CN103566353A - Method for constructing asthmatic animal model - Google Patents
Method for constructing asthmatic animal model Download PDFInfo
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- CN103566353A CN103566353A CN201310541794.8A CN201310541794A CN103566353A CN 103566353 A CN103566353 A CN 103566353A CN 201310541794 A CN201310541794 A CN 201310541794A CN 103566353 A CN103566353 A CN 103566353A
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Abstract
The invention provides a method for constructing an asthmatic animal model. The method comprises the following steps: grinding 15-20g of fresh silkworm chrysalis into powder with liquid nitrogen; adding 25ml of PBS (phosphate buffer solution) to wash silkworm protein, and grinding the silkworm protein for 1-2 hours in a homogenizer; taking out the silkworm protein in the homogenizer, and centrifuging at 4 DEG C at 8,000rpm for 15 minutes; repeatedly centrifuging supernatant for three times; assaying whether protein concentration reaches standard or not by adopting a BCA (bicinchoninic acid) method to obtain the silkworm protein. The silkworm protein is applied to a Babl/c mouse at 5-6 weeks, injecting 50mu g of silkworm protein into a mouse in an intraperitoneal manner, and adding 4mg of aluminum hydroxide for sensitization once a week, and continuously performing intraperitoneal injection for three weeks; continuously exciting for 1 week (50mu g of silkworm crude protein is inhaled in a nasal manner for a mouse) after the last time of sensitization; performing airway hyperreactivity assay and pathological examination in 24 hours after nasal inhalation in the last day. According to the method, the problem of asthmatic animal model construction difficulty can be solved, and the animal model is stable and controllable and can be used for studying asthma pathogenesis and curative effect evaluation of asthma treatment medicaments.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to a kind of method that builds animal model in asthma.
Background technology
Allergic asthma is to have a strong impact on human health, and its sickness rate is every year with 5% speed increment.According to the estimation of World Health Organization, asthmatic patient reaches 300,000,000 more than in world wide, and feeds through in the crowd who comprises all ages and classes or different economic situations at all levels.In the past twenty years, as anaphylactic diseases such as asthma, be stable ascendant trend.This not only affects people's quality of life, particularly Infantile asthma, has directly affected the normal development of children's torso, also the development of national economy is caused to certain inhibition.
At present, both at home and abroad the research of allergic asthma is deepened day by day, but but relatively very few for the effective specific treatment means of allergic asthma.Now general allergic asthma animal model only has the mouse asthmatic model of chicken ovalbumin (OVA) induction in the world.Due to chicken ovalbumin be not one at the diseases induced anaphylactogen of human body, the result of study obtaining by this model, can not reflect the pathological state of asthma in human body well.Therefore, set up a kind of animal model that can simulate human body asthma pathological state, can be used for Claritin screening and research asthma pathogenesis have important scientific meaning and potential clinical value.
Although Adrenal Glucocorticoid remains relatively the medicine of effectively treatment and Polyglucan asthma attack, the side effects limit that it is larger clinical application.In view of directly carrying out the limitation of human trial, the determining of the relevant cause of disease, pathogenetic exploration, the evaluation of new treatment, the research and development of new drug all need to be undertaken by zoopery to a great extent.Therefore, animal model in asthma is of great significance for the research tool of asthma.
Summary of the invention
A kind of method that the object of this invention is to provide structure animal model in asthma with the protein induced formation of silkworm, stablizes animal model controlled, for the research of Pathogenesy of Asthma and the therapeutic evaluation for the treatment of asthma medicine.
Technical scheme of the present invention is: a kind of method that builds animal model in asthma, is characterized in that the protein induced formation of the method use silkworm.
The further technical scheme of the present invention is: by silkworm pupa 15-20 gram, add liquid nitrogen and be ground to and become powder, then the PBS pupa albumen that adds 25ml rinses, in homogenizer, grind 1-2h, take out the pupa albumen in homogenizer, after low-temperature centrifugation, get supernatant, obtain silkworm albumen.
The present invention further technical scheme is: silkworm pupa is fresh silkworm pupa.
The present invention further technical scheme is: after pupa albumen repeated centrifugation in homogenizer three times, get supernatant, obtain silkworm albumen.
The present invention is with the animal model in asthma of the protein induced formation of silkworm, efficiently solve the problem that builds animal model in asthma difficulty, compare with the mouse asthmatic model of existing chicken ovalbumin (OVA) induction, the present invention stablizes controlled with the animal model in asthma of the protein induced formation of silkworm, the pathological state that can reflect well asthma in human body, can be used for the research of Pathogenesy of Asthma and the therapeutic evaluation for the treatment of asthma medicine.
The specific embodiment
Below in conjunction with the specific embodiment, the detailed structure of invention is further described.
1, the preparation of silkworm albumen:
Get fresh silkworm pupa 15-20 gram, adding liquid nitrogen is ground to and becomes powder, the PBS pupa albumen that adds 25ml rinses, in homogenizer, grind 1-2h, take out the pupa albumen in homogenizer, 4 ℃ of centrifugal 8000rpm/15min, get supernatant, repeated centrifugation three times, it is up to standard that BCA method is surveyed protein concentration, obtains silkworm albumen.
2, modeling:
Application 5-6 Babl/c mice in age in week, a lumbar injection pupa albumen 50 μ g/ adding aluminum hydroxide 4 mg sensitization, weekly, lumbar injection is three weeks continuously.After last sensitization, continuous agitation one week (Pupa bombycis crude protein 50 μ g/ every day collunarium).Last exciting for 1 time after 24 h, carries out airway hyper-reaction detection and pathological examination.
3, the evaluation of model:
The animal model in asthma that the present invention builds be take and occurred that typical asthma sample outbreak, pathological tissue occur that inflammation cellular infiltration (comprising acidophil, lymphocyte, neutrophilic granulocyte, macrophage) is feature; Its clinical typical characteristic has: airway inflammation and airway hyperreactivity are main.
1. the reaction asthma mouse audible of model group after exciting and significantly stridulating, after sucking pupa albumen, there is obviously grabbing nose, dysphoria, and then there is tachypnea, and abdominal muscle is twitched, and extremity are weak and limp, bradykinesia, some appearance mouth breathings, and lie prostrate gradually together, after taking-up animal, leave over as seen more Excreta.There is not above-mentioned performance in matched group Mus, the accidental nose of grabbing, and keep normality always.
2. leukocyte differential count situation and matched group comparison in model group BALF, in model group BALF, neutrophil cell, lymphocyte, eosinophilic granulocyte's percentage ratio matched group obviously raise, and difference all has statistical significance (p<0.05).
3. the model group Mus lung tissue disease pathologic that changes of science shows that asthma group bronchus of rat mucosa injury is obvious, and bronchus, blood vessel around eosinophils significantly increase; Matched group bronchiolar epithelium is complete, and bronchus tube wall and blood vessel are around without eosinophils.
4. there is obvious airway hyper-reaction in model group.
Claims (5)
1. build a method for animal model in asthma, it is characterized in that the protein induced formation of the method use silkworm.
2. the method for structure animal model in asthma according to claim 1, it is characterized in that by silkworm pupa 15-20 gram, adding liquid nitrogen is ground to and becomes powder, the PBS pupa albumen that adds again 25ml rinses, in homogenizer, grind 1-2h, take out the pupa albumen in homogenizer, after low-temperature centrifugation, get supernatant, obtain silkworm albumen.
3. the method for structure animal model in asthma according to claim 1 and 2, is characterized in that silkworm pupa is fresh silkworm pupa.
4. the method for structure animal model in asthma according to claim 1 and 2, is characterized in that getting supernatant after pupa albumen repeated centrifugation in homogenizer three times, obtains silkworm albumen.
5. the method for structure animal model in asthma according to claim 3, is characterized in that getting supernatant after pupa albumen repeated centrifugation in homogenizer three times, obtains silkworm albumen.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201310541794.8A CN103566353A (en) | 2013-11-06 | 2013-11-06 | Method for constructing asthmatic animal model |
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| CN201310541794.8A CN103566353A (en) | 2013-11-06 | 2013-11-06 | Method for constructing asthmatic animal model |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210981A (en) * | 2015-09-15 | 2016-01-06 | 中国科学院生物物理研究所 | Set up method and the application thereof of the ferret model that can be applicable to human diseases research |
| CN111820188A (en) * | 2020-08-04 | 2020-10-27 | 中山大学附属第一医院 | Establishment method and application of asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model |
| CN115349497A (en) * | 2022-08-30 | 2022-11-18 | 江西中洪博元生物技术有限公司 | Pig roundworm asthma-induced mouse model and construction method thereof |
-
2013
- 2013-11-06 CN CN201310541794.8A patent/CN103566353A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105210981A (en) * | 2015-09-15 | 2016-01-06 | 中国科学院生物物理研究所 | Set up method and the application thereof of the ferret model that can be applicable to human diseases research |
| CN111820188A (en) * | 2020-08-04 | 2020-10-27 | 中山大学附属第一医院 | Establishment method and application of asthma-like slow obstructive pulmonary overlapping airway inflammation mouse model |
| CN115349497A (en) * | 2022-08-30 | 2022-11-18 | 江西中洪博元生物技术有限公司 | Pig roundworm asthma-induced mouse model and construction method thereof |
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Application publication date: 20140212 |