CN115349497A - Pig roundworm asthma-induced mouse model and construction method thereof - Google Patents
Pig roundworm asthma-induced mouse model and construction method thereof Download PDFInfo
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- CN115349497A CN115349497A CN202211057494.8A CN202211057494A CN115349497A CN 115349497 A CN115349497 A CN 115349497A CN 202211057494 A CN202211057494 A CN 202211057494A CN 115349497 A CN115349497 A CN 115349497A
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Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/10—Animals modified by protein administration, for non-therapeutic purpose
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0387—Animal model for diseases of the immune system
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a pig roundworm asthma-induced mouse model and a construction method thereof, wherein the pig roundworm asthma-induced mouse model comprises the following steps: 1) Anaesthetizing the mouse, exposing the trachea through an operation, injecting a ascaris suum protein sensitizer between the 3 rd and 4 th cartilages below the throat, inclining an operating table to lift the head of the mouse, and suturing after keeping the head for 30 seconds; 2) And (3) dripping the ascaris suum protein excitant into the nose of the mouse 8 days after suturing, dripping the nose once every day for 5 days, and observing the respiratory state of the mouse after the nose dripping is finished. The invention uses commercial ascaris suum protein as antigen, thus avoiding the risk of environmental pollution or injury to experimenters caused by ascaris or ascaris ova; the common Balb/c mouse has lower price and definite genetic background, and is convenient for discussing the immune genetic mechanism generated by the inflammatory reaction of the asthma airway and the airway hyperresponsiveness; the nasal drop solvent selects a near neutral PBS buffer solution, so that the additional influence on the lung is reduced, and the pathogenesis is closer to clinical.
Description
Technical Field
The invention belongs to the technical field of animal models, and relates to a pig roundworm asthma-induced mouse model and a construction method thereof.
Background
Allergic asthma (asthma) is a chronic airway inflammation involving a variety of cells, particularly mast cells, eosinophils and T lymphocytes, which in susceptible individuals can cause recurrent wheezing, shortness of breath, chest tightness and/or cough, which often occurs during the night and/or early morning hours and increases the airway responsiveness to a variety of stimuli.
When the same allergen contacts with the body in the sensitized state again, the allergen is combined with IgE on the sensitized mast cell membrane surface Fc epsilon RI, receptor cross-linking can trigger the degranulation of mast cells, and further release of other intermediate mediums. This leads to the formation of the allergic asthmatic prophase reaction. The antigen-specific T cells initiate eosinophil to move and gather to the lung, and activate Th2 cells to produce IL-4 and ICOS-L, so that activated B cells secrete more IgE, and further tissue damage is caused.
At present, common allergens for molding comprise Ovalbumin (OVA), house dust mites, pollen and the like, and different model effects can be achieved through different sensitization ways and excitation ways.
Disclosure of Invention
The invention aims to provide a mouse model with asthma caused by ascaris suum and a construction method thereof, wherein the mouse model with allergic asthma is successfully induced and constructed by taking ascaris suum protein as an antigen.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
the invention provides a construction method of a mouse model for treating asthma caused by ascaris suum, which comprises the following steps:
1) Anaesthetizing the mouse, exposing the trachea through an operation, injecting a ascaris suum protein sensitizer between the 3 rd and 4 th cartilages below the throat, inclining an operating table to lift the head of the mouse, and suturing after keeping the head for 30 seconds;
2) And (3) dripping the ascaris suum protein excitant into the nose of the mouse 8 days after suturing, dripping the nose once every day for 5 days, and observing the respiratory state of the mouse after the nose dripping is finished.
Preferably, the mice are SPF grade Balb/c mice, half male and half female, and 7-8 weeks old.
Preferably, step 1) further comprises anesthetizing the mice after acclimatizing the mice for 7 days.
Preferably, the mass volume concentration of the ascaris suum protein sensitizer is 40 mug/ml.
More preferably, 25 μ l of the ascaris suum protein sensitiser is injected with the insulin syringe in step 1).
More preferably, the ascaris suum protein sensitiser is formulated by: weighing 10 μ l of ascaris suum protein solution 5mg/ml and 1.24ml PBS to prepare sensitizer 40 μ g/ml, 1.25ml in total.
Preferably, in step 1), the operating table is tilted by 25-35 °.
Preferably, the concentration of the ascaris suum protein activator is 0.25mg/ml.
More preferably, the mice in step 2) are instilled with 50 μ L of ascaris suum protein elicitor via nose.
More preferably, the ascaris suum protein elicitor is formulated by: 100 mul of 10mg/ml ascaris suum protein solution and 3.9ml PBS are weighed to prepare 0.25mg/ml excitant, and the total volume is 4ml.
Preferably, model verification is further included: 24 hours after the last excitation is finished, lung tissues are taken after the animals are euthanized, after the animals are fixed by 10 percent formalin, the tissues are sliced by conventional paraffin embedding, pathological examination and HE staining are carried out, inflammation evaluation is carried out on the tissue slices, inflammatory cell infiltration of lung tissues of a model group, pulmonary alveoli expansion, lung septal rupture, bronchial and vascular muscle layer thickening are carried out, and the model building is successful.
The invention also provides a pig roundworm asthma-induced mouse model constructed by the construction method.
The ascaris suum protein is used as an antigen to induce the mouse allergic asthma model, and has the following advantages:
the commercial ascaris suum protein is used as an antigen, so that the risk of environmental pollution or injury to experimenters caused by ascaris or ascaris ova is avoided.
And (II) the mouse product is a common Balb/c mouse, the price is low, the genetic background is clear, and the immune genetic mechanism generated by asthma airway inflammatory reaction and Airway Hyperresponsiveness (AHR) is convenient to discuss.
And (III) the nasal drop solvent selects a near-neutral PBS buffer solution, so that the additional influence on the lung is reduced, and the pathogenesis is closer to the clinic.
Drawings
FIG. 1 is the HE staining results of the normal group and the model group in the examples of the present invention.
Detailed Description
In order to more clearly illustrate the present invention, the present invention will be further described in detail with reference to the following examples and drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Examples
(I) preparation of the experiment
1. Animals:
SPF grade Balb/c mice, male and female half, 7-8 weeks old.
2. Reagents and consumables:
ascaris suum protein; phosphate Buffered Saline (PBS) at pH 7.2-7.4; physiological saline; 10 muL/100 muL/1 mL pipette; an insulin syringe; an analytical balance; 1 pair of surgical scissors with the length of 10cm, 2 pairs of forceps with the length of 10cm, 1 pair of micro forceps with the bend, 1 pair of needle holders, 4-0 suture lines and iodophors.
3. The preparation method of the main reagent comprises the following steps:
preparing a sensitizer: weighing 10 mu l of ascaris suum protein solution with the concentration of 5mg/ml and 1.24ml of PBS to prepare sensitizer with the concentration of 40 mu g/ml, which is 1.25ml;
preparing an exciting agent: 100 mul of 10mg/ml ascaris suum protein solution and 3.9ml PBS are weighed to prepare 0.25mg/ml excitant, and the total volume is 4ml.
(II) Molding operation
1. Sensitization: after the animals are adaptively raised for 7 days, anesthetizing the mice, exposing the trachea by operation, injecting 25 mul of sensitizer between the cartilage of the 3 rd section and the cartilage of the 4 th section below the throat by using an insulin syringe, inclining an operating table by about 30 degrees to lift the head of the mice, and suturing after keeping for 30 s;
2. excitation: on day 8 after suturing, mice were instilled with 50 μ L/mouse nasal elicitor once a day for 5 days, and after completion of nasal instillation, the respiratory state of the mice was observed.
(III) model verification
24h after the final excitation is finished, lung tissues are taken after the animals are euthanized, after 10% formalin fixation, the lung tissues are sliced in a conventional paraffin embedding way, pathological examination and HE staining are carried out, and inflammation evaluation is carried out on the tissue slices.
As shown in fig. 1, HE staining results indicated that the lung tissue structure was intact in the normal group; the model group has the advantages of lung tissue inflammatory cell infiltration, alveolar dilatation, lung space rupture, bronchial and vascular muscularis thickening and successful model building.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all the embodiments of the present invention are not exhaustive, and all the obvious variations or modifications which are introduced in the technical scheme of the present invention are within the scope of the present invention.
Claims (10)
1. A method for constructing a mouse model of asthma caused by ascaris suum comprises the following steps:
1) Anaesthetizing the mouse, exposing the trachea through an operation, injecting a ascaris suum protein sensitizer between the 3 rd and 4 th cartilages below the throat, inclining an operating table to lift the head of the mouse, and suturing after keeping the head for 30 seconds;
2) And (3) dripping the ascaris suum protein excitant into the nose of the mouse 8 days after suturing, dripping the nose once every day for 5 days, and observing the respiratory state of the mouse after the nose dripping is finished.
2. The method for constructing the asthma-induced mouse model of ascaris suum according to claim 1, wherein the mouse is an SPF-grade Balb/c mouse, half female and half male, and 7-8 weeks old.
3. The method for constructing the asthma mouse model caused by ascaris suum according to claim 1, wherein the step 1) further comprises performing mouse anesthesia after adaptively feeding the mouse for 7 days.
4. The method for constructing the asthma-induced mouse model by using ascaris suum according to claim 1, wherein the mass volume concentration of the ascaris suum protein sensitizer is 40 μ g/ml.
5. The method for constructing the asthma-induced mouse model by using ascaris suum according to claim 4, wherein 25 μ l of ascaris suum protein sensitizer is injected by using an insulin syringe in the step 1).
6. The method for constructing the asthma mouse model caused by ascaris suum according to claim 1, wherein in the step 1), the operating table is inclined by 25-35 °.
7. The method for constructing the asthma-induced mouse model by using ascaris suum according to claim 1, wherein the concentration of the ascaris suum protein excitant is 0.25mg/ml by mass and volume.
8. The method for constructing the asthma-induced mouse model by using ascaris suum according to claim 7, wherein 50 μ L/mouse of ascaris suum protein excitant is instilled through nose in the mouse in the step 2).
9. The method for constructing the asthma mouse model caused by ascaris suum according to claim 1, further comprising model verification: 24 hours after the last excitation is finished, lung tissues are taken after the animals are euthanized, after the animals are fixed by 10% formalin, the tissues are sliced by conventional paraffin embedding, pathological examination and HE staining are carried out, inflammation evaluation is carried out on the tissue slices, inflammatory cell infiltration of lung tissues of a model group, pulmonary alveoli are expanded, lung intervals are broken, and bronchial and vascular muscle layers are thickened, thus indicating that the model building is successful.
10. The ascaris suum asthma-induced mouse model constructed by the construction method of the ascaris suum asthma-induced mouse model according to any one of claims 1 to 9.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211057494.8A CN115349497A (en) | 2022-08-30 | 2022-08-30 | Pig roundworm asthma-induced mouse model and construction method thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1898564A (en) * | 2003-12-24 | 2007-01-17 | 惠氏公司 | Methods of treating asthma |
| CN102895657A (en) * | 2012-10-15 | 2013-01-30 | 中国人民解放军第三军医大学第二附属医院 | Method for constructing asthma model of nonhuman mammal |
| CN103566353A (en) * | 2013-11-06 | 2014-02-12 | 刘志刚 | Method for constructing asthmatic animal model |
-
2022
- 2022-08-30 CN CN202211057494.8A patent/CN115349497A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1898564A (en) * | 2003-12-24 | 2007-01-17 | 惠氏公司 | Methods of treating asthma |
| CN102895657A (en) * | 2012-10-15 | 2013-01-30 | 中国人民解放军第三军医大学第二附属医院 | Method for constructing asthma model of nonhuman mammal |
| CN103566353A (en) * | 2013-11-06 | 2014-02-12 | 刘志刚 | Method for constructing asthmatic animal model |
Non-Patent Citations (4)
| Title |
|---|
| D.M.ITAMI等: "Modulation of murine experimental asthma by Ascaris suumcomponents", CLIN EXP ALLERGY, pages 873 - 879 * |
| 戴继宏,谭毅,符州: "哮喘动物模型的研究现状", 中国实验动物学杂志, no. 03 * |
| 王玉梅;罗元平;罗莉;: "STAT5、STAT6在哮喘肺组织的表达", 中国临床研究, no. 11, pages 987 - 988 * |
| 陈子,李庆生,淤泽溥: "抗过敏实验方法及过敏反应动物模型研究进展", 云南中医学院学报, no. 03 * |
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