CN107982516A - A kind of preparation method of new pyemia animal model - Google Patents
A kind of preparation method of new pyemia animal model Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of new pyemia animal model, and using 8 week old Balb/c mouse of SPF levels, 25 30g of weight, after adaptability is raised 1 week, is tested, it is characterised in that include the following steps；Step 1：Adaptability is raised and the modeling stage, the water inlet of mouse ad lib, and using the illumination/dark alternately illumination at 12h intervals, humiture is suitable, 60 standard compliant Balb/c mouse is marked, half male and half female, and the corresponding sequence number of every mouse is fixed and uniquely.Female, 2 big group of male are divided into using random digits table, each big group is divided into A again（N=6）、B（N=8）、C（N=8）、D（N=8）4 groups；New model with pharmaceutical interventions such as ciclosporin As, causes the low state of Immune Function In Animals in advance, this is consistent with because the other reasons such as operation, age cause immunocompromised crowd to suffer from pyemic situation, preferably simulates the natural process of pyemia occurrence and development.
Description
Technical field
The present invention relates to a kind of animal medicine model field, more particularly, to a kind of preparation of new pyemia animal model
Method.
Background technology
Pyemia refers to the systemic inflammatory response syndrome as caused by infection, and incidence remains high, and the patient
Easily concurrent multiple organs dysfunction.Pathogenesis of sepsis mechanism is complicated, is related to inflammatory reaction, endothelial injuries, blood coagulation disorders etc. one
Series of problems, is that the state such as the number one killer of critical illness care unit death, America and Europe has hundreds of thousands of them to die of pyemia every year.
The pyemia animal model of standardization is established, is that exploration severe sepsis, infectious shock and multiple organ dysfunction are comprehensive
Close the pathophysiological mechanism of disease and the needs of control measure.
Researcher has been set up a variety of pyemia animal models, can replicate some symptoms of clinical sepsis patient
And laboratory examination results.These models are roughly divided into infection model, Intravascular infusions Model of Bacterial, endotoxicosis model.
Injection dosage, injection system by varying bacterial/endotoxin（Abdominal cavity/vein etc.）Deng researcher can send out pyemia
The internal organs Pathologic changes such as hemodynamic responses after being ill, blood inflammatory cytokines levels in non-diabetic change, liver lung kidney are studied, and then
Understand the mechanism of pathogenesis of sepsis.Cecal ligation and perforation（CLP）Model is that current research pyemia and septic shock should
With a kind of most wide model.The exploitation for the treatment of of sepsis medicine is carried out using the model, some medicine animal experiment stages are shown
The effect of preferable, but in follow-up II phase and III clinical trial phase, the effect do not envisioned, some can even add
Weight disease.The one of the major reasons for causing this phenomenon is that existing pyemia animal model cannot simulate mankind's septicopyemia well
The natural process of disease morbidity.
Clinical research finds that pyemic incidence and age are closely related, and the age often increases by ten years old, pyemic morbidity
Rate is exponentially increased, and suffers from 13.1 times that pyemic patient is less than 65 years old patient in over-65s crowd, and the elderly's purulence
Toxication is occurred frequently to reduce close relation with its body's immunity.Pyemia animal model is established in the past, and selection animal is
Young, health animal, deviates, the achievement which results in Research of Animal Model for Study is difficult to control in clinical pyemia with clinical practice
It is applied in treatment.Therefore, the experimental animal model of more approach clinic pathogenesis of sepsis process how is established, is pyemia base
Plinth is studied and medicine exploitation urgent problem.The present invention is with the adult mice of 30g or so, after multiple immunosupress
The state of immunologic hypofunction is simulated, establishes a kind of new sepsis model, for further investigation person in middle and old age's sepsis patient
Pathogenesis and exploitation clinical treatment medicine provide new cast material.
The content of the invention
The technical problem to be solved in the present invention is overcome the defects of existing, there is provided a kind of system of new pyemia animal model
Preparation Method, so as to solve the above problems.
To achieve the above object, the present invention provides following technical solution：A kind of preparation side of new pyemia animal model
Method, using 8 week old Balb/c mouse of SPF levels, weight 25-30g, after adaptability is raised 1 week, is tested, it is characterised in that bag
Include following steps；
Step 1：Adaptability is raised and the modeling stage, the water inlet of mouse ad lib, is alternately shone using the illumination/dark at 12h intervals
Bright, humiture is suitable, 60 standard compliant Balb/c mouse is marked, half male and half female, the corresponding sequence number of every mouse
It is fixed and unique.Female, 2 big group of male are divided into using random digits table, each big group is divided into A again（N=6）、B
（N=8）、C（N=8）、D（N=8）4 groups；
Step 2：A groups：Control group, in each injection time point, give with medicine and（Or）The identical physiology salt of bacterium volume
Water；
Step 3：B groups：Ciclosporin A injection group：At the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg weight
Dosage give ciclosporin A intraperitoneal injection, at the 0th day, give identical with bacterium volume physiological saline intraperitoneal injection；
Step 4：C groups：Bacterial injections group：At the -5th day, -3 days, -1 day, each mouse of weighing, gave physiological saline abdominal cavity note
Penetrate, at the 0th day, every mouse peritoneal injection was resuspended in 0.5-5 × 10 of physiological saline9Cfu bacteriums；
Step 5：D groups：Ciclosporin A+bacterium group, at the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg bodies
The dosage of weight gives ciclosporin A intraperitoneal injection, at the 0th day, every mouse peritoneal injection be resuspended in the 0.5-5 of physiological saline ×
109Cfu bacteriums；
Step 6：Collection of specimens：Each group animal, in the 0th day bacterial injection or physiological saline, injection 12h started, and observes animal
Survival condition, mouse hair is upright, belly body temperature is lower, touches do not react when, excision eyeball takes blood and separates serum；
De- neck puts to death mouse, and tissue, the physiological saline such as the liver of quick separating mouse, kidney, lung, spleen rinse twice, and blotting paper sucks residual
After buffer solution, tissue is soaked in 4% paraformaldehyde solution fixed.
Step 7：Each group mice serum Indexs measure and pathological analysis：To mice serum, ELISA method detection serum is utilized
Inflammatory Factors Contents；Utilize Biochemical Analyzer detection Mouse Liver, Renal tissues damage index；The organs such as the liver of each group mouse, kidney, lung
Paraffin section after HE is dyed, tissue degree of impairment is compared under microscope.
As a preferred technical solution of the present invention, the ciclosporin A injection uses appropriate ciclosporin A powder, molten
In physiological saline, 2mg/mL ciclosporin A parenteral solutions are made into, it is spare in -20 DEG C of packing after ultrasonic wave mixes.
As a preferred technical solution of the present invention, the injection dosage of the ciclosporin A is 10mg/kg weight.
As a preferred technical solution of the present invention, the bacterium often uses escherichia coli cloning bacterium for bioengineering field
Strain DH5 α, bacterium are collected by centrifugation after the amplification of LB medium cultures, are resuspended after PBS buffer is washed standby to appropriate concentration
With.
As a preferred technical solution of the present invention, the injection dosage of the DH5 α is 1 × 109Cfu/ mouse.
As a preferred technical solution of the present invention, 4% paraformaldehyde solution is dissolved in 900mL using 40g paraformaldehydes and steams
Distilled water, pH is 7.4 or so for adjustment（±0.05）, 1000mL is settled to distilled water, room temperature is kept in dark place.
As a preferred technical solution of the present invention, the collection of specimens mainly includes mice serum, liver, lung, kidney, the heart
The dirty collection for waiting tissue.
As a preferred technical solution of the present invention, serological index detection, mainly including Serum TNF-α, IL-6, is adopted
With the expression of ELISA method detection each group mice serum inflammatory factor.
As a preferred technical solution of the present invention, the Mouse Liver, Renal tissues damage index, mainly including Gu Bingzhuan
Ammonia enzyme, glutamic-oxalacetic transaminease, creatinine, urea nitrogen.
As a preferred technical solution of the present invention, pathological analysis, main liver, lungs, the kidney for including each group mouse
The pathological change of dirty tissue, tissues observed structure is dyed using Hematoxylin-eosin.
Compared with prior art, the beneficial effects of the invention are as follows：The preparation method of this kind of new pyemia animal model, system
Preparation Method is simple, the animal of adaptive immune hypofunction is pre-processed by ciclosporin A, such animal is through Escherichia coli heavy dose abdomen
Chamber is injected, you can and preferable pyemia animal model is obtained, is compared with existing CLP models, without special equipment and technology,
And reproducible, the main work being related to is medicine intraperitoneal injection and Bacteria Culture and injection, medicine weighs, bacterium training
Foster grade can be by instrument quantitatives such as precision balance, spectrophotometers, and process control is strong, and man's activity is small, and animal used is
SPF grades of Balb/c mouse, hereditary information is clear and definite, and difference is small between animal so that the pyemia animal model of preparation has good weight
Renaturation, new model can preferably simulate the immunocompromised state of clinical sepsis patient premorbid, and new model uses ciclosporin A in advance
Etc. pharmaceutical intervention, the low state of Immune Function In Animals is caused, this is with causing immunocompromised because of other reasons such as operation, ages
Crowd suffers from pyemic situation and is consistent, and preferably simulates the natural process of pyemia occurrence and development.
Brief description of the drawings
Attached drawing is used for providing a further understanding of the present invention, and a part for constitution instruction, the reality with the present invention
Apply example to be used to explain the present invention together, be not construed as limiting the invention.In the accompanying drawings：
Fig. 1 is the blood bacterial culture schematic diagram of different group mouse prepared by the present invention；
Fig. 2 is the animal pattern each group mice serum transaminase of the invention prepared, creatinine, urea nitrogen levels schematic diagram；
Fig. 3 is animal pattern each group mice serum inflammatory Cytokines Expression level schematic diagram prepared by the present invention.
Embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments, is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work
Embodiment, belongs to the scope of protection of the invention.
- 3 are please referred to Fig.1, the present invention provides a kind of technical solution：A kind of preparation method of new pyemia animal model,
Using 8 week old Balb/c mouse of SPF levels, weight 25-30g, after adaptability is raised 1 week, is tested, it is characterised in that including
Following steps；
Step 1：Adaptability is raised and the modeling stage, the water inlet of mouse ad lib, is alternately shone using the illumination/dark at 12h intervals
Bright, humiture is suitable, 60 standard compliant Balb/c mouse is marked, half male and half female, the corresponding sequence number of every mouse
It is fixed and unique.Female, 2 big group of male are divided into using random digits table, each big group is divided into A again（N=6）、B
（N=8）、C（N=8）、D（N=8）4 groups；
Step 2：A groups：Control group, in each injection time point, give with medicine and（Or）The identical physiology salt of bacterium volume
Water；
Step 3：B groups：Ciclosporin A injection group：At the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg weight
Dosage give ciclosporin A intraperitoneal injection, at the 0th day, give identical with bacterium volume physiological saline intraperitoneal injection；
Step 4：C groups：Bacterial injections group：At the -5th day, -3 days, -1 day, each mouse of weighing, gave physiological saline abdominal cavity note
Penetrate, at the 0th day, every mouse peritoneal injection was resuspended in 0.5-5 × 10 of physiological saline9Cfu bacteriums；
Step 5：D groups：Ciclosporin A+bacterium group, at the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg bodies
The dosage of weight gives ciclosporin A intraperitoneal injection, at the 0th day, every mouse peritoneal injection be resuspended in the 0.5-5 of physiological saline ×
109Cfu bacteriums；
Step 6：Collection of specimens：Each group animal, in the 0th day bacterial injection or physiological saline, injection 12h started, and observes animal
Survival condition, mouse hair is upright, belly body temperature is lower, touches do not react when, excision eyeball takes blood and separates serum；
De- neck puts to death mouse, and tissue, the physiological saline such as the liver of quick separating mouse, kidney, lung, spleen rinse twice, and blotting paper sucks residual
After buffer solution, tissue is soaked in 4% paraformaldehyde solution fixed.
Step 7：Each group mice serum Indexs measure and pathological analysis：To mice serum, ELISA method detection serum is utilized
Inflammatory Factors Contents；Utilize Biochemical Analyzer detection Mouse Liver, Renal tissues damage index；The organs such as the liver of each group mouse, kidney, lung
Paraffin section after HE is dyed, tissue degree of impairment is compared under microscope.
Ciclosporin A injection uses appropriate ciclosporin A powder, is dissolved in physiological saline, is made into the injection of 2mg/mL ciclosporin As
Liquid, after ultrasonic wave mixes, spare in -20 DEG C of packing, the injection dosage of ciclosporin A is 10mg/kg weight, and bacterium is bioengineering
Escherichia coli cloning bacterial strain DH5 α are often used in field, and bacterium is collected by centrifugation, is washed through PBS buffer after the amplification of LB medium cultures
Resuspension is spare to appropriate concentration after washing, and the injection dosage of DH5 α is 1 × 109Cfu/ mouse, 4% paraformaldehyde solution use
40g paraformaldehydes are dissolved in 900mL distilled water, and pH is 7.4 or so for adjustment（±0.05）, 1000mL, room temperature are settled to distilled water
It is kept in dark place, collection of specimens mainly includes the collection of the tissues such as mice serum, liver, lung, kidney, heart, and serological index detection is main
To include Serum TNF-α, IL-6, using the expression of ELISA method detection each group mice serum inflammatory factor, Mouse Liver, kidney
Tissue damage index, mainly including glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine, urea nitrogen, pathological analysis, mainly including each group
The pathological change of the liver, lungs, renal tissue of mouse, tissues observed structure is dyed using Hematoxylin-eosin.
Embodiment 1
1. experimental animal and raising
Using 8 week old Balb/c mouse of SPF levels, weight 25-30g.Experimental animal is by Military Medical Science Institute's Experimental Animal Center
There is provided, credit number：SCXK（Capital）2014-0013.Animal is after adaptability is raised 1 week, then row experiment.Adaptability is raised and mould
Type establishment stage, the water inlet of mouse ad lib, is alternately illuminated, humiture is suitable using the illumination/dark at 12h intervals.
2. test other main materials
Ciclosporin A（MCE companies）, bacillus coli DH 5 alpha（This laboratory preserves）, injection physiological saline（The glad medicine company share of occasion has
Limit company）, disposable syringe（Sheng Guang Medical Products Co., Ltd.s）, tryptone（Oxoid companies）, dusty yeast（Oxoid is public
Department）, sodium chloride（Chinese medicines group chemical reagent Beijing Co., Ltd）.
3. preparation method：
3.1 in the -5th day, and precision weighs ciclosporin A dry powder 10mg, it is used 5mL and injection physiological saline solution, is being vortexed
After vibrating 30s on mixer, 10min is handled with low-frequency ultrasonic waves, it is in uniform suspension to make ciclosporin A；
3.2 respectively at the -5th day, -3 days, -1 day, and each mouse of weighing, ciclosporin A abdominal cavity is given by the dosage of 10mg/kg weight
Injection, at the 0th day, every mouse peritoneal injection was resuspended in the 1 × 10 of physiological saline9Cfu bacteriums, while set up physiological saline pair
According to group, independent ciclosporin A injection group, independent bacterial injections group, it is grouped as follows in detail：
A groups：Control group, in each injection time point, give with medicine and（Or）The identical physiological saline of bacterium volume；
B groups：Ciclosporin A injection group：At the -5th day, -3 days, -1 day, each mouse of weighing, was given by the dosage of 10mg/kg weight
Ciclosporin A is injected intraperitoneally, and at the 0th day, gives the physiological saline intraperitoneal injection identical with bacterium volume；
C groups：Bacterial injections group：At the -5th day, -3 days, -1 day, each mouse of weighing, gave physiological saline intraperitoneal injection, the 0th
My god, every mouse peritoneal injection is resuspended in the 1 × 10 of physiological saline9Cfu bacteriums；
D groups：Ciclosporin A+bacterium group, at the -5th day, -3 days, -1 day, each mouse of weighing, was given by the dosage of 10mg/kg weight
Ciclosporin A is given to be injected intraperitoneally, at the 0th day, every mouse peritoneal injection was resuspended in the 1 × 10 of physiological saline9Cfu bacteriums；
3.3 after physiological saline or bacterial injections 12h is given, and starts to observe animal existing state, and takes the circumstances into consideration to start collection respectively
The tissue specimens such as the whole blood and liver, kidney, lung of group animal.
The survivorship curve of 3.4 each group animals, A groups, B group animals have no dead；After 10h, C groups, D groups mortality of animals with
Bacterial injections time lengthening is significantly raised, and further, mortality of animals is compared, the significantly raised C groups of D groups.
Embodiment 2
1. collection of specimens
Each four subgroups of part of two animals organized greatly of male and female, each subgroup was in the 0th day bacterial injection or physiological saline, after injection
12h starts, and observes the survival condition of animal, when mouse web portion body temperature is lower, hair is upright, shake-up is not reacted, gives
After 5mg/kg chloraldurate deep anaesthesias, draw blood from heart, a part of whole blood, which is added in advance, has sterile heparin sodium tube sealing to inject
In the sterile collection tube of 50 μ L of liquid, for blood bacterial culture, another part whole blood is added in 1.5mL collecting pipes, and room temperature is placed
2h, 8000rpm centrifuge 5min, and separation serum is detected for tissue damage serological index, takes the mouse after blood, take off neck and put to death,
The tissue such as the liver of quick separating mouse, kidney, lung, spleen, takes left lobe of liver, left kidney, left lung, ensures that materials position is consistent as far as possible；Tissue
In physiological saline rinsing twice, after blotting paper sucks residual buffer solution, it is soaked in 4% paraformaldehyde solution；The other half tissue takes out
And after equally washing, precise weighing, in -80 DEG C of preservations.
2. blood bacterial culture content detection
Mouse anticoagulation, after sterile 10 times of gradient doubling dilutions of physiological saline, is spread evenly across broth cultivation full of nutrition
Support on base solid plate, 37 DEG C of culture carton upside down overnight incubations, calculate the clump count being incubated overnight on tablet, compare each group mouse
Blood bacterial content, it can be seen that for A groups with having no bacterium in the mouse blood of B groups, two groups of C, D has different degrees of bacteremia
Occur, D group mouse blood bacteriums are slightly more than C groups, difference unobvious.
3. serum levels of inflammatory cytokines content detection
Using commercially available IL-6, IL-10, TNF-α, IFN-γ detection kit, respectively to the inflammation of above-mentioned each group mouse
Factor content carries out quantitative detection.
4. serum liver renal damage index
Specification is detected according to kits such as glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, urea nitrogen, creatinines, detects serum Liver and kidney function
Index.
5. histopathology result
The liver of each group mouse, kidney, lung tissue, it is, fixed in 4% paraformaldehyde after removing residual blood with normal saline flushing twice
24-48h, then carries out trimming and the materials of tissue, dyed through paraffin embedding, section, HE and etc., after dyeing, in
Property resin mounting, the tissues observed pathological change under high-power microscope.
Concrete principle：Mouse feeder：In environment adaptation and whole experiment process, carried out using maintenance level mouse feed
Feed, during which free water and diet, ciclosporin A pre-treatment：It is random to take out the small of half quantity after adaptability is raised
Mouse, is injected intraperitoneally ciclosporin A, injects every other day once, altogether three times, Escherichia coli impact：Mouse without ciclosporin A processing, point
For two groups, one group is Normal group（A groups）, another group of freshly prepared Escherichia coli of injection（C groups）；Ciclosporin A pre-treatment
Mouse, be randomly divided into two groups, one group as control（B groups）, the Escherichia coli of another group of injection quantity identical with C groups（D groups）；
Preparation method of the present invention is using Balb/c mouse as experimental subjects, and convenient sources are easy to get, and cost is relatively low；It is immune to use ciclosporin A
Chaff interferent, carries out animal intervention by the way of intraperitoneal injection, and medicament sources are convenient, quality controllable, and immune interference effect can be with
Detected by immune factor, immunocyte content etc.；The purulence of immunocompromised animal is prepared by the way that Escherichia coli impact is injected intraperitoneally
Toxication model, bacterial number is controllable, easy to operate；Relative to Cecal Ligation art establish model, new method need not open abdomen into
Row surgical procedure, individual homogeneity is good, reduces difference caused by manual operation.
The preparation method of this kind of new pyemia animal model, preparation method is simple, is exempted from by ciclosporin A pretreatment
The animal of epidemic disease hypofunction, such animal are injected intraperitoneally through Escherichia coli are heavy dose of, you can obtain preferable pyemia animal mould
Type, is compared with existing CLP models, without special equipment and technology, and it is reproducible, the main work being related to is
Medicine is injected intraperitoneally and Bacteria Culture and injection, medicine weighing, Bacteria Culture etc. can be by instrument such as precision balance, spectrophotometers
Device quantifies, and process control is strong, and man's activity is small, and animal used is SPF grades of Balb/c mouse, and hereditary information is clear and definite, animal
Between difference it is small so that the pyemia animal model of preparation has good repeatability, and new model can preferably simulate clinical pyemia
With pharmaceutical interventions such as ciclosporin As, it is low to cause Immune Function In Animals in advance for immunocompromised state before morbidity, new model
State, this is consistent with because the other reasons such as operation, age cause immunocompromised crowd to suffer from pyemic situation, preferably mould
The natural process of pyemia occurrence and development is intended.
Finally it should be noted that：The foregoing is only a preferred embodiment of the present invention, is not intended to limit the invention,
Although the present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still may be used
To modify to the technical solution described in foregoing embodiments, or equivalent substitution is carried out to which part technical characteristic,
Within the spirit and principles of the invention, any modification, equivalent replacement, improvement and so on, should be included in the present invention's
Within protection domain.
Claims (10)
1. a kind of preparation method of new pyemia animal model, using 8 week old Balb/c mouse of SPF levels, weight 25-30g, is fitted
After answering property is raised 1 week, tested, it is characterised in that include the following steps；
Step 1：Adaptability is raised and the modeling stage, the water inlet of mouse ad lib, is alternately shone using the illumination/dark at 12h intervals
Bright, humiture is suitable, 60 standard compliant Balb/c mouse is marked, half male and half female, the corresponding sequence number of every mouse
It is fixed and unique；
Female, 2 big group of male are divided into using random digits table, each big group is divided into A again（N=6）、B（N=8）、
C（N=8）、D（N=8）4 groups；
Step 2：A groups：Control group, in each injection time point, give with medicine and（Or）The identical physiology salt of bacterium volume
Water；
Step 3：B groups：Ciclosporin A injection group：At the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg weight
Dosage give ciclosporin A intraperitoneal injection, at the 0th day, give identical with bacterium volume physiological saline intraperitoneal injection；
Step 4：C groups：Bacterial injections group：At the -5th day, -3 days, -1 day, each mouse of weighing, gave physiological saline abdominal cavity note
Penetrate, at the 0th day, every mouse peritoneal injection was resuspended in 0.5-5 × 10 of physiological saline9Cfu bacteriums；
Step 5：D groups：Ciclosporin A+bacterium group, at the -5th day, -3 days, -1 day, each mouse of weighing, by 5-30mg/kg bodies
The dosage of weight gives ciclosporin A intraperitoneal injection, at the 0th day, every mouse peritoneal injection be resuspended in the 0.5-5 of physiological saline ×
109Cfu bacteriums；
Step 6：Collection of specimens：Each group animal, in the 0th day bacterial injection or physiological saline, injection 12h started, and observes animal
Survival condition, mouse hair is upright, belly body temperature is lower, touches do not react when, excision eyeball takes blood and separates serum；
De- neck puts to death mouse, and tissue, the physiological saline such as the liver of quick separating mouse, kidney, lung, spleen rinse twice, and blotting paper sucks residual
After buffer solution, tissue is soaked in 4% paraformaldehyde solution fixed；
Step 7：Each group mice serum Indexs measure and pathological analysis：To mice serum, ELISA method detection inflammation is utilized
Factor content；Utilize Biochemical Analyzer detection Mouse Liver, Renal tissues damage index；The stone of the organs such as the liver of each group mouse, kidney, lung
Wax is cut into slices after HE is dyed, and tissue degree of impairment is compared under microscope.
A kind of 2. preparation method of new pyemia animal model according to claim 1, it is characterised in that the ring spore
Plain A injections use appropriate ciclosporin A powder, are dissolved in physiological saline, are made into 2mg/mL ciclosporin A parenteral solutions, ultrasonic wave mixes
It is spare in -20 DEG C of packing after even.
A kind of 3. preparation method of new pyemia animal model according to claim 1, it is characterised in that the ring spore
The injection dosage of plain A is 10mg/kg weight.
A kind of 4. preparation method of new pyemia animal model according to claim 1, it is characterised in that the bacterium
Escherichia coli cloning bacterial strain DH5 α are often used for bioengineering field, bacterium is collected by centrifugation after the amplification of LB medium cultures, passes through
It is resuspended after PBS buffer washing spare to appropriate concentration.
A kind of 5. preparation method of new pyemia animal model according to claim 4, it is characterised in that the DH5 α
Injection dosage be 1 × 109Cfu/ mouse.
A kind of 6. preparation method of new pyemia animal model according to claim 1, it is characterised in that 4% poly first
Aldehyde solution is dissolved in 900mL distilled water using 40g paraformaldehydes, and pH is 7.4 or so for adjustment（±0.05）, it is settled to distilled water
1000mL, room temperature are kept in dark place.
A kind of 7. preparation method of new pyemia animal model according to claim 1, it is characterised in that the sample
Collection mainly includes the collection of the tissues such as mice serum, liver, lung, kidney, heart.
8. the preparation method of a kind of new pyemia animal model according to claim 1, it is characterised in that serology refers to
Mark detection, mainly including Serum TNF-α, IL-6, using the expression of ELISA method detection each group mice serum inflammatory factor.
A kind of 9. preparation method of new pyemia animal model according to claim 1, it is characterised in that the mouse
Liver, Renal tissues damage index, mainly including glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, creatinine, urea nitrogen.
A kind of 10. preparation method of new pyemia animal model according to claim 1, it is characterised in that pathology point
Analysis, the pathological change of main liver, lungs, renal tissue including each group mouse, tissues observed is dyed using Hematoxylin-eosin
Structure.
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| CN111418558A (en) * | 2019-09-22 | 2020-07-17 | 山西大学 | Construction method of ablation metabolic urinary calculus animal model |
| CN112029769A (en) * | 2020-09-11 | 2020-12-04 | 中国人民解放军陆军特色医学中心 | Construction method of Cyp1a1 gene knockout mouse model and application of model in sepsis |
| CN114304063A (en) * | 2021-12-13 | 2022-04-12 | 中国人民解放军联勤保障部队第九二〇医院 | Method for constructing mouse sepsis model under simulated real battlefield environment |
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| CN109481666A (en) * | 2018-12-21 | 2019-03-19 | 江苏省中医院 | A kind of method for building up of blood of human body tumour PDX model |
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| CN112029769A (en) * | 2020-09-11 | 2020-12-04 | 中国人民解放军陆军特色医学中心 | Construction method of Cyp1a1 gene knockout mouse model and application of model in sepsis |
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