CN108310368A - A method of structure liver humanized mouse model - Google Patents
A method of structure liver humanized mouse model Download PDFInfo
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- CN108310368A CN108310368A CN201710027143.5A CN201710027143A CN108310368A CN 108310368 A CN108310368 A CN 108310368A CN 201710027143 A CN201710027143 A CN 201710027143A CN 108310368 A CN108310368 A CN 108310368A
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Abstract
The invention belongs to biomedical sectors, it is related to a kind of construction method of humanization liver mosaic mouse model, including, building has the ADSB mouse of inducible liver damage and immunodeficient pheno, and after so that the experiment mice is formed liver damage by diphtheria toxin intervention, carry out that human liver cell is chimeric to build to obtain people's liver chimeric mouse model；The method of the present invention constructs the ADSB mouse with inducible liver damage and immunodeficient pheno, feasible animal model support can be provided for liver transfer operation research by building the humanization liver mouse model obtained, it can the mechanism such as further investigated hepatocyte transplantation and liver regeneration, and the relevant basic research of infection such as can be used for human viral's hepatitis such as HBV, HCV, there is important theory and application value to associated treatment medicament research and development and assessment.
Description
Technical field
The invention belongs to field of biomedicine technology, are related to the method for building up of experimental animal model, and in particular to a kind of structure
The method for building people's liver chimeric mouse model, more particularly to a kind of laggard pedestrian's liver inosculating model of derivable specific hepar damnification
Construction method.
Background technology
Data discloses liver diseases and has become to one of maximum disease of mankind's threat, in addition to virus hepatitis, liver
The harm of hepatic sclerosis, liver cancer etc. is quite serious in disease；According to statistics, currently, China is every year for the direct of hepatitis and hepatopathy
Medical expense is up to more than 1,000 hundred million yuans.Clinic is in the intervening measure of liver diseases, liver transplantation is for solution
Certainly liver system disease and liver metabolism disorder etc. gradually become a kind of one of very important intervention means.
Know together in the industry, humanization liver, immune system double chimeric mouse models can be used for studying and associated viral replication and exempt from
Epidemic disease response situation can be the research of the great infectious liver diseases such as HBV, HCV infection, and can be fatty liver, hepatic sclerosis, liver cancer
The research of equal liver diseases provides humanization research platform.
It is related studies have shown that for hepatitis type B virus, due to its special thermophilic people liver property, be not easy in other animals
Model reappears its course of infection, therefore, lacks effective small animal model at present, foundation can effectively simulate human body HBV infection and
The experimental animal model of immune response is just becoming current and is deeply promoting HBV researchs great and urgent need.
Present situation based on the prior art, present inventor is quasi- to provide a kind of side of structure liver humanized mouse model
Method, the especially construction method of the laggard pedestrian's liver inosculating model of derivable specific hepar damnification, it is chimeric to obtain people liver
Mouse model.Humanization research platform is provided for great infectious liver disease and for liver diseases such as liver cancer.
Invention content
It is an object of the invention to overcome defect of the existing technology, a kind of structure liver humanized mouse model is provided
Method, the construction method of the especially derivable specific laggard pedestrian's liver inosculating model of hepar damnification, to obtain people liver
Chimeric mouse model；The model can be used for the research of the great infectious liver disease such as HBV, HCV infection and hard for fatty liver, liver
The research of the liver diseases such as change, liver cancer provides humanization research platform.
The present invention provides a kind of construction methods of humanization liver mosaic mouse model, including structure has can
Induction type liver damages the ADSB mouse with immunodeficient pheno, and so that the experiment mice is formed liver damage by diphtheria toxin intervention
Afterwards, progress human liver cell is chimeric builds to obtain people's liver chimeric mouse model；The humanization liver mouse mould that the method for the present invention structure obtains
Type can be studied for liver transfer operation and provide the support of feasible animal model, can the mechanism such as further investigated hepatocyte transplantation and liver regeneration,
And the relevant basic research of infection such as can be used for human viral's hepatitis such as HBV, HCV, to associated treatment medicament research and development and assessment
With important theory and application value.
The present invention is based on the appearance for the related particular line animal known in the industry and the mankind for relying on them to build are great
Disease animal model is the basis of the required tool of infectious diseases translational medicine research, in the derivable specificity of acquisition
It is advanced optimized as Alb-cre/DTR/SCID- on the basis of hepar damnification mouse model (number of patent application 201510153724.4)
beige(Alb-cre：Liver specific expression's Cre enzymes；DTR：Conditionity is transferred to people's diptheria toxin receptor gene, expression by
Cre/LoxP systems control；SCID-beige：Immunodeficient mouse) mouse model, referred to as ADSB mouse；In the method for the present invention
Intervening ADSB mouse using diphtheria toxin causes the ADSB mouse that hepar damnification occurs, then to the ADSB of the hepar damnification
Mouse carries out human liver cell transplanting, after January observation detect human liver cell to succeed in Mice Body to be colonized, build acquisition
Humanization liver mouse model.
Specifically, a kind of construction method of humanization liver chimeric mouse model of the present invention, which is characterized in that be based on
Tri- transgenic mices of ADSB carry out human liver cell transplanting comprising step on the basis of specific hepar damnification：
Acquisition, breeding and the identification of tri- transgenic mices of 1.ADSB
Alb-cre/DTR bi-transgenic mices will be obtained after Alb-cre and DTR mouse hybrids, then by itself and SCID-
Beige mouse hybrids obtain tri- transgenic mices of ADSB；
2. liver damage model is established,
Diphtheria toxin is carried out to ADSB mouse and intervenes to obtain liver damage modeling；
3. establishing human liver cell chimeric mouse model.
In the method for the present invention, using ADSB mouse as modeling experimental mouse；
In the method for the present invention, to tri- transgenic mices of ADSB of acquisition, hybridization is generated by the method for PCR and streaming
Filial generation carries out genetic typing, determines that it contains three of the above genotype；
In the method for the present invention, diphtheria toxin is carried out to ADSB mouse and intervenes to obtain liver damage modeling；One embodiment of the present of invention
In, the modeling by the way of by intraperitoneal injection, using through approach is injected intraperitoneally by the PBS solution containing diphtheria toxin to ADSB
Experimental mouse；Diphtheria toxin dosage used in modeling is 2~5ng/g mouse weights；
As a contrast with same week old C57BL/6 mouse；The variation for detecting liver damage related biochemical indicator damages modeling to evaluate liver
Success or not；
In the method for the present invention, the weight of mouse is carried out daily after diphtheria toxin is injected intraperitoneally to continue observation, and take a blood sample
Virus monitory ALT values are detached, ALT values notable raising person compared with C57BL/6 mouse is determined as liver damage successful model.
In the method for the present invention, human liver cell chimeric mouse model is established, in one embodiment of the present of invention, by being noted in spleen
The mode penetrated is by 0.5-1 × 106Human liver cell is transplanted in second day Mice Body of hepar damnification, after transplanting four weeks
ELISA detects the expression of mice serum human albumin, and expression is fitted into small compared with C57BL/6 mouse significantly raising person, determination for people liver
Mouse model.
The humanization liver mouse model that the method for the present invention structure obtains will provide a kind of feasible dynamic for liver transfer operation research
Object model support has important theory significance for researchs such as further investigated hepatocyte transplantation and liver regenerations and applies valence
Value, is alternatively arranged as humanized animal's model platform of other hepatopathy researchs.
Description of the drawings
Fig. 1 show the genetic typing to ADSB mouse as a result,
Wherein, ADSB mouse are the immunodeficient mouses containing Alb-cre transgenosis and DTR transgenosis simultaneously.
Fig. 2 shows ADSB and C57BL/6 mouse weight change curves,
Wherein, there is significant decline in the weight of ADSB mouse after injecting diphtheria toxin, and the weight of C57BL/6 with
No significant change is compared before injecting diphtheria toxin.
Fig. 3 shows ADSB and C57BL/6 mouse ALT change curves,
Wherein, there is significant rising in the ALT values of ADSB mouse after injecting diphtheria toxin, and the ALT values of C57BL/6
No significant change compared with before injection diphtheria toxin.
Fig. 4 shows liver organization gross examination of skeletal muscle and pathology figure after the damage of ADSB mouse livers,
Wherein, there is apparent lesion in the liver organization of ADSB mouse, and tissue turned pale has blutpunkte, HE to show liver
Damage, and there is not exception in the liver organization of C57BL/6 mouse.
Fig. 5 shows the ELISA detections of ADSB and C57BL/6 mouse human albumins,
Wherein, the albumin content of 4th week after the transfer, ADSB mouse is significantly higher than C57BL/6 control mices (* *
Represent P<0.01).
Specific implementation mode
Embodiment 1 builds humanization liver mosaic mouse model
Building has inducible liver damage and the ADSB mouse of immunodeficient pheno, and is made by diphtheria toxin intervention described
After experiment mice forms liver damage, progress human liver cell is chimeric to build to obtain people's liver chimeric mouse model, including step：
1) breeding and identification of tri- transgenic mices of ADSB
After Alb-cre and DTR mouse hybrids, Alb-cre/DTR bi-transgenic mices are obtained, then small with SCID-beige
Mouse hybridization obtains ADSB mouse, and carries out genetic typing to mouse obtained by hybridization as the method for PCR and streaming；Fig. 1 is shown
The present invention is to the genetic typing of ADSB mouse as a result, the results show that the ADSB mouse of structure are to contain Alb-cre transgenosis simultaneously
With the immunodeficient mouse of DTR transgenosis；
2) liver damage model is established,
Diphtheria toxin is carried out to ADSB mouse and intervenes to obtain liver damage modeling；
In the present embodiment, the diphtheria toxin of 2~5ng/g mouse weight dosage is dissolved in the PBS solution of 200 μ l, is used
Modeling is by the PBS solution containing diphtheria toxin to ADSB experimental mouses by way of intraperitoneal injection；Diphtheria toxin agent used in modeling
Amount is 2~5ng/g mouse weights；
As a contrast with same week old C57BL/6 mouse；The variation for detecting liver damage related biochemical indicator damages modeling to evaluate liver
Success or not；
In the present embodiment, the weight of mouse is carried out daily after diphtheria toxin is injected intraperitoneally to continue observation, and take a blood sample and divide
From Virus monitory ALT values, ALT values notable raising person compared with C57BL/6 mouse is determined as liver damage successful model；
Fig. 2 shows ADSB and C57BL/6 mouse weight change curves, wherein the ADSB mouse after injecting diphtheria toxin
Weight there is significant decline, and the weight of C57BL/6 no significant change compared with before injection diphtheria toxin；
Fig. 3 shows ADSB and C57BL/6 mouse ALT change curves, wherein the ADSB mouse after injecting diphtheria toxin
There is significant rising in ALT values, and the ALT values of C57BL/6 no significant change compared with before injection diphtheria toxin；
Fig. 4 shows liver organization gross examination of skeletal muscle and pathology figure after the damage of ADSB mouse livers, wherein the liver of ADSB mouse
There is apparent lesion in dirty tissue, tissue turned pale, there is a blutpunkte, and HE shows hepar damnification, and the liver group of C57BL/6 mouse
It knits and exception does not occur；
3) human liver cell chimeric mouse model is established,
In the present embodiment, by 0.5-1 × 10 by way of intrasplenic injection6Human liver cell is transplanted to the hepar damnification
In second day Mice Body, transplant four weeks after ELISA detection mice serum human albumin expression, Fig. 5 show ADSB and
C57BL/6 mouse human albumins ELISA detections, wherein the albumin content of 4th week after the transfer, ADSB mouse is significantly high
In C57BL/6 control mices, (* * represent P<0.01), the results show that successfully building people's liver chimeric mouse model.
Result of this example indicate that this method structure obtains humanization liver mouse model, which can be that liver moves
It plants research and feasible animal model support is provided, can be used for the relevant basis of the infection such as human viral's hepatitis such as HBV, HCV and grind
Study carefully, and there is important theory and application value to associated treatment medicament research and development and assessment.
Claims (6)
1. a kind of construction method of humanization liver mouse model, which is characterized in that it includes that structure has inducible liver damage
With the ADSB mouse of immunodeficient pheno, and after being intervened by diphtheria toxin, carry out human liver cell it is chimeric build people liver be fitted into it is small
Mouse model；It includes step：
1) acquisition, breeding and the identification of tri- transgenic mices of ADSB
Alb-cre/DTR bi-transgenic mices will be obtained after Alb-cre and DTR mouse hybrids, then it is small with SCID-beige
Mouse hybridizes, and obtains tri- transgenic mices of ADSB；
2) liver damage model is established,
Diphtheria toxin is carried out to ADSB mouse and intervenes to obtain liver damage modeling；
3) human liver cell chimeric mouse model is established.
2. the construction method of humanization liver mouse model as described in claim 1, which is characterized in that described to obtain three transgenosis
Mouse is tri- transgenic mices of Alb-cre/DTR/SCID-beige (ADSB mouse).
3. the construction method of humanization liver mouse model as described in claim 1, which is characterized in that the ADSB tri- of acquisition
Transgenic mice carries out genetic typing to the filial generation that hybridization generates by the method for PCR and streaming, determines that it contains described three
Kind genotype.
4. the construction method of humanization liver mouse model as described in claim 1, which is characterized in that carried out to ADSB mouse
Diphtheria toxin intervene liver damage modeling in, diphtheria toxin dosage used in modeling be 2~5ng/g mouse weights.
5. by the construction method of the humanization liver mouse model described in claim 1 or 4, which is characterized in that ADSB mouse
Carry out diphtheria toxin intervene liver damage modeling in, as a contrast with same week old C57BL/6 mouse；It detects liver and damages related biochemical indicator
Variation, with evaluate liver damage modeling success or not；The liver damages related biochemical indicator ALT values and compares C57BL/6 mouse phases
Than significantly raising person, it is determined as liver damage successful model.
6. the construction method of humanization liver mouse model as described in claim 1, which is characterized in that it is embedding to establish human liver cell
It closes in mouse model, by 0.5-1 × 106Human liver cell migrates to the mouse that the liver damages second day, transplants ELISA after four weeks
The expression of mice serum human albumin is detected, expression relatively control C57BL/6 mouse significantly raising person determines to be people's liver gomphosis mouse
Model.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109735485A (en) * | 2019-01-29 | 2019-05-10 | 深圳市拓普生物科技有限公司 | Humanization liver animal model and its construction method and application |
| CN112575037A (en) * | 2019-09-29 | 2021-03-30 | 上海市公共卫生临床中心 | Construction method of humanized transgenic mouse model of chimeric human HLA-DP genome region |
| WO2022111696A1 (en) * | 2020-11-30 | 2022-06-02 | 江苏大学 | Maintenance of programmed chronic liver injury of fah gene-deficient animals and application thereof in preparation of heterologous liver models |
| CN115299406A (en) * | 2022-08-19 | 2022-11-08 | 中南大学湘雅二医院 | Animal model construction method and method for ablating fat cells |
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