CN106442960B - The method for evaluating injury of lungs therapeutic agent and derivant with zebra air bladder damage inflammatory model - Google Patents
The method for evaluating injury of lungs therapeutic agent and derivant with zebra air bladder damage inflammatory model Download PDFInfo
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Abstract
The present invention provides the method for building up that zebra air bladder damages inflammatory model, and the method for application the zebra air bladder damage inflammatory model evaluation injury of lungs therapeutic agent and derivant effect.Zebra air bladder provided by the invention damages inflammatory model, has many advantages, such as easy, quick, economical, efficient, high-throughput；The truth of injury of lungs therapy apparatus or derivant in vivo can be accurately reflected, it may be implemented in the effect of high-throughput evaluation drug is to treatment acute lung injury inflammation in vivo, its method for evaluating efficacy of drugs has many advantages, such as reliable, quick, efficient, cheap, high performance-price ratio, significant.
Description
Technical field
The invention belongs to the technical fields of drug evaluation and screening, and in particular to a kind of easy, economic, high-throughput zebra
Air bladder damages the application of inflammatory model, especially with zebra fish model evaluation injury of lungs therapeutic agent effect and derivant toxicity
Method.
Background technology
Acute and chronic lung disease, especially injury of lungs, lung inflammation, lung tumors be seriously endanger people come healthy disease it
One.Airborne fine particulate matter (PM2.5), smoking, radiation, the physical chemical factors such as chemotherapy cause acute and chronic injury of lungs and lung cancer
Risk factor, wherein PM2.5The chronic obstructive injury of lungs caused by reasons such as/smoking be it is a kind of it is not fully reversible, to harmful
Grain and gas generate abnormal inflammatory response, air-flow restrictive respiration systemic disease, can increase lung cancer generation risk, now at
For one of higher disease of incidence.The World Health Organization (World Health Organization, WHO) report point out,
In global range, air pollution results in the generation of 5% tracheocarcinoma, bronchiolar carcinoma and lung cancer, 2% heart death rate, with
And 1% infection in respiratory system the death rate, be equivalent to 800,000 people's death and 7,900,000 people's injury of lungs.PM2.5Adsorption is a large amount of
Poisonous and harmful substance can be deposited on alveolar by breathing, cause injury of lungs.The World Health Organization, Environmental Protection Agency etc.
Many mechanisms have announced PM2.5It is maximum to human health damage and representative strongest atmosphere pollution.Injury of lungs at present
Treatment mainly has：Ventilation oxygen-supplying selects antibiotic, glucocorticoid, exogenous pulmonary surfactant, vasodilator, phase
Close enzyme etc..But existing injury of lungs medicine there is or unsatisfactory curative effect or the problems such as larger or costly toxic side effect, because
This develops new injury of lungs medicine and is very necessary[1-2]。
Drug screening is to find, develop an important link in pharmaceutical procedures, tests the foundation of model of lung injury to commenting
Valence and screening injury of lungs medicine are most important.Injury of lungs animal model is mainly used to the mechanism of research injury of lungs at present,
But most of these animal models are replicated according to the known risk factor for inducing injury of lungs, such as septicopyemia, secondary
In the fat embolism of fracture, sucking acidic materials, the ischemia-reperfusion of lung or tail vein bed and other clinical risks because
Element, but all these models are impossible to cmpletely replicate the feature of mankind's injury of lungs.Ideal lung injury experiment animal mould
Type should be able to replicate the mechanism and consequence of the generation of mankind's injury of lungs.But none of experimental animal model can so far
To replicate all features of mankind's injury of lungs completely, most lung injury experiment animal model is just for the single of mankind's injury of lungs
Or a few pathophysiological features is replicated, such as ventilatory exception, lung compliance decline, pulmonary parenchyma injury and alveolar hair
Thin blood vessel membrane permeability increase etc., and there is modeling complicated for operation, technology requirement height, operation wound in these animal models
Greatly, the shortcomings of injury of lungs incidence is low, complication and the death rate are high, model foundation is unstable, repeatable difference[3-4], it is unfavorable for
The evaluation of follow-up injury of lungs medicine and screening operation, and experimental period is long, costly, heavy workload.In vitro cell model
Lack drug in biological whole metabolic conversion and internal loop distribution, cannot reflect the truth of drug in vivo.Cause
This establishes a kind of good process of aids drug in vivo of energy, and can quickly and easily evaluate and screening injury of lungs medicine
Animal model have important application value.
Alveolar is more sensitive to the attack of extraneous pathogenic bacteria as a basic unit[5], bacterium and virus are to alveolar sense
It contaminates and can cause ALI/ARDS diseases into the particle in the pollution air of alveolar[6-8], and after alveolar is infected, it can cause
The aggregation of neutrophil leucocyte, subsequent neutrophil leucocyte can discharge the substances such as granule protein or reactive oxygen species (ROS)[9], these substances
The process of ALI/ARDS can be further speeded up.The study found that in animal acute lung injury disease model, by interfering neutral grain
The behaviors such as cell chemotaxis, migration, adherency and cell exudation, can alleviate the disease progression of animal[10-12].Acute lung is damaged at present
Though the research of wound has the history of decades, the mechanism infiltrated in acute lung injury process for neutrophil leucocyte is still unknown
Really, but to a certain extent, being primarily due to animal model can not be provided for researcher to alveolar neutrophil leucocyte behavior progress
The convenience of dynamic observation[13]。
Zebra fish is a kind of novel vertebra model animal, is up to 85% with human gene homology, signal transduction is logical
Road is substantially approximate with the mankind, and biological structure and physiological function are similar to mammal height, and (microwell plate point can be used with small
Analysis), growth cycle short, in vitro fertilization, transparent (can directly with the naked eye be observed with disecting microscope), the spies such as single laying is higher
Point[14].Zebra fish model not only has the advantages such as experiment in vitro is quick, efficient, cheap, dosage is small, but also has mammal
The advantages that experiment is predictive strong, observable high than degree multiple organs, obtains in compound drug effect, toxicity assessment in recent years
Extensive use[15,16]。
Vitals of the air bladder as fish buoyancy adjustment are largely studied in anatomy[20], morphology[21]With
Transcription group[22]Equal different levels confirm that the lung with terrestrial animal is homologous organs.2013 and 2014, Cass and
Robertson etc. detects pulmonary surfactant on zebra air bladder respectively[21,23]；Although it has been reported that spot
Horse air bladder can be applied to the basic research of some human diseases, such as 2013, and Gratacap et al. is by studying zebra fish
To the reaction after fish glue monilial infection air bladder, the interaction between Candida albicans, epithelial cell and immunocyte three has been probed into
Relationship；Voelz in 2015 et al., to the ventricles of the brain after mucor spore infection zebra fish and the reaction after fish glue, is explored by zebra fish
The reciprocation of mucor spore and body immune system；However, so far, zebra air bladder is used to carry out lung as animal model
There is not been reported for the pertinent literature of disease research.Therefore the method for evaluating injury of lungs therapeutic agent and derivant at present, still it is based on
Traditional experimental animal model (such as muroid, canine) is commented there is not yet carrying out drug based on zebra air bladder damage inflammatory model
The document report of valence.
Invention content
It is an object of the invention to：A kind of method for building up of zebra air bladder damage inflammatory model is provided, while one kind being provided
Utilize the method for the model evaluation injury of lungs therapeutic agent and derivant effect.Method provided by the invention has easy, quick, warp
The advantages that helping, be efficient, is high-throughput.
For achieving the above object, this invention takes following technical schemes：
Content one：A kind of method for building up of zebra air bladder damage inflammatory model, includes the following steps：
(1) determination of zebra fish best stage of development
4~5 pairs of neutrophil leucocyte green fluorescent transgenic strain zebra fish (MPO) parent mating are taken, according to
Westerfield[26]Method hatched blastocyst；The zebra fish of selection different developmental phases, which is placed under disecting microscope, to be observed, and is chosen
It takes normotrophic zebra fish to move into 6,12,24,48,96 or 384 hole microwell plates, different number is put into according to microwell plate specification
Zebra fish；
Corresponding microwell plate is set as 3 experimental groups according to the different stages of development：1 is derivant processing group, should
The derivant of single concentration is injected in group to zebra air bladder chamber；1 is solvent processing group, to bodies such as zebra air bladder chamber injections in the group
Long-pending PBS；1 is blank control group, to zebra fish without any processing in the group；Then, each experimental group is added identical
The fish culture water of volume, at the same temperature constant temperature incubation；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and protected with three-dimensional fluorescence microscope
It deposits, qualitative and quantitative assessment is carried out to the neutrophil leucocyte quantity of zebra air bladder position aggregation, determines the best development of zebra fish
Stage；
The zebra fish of the different developmental phases is the zebra fish of 4.5~6dpf；The zebra fish of the best stage of development
For the zebra fish of 5dpf；
(2) determination of the best administration concentration of derivant
The zebra fish of 5dpf is taken to move into microwell plate；
The derivant processing group of blank control group, solvent injection group and different dosing concentration is set：Blank control group is to spot
Horse fish injects PBS without any processing, solvent injection group to zebra air bladder chamber, and derivant processing group distinguishes zebra air bladder chamber
Inject isometric but different dosing concentration derivant；Then, the breeding water of same volume is added in each experimental group, identical
At a temperature of constant temperature incubation；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and protected with three-dimensional fluorescence microscope
It deposits, qualitative and quantitative assessment is carried out to the neutrophil leucocyte quantity of zebra air bladder position aggregation, determines the best administration of derivant
Concentration；
The administration concentration of the derivant is 1~10mg/mL, and the best administration concentration of the derivant is 10mg/mL；
(3) structure zebra air bladder damages inflammatory model
The zebra fish of 5dpf is taken to move into microwell plate；
3 experimental groups are set：1 is derivant processing group, injects the induction of 10mg/mL in the group to zebra air bladder chamber
Agent, 1 is solvent injection group, and isometric PBS is injected to zebra air bladder chamber in the group；Then, each experimental group is added identical
The breeding water of volume, at the same temperature constant temperature incubation；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and protected with three-dimensional fluorescence microscope
It deposits, for statistical analysis to the neutrophil leucocyte quantity of zebra air bladder position aggregation, according to statistical result, judgement has been established
Zebra air bladder damages inflammatory model.
Preferably, fish culture water condition is：Dissolved oxygen mass concentration is 6-8mg/L, and water temperature is 28 DEG C, pH 7.2-7.6,
Total hardness is 200-250mg/L.
Preferably, constant temperature incubation condition is：Microwell plate is placed in constant incubator, and 4h is cultivated at 28 DEG C.
Content two：A method of using zebra air bladder damage inflammatory model, evaluation injury of lungs derivant toxicity, including such as
Lower step：
(1) zebra fish is chosen
Zebra fish in 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into microwell plate
In, the zebra fish of different number is put into according to microwell plate specification；Specification can be 6,12,24,48,96 or 384 holes.
(2) derivant is handled
Experimental group is set：The compound processing group of 1 solvent control group, 1 blank control group, different injection dosages；It is empty
White control group is to zebra fish without any processing, and solvent injection group injects PBS to zebra air bladder chamber, and compound processing group is to spot
Horse air bladder chamber injects the derivant of 0.1,10,100,500 and 1000ng respectively；Then, same volume is added in each experimental group
Breeding water, at the same temperature constant temperature incubation；
(3) qualitative and quantitative assessment is carried out to fish glue position neutrophil accumulation quantity
After derivant is handled to experimental endpoints, is taken pictures and preserved with three-dimensional fluorescence microscope；Observation compares each experimental group
Zebra air bladder position neutrophil accumulation, the fish glue damage of qualitative evaluation injury of lungs derivant induction；And it acquires, count zebra fish
Neutrophil leucocyte quantity, fish glue degree of injury caused by quantitative assessment injury of lungs derivant, fish glue degree of injury are induced times with fish glue inflammation
Number indicates that calculation formula (b) is：
Fish glue damages inflammation and induces multiple=(NCompound group-NSolvent control group)/NSolvent control group
(4) toxic degree of statistical analysis injury of lungs derivant
Statistical procedures result withIt indicates, more comparison among groups use variance analysis, two comparison among groups to use
Dunnett ' s T-, which are examined, carries out statistical procedures, p<0.05 is notable for otherness.According to statistical result, evaluation injury of lungs lures
It leads fish glue caused by agent and damages toxic degree.
Preferably, derivant is the drug or chemical reagent that can induce injury of lungs, including but not limited to LPS, Poly I/
C。
Content three：A method of using zebra air bladder damage inflammatory model, evaluation injury of lungs therapeutic agent effect, including such as
Lower step：
(1) zebra fish is chosen
Zebra fish in 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into microwell plate
In, the zebra fish of different number is put into according to microwell plate specification；Specification can be 6,12,24,48,96 or 384 holes.
(2) derivant is handled
Blank control group is without any processing to zebra fish, and solvent control group is other to zebra air bladder chamber injection 5nL PBS
The equal fish glue chamber of group zebra fish injects 50ng derivants；
(3) injury of lungs therapeutic agent is handled
Experimental group is set：1 blank control group, 1 solvent control group, 1 model control group, the compound of various concentration
Processing group；Zebra fish gives 0.1 μM, 1 μM, 10 μM, 100 μM, 1000 μM in a manner of water-soluble or injection in compound processing group
Therapeutic agent；Model control group, solvent control group and blank control group zebra fish are without any processing；Then, each experimental group is equal
The breeding water of same volume is added, at the same temperature constant temperature incubation；
(4) qualitative and quantitative assessment is carried out to fish glue position neutrophil accumulation quantity
After therapeutic agent is handled to experimental endpoints, is taken pictures and preserved with three-dimensional fluorescence microscope；Observation compares each experimental group
Zebra air bladder position neutrophil accumulation, the anti-inflammatory effect of qualitative evaluation injury of lungs therapeutic agent；And it acquires, count in zebra fish
Property granulocyte quantity, the anti-inflammatory effect of quantitative assessment injury of lungs therapeutic agent, calculation formula (a) are：
Anti-inflammatory effect (%)=(NModel control group-NCompound processing group)/NModel control group× 100%；
(5) therapeutic efficiency of statistical analysis injury of lungs therapeutic agent
Statistical procedures result withIt indicates, more comparison among groups use variance analysis, two comparison among groups to use
Dunnett ' s T-, which are examined, carries out statistical procedures, p<0.05 is notable for otherness.According to statistical result, evaluation injury of lungs is controlled
Treat the therapeutic efficiency of agent.
Preferably, therapeutic agent is the drug or chemical reagent that can treat injury of lungs, including but not limited to PD98059,
LY294002、sp600125、PHPS1。
Compared with prior art, the zebra air bladder that the present invention establishes damages inflammatory model, has the following advantages that：
(1) in vivo：Experiment material is zebra fish, and as a kind of vertebrate, screening model category In vivo model can
The absorption,distribution,metabolism,excretion of true reflection drug in vivo, really reflects the whole bioactivity of drug.
(2) intuitive：Experiment material be neutrophil leucocyte green fluorescent transgenic zebra fish, zebra fish after drug-treated,
It, can be with hurtless measure in the behavior of any time point realtime dynamic observation neutrophil leucocyte of experimental setup by fluorescence microscope
Feature；And mammal may not complete this operation.
(3) high-throughput：Zebra fish juvenile fish very little, only 1-4 millimeters, can the 6 of a standard, 12,24,48,96 or
Analysis and experimental period are carried out in 384 orifice plates short to be made zebra fish become a kind of to carry out high throughput automated internal drug sensitization
The ideal model of evaluation.
(4) economical：Required expense is low, is expended daily as the screening experiment every of experimental vehicle using canine and is more than 10 dollars,
It is expended daily as the screening experiment every of experimental vehicle using cavy and is more than 1 dollar, and it is real by the screening of experimental vehicle of zebra fish
It tests every and expends daily and be less than 0.01 dollar.
(5) compound amount is few：Detection compound dosage is few, usually only needs several milligrams, and traditional screening experiment then needs
Several milligrams or more of compound.
(6) easy：Experimentation is easy to operate, and zebra fish is after drug-treated, you can quantify, analyzes the drug
Sensitization, and conventional animal experimental implementation process is complicated, judge index is subjective, easy tos produce false positive results.
(7) quickly：Experimental period is short, can be completed in 1 day；And cavy often needs the time of several weeks to several months, dog often needs
Want the time of several months to several years.Zebra fish completes embryonic development within 72 hours at first.Most internals, including the heart
Vascular system, intestines, liver and kidney, the rapid shaping in 24-48 hours, traditional experimental vehicle mouse and monkey are then respectively necessary for
It can complete embryonic development within 21 days and 9 months.
(8) reliable predictive：The gene of zebra fish and the similarity of human gene are up to 85% or so, biology work(
Can be similar to mammal and mankind's height, experimental result comparativity is strong, predictive good.
(9) stability is high, reproducible：The present invention repeats experiment ten several times, and obtained experimental result is essentially identical.
Compared with prior art, evaluation injury of lungs therapeutic agent and the induction of inflammatory model are damaged the present invention is based on zebra air bladder
The method of agent, has the following advantages that：
(1) injury of lungs animal model is mainly used to study the mechanism of injury of lungs, but most of these animal models at present
It is to be replicated according to the known risk factor for inducing injury of lungs, and these animal models exist that modeling is complicated for operation, skill
Art requirement is high, operation wound is big, injury of lungs incidence is low, complication and the death rate are high, model foundation is unstable, repeatable difference
The shortcomings of, it is unfavorable for evaluation and the screening operation of follow-up injury of lungs medicine, and experimental period is long, costly, heavy workload；
And In vitro cell model lacks drug in biological whole metabolic conversion and internal loop distribution, cannot reflect drug in vivo
Truth.
(2) in the present invention, inventor provides a kind of profit on the basis of establishing zebra air bladder damage inflammatory model
With the method for model evaluation the injury of lungs derivant toxicity and therapeutic agent effect, this is a kind of completely new drug evaluation model and comment
Valence method can accurately reflect the truth of drug in vivo, it can be achieved that high-throughput evaluation drug is to treating acute lung in vivo
The effect of damaging inflammation, has many advantages, such as reliable, quick, efficient, cheap, high performance-price ratio.
(3) evaluation method of the invention can be not only used for the poison that evaluation discloses drug (as is known derivant, therapeutic agent)
Property or effect, it may also be used for screening have undisclosed drug (compound that such as structure is completely new, drug effect is unknown, be usually used in first,
The new drug development of me-better, me-too etc.) toxicity or effect, be widely used, it is significant.
Description of the drawings
Fig. 1：Image qualitative evaluation determines the zebra fish best stage of development
Fig. 2：LPS damages inflammatory reaction quantitative assessment to the fish glue that different phase zebra fish induces
Fig. 3：Pass through the qualitative determining zebra air bladder inflammatory model of image analysis
Fig. 4：The fish glue of the LPS of dosage and time dependence inductions damages inflammatory reaction quantitative analysis
Fig. 5：Zebra air bladder om observation
Fig. 6：Zebra air bladder Electronic Speculum observes epithelial cell damage
Fig. 7：Zebra air bladder Electronic Speculum observes injury of mitochondria
Fig. 8：LPS influences of the different time points to LI-1 β expressions after injection
Fig. 9：LPS influences of the different time points to LI-6 expressions after injection
Figure 10：Inflammatory model is damaged by the qualitative determining zebra air bladder of image analysis
Figure 11：The fish glue of 50ng LPS inductions damages inflammation quantitative analysis
Figure 12：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining PD98059 of image analysis
Figure 13：PD98059 significantly inhibits the zebra air bladder damage inflammatory reaction of LPS inductions
Figure 14：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining LY294002 of image analysis
Figure 15：LY294002 significantly inhibits the zebra air bladder inflammatory reaction of LPS inductions
Figure 16：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining sp600125 of image analysis
Figure 17：Sp600125 significantly inhibits the zebra air bladder inflammatory reaction of LPS inductions
Figure 18：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining PHPS1 of image analysis
Figure 19：PHPS1 significantly inhibits the zebra air bladder damage inflammatory reaction of LPS inductions
Figure 20：Zebra air bladder is caused to damage inflammatory reaction by the qualitative determining Poly I/C of image analysis
Figure 21：Poly I/C can induce zebra air bladder inflammatory reaction
Specific implementation mode
The following is specific embodiments of the present invention, is further described to technical scheme of the present invention, but of the invention
Protection domain be not limited to these examples.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection domain.
Embodiment 1 determines the zebra fish best stage of development
1 zebra fish is chosen
4~5 pairs of neutrophil leucocyte green fluorescent transgenic strain zebra fish (MPO) parent mating are taken, according to
Westerfield[26]Method hatched blastocyst.When zebra fish development is to 4.5dpf, preceding fish glue has been inflated to be formed.By 4.5dpf,
The zebra fish of 5dpf, 6dpf are placed under disecting microscope and observe, and the normotrophic zebra fish of picking moves into three 6 orifice plates respectively
In, per 30 tail of hole.(note：Dpf=day post fertilization in the present invention, Chinese refer to that zebra fish will be fertilized the day after tomorrow
Number, as 5dpf refers to zebra fish after fertilization five days.)
2 derivant LPS processing
The 3 experimental groups zebra fish of 4.5dpf, 5dpf, 6dpf (every group be respectively) are set, and each experimental group is lured including 1
Lead agent processing group, 1 solvent control group and 1 blank control group.Zebra air bladder chamber injection 50ng LPS in derivant processing group；
Zebra air bladder chamber injects isometric PBS (i.e. PBS and LPS equivalent) in solvent control group；Zebra fish is not appointed in blank control group
It manages where；The breeding water of equal volume is added according to the specification of microwell plate for each experimental group, and microwell plate is placed in 28 DEG C
4h is cultivated in constant incubator.
3 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS processing zebra fish to experimental endpoints, a processing group zebra fish is clapped using three-dimensional fluorescence microscope
According to and preserve.On the one hand neutrophil accumulation degree qualitative determining zebra in each experimental group zebra air bladder position is compared by observation
The fish best stage of development (see Fig. 1) is on the other hand carried out using Nikon NIS-Elements D3.10 high vision processing softwares
Image analysis acquires and counts zebra air bladder position neutrophil leucocyte quantity, and fish glue damages journey caused by quantitative assessment derivant LPS
Degree, fish glue degree of injury calculation formula are shown in (b).
Blank control group zebra air bladder position neutrophil leucocyte quantity does not have substantially.With the increase at zebra fish age, lure
It leads the neutrophil leucocyte quantity presentation of agent LPS processing group zebra air bladders position and first increases the trend reduced afterwards, 5dpf zebra air bladders damage
It is maximum (see Fig. 2) to hinder degree.
Statistical procedures result is shown：The zebra air bladder damage ratio of 4.5dpf, 5dpf, 6dpf be respectively (69.97 ±
3.69) %, (45.18 ± 2.98) %, (30.39 ± 1.23) % (see Fig. 2), this shows the increase with the zebra fish age, fish glue
Damage ratio presentation first increases the trend reduced afterwards, the zebra air bladder damage ratio highest of 5dpf.Pass through variance analysis, derivant processing
The fish glue damage ratio of group is apparently higher than solvent control group, the statistically significant (p of difference<0.05)；The zebra fish of 5dpf and 4.5dpf
It is compared with the zebra fish myelin damage rate of 6dpf, p<0.05.
Fig. 1：Image qualitative evaluation determines the zebra fish best stage of development
Wherein, Control is blank control group, and Vehicle is solvent control group；5dpf zebra fish is handled through derivant
Afterwards, fish glue chamber neutrophil accumulation is most intensive, and it is the fiercest to induce intensity, when 4.5dpf and 6dpf, neutrophil accumulation phase
To sparse, induction intensity is relatively mild.
Fig. 2：LPS damages inflammatory reaction quantitative assessment to the fish glue that different phase zebra fish induces
Compared with model control group, * p ＜ 0.001.Control is blank control group, and Vehicle is solvent control group；
For 5dpf zebra fish after derivant is handled, fish glue chamber neutrophil accumulation is most intensive, and induction intensity is the fiercest, and it is high to induce multiple
Up to 13.8 times；When 4.5dpf and 6dpf, neutrophil accumulation is relatively sparse, and induction intensity is relatively mild, induces multiple difference
For 5.3 and 3.4.
To sum up, determine that the best stage of development of zebra fish is 5dpf.
Embodiment 2 determines derivant LPS optimization process time span and injection dosage
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into five 6 respectively
In orifice plate, per 30 tail of hole.
The processing of 2 compounds
6 experimental groups are set：4 derivant LPS processing groups, 1 solvent injection group, 1 blank control group.Derivant group
Zebra air bladder chamber injects the LPS (lipopolysaccharides) of various dose respectively, and injection dosage is respectively 5ng, 12.5ng, 25ng and 50ng
(concentration is respectively the LPS difference fish glue chamber injections 5nL of 1mg/mL, 2.5mg/mL, 5mg/mL and 10mg/mL)；Solvent injection group spot
Horse air bladder chamber injects 5nL PBS；Blank control group zebra fish is without any processing.Each experimental group is added according to microwell plate specification
The breeding water of same volume, and by microwell plate be placed in 28 DEG C of constant incubators cultivate different time (such as 1h, 2h, 4h,
8h, 16h, for 24 hours etc.).
3 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS processing zebra fish to different experiments terminal, using three-dimensional fluorescence microscope to zebra air bladder position
It takes pictures and preserves；The neutrophil leucocyte quantity (see Fig. 3) for observing and counting the aggregation of zebra air bladder position, lures various dose
Lead agent LPS different time points (x hpi after injection：X hours post injection, x hours after injection) fish glue that induces is scorching
Disease reaction carries out quantitative assessment (see Fig. 4).
Fig. 3：Pass through the qualitative determining zebra air bladder inflammatory model of image analysis
Lps injection group neutrophil accumulation feature：1hpi~4hpi nearby bleeding pipe end neutrophil leucocytes quantity gradually becomes more,
Gradually migrated to fish glue center；4hpi and 8hpi peak；The pi neutrophil leucocytes of 16hpi~for 24 hours are gradually decreased in middle position,
Finally in the close disappearance of nearby bleeding tube end position.
Fig. 4：The fish glue of the LPS of dosage and time dependence inductions damages inflammatory reaction quantitative analysis
Compared with solvent control group, * p ＜ 0.001.1hpi~4hpi zebra air bladders position neutrophil leucocyte dramatically increases,
Dosage positive correlation is presented；4hpi and 8hpi peak；The pi of 16hpi~for 24 hours is gradually decreased, but compared with solvent control group,
Still there is significant difference.
4 histopathologies (om observation)
Derivant LPS processing zebra fish to 1hpi, 4hpi and for 24 hours after pi, acquires zebra fish sample, is used in combination respectively respectively
4% paraformaldehyde is fixed for 24 hours, and routine paraffin wax embedding, slice are carried out, and H＆E dyeing is observed and taken pictures, under the microscope to zebra
Fish carries out fish glue pathological analysis (see Fig. 5).
Fig. 5：Zebra air bladder om observation
In 1hpi and 4hpi, inflammatory cell increases 50ng lps injection group zebra fish, and pi fish glues wall thickening, inflammation are thin for 24 hours
Born of the same parents are reduced.
5 Ultrastructural Pathologies detect (Electronic Speculum observation)
Derivant LPS processing zebra fish to 1hpi, 4hpi and for 24 hours after pi, acquires zebra fish sample, uses respectively respectively
Before 2.0% (volume fraction) paraformaldehyde and 2.0% (volume fraction) glutaraldehyde solution after fixed (4 DEG C) for 24 hours, phosphoric acid buffer is used
Liquid (pH 7.12) rinses, fixed 2h after being subsequently placed in 1% (volume fraction) osmic acid, distilled water serial dehydration of alcohol after rinsing,
Embedding, ultramicrotome slice (60~90nm), acetic acid uranium-lead citrate are redyed, observe and take pictures under transmission electron microscope, to zebra
Fish carries out fish glue Electronic Speculum Ultrastructural Pathology analysis (see Fig. 6 and Fig. 7).
Fig. 6：Zebra air bladder Electronic Speculum observes epithelial cell damage
Derivant 50ng LPS cause different degrees of epithelial cell damage (arrow in 1hpi, 4hpi and pi for 24 hours
It is shown).
Fig. 7：Zebra air bladder Electronic Speculum observes injury of mitochondria
Derivant 50ng LPS cause different degrees of injury of mitochondria in 4hpi and pi for 24 hours (shown in arrow).
6 inflammatory factor IL-1 β and IL-6 expressions are analyzed
After derivant LPS processing zebra fish to different experiments terminal, as 1hpi (hours post injection),
2hpi, 3hpi, 4hpi, 8hpi etc., collecting sample, using the expression of q-PCR methods detection IL-1 β and IL-6 (see Fig. 8
With Fig. 9).
Fig. 8：LPS influences of the different time points to LI-1 β expressions after injection
Compared with solvent control group, * * p ＜ 0.001.0-1 hours after 50ng lps injections, IL-1 β expression, which is presented, to increase sharply
Gesture, and reach peak value in 1hp, downward trend is presented in subsequent 1-24hpi, except compared with solvent control group, still there is system in 4hpi
Meter learns difference (p ＜ 0.001) outside, at remaining time point equal no difference of science of statistics (p ＞ 0.05).
Fig. 9：LPS influences of the different time points to LI-6 expressions after injection
Compared with solvent control group, * p ＜ 0.01.0-1 hours after 50ng lps injections, surge trend is presented in IL-6 expression,
And reaching peak value in 1hp, downward trend is presented in subsequent 1-24hpi, except compared with solvent control group, still there is statistics in 4hpi
Difference (p ＜ 0.001) outside, remaining time point equal no difference of science of statistics (p ＞ 0.05).
7 statistical analysis
Using JMP8.0 softwares to fish glue position neutrophil leucocyte quantity, the inflammatory factor of above-mentioned image analysis statistics gained
IL-1 β and LI-6 expressions are for statistical analysis.Statistical procedures result withIt indicates, more comparison among groups use sides
Difference is analysed, and two comparison among groups are examined using Dunnett ' s T- and carry out statistical procedures, p<0.05 is notable for otherness.
To sum up, determine that the best administration concentration of derivant LPS is 10mg/mL (it is 50ng to be equivalent to optimal injection dosage),
The optimization process time is 4h.
3 zebra air bladder of embodiment damages the construction method of inflammatory model
Based on zebra fish best stage of development, compound optimization process time span and the injection in Examples 1 and 2
Amount, the concentration by optimizing LPS establish zebra fish acute lung injury inflammatory model.Design scheme is as follows：
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into six orifice plates respectively
In, per 30 tail of hole.
2 derivant LPS processing
3 experimental groups are set：1 derivant processing group, 1 solvent injection group, 1 blank control group.Derivant group spot
Horse air bladder chamber injects 50ng LPS respectively (10mg/mL concentration injects 5nL)；Solvent control group zebra air bladder chamber injects 5nL PBS；
Normal group zebra fish is without any processing.The breeding water of equal volume is added in each experimental group according to microwell plate specification,
And microwell plate is placed in 28 DEG C of constant incubators and is cultivated 4 hours.
Zebra air bladder damage inflammation has been established in the neutrophil accumulation quantity of 3 statistical analysis zebra air bladder positions, confirmation
Model
After derivant LPS processing zebra fish to 4h, zebra air bladder position is taken pictures and preserved using three-dimensional fluorescence microscope；
The neutrophil leucocyte quantity (Figure 10) for observing and counting the aggregation of zebra air bladder position, to the neutrophil leucocyte number of fish glue position aggregation
Measure it is for statistical analysis, according to statistical procedures as a result, judge had been established zebra air bladder damage inflammatory model (Figure 11).
Figure 10：Inflammatory model is damaged by the qualitative determining zebra air bladder of image analysis
4 hours after the LPS fish glue chambers injection of 50ng, induces zebra air bladder position neutrophil leucocyte and largely assemble.
Figure 11：The fish glue of 50ng LPS inductions damages inflammation quantitative analysis
Compared with solvent control group, * p ＜ 0.001.It is a large amount of that 50ng LPS induce zebra air bladder position neutrophil leucocyte quantity
Aggregation, fold induction is up to 10 times, with notable statistics difference compared with solvent control group.
The effect of embodiment 4 damages injury of lungs therapeutic agent known to inflammatory model evaluation with zebra air bladder
Experiment one：The anti-inflammatory effect of the known injury of lungs therapeutic agent PD98059 of evaluation
PD98059 can be used for inflammatory reaction caused by treating acute lung injury, be known injury of lungs therapeutic agent.This experiment
Inflammatory model, the anti-inflammatory effect of evaluation injury of lungs therapeutic agent PD98059 are damaged by the zebra air bladder that embodiment 3 is built.
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into 6 orifice plates, often
30 tail of hole.
2 LPS processing
Normal group zebra fish is without any processing, and solvent control group zebra air bladder chamber injects 5nL PBS,
The equal fish glue chamber of other groups of zebra fish injects 50ng LPS, immediately carries out step 3.
3 PD98059 processing
8 experimental groups are set：5 compound processing groups, 1 model control group, 1 solvent control group, 1 blank control
Group.5 PD98059 processing groups are respectively a concentration of 6.25,12.5,25,50 and 100 μM of PD98059, each experimental group liquid
Volume is 3mL；Model control group, solvent control group and blank control group zebra fish are without any processing, and each experimental group adds
Enter 3mL breeding waters.
4 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS and drug PD98059 is jointly processed by zebra fish to 4h, using three-dimensional fluorescence microscope to zebra fish
It takes pictures and preserves in fish glue position；The neutrophil leucocyte quantity N (Figure 12) for observing and counting the aggregation of zebra air bladder position, to PD98059
The anti-inflammatory effect amount of the progress analysis (Figure 13) of inflammatory model is damaged to the zebra air bladder of LPS inductions, and calculates anti-inflammatory effect, is resisted
Inflammation effect formula is shown in (a).
Figure 12：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining PD98059 of image analysis
Wherein, Control is Normal group, and Vehicle is solvent control group, and Model is model control group.
Five concentration group zebra air bladder position neutrophil leucocyte quantity of PD98059, significantly reduce compared with model control group zebra fish.
Figure 13：PD98059 significantly inhibits the zebra air bladder damage inflammatory reaction of LPS inductions
Compared with model control group, * p ＜ 0.001.Each concentration group zebra air bladder position neutrophil leucocyte quantity of PD98059
(figure A) is relatively substantially reduced with model group, and each concentration groups of PD98059 is prompted to significantly inhibit the zebra air bladder inflammation of LPS inductions
Reaction, and inhibiting rate is presented dosage correlation shown in upper figure and increases (figure B).
Experiment two：The anti-inflammatory effect of the known injury of lungs therapeutic agent LY294002 of evaluation
LY294002 can be used for inflammatory reaction caused by treating acute lung injury, be known injury of lungs therapeutic agent.This reality
It tests and inflammatory model, the anti-inflammatory effect of evaluation injury of lungs therapeutic agent LY294002 is damaged by the zebra air bladder that embodiment 3 is built.
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into 6 orifice plates, often
30 tail of hole.
2 derivant LPS processing
Normal group zebra fish is without any processing, and solvent control group zebra air bladder chamber injects 5nL PBS,
The equal fish glue chamber of other groups of zebra fish injects 50ng LPS, immediately carries out step 3.
3 LY294002 processing
6 experimental groups are set：3 compound combination processing groups, 1 model control group, 1 solvent control group, 1 blank
Control group.3 LY294002 processing groups are respectively a concentration of 37.5,75 and 150 μM of LY294002, each experimental group liquid body
Product is 3mL；Model control group, solvent control group and blank control group zebra fish are without any processing, and each experimental group is added
3mL breeding waters.
4 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS and LY294002 are jointly processed by zebra fish to 4h, using three-dimensional fluorescence microscope to zebra air bladder
It takes pictures and preserves in position；The neutrophil leucocyte quantity N (Figure 14) for observing and counting the aggregation of zebra air bladder position, to LY294002
The anti-inflammatory effect amount of the progress analysis (Figure 15) of inflammatory model is damaged to the zebra air bladder of LPS inductions, and calculates anti-inflammatory effect,
The middle same a of anti-inflammatory effect formula.
Figure 14：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining LY294002 of image analysis
Wherein, Control is Normal group, and Vehicle is solvent control group, and Model is model control group.
Tri- concentration group zebra air bladder position neutrophil leucocyte quantity of LY294002, significantly reduce compared with model control group zebra fish.
Figure 15：LY294002 significantly inhibits the zebra air bladder inflammatory reaction of LPS inductions
Compared with model control group, * p ＜ 0.001.Each concentration group zebra air bladder position neutrophil leucocyte quantity of LY294002
(figure A) is relatively substantially reduced with model group, and each concentration groups of LY294002 is prompted to significantly inhibit the zebra air bladder inflammation of LPS inductions
Reaction, and inhibiting rate is presented dosage correlation shown in upper figure and increases (figure B).
Experiment three：The anti-inflammatory effect of the known injury of lungs therapeutic agent sp600125 of evaluation
Sp600125 can be used for inflammatory reaction caused by treating acute lung injury, be known injury of lungs therapeutic agent.This reality
It tests and inflammatory model, the anti-inflammatory effect of evaluation injury of lungs therapeutic agent sp600125 is damaged by the zebra air bladder that embodiment 3 is built.
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into 6 orifice plates, often
30 tail of hole.
2 derivant LPS processing
Normal group zebra fish is without any processing, and solvent control group zebra air bladder chamber injects 5nL PBS, other groups of spots
The equal fish glue chamber of horse fish injects 50ng LPS, immediately carries out step 3.
3 sp600125 processing
6 experimental groups are set：3 compound combination processing groups, 1 model control group, 1 solvent control group, 1 blank
Control group.3 sp600125 processing groups are respectively a concentration of 37.5,75 and 150 μM of sp600125, each experimental group liquid body
Product is 3mL；Model control group, solvent control group and blank control group zebra fish are without any processing, and each experimental group is added
3mL breeding waters.
4 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS and sp600125 are jointly processed by zebra fish to 4h, using three-dimensional fluorescence microscope to zebra air bladder
It takes pictures and preserves in position；The neutrophil leucocyte quantity N (Figure 16) for observing and counting the aggregation of zebra air bladder position, to sp600125
The anti-inflammatory effect amount of the progress analysis (Figure 17) of inflammatory model is damaged to the zebra air bladder of LPS inductions, and calculates anti-inflammatory effect,
The middle same a of anti-inflammatory effect formula.
Figure 16：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining sp600125 of image analysis
Wherein, Control is Normal group, and Vehicle is solvent control group, and Model is model control group.
Three dense set of measurements zebra air bladder position neutrophil leucocyte quantity of sp600125, significantly reduce compared with model control group zebra fish.
Figure 17：Sp600125 significantly inhibits the zebra air bladder inflammatory reaction of LPS inductions
Compared with model control group, * p ＜ 0.001.Each concentration group zebra air bladder position neutrophil leucocyte quantity of sp600125
(figure A) is relatively substantially reduced with model group, and each concentration groups of sp600125 is prompted to significantly inhibit the zebra air bladder inflammation of LPS inductions
Reaction, and inhibiting rate is presented dosage correlation shown in upper figure and increases (figure B).
Embodiment 5 damages the anti-inflammatory effect of inflammatory model evaluation PHPS1 with zebra air bladder
PHPS1 is the inhibitor of Protein-tyrosine-phosphatase Shp2, but whether it has therapeutic effect to acute lung injury,
There is not been reported.The known injury of lungs therapeutic agent of embodiment 4 demonstrates the evaluation therapeutic agent that inflammatory model is damaged with zebra air bladder
The feasibility of effect, therefore the present embodiment has judged whether it has lung damage using the anti-inflammatory effect of model evaluation PHPS1
The effect of wound treatment.
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, in the normotrophic zebra fish difference microwell plate of picking.
2 derivant LPS processing
Normal group zebra fish is without any processing, and other equal fish glue chambers of experimental group zebra fish inject 50ng LPS, later
Step 3 is carried out immediately.
3 drug PHPS1 processing to be measured
8 experimental groups are set：3 PHPS1 processing groups, 1 model control group, 1 Normal group.3 PHPS1 processing
Group is respectively 3.75,7.5 and 15ng PHPS1 (intravenous injection)；Model control group and Normal group zebra fish do not do any place
Reason.The breeding water of equal volume is added according to the specification of microwell plate for each experimental group, and microwell plate is placed in 28 DEG C of constant temperature incubations
4h is cultivated in case.
4 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After derivant LPS and drug PHPS1 is jointly processed by zebra fish to 4h, using three-dimensional fluorescence microscope to zebra air bladder
It takes pictures and preserves in position；The neutrophil leucocyte quantity N (Figure 18) for observing and counting the aggregation of zebra air bladder position, to PHPS1 couples
The anti-inflammatory effect amount of the progress analysis (Figure 19) of the zebra air bladder damage inflammatory model of LPS inductions, and anti-inflammatory effect is calculated, it calculates
The same a. of formula
Figure 18：The anti-inflammatory effect of inflammatory model is damaged to zebra air bladder by the qualitative determining PHPS1 of image analysis
PHPS1 significantly inhibits the aggregation of the fish glue position neutrophil leucocyte of LPS inductions.
Figure 19：PHPS1 significantly inhibits the zebra air bladder damage inflammatory reaction of LPS inductions
Compared with model control group, * p ＜ 0.001.Each dosage injection group zebra air bladder position neutrophil leucocyte numbers of PHPS1
Amount is relatively substantially reduced with model group, prompts the zebra air bladder inflammation that each dosage groups of PHPS1 can significantly inhibit LPS inductions anti-
It answers, and inhibiting rate is presented dosage correlation shown in upper figure and increases.
Embodiment 6 damages the induction inflammatory effect of inflammatory model evaluation Poly I/C with zebra air bladder
Poly I/C be artificial synthesized double-stranded RNA [polyriboinosinic polyribocytidy-lic acid,
poly(I:C)], it is often used to the correlative study of simulation picornavirus infection and viral pathogenesis mechanism, but can it induce acute lung
Inflammation is damaged, there is not been reported.The known injury of lungs derivant of embodiment 3 demonstrates constructed zebra air bladder damage inflammation
Model is effective, therefore the present embodiment, using the induction inflammatory effect of model evaluation Poly I/C, Poly I/C are to spot for evaluation
The inflammatory reaction degree (i.e. the toxicity assessment of derivant) that horse air bladder induces.
1 zebra fish is chosen
The zebra fish of 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into 6 orifice plates, often
30 tail of hole.
2 Poly I/C processing
6 experimental groups are set：4 compound combination processing groups, 1 solvent control group, 1 blank control group.4 chemical combination
Object processing group is respectively the Poly I/C that injection dosage is 10,20,40 and 80ng；3mL breeding waters are added in each experimental group.
3 pairs of fish glue position neutrophil accumulation quantity carry out qualitative and quantitative assessment
After Poly I/C processing zebra fish to 4h or different time sections (1h, 4h, 8h etc.), three-dimensional fluorescence microscope pair is utilized
It takes pictures and preserves in zebra air bladder position；The neutrophil leucocyte quantity N (Figure 20) of zebra air bladder position aggregation is observed and counts, it is right
Zebra air bladder inflammatory reaction caused by Poly I/C carries out quantitative analysis (Figure 21), and calculates fish glue damage inflammation and induce multiple, meter
Calculate the same b of formula.
Figure 20：Zebra air bladder is caused to damage inflammatory reaction by the qualitative determining Poly I/C of image analysis
Control is Normal group, and Vehicle is solvent control group, four dosage group zebra air bladders of Poly I/C5
Position neutrophil leucocyte quantity is presented the time in 1-4hpi compared with solvent control group zebra fish and dosage correlation increases, is in then
Existing temporal correlation is reduced.
Figure 21：Poly I/C can induce zebra air bladder inflammatory reaction
Compared with solvent control group, * * p ＜ 0.05, * * * p ＜ 0.001.Dosage and temporal correlation is presented in 0~4hpi
Increase (p ＜ 0.001), and reach peak value in 4hpi, is 2.6~4.4 times of solvent control group；In 4hpi~pi presentations for 24 hours
Dosage and temporal correlation reduce (p ＜ 0.001 or 0.05), each injection group and the more equal nothing of solvent PBS injection groups in pi for 24 hours
Significant difference (p ＞ 0.05).
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Claims (7)
1. a kind of method for building up of zebra air bladder damage inflammatory model, includes the following steps：
(1) determination of zebra fish best stage of development
Neutrophil leucocyte green fluorescent transgenic strain zebra fish parent mating, hatching are taken, the zebra of different developmental phases is selected
Fish moves into microwell plate respectively；
Corresponding microwell plate is set as 3 experimental groups according to the different stages of development：1 is derivant processing group, in the group
The derivant of single concentration is injected to zebra air bladder chamber；1 is solvent processing group, is injected in equal volume to zebra air bladder chamber in the group
PBS；1 is blank control group, to zebra fish without any processing in the group；Then, same volume is added in each experimental group
Fish culture water, constant temperature incubation at the same temperature；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and is preserved with three-dimensional fluorescence microscope,
Qualitative and quantitative assessment is carried out to the neutrophil leucocyte quantity of zebra air bladder position aggregation, determines the best development rank of zebra fish
Section；
The zebra fish of the different developmental phases is the zebra fish of 4.5~6dpf；The zebra fish of the best stage of development is
The zebra fish of 5dpf；
(2) determination of the best administration concentration of derivant
The zebra fish of 5dpf is taken to move into microwell plate；
The derivant processing group of blank control group, solvent injection group and different dosing concentration is set：Blank control group is to zebra fish
Without any processing, solvent injection group injects PBS to zebra air bladder chamber, and derivant processing group injects zebra air bladder chamber respectively
Isometric but different dosing concentration derivant；Then, the breeding water of same volume is added in each experimental group, mutually synthermal
Lower constant temperature incubation；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and is preserved with three-dimensional fluorescence microscope,
Qualitative and quantitative assessment is carried out to the neutrophil leucocyte quantity of zebra air bladder position aggregation, determines that the most preferably administration of derivant is dense
Degree；
The administration concentration of the derivant is 1~10mg/mL, and the best administration concentration of the derivant is 10mg/mL；
(3) structure zebra air bladder damages inflammatory model
The zebra fish of 5dpf is taken to move into microwell plate；
3 experimental groups are set：1 is derivant processing group, injects the derivant of 10mg/mL in the group to zebra air bladder chamber, 1
For solvent injection group, isometric PBS is injected to zebra air bladder chamber in the group；Then, same volume is added in each experimental group
Breeding water, at the same temperature constant temperature incubation；
After derivant handles zebra fish to experimental endpoints, zebra air bladder position is taken pictures and is preserved with three-dimensional fluorescence microscope,
For statistical analysis to the neutrophil leucocyte quantity of zebra air bladder position aggregation, according to statistical result, spot has had been established in judgement
Horse air bladder damages inflammatory model.
2. the method for building up of zebra air bladder damage inflammatory model according to claim 1, which is characterized in that described breeds fish
Watering condition is：Dissolved oxygen mass concentration is 6-8mg/L, and water temperature is 28 DEG C, pH 7.2-7.6, total hardness 200-250mg/
L。
3. the method for building up of zebra air bladder damage inflammatory model according to claim 1, which is characterized in that the constant temperature
Condition of culture is：Microwell plate is placed in constant incubator, and 4h is cultivated at 28 DEG C.
4. a kind of purposes of zebra air bladder damage inflammatory model as described in claims 1 to 3 is any, which is characterized in that described
Model is drug or the chemistry examination that can induce injury of lungs for evaluating the derivant for causing injury of lungs toxicity, the derivant
Agent, including but not limited to LPS, Poly I/C.
5. it is a kind of with as claims 1 to 3 is any the zebra air bladder damages inflammatory model evaluates cause injury of lungs toxicity
The method of derivant, includes the following steps：
(1) zebra fish is chosen
Zebra fish in 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into microwell plate,
The zebra fish of different number is put into according to microwell plate specification；Specification is 6,12,24,48,96 or 384 holes；
(2) derivant is handled
Experimental group is set：The compound processing group of 1 solvent control group, 1 blank control group, different injection dosages；Blank pair
According to group to zebra fish without any processing, solvent injection group injects PBS to zebra air bladder chamber, and compound processing group is to zebra fish
Fish glue chamber injects the derivant of 0.1,10,100,500 and 1000ng respectively；Then, the cultivation of same volume is added in each experimental group
With water, constant temperature incubation at the same temperature；
(3) qualitative and quantitative assessment is carried out to fish glue position neutrophil accumulation quantity
After derivant is handled to experimental endpoints, is taken pictures and preserved with three-dimensional fluorescence microscope；Observation compares each experimental group zebra
Air bladder position neutrophil accumulation, the fish glue damage of qualitative evaluation injury of lungs derivant induction；And it acquires, count zebra fish neutrality
Granulocyte quantity, fish glue degree of injury caused by quantitative assessment injury of lungs derivant, fish glue degree of injury induce multiple table with fish glue inflammation
Show, calculation formula (b) is：
Fish glue damages inflammation and induces multiple=(NCompound group-NSolvent control group)/NSolvent control group；
According to statistical result, evaluates fish glue caused by injury of lungs derivant and damage toxic degree.
6. a kind of purposes of zebra air bladder damage inflammatory model as described in claims 1 to 3 is any, which is characterized in that described
Model is used to evaluate the therapeutic agent for the treatment of injury of lungs, and the therapeutic agent is the drug or chemical reagent that can treat injury of lungs,
Including but not limited to PD98059, LY294002, sp600125, PHPS1.
7. a kind for the treatment of with the zebra air bladder damage inflammatory model evaluation treatment injury of lungs as described in claims 1 to 3 is any
The method of agent, includes the following steps：
(1) zebra fish is chosen
Zebra fish in 5dpf is placed under disecting microscope and is observed, the normotrophic zebra fish of picking moves into microwell plate,
The zebra fish of different number is put into according to microwell plate specification；Specification is 6,12,24,48,96 or 384 holes；
(2) derivant is handled
Blank control group is without any processing to zebra fish, and solvent control group injects 5nL PBS, other groups of spots to zebra air bladder chamber
The equal fish glue chamber of horse fish injects 50ng derivants；
(3) injury of lungs therapeutic agent is handled
Experimental group is set：1 blank control group, 1 solvent control group, 1 model control group, the processing of the compound of various concentration
Group；Zebra fish is given 0.1 μM, 1 μM, 10 μM, 100 μM, 1000 μM in a manner of water-soluble or injection and controlled in compound processing group
Treat agent；Model control group, solvent control group and blank control group zebra fish are without any processing；Then, each experimental group is added
The breeding water of same volume, at the same temperature constant temperature incubation；
(4) qualitative and quantitative assessment is carried out to fish glue position neutrophil accumulation quantity
After therapeutic agent is handled to experimental endpoints, is taken pictures and preserved with three-dimensional fluorescence microscope；Observation compares each experimental group zebra
Air bladder position neutrophil accumulation, the anti-inflammatory effect of qualitative evaluation injury of lungs therapeutic agent；And it acquires, count zebra fish neutrality grain
Cell quantity, the anti-inflammatory effect of quantitative assessment injury of lungs therapeutic agent, calculation formula (a) are：
Anti-inflammatory effect (%)=(NModel control group-NCompound processing group)/NModel control group× 100%；
According to statistical result, the therapeutic efficiency of injury of lungs therapeutic agent is evaluated.
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