosuppressants once in week 4, with each treatment dose being 1-10 mg / kg.

5. The method as described in claim 4, characterized in that, In step S2, the mice are given four immunosuppressants on days 1, 3, 5, and 7 of week 1; two immunosuppressants are given on days 2, 4, and 6 of week 2; three immunosuppressants are given on days 2 and 5 of week 3; and one immunosuppressant is given on days 3 and 6 of week 4.

6. The method as described in claim 1, characterized in that, In step S2, the dose of immunosuppressant treated mice during weeks 1-2 was 30 mg / mL each time.

7. The method as described in claim 1, characterized in that, In step S2, the immunosuppressant dose for each treatment of mice during weeks 3-4 was 3.3 mg / mL.

8. A mouse immunosuppressive model that maintains a prolonged state of immunosuppression, characterized in that, It is constructed using the method described in any one of claims 1 to 7.

9. The application of the mouse immunosuppression model of claim 8 in immunotherapy-related diseases.

10. The application of the mouse immunosuppression model of claim 8 in the screening of immune response-related vaccines.

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