CN109646453A - A kind of foundation and detection method of hepatic injury er stress differential expression model mice - Google Patents
A kind of foundation and detection method of hepatic injury er stress differential expression model mice Download PDFInfo
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Abstract
The present invention provides the foundation and detection method of a kind of hepatic injury er stress differential expression model mice, belong to animal model technical field.By 2 groups of gender, growth week old and weight identical BALB/c mouse point, acute group is administered once, and chronic group of weekly administration twice, is divided into 72h between being administered twice, and successive administration 12~16 weeks；2 groups of mouse are put to death after administration, extract the hepatic tissue of two groups of mouse respectively；GRP78 and CHOP expression in murine liver tissue is measured respectively, calculates the ratio of two groups of CHOP/GRP78；When chronic group of CHOP/GRP78 ratio is significantly higher than acute group, chronic group of mouse is hepatic injury er stress differential expression model mice.The present invention provides raw material to study the pathogenesis of acute and chronic hepatic injury and disease progression, provides fundamental basis for clinical preventing, treating for hepatopathy, has the advantages that method is easy, modeling success rate is high, favorable repeatability.
Description
Technical field
The invention belongs to animal model constructing technology fields, and in particular to a kind of hepatic injury er stress differential expression
The foundation and detection method of model mice.
Background technique
Hepatic injury is caused by Different types of etiopathogenises
The clinical syndrome of the liver dysfunction of feature, the death rate are high.And necrosis of liver cells is the common pathological characteristic of various hepatic injury,
It include: necrosis of liver cells (necrosis), procedural downright bad (necroptosis), apoptosis (apoptosis) and autophagy
(autophagyd).Liver disease progress, i.e., hepatocellular injury is related with many factors, under the effect of various pathogenic factors, liver
Cell maintains cell homeostasis by starting a variety of protection mechanisms, promotes cell survival.Therefore the antibody Monoclonal of liver cell itself
Ability and impairment factor codetermine the final result of hepatopathy.
The treatment method that most patients with liver deficiencies preferentially select is Experience of Combined Treatment of Internal Medicine, however, due to pathogenesis
It is unclear that medical treatment effect is limited to, the death rate is 50~80%.Experimental animal hepatic injury er stress otherness table
It is an indispensable tool in the pathogenesis and therapeutic agent screening for study various liver diseases up to model.State in recent years
Inside and outside manufacture Experimental Hepatic Damage Animal models are concentrated mainly on chemical liver injury and Immune liver injury.
Immune liver injury mostly uses the modelings such as concanavalin A (Con), lipopolysaccharides (BCG+LPS)；Chemical liver injury master
If by carbon tetrachloride (CCl4), D-Gal (D-Gal), ethyl alcohol, cause the induction such as flavine.In addition, public in the prior art
A kind of novel modeling method is opened, such as the patent of publication number CN103735561A is disclosed using tunicamycin dimethyl sulfoxide
As er stress inducer, to intragastric administration on mice, the foundation of completion animal model after 8~48h is administered in solution, though the method
It so will appreciate that self regulating and control ability of the cell under stress situation, moreover it is possible to hepatopathy pathogenesis is further appreciated that, thus to formulation
New intervention, remedy measures help with higher.But this method does not provide the mouse using the processing of above-mentioned medication
Whether mouse liver injury model is really formed, using the enzyme activity of AST and ALT in measurement administration mouse liver, measures liver
Weight and liver index and do pathological section, it is seen that although above scheme discloses the method for building up of mouse liver injury models, but
It is that its subsequent verifying step is more complex, measurement content is larger, and the judgment method of measurement result does not provide specific judgement
Standard, the difficult judgment for unfamiliar researcher.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of novel hepatic injury er stress differential expression models
The foundation and detection method of mouse, the method is not only easy to operate, it is low to require operator, but also it is high to model success rate.
The foundation and detection method of a kind of hepatic injury er stress differential expression model mice provided by the invention, packet
Include following steps:
1) BALB/c mouse for randomly selecting identical gender, similar growth week old and weight is divided into 2 groups, according to medication
It is divided into acute group or chronic group；Acute group is administered once, and mouse is put to death in administration afterwards for 24 hours；Chronic group of weekly administration twice, is given twice
The interval time of medicine is 72h, and successive administration put to death mouse after 12~16 weeks；The drug of administration is er stress inducer；
2) respectively extraction step 1) in two groups of mouse hepatic tissue, obtain acute group of murine liver tissue and chronic group of Mouse Liver
Tissue；
3) the expression water of GRP78 and CHOP in the acute group of murine liver tissue and chronic group of murine liver tissue is measured respectively
It is flat, calculate the ratio of acute group of CHOP/GRP78 and the ratio of chronic group of CHOP/GRP78；
4) acute group of CHOP/GRP78 ratio situation of change compared with the ratio of chronic group of CHOP/GRP78 is analyzed, when chronic
When group CHOP/GRP78 ratio is significantly higher than the ratio of acute group of CHOP/GRP78, chronic group of mouse is hepatic injury er stress
Differential expression model mice.
Preferably, er stress inducer is CCl in step 1)4Olive oil mixed solution；The CCl4Olive oil mixing
CCl in solution4Volume ratio with olive oil is 1: 3~5.
Preferably, the CCl4The dosage of olive oil mixed solution is 5~10ml/Kg mouse.
Preferably, in step 3) in acute group of murine liver tissue and chronic group of murine liver tissue GRP78 and CHOP expression
Horizontal measuring method includes that GRP78 or CHOP is formed band using immunoblotting, measurement GRP78 or CHOP band
Gray value indicates acute group of CHOP/GRP78 expression ratio and chronic group with the gray value ratio of the CHOP and GRP78
The ratio of CHOP/GRP78 expression.
Preferably, the weight of BALB/c mouse includes 23~27g in step 1).
Preferably, in step 1) in acute group or chronic group every group be not less than 8 BALB/c mouses.The present invention provides
A kind of foundation and detection method of hepatic injury er stress differential expression model mice, are divided into acute group according to administration time
Hepatic injury and chronic group of hepatic injury, this is the experimental animal of the unmentioned hepatic injury er stress differential expression of the prior art
Then model detects GRP78 and CHOP albumen and is expressed as central evaluation index, wherein GRP78 albumen is that growth-promoting deposits mark egg
White, expression indication hepatic injury is improved, and CHOP albumen is apoptosis marker protein, and expression indication hepatic injury aggravates；Together
When set the judgment criteria for being successfully established hepatic injury er stress differential expression model mice as chronic group of CHOP/
When GRP78 ratio is significantly higher than the ratio of acute group of CHOP/GRP78, chronic group of mouse is hepatic injury er stress otherness
Expression model mouse.Method provided by the invention has the characteristics that Detection accuracy 100%, to establish hepatic injury er stress
Differential expression model mice provides new approaches, while to study the pathogenesis of acute and chronic hepatic injury and disease progression
Experimental tool is provided, is provided fundamental basis for clinical preventing, treating for hepatopathy.
Meanwhile foundation provided by the invention and detection method, this there is condition to require low, method is easy, stability is good, builds
The advantages that mould success rate height, favorable repeatability；With the expression of Western blot detection GRP78 and CHOP, there is operation
The characteristics of simplicity, favorable repeatability.
Detailed description of the invention
Fig. 1 is to establish CCl in embodiment4The flow chart of guidance model mouse er stress differential expression；
Fig. 2 is immunoblotting (Western blot) result figure and statistical chart of model group in embodiment；
Fig. 3 is GRP78 and CHOP expressing quantity and the column diagram of CHOP/GRP78 value in model group in embodiment.
Specific embodiment
The foundation and detection method of a kind of hepatic injury er stress differential expression model mice provided by the invention, packet
Include following steps:
1) BALB/c mouse for randomly selecting identical gender, similar growth week old and weight is divided into 2 groups, according to medication
It is divided into acute group or chronic group；Acute group of medication is that administration number of times is that once, mouse is put to death in administration afterwards for 24 hours；Chronic group
Medication be weekly administration twice, the interval time being administered twice is 72h, and successive administration puts to death mouse after 12~16 weeks；
The drug of administration is er stress inducer；
2) respectively extraction step 1) in two groups of mouse hepatic tissue, obtain acute group of murine liver tissue and chronic group of Mouse Liver
Tissue；
3) the expression water of GRP78 and CHOP in the acute group of murine liver tissue and chronic group of murine liver tissue is measured respectively
It is flat, calculate the ratio of acute group of CHOP/GRP78 and the ratio of chronic group of CHOP/GRP78；
4) acute group of CHOP/GRP78 ratio situation of change compared with the ratio of chronic group of CHOP/GRP78 is analyzed, when chronic
When group CHOP/GRP78 ratio is significantly higher than the ratio of acute group of CHOP/GRP78, chronic group of mouse is hepatic injury er stress
Differential expression model mice.
The present invention provides the BALB/c mouses for randomly selecting identical gender, similar growth week old and weight to be divided into 2 groups, root
It is divided into acute group or chronic group according to medication；Acute group of medication is that administration number of times is once, after administration for 24 hours；It is chronic
Group medication be weekly administration twice, the interval time being administered twice be 72h, successive administration 12~16 weeks；The medicine of administration
Object is er stress inducer.
In the present invention, er stress inducer is preferably CCl4Olive oil mixed solution；The CCl4Olive oil mixing
CCl in solution4Volume ratio with olive oil is preferably 1: 3~5, and more preferably 1: 4.The CCl4Olive oil mixed solution is preferred
With 0.22 micron of bacteria filter bacteriological filtration.The CCl4The dosage of olive oil mixed solution is preferably 5~10ml/Kg mouse.
In the present invention, the medication preferably includes following steps: at mouse lower abdomen is from hunter's line about 0.5cm
Injection is needled into subcutaneously, along subcutaneously 3~5cm is pushed ahead, then makes syringe needle and mouse web portion about at 30 ° of piercing abdominal cavities, needle
The speed that head is pierced into is fast, disappears moment wait resist sense, illustrate syringe needle benefit enter it is intraperitoneal, generally not into the syringe needle behind abdominal cavity
More than 1cm, mouse head is in low level when injection, and tail portion lifts slightly, so that internal organ is moved forward, avoid being injected into internal organs.Injection is matched at this time
The CCl made4Olive oil mixed solution, nothing by mouth 12h before being administered.
In the present invention, the weight of BALB/c mouse preferably includes 23~27g.Preferably every group in acute group or chronic group
Not less than 8 BALB/c mouses.
After injection, the present invention puts to death 2 groups of BALB/c mouses, extracts two groups of murine liver tissues respectively, obtains acute group of mouse
Hepatic tissue and chronic group of murine liver tissue.
In the present invention, the method for the execution preferably after extracing eyeball and draining blood put to death by cervical dislocation.It mentions
The method for taking hepatic tissue is as follows: fresh liver tissue block 50mg, is dissolved in 5ml lysate, sufficiently grinds even with homogenizer, in 4 DEG C of items
The revolving speed of 12000r/min is centrifuged 5min under part, extracts supernatant and mixes with the volume ratio of albumen sample-loading buffer 4: 1, boiling water boiling
3~5min obtains hepatic injury albumen.
After obtaining hepatic injury albumen, the present invention measures the acute group of murine liver tissue and chronic group of murine liver tissue respectively
The expression of middle GRP78 and CHOP calculates the ratio of acute group of CHOP/GRP78 and the ratio of chronic group of CHOP/GRP78.
In the present invention, marker protein (GRP78) and apoptosis are deposited using Western blot method normal process detection growth-promoting
The level of marker protein (CHOP).The case where GRP78 indicates liver regeneration, and CHOP indicates apoptosis, both liver itself has
Have, when liver is in er stress, their expression can change, and mouse hepatopathy process is prompted to improve or become
It is bad.It is preferred that with immunoblotting (Western blot) detection GRP78 and CHOP expression, measure each group GRP78 or
The gray value of CHOP protein band, with the gray value ratio of each group CHOP and GRP78 indicate acute group of CHOP/GRP78 ratio and
The ratio of chronic group of CHOP/GRP78.
After obtaining the ratio of acute group of CHOP/GRP78 ratio and chronic group of CHOP/GRP78, the present invention analyzes acute group
CHOP/GRP78 ratio situation of change compared with the ratio of chronic group of CHOP/GRP78, when chronic group of CHOP/GRP78 ratio is significant
When ratio higher than acute group of CHOP/GRP78, chronic group of mouse is hepatic injury er stress differential expression model mice.
When chronic group of CHOP/GRP78 ratio is significantly higher than the ratio of acute group of CHOP/GRP78, chronic liver injury endoplasmic reticulum is prompted to answer
Swash and promote apoptotic response enhancing, completes the foundation and detection of hepatic injury er stress differential expression model mice.
Accurate testing result in order to obtain, the above experimental procedure are preferably repeated 3 times.It reacts from mouse activity as it can be seen that acute
The activity of group mouse, reaction are as usual, and appetite is as usual, and breathing is steady, no death, and chronic group of mouse shows as activity, slow in reacting,
Appetite is deteriorated, and ascites occurs, or even dead；CCl is injected as seen from Figure 24For 24 hours, GRP78 and CHOP are expressed olive oil mixed solution
It increases, injects CCl4Olive oil mixed solution 12~16 weeks, GRP78 expression significant raising of CHOP expression without significant change, than
Compared with CHOP/GRP78 ratio variation, chronic liver injury group CHOP/GRP78 ratio be apparently higher than acute liver damage group (P≤
0.05), prompt chronic liver injury er stress promotees apoptotic response enhancing, exists and promotees survival signaling to the difference for promoting apoptotic signal
Property conversion.
Below with reference to embodiment to a kind of hepatic injury er stress differential expression model mice provided by the invention
It establishes and detection method is described in detail, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
(1) mouse selects
Subsidiary company buys the BALB/c mouse that a collection of weight is 23~27g, is divided into three groups, and every group includes 24,24
BALB/c mouse is divided into acute group, chronic group and control group again.
(2)CCl4Olive oil mixed solution is prepared
By 1ml CCl4It is completely dissolved in 4ml olive oil, and with 0.22 micron of bacteria filter bacteriological filtration, under the conditions of room-temperature sterile
It stores spare.
(3) administration group is administered
12h fasting before administration.Acute group of medication is injection CCl4Olive oil mixed solution (CCl4And olive oil
Volume ratio is 1: 4) once, cervical dislocation puts to death mouse after extracing eyeball and draining blood afterwards for 24 hours for administration；Chronic group of administration
Twice for weekly administration, the interval time being administered twice is 72h to method, successive administration 12~16 weeks, is administered after terminating through extracing
Cervical dislocation puts to death mouse after eyeball drains blood；Control group is not to be administered, the cervical dislocation after extracing eyeball and draining blood
Method puts to death mouse.The method of the administration is as follows: injection is needled into subcutaneously from hunter's line about 0.5cm in mouse lower abdomen,
Along subcutaneously 3~5cm is pushed ahead, syringe needle and mouse web portion is then made about to be pierced into abdominal cavity at 30 ° of angles, the speed that syringe needle is pierced into is wanted
Fastly, it disappears moment wait resist sense, illustrates that benefit enters intraperitoneal syringe needle, is usually no more than 1cm into the syringe needle behind abdominal cavity, when injection
Mouse head is in low level, and tail portion lifts slightly, so that internal organ is moved forward, avoid being injected into internal organs.
4) the extractant measurement of marker protein
Drug is injected for 24 hours and after 12~16 weeks, cervical dislocation is put to death above-mentioned mouse and taken after extracing eyeball and draining blood
Fresh liver tissue block 50mg, is dissolved in 5ml lysate, sufficiently grinds even with homogenizer, in 4 DEG C of centrifugation 5min (12000r/min),
It extracts supernatant to mix with the volume ratio of albumen sample-loading buffer 4: 1, boiling water boiling to 3~5min.By Western blot method mark
Quasi- process detection growth-promoting deposits the level of marker protein (GRP78) and apoptosis marker protein (CHOP), and wherein GRP78 indicates liver again
The case where raw, and CHOP indicates apoptosis, both liver itself has, when liver is in er stress, they
Expression can change, and prompt mouse hepatopathy process to improve or degenerate, using β-Acatin albumen as internal reference.Use Western blotting
Method (Western blot) detection, by analyzing corresponding protein band gray value, compare the ratio variation of CHOP/GRP78.With
Upper experiment is repeated 3 times.It the results are shown in Table 1.
The expression quantity of 1 different time marker protein of table
By acute group and chronic group of CHOP/GRP78 mean value discovery for statistical analysis, chronic liver injury group CHOP/
GRP78 ratio is apparently higher than acute liver damage group (P≤0.05), is prompted chronic liver injury er stress to promote apoptotic response and is increased
By force, the foundation and detection of hepatic injury er stress differential expression model mice are completed.
From mouse activity reaction as it can be seen that acute group of mouse activity, reaction are as usual, appetite is as usual, and breathing is steady, no death,
And chronic group of mouse shows as activity, slow in reacting, appetite variation, ascites occurs, or even dead；CCl is injected as seen from Figure 24
For 24 hours, GRP78 and CHOP expression increase olive oil mixed solution, inject CCl4Olive oil mixed solution 12~16 weeks, GRP78 table
Up to no significant change, the significant raising of CHOP expression, the ratio for comparing CHOP/GRP78 change, chronic liver injury group CHOP/
GRP78 ratio is apparently higher than acute liver damage group (P≤0.05), is prompted chronic liver injury er stress to promote apoptotic response and is increased
By force, exist and promote survival signaling to the otherness conversion for promoting apoptotic signal.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (6)
1. a kind of foundation and detection method of hepatic injury er stress differential expression model mice, which is characterized in that including
Following steps:
1) BALB/c mouse for randomly selecting identical gender, similar growth week old and weight is divided into 2 groups, is divided into according to medication
Acute group or chronic group；Acute group is administered once, and mouse is put to death in administration afterwards for 24 hours；Chronic group of weekly administration twice, is administered twice
Interval time is 72h, and successive administration put to death mouse after 12~16 weeks；The drug of administration is er stress inducer；
2) respectively extraction step 1) in two groups of mouse hepatic tissue, obtain acute group of murine liver tissue and chronic group of Mouse Liver group
It knits；
3) expression of GRP78 and CHOP in the acute group of murine liver tissue and chronic group of murine liver tissue is measured respectively,
Calculate the ratio of acute group of CHOP/GRP78 and the ratio of chronic group of CHOP/GRP78；
4) acute group of CHOP/GRP78 ratio situation of change compared with the ratio of chronic group of CHOP/GRP78 is analyzed, when chronic group
When CHOP/GRP78 ratio is significantly higher than the ratio of acute group of CHOP/GRP78, chronic group of mouse is that hepatic injury er stress is poor
Anisotropic expression model mouse.
2. foundation according to claim 1 and detection method, which is characterized in that er stress inducer is in step 1)
CCl4Olive oil mixed solution；The CCl4CCl in olive oil mixed solution4Volume ratio with olive oil is 1: 3~5.
3. foundation according to claim 2 and detection method, which is characterized in that the CCl4Olive oil mixed solution is given
Dose is 5~10ml/Kg mouse.
4. foundation according to claim 1 and detection method, which is characterized in that in step 3) acute group of murine liver tissue and
The measuring method of the expression of GRP78 and CHOP includes using immunoblotting by GRP78 in chronic group of murine liver tissue
Or CHOP forms band, measures the gray value of GRP78 or CHOP band, indicates acute with the gray value ratio of CHOP and GRP78
The ratio of group CHOP/GRP78 expression ratio and chronic group of CHOP/GRP78 expression.
5. foundation and detection method described in any one according to claim 1~4, which is characterized in that BALB/c in step 1)
The weight of mouse includes 23~27g.
6. foundation and detection method described in any one according to claim 1~4, which is characterized in that acute in step 1)
It is not less than 8 BALB/c mouses for every group in group or chronic group.
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| CN113368094A (en) * | 2021-05-11 | 2021-09-10 | 广州中医药大学(广州中医药研究院) | Method for establishing chronic jaundice model |
| CN116270603A (en) * | 2023-03-24 | 2023-06-23 | 黑龙江八一农垦大学 | Experimental method for relieving liver injury caused by cold stress by using daidzein |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113368094A (en) * | 2021-05-11 | 2021-09-10 | 广州中医药大学(广州中医药研究院) | Method for establishing chronic jaundice model |
| CN116270603A (en) * | 2023-03-24 | 2023-06-23 | 黑龙江八一农垦大学 | Experimental method for relieving liver injury caused by cold stress by using daidzein |
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