CN108668998A - Staphylococcal enterotoxin B aerosol lung delivers the preparation method of infecting mouse model - Google Patents
Staphylococcal enterotoxin B aerosol lung delivers the preparation method of infecting mouse model Download PDFInfo
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Classifications
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- 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
- A01K67/02—Breeding vertebrates
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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)
- Medicinal Preparation (AREA)
Abstract
The invention discloses the preparation methods that a kind of staphylococcal enterotoxin B aerosol lung delivers infecting mouse model.The method provided by the present invention for preparing staphylococcal enterotoxin B aerosol lung delivering infected animal model, includes the following steps：(1) staphylococcal enterotoxin B is delivered to the lung of animal by way of liquid aersol；(2) animal processed to step (1) carries out lipopolysaccharides sensitization again, obtains animal model.Transtracheal carries out lung delivering and SEB samples is directly reached lung's target organ, effect is direct, save sample, the present invention is using BALB/c mouse as model experiment animal, SEB aerosol lungs delivering infection model is successfully constructed, which is the ideal infection model studied SEB sucking pneumonia pathogenic mechanisms and screen medicine.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of staphylococcal enterotoxin B aerosol lung delivering infection is small
The preparation method of mouse model.
Background technology
Biological threats and its defence are one of most important national security concern problems in current countries in the world, and military doctor
Learn one of the key areas of research.Biological threats include mainly biological war and bio-terrorism, refer to utilizing biological agent (packet
Include war agent) closely related other organisms of deliberately living to specific objective crowd and with human being's production (such as animal, plant)
Start the behavior attacked with facility etc..Therefore, biological threats not only can largely kill people, animal, and caused by common people's psychology
Panic, social disturbances and economic loss etc. are all very huge.The essential element for constituting biological threats is biological agent, and is given birth to
It is most importantly strong pathogen and its toxic product (toxin) in agent.
Staphylococcal enterotoxin B (Staphylococcal enterotoxin B, SEB) is Staphylococcus aureus
A kind of exotoxin that bacterium generates, is important bacterial superantigen, a large amount of T cells can be activated under the conditions of very low concentrations, and stimulate
Cell factor is discharged, the poisoning symptoms such as humans and animals vomiting, diarrhea, abdominal pain can be caused, or even cause the irreversible lesion of body
Lead to death.The SEB of 1.7 μ g can kill an adult, be to be put into the United Nations《Biological Weapons Convention》It verifies clear
Single one of 11 kinds of biotoxins.
Toxic shock syndrome, TSS (TSS) is that SEB infects an important clinical symptoms, is not currently fully understood TSS's
Immune pathogenic mechanism, also lacks effective treatment means.It is that TSS is immunized in pathogenic mechanism research to lack ideal animal model
One key factor.SEB suckings can induce acute systemic lung inflammation.The researchs such as Neumann B. find mouse nasal cavity drop
Note SEB can induce mucous membrane and air flue recruits polymorphonuclear cell and monocyte, and along with IL-4 in bronchoalveolar lavage fluid and TNF α
Content increase.The exposure of low concentration SEB (20ng) mouth and nose can induce Eosinophilic's Airway inflammatory response and acidophil granules
Cell degranulation, and the collunarium of high concentration exposure (2000ng) causes neutrophil leucocyte airway inflammation, the airway destruction of duration,
Toxicogenic shock and death.These researchs use the mode of infection of mouth and nose exposure or collunarium, although to experimental animal hurtless measure,
Can not accurate quantification dosage, but sample is lost larger and is easy to happen sedimentation in exposure tower, and relevant instrument is set
Standby volume is big, expensive.
Invention content
The object of the present invention is to provide the preparation sides that a kind of staphylococcal enterotoxin B aerosol lung delivers infecting mouse model
Method.
In a first aspect, a kind of claimed staphylococcal enterotoxin B aerosol lung for preparing delivers infection animal mould
The method of type.
The method provided by the present invention for preparing staphylococcal enterotoxin B aerosol lung delivering infected animal model, specifically
It may include following steps：(1) staphylococcal enterotoxin B is delivered to the lung of animal by way of liquid aersol；(2) again
The animal processed to step (1) carries out lipopolysaccharides (LPS) sensitization, obtains animal model.
Further, the animal can be mouse, and such as BALB/c mouse is specific such as BALB/c female mices.
Further, the delivering amount of the staphylococcal enterotoxin B can be the staphylococcal enterotoxin B to the animal
0.5-2.5 times of half lethal dose (LD50).In the mouse experiment of the present invention, the staphylococcal enterotoxin B is passed
The amount of sending is specially that 0.2-1.0 μ g/ weight is 0.02kg.Such as every mouse 0.2-1.0 μ g (every mouse weight 0.02kg).
Further, in step (1), it is described staphylococcal enterotoxin B is delivered to by way of liquid aersol it is dynamic
The lung of object can be the lung that the solution of staphylococcal enterotoxin B is delivered to the animal by liquid aersol lung delivery apparatus
Portion.
In one embodiment of the invention, it specially under laryngoscope auxiliary, is passed after the animal holonarcosis with lung
The lung for sending device Penn-Century high-pressure sprayers that the solution of the staphylococcal enterotoxin B is delivered to the animal.
Wherein, staphylococcal enterotoxin B is concretely dissolved in containing 0.05- by the solution of the staphylococcal enterotoxin B
The solution obtained after in the physiological saline of 0.2% (such as 0.05%, % indicates volumn concentration) poloxamer.
Wherein, the poloxamer is PLURONICS F87, specially PLURONICS F87 (P5556-100ML, Sigma).
Correspondingly, it is 0.02kg that the delivering amount of the solution of the staphylococcal enterotoxin B, which can be 50 μ L/ weight,.Such as every
50 μ L (every mouse weight 0.02kg) of mouse.Wherein, 0.2-1.0 μ are contained in the solution of staphylococcal enterotoxin B described in 50 μ L
The staphylococcal enterotoxin B of g.
Further, in step (2), the animal processed to step (1) carries out lipopolysaccharides (LPS) sensitization as to institute
State animal intraperitoneal injection of LPS (LPS).
Further, to the dosage of the animal intraperitoneal injection of LPS (LPS), concretely 75 μ g/ weight are
0.02kg.Such as every 75 μ g (every mouse weight 0.02kg) of mouse.
Further, step (2) is that 1-4 hours (such as 4 hours) carry out after the completion of step (1).
Second aspect, animal model prepared by claimed method previously it is following it is any in application：
(A1) medicine or vaccine of staphylococcal enterotoxin B associated diseases are screened；
(A2) pathogenesis of staphylococcal enterotoxin B associated diseases is studied.
Wherein, the staphylococcal enterotoxin B associated diseases can be toxic shock syndrome, TSS (TSS), staphylococcus intestines poison
Plain B aspiration pneumonias etc..
The third aspect, it is dynamic that claimed one kind being used to prepare the delivering infection of staphylococcal enterotoxin B aerosol lung
The kit of object model.
The reagent provided by the present invention for being used to prepare staphylococcal enterotoxin B aerosol lung delivering infected animal model
Box can contain staphylococcal enterotoxin B, lipopolysaccharides (LPS) and liquid aersol lung delivery apparatus.
Wherein, the staphylococcal enterotoxin B can exist in the form of a solution.The solution is concretely by staphylococcus intestines
Toxin B is dissolved in containing 0.05-0.2% (such as 0.05%, % indicates volumn concentration) poloxamer (such as PLURONICS F87)
The solution obtained after in physiological saline.
The liquid aersol lung delivery apparatus can be lung delivery apparatus Penn-Century high-pressure sprayers.
Fourth aspect, the claimed kit are preparing the delivering sense of staphylococcal enterotoxin B aerosol lung
Contaminate the application in animal model.
Wherein, the animal can be mouse, and such as BALB/c mouse is specific such as BALB/c female mices.
Transtracheal carries out lung delivering and SEB samples is directly reached lung's target organ, and effect is direct, saves sample, the present invention
Using BALB/c mouse as model experiment animal, SEB aerosol lungs delivering infection model is successfully constructed, which is research SEB
It sucks pneumonia pathogenic mechanism and screens the ideal infection model of medicine.
Description of the drawings
Fig. 1 is SEB lungs delivering sucking infecting mouse dosis-mortality curve.
Fig. 2 is LPS sensitization BABL/c mouse, and mouse lung pathology after 6h is infected in the delivering sucking of row SEB lungs.
Fig. 3 is LPS sensitization BABL/c mouse, lung pathologies after the delivering sucking infection for 24 hours of row SEB lungs.
Fig. 4 is LPS sensitization BABL/c mouse, and serum and bronchoalveolar lavage fluid in different time are infected in the delivering sucking of row SEB lungs
(BALF) cytokine content in.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Experimental animal uses BALB/c mouse, female, 6-8 week old SPF grades, weight 20g or so, to purchase in following embodiments
From Beijing Vital River Experimental Animals Technology Co., Ltd..
The staphylococcal enterotoxin B (SEB) used in following embodiments derives from military medical research institute of Academy of Military Sciences.
The lipopolysaccharides (LPS) used in following embodiments is sigma Products, article No. L2880.
The PLURONICS F87 used in following embodiments is sigma Products, article No. P5556-100ML.
Embodiment 1, the structure of liquid aersol lung delivering sucking SEB infecting mouse models and identification
One, the preparation of liquid aersol SEB mother liquors
SEB freeze-dried powders are dissolved in the physiological saline containing 0.05% (volumn concentration) PLURONICS F87, final concentration
1mg/mL。
Two, the structure of liquid aersol lung delivering SEB infection (LPS sensitization) mouse models
This experiment attacks malicious mouse (biotechnology company of tonneau China is tieed up in BALB/c, Beijing) with lung route of delivery, is divided into SEB realities
Test group and physiological saline (containing 0.05% PLURONICS F87) control group.1% yellow Jackets anesthetized mice is first injected intraperitoneally, so
Row lung delivering sucking infection experiment afterwards, lung delivering sucking infection method are as follows：
SEB experimental groups：After mouse holonarcosis, under laryngoscope auxiliary, with lung delivery apparatus Penn-Century high pressures
SEB liquid (containing 0.05% PLURONICS F87) is delivered to mouse lung by sprayer；It is every 50 μ of mouse that lung, which delivers amount of liquid,
L.Mouse is put back into mouse cage, pay attention to it is warming, mouse generally anesthesia 3 hours or so revive.After infecting 4h, intraperitoneal injection of mice
LPS carries out sensitization, 75 μ g/, 100 μ L.
Physiological saline (containing 0.05% PLURONICS F87) control group：After mouse holonarcosis, under laryngoscope auxiliary, use
Saline fluid (containing 0.05% PLURONICS F87) is delivered to mouse by lung delivery apparatus Penn-Century high-pressure sprayers
Lung；It is every 50 μ L of mouse that lung, which delivers amount of liquid,；Mouse is put back into mouse cage, pay attention to it is warming, mouse generally anaesthetize 3 hours left sides
Right revival.After infecting 4h, intraperitoneal injection of mice LPS carries out sensitization, 75 μ g/, 100 μ L.
Three, it detects
1, the half lethal dose of SEB lungs delivering sucking infecting mouse is determined
6 dosage groups are set, SEB infective doses are respectively 0mg/kg (5 mouse), 0.0005mg/kg (5 mouse),
0.0025mg/kg (6 mouse), 0.0125mg/kg (10 mouse), 0.05mg/kg (8 mouse), 0.25mg/kg is (5 small
Mouse), it observes and records lung delivering sucking and infects mouse survival situation in 5 days, calculate every group of the death rate, draw the delivering of SEB lungs and inhale
Enter the dosis-mortality curve of infection.The results are shown in Figure 1, and the half that the delivering sucking infection of SEB lungs is calculated according to the curve causes
Dead dosage is LD50=0.02mg/kg.
2, zootomy and sample treatment
The half lethal dose of sucking infection is delivered according to the SEB lungs of calculating, and two SEB infective doses are set：0.2μg/
Only only (mouse weight is 20 grams or so) with 1 μ g/, respectively 0.5 times of LD50With 2.5 times of LD50.It infects 4h pneumoretroperitoneums and injects mouse
LPS carries out sensitization, 75 μ g/, 100 μ L.And the 6h after infecting SEB and for 24 hours respectively, serum, lung-douching fluid and internal organs are acquired,
It is respectively used to cytokine content measurement and Histopathological Studies.The specific method is as follows：
(1) serum is acquired：It plucks eyeball and takes blood, after being placed at room temperature for 2h, 3000rpm centrifuges 20min, draws supernatant, -80 DEG C of jellies
Deposit standby inspection.
(2) lung-douching fluid：Tracheostomize is inserted into 1mL pipettors tip of nesting 10 μ Ltip in tracheae, and will
400 μ L PBS liquid inject in lung cavity, then draw.3000rpm is centrifuged in 10min, draws supernatant, and -80 DEG C freeze standby inspection.
(3) tissue sample：Solution takes the lungs of different time points, is fixed, is sent with 10% formalin solution/PBS solution
Beijing Xuebang pathology company carries out organization embedding, slice, H＆E dyeing.
(4) cell factor ELISA assay methods (ExCell products, article No. are EM004-96 and EM008-96)：
1. taking out lath needed for experiment from having balanced into the hermetic bag of room temperature, extraction gets out sample, standard items and life
Object element antibody working solution.
2. dissolving standard items with sample diluting liquid, sample diluting liquid 2 times of gradient dilutions (1000pg/ml, 500pg/ are used in combination
ml,250pg/ml,125pg/ml,62.5pg/ml,31.25pg/ml,15.62pg/ml)。
3. blood serum sample, lung-douching fluid sample after being diluted with sample diluting liquid, take 20 μ l to be added in sample well and are tried with 20 μ l
Dilution agent liquid mixes；40 μ l standard dilutions are added in standard items.Then 50 μ l biotinylated antibody working solutions are added per hole,
Reacting hole is sealed with sealing plate glue.
4. incubation at room temperature 120 minutes prepares enzyme conjugates working solution in 30 minutes in advance, room temperature is kept in dark place.
5. board-washing 5 times is added 100 holes μ l/ of enzyme conjugates working solution, reacting hole is sealed with glue sealing board.
6. being incubated at room temperature board-washing 5 times after sixty minutes.
7. 100 holes μ l/ of chromogenic substrate are added, room temperature is protected from light incubation 15 minutes.
8. 100 holes μ l/ of terminate liquid are added, OD450 is measured after mixing, OD630 is used in combination to correct wavelength.
(5) tissue pathological slice, colouring method：
A. film-making process
Tissue samples, materials 3mm is thick, gradient alcohol dehydration 70%, 80%, 95%, 100% each 30 minutes, dimethylbenzene two
Each 20 minutes of bottle, each 12 minutes of two cylinder of paraffin waxdip, embedding are sliced 4 microns, bake piece.
B. it dyes
It dyes in haematoxylin Yihong (hematoxylin eosin, HE)：(1) it dewaxes, three bottles of every bottle of dimethylbenzene dewax 8 minutes；
Two bottle of 100% every bottle of alcohol 8 minutes；Each 8 minutes of 90% alcohol, 80% alcohol, 60% alcohol.(2) haematoxylin dyeing 4 minutes,
Flowing water cleans；(3) hydrochloride alcohol breaks up 2-3 seconds, flowing water cleaning；(4) 0.5% ammonium hydroxide 20 seconds, flowing water cleaning, upper sem observation.(5)
0.5% eosin stains 1 minute；(6) 80% alcohol, 90% alcohol respectively break up 3-5 seconds；95% alcohol 5 minutes；Three bottle of 100% wine
Each 5 minutes of essence；Each 5 minutes of two bottles of dimethylbenzene.(7) resinene glue sealing；Om observation and microphotograph.
3, SEB lungs delivering sucking infection pathological examination
As shown in Figure 2：After SEB lungs delivering sucking infection 6h, 0.05% poloxamer of control group, alveolar structure is substantially just
Often；0.2 μ g SEB/0.05% poloxamer groups, local pulmonary bubble out existing inflammatory cell infiltration and alveolar hyperemia phenomenon；1μg SEB/
There is diffusivity bleeding in 0.05% poloxamer group, alveolar.
As shown in Figure 3：After the delivering sucking infection for 24 hours of SEB lungs, 0.05% poloxamer of control group, alveolar structure is substantially just
Often；There is inflammatory cell infiltration, congested and alveolar wall thickening phenomenon in 0.2 μ g SEB/0.05% poloxamer groups, alveolar；1μg
SEB/0.05% poloxamer groups, alveolar wall seriously thicken, and alveolar becomes smaller, and alveolar is largely congested, and it is thin that a large amount of inflammation occurs in alveolar
Born of the same parents.
4, SEB lungs delivering sucking infection inflammation is because of subcase
TNF α, IL-6 are the relevant important inflammatory factors of SEB Induced By Exposures shock, and sucking infecting mouse is delivered to SEB lungs
The mice serum and lung-douching fluid of different time have carried out the measurement of Cellular inflammatory factor content.As shown in Figure 4：After SEB exposures
Systemic inflammatory responses are quickly induced, but substantially return to level before SEB exposures for 24 hours, it was demonstrated that SEB lungs delivering sucking infection-induced
Serious Acute pulmonary inflammation.
It is above-mentioned the experimental results showed that SEB lungs delivering sucking infection can induce chmice acute lung inflammation reaction, the mouse
Model can be used for the evaluation study of SEB vaccines or medicine.
The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is above-mentioned
Embodiment.To those skilled in the art, the present invention can be carried out under the premise of without prejudice to inventive spirit etc.
Same modifications and substitutions, these equivalent modifications and substitutions are all contained in the application claim limited range.
Claims (10)
1. a kind of method preparing staphylococcal enterotoxin B aerosol lung delivering infected animal model, includes the following steps：(1)
Staphylococcal enterotoxin B is delivered to the lung of animal by way of liquid aersol；(2) processed to step (1) again
The animal carries out lipopolysaccharides sensitization, obtains animal model.
2. according to the method described in claim 1, it is characterized in that：The animal is mouse.
3. method according to claim 1 or 2, it is characterised in that：The delivering amount of the staphylococcal enterotoxin B is described
0.5-2.5 times to the half lethal dose of the animal of staphylococcal enterotoxin B.
4. according to any method in claim 1-3, it is characterised in that：The delivering amount of the staphylococcal enterotoxin B
It is 0.02kg for 0.2-1.0 μ g/ weight.
5. method according to any one of claims 1-4, it is characterised in that：It is described to pass through liquid aersol in step (1)
Mode by staphylococcal enterotoxin B be delivered to animal lung be by the solution of staphylococcal enterotoxin B it is molten by liquid gas
Glue lung delivery apparatus is delivered to the lung of the animal；
And/or
In step (2), it is more to animal intraperitoneal injection fat that the animal processed to step (1), which carries out lipopolysaccharides sensitization,
Sugar；
And/or
Step (2) is to carry out for 1-4 hours after the completion of step (1).
6. according to the method described in claim 5, it is characterized in that：The solution of the staphylococcal enterotoxin B is by grape ball
The solution that bacterium enterotoxin B obtains after being dissolved in the physiological saline containing the poloxamer that volumn concentration is 0.05-0.2%；
And/or
Dosage to the animal intraperitoneal injection of LPS is that 75 μ g/ weight are 0.02kg.
7. according to the method described in claim 6, it is characterized in that：The poloxamer is PLURONICS F87.
8. the animal model that in claim 1-7 prepared by any the method it is following it is any in application：
(A1) medicine or vaccine of staphylococcal enterotoxin B associated diseases are screened；
(A2) pathogenesis of staphylococcal enterotoxin B associated diseases is studied.
9. a kind of kit being used to prepare staphylococcal enterotoxin B aerosol lung delivering infected animal model, contains grape ball
Bacterium enterotoxin B, lipopolysaccharides and liquid aersol lung delivery apparatus.
10. the kit described in claim 9 is in preparing staphylococcal enterotoxin B aerosol lung delivering infected animal model
Using.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109939221A (en) * | 2019-04-04 | 2019-06-28 | 中国人民解放军军事科学院军事医学研究院 | A method for establishing an animal model of acute lung injury caused by ricin |
| CN111616106A (en) * | 2020-06-23 | 2020-09-04 | 成都欧林生物科技股份有限公司 | A method for establishing an animal model of fracture-related Staphylococcus aureus infection |
| CN112970670A (en) * | 2021-02-06 | 2021-06-18 | 澎立生物医药技术（上海）有限公司 | Construction method of intestinal mucositis animal model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102101062A (en) * | 2010-11-26 | 2011-06-22 | 中国人民解放军军事医学科学院微生物流行病研究所 | Aerosol microenvironment enclosed cabin for aerosol experiment |
| CN107753144A (en) * | 2016-08-18 | 2018-03-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | Hand-held liquid aerosol lung delivery apparatus |
-
2018
- 2018-04-20 CN CN201810358553.2A patent/CN108668998A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102101062A (en) * | 2010-11-26 | 2011-06-22 | 中国人民解放军军事医学科学院微生物流行病研究所 | Aerosol microenvironment enclosed cabin for aerosol experiment |
| CN107753144A (en) * | 2016-08-18 | 2018-03-06 | 中国人民解放军军事医学科学院微生物流行病研究所 | Hand-held liquid aerosol lung delivery apparatus |
Non-Patent Citations (3)
| Title |
|---|
| 吕宝谱: "不同剂量胸腺肤-ａ1在肠毒素B(SEB)与内毒素(LPS)复合诱导小鼠腺毒症模型中的免疫干预作用研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
| 李才: "《疾病模型与实验病理学》", 31 December 2002, 吉林大学出版社 * |
| 温占波: "分枝杆菌噬菌体D29气溶胶特性和动物气溶胶暴露系统的初步研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109939221A (en) * | 2019-04-04 | 2019-06-28 | 中国人民解放军军事科学院军事医学研究院 | A method for establishing an animal model of acute lung injury caused by ricin |
| CN111616106A (en) * | 2020-06-23 | 2020-09-04 | 成都欧林生物科技股份有限公司 | A method for establishing an animal model of fracture-related Staphylococcus aureus infection |
| CN112970670A (en) * | 2021-02-06 | 2021-06-18 | 澎立生物医药技术（上海）有限公司 | Construction method of intestinal mucositis animal model |
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