CN106420692A - Method for establishing model of aflatoxin inducing macaque liver fibrosis - Google Patents
Method for establishing model of aflatoxin inducing macaque liver fibrosis Download PDFInfo
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- CN106420692A CN106420692A CN201610692028.5A CN201610692028A CN106420692A CN 106420692 A CN106420692 A CN 106420692A CN 201610692028 A CN201610692028 A CN 201610692028A CN 106420692 A CN106420692 A CN 106420692A
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Abstract
The invention relates to a method for establishing a model of aflatoxin inducing macaque liver fibrosis. The method comprises the following steps: with macaque as a model animal, during the model making, adding 0.5mg/kg aflatoxin to the drinking water of macaque for sucking, twice every month at an interval of 2 weeks for 8 successive months; feeding the macaque with macaque commercial maintenance-period feed containing more than 19% of protein and more than 4% of crude fat; performing pathological analysis every 2 months; and 8 months later, performing pathological detection to show obvious liver fibrosis symptoms. The method provided by the invention is safe and reliable, shortens the model making time of an animal model, and is of great significance to screening and predicting effective drugs for controlling liver fibrosis and even liver cancer as an experimental foundation.
Description
Technical field
The invention belongs to the method for building up of animal model, be specifically related to the construction method of Liver Fibrosis Model.
Background technology
Liver fibrosis refers to fibroplasia in liver under various cause of disease effect, can be with fiber every formation.Liver fibrosis is mainly
A kind of pathological concept, means hepatic tissue extracellular matrix composition hyperplasia and abnormal deposition, the liver structure being caused and/
Or the pathological change that dysfunction changes.Its pathomechanism is also mostly is owing to hepatic parenchymal cells is downright bad, cause liver network
Subside and formed, be a kind of passive and irreversible pathologic process.Build similar to mankind's liver fibrosis cause of disease, the course of disease and pathology
Experimental animal model, be screening and prediction preventing and treating the liver fibrosis even active drug of liver cancer basis, in this basis, pass through
Laboratory test includes zooperal qualitative or quantitative data, it was demonstrated that medicine preventing and treating liver fibrosis on medical usage,
Pharmacological action.The making of current hepatic fibrosis animal model mainly has carbon tetrachloride and N-nitrosodimethylamine two kinds chemically liver fine
The foundation of dimensionization animal model, is mainly used on the animals used as test such as big mouse and rabbit；Next to that use heterogenous animal serum, egg
Bai Zuowei antigen injects the foundation that animal makes fibrosis model repeatedly；Additionally also utilize alcohol, cholestasis, disease
The induction hepatic fibrosis animal model such as poison and parasite.But, moving of the chemicals such as carbon tetrachloride, N-nitrosodimethylamine induction
There is very big difference with the cause of disease of mankind's liver fibrosis natural occurrence, the course of disease and pathology in thing model；Fibrosis model
Need to repeatedly inject animal, lack the human care necessary to macaque；Although alcohol induction primate liver fibrosis and
Mankind's AML is sufficiently close to, but the modeling cycle needs 3～5 years, and the time is very long.
Aflatoxin is a kind of chemical substance having strong bio-toxicity, is often being gone mouldy by aspergillus flavus and other several mould
Food in produce, such as rice, beans, peanut etc., have very strong carcinogenesis.But Aflatoxin carcinogenesis and liver fibrosis
The cause of disease, the course of disease and the pathology that are formed there is no positive evidence and understanding.
Content of the invention
It is an object of the invention to provide that a kind of modeling time is short, modeling success rate and the high liver fibrosis of modeling efficiency are moved
The method for building up of thing model, the i.e. method with aflatoxins induction macaque liver fibrosis model.
The present invention realizes the technical scheme of its purpose：
Randomly selecting healthy macaque as animal pattern, during modeling, the aflatoxins with 0.5mg/kg adds macaque respective
Sucking for macaque in drinking water, monthly 2 times, every minor tick 2 weeks, continuous 8 months, through pathology detection, macaque performance is significantly
Liver fibrosis symptom.
Further, described drinking water preparation is to weigh 10mg aflatoxins, and piping and druming repeatedly makes it be substantially dissolved in 1mL
DMSO in, in advance preheat 50 DEG C～60 DEG C of physiological saline, contain the DMSO to 100mL of aflatoxins with normal saline dilution,
It is dispensed in the respective special drinking bottle of macaque by 0.5mg/kg the weight of animals and suck for macaque.
Further, described rhesus macaque is 10～14 years old, body weight 5～8 kilograms male or female, and feed macaque business
Industry maintains phase feed.
Further, the nutritional labeling of described business maintenance phase feed is：Thick protein：19.5%th, coarse ash is the 6.88%th,
Crude fat the 4.28%th, crude fibre the 3.1%th, moisture 8.2%.
Advantages of the present invention and good effect are：
1st, reagent is periodically added in animal drinking water during modeling or supplies it with in respective special drinking bottle by the present invention
The method safety that animal model is set up in sucking is reliable, within 8 months, gets final product modeling, decreases the model modeling time of animal, improve
Modeling success rate and modeling efficiency, be of great practical significance.
2nd, pass through hepatic fibrosis animal model, build the experiment similar to mankind's liver fibrosis cause of disease, the course of disease and pathology and move
Thing model, in this basis, includes zooperal qualitative or quantitative data, research liver fibrosis and liver by laboratory test
The mechanism of cancer, the active drug of screening and prediction preventing and treating liver fibrosis, it was demonstrated that medicine in preventing and treating liver fibrosis for the medicine
Purposes, pharmacological action, evaluate the drug effect to disease for the related drugs.
Brief description
Fig. 1 is serum biochemical markers thing hyaluronic acid（HA）Serological index test result.
Fig. 2 is serum biochemical markers nitride layer Fibronectin（LN）Serological index test result.
Fig. 3 is serum biochemical markers thing type III precollagen V end peptide（PIIINP) serological index test result.
Fig. 4 is serum biochemical markers thing glutamic-oxalacetic transaminease (AST) serological index test result.
Fig. 5 is serum biochemical markers thing glutamic-pyruvic transaminase (ALT) serological index test result.
Fig. 6 is serum biochemical markers thing total protein（TP）Serological index test result.
Fig. 7 is the ultrasonic figure of normal liver tissue.
Fig. 8 is the ultrasonic figure of Liver fibrosis tissue.
Fig. 9 is light microscopic（200×）Lower observation masson dyeing normal hepatocytes section, liver cell is normal, hence it is evident that visible cell core,
Kytoplasm and film.
Figure 10 is light microscopic（200×）The liver cell of lower observation HE dyeing normal hepatocytes section is centered on central vein, to week
Enclose radially.
Figure 11 is light microscopic（200×）There is liver cirrhosis pathology pathology in lower observation masson dyeing liver cell.
Figure 12 is light microscopic（400×）There is liver cirrhosis pathology pathology in lower observation masson dyeing liver cell.
Being further described the present invention below in conjunction with drawings and Examples, specific embodiment discussed below includes
But do not limit scope.
Detailed description of the invention
（One）The foundation of model
Embodiment 1：
Randomly select from healthy macaque 2 male and 1 female, 12 years old age, each 1 of male Body Weight 8.1Kg, 7.9Kg, 1
Female Body Weight 5.3Kg is as animal pattern.With the aflatoxins of 0.5mg/kg during modeling（Aflatoxins Aflatoxin
B1 manufacturer：Sigma article No.：A6636 specification：50mg powder）It is directly added in the respective drinking water of macaque and suck for macaque
Inhale, monthly 2 times, every minor tick 2 weeks, continuous 8 months, continuous 8 months, and by monthly 2 times, every minor tick 2 weeks, per the end of month enters
Row serology Indexs measure, after modeling 1st month, 3 months, 6 months, serum result such as Fig. 1 of 8 months.Through pathology detection,
The obvious liver fibrosis symptom of macaque performance.Meanwhile, randomly select from healthy rhesus macaque 2 male and 1 female as right
According to the age, body weight was 5～8 Kg, gavages DMSO according to body weight at 12 years old.During modeling, rhesus macaque maintains the nutrition of phase feed
Composition is：Thick protein：19.5%th, coarse ash the 6.88%th, crude fat the 4.28%th, crude fibre the 3.1%th, moisture 8.2%.
Embodiment 2：
The drinking water preparation method of embodiment 1 is：Weighing 10mg aflatoxins, piping and druming repeatedly makes it be substantially dissolved in 1mL's
In DMSO, preheat 50 DEG C～60 DEG C of physiological saline in advance, contain the DMSO to 100mL of aflatoxins with normal saline dilution, press
0.5mg/kg the weight of animals is dispensed in the respective special sterile water bottle of macaque to be sucked for macaque.Remaining is with embodiment 1 one
Cause.
（Two）The evaluation method of model foundation and result
Evaluation method：Liver fibrosis diagnosis pattern mainly includes that serum biochemical markers thing, iconography, Histopathology etc. are comprehensive and examines
Disconnected.
1st, serum biochemical markers thing includes hyaluronic acid（HA）, laminin（LN）, type III precollagen V end peptide
（PIIINP), glutamic-oxalacetic transaminease (AST), glutamic-pyruvic transaminase (ALT), total protein（TP）Deng.Serological index test result.
During modeling, the 1st, respectively at the 3rd, 6 with 8 months, serum is carried out to modeling animal and separate detection, right before collecting blood sample
Experiment macaque fasting 12 hours, then under the conditions of ketamine 10mg/kg neuromuscular block, hind leg vein is taken a blood sample 5 milliliters,
3000rpm centrifuges 15 minutes, separates serum, test sera HA, LN, PIIINP, AST, ALT, TP concentration.Modeling the 1st, 3 and 6
After Yue, hyaluronic acid（HA）Index test result shows, liver fibrosis neuropathy model monkey and Normal group significant difference（P<
0.05）, and after 8th month, recovery is extremely and comparison does not has difference.Other indexs such as laminin（LN）, type III precollagen V end
Peptide（PIIINP), glutamic-oxalacetic transaminease (AST), glutamic-pyruvic transaminase (ALT) and total protein（TP）Test value each stage model monkey
And difference is not notable (P ＞ 0.05) between Normal control animals.（See Fig. 1）
2nd, ultrasound diagnosis.
Under the conditions of ketamine 10mg/kg neuromuscular block, utilize GE portable type b ultrasonic, use 3.5MHz electronics convex array probe
Hepatic disease position is scanned, preserves picture and be analyzed.Ultrasonic testing results：
The ultrasonic figure of normal liver tissue（See Fig. 2）.Liver tunicle is neatly smooth, the strong echo very thin in wire, returns with peritonaeum wire
There is small gap between sound.Liver parenchyma luminous point is fine, be evenly distributed；Liver duct system traveling is normal, and texture understands, entrant sound
Well.
The ultrasonic figure of Liver fibrosis tissue（See Fig. 3）.Liver surface is irregular, and rugged and rough, liver parenchyma Echo heterogenicity is even,
There is necrosis region.
3rd, liver histopathological analysis.Under the conditions of ketamine 10mg/kg neuromuscular block, B ultrasonic induces row biopsy hepatic tissue,
10% neutral formalin is fixed, FFPE, 4um section, prepares liver histological section, for HE and Masson dyeing, adopts
Observe liver organization collagen fiber hyperplasia situation with Leica microscope.Analysis result：
Normal hepatocytes is cut into slices, and liver cell is normal, hence it is evident that visible cell core, kytoplasm and film【See Fig. 4（200×）（Masson dyes）】,
Liver cell is centered on central vein, to surrounding radially【See Fig. 5（200×）（HE dyes）.
Liver cirrhosis pathology pathology：Light Microscopic observation, there are downright bad widely denaturation, a large amount of necrosiss of liver cells in liver cell
After, original network collagenzation in lobuli hepatis, and produce collagenous fibres, fibroplasia causes liver fibrosis, forms portal vein
Fibrous septum between district and portal vein district.【Fig. 6（200×）, masson dyes】；Light Microscopic observation, liver cell occurs extensively
Downright bad denaturation, and in lobuli hepatis stretch, fibroplasia
Become liver fibrosis.【Fig. 7（400×）, masson dyes】.
Claims (5)
1. the method for aflatoxins induction macaque liver fibrosis model, is characterized in that：Randomly select healthy macaque to move as model
Thing, sucks for macaque, monthly 2 times, often during modeling in the aflatoxins addition respective drinking water of macaque of 0.5mg/kg
Minor tick 2 weeks, continuous 8 months, through pathology detection, the obvious liver fibrosis symptom of macaque performance.
2. the method for claim 1, is characterized in that：Described drinking water preparation is to weigh 10mg aflatoxins, repeatedly
Piping and druming makes it be substantially dissolved in the DMSO of 1mL, preheats 50 DEG C～60 DEG C of physiological saline in advance, contains Huang with normal saline dilution
The DMSO to 100mL of aspergillin, is dispensed in the respective special drinking bottle of macaque by 0.5mg/kg the weight of animals and suckes for macaque
Inhale.
3. method as claimed in claim 1 or 2, is characterized in that：Described rhesus macaque is 10～14 years old, body weight 5～8 kilograms
Male or female, and feed macaque business maintenance phase feed.
4. method as claimed in claim 1 or 2, is characterized in that：The nutritional labeling of described business maintenance phase feed is：Thick egg
White matter：19.5%th, coarse ash the 6.88%th, crude fat the 4.28%th, crude fibre the 3.1%th, moisture 8.2%.
5. method as claimed in claim 3, is characterized in that：The nutritional labeling of described business maintenance phase feed is：Crude protein
Matter：19.5%th, coarse ash the 6.88%th, crude fat the 4.28%th, crude fibre the 3.1%th, moisture 8.2%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110163195A (en) * | 2018-02-14 | 2019-08-23 | 中国医药大学附设医院 | Liver cancer divides group's prediction model, its forecasting system and liver cancer to divide group's judgment method |
| CN111631188A (en) * | 2019-03-01 | 2020-09-08 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
-
2016
- 2016-08-19 CN CN201610692028.5A patent/CN106420692A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110163195A (en) * | 2018-02-14 | 2019-08-23 | 中国医药大学附设医院 | Liver cancer divides group's prediction model, its forecasting system and liver cancer to divide group's judgment method |
| CN111631188A (en) * | 2019-03-01 | 2020-09-08 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
| CN111631188B (en) * | 2019-03-01 | 2022-04-22 | 广西中医药大学 | Method for rapidly inducing alcoholic fatty liver model by single factor |
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