阅读说明：本技术 使用豚草花粉诱导的小鼠过敏性结膜炎模型及造模方法 (Mouse allergic conjunctivitis model induced by ragweed pollen and molding method ) 是由 江斌 李焱 袁永明 于 2021-08-18 设计创作，主要内容包括：本发明提供了使用豚草花粉诱导的小鼠过敏性结膜炎模型及造模方法,方法包括：将豚草花粉跗关节注射液注射进小鼠跗关节皮下,注射体积为65-85μl,注射后第10天开始,将豚草花粉滴眼液滴至小鼠右眼,每只10-15μl,每天一次,共4次。本发明成模率高,致敏反应明显。(The invention provides a mouse allergic conjunctivitis model induced by ragweed pollen and a molding method, wherein the method comprises the following steps: injecting the ragweed pollen tarsal joint injection into the mouse tarsal joint subcutaneously with an injection volume of 65-85 μ l, starting on the 10 th day after injection, and dropping the ragweed pollen eye drops to the right eye of the mouse, each 10-15 μ l, once a day for 4 times. The invention has high molding rate and obvious sensitization reaction.)

1. A modeling method using a ragweed pollen-induced mouse allergic conjunctivitis model, comprising:

injecting the ragweed pollen tarsal joint injection into the mouse tarsal joint subcutaneously with an injection volume of 65-85 μ l, starting on the 10 th day after injection, and dropping the ragweed pollen eye drops to the right eye of the mouse, each 10-15 μ l, once a day for 4 times.

2. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 1, characterized in that the ragweed pollen tarsal joint injection is formulated by the following method: taking 2.0-3.0mg of ragweed pollen, adding 650 mu l of alum adjuvant, and mixing for use.

3. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 2, characterized in that the ragweed pollen eye drops are formulated by the following method: taking 15-20mg of ragweed pollen, dissolving in 100 mul PBS buffer solution, and preparing into 150-200mg/ml working solution.

4. The method of modeling using a ragweed pollen-induced mouse model of allergic conjunctivitis according to claim 1, wherein injection is performed in both hind limbs when 65-85 μ l of injection cannot be subcutaneously accommodated in the unilateral tarsal joint.

5. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 1, further comprising adaptively feeding the mouse for 3d before the modeling.

6. The modeling method of the mouse model of allergic conjunctivitis induced by ragweed pollen according to claim 1, wherein the mouse is a 6 w-old Balb/c mouse, and is male and female unlimited.

7. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 1, further comprising performing model validation after the last challenge.

8. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 7, wherein said model validation comprises: and completing the photographing and observation of the eye microscopic picture within 20min after the last excitation.

9. The modeling method using ragweed pollen-induced mouse allergic conjunctivitis model according to claim 8, wherein said model validation further comprises: sampling is completed within 30min after the last excitation, blood is taken for death after the animals are anesthetized, the right eyes of the mice are taken out along with the eyelids, the mice are fixed by 10% formalin, the sections are embedded by normal paraffin, and the conjunctival morphology and the eye-shaped cell infiltration condition are observed by HE staining.

10. The ragweed pollen-induced mouse allergic conjunctivitis model obtained by the molding method of the ragweed pollen-induced mouse allergic conjunctivitis model according to any one of claims 1 to 9.

Technical Field

The invention belongs to the technical field of construction of an allergic conjunctivitis animal model, and relates to a ragweed pollen-induced mouse allergic conjunctivitis model and a molding method.

Background

Allergic conjunctivitis is a common ocular surface disease with seasonal and periodic episodes, and hypersensitivity inflammation due to exposure of ocular tissues to drugs or other allergens in the environment. Common clinical symptoms include redness, itching, tearing, and edema of the conjunctiva. When the same allergen contacts with the body in the sensitized state again, the allergen is combined with IgE on the sensitized Fc epsilon RI on the surface of the conjunctiva mast cell membrane, receptor cross-linking can trigger the degranulation of the conjunctiva mast cell, and further release of other intermedium can cause the formation of allergic conjunctivitis prophase phase reaction, which is mainly characterized by vasodilation, increased permeability of the vascular wall and pruritus, and the period lasts for 20-30 min. The chemokines released by mast cells initiate late phase reactions by inducing locally activated vascular endothelial cells to express new adhesion molecules, typically occurring 6-8 hours after allergen exposure, during which infiltration of various inflammatory cells, particularly eosinophils, is the major histological manifestation of the late phase reactions. Antigen-specific T cells initiate eosinophil mobilization to the conjunctiva, which in turn leads to tissue damage.

Although various drugs such as vasoconstrictors, local mast cell stabilizers, antihistamines, glucocorticoids, non-glucocorticoid anti-inflammatory drugs and the like are clinically selected for treating allergic conjunctivitis, no drug is completely satisfactory at present. Therefore, it is necessary to develop a novel anti-allergic conjunctivitis ophthalmic drug with better curative effect and less side effect. The evaluation of the curative effect of the new drug and the research of the pathophysiology of the allergic conjunctivitis require the establishment of an animal allergic conjunctivitis model.

Disclosure of Invention

The invention aims to provide a mouse allergic conjunctivitis model induced by ragweed pollen and a molding method, which have high molding rate and obvious allergic reaction.

In order to achieve the purpose, the invention adopts the technical scheme that:

a modeling method using a ragweed pollen-induced mouse allergic conjunctivitis model, comprising:

injecting the ragweed pollen tarsal joint injection into the mouse tarsal joint subcutaneously with an injection volume of 65-85 μ l, starting on the 10 th day after injection, and dropping the ragweed pollen eye drops to the right eye of the mouse, each 10-15 μ l, once a day for 4 times.

Preferably, the ragweed pollen tarsal joint injection is prepared by the following method: taking 2.0-3.0mg of ragweed pollen, adding 650 mu l of alum adjuvant, and mixing for use.

Preferably, the ragweed pollen eye drops are prepared by the following method: taking 15-20mg of ragweed pollen, dissolving in 100 mul PBS buffer solution, and preparing into 150-200mg/ml working solution.

Preferably, the injection is performed in both hind limbs when 65-85 μ l of the injection cannot be subcutaneously accommodated in the unilateral tarsal joint.

Preferably, the method further comprises adaptively feeding the mice for 3d before the molding.

Preferably, the mice are 6 w-age Balb/c mice, and the sex is not limited.

Preferably, model verification is performed after the last excitation.

More preferably, the model verification comprises: and completing the photographing and observation of the eye microscopic picture within 20min after the last excitation.

More preferably, the model verification further comprises: sampling is completed within 30min after the last excitation, blood is taken for death after the animals are anesthetized, the right eyes of the mice are taken out along with the eyelids, the mice are fixed by 10% formalin, the sections are embedded by normal paraffin, and the conjunctival morphology and the eye-shaped cell infiltration condition are observed by HE staining.

The invention also provides a mouse allergic conjunctivitis model obtained by the molding method.

The invention has the following beneficial effects:

1. the used mouse product is a common balb/c mouse, the price is lower, and the molding rate is more than 83 percent.

2. The eye drop excitation solvent selects the PBS buffer solution close to neutrality, so that the additional influence of the aluminum hydroxide adjuvant on eyes in the traditional method is reduced, and the pathogenesis is closer to clinic.

3. In contrast to other methods, the method (this method) which was triggered 10-13 days after the subcutaneous injection of the tarsal joint could observe the late phase reaction of allergic conjunctivitis by pathological section and HE staining.

Drawings

FIG. 1 shows a general observation of an embodiment of the present invention.

FIG. 2 shows the HE staining results of examples of the invention.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and the accompanying 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

First, preparation of experiment

Animals: 6 w-age Balb/c mice, male and female are not limited.

Reagents and consumables: ragweed pollen (RW, GRER, lot:314555), Alum adjuvant (Imject Alum, Thermo science, lot: WA313000), phosphate buffered solution (pH 7.4).

The preparation method of the main reagent comprises the following steps:

ragweed pollen tarsal joint injection: taking 2.0mg (2.0-3.0 mg) of ragweed pollen, adding 650 μ l of alum adjuvant, and mixing.

Ragweed pollen eye drops: dissolving 15mg (15-20mg all) of ragweed pollen in 100 μ l PBS buffer solution to obtain 150mg/ml working solution.

Second, molding operation method

And (3) carrying out animal adaptive feeding for 3d, starting to carry out molding, and injecting the prepared ragweed pollen tarsal joint injection into the subcutaneous tissues of the tarsal joints of the mice in a volume of 65 mu l (65-85 mu l), wherein if 65 mu l cannot be accommodated in the subcutaneous tissues of the single tarsal joints, the ragweed pollen tarsal joint injection can be injected into both hind limbs. Starting on day 10 after injection, the prepared ragweed pollen eye drops are dropped to the right eye of the mouse, 10 μ l each (10-15 μ l each) once a day for 4 times.

Third, model verification method

General observations were: after the last excitation, the eye microscopic photograph was taken and observed within 20 minutes, as shown in fig. 1, it can be seen that the eye secretion of the model group mouse is increased and the eye blood vessels are more full compared with the normal control group mouse.

And (3) model verification: sampling is completed within 30min after the last excitation, blood is taken to kill after the animals are anesthetized, the right eyes of the mice are taken out along with the eyelids, the mice are fixed by 10% formalin, the sections are embedded by normal paraffin, and the conjunctiva morphology and the eye-shaped cell infiltration condition are observed by HE staining, as shown in figure 2. HE staining results indicated inflammatory cell infiltration in the conjunctiva of 83.3% of mice and a change in conjunctival morphology, with edema thickening and hyperemia in the conjunctiva of 30% of mice.

In the replication process of a laboratory, the ovalbumin excitation method after injecting the ovalbumin into the abdominal cavity, the excitation method after injecting the ragweed pollen and the aluminum hydroxide adjuvant into the abdominal cavity and the eye drop excitation method after the ragweed pollen is insufflated into the nasal cavity for sensitization can not observe obvious eye phenomena and related pathological characteristics. Compared with the intraperitoneal injection and aerosol inhalation, the method for injecting the antigen to the tarsal joints of the feet of the mice can achieve better effect.

Fourth, application

The allergic conjunctivitis mouse model can be applied to the research and development of a medicine for treating conjunctivitis, and if a medicine research and development company prepares a certain medicine, the mouse model can finally detect whether the medicine is effective or not by designing different groups.

The group design is as follows:

1. normal control group (pure synchronous feeding)

2. Sham operation (and modeling with general basic operations, but not related to the operations leading to the disorder)

3. Model set (allergic conjunctivitis)

4. Positive drug combination (reference drug, effective treatment)

5. Negative drug group (administration generally regarded as non-therapeutic)

6. Vehicle and excipient group (the group has no or partial treatment effect)

7. Low dose group of test drugs

8. Medium dose group of test drugs

9. High dose group of test drugs

The sham, negative control and vehicle groups can be eliminated in some groups depending on the particular requirements of the customer and the nature of the model.

After the allergic conjunctivitis animal model is constructed according to the invention, the treatment scheme can be executed for the animal according to groups, and after the treatment scheme period is finished, whether the treatment medicine is effective or not is confirmed through pathology, biochemistry and other related detection.

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.