CN109735485B - Humanized liver animal model and construction method and application thereof - Google Patents
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Abstract
The invention relates to a humanized liver animal model and a construction method and application thereof. The construction method of the humanized liver animal model comprises the following steps: injecting the human liver progenitor cells into the immunodeficiency animals with partially excised livers to obtain the humanized liver animal model. The construction method of the humanized liver animal model is simple and convenient, and the problem that liver injury drugs, viruses, bacteria and the like are used in the construction process to cause interference to the application in the later modeling period is solved.
Description
Technical Field
The invention relates to a humanized liver animal model and a construction method and application thereof.
Background
The liver disease refers to pathological changes occurring in the liver, the liver diseases are various and comprise hepatitis A, hepatitis B, hepatitis C, adiposis, liver cancer, cirrhosis, alcoholic or non-alcoholic liver diseases and the like, and the liver disease is a common disease which has great harm to the life of human beings. China is one of the countries with the largest incidence rate of liver diseases, and data statistics shows that in recent years, the liver diseases such as hepatitis, fatty liver, drug-induced liver damage, liver cancer cirrhosis and the like threaten the healthy life of human beings, wherein the incidence rate of viral hepatitis is the highest, and the incidence rate is increased year by year. According to the Worldwide Health Organization (WHO) research on mortality of liver diseases, the total number of deaths due to cirrhosis and liver cancer worldwide has increased by 5000 ten thousand per year over the last 20 years. After years of research, the liver disease spectrum of our country has the following characteristics: firstly, the liver diseases are abundant in variety, and at least more than 100 types of liver diseases are available; secondly, the specific gravity of the infectious liver disease is still high; thirdly, the age of the infected liver disease is younger; fourth, the types of non-infected liver diseases show a significantly rising tendency.
Aiming at the hepatopathy, a great deal of manpower and material resources are actively invested by medicine enterprises and scientific research units all over the world, and the medicine with good treatment effect is developed. However, in the prior stage of liver disease drug development, the primary screening and drug effect evaluation of drugs are mainly carried out by using a common animal model, such as a mouse model, but after all, the liver of an animal is quite different from the liver of a human, so that the reaction of the liver of the human to the drugs cannot be well simulated.
Disclosure of Invention
Therefore, a method for constructing a humanized liver animal model is needed.
In addition, a humanized liver animal model and application thereof are also provided.
A construction method of a humanized liver animal model comprises the following steps:
injecting the human liver progenitor cells into the immunodeficiency animals with partially excised livers to obtain the humanized liver animal model.
According to the method for constructing the humanized liver animal model, the immunodeficiency animal with the partially excised liver is used as a model constructing object to inject the human liver progenitor cells to construct the humanized liver animal model, so that the application limitation after the model construction is finished due to the immunosuppression of cyclosporin A, radioactive rays and the like on the animal caused by the xenogenic immune rejection after the human liver progenitor cells are injected into the animal body is avoided; on the other hand, the interference caused by using drugs, viruses, bacteria and the like to the application after modeling after liver injury is avoided. Moreover, the humanized liver animal model obtained according to the construction method is proved to have a human bile duct structure, which shows that the constructed humanized liver animal model has the liver function, can provide a feasible animal model support for clinical liver disease research or new drug metabolism research, and has important significance for liver regeneration and related research.
In one embodiment, in the step of injecting human hepatic progenitors into the partially resected liver immunodeficient animal, the human hepatic progenitors are administered in an amount of 1X 1061X 10 animals/animal7One animal per animal.
In one embodiment, the method further comprises the step of obtaining the human hepatic progenitors:
inducing and differentiating human stem cells in a hepatocyte differentiation medium, wherein the hepatocyte differentiation medium comprises hepatocyte growth factor, fiber growth factor and epidermal growth factor.
In one embodiment, the mass volume concentration of the hepatocyte growth factor is 10 to 40 mug/L; the mass volume concentration of the fiber growth factor is 10 mug/L-20 mug/L; the mass volume concentration of the epidermal growth factor is 10 mug/L-20 mug/L.
In one embodiment, the mass of the resected liver of the immunodeficient animal is 50% to 70% of the mass of the original liver of the immunodeficient animal.
In one embodiment, the immunodeficient animal is an immunodeficient mouse or an immunodeficient rat.
In one embodiment, the immunodeficient mouse is selected from one of a Nude mouse, a SCID mouse, a NOD-SCID mouse, an NSI mouse, an NCG mouse, a Rag2 mouse, and an IL2RG mouse.
In one embodiment, the immunodeficient animal is a NCG mouse.
A humanized liver animal model is obtained by the construction method of the humanized liver animal model.
The humanized liver animal model is applied to screening of liver disease drugs and evaluation of drug effects.
Drawings
FIG. 1 is an immunofluorescence plot of human hepatic progenitors of example 1;
FIG. 2 is a schematic representation of the NCG mouse of example 2 being immobilized and having an abdominal opening;
FIG. 3 is a resected portion of the liver of the NCG mouse of example 2;
FIG. 4 is a schematic view of the injection of human hepatic progenitors into the spleen of NCG mice of example 2;
FIG. 5 is a graph of the results of the immunohistochemistry of the liver in the humanized liver animal model of example 2.
Detailed Description
To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Some embodiments of the invention are presented in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The method for constructing the humanized liver animal model of the embodiment comprises the following steps:
s110, cutting off the part of the liver of the immunodeficiency animal after surgery to obtain the liver injury animal.
An immunodeficient animal is one in which one or more components of the immune system are deficient due to congenital genetic mutation or by artificial means. In this embodiment, the immunodeficient animal is a non-human mammal. Preferably, the immunodeficient animal is a rodent. Further, the immunodeficient animal is an immunodeficient rat or an immunodeficient mouse. In one embodiment, the immunodeficient mouse is selected from one of a Nude mouse, a SCID mouse, a NOD-SCID mouse, an NSI mouse, an NCG mouse, a Rag2 mouse, and an IL2RG mouse.
Specifically, the step of removing the operated part of the liver of the immunodeficient animal specifically comprises the following steps:
the method comprises the steps of opening the abdomen of an immunodeficient animal, extruding the liver of the immunodeficient animal out of the opening, ligating the extruded liver of the immunodeficient animal and cutting off, wherein the mass of the cut-off liver is 50% -70% of the mass of the original liver of the immunodeficient animal. Further, the mass of the excised liver is 50%, 55%, 60%, 65% or 70% of the mass of the liver of the immunodeficient animal.
After the immunodeficiency animals with partially resected livers are injected into human liver progenitor cells, drugs such as cyclosporin A can be avoided, animals are subjected to immunosuppression treatment to weaken xenogeneic immunological rejection, and the applications of subsequent animals such as drug screening and drug effect evaluation after construction are not influenced. In addition, the survival rate of the animals can be improved because the above-mentioned immunodeficient animals having partially excised livers are prevented from being treated with immunosuppression.
In some embodiments, immunodeficient animals with partially resected liver are commercially available.
S130, injecting the human liver progenitor cells into the immunodeficient animal with the liver partially cut off to obtain the humanized liver animal model.
Specifically, the step of injecting the human hepatic progenitors into the immunodeficient animal with the liver partially excised to obtain the humanized liver animal model comprises the following operations:
s131, preparing human hepatic progenitor cells.
Hepatic progenitor cells are also called small liver cells and bile duct epithelioid cells, and are heterogeneous cells distributed in adult livers and have bidirectional differentiation potential. The human hepatic progenitor cells are human hepatic progenitor cells, and are formed by differentiation of human stem cells. Among them, the human stem cell is a human stem cell cultured separately or a human stem cell commercialized. Further, the human stem cell is selected from one of human umbilical cord mesenchymal stem cell, IPS cell and embryonic stem cell.
Specifically, the human stem cells are induced to differentiate in a hepatocyte differentiation medium, wherein the hepatocyte differentiation medium comprises hepatocyte growth factor, fiber growth factor and epidermal growth factor. Stem cells are pluripotent and, without committed differentiation, are difficult to form into hepatic progenitors. Furthermore, the mass volume concentration of the hepatocyte growth factor in the cell differentiation culture medium is 10 mug/L-40 mug/L; the mass volume concentration of the fiber growth factor is 10 mug/L-20 mug/L; the mass volume concentration of the epidermal growth factor is 10 mug/L-20 mug/L. Preferably, the concentration of hepatocyte growth factor by mass volume is 12. mu.g/L, 16. mu.g/L, 20. mu.g/L, 25. mu.g/L, 30. mu.g/L or 35. mu.g/L; the mass volume concentration of the fiber growth factor is 12 mug/L, 14 mug/L, 16 mug/L or 18 mug/L; the mass volume concentration of the epidermal growth factor is 12 mug/L, 14 mug/L, 16 mug/L or 18 mug/L. Further, the mass volume concentration ratio of the hepatocyte growth factor to the fiber growth factor is 2: 0.5 to 4. The mass volume concentration ratio of the hepatocyte growth factor to the epidermal growth factor is 1: 0.5 to 2.
S133, injecting the human liver progenitor cells into the immunodeficient animal with the liver partially cut off to obtain the humanized liver animal model.
Specifically, the injection mode is selected from one of subcutaneous injection, enveloped injection, intravenous injection, intraperitoneal injection and spleen injection. Preferably, the injection is spleen injection. The human hepatic progenitor cells are injected into the spleen, transported into the liver through blood circulation, and repair the damaged liver. The human liver progenitor cells are supplemented after the liver is cut by utilizing the super-strong repair capability of the liver, so that the liver of the immunodeficient animal with the partially cut liver can quickly repair the injury by utilizing the exogenous human liver progenitor cells, and the generated humanized liver mouse model is formed.
In one embodiment, the human hepatic progenitors are administered in an amount of 1X 106Animal 1X 107Animals were used. Further, the amount of human hepatic progenitor cells is 1X 1063X 10 animals/animal65X 10 animals/animal6Animal, 7X 106Animals or 1X 1 animals with a number of 07Animals were used. When injected according to the dosage, the human hepatic progenitor cells can grow and differentiate in the liver of the mouse more easily to form the bile duct.
The amount of human hepatic progenitors used is critical to affect the survival of human hepatic progenitors in a partially resected liver immunodeficient animal. Too high cell amount can cause the blockage of animal blood vessels and cause the death of animals; too low a cell amount does not give any effect. According to the dosage, the human hepatic progenitor cells can grow and differentiate in the liver of the liver-damaged animal and repair the damaged liver.
The construction method of the humanized liver animal model is simple and convenient to operate, short in period and convenient for large-scale production. The humanized liver animal model obtained by the construction method of the humanized liver animal model has the formation of humanized bile ducts, has no interference of other medicines, viruses, bacteria and the like, and is beneficial to screening of liver disease medicines and evaluation of drug effect.
The humanized liver animal model is applied to screening of liver disease drugs and evaluation of drug effects.
A method for evaluating the drug effect of a liver disease drug comprises the following steps:
injecting a liver disease drug into the humanized liver animal model; and evaluating the drug effect of the liver disease drug.
A screening method of a liver disease drug comprises the following steps:
injecting a liver disease drug into the humanized liver animal model; and screening liver disease medicine.
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The following detailed description is given with reference to specific examples. The examples, which are not specifically illustrated, employ drugs and equipment, all of which are conventional in the art. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those in the literature, in books, or as recommended by the manufacturer.
Example 1 isolation and Induction of hepatic progenitors of adult origin
(1) Cutting human umbilical cord (from southern mountain hospital) along center under sterile condition, separating blood vessel, and cutting the rest tissue to about 2mm3The small pieces were then placed inside in 10cm cell culture dishes previously wetted with 1ml of stem cell complete medium (human umbilical cord mesenchymal stem cell medium, Youkang biology). Then placing the mixture in CO with the volume fraction of 5 percent at the temperature of 37 DEG C2Carbon dioxide incubatorAnd (4) medium culture for 2h, and adding 8ml of complete culture medium (human umbilical cord mesenchymal stem cell culture medium, Youkang biology) after the tissues are attached tightly. Changing the liquid every 3 days, and observing the climbing condition of adherent cells around the patch; and culturing for 2 weeks to obtain the human primary umbilical cord mesenchymal stem cells.
(2) Collecting the separated human-derived primary umbilical cord mesenchymal stem cells, carrying out induced differentiation on the human-derived primary umbilical cord mesenchymal stem cells by using a liver directed differentiation culture medium (Seike: HUXMX-90101) to obtain human-derived hepatic progenitor cells, and carrying out induced differentiation for 14 days to obtain the human-derived hepatic progenitor cells after being identified by an immunofluorescence method. The identification result is shown in figure 1, and the bright white part in figure 1 is human hepatic progenitor cells.
Example 2 establishment of humanized liver animal model
(1) As shown in fig. 2 to 3, NCG mice (model animal research institute of Nanjing university) were anesthetized, fixed in a supine position, and a 1 cm-long incision was opened in the abdomen of the mice, the liver was extruded from both sides of the opening toward the middle, and the extruded liver was ligated to the proximal end, 50% of the total liver volume was excised after ligation, and the remaining liver was returned to the abdomen, and the mice were housed in a cage for 2 days to obtain liver-injured mice.
(2) As shown in FIG. 4, human hepatic progenitors were cultured at 1X 107Injecting one or more of the cells into the spleen of the liver injury mouse obtained in the step (1), detecting human ALB marker protein of the liver through immunohistochemistry detection 1 week, 2 weeks and 4 weeks after the injection, and determining the establishment of an immune humanized liver animal model according to tissue slice immunohistochemistry standards. Among them, the results of liver examination 4 weeks after injection are shown in fig. 5. In fig. 5, dark gray (brown in the actual color photograph) is the ALB marker protein, black (dark blue in the actual color photograph) is the bile duct, and the chimera ratio of the human liver in the mouse liver is 60%. As can be seen in fig. 5, the establishment of the humanized liver animal model.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (7)
1. A construction method of a humanized liver animal model is characterized by comprising the following steps:
injecting human liver progenitor cells into an immunodeficient animal with partially excised liver to obtain a humanized liver animal model, wherein the immunodeficient animal is an immunodeficient mouse, the mass of the excised liver of the immunodeficient animal is 50% -60% of the mass of the original liver of the immunodeficient animal, and the dosage of the human liver progenitor cells is 1 x 107One animal per animal.
2. The method of constructing a humanized liver animal model according to claim 1, further comprising the step of obtaining the human hepatic progenitors:
inducing and differentiating human stem cells in a hepatocyte differentiation medium, wherein the hepatocyte differentiation medium comprises hepatocyte growth factor, fiber growth factor and epidermal growth factor.
3. The method for constructing a humanized liver animal model according to claim 2, wherein the mass volume concentration of the hepatocyte growth factor is 10 μ g/L to 40 μ g/L; the mass volume concentration of the fiber growth factor is 10 mug/L-20 mug/L; the mass volume concentration of the epidermal growth factor is 10 mug/L-20 mug/L.
4. The method of claim 3, wherein the ratio of the mass-to-volume concentration of the hepatocyte growth factor to the fiber growth factor is 2: 0.5-4, wherein the mass volume concentration ratio of the hepatocyte growth factor to the epidermal growth factor is 1: 0.5 to 2.
5. The method of claim 1, wherein the immunodeficient mouse is selected from the group consisting of a Nude mouse, a SCID mouse, a NOD-SCID mouse, an NSI mouse, an NCG mouse, a Rag2 mouse, and an IL2RG mouse.
6. The method of claim 5, wherein the immunodeficient animal is a NCG mouse.
7. The use of the humanized liver animal model of any one of claims 1 to 6 in screening of liver disease drugs and evaluation of drug efficacy.
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