CN113647359A - Construction method of tunicamycin injection induced acute liver injury mouse model - Google Patents
Construction method of tunicamycin injection induced acute liver injury mouse model Download PDFInfo
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- 206010067125 Liver injury Diseases 0.000 title claims abstract description 27
- YJQCOFNZVFGCAF-UHFFFAOYSA-N Tunicamycin II Natural products O1C(CC(O)C2C(C(O)C(O2)N2C(NC(=O)C=C2)=O)O)C(O)C(O)C(NC(=O)C=CCCCCCCCCC(C)C)C1OC1OC(CO)C(O)C(O)C1NC(C)=O YJQCOFNZVFGCAF-UHFFFAOYSA-N 0.000 title claims abstract description 26
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- CDVAIHNNWWJFJW-UHFFFAOYSA-N 3,5-diethoxycarbonyl-1,4-dihydrocollidine Chemical compound CCOC(=O)C1=C(C)NC(C)=C(C(=O)OCC)C1C CDVAIHNNWWJFJW-UHFFFAOYSA-N 0.000 description 1
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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
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D7/00—Devices or methods for introducing solid, liquid, or gaseous remedies or other materials into or onto the bodies of animals
-
- 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
- A01K2207/00—Modified animals
- A01K2207/20—Animals treated with compounds which are neither proteins nor nucleic acids
-
- 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
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a construction method of a mouse model for inducing acute liver injury by injecting tunicamycin, which comprises the steps of injecting a prepared 0.1 mu g/mu l Tunicamycin (TM) solution into a mouse body in an intraperitoneal injection mode, killing the mouse by adopting a cervical vertebra dislocation method after 12h, and collecting blood and liver tissues for morphological, pathological and biochemical index analysis. The method has the advantages of simple operation, short modeling period, high modeling success rate and high bioavailability, provides a new idea for establishing the acute liver injury model mouse, provides an experimental tool for researching the pathogenesis of liver injury and disease progression, and provides a theoretical basis for preventing and treating clinical liver diseases.
Description
Technical Field
The invention belongs to the technical field of animal model construction, and particularly relates to a method for constructing a mouse model for inducing acute liver injury by intraperitoneal injection.
Background
Liver disease is one of the major health-threatening diseases, and has high morbidity and mortality. The liver injury can be caused to a certain extent by taking medicaments for a long time, inflammatory liver diseases (such as viral hepatitis, fatty hepatitis or alcoholic hepatitis), unhealthy living eating habits (long-term staying up all night, excessive drinking and high working pressure) and the like, and the liver cirrhosis, the liver cancer and even the death can be caused if the long-term liver injury is not effectively treated. The mouse is used as a conventional experimental animal, so that the establishment of a mouse model of acute liver injury is very important in the research of pathogenesis of liver diseases and the research and development of drugs.
Currently, the commonly used mouse models of acute liver injury include liver injury models induced by carbon tetrachloride (CCl4), alpha-naphthalene isothiocyanate (ANIT), 3, 5-diethoxycarbonyl-1, 4-dihydro-2, 4, 6-trimethylpyridine (DDC), Lipopolysaccharide (LPS)/D-galactosamine (D-GalN), acetaminophen (APAP) and the like. Different acute liver injury animal models have different biochemical and liver pathological characteristics, each animal model has advantages and disadvantages, and no completely ideal acute liver injury animal model exists. In addition, a novel modeling method for mice with endoplasmic reticulum stress-type liver injury is disclosed in the prior art, for example, a patent publication No. CN103735561A discloses that tunicamycin dimethyl sulfoxide solution is adopted as an endoplasmic reticulum stress inducer to irrigate the stomach of the mice, although the method can induce the occurrence of acute liver injury after administration for 8-48 h, the method adopts the method of intragastric administration, and the phenomenon that the bioavailability is reduced due to the first pass metabolism of the gastrointestinal tract can occur when the endoplasmic reticulum stress inducer is absorbed by the gastrointestinal tract.
Disclosure of Invention
The invention aims to provide a novel method for constructing a mouse model for inducing acute liver injury by injecting a tunicamycin solution into an abdominal cavity, which is simple and convenient to operate, short in modeling period, high in modeling success rate and high in mouse bioavailability. The technical scheme comprises the following steps:
1. preparing tunicamycin solution;
preparation of 150mM glucose solution: 810.72mg of glucose was precisely weighed and dissolved in 30ml of sterilized water, and dissolved by shaking.
② 0.1 μ g/μ l Tunicamycin (TM) solution preparation: 1mg of tunicamycin powder was precisely weighed and dissolved in 100. mu.l of Dimethylsulfoxide (DMSO), and 10ml of a 150mM glucose solution was added thereto, dissolved by shaking, and stored at 4 ℃.
Preparation of 1% DMSO control solution: mu.l of dimethyl sulfoxide (DMSO) solution was added to 10ml of 150mM glucose solution, dissolved with shaking, and stored at 4 ℃.
And fourthly, randomly dividing the 7-week-old C57BL/6 mice into a control group and an experimental group according to the weight, wherein each group comprises 12 mice, the experimental group starts to inject the TM solution (100 mu l/10g) prepared in the step III into the abdominal cavity, meanwhile, the control group injects the 1% DMSO control solution (100 mu l/10g) prepared in the step III into the abdominal cavity, and the mice are sacrificed and taken after 12 hours.
Observing the liver shape and pathological diagram and detecting the biochemical indexes related to liver damage.
Advantageous effects
The invention is a method for constructing mice model for inducing acute liver injury by intraperitoneal injection of tunicamycin solution, and the method better avoids the first-pass metabolism phenomenon of gastrointestinal tract due to the adoption of the intraperitoneal injection method, so that the establishment of the mice model for acute liver injury can be completed by using lower dose of tunicamycin (1 microgram/g) within a short period (12 hours), a new thought is provided for establishing the mice model for acute liver injury, an experimental tool is provided for researching the pathogenesis of liver injury and disease progression, and a theoretical basis is provided for the prevention and treatment of clinical liver diseases.
Detailed Description
The invention relates to a method for constructing a mouse model for inducing acute liver injury by injecting a tunicamycin solution into an abdominal cavity, which comprises the following specific implementation modes:
preparation of a solution:
preparation of 150mM glucose solution: 810.72mg of glucose powder were precisely weighed into a 50ml sterile EP tube using an analytical balance, and 30ml of sterilized water was added to the EP tube and dissolved with shaking.
Preparation of 0.1. mu.g/. mu.l Tunicamycin (TM) solution: 1mg of tunicamycin powder was precisely weighed on an analytical balance and dissolved in 100. mu.l of dimethyl sulfoxide (DMSO), 10ml of a 150mM glucose solution was added, and the solution was dissolved by shaking and stored at 4 ℃.
Preparation of 1% DMSO control: mu.l of dimethyl sulfoxide (DMSO) solution was added to 10ml of 150mM glucose solution, dissolved with shaking, and stored at 4 ℃.
② selection of mice
A batch of 6-week-old C57BL/6 mice are purchased from a company, the weight of the mice is 18g-20g, the mice are all SPF-grade, the mice are raised in an SPF-grade animal room for one week, the raising environment is 25 ℃, the humidity is 50% -55%, the mice are 12h dark under light for 12h, and the mice can freely eat and drink water. One week later, the animals were divided into two groups of 12 animals by body weight.
③ Abdominal injection
The drug is aspirated and the air bubbles are removed. The needle head can be upwards used for sucking a section of gas, and then the gas is slowly discharged, so that the effect of taking out bubbles is achieved. The tail of the mouse is held by one hand and pulled backwards, and the scalp and back fur of the mouse are held by the thumb and the forefinger of the other hand, and the abdominal cavity is upward. The needle point of the syringe which absorbs liquid in advance faces upwards, and after the syringe is parallelly pricked into the skin, the syringe is pricked into the abdominal cavity at an angle of 45 degrees with the abdominal cavity, and the sample is injected after the needle point part is sensed to be movable.
Administration to control group
Control groups each mouse was weighed and body weight recorded and dosed by body weight, 100 μ l of a formulated 1% DMSO control solution was i.p. per 10g weight. After administration for 12h, mice were sacrificed by cervical dislocation after removal of the eyeball to collect blood, and the mouse liver was collected.
Administration of experimental group
Experimental groups Each mouse was weighed and body weight was recorded, and administered according to body weight by intraperitoneal injection of 100. mu.l of a prepared TM solution at a concentration of 0.1. mu.g/. mu.l per 10g weight. After administration for 12h, mice were sacrificed by cervical dislocation after removal of the eyeball to collect blood, and the mouse liver was collected.
Liver analysis
Liver morphology: the collected mouse livers were washed with pre-cooled PBS buffer and then photographed on a petri dish to record morphology.
Liver pathology: the middle part of the fresh liver of the mouse is firstly put into an embedding box and soaked in 4 percent PFA overnight, and after the overnight, the mouse is dehydrated by an ethanol gradient dehydration method according to the following procedure.
Next, the dehydrated tissue was taken out, embedded in a paraffin embedding machine, and then cut into a tissue having a thickness of 4 μm with a paraffin microtome. Hematoxylin-eosin (H & E) staining was performed according to the following table procedure.
And finally, sealing the piece by using neutral gum after dyeing, wherein air bubbles are avoided in the sealing process, and the piece is dried by blowing, so that the piece can be observed under a microscope and photographed.
And (3) detecting blood biochemical indexes: mu.l of heparin sodium was added to 1.5ml of sterilized EP in advance, and the tube bottom was flicked with a finger before use to uniformly disperse the heparin sodium on the tube wall. Blood is taken by an eyeball-picking method before the mice are taken, so that the blood coagulation phenomenon is avoided. Standing on ice for 30min after blood is taken out, centrifuging at 4 ℃ for 15min at 3000rpm, sucking supernatant fluid to obtain blood plasma, and detecting ALT and AST according to the method steps of the blood plasma alanine Aminotransferase (ALT) and glutamic acid Aminotransferase (AST) kit specification.
According to the final analysis of the invention, the liver of the experimental group is yellow, large and soft in shape, and part of the liver is in a yellow-white stripe shape; h & E staining pathology shows that the liver cord of the experimental group is disordered and the cytoplasm is sparse; the biochemical indexes show that AST and ALT in blood plasma are obviously increased. Therefore, the mouse model of acute liver injury is successfully constructed by injecting the tunicamycin solution into the abdominal cavity.
Therefore, the construction method of the mice model for inducing acute liver injury by intraperitoneal injection of tunicamycin according to the method is simple and convenient to operate, short in modeling period, high in modeling success rate and high in bioavailability of the mice.
The above description is only one embodiment of the present invention, and it should be noted that, for those skilled in the art, several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. The construction method of the tunicamycin injection induced acute liver injury mouse model is characterized by comprising the following steps:
1) preparing tunicamycin solution;
1-1) preparation of a 150mM glucose solution: accurately weighing 810.72mg of glucose, dissolving in 30ml of sterilized water, and shaking for dissolution;
1-2) preparation of 0.1. mu.g/. mu.l tunicamycin TM solution: precisely weighing 1mg tunicamycin powder, dissolving in 100 μ l dimethyl sulfoxide (DMSO), adding 10ml 150mM glucose solution, shaking for dissolving, and storing at 4 deg.C;
1-3) preparation of 1% DMSO control: adding 100 μ l dimethyl sulfoxide DMSO solution into 10ml 150mM glucose solution, shaking for dissolving, and storing at 4 deg.C;
2) randomly dividing 7-week-old C57BL/6 mice into a control group and an experimental group according to the weight, wherein the quantity of each group is distributed as required, the experimental group starts to inject 100 mu l/10g of TM solution prepared in the step 1-2) into the abdominal cavity, meanwhile, the control group injects 100 mu l/10g of 1% DMSO control solution prepared in the step 1-3) into the abdominal cavity, and the materials are sacrificed after 12 hours;
3) observing the liver morphology and pathological diagram and detecting the biochemical indexes related to the liver injury.
2. The method according to claim 1, wherein the mice in step 2) have a body weight of 18g to 20g, all of SPF grade, and are kept in an SPF grade animal room for one week at a temperature of 25 ℃, a humidity of 50% to 55%, 12h in the dark and 12h in the light, free access to food and water, and randomly divided into control groups by body weight after one week.
3. The method of claim 1, wherein the step of intraperitoneally injecting in step 2) comprises the steps of: sucking the medicine and removing bubbles; the needle head is upward, after a section of gas is sucked, the gas is slowly discharged, so that the effect of taking out bubbles is achieved; gripping the tail of the mouse with one hand and pulling backwards, gripping the scalp and back fur of the mouse with the thumb and the forefinger of the other hand, and making the abdominal cavity upwards; after the needle point of the syringe which previously sucks liquid is upwards arranged and is parallelly pricked into the skin, the syringe is pricked into the abdominal cavity at an angle of 45 degrees with the abdominal cavity, and the needle point part is sensed to move, so that a sample is injected.
4. The method of claim 1, wherein in step 2), the control group is administered as follows:
each mouse was weighed and body weight recorded, dosed according to body weight, 100. mu.l of a formulated DMSO control solution of 1% concentration was intraperitoneally injected per 10g weight, the mice were sacrificed by cervical dislocation after removal of eyeball to collect blood 12 hours after dosing, and mouse livers were collected.
5. The method of claim 1, wherein the experimental group is administered in step 2) as follows:
each mouse was weighed and body weight was recorded, and administered according to the body weight, 100. mu.l of a prepared TM solution having a concentration of 0.1. mu.g/. mu.l was intraperitoneally injected per 10g weight, and the mice were sacrificed by cervical dislocation after blood collection by removing eyeballs after 12 hours of administration, and mouse livers were collected.
6. The method of claim 1, wherein in step 3), the liver analysis is specifically as follows;
cleaning the collected mouse liver with precooled PBS buffer solution, and then placing the washed mouse liver on a culture dish to photograph and record the shape;
liver pathology: firstly, filling the middle part of a fresh liver of a mouse into an embedding box, soaking the middle part of the fresh liver of the mouse in 4% PFA overnight, dehydrating the middle part of the fresh liver of the mouse by adopting an ethanol gradient dehydration method after the overnight, and dehydrating the middle part of the fresh liver of the mouse according to the procedure in the following table 1;
TABLE 1
Secondly, taking out the dehydrated tissue, embedding the tissue by using a paraffin embedding machine, and cutting the tissue with the thickness of 4 mu m by using a paraffin slicer; hematoxylin-eosin H & E staining was performed according to the following table 2 steps;
TABLE 2
And finally, sealing the piece by using neutral gum after dyeing, wherein air bubbles are avoided in the sealing process, drying the piece by blowing, and observing and taking a picture under a microscope.
7. The method of claim 1, wherein in step 3), the blood biochemical indicator is detected as follows:
adding 5 μ l heparin sodium into sterilized 1.5ml EP in advance, and flicking the tube bottom with finger before use to uniformly disperse heparin sodium on the tube wall; blood is taken by an eyeball picking method before the mice are taken, so that the blood coagulation phenomenon is avoided; standing on ice for 30min after blood is taken out, centrifuging at 4 ℃ for 15min at 3000rpm, sucking supernatant fluid to obtain blood plasma, and detecting ALT and AST according to the method steps of the blood plasma alanine ALT and glutamic acid aspartate AST kit specification.
8. The method of claim 1, wherein the observation of liver morphology in step 3) is specifically as follows: from the aspect of liver morphology, the liver of the experimental group is yellow, large and soft, and part of the liver is in a yellow-white alternate stripe shape; h & E staining pathology shows that the liver cord of the experimental group is disordered and the cytoplasm is sparse; the biochemical indexes show that AST and ALT in blood plasma are obviously increased, and at the moment, the mouse model for inducing acute liver injury is successfully constructed by injecting the tunicamycin solution into the abdominal cavity.
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