A method of constructing a model of asthma immune tolerance

Abstract

The present application relates to the field of animal model construction, and discloses a method for constructing an asthma immune tolerance model, comprising the following steps: A. establishing an immune tolerance mouse model: let the young mice drink sterile water containing 0.5-1.5% OVA by mass fraction every day for 7 consecutive days, replace the drinking water every 24 hours, and start to drink sterile water on the eighth day; on the 15th day, inject 125ul of OVA with a mass fraction of 0.5-1.5% into the abdominal cavity of the sensitized mice, and start to atomize and excite the mice with OVA with a mass fraction of 0.5-1.5% for 1 hour on the 22nd day, for 7 consecutive days; B. identification of the immune tolerance mouse model. The present application directly obtains the immune tolerance to OVA by orally administering OVA to the weaned mice, significantly shortens the time for constructing the asthma immune tolerance model, reduces the steps for constructing the model, and greatly improves the efficiency. The present application solves the technical problems in the prior art, such as the very long time for constructing the breast milk tolerance model, the very cumbersome steps such as atomizing the mother mice and atomizing the mice in the later stage, and the high cost.

Description

Technical Field

[0001] This invention relates to the field of animal model construction, and more particularly to a method for constructing an asthma immune tolerance model. Background Technology

[0002] The internationally accepted method for constructing an asthma immune tolerance model using ovalbumin (OVA) is mostly based on breast milk tolerance. This involves nebulizing OVA into mother mice that have just given birth, allowing the mice to indirectly acquire immune tolerance to OVA through their mother's milk. After weaning, mice that have acquired OVA immune tolerance will not develop asthma upon re-exposure to OVA allergens, while mice that have not acquired OVA immune tolerance will develop asthma upon re-exposure to OVA allergens. Summary of the Invention

[0003] This invention addresses the shortcomings of existing technologies by providing a method for constructing an asthma immune tolerance model.

[0004] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0005] A method for constructing an asthma immune tolerance model includes the following steps:

[0006] A. Establishing an immune-tolerant mouse model: Young mice were given sterile water containing 0.5-1.5% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 0.5-1.5% OVA. On the 22nd day, the mice were challenged by nebulization with 0.5-1.5% OVA for 1 hour for 7 consecutive days.

[0007] B. Identification of immune-tolerant mouse models.

[0008] This invention directly induces immune tolerance to OVA in newly weaned mice by orally administering ovalbumin (OVA), which significantly shortens the time required to construct an asthma immune tolerance model, reduces the number of steps involved in the model construction, and greatly improves efficiency.

[0009] Preferably, in step A, the young mice are 3-4 weeks old.

[0010] Preferably, in step A, the young mice are 3-4 week old SPF-grade BALB / c female mice.

[0011] Preferably, in step A, the mice drink sterile water containing 1% OVA daily.

[0012] Preferably, in step A, on day 15, mice are sensitized by intraperitoneal injection of 125 μL containing 1% OVA.

[0013] Preferably, in step A, mice are stimulated for 1 hour by nebulization with 1% OVA starting on day 22.

[0014] Preferably, in step B, the identification of the immune-tolerant mouse model includes measuring the OVA-specific IgE content.

[0015] Preferably, in step B, the identification of the immune-tolerant mouse model includes,

[0016] a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0017] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0018] Preferably, in step B, the left lung tissue is subjected to hematoxylin-eosin and periodic acid-Schiff pathological staining.

[0019] This invention directly induces immune tolerance to OVA in newly weaned mice through oral administration, significantly shortening the time required to construct an asthma immune tolerance model and reducing the number of steps involved, thus greatly improving efficiency. This invention solves the technical problem of existing technologies where constructing a breast milk tolerance model is extremely time-consuming, requiring mating, pregnancy, and farrowing of the mother mouse, followed by nebulization of the mother mouse and subsequent nebulization of the mice – a highly cumbersome, costly, and inefficient process. The shortened period for constructing the asthma immune tolerance model in the research process not only prepares the material for later experiments but also facilitates subsequent research by reducing the model culture time; it saves research costs and is more conducive to scientific research. Furthermore, oral immune tolerance has a high success rate and is more modifiable, making it more suitable for future clinical research. The applicable population is not limited to breastfed infants and can be used for all age groups. Attached Figure Description

[0020] Figure 1 This refers to the determination of OVA-specific IgE content in Example 3.

[0021] Figure 2 This is a graph showing the total number of cells and the number of inflammatory cells in the mouse BALF in Example 3.

[0022] Figure 3 This is a diagram showing the expression of Th2 cytokine mRNA in mouse lungs in Example 3.

[0023] Figure 4 This is a diagram of lung inflammation in mice from Example 3.

[0024] Figure 5This is a diagram of airway hyperresponsiveness after oral administration of OVA in Example 3. Detailed Implementation

[0025] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments.

[0026] Example 1

[0027] A method for constructing an asthma immune tolerance model includes the following steps:

[0028] A. Establishing an immune-tolerant mouse model: Young mice were given sterile water containing 0.5% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 0.5% OVA. On the 22nd day, the mice were challenged by nebulization with 0.5% OVA for 1 hour for 7 consecutive days.

[0029] B. Identification of immune-tolerant mouse models.

[0030] Example 2

[0031] A method for constructing an asthma immune tolerance model includes the following steps:

[0032] A. Establishing an immune-tolerant mouse model: 3-4 week old SPF-grade female BALB / c mice were given sterile water containing 1.5% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 1.5% OVA. On the 22nd day, the mice were challenged by nebulization with 1.5% OVA for 1 hour for 7 consecutive days.

[0033] B. Identification of immune-tolerant mouse models.

[0034] Example 3

[0035] A method for constructing an asthma immune tolerance model includes the following steps:

[0036] A. Establishing an immune-tolerant mouse model: 3-4 week old SPF-grade female BALB / c mice were given sterile water containing 1% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 1% OVA. On the 22nd day, the mice were challenged by nebulization with 1% OVA for 1 hour for 7 consecutive days.

[0037] B. Identification of immune-tolerant mouse models.

[0038] In step B, such as Figure 1-5As shown, the identification of the immune-tolerant mouse model includes:

[0039] (1) Determine the OVA-specific IgE content.

[0040] Specific methods for measuring mouse serum IgE:

[0041] ① Coating: Dilute the capture antibody in Coating Buffer, and add 100 μL to each well. After sealing the ELISA plate, place it directly in a 4°C freezer overnight.

[0042] ② Wash and discard the coating solution. Wash with washing buffer (PBST). The method is to add 250ul of PBST to each well, shake it a few times by hand, discard the washing solution, pat it on absorbent paper, and then refill it. Repeat this washing process 3-5 times.

[0043] ③ After the final wash, repeatedly pat the sealant onto absorbent paper to ensure no washing liquid residue remains in each well. Add 250ul of sealing solution to each well and let it stand at room temperature for 2 hours.

[0044] ④ After washing and sealing, wash the ELISA plate using the same method as in step 2.

[0045] ⑤ Add the standard and sample gradient dilution standard, select 7 wells to dilute the concentration by 2 times in sequence, and use the last well as a blank control. At the same time, dilute the prepared mouse serum 50 times and add 100 μL to each well. Incubate at room temperature for 2 hours.

[0046] ⑥ The washing process is the same as step ④.

[0047] ⑦ Add secondary antibody: After diluting the detection antibody in Assay Buffer, add 100 μL to each well, seal, and incubate at room temperature for 1 hour.

[0048] ⑧ The specific steps for preparing the detergent are the same as in step ④.

[0049] ⑨ Add enzyme-labeled antibody: Dilute the HRP-labeled antibody and add 100 μL to each well, then incubate at room temperature for half an hour.

[0050] ⑩ The washing process is the same as step ④.

[0051] Add 100 μL of colorimetric reagent to each well of the ELISA plate. Then, plate the ELISA plate at room temperature in the dark.

[0052] Let it sit for about 15 minutes.

[0053] The reaction is terminated by adding a stop solution (2 mol / L sulfuric acid solution) to each well of the ELISA plate.

[0054] OD value measurement: Read the value on a microplate reader at a wavelength of 450 nm. Then analyze it using software.

[0055] against Figure 1 Analysis: Serum ELISA results showed that IgE was significantly elevated in the asthma group, while there was no significant statistical difference between the Control group and the Tolerance group.

[0056] Note: n=6, data were processed using GraphPad Prism 5 statistical software, and the data are expressed as Mean±SEM, two groups.

[0057] Independent samples t-tests were used for comparisons between data points, and two-way ANOVA was used for comparisons among multiple groups of continuous data.

[0058] Statistical significance was defined as a p-value < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001 vs Control group; △P < 0.05, △△P < 0.01, △△△P < 0.001 vs Tolerance group.

[0059] (2)a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0060] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0061] I. Methods for cell counting and staining of bronchoalveolar lavage fluid:

[0062] (1) Sample collection: After anesthetizing the mice, fix the mice on the operating table, put the mice in supine position and perform tracheal intubation, irrigate the alveoli with 1ml PBS using a needle, and aspirate twice repeatedly, being careful not to break the alveoli, and the movements should be gentle and slow to ensure that the respiration rate is above 80%.

[0063] (2) Immediately centrifuge the bronchoalveolar lavage fluid at 4°C, 500g for 5 min, discard the supernatant, resuspend the cells in 1 ml PBS, and drop 10 μl onto a cell counting plate for microscopic cell counting. Centrifuge again at 4°C, 500g for 5 min, discard the supernatant, resuspend the cells in 200 μl PBS, and add all the cells to the cell collection wells of the cell smear centrifuge. Mark the wells with a pen to facilitate accurate location during staining, then centrifuge at 300 rpm for 5 min. The cells are then immediately transferred to a glass slide.

[0064] (3) Staining: Carefully add Wright-Giemsa A solution (about 0.5-0.8 ml) to the smear, allowing the staining solution to cover the circled area of ​​the specimen smear for 2 minutes; then add Wright-Giemsa B solution on top of solution A (the amount is 2-3 times that of solution A); gently blow with your mouth to mix the two solutions thoroughly, and stain for about 5-10 minutes; carefully wash the slide horizontally with water, then let it air dry before microscopic examination, and classify and count the cells according to their morphology.

[0065] Figure 2 Analysis: Wright-Giemsa staining was performed on cells in bronchoalveolar lavage fluid (BALF) of mice. Based on the morphology, size, and nuclear morphology of the stained cells, they were classified into eosinophils, macrophages, lymphocytes, and neutrophils. Statistical analysis revealed that the total number of cells and the number of each of the above cell types in the BALF of the OVA group were significantly higher than those in the Control and Tolerance groups, while there was no significant difference between the latter two groups.

[0066] II. Extraction of total RNA from lung tissue

[0067] ① Take the whole lung of a mouse (about 200-300mg) into a 1.5ml EP tube, add 800ul Trizol reagent, put one large and one small bead into each EP tube, put it into a high-throughput tissue homogenizer, make sure to balance it, homogenize at a frequency of 70HZ for 120 seconds and then take it out.

[0068] ② Aspirate 200 μL of lung tissue homogenate from the above EP tube into a new EP tube, then add 40 μL of chloroform and 20 μL of RNase-free ddH2O, cap the EP tube, shake vigorously in your hand for 15 seconds, let stand at room temperature for 2-3 minutes, and then centrifuge at 12000g, 4℃ for 15 minutes.

[0069] ③ Take the upper aqueous phase and place it in a new EP tube. Add an equal volume of isopropanol and mix thoroughly. Let it stand at room temperature for 30 minutes, then centrifuge at 12000g, 4℃ for 10 minutes.

[0070] ④ Discard the supernatant, add 800ul of freshly prepared 75% ethanol for washing, shake it up and down in your hand several times until you see white flocculent matter floating in the alcohol, centrifuge at 10000g, 4℃ for 5 minutes and discard the supernatant; repeat the washing once.

[0071] ⑤ Aspirate as much of the remaining supernatant as possible, and then let the precipitated RNA air dry at room temperature;

[0072] ⑥ Dissolve the precipitate with RNase-free water (approximately 80-100 μL) and store in a -80°C refrigerator.

[0073] III. First-strand cDNA synthesis:

[0074] ① Dissolve the above RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-Free Water and place on ice for later use.

[0075] ②Prepare the reaction system

[0076] Table 1. cDNA First-Strand Synthesis Reaction System

[0077] reagents 20ul reaction system Final concentration dNTP Mix 4ul 500uM Primer Mix 2ul RNA x ul 5pg-5ug 5x RT Buffer 4ul 1x DTT 2ul 10nM HiFiScript 1ul RNase-Free Water up to 20ul

[0078] ③ Vortex oscillation, centrifugal force.

[0079] ④ Program settings: 42℃ for 30 min, 85℃ for 5 min to complete the reaction, cool on ice and set aside.

[0080] IV. Real-time PCR:

[0081] Primer synthesis (Shanghai Sangon Biotech), primer sequences used for each gene are shown in Table 2 below:

[0082] Table 2. Primers for each gene

[0083]

[0084] The composition of the reaction system is as follows:

[0085] ①Reaction system

[0086] Table 3. Real-time PCR reaction system

[0087] reagents 10ul reaction system 2xUltra SYBRMixture(With High ROX) 5ul Forward Primer 0.3ul Reverse Primer 0.3ul DNA 2ul RNase-Free Water up to 10ul

[0088] ②Reaction procedure:

[0089] Table 4. Real-time PCR reaction procedure

[0090]

[0091] After the reaction, the amplification and melting curves were confirmed. The cycle threshold (CT) value, which represents the number of cycles required for the fluorescence signal in each reaction tube to reach the set threshold, was used as an internal reference to correct the Ct value of the target gene in a single sample, i.e., the ΔCt value (Ct). 目的基因 -Ct 管家基因 Each sample was repeated in triplicate. The relative expression values ​​of the target gene between the control and experimental groups were calculated by multiplying the values ​​by 2. －△△Ct It means that △△Ct = experimental group (Ct) 目的基因 -Ct 管家基因 )-Control group (Ct)目的基因 -Ct 管家基因 ).

[0092] Figure 3 Analysis: Quantitative real-time PCR showed that the expression of asthma-related genes (IL-4, IL-5, IL-13, and IL-17A) was significantly upregulated in the lungs of mice in the asthma group compared with those in the mock and tolerance groups, while there was no significant statistical difference between the latter two groups.

[0093] (3) The left lung tissue was stained with hematoxylin-eosin and periodic acid-Schiff stain.

[0094] I. Preparation of lung tissue pathological sections:

[0095] Materials Collection: According to the experimental groups, on the 7th day after OVA nebulization, each mouse was weighed. 80ul / 20g of 2% sodium pentobarbital was injected into a 1ml syringe. The animal was held firmly with its abdomen facing upwards, and to avoid puncturing internal organs, the head could be lowered to move the internal organs towards the diaphragm. The needle of the syringe was inserted into the skin with the right hand at the midpoint of the line connecting the groin and the linea alba. After the needle reached the subcutaneous tissue, it was advanced 3-5mm further. After complete anesthesia, the mouse was euthanized by cervical dislocation. The mouse was then fixed on a foam frame with its face upwards using the injection needle. The thoracic rim was cut open to expose the hair and skin, and the clavicle and sternum were severed to fully expose the heart and lung tissue. The lower lobe of the left lung of each mouse was harvested and fixed by immersion in 4% paraformaldehyde solution for 24 hours.

[0096] Dehydration: 70% ethanol 20 min → 80% ethanol 20 min → 95% ethanol I 10 min → 95% ethanol II 10 min → 100% ethanol I 10 min → 10% ethanol II 10 min

[0097] Transparent: Xylene I 10 min → Xylene II 10 min

[0098] Wax impregnation: benzene wax 30 min → paraffin wax 30 min

[0099] Sectioning: Freeze the paraffin-embedded block at -20℃ for 5 minutes, then section it to a thickness of 4μm. Flatten the sections in warm water at 45~55℃, and then lift the paraffin sections with a glass slide.

[0100] Baking with wax: 62℃ for more than 1 hour until the paraffin melts.

[0101] Dewaxing: While still hot, sequentially add xylene I for 10 min → xylene II for 10 min → 100% ethanol I for 10 min → 100% ethanol II for 10 min → 95% ethanol I for 10 min → 95% ethanol II for 10 min → 80% ethanol for 10 min → 70% ethanol for 10 min → rinse with tap water.

[0102] HE staining: Hematoxylin staining for 5 min → differentiation with 1% hydrochloric acid ethanol for a few seconds (5-10 s) → rinse with tap water to turn blue → eosin staining for 1 min → rinse with tap water for 5-10 s.

[0103] Dehydration: 70% ethanol 2 min → 80% ethanol 2 min → 95% ethanol I 2 min → 95% ethanol II 2 min → 100% ethanol I 2 min → 100% ethanol II 2 min

[0104] Transparent: Xylene I 2 min → Xylene II 2 min

[0105] Covering: Apply neutral resin → Cover slide

[0106] Figure 4 Analysis: Pathological sections of mouse lungs and HE staining showed that, compared with the Control and Tolerance groups, mice in the OVA group all exhibited thickening of pulmonary blood vessels and bronchial walls, accompanied by extensive inflammatory cell infiltration. There was no significant statistical difference between the first two groups.

[0107] II. Method for measuring airway responsiveness in mice:

[0108] ① Airway responsiveness in mice was determined using the Buxco mouse pulmonary function instrument invasive pulmonary impedance method within 24 hours after the last challenge. Mice were anesthetized with 2% sodium pentobarbital (30 mg / kg), endotracheal intubation was performed, and a small animal ventilator was connected. The ventilator frequency was set to 120 r / min, and the flow rate was adjusted to ensure that the tidal volume of the mice reached 0.13–0.15 ml. The changes in airway airflow and pressure were measured to obtain the changes in the resistance index (RI) of the mice.

[0109] ② Record the baseline value of airway resistance in mice for 1 minute as the baseline value; then measure the change in airway resistance after nebulizing mice with a 10 μl dose of methacholine (Mch). Each nebulization lasts 30 seconds, and the data is recorded for 3 minutes. After a 4-minute interval, the results are analyzed using software. The statistical results are expressed as the average increase in airway resistance (RI) per unit time (compared to the baseline value) under different concentrations of Mch stimulation, denoted as RI avg (%Change). A higher RI value indicates higher airway reactivity, and vice versa.

[0110] Figure 5Data analysis: Within 24 hours of the last challenge, mice were connected to a Buxco invasive respiratory function monitoring system, and changes in airway resistance (RI) were recorded. Airway resistance (RI) in the Control, Tolerance, and OVA groups all showed a trend of increasing with increasing Mch concentration. The baseline RI in the OVA group was (2.03±0.93) H2O·s / m, which increased to (8.15±3.80) cmH2O·s / ml after Mch stimulation. The baseline RI in the Control group was (1.62±1.15) cmH2O·s / ml, which increased to (4.36±2.00) cmH2O·s / ml after Mch stimulation. The baseline RI in the Tolerance group was (1.73±1.33) H2O·s / m, which increased to (5.15±2.80) cmH2O·s / ml after Mch stimulation. The changes in RI average (% change) in each group after stimulation with different concentrations of Mch are as follows.

[0111] Table 5. Percentage increase in RI from baseline in each group of mice

[0112] Mch (mg / ml) 0 3.125 6.25 12.5 Tolerance (%) 0 12.38±9.72** 58.21±22.20** 99.76±15.90** OVA (%) 0 54.00±12.96 135.60±22.80 213.22±44.67 Control (%) 0 <![CDATA[17.63±8.06 △△ ]]> <![CDATA[64.12±6.29 △△ ]]> <![CDATA[80.56±4.83 △△△ ]]>

[0113] Example 4

[0114] A method for constructing an asthma immune tolerance model includes the following steps:

[0115] A. Establishment of an immune-tolerant mouse model: Three-week-old SPF-grade female BALB / c mice were given sterile water containing 0.6% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 0.6% OVA. On the 22nd day, the mice were challenged by nebulization with 0.6% OVA for 1 hour for 7 consecutive days.

[0116] B. Identification of immune-tolerant mouse models.

[0117] In step B, the identification of the immune-tolerant mouse model includes measuring the OVA-specific IgE content.

[0118] Step B, the identification of the immune-tolerant mouse model, includes,

[0119] a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0120] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0121] Example 5

[0122] A method for constructing an asthma immune tolerance model includes the following steps:

[0123] A. Establishment of an immune-tolerant mouse model: Four-week-old SPF-grade female BALB / c mice were given sterile water containing 1.4% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 1.4% OVA. On the 22nd day, the mice were challenged by nebulization with 1.4% OVA for 1 hour for 7 consecutive days.

[0124] B. Identification of immune-tolerant mouse models.

[0125] In step B, the identification of the immune-tolerant mouse model includes measuring the OVA-specific IgE content.

[0126] Step B, the identification of the immune-tolerant mouse model, includes,

[0127] a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0128] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0129] Example 6

[0130] A method for constructing an asthma immune tolerance model includes the following steps:

[0131] A. Establishing an immune-tolerant mouse model: 3-4 week old SPF-grade female BALB / c mice were given sterile water containing 0.8% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 0.8% OVA. On the 22nd day, the mice were challenged by nebulization with 0.9% OVA for 1 hour for 7 consecutive days.

[0132] B. Identification of immune-tolerant mouse models.

[0133] In step B, the identification of the immune-tolerant mouse model includes measuring the OVA-specific IgE content.

[0134] Step B, the identification of the immune-tolerant mouse model, includes,

[0135] a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0136] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0137] In step B, the left lung tissue was subjected to hematoxylin-eosin and periodic acid-Schiff pathological staining.

[0138] Example 7

[0139] A method for constructing an asthma immune tolerance model includes the following steps:

[0140] A. Establishing an immune-tolerant mouse model: 3-4 week old mice were given sterile water containing 1.5% OVA daily for 7 consecutive days, with the drinking water changed every 24 hours. On the eighth day, they were given sterile water. On the 15th day, the mice were sensitized by intraperitoneal injection of 125 μL containing 1.2% OVA. On the 22nd day, the mice were challenged by nebulization with 1.1% OVA for 1 hour for 7 consecutive days.

[0141] B. Identification of immune-tolerant mouse models.

[0142] Step B, the identification of the immune-tolerant mouse model, includes,

[0143] a. Perform bronchoalveolar lavage, cell counting and classification, and determine the levels of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid;

[0144] b. RNA was extracted from the homogenate of the right lung tissue, and the expression of cytokine mRNA in step a was determined by real-time quantitative PCR.

[0145] In summary, the above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be covered by the present invention.

Claims

1. A method of constructing a model of immune tolerance in asthma, characterized by, Comprising the following steps, A. Establishing the immune tolerance mouse model: let the young mice drink sterile water containing 0.5-1.5% OVA by mass fraction every day for 7 consecutive days, change the drinking water every 24 hours, and start drinking sterile water on the eighth day; wherein the young mice are 3-4 week old mice; on the 15th day, inject 125ul of 0.5-1.5% OVA by mass fraction into the abdominal cavity of the sensitized mice, and start to aerosolize the mice with 0.5-1.5% OVA by mass fraction for 1 hour on the 22nd day, for 7 consecutive days; B. Identification of the immune tolerance mouse model; the identification of the immune tolerance mouse model includes determining the content of OVA-specific IgE; also including: a. Perform bronchoalveolar lavage, perform cell counting and classification, and determine the content of IL-4, IL-5, IL-13 and IFN-γ cytokines in the bronchoalveolar lavage fluid; b. Extract RNA from right lung tissue homogenate, and determine the expression of mRNA of the cytokines in step a by real-time quantitative PCR; perform hematoxylin-eosin and periodic acid-schiff's pathological staining on the left lung tissue; Wherein the extraction of total RNA from lung tissue: ① Take 200-300 mg of mouse lung into a 1.5 ml EP tube, add 800 ul of Trizol reagent, put a large and small bead in each EP tube, balance, grind for 120 seconds at a frequency of 70 Hz, and then take out; ② Absorb 200 ul of lung homogenate in the above EP tube into a new EP tube, then add 40 ul of chloroform and 20 ul of RNase-free ddH2O, cover the EP tube cap, shake vigorously for 15 seconds, and then stand at room temperature for 2-3 minutes, then centrifuge at 12000g, 4°C for 15 minutes; ③ Absorb the upper water phase into a new EP tube, add an equal volume of isopropanol, mix thoroughly, stand at room temperature for 30 minutes, then centrifuge at 12000g, 4°C for 10 minutes; ④ Discard the supernatant, add 800 ul of 75% ethanol prepared on site for washing, shake up and down in the hand until white flocculent material is seen floating in the alcohol, then centrifuge at 10000g, 4°C for 5 minutes to discard the supernatant; repeat the washing once; ⑤ Try to absorb the remaining supernatant, then place the precipitated RNA at room temperature to dry naturally; ⑥ Dissolve the precipitate with Rnase-free water, 80-100 ul, and store in a -80°C refrigerator.

2. The method of constructing a model of immune tolerance to asthma according to claim 1, wherein: In step A, the young mice are 3-4 week old SPF BALB / c female mice.

3. The method of constructing a model of immune tolerance to asthma according to claim 1, wherein: In step A, the mice drink sterile water containing 1% OVA by mass fraction every day.

4. The method of constructing a model of immune tolerance to asthma according to claim 1, wherein: In step A, on the 15th day, inject 125ul of 1% OVA by mass fraction into the abdominal cavity of the sensitized mice.

5. The method of constructing a model of immune tolerance to asthma according to claim 1, wherein: In step A, start to aerosolize the mice with 1% OVA by mass fraction for 1 hour on the 22nd day.

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