CN110278914A - A kind of alcoholic fibrosis and inflammatory animal model and its construction method and purposes - Google Patents
A kind of alcoholic fibrosis and inflammatory animal model and its construction method and purposes Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
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Abstract
The present invention relates to biological medicine research fields, specifically provide the construction method of a kind of alcoholic fibrosis and inflammatory animal model, including alcohol induced to animal alcohol diet induced and periodically intraperitoneal injection, obtain alcoholic fibrosis and inflammatory animal model, the present invention plays synergistic effect by two kinds of induction modes, building has obtained the mouse alcoholic fibrosis and inflammatory model for the pathogenesis for being conducive to further investigate alcoholic fibrosis, animal model needed for the model can be used as medicament research and development, especially as alcoholic liver disease with the particular animals model of liver fibrosis and inflammation, effective prevention and treatment alcoholic liver disease is screened with the drug and method of liver fibrosis and inflammation, with apparent application advantage and high scientific research value, overcome that alcohol dosage is excessive to will lead to animal dead, it is too small to will lead to wine The disadvantages of essence hepatic injury is small, fibrosis and unobvious inflammation has building process simplicity, easy to operate, the low advantage of construction cost.
Description
Technical field
The present invention relates to biological medicine research fields, and in particular to a kind of alcoholic fibrosis and inflammatory animal model and
Its construction method and purposes.
Background technique
Alcoholic liver disease (Alcoholic liver disease, ALD) is a kind of liver as caused by long-term heavy drinking
Dirty disease, initial stage is usually expressed as fatty liver, and then can develop into alcoholic hepatitis, liver fibrosis and cirrhosis.It is some relatively light
ALD can be reversed after abstinence from alcohol, and some serious ALD, such as cirrhosis are fatal and irreversible.In west such as America and Europes
Square developed country, ALD are still the primary cause of disease for leading to cirrhosis.In China, ALD is the second largest liver after virus hepatitis
Dirty illness, the ratio that ALD accounts for same period hepatopathy inpatient in recent years are constantly rising, are surpassing in the constituent ratio of causes of cirrhosis
Cross 25%.ALD has become a kind of disease for seriously endangering human life and health.
ALD still lacks special effective treatment means at present, and other treatment methods are mainly aided on the basis of abstinence from alcohol,
Such as confrontation or improvement alcohol metabolism, lipid-loweringing, anti-inflammatory anti-oxidant, immunological regulation, anti-hepatic fibrosis and liver transfer operation etc..ALD disease
It is related to many factors such as gender, obesity, heredity, diet and drinking type because mechanism is sufficiently complex, but its specific mechanism
Be not yet it is clear that may be by multiple types cell (such as neutrophil leucocyte, endothelial cell, Kupffer Cell, liver cell) and
Impairment factor (such as endotoxin, oxidative stress and protease) coefficient result.Drinking type is to lead to ALD progression of disease
One of key factor, but its progress for how influencing ALD is unclear.Currently, mankind ALD can be simulated completely due to lacking
Animal model, greatly limit the research of its pathogenesis and control measure.Therefore, finding one kind can simulate
The reliable and stable animal model of mankind ALD becomes an important directions of disease areas research, to disease development machine
System research and drug screening have a very important role.
The middle of last century, people start with toy such as rodent model to study ALD, the seventies and this century
The first half has respectively reached peak.Currently, several rodent ALD models are had been set up, including alcohol stomach-filling model,
Lieber-DeCarli alcohol liquid feed model and Tsukamato-French model.Lieber-DeCarli alcohol liquid is raised
Only there is slight Serum ALT and increases, is not the ideal of alcoholic fibrosis and inflammation in the hepatic injury lesser extent for expecting model
Model.The continuous intragastric infusion alcohol method that Tsukamato and French uses operation implantation stomach tube establishes Tsukamato-
French model, the model can induce serious steatosis, slight liver inflammation and slight fibrosis, but to experiment skill
The requirement of art and experimental animal is stringent, laborious, somewhat expensive, therefore is difficult to promote the use of.Acute alcohol stomach-filling model is only capable of inducing
Hepatic steatosis and slight Serum ALT and AST are increased.
The new model established in the recent period by U.S. National Institutes alcohol abuse and hepatopathy research department, alcoholism research institute, i.e.,
Chronic Alcohol feeds the alcoholic liver disease mouse model (NIAAA model or Gao-Binge model) of extra urgaent dispatch alcohol stomach-filling, with multiple
Drinking behavior and slow extra urgaent dispatch hepatic injury of the frequent chronic drinking with excessive drinking person in preparing alcohol hepatitis.This mould
Although type has the characteristics that the time is short, expense is low, easy to operate, add continuous several times acute even if Chronic Alcohol is fed 4-6 weeks
, also only there is the extensive steatosis of liver in alcohol stomach-filling, and it is fine to have no apparent liver for the horizontal slight raising of Serum ALT, AST
Dimensionization.
Therefore, it needs to establish that a kind of building process is simple, easy to operate, construction cost is low at present, has apparent liver fine
The alcoholic fibrosis and inflammatory animal model of dimensionization.
Summary of the invention
Therefore, the technical problem to be solved in the present invention is that overcoming defect in the prior art, provide building process it is simple,
It is easy to operate, construction cost is low, with apparent liver fibrosis alcoholic fibrosis and inflammatory animal model.
The present invention provides the construction methods of a kind of alcoholic fibrosis and inflammatory animal model, including, to animal wine
Smart diet induced and periodically intraperitoneal injection are alcohol induced, obtain alcoholic fibrosis and inflammatory animal model.
The process of the alcohol diet induced is to use the continuous nutrition purposes, especially for feeding animals of ethanol liquid feed 20~80 days；
The alcohol induced process of the periodically intraperitoneal injection is during alcohol diet induced, every 3~10 days to animal
Ethyl alcohol is injected intraperitoneally.
The process of the alcohol diet induced is to use the continuous nutrition purposes, especially for feeding animals of ethanol liquid feed 30~60 days；
The alcohol induced process of the periodically intraperitoneal injection is during alcohol diet induced, every 4~6 days to animal
Ethyl alcohol is injected intraperitoneally.
The weight containing ethyl alcohol is 50~55g in every liter of ethanol liquid feed.
During the alcohol diet induced, the feeding volume of daily ethanol liquid feed is calculated according to the content of ethyl alcohol, is moved
The weight of the every kg body weight per day intake ethyl alcohol of object is 20~25g.
During the periodically intraperitoneal injection is alcohol induced, the weight of ethyl alcohol is injected intraperitoneally in the every kg body weight of animal every time
For 4~5g.
During the periodically intraperitoneal injection is alcohol induced, second is injected intraperitoneally in the every kg body weight of preceding 1~4 animal every time
The weight of alcohol is 4g, and the weight that ethyl alcohol is injected intraperitoneally in the later every kg body weight of animal every time is 5g.
The animal is selected from one of mouse, rat, beasle dog, rhesus macaque.
The alcoholic fibrosis and inflammation being prepared the present invention also provides any of the above-described construction method are dynamic
Object model.
The alcoholic fibrosis and inflammation being prepared the present invention also provides any of the above-described construction method are dynamic
Object model is used to screening or identifying prevention or treat the purposes of alcoholic liver medicine,
Preferably, the alcoholic liver disease is the alcoholic liver disease with liver fibrosis and inflammation.
Technical solution of the present invention has the advantages that
1. the construction method of alcoholic fibrosis provided by the invention and inflammatory animal model, by being drunk to animal alcohol
Food induction is the chronic relatively large ethyl alcohol of nursing, the more preferable long-term heavy drinking mode of simulation people；And pass through periodically intraperitoneal injection
Alcohol induced, directly intraperitoneal injection alcohol, more preferable simulation people periodicity crapulence alcohol drinking patterns, the present invention pass through two kinds of induction sides
Formula plays synergistic effect, and building has obtained the mouse alcoholic liver for the pathogenesis for being conducive to further investigate alcoholic fibrosis
Fibrosis and inflammatory model can imitate mankind's alcoholic fibrosis pathological process, compared to existing method to the maximum extent
Building obtains model, and not only building process is easy for mouse alcoholic fibrosis of the invention and inflammatory model, easy to operate, structure
Build it is at low cost, and with apparent alcoholic fibrosis pathological manifestations, as hepatic cell fattydegeneration, cell karyorrhexis,
Cell dissolution, collagen fiber hyperplasia and inflammation change, and as the modeling time extends, hepatic injury and degree of hepatic fibrosis are aggravated, and make
The model can be used as medicament research and development alcoholic liver disease needed for animal model, especially as alcoholic liver disease with liver fiber
Change and the particular animals model of inflammation, screen effective prevention and treatment alcoholic liver disease with the drug of liver fibrosis and inflammation and
Method has apparent application advantage and high scientific research value, overcomes that alcohol dosage is excessive to will lead to animal dead, too small
It is small to will lead to alcoholic liver injury, the disadvantages of fibrosis and unobvious inflammation.
2. the construction method of alcoholic fibrosis provided by the invention and inflammatory animal model, the alcohol diet induced
Process be use the continuous nutrition purposes, especially for feeding animals of ethanol liquid feed 30~60 days；Alcohol induced process is injected intraperitoneally in the periodicity
During 30~60 days alcohol diet induceds, ethyl alcohol is injected intraperitoneally to animal every 4~6 days, wherein experiment shows by above-mentioned side
There is minor injury, inflammatory features and fibrosis in observation hepatic tissue after method control handles mouse 30 days, after processing 60 days
To alcoholic fibrosis and inflammatory model with obvious liver fibrosis.
3. screened the continuous alcohol amount for feeding ethanol liquid feed early period of the invention and the interval of ethyl alcohol be injected intraperitoneally
Between and alcohol amount, the alcohol amount of discovery first chronic nursing and intraperitoneal injection is excessive to will lead to animal dead, too small to will lead to alcohol
Property hepatic injury it is small, the disadvantages of fibrosis and unobvious inflammation, and present invention employs be higher than other mouse ALD moulds reported at present
The dosage of type, in conjunction with periodically intraperitoneal injection alcohol and the continuous feeding pattern of ethyl alcohol, two kinds of induction modes play synergistic effect, structure
It builds to have obtained a kind of mouse model with Alcoholic chronic hepatic fibrosis and inflammation, feeds ethyl alcohol daily by controlling respectively
Weight is 20~25g/kg the weight of animals, and the weight that ethyl alcohol is injected intraperitoneally is 4~5g/kg the weight of animals, successfully overcomes alcohol
Measure it is excessive will lead to animal dead, the disadvantages of too small alcoholic liver injury that will lead to of alcohol amount is small, fibrosis and unobvious inflammation,
The animal model for constructing with apparent hepatic injury, liver fibrosis and liver inflammation.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is that the liver of embodiment 1 obtains in experimental example 1 of the present invention alcoholic fibrosis and inflammatory animal model is super
Sound spectrogram, wherein P is liver parenchyma echo signal；
Fig. 2 is treated the Normal group of embodiment 1 and embodiment 1 and embodiment 2 difference in experimental example 1 of the present invention
The HE colored graph (100X) of the hepatic pathology section of obtained alcoholic fibrosis and inflammatory animal model；Wherein 2-a is positive
Normal control group, 2-b are the alcoholic fibrosis and inflammatory animal model that embodiment 2 obtains, and 2-c~2-g obtains for embodiment
Alcoholic fibrosis and inflammatory animal model；Wherein in Fig. 2-a, LCol is the hepatic cell cords of normal alignment, in Fig. 2-b,
LC is the liver cell of irregular alignment, and M is macrophage, and in Fig. 2-c, M is macrophage, and LCol is the liver of irregular alignment
Cell, LC are the liver cell of steatosis vacuolation, and SC be the hepatic stellate cells activated, and in Fig. 2-d, V is central vein, and C is
Capsule gap, F are steatosis, and I is liver cell irregular alignment, and in Fig. 2-e, L be the cytoplasm of dissolution, N be denaturalized it is thin
Karyon, in Fig. 2-f, 1 is normal hepatic stellate cells, and 2 be abnormal shape hepatic stellate cells, and 3 be lymphocyte, in Fig. 2-g, G
For the giant cell in liver；
Fig. 3 is treated the Normal group of embodiment 1 and embodiment 1 and embodiment 2 difference in experimental example 1 of the present invention
The quantized result of the sirius red stains of the hepatic pathology section of obtained alcoholic fibrosis and inflammatory animal model and
The quantized result figure of VCAM-1 dyeing；
Fig. 4 is treated the Normal group of embodiment 1 and embodiment 1 and embodiment 2 difference in experimental example 1 of the present invention
The sirius red stains figure of the hepatic pathology section of obtained alcoholic fibrosis and inflammatory animal model；Wherein 4-a is positive
Normal control group, 4-b are the alcoholic fibrosis and inflammatory animal model that embodiment 2 obtains, and 4-c~4-g is at embodiment 1
Normal group and alcoholic fibrosis and inflammatory animal model after reason；Wherein in 4-b, F indicates liver fibrosis, in 4-c,
F indicates that liver fibrosis increases sharply, and in 4-d, E indicates the extension of phlebofibrosis, and PV indicates portal vein, and in Fig. 4-e and 4-f, P is indicated
Hepatocyte parenchyma liver fibrosis, in 4-g, V indicates that central vein, E indicate the extension of central vein fibrosis；
Fig. 5 is the alcoholic fibrosis and inflammation that embodiment 1 obtains in experimental example 1 of the present invention in experimental example 1 of the present invention
The VCAM-1 immunohistochemical staining figure (100X) of animal model hepatic pathology section, V indicate portal vein stained positive.
Specific embodiment
There is provided following embodiments is to preferably further understand the present invention, it is not limited to the best embodiment party
Formula is not construed as limiting the contents of the present invention and protection scope, anyone under the inspiration of the present invention or by the present invention and its
The feature of his prior art is combined and any and identical or similar product of the present invention for obtaining, all falls within of the invention
Within protection scope.
Specific experiment step or condition person are not specified in embodiment, according to the literature in the art described routine experiment
The operation of step or condition can carry out.Reagents or instruments used without specified manufacturer, being can be by commercially available acquisition
Conventional reagent product.
Wherein, Rodent Liquid Diet, Lieber-DeCarli'82 that liquid feed is provided by Bio-Serv,
Shake and Pour is subdivided into two styles of ethyl alcohol and control.Ethanol liquid feed: Rodent Liquid Diet,
Lieber-DeCarli'82, Shake and Pour, Ethanol, lot number: 217196.Compare liquid feed (hereinafter referred to as grape
Liquid glucose body feedstuff): Rodent Liquid Diet, Lieber-DeCarli'82, Shake and Pour, Control, batch
Number: 217066.Ethanol liquid feed can be added absolute ethanol by products instruction and mix.Ethyl alcohol is by VWR
Chemicals is provided, lot number: 17J044036.Glucose (D-Glucose Powder) is by Dextro Energy Bmbh&
Co.KG is provided, lot number: L7283 07:21.During alcohol diet induced, the feeding volume of daily ethanol liquid feed is according to second
The content of alcohol calculates.
Embodiment 1
The construction method of a kind of alcoholic fibrosis and inflammatory animal model is present embodiments provided, including is walked as follows
It is rapid:
(1) ICR mouse 16 are chosen, mouse is divided into 8 model groups and 8 Normal groups at random by male, 8 week old,
And individually raise model group in the cage for having water and ethanol liquid feed, Normal group is individually raised in water and grape
In the cage of liquid glucose body feedstuff, temperature is 22 ± 2 DEG C, illumination in 12 hours and dark cycle.
(2) model group be given once daily ethyl alcohol containing 50.3g/liter ethanol liquid feed, the nursing of daily ethanol liquid feed
Amount is calculated according to the content of ethyl alcohol, and the weight of animal every kg body weight intake ethyl alcohol is 21.25g, is raised 60 days, and from first
It starts, every the ethyl alcohol of intraperitoneal injection in 5 days, preceding 4 injection 4g ethyl alcohol/kg the weight of animals, remaining injection 5g ethyl alcohol/kg
The weight of animals, Normal group give the glucose of heat identical as model group, take 7.47g glucose/kg animal first four times
Weight, remaining takes 9.3g glucose/kg the weight of animals, and model group and Normal group are raised 60 days, wherein at model group
Reason obtained alcoholic fibrosis and inflammatory animal model after 60 days.
Embodiment 2
The construction method for present embodiments providing a kind of alcoholic fibrosis and inflammatory animal model, in embodiment 1
The difference of model group animal is only that processing number of days is different, and the processing number of days of the present embodiment is 30 days, includes the following steps:
ICR mouse, male are chosen, 8 week old at random individually raise 8 mouse in the cage for having water and ethanol liquid feed
In son, temperature is 22 ± 2 DEG C, illumination in 12 hours and dark cycle, be then given once daily ethyl alcohol containing 50.3g/liter ethanol liquid
The feeding volume of feed, daily ethanol liquid feed is calculated according to the content of ethyl alcohol, the weight of the every kg body weight intake ethyl alcohol of animal
It for 21.25g, raises 30 days, and from daystart, every the ethyl alcohol of intraperitoneal injection in 5 days, preceding 4 injection 4g ethyl alcohol/
Kg the weight of animals, remaining injection 5g ethyl alcohol/kg the weight of animals is (i.e. respectively at the 1st day, 6 days, 11 days, 16 days, 21 days, 26 days abdomens
Chamber injects ethyl alcohol, wherein the 1st day, 6 days, 11 days, 16 days injection 4g ethyl alcohol/kg the weight of animals, remaining injection 5g ethyl alcohol/kg animal
Weight), it handles 30 days, obtains alcoholic fibrosis and inflammatory animal model.
Embodiment 3
The construction method of a kind of alcoholic fibrosis and inflammatory animal model is present embodiments provided, including is walked as follows
It is rapid:
The construction method of a kind of alcoholic fibrosis and inflammatory animal model is present embodiments provided, including is walked as follows
It is rapid:
ICR mouse, male are chosen, 8 week old at random individually raise 8 mouse in the cage for having water and ethanol liquid feed
In son, temperature is 22 ± 2 DEG C, illumination in 12 hours and dark cycle, be then given once daily ethyl alcohol containing 50.3g/liter ethanol liquid
The feeding volume of feed, daily ethanol liquid feed is calculated according to the content of ethyl alcohol, the weight of the every kg body weight intake ethyl alcohol of animal
It for 25g, raises 20 days, and from daystart, every the ethyl alcohol of intraperitoneal injection in 3 days, per injection 5g ethyl alcohol/kg animal
Weight raises 20 days, obtains alcoholic fibrosis and inflammatory animal model.
Embodiment 4
The construction method of a kind of alcoholic fibrosis and inflammatory animal model is present embodiments provided, including is walked as follows
It is rapid:
The construction method of a kind of alcoholic fibrosis and inflammatory animal model is present embodiments provided, including is walked as follows
It is rapid:
ICR mouse, male are chosen, 8 week old at random individually raise 8 mouse in the cage for having water and ethanol liquid feed
In son, temperature is 22 ± 2 DEG C, illumination in 12 hours and dark cycle, be then given once daily ethyl alcohol containing 55g/liter ethanol liquid raise
The feeding volume of material, daily ethanol liquid feed is calculated according to the content of ethyl alcohol, and the weight of the every kg body weight intake ethyl alcohol of animal is
20g is raised 80 days, and from daystart, every the ethyl alcohol of intraperitoneal injection in 10 days, per injection 4g ethyl alcohol/kg animal
Weight raises 80 days, obtains alcoholic fibrosis and inflammatory animal model.
1 HE of experimental example dyeing
Alcoholic fibrosis made from Example 1 and inflammatory animal model carry out ultrasound image detection, such as Fig. 1 institute
Show occur the patch of specific compact area in liver parenchyma.Alcoholic liver fiber made from Example 1 and embodiment 2
Change and inflammatory animal model and embodiment 1 treated that Normal group carries out dislocation of cervical vertebra execution, then dissection obtains liver
It is dirty, it is fixed on after liver is cut into small pieces in 10% formalin buffer, 10% formalin of liver samples of excision
It is fixed, it immerses in graded ethanol and is dehydrated, dimethylbenzene cleaning is embedded in paraffin, cuts the paraffin section of 6 μ m-thicks and be installed in
It on glass slide, is dyed with h and E (HE), then carries out pathology of hepar observation.
H and E (HE) dyeing is conventional method, specific steps are as follows: (1) dewax 5 minutes in dimethylbenzene.(2) two
It is impregnated 5 minutes in toluene and absolute alcohol (1: 1) mixed liquor.
(3) 100%, 95%, 85%, 70% alcohol, it is each to impregnate 5 minutes.(4) hematoxylin dye liquor dyes 10 minutes.(5) water
It washes away except extra dye liquor, 0.5% hydrochloride alcohol (70% alcohol) color separation a moment.(6) flowing water rinses 20 minutes.(7) it distills
Water is short to be washed.(8) 0.3% eosin stains dye 1~5 minute.(9) successively through 70%, 85%, 95%, 100% dehydration of alcohol, respectively
2 minutes.(10) transparent 2 times of dimethylbenzene, 5 minutes every time.(11) appropriate neutral gum, mounting is added dropwise.
As a result as shown in Figure 2, Normal group hepatic tissue is regularly arranged, has no inflammatory cell infiltration (see Fig. 2-a).Implement
Through treated within 30 days, mouse model liver pathological section shows liver cell irregular alignment to example 2, and inflammatory cell infiltration is (see Fig. 2-
b).Embodiment 1 through treated in 60 days mouse model liver pathological section the shows serious irregular alignment of liver cell (see Fig. 2-c and
2-d), and there is macrophage, stellate cell activator and hepatic cell fattydegeneration and form big blown-out shot (see 2-b, 2-c and 2-f),
Also there is cell nuclear degeneration, cell karyorrhexis and cell dissolution (see Fig. 2-e), in addition, embodiment 1 is through treated in 60 days mouse
The lymphocyte of model is diffused into entire liver (see Fig. 2-f), and macrophage and giant cell occurs (see Fig. 2-g).
At 1500 μm2Random areas in, by under 400x amplification factor observe dead cell number, calculate average death
Cell number, Mean Death cell number is 3.2 (s.d.=2.4) in Normal group, and in treated the model of embodiment 1
The Mean Death cell number of group is 14 (s.d.=1.5), hence it is evident that is greater than Normal group, illustrates that hepatocellular injury is serious.
2 sirius red stains of experimental example
Treated that Normal group carries out cervical vertebra is de- for Example 1 and embodiment 2 treated model group and embodiment 1
Position is put to death, and then dissection obtains liver, is fixed in 10% formalin buffer after liver is cut into small pieces, the liver of excision
Dirty sample is fixed with 10% formalin, is immersed in graded ethanol and is dehydrated, and dimethylbenzene cleaning is embedded in paraffin, cuts 6 μ m-thicks
Paraffin section is simultaneously installed on glass slide, carries out stock-dye to the collagen in liver section with sirius red stains method,
Collagen deposition situation is observed by the whole optical density of measurement slice.
Sirius red stains method is conventional method, the steps include: that routinely dewaxing adds sirius red stains to water to paraffin section
30min, sets wet box, and absolute alcohol breaks up 2s, dehydration, transparent, neutral gum sealing.As a result as shown in Figure 4, the liver of Normal group
Pathological section has no fibrosis (see Fig. 4-a), the light of the hepatic pathology section entirety of mouse model of the embodiment 2 through processing in 30 days
Density dramatically increases (p < 0.05) (see Fig. 4-b), and the hepatic pathology section of mouse model of the experimental example 1 through processing in 60 days is whole
Optical density obviously increase (p < 0.01) (Fig. 4-c), and visible near entire liver and central vein have collagen staining
Small patch, the collagen staining near portal vein is the most obvious, or even some collagenous fibres occurs to can connect two centers quiet
Arteries and veins (Fig. 4-c to Fig. 4-g).
Day wolf made from Example 1 and embodiment 2 treated model group and embodiment 1 treated Normal group
Star red stained sections carry out quantitative analysis respectively, and slice is taken to amplify 400 times of microscopic observations and recording colored light in same area
The relative value of density, at sirius red stains relative value (%)=embodiment 1, embodiment 2 treated model group or embodiment 1
Dyeing optical density/reference dyeing optical density × 100% that Normal group after reason measures passes through the whole light of measurement slice
Density observes collagen deposition situation.The reading of completely black part is wherein first measured as reference.As a result as shown in Figure 3, through quantitative
Statistics, the optical density (37%) of the hepatic pathology section entirety of the mouse model through processing in 30 days is with respect to Normal group (17%)
It dramatically increases (p < 0.05) (see Fig. 3), and the optical density of the hepatic pathology section entirety of the mouse model through processing in 60 days
(59%) opposite Normal group (17%) obviously increases (p < 0.01) (Fig. 3).
3 immunohistochemical analysis of experimental example
Vascular cell adhesion molecule 1 (VCAM-1) is to participate in inflammation adjusting, cellullar immunologic response reaction, fibrotic processes
One of important regulating and controlling factor.Example 1 and embodiment 2 treated model group and embodiment 1 treated Normal group
Model group that treated carries out dislocation of cervical vertebra execution, and then dissection obtains liver, carries out experimental record, is fixed on after being cut into small pieces
In 10% formalin buffer, the liver samples of excision are fixed with 10% formalin, are immersed in graded ethanol and are dehydrated, and two
Toluene cleaning, is embedded in paraffin, cuts the paraffin section of 6 μ m-thicks and be installed on glass slide, dewaxed with dimethylbenzene, gradient
After alcohol rehydration, slice is being placed in the 10mM boiled, boils 20min in the sodium citrate buffer solution of pH6.8.Just with 10%
Changshan sheep blood serum and 1% bovine serum albumin(BSA) phosphate buffer (PBS) in be incubated for closing, second day, in biotin mark
After being cultivated 1 hour in the goat anti-rabbit antibody of note, after being cultivated 1 hour in the horseradish peroxidase HRP of marked by streptavidin, add
Enter 3,3 '-diaminobenzidines (DAB) substrate.It after reaction stops, being dehydrated, dimethylbenzene cleaning and DPX mounting.Such as Fig. 5 institute
Show (by taking treated the model group of embodiment 1 as an example), no matter quiet the liver vessel VCAM-1 stained positive when amplifying 100 times is
There are VCAM-1 positive findings, mainly portal vein and veinlet in arteries and veins or artery, slice dyeing.
Made from Example 1 and embodiment 2 treated model group and embodiment 1 treated Normal group
VCAM-1 stained slice carries out quantitative analysis respectively, takes slice that the liver blood vessel at VCAM-1 positive position is amplified 100 times, then
Observe and record every 0.34mm2In in 30 random areas VCAM-1 positive cell blood vessel number and total blood vessel number,
Blood vessel number relative value=VCAM-1 positive cell blood vessel number of VCAM-1 positive cell/total blood vessel number × 100%), as a result
As shown in Figure 3, there is the quantity of VCAM-1 positive liver blood vessel in the hepatic pathology section of mouse model of the experimental example 1 through processing in 60 days
It is 90, the quantity of the hepatic pathology section discovery VCAM-1 positive liver blood vessel of mouse model of the embodiment 2 through processing in 30 days is
90, and Normal group is 15.5.Therefore, the mouse model handled compared to Normal group, embodiment 2 through 30 days
The quantity of the hepatic pathology section VCAM-1 positive vessels of the mouse model of hepatic pathology section and experimental example 1 through processing in 60 days is aobvious
It writes and increases, the difference of two group model groups and Normal group is statistically significant.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. the construction method of a kind of alcoholic fibrosis and inflammatory animal model, which is characterized in that including being drunk to animal alcohol
Food induction and periodically intraperitoneal injection are alcohol induced, obtain alcoholic fibrosis and inflammatory animal model.
2. construction method according to claim 1, which is characterized in that
The process of the alcohol diet induced is to use the continuous nutrition purposes, especially for feeding animals of ethanol liquid feed 20~80 days；
The alcohol induced process of the periodically intraperitoneal injection is during alcohol diet induced, every 3~10 days to animal abdominal cavity
Inject ethyl alcohol.
3. construction method according to claim 1 or 2, which is characterized in that the process of the alcohol diet induced is to use
The continuous nutrition purposes, especially for feeding animals of ethanol liquid feed 30~60 days；
It is described alcohol induced process to be injected intraperitoneally periodically to infuse every 4~6 days to animal abdominal cavity during alcohol diet induced
Penetrate an ethyl alcohol.
4. construction method according to claim 2 or 3, which is characterized in that the weight containing ethyl alcohol in every liter of ethanol liquid feed
Amount is 50~55g.
5. according to the construction method any in claim 2~4, which is characterized in that during the alcohol diet induced,
The feeding volume of daily ethanol liquid feed is calculated according to the content of ethyl alcohol, and the weight of the every kg body weight per day intake ethyl alcohol of animal is
20~25g.
6. according to the construction method any in claim 2~5, which is characterized in that the periodically intraperitoneal injection ethyl alcohol
In Induction Process, the weight that ethyl alcohol is injected intraperitoneally in the every kg body weight of animal every time is 4~5g.
7. according to the construction method any in claim 2~6, which is characterized in that the periodically intraperitoneal injection ethyl alcohol
In Induction Process, the weight that ethyl alcohol is injected intraperitoneally in the every kg body weight of preceding 1~4 animal every time is 4g, every kilogram of body of later animal
The weight that ethyl alcohol is injected intraperitoneally in weight every time is 5g.
8. any construction method according to claim 1~7, which is characterized in that the animal be selected from mouse, rat,
One of beasle dog, rhesus macaque.
9. any construction method is prepared in claim 1~8 alcoholic fibrosis and inflammatory animal model.
10. any construction method is prepared in claim 1~8 alcoholic fibrosis and inflammatory animal model
For screening or identifying prevention or treat the purposes of alcoholic liver medicine,
Preferably, the alcoholic liver disease is the alcoholic liver disease with liver fibrosis and inflammation.
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| CN115701351A (en) * | 2021-08-02 | 2023-02-10 | 中南民族大学 | Ethosome preparation and method for applying ethosome preparation to preparation of alcoholic liver disease animal model |
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| CN102972345B (en) * | 2012-12-12 | 2014-12-17 | 贵州大学 | Modeling processing method of non-alcoholic fatty liver mouse model |
| CN105901380B (en) * | 2015-09-29 | 2020-04-21 | 中南民族大学 | Feed for preparing animal model with alcoholic liver disease |
| CN107693507B (en) * | 2017-09-29 | 2020-07-31 | 四川省人民医院 | Method for establishing rhesus monkey hepatic fibrosis model by intraperitoneal injection method |
| CN110278914A (en) * | 2019-06-10 | 2019-09-27 | 漳州片仔癀药业股份有限公司 | A kind of alcoholic fibrosis and inflammatory animal model and its construction method and purposes |
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| CN115701351A (en) * | 2021-08-02 | 2023-02-10 | 中南民族大学 | Ethosome preparation and method for applying ethosome preparation to preparation of alcoholic liver disease animal model |
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