CN113647359A

CN113647359A - Construction of a mouse model of acute liver injury induced by tunicamycin

Info

Publication number: CN113647359A
Application number: CN202110933719.0A
Authority: CN
Inventors: 杨兰香; 黄昭; 郭豪杰; 程旺开; 熊冉; 张佳佳
Original assignee: Wuhu Institute of Technology
Current assignee: Wuhu Institute of Technology
Priority date: 2021-08-15
Filing date: 2021-08-15
Publication date: 2021-11-16

Abstract

本发明公开了注射衣霉素诱导急性肝损伤小鼠模型的构建方法，将配置好的浓度为0.1μg/μl衣霉素(TM)溶液通过腹腔注射的给药方式注射到小鼠体内，12h后采用颈椎脱臼方法处死小鼠并收集血液、肝脏组织进行形态、病理及生化指标分析。该方法不仅操作简便、建模周期短、建模成功率高、生物利用率高，而且为建立急性肝损伤模型小鼠提供了新思路，同时为研究肝损伤及病情进展的发病机制提供实验工具，为临床肝病的预防、治疗提供理论基础。The invention discloses a method for constructing a mouse model of acute liver injury induced by injection of tunicamycin. The prepared concentration of tunicamycin (TM) solution of 0.1 μg/μl is injected into the mouse body by intraperitoneal injection for 12 hours. Afterwards, the mice were sacrificed by cervical dislocation, and blood and liver tissues were collected for morphological, pathological and biochemical analysis. This method is not only easy to operate, has a short modeling period, high modeling success rate, and high bioavailability, but also provides a new idea for establishing acute liver injury model mice, as well as an experimental tool for studying the pathogenesis of liver injury and disease progression. , to provide a theoretical basis for the prevention and treatment of clinical liver disease.

Description

Construction method of tunicamycin injection induced acute liver injury mouse model

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

1. the construction method of the mouse model of acute liver injury induced by injection tunicamycin is characterized in that, the following steps are required:

1) Preparation of tunicamycin solution;

1-1) Preparation of 150 mM glucose solution: Precisely weigh 810.72 mg of glucose and dissolve it in 30 ml of sterilized water, and shake to dissolve;

1-2) Preparation of 0.1 μg/μl tunicamycin TM solution: Precisely weigh 1 mg of tunicamycin powder and dissolve it in 100 μl dimethyl sulfoxide (DMSO), then add 10 ml of 150 mM glucose solution, shake to dissolve, and store at 4°C ;

1-3) Preparation of 1% DMSO control solution: add 100 μl of dimethyl sulfoxide DMSO solution to 10 ml of 150 mM glucose solution, shake to dissolve, and store at 4°C;

2) The 7-week-old C57BL/6 mice were randomly divided into two groups according to their body weight, the control group and the experimental group, and the number of each group was allocated as needed. At the same time, the control group was intraperitoneally injected with 100 μl/10 g of the 1% DMSO control solution prepared in steps 1-3), and sacrificed 12 hours later to collect the materials;

3) Observe the liver morphology, pathological map and detect the biochemical indicators related to liver injury.

2. method as claimed in claim 1, is characterized in that, the weight of mouse is 18g-20g in step 2), is SPF grade, is raised in SPF grade animal room for one week, and raising environment is that temperature 25 ℃, humidity are 50 ℃ %-55%, 12 hours of light and 12 hours of darkness, free food and water, and randomly divided into control group according to body weight after one week.

3. method as claimed in claim 1, it is characterised in that step 2) in the step during intraperitoneal injection: suction medicine, and remove air bubbles; needle upward, after drawing a section of gas, slowly exhaust gas to achieve the removal of air bubbles Grab the tail of the mouse with one hand and pull it back, while the thumb and index finger of the other hand grab the fur on the back of the mouse's head, with the abdominal cavity facing upwards; place the needle of the syringe with the liquid that has been drawn in advance on the plane and parallel to the top. After being inserted into the skin, the syringe is inserted into the abdominal cavity at an angle of 45 degrees to the abdominal cavity, and the needle tip is felt to move, and the sample is injected.

4. method as claimed in claim 1, is characterized in that, in step 2) in, control group administration is as follows:

Each mouse was weighed and recorded the body weight, and the mice were administered according to their body weight. 100 μl of the prepared DMSO control solution with a concentration of 1% was injected intraperitoneally for each 10 g of weight. mice, and the mouse livers were collected.

5. method as claimed in claim 1 is characterized in that, in step 2), experimental group administration is specifically as follows:

Each mouse was weighed and recorded the body weight. The administration was based on body weight. 100 μl of the prepared TM solution with a concentration of 0.1 μg/μl was injected intraperitoneally for every 10 g of weight. Mice were sacrificed, and mouse livers were collected.

6. The method of claim 1, wherein in step 3), the liver analysis is as follows;

After the collected mouse liver was washed with pre-cooled PBS buffer, it was placed on a petri dish to take pictures and record the morphology;

Liver pathology: First, take the middle part of the fresh liver of mice and put it into a embedding box and soak it in 4% PFA overnight. After overnight, dehydrate by ethanol gradient dehydration method, and dehydrate it according to the procedure in Table 1;

Table 1

Next, take out the dehydrated tissue, embed the tissue with a paraffin embedding machine, and then cut the tissue with a thickness of 4 μm with a paraffin microtome; perform hematoxylin-eosin H&E staining according to the steps in Table 2;

Table 2

Finally, after dyeing, seal the slides with neutral gum. Pay attention to avoid air bubbles during the process of sealing, just blow dry, observe and take pictures under a microscope.

7. method as claimed in claim 1, is characterized in that, in step 3) in, blood biochemical index detection is as follows:

Add 5 μl of heparin sodium to sterilized 1.5 ml of EP in advance, flick the bottom of the tube with your finger before use, so that the heparin sodium is evenly dispersed on the wall of the tube; before the mice are drawn, the eyeball method is used to collect blood to avoid coagulation; After the blood was finished, let it stand on ice for 30min, centrifuge at 3000rpm for 15min at 4°C, and draw the supernatant to obtain the plasma. ALT and AST were carried out according to the method steps of the plasma glutamate ALT and glutamate AST kit instructions. detection.

8. method as claimed in claim 1, it is characterised in that, in step 3), observe liver morphology as follows: from liver morphology, experimental group liver is yellow, large and soft, and some areas are yellow and white striped The pathological results of H&E staining showed that the liver cord was disordered and the cytoplasm was sparse in the experimental group. Biochemical indexes showed that the plasma AST and ALT increased significantly. At this time, the mouse model of acute liver injury induced by intraperitoneal injection of tunicamycin solution was successfully established.