CN112352739A - Non-alcoholic fatty liver disease mouse model and construction method thereof - Google Patents
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Abstract
本发明公开了一种非酒精性脂肪肝小鼠模型及其构建方法，具体构建步骤为：步骤一：转染小鼠胚胎干细胞，将包含ghr基因第4和第5外显子片段利用Pfu Turbo DNA聚合酶扩增并克隆到pCR Blunt II‑TOPO载体中。本发明通过此方法建模的小鼠在8周龄时，肝脏体积显著增加，H&E染色和油红染色结果均呈脂肪肝病变，肝脏细胞脂肪合成显著增加，脂肪分解明显减少，用该方法建立的小鼠模型，有稳定遗传性，临床症状与人类情况类似，成模时间短、效率高，死亡率低，不需要饮食诱导即可成模，具有较大的应用推广前景，是药物筛选和药理学研究的理想的非酒精性脂肪肝动物模型。
The invention discloses a non-alcoholic fatty liver mouse model and a construction method thereof. The specific construction steps are as follows: Step 1: Transfect mouse embryonic stem cells, and use Pfu Turbo for fragments containing the fourth and fifth exons of the ghr gene DNA polymerase amplified and cloned into the pCR Blunt II-TOPO vector. At the age of 8 weeks, the liver volume of the mice modeled by this method in the present invention increased significantly, the results of H&E staining and oil red staining showed fatty liver lesions, liver cell fat synthesis was significantly increased, and lipolysis was significantly reduced. The mouse model has stable heredity, clinical symptoms are similar to those of humans, the modeling time is short, the efficiency is high, and the mortality rate is low. Ideal non-alcoholic fatty liver animal model for pharmacological studies.
Description
Technical Field
The invention relates to the technical field of a molding process of a medical animal model, in particular to a non-alcoholic fatty liver mouse model and a construction method thereof.
Background
Non-alcoholic fatty liver disease (NAFLD), a type of chronic liver disease characterized by diffuse hepatocellular hypertrophy and vesicular hepatic steatosis, has increased year by year in recent years, and has seriously threatened human health, and chinese guidelines for fatty liver prevention and treatment (2018 edition): the prevalence rate of the Chinese non-alcoholic fatty liver disease is up to 25%, wherein 15% of the Chinese non-alcoholic fatty liver diseases are developed into non-alcoholic steatohepatitis, a small number of patients develop liver cirrhosis and liver cancer, the non-alcoholic fatty liver disease is relatively complex, if the non-alcoholic fatty liver disease is not controlled in time, inflammatory reaction and liver fibrosis can be caused, the non-alcoholic fatty liver disease is a pathological syndrome which has no excessive drinking history and is characterized by hepatocyte steatosis and the like, and comprises 4 pathological processes of simple fatty liver, steatohepatitis, fatty fibrosis and fatty liver cirrhosis.
At present, the non-alcoholic fatty liver animal model for scientific research is very limited, and no non-alcoholic fatty liver model mouse with stable inheritance exists in the market, the non-alcoholic fatty liver animal model mouse needs to be cultured and modified in the future, diet and carbon tetrachloride injection induction are mostly adopted, the modeling time is long, the modeling success rate is low, and the death rate is high.
Therefore, it is necessary to invent a mouse model of non-alcoholic fatty liver disease and a method for constructing the same to solve the above problems.
Disclosure of Invention
The invention aims to provide a non-alcoholic fatty liver mouse model and a construction method thereof, and aims to solve the problems that the existing non-alcoholic fatty liver animal model for scientific research is very limited, no non-alcoholic fatty liver model mouse with stable inheritance exists in the market, the non-alcoholic fatty liver model mouse needs to be cultured and modified in the next day, the modeling time is long, the modeling success rate is low, and the mortality rate is high.
In order to achieve the above purpose, the invention provides the following technical scheme: a non-alcoholic fatty liver mouse model and a construction method thereof, the concrete construction steps are as follows:
the method comprises the following steps: transfecting a mouse embryonic stem cell, amplifying and cloning a fragment (with the size of 10.8kb) containing exon 4 and exon 5 of the ghr gene into a pCR Blunt II-TOPO vector by utilizing Pfu Turbo DNA polymerase, cutting an 8kb fragment from the fragment by utilizing BamHI and XbaI, connecting the fragment to a PL253 vector containing a Thymidine kinase cassette (Thymidine kinase cassette) by utilizing T4 ligase, recombining and exchanging a subclone genome region by utilizing PL452 and PL451 vectors containing neomycin (neo) cassettes, inserting 2 Loxp sites and a Frt-neo-Frt-LoxP cassette to obtain a conditional targeting vector, linearizing the conditional targeting vector by utilizing Not I restriction endonuclease digestion, and transfecting the mouse embryonic stem cell by utilizing an electroporation method;
step two: performing genotype verification on the embryonic stem cells, namely identifying and determining target clones including a first LoxP site, a ghr exon 4, two Frt sites and a second LoxP site by using the transfected mouse embryonic stem cells in the step one through PCR (polymerase chain reaction) genotype;
step three: constructing a GHR flox mouse model, injecting the embryonic stem cells with accurate genotype verified in the second step into C57BL/6J mouse blastocysts, and removing the neo cassette by crossing with a high-efficiency FLPo-deleter mouse to obtain the GHR flox (GHR)fl/fl) A mouse;
step four: obtaining liver tissue specificity knockout GHR mouse (LiGHRKO), and using Cre-LoxP hybridization technique to obtain GHR flox (GHR) obtained from the third stepfl/fl) Mouse and liver cellCrossing with an opposite promoter Albumin-cre (Alb-cre) mouse to obtain a liver tissue specific knockout ghr mouse (LigHRKO).
Preferably, the GHR flox (GHR)fl/fl) Mice contain two LoxP sites flanking exon 4 of the ghr gene in their ghr gene.
Preferably, the non-alcoholic fatty liver mouse model is a liver tissue-specific knockout ghr mouse (light rko).
Preferably, the mouse model of non-alcoholic fatty liver disease is preferably a male mouse, and the mouse model of non-alcoholic fatty liver disease is normally fed on a diet in an animal room.
Preferably, the temperature in the animal room is kept at 23 +/-2 ℃ and the relative humidity is kept at 50% +/-10%, so that the environment is clean and the air circulation is ensured.
Preferably, the pCR Blunt II-TOPO vector is available from Invitrogen Life technologies, Inc. USA.
In the technical scheme, the invention provides the following technical effects and advantages:
when a mouse modeled by the method is 8 weeks old, the liver volume is remarkably increased, the color is yellowish, the results of H & E staining and oil red staining show fatty liver pathological changes, the fat synthesis of liver cells is remarkably increased, the fat decomposition is remarkably reduced, a non-alcoholic fatty liver mouse disease model is obtained, the modeling rate is 100%, no mouse dies during modeling, the death rate of modeling is greatly reduced, and the ideal state that the death rate of modeling is 0% is achieved.
Drawings
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
FIG. 1 is a schematic diagram of a liver-specific ghr gene (LigghRKO) knockout mouse construction technique of the present invention;
FIG. 2 shows the genotype identification PCR results of the LiGHRKO mouse of the present invention: a is the mouse rat tail ghr LoxP locus genotype identification result; b is mouse rat tail Alb-cre gene PCR result;
FIG. 3 is a bar graph of the ghr mRNA levels in liver tissues of control mice and light RKO mice under normal diet conditions in accordance with the present invention;
FIG. 4 is an appearance diagram of a control group mouse (left side) and a light RKO mouse (right side) under a normal diet condition according to the present invention;
FIG. 5 is a histogram of tissue weights of control mice and light rko mice under normal diet conditions in accordance with the present invention;
FIG. 6 is an appearance diagram of the liver of a control group mouse (panel A) and the liver of a light RKO mouse (panel B) under a normal diet condition according to the present invention;
FIG. 7 shows the results of H & E staining of liver tissues of control mice (panel A) and H & E staining of liver tissues of light RKO mice (panel B) under normal diet conditions according to the present invention;
FIG. 8 shows the results of oil red staining of liver tissue of control mice (panel A) and the results of oil red staining of liver tissue of light RKO mice (panel B) under normal diet conditions according to the present invention.
Detailed Description
In order to make the technical solutions of the present invention better understood, those skilled in the art will now describe the present invention in further detail with reference to the accompanying drawings.
Example 1:
the invention provides a non-alcoholic fatty liver mouse model and a construction method thereof as shown in figures 1-3, and the specific construction steps are as follows:
the method comprises the following steps: transfecting mouse embryonic stem cells, amplifying and cloning a fragment (10.8 kb in size) including the 4 th and 5 th exons of the ghr gene by Pfu Turbo DNA polymerase into a pCR Blunt II-TOPO vector purchased from Invitrogen life technologies ltd (Invitrogen), excising the 8kb fragment therefrom with BamHI and XbaI, ligating the 8kb fragment to a PL253 vector containing a Thymidine kinase cassette (thymidin kinase cassette) using T4 ligase, recombinantly interchanging the subcloned genomic region with PL452 and PL451 vectors containing neomycin (neo) cassettes, inserting 2 Loxp sites and a Frt-neo-Frt-Loxp cassette to obtain a conditional targeting vector, linearizing the conditional targeting vector by Not I restriction endonuclease digestion, and transfecting the mouse embryonic stem cells using electroporation;
step two: performing genotype verification on the embryonic stem cells, namely identifying and determining target clones including a first LoxP site, a ghr exon 4, two Frt sites and a second LoxP site by using the transfected mouse embryonic stem cells in the step one through PCR (polymerase chain reaction) genotype;
step three: constructing a GHR flox mouse model, injecting the embryonic stem cells with accurate genotype verified in the second step into C57BL/6J mouse blastocysts, and removing the neo cassette by crossing with a high-efficiency FLPo-deleter mouse to obtain the GHR flox (GHR)fl/fl) Mouse, the GHR flox (GHR)fl/fl) The ghr gene of the mouse comprises two LoxP sites located on both sides of exon 4 of the ghr gene;
step four: obtaining liver tissue specificity knockout GHR mouse (LiGHRKO), and using Cre-LoxP hybridization technique to obtain GHR flox (GHR) obtained from the third stepfl/fl) And hybridizing the mouse with a liver cell specific promoter Albumin cre (Alb-cre) mouse to obtain a liver tissue specific knockout ghr mouse (LigHRKO), wherein the non-alcoholic fatty liver mouse model is the liver tissue specific knockout ghr mouse (LigHRKO).
Further, the non-alcoholic fatty liver disease mouse model is a male mouse, the control group mouse is a ghr flox male mouse, the non-alcoholic fatty liver disease mouse model and the control group mouse are both fed in a normal diet in an animal room, the temperature in the animal room is kept at 23 +/-2 ℃, the relative humidity is kept at 50% +/-10%, and the environment cleanness and the air circulation are ensured.
The modeling rate of the non-alcoholic fatty liver disease mouse prepared in the embodiment is 100%, ten mice with the liver tissue specificity knockout ghr (LiRKO) are randomly selected, the mouse tail of the LiGHRKO mouse is subjected to genotype identification, ghR LoxP site genes and Alb-cre genes are identified, the identification result is shown in figure 2, all the non-alcoholic fatty liver disease mice prepared in the embodiment are liver tissue specificity knockout ghR mice (LiRKO), ten mice with the liver tissue specificity knockout ghRKO male mice of 8 weeks and control mice are randomly selected, RNA extraction and reverse transcription are respectively carried out on the liver tissues of the LiGHRKO male mice of 8 weeks and the control mice to obtain cDNA, relevant gene expression detection is carried out through qRT-PCR experiments, the result is shown in figure 3, the result shows that the mice with the liver tissue specificity knockout ghr, namely the non-alcoholic fatty liver disease mice are successfully constructed, and in the modeling process, none of the mice are dead rate of the mice is greatly reduced, and the ideal state that the molding death rate is 0 percent is achieved.
Example 2:
the invention provides a non-alcoholic fatty liver mouse model and a construction method thereof as shown in figures 1-6, and the specific construction steps are as follows:
the method comprises the following steps: transfecting mouse embryonic stem cells, amplifying and cloning a fragment (10.8 kb in size) including the 4 th and 5 th exons of the ghr gene by Pfu Turbo DNA polymerase into a pCR Blunt II-TOPO vector purchased from Invitrogen life technologies ltd (Invitrogen), excising the 8kb fragment therefrom with BamHI and XbaI, ligating the 8kb fragment to a PL253 vector containing a Thymidine kinase cassette (thymidin kinase cassette) using T4 ligase, recombinantly interchanging the subcloned genomic region with PL452 and PL451 vectors containing neomycin (neo) cassettes, inserting 2 Loxp sites and a Frt-neo-Frt-Loxp cassette to obtain a conditional targeting vector, linearizing the conditional targeting vector by Not I restriction endonuclease digestion, and transfecting the mouse embryonic stem cells using electroporation;
step two: performing genotype verification on the embryonic stem cells, namely identifying and determining target clones including a first LoxP site, a ghr exon 4, two Frt sites and a second LoxP site by using the transfected mouse embryonic stem cells in the step one through PCR (polymerase chain reaction) genotype;
step three: constructing a ghr flox mouse model, and verifying the step twoInjecting embryo stem cell with accurate genotype into C57BL/6J mouse blastocyst, removing neo cassette by crossing with high efficiency FLPo-deleter mouse to obtain GHR flox (GHR)fl/fl) Mouse, the GHR flox (GHR)fl/fl) The ghr gene of the mouse comprises two LoxP sites located on both sides of exon 4 of the ghr gene;
step four: obtaining liver tissue specificity knockout GHR mouse (LiGHRKO), and using Cre-LoxP hybridization technique to obtain GHR flox (GHR) obtained from the third stepfl/fl) And hybridizing the mouse with a liver cell specific promoter Albumin-cre (Alb-cre) mouse to obtain a liver tissue specific knockout ghr mouse (LigHRKO), wherein the non-alcoholic fatty liver mouse model is the liver tissue specific knockout ghr mouse (LigHRKO).
Further, the non-alcoholic fatty liver disease mouse model is a male mouse, the control group mouse is a ghr flox male mouse, the non-alcoholic fatty liver disease mouse model and the control group mouse are both fed in a normal diet in an animal room, the temperature in the animal room is kept at 23 +/-2 ℃, the relative humidity is kept at 50% +/-10%, and the environment cleanness and the air circulation are ensured.
The non-alcoholic fatty liver disease mouse model prepared in the embodiment is obviously similar to the human condition in clinical symptoms, can be modeled without diet induction, is randomly selected from ten mice of 8-week-old Ligrko male mice and ten mice of a control group, is respectively subjected to appearance photographing on the mice of 8-week-old Ligrko male mice and the mice of the control group, is subjected to anatomical material drawing, is weighed, is subjected to liver tissue photographing, and is subjected to anatomical and histomorphological comparison, as shown in fig. 4-6, the results show that the livers of the Ligrko mice are remarkably increased in volume and are yellowish in color compared with the mice of the control group, and the non-alcoholic fatty liver disease mouse model is obtained.
Example 3:
the invention provides a non-alcoholic fatty liver mouse model and a construction method thereof as shown in figures 1-8, and the specific construction steps are as follows:
the method comprises the following steps: transfecting mouse embryonic stem cells, amplifying and cloning a fragment (10.8 kb in size) including the 4 th and 5 th exons of the ghr gene by Pfu Turbo DNA polymerase into a pCR Blunt II-TOPO vector purchased from Invitrogen life technologies ltd (Invitrogen), excising the 8kb fragment therefrom with BamHI and XbaI, and then ligating the 8kb fragment to a PL253 vector containing a Thymidine kinase cassette (thymidin kinase cassette) using T4 ligase, and then recombinantly interchanging the subcloned genomic region with PL452 and PL451 vectors containing neomycin (neo) cassettes, inserting 2 Loxp sites and a Frt-neo-T-Loxp cassette to obtain a conditional targeting vector, linearizing the conditional targeting vector by Not I restriction endonuclease cleavage, and transfecting the mouse embryonic stem cells using electroporation;
step two: performing genotype verification on the embryonic stem cells, namely identifying and determining target clones including a first LoxP site, a ghr exon 4, two Frt sites and a second LoxP site by using the transfected mouse embryonic stem cells in the step one through PCR (polymerase chain reaction) genotype;
step three: constructing a GHR flox mouse model, injecting the embryonic stem cells with accurate genotype verified in the second step into C57BL/6J mouse blastocysts, and removing the neo cassette by crossing with a high-efficiency FLPo-deleter mouse to obtain the GHR flox (GHR)fl/fl) Mouse, the GHR flox (GHR)fl/fl) The ghr gene of the mouse comprises two LoxP sites located on both sides of exon 4 of the ghr gene;
step four: obtaining liver tissue specificity knockout GHR mouse (LiGHRKO), and using Cre-LoxP hybridization technique to obtain GHR flox (GHR) obtained from the third stepfl/fl) And hybridizing the mouse with a liver cell specific promoter Albumin cre (Alb-cre) mouse to obtain a liver tissue specific knockout ghr mouse (LigHRKO), wherein the non-alcoholic fatty liver mouse model is the liver tissue specific knockout ghr mouse (LigHRKO).
Further, the non-alcoholic fatty liver disease mouse model is a male mouse, the control group mouse is a ghr flox male mouse, the non-alcoholic fatty liver disease mouse model and the control group mouse are both fed in a normal diet in an animal room, the temperature in the animal room is kept at 23 +/-2 ℃, the relative humidity is kept at 50% +/-10%, and the environment cleanness and the air circulation are ensured.
The non-alcoholic fatty liver disease mouse model prepared in the embodiment is similar to a human condition in clinical symptoms, can be modeled without diet induction, and is characterized in that ten mice of 8-week-old Ligrkro male mice and ten mice of a control group are randomly selected, and liver tissue H & E staining and liver tissue oil red staining are performed on the 8-week-old Ligrkro male mice and the mice of the control group, as shown in fig. 7-8, the results of the liver tissue H & E staining and the liver tissue oil red staining are fatty liver lesions, the fat synthesis of liver cells is remarkably increased, the fat decomposition is remarkably reduced, the non-alcoholic fatty liver mouse disease models are obtained, and in the modeling process, none of the mice dies.
The embodiment shows that when a mouse modeled by the method is 8 weeks old, the liver volume is obviously increased, the color is yellowish, the results of H & E staining and oil red staining show fatty liver pathological changes, the fat synthesis of liver cells is obviously increased, the lipolysis is obviously reduced, non-alcoholic fatty liver mouse disease models are obtained, the modeling rate is 100%, no mouse dies in the modeling process, the death rate of modeling is greatly reduced, and the ideal state that the death rate of modeling is 0% is achieved.
While certain exemplary embodiments of the present invention have been described above by way of illustration only, it will be apparent to those of ordinary skill in the art that the described embodiments may be modified in various different ways without departing from the spirit and scope of the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive, and any modifications, equivalents, improvements and the like that come within the spirit and principles of the invention are intended to be included within the scope of the invention.
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| CN117256563A (en) * | 2023-04-19 | 2023-12-22 | 青岛大学 | Method for constructing non-alcoholic fatty liver animal model |
| CN117887768A (en) * | 2023-12-16 | 2024-04-16 | 上海市同济医院 | A method for establishing a mouse non-alcoholic fatty liver disease model based on the autophagy-lysosome pathway |
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