Construction method and application of humanized double-sensitized animal model and systemic allergy animal model

Abstract

The application provides a construction method of a humanized double-sensitization animal model. The PBMCs of a peanut and pollen co-allergic patient are transplanted to an immunodeficient mouse, and a double-allergen sensitization humanized mouse model is constructed by combining the design of peanut sensitization and pollen-induced sensitization model. The application further provides a construction method of a systemic allergic animal model, which comprises the step of sensitizing and exciting the double-allergen sensitization humanized mouse model with crude peanut extract. The double-allergen sensitization humanized mouse model has high molding rate, stable model, no toxicity and harmlessness, and has specificity; the systemic allergic reaction model can be effectively constructed by food allergen excitation, the multiple allergic characteristics of the human body are simulated, the clinical relevance is strong, and important choices and bases are provided for basic research clinicalization.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for constructing a humanized double-sensitized animal model and a systemic allergic animal model and application thereof. BACKGROUND

[0002] Human body often suffers from multiple allergic diseases at the same time, such as food allergy and allergic asthma, which are complicatedly related to each other and involve a wide range of symptoms and can affect multiple systems of the whole body. In addition to pollen, some allergic asthma patients can also be induced by peanuts; many patients with food allergy can also gradually develop into allergic asthma in later life, and potential asthma patients are also more likely to experience near-fatal food-induced systemic allergic reactions.

[0003] Although epidemiological studies have determined the relationship between food allergy and allergic asthma, the interaction between digestive system and respiratory system allergic inflammation is not very clear. The currently known allergic animal model researches are mostly focused on one allergen and its related clinical phenotypes, and the interaction between different allergens is rarely studied. At the same time, due to the differences between species or the differences between the applied allergens and the allergens of the sensitized humans, many related animal models cannot express specific human therapeutic targets, resulting in that many drugs or vaccines cannot be effectively targeted at human diseases and cannot be applied to the clinic.

[0004] Therefore, establishing a humanized animal model with multiple allergic characteristics will help to explore the synergistic sensitization mechanism of multiple allergens, facilitate in-depth research on the pathogenesis and diagnosis and treatment of the corresponding diseases, and at the same time, will not bring treatment risks to clinical subjects, which has very important significance. SUMMARY

[0005] The present application aims to provide a humanized double-sensitized animal model which is allergic to both food (peanuts) and pollen.

[0006] The present application provides a method for constructing a humanized double-sensitized animal model, comprising the following steps:

[0007] (1) injecting PBMCs of a co-allergic patient of peanuts and pollen, human recombinant IL-4 and crude peanut extract into an immunodeficient animal;

[0008] (2) after the injection of step (1) is completed, injecting crude peanut extract for 2 consecutive weeks;

[0009] (3) injecting human recombinant IL-4 and birch pollen 1 week after the injection of step (2) is completed;

[0010] (4) after the injection of step (3) is completed, injecting birch pollen for 2 consecutive weeks.

[0011] Further, the injection method of step (1) is intraperitoneal injection, and the injection dose of PBMCs is (1-3) x 10 7 6 / each, preferably 2 x 10 7 6 / each; the injection dose of IL-4 is 800-1200 U / each, preferably 1000 U / each; and the injection dose of crude peanut extract is 100-200 μg / each, preferably 150 μg / each.

[0012] Further, the injection method of step (2) is intraperitoneal injection, and the injection dose of crude peanut extract is 100-200 μg / each, preferably 150 μg / each, once a week.

[0013] Further, the injection method of step (3) is intraperitoneal injection, and the injection dose of IL-4 is 800-1200 U / each, preferably 1000 U / each; and the injection dose of birch pollen is 10-30 μg / each, preferably 20 μg / each.

[0014] Further, the injection method of step (4) is intraperitoneal injection, and the injection dose of birch pollen is 10-30 μg / each, preferably 20 μg / each, once a week.

[0015] The present application also provides a method for constructing a systemic allergic animal model, which comprises the step of sensitizing and exciting the above-mentioned humanized double-sensitized animal model with crude peanut extract.

[0016] Further, the exciting method is that the crude peanut extract is administered by gavage for 2 weeks, and the gavage dose is 200-300 μg / each, preferably 250 μg / each, once a week.

[0017] Further, the exciting method is that the crude peanut extract is injected intravenously for 2 weeks, and the injection dose is 100-200 μg / each, preferably 150 μg / each, once a week.

[0018] Further, the animal is any one of mouse, rat, dog, pig, monkey and ape, preferably mouse, and more preferably NSG immunodeficient mouse.

[0019] The present application also provides the application of the animal model constructed by the above-mentioned method in the research of allergic diseases caused by multiple allergens, and the research is for the purpose of non-disease diagnosis or treatment.

[0020] Preferably, the research is the research of the synergistic sensitization mechanism of multiple allergens, or the research of screening drugs for preventing and treating allergic diseases caused by multiple allergens; more preferably, the allergic disease is allergic asthma.

[0021] The experimental results show that the peanut and pollen double sensitized animal model can be successfully constructed by the method, the forming rate is high, the model is stable, harmless and specific; the food allergen is used to stimulate the double sensitized animal model, so that the systemic allergic animal model with the double characteristics of food allergy and airway allergic reaction can be successfully induced; the animal model constructed by the method has strong clinical relevance, and provides important selection and basis for the clinical research of the multiple allergen synergistic sensitization mechanism and systemic allergy.

[0022] The beneficial effects of the present application are: 1. In clinical practice, multiple sensitized individuals often first show food allergy reaction, and then further develop asthma. The sensitization sequence of the present application, first sensitizing with CPE and then further sensitizing with pollen to construct a double sensitized animal model, is close to the clinical practice and has good clinical reference value.

[0023] 2. In clinical practice, food can induce severe systemic allergic reactions (including airway allergic reactions), but in the construction of animal models, only using food to induce stimulation often only leads to the increase of airway inflammatory factors or cells, and it is difficult to induce asthma characteristics and simulate clinical practice. The present application successfully constructs a systemic allergic animal model with the double characteristics of food allergy and airway allergic reaction by using food allergen stimulation on the basis of the double allergen sensitized model, which has strong clinical relevance and is more conducive to clinical research.

[0024] The PBMCs of the present application refer to peripheral blood mononuclear cells.

[0025] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the present application.

[0026] The above content of the present application is further described in detail by the following embodiment form. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a peanut and pollen double allergic humanized mouse model construction and sensitization stimulation mode diagram.

[0028] Figure 2 is the allergen specific IgE expression of mouse serum.

[0029] Figure 3 is the IL-4 expression in mouse BALF (A), the eosinophil count in BALF (B) and the detection of airway hyperresponsiveness in mice (C).

[0030] Figure 4 Figure 1 is the mouse plasma histamine release level (A) and systemic anaphylaxis symptom score (B). DETAILED DESCRIPTION

[0031] Unless otherwise specified, the raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products. The crude peanut extract used in the present application is prepared by the following method: fresh roasted peanuts are purchased on the market, 25 g of ground crude peanuts are mixed with 250 ml of 20 mM Tris buffer, and stirred for about 3 hours, then centrifuged at 3000 g for 30 min, after centrifugation, the supernatant is transferred to a new 50 ml centrifuge tube and centrifuged again at 10000 g for 30 min, the residual trace fat and insoluble particles are removed, and the supernatant is collected as the crude peanut extract. The birch pollen used for injection in the present application is a PBS solution of birch pollen with a concentration of 0.2 μg / μL, and the injection dose is 20 μg, which refers to the mass of birch pollen, i.e. the volume of the PBS solution of birch pollen injected is 100 μL.

[0032] Example 1, construction of animal model of the present application and sensitization and challenge

[0033] Construction of humanized double-sensitized animal model: (1) Select 6-8 week-old, SPF female NOD-scidIL2Rgamma null (NSG) immunodeficient mice.

[0034] (2) Isolate peripheral blood mononuclear cells (PBMCs) of peanut and pollen co-allergic patients and non-allergic controls by Ficoll method, and perform intraperitoneal injection of the NSG mice at the 0thweek, with a dose of 2×10 7 / each; at the same time, the mice are intraperitoneally injected with crude peanut extract (CPE), with a dose of 150 μg / each once a week, for 3 weeks (from the 0thweek to the 2ndweek), and at the 0thweek, the mice are intraperitoneally injected with human recombinant IL-4, with a dose of 1000 U / each.

[0035] (3) From the 3rdweek to the 5thweek, the mice are intraperitoneally injected with birch pollen, with a dose of 20 μg / each once a week, for 3 weeks, and at the 3rdweek, the mice are intraperitoneally injected with human recombinant IL-4, with a dose of 1000 U / each.

[0036] The sensitization and challenge of the humanized double-sensitized animal model induces a systemic allergic animal model: (4) The mice are challenged by CPE gavage or tail vein injection from the 6th week to the 7th week, the gavage dose is 250 μg per mouse once a week, and the gavage is continued for 2 weeks; the tail vein injection dose is 150 μg per mouse once a week, and the injection is continued for 2 weeks.

[0037] The flow chart of the modeling and sensitization and challenge of the present application is shown in Figure 1 .

[0038] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0039] Experimental Example 1, Detection of Serum Allergen-specific IgE Expression

[0040] 1. Experimental Method

[0041] The mice (5) modeled according to the method of Example 1 are used as the model group, and a model control group and a normal control group are set up, and the method is as follows:

[0042] Model control group (5): The PBS solution is used instead of the crude peanut extract for intraperitoneal injection in step (2) of Example 1, the PBS solution is used instead of the birch pollen for intraperitoneal injection in step (3), the PBS solution is used instead of the crude peanut extract for allergen challenge in step (4), and the remaining method steps are the same as those of Example 1.

[0043] Model control group + pollen (5): The PBS solution is used instead of the crude peanut extract for intraperitoneal injection in step (2) of Example 1, and the remaining method steps are the same as those of Example 1.

[0044] Model control group + peanut (5): The PBS solution is used instead of the birch pollen for intraperitoneal injection in step (3) of Example 1, and the remaining method steps are the same as those of Example 1.

[0045] Normal control group (5): The PBMCs of the peanut and pollen co-allergic patient in step (2) of Example 1 are replaced by the PBMCs of the non-allergic control, and the remaining method steps are the same as those of Example 1.

[0046] After the mice in each group are sensitized and modeled at the 6th week, blood is taken from the tail vein before challenge, and the serum allergen-specific IgE expression is detected by ELISA or ImmunoCAP analyzer.

[0047] 2. Experimental Results

[0048] Compared with the model control group (green) and the normal control group (blue), the model group (red) can detect the expression of human peanut-specific IgE and pollen-specific IgE in the serum after continuous sensitization for 6 weeks, and the expression level has a significant difference; at the same time, the model control group sensitized only with pollen (yellow) cannot detect the expression of peanut-specific IgE, and the model control group sensitized only with CPE (purple) cannot detect the expression of pollen-specific IgE (p<0.05, see Figure 1 Figure 2 A and 2B).

[0049] The above results show that only the model derived from the PBMCs of a co-allergic patient, and receiving double allergen sensitization, can produce peanut and pollen-specific IgE, that is, can cause double allergic reactions of food and airway allergy. It is proved that the method of the present application successfully and specifically constructs a double sensitization model.

[0050] Experimental Example 2: Detection of plasma histamine release level of mice

[0051] 1. Experimental method

[0052] The modeling of the model group, the model control group and the normal control group is the same as that in Experimental Example 1, and the plasma histamine release level of each group of mice is detected by ELISA after 30 minutes of challenge at the 6th week.

[0053] 2. Experimental results

[0054] Compared with the model control group (green) and the normal control group (blue), the plasma histamine release level of the model group (red) is significantly increased after 30 minutes of challenge at the 6th week (p<0.05, see Figure 2 Figure 4 A). Plasma histamine is the main medium of systemic allergic reaction, and the results show that the model control group and the normal control group cannot effectively trigger an allergic reaction, while the double sensitization model group constructed by the method of the present application can be successfully sensitized and triggered by food allergens.

[0055] Experimental Example 3: Systemic allergic symptoms of the animal model constructed by the method of the present application

[0056] 1. Detection of airway hyperresponsiveness of mice

[0057] 1.1 Experimental method

[0058] The setting of the model group, the model control group and the normal control group is the same as that in Experimental Example 1.

[0059] After 24 hours of challenge of each group of mice at the 6th week, the airway hyperresponsiveness of each group of mice is detected by using a Buxco small animal lung function instrument, tracheal intubation is performed after general anesthesia, different concentrations of acetylcholine are atomized, and airway resistance index (RI) is used to evaluate airway hyperresponsiveness.

[0060] 1.2 Experimental results

[0061] Airway hyperresponsiveness is an important feature of allergic asthma. Compared with the model control group (green) and the normal control group (blue), the model group (red) showed significantly increased airway hyperresponsiveness after 24 hours of challenge at week 6 (p<0.05, see Figs. Figure 3 C).

[0062] 2. Expression of IL-4 and eosinophil count in BALF of mice

[0063] 2.1 Experimental method

[0064] The model group, the model control group, and the normal control group were set up as in Experimental Example 1.

[0065] At week 7, the mice in each group were anesthetized by intraperitoneal injection of 3% chloral hydrate at a dose of 10 ml / kg after challenge, and then both lungs were exposed, the trachea was isolated and cannulated with a 5 ml needle, and 0.5 ml PBS was used to lavage the right lung 3 times to recover BALF. The BALF cell pellet was collected, resuspended after PBS washing, and then counted under a microscope. After Giemsa staining, 200 cells per field were counted under an oil immersion lens for cell differential count. The BALF supernatant was subjected to ELISA to detect IL-4 expression.

[0066] 2.2 Experimental results

[0067] The IL-4 inflammatory factor can reflect allergic inflammatory response. Compared with the model control group (green) and the normal control group (blue), the model group (red) showed increased eosinophil infiltration and elevated IL-4 expression in BALF after challenge at week 7 (p<0.05, see Figs. Figure 3 A and B).

[0068] 3. Systemic allergic symptom score of mice

[0069] 3.1 Experimental method

[0070] The model group, the model control group, and the normal control group were set up as in Experimental Example 1.

[0071] At week 7, the systemic allergic symptoms of the mice were recorded 30 minutes after challenge and quantitatively scored. The scoring criteria are shown in Table 1.

[0072] Table 1. Systemic allergic symptom score

[0073]

[0074] 3.2 Experimental results

[0075] Compared with the model control group (green) and the normal control group (blue), the model group (red) showed significantly increased systemic allergic symptom score after challenge for 30 minutes at week 7 (p<0.05, see Figs.Figure 4 B).

[0076] The above experimental results show that the humanized dual-sensitization animal model constructed by the method of the present invention, after food allergen sensitization and stimulation, produces a systemic allergic reaction model with obvious airway hyperresponsiveness, allergic inflammatory response and systemic allergic symptoms, confirming that it effectively induces the characteristics of systemic allergic reaction, which is beneficial for further research on common food-induced systemic allergic reactions in clinical practice.

[0077] In summary, this invention provides a method for constructing a humanized animal model with dual sensitization to peanuts and pollen. This method boasts a high success rate, model stability, non-toxicity, and specificity. By using food allergens to challenge the aforementioned dual-sensitized animal model, a systemic allergic animal model exhibiting both food allergy and airway allergic reactions can be successfully induced. The animal model constructed using this method overcomes the shortcomings of existing single-allergen and related clinical phenotype allergic animal models, enabling research on the interaction mechanisms between different allergens. It also helps to express specific human therapeutic targets, allowing for research on the pathogenesis and treatment of corresponding diseases without posing treatment risks to clinical subjects. Furthermore, it demonstrates strong clinical relevance, providing an important option and basis for research on the synergistic sensitization mechanisms of multiple allergens and the clinical application of basic research on systemic allergies.

Claims

1. A method for constructing a humanized dual-sensitization animal model, characterized in that, Includes the following steps: (1) Injecting PBMCs, recombinant human IL-4 and crude peanut extract into immunodeficient animals that were co-allergic to peanuts and pollen; (2) After step (1) is completed, inject crude peanut extract for two consecutive weeks; (3) One week after the injection in step (2), human recombinant IL-4 and birch pollen are injected; (4) After step (3) is completed, birch pollen is injected for two consecutive weeks; The injection dose of PBMCs is 2 × 10⁻⁶. 7 / animal, the injection dose of IL-4 is 1000U / animal, the injection dose of crude peanut extract is 150μg / animal, and the injection dose of birch pollen is 20μg / animal; The injection dose of the crude peanut extract in step (2) is once a week, and the injection dose of the birch pollen in step (4) is once a week.

2. The method as described in claim 1, characterized in that, The injection method described in step (1) is intraperitoneal injection.

3. The method as described in claim 1, characterized in that, The crude peanut extract described in step (2) is injected intraperitoneally.

4. The method as described in claim 1, characterized in that, The injection method described in step (3) is intraperitoneal injection.

5. The method as described in claim 1, characterized in that, The method of injecting birch pollen in step (4) is intraperitoneal injection.

6. The method according to any one of claims 1 to 5, characterized in that, The immunodeficient animal is any one of the following: immunodeficient mouse, rat, dog, pig, monkey, or ape.

7. The method as described in claim 6, characterized in that, The immunodeficient animal is an immunodeficient mouse.

8. The method as described in claim 7, characterized in that, The immunodeficient mice are NSG immunodeficient mice.

9. A method for constructing a systemic allergy animal model, characterized in that, Includes the following steps: (1) Construct a humanized dual-sensitization animal model according to the method described in any one of claims 1 to 8; (2) Sensitization stimulation was performed using crude peanut extract.

10. The method as described in claim 9, characterized in that, The stimulation method is as follows: crude peanut extract is administered by gavage for 2 weeks, with a gavage dose of 200-300 μg / animal once a week.

11. The method as described in claim 10, characterized in that, In the aforementioned stimulation method, 250 μg / animal is administered via gavage each time.

12. The method as described in claim 9, characterized in that, The stimulation method is as follows: intravenous injection of crude peanut extract for 2 weeks, with an injection dose of 100~200μg / animal once a week.

13. The method as described in claim 12, characterized in that, In the aforementioned activation method, 150 μg / animal is injected each time.

14. The application of the animal model constructed by the method of any one of claims 1 to 13 in the study of allergic diseases caused by multiple allergens, wherein the study is for the purpose of non-disease diagnosis or treatment.

15. The application as described in claim 14, characterized in that, The research focuses on the synergistic sensitization mechanism of multiple allergens, or on screening drugs to prevent and treat allergic diseases caused by multiple allergens.

16. The application as described in claim 15, characterized in that, The allergic disease mentioned is allergic asthma.

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