JP7233039B2 - Human non-alcoholic steatohepatitis model - Google Patents

Description

The present invention provides a human non-alcoholic steatohepatitis model obtained by subjecting a rodent to a specific treatment, and a method of using a rodent subjected to a specific treatment as a human non-alcoholic steatohepatitis model. , a method for producing this model, and a method for screening a preventive, ameliorating, or therapeutic agent for human non-alcoholic steatohepatitis using this model.

Causes of liver disease include hepatitis virus, alcohol, autoimmunity, primary biliary cholangitis, obesity, diabetes, hypertriglyceridemia, excessive nutritional intake due to long-term parenteral nutrition, endocrine disorders, and low beta Lifestyle habits and lifestyle-related diseases such as lipoproteinemia and starvation-replenishment syndrome are known. In recent years, non-alcoholic fatty liver disease (NAFLD) due to lifestyle habits has increased and become a problem. NAFLD includes simple fatty liver, in which fat is deposited in hepatocytes, and non-alcoholic steatohepatitis (NASH), in which inflammation and fibrosis progress due to liver steatosis. Unlike simple fatty liver, NASH progresses to liver cirrhosis and further to liver cancer. Therefore, elucidation of the progression mechanism from simple fatty liver to NASH and accurate differentiation between the two are particularly important issues.

Animal models exhibiting NASH symptoms are necessary for research on NASH onset mechanisms and studies on prevention or treatment of NASH. Conventionally, as an animal model of NASH, rats fed with a choline- and methionine-deficient diet are known (Non-Patent Document 1). However, it is difficult to use for continuous observation of NASH symptoms and drug efficacy evaluation because body weight decreases due to decreased muscle mass.
It is also known that feeding rats with a choline-deficient methionine-containing diet can induce NASH. However, the influence of species difference is strong, and fibrosis characteristic of NASH is not observed even when this diet is given to mice (Non-Patent Document 1).
In addition, since the livers of these model animals are non-human animal livers, they are not animal models that completely reproduce human NASH, and the metabolic activity in the liver is also of the non-human animal type. Therefore, it is difficult to say that this model is suitable for evaluating the efficacy of NASH in humans.

Here, in Patent Document 1, a primary chimeric non-human animal is produced by transplanting human hepatocytes into a liver-damaged immunodeficient non-human animal, and human hepatocytes are separated from the liver of this primary chimeric non-human animal by a collagenase perfusion method. However, in chimeric non-human animals obtained by transplantation into new liver-damaged immunodeficient non-human animals, macrovesicular fat deposits and swelling of human hepatocytes were observed in human hepatocytes. Inflammatory cells centering on the neutrophils accumulate, fibrosis is also observed, and it is disclosed that symptoms of NASH are exhibited. The serially transplanted chimeric non-human animal taught by Patent Document 1 has a large portion of its liver replaced with human hepatocytes, so it was expected that it could be used as an animal model that reproduces human NASH.
However, the NASH symptoms of the chimeric non-human animal taught by Patent Document 1 have low reproducibility, and are difficult to be practically used as an animal model for wide use in screening of therapeutic agents for NASH. Moreover, in this method, since NASH is produced by transplanting human hepatocytes, there is also a problem that a non-NASH group in which human hepatocytes are transplanted, that is, a control group cannot be established.

WO2008/001614

Folia Pharmacol.Jpn. 144, 69-74 (2014)

The present invention provides a rodent animal model that stably exhibits human NASH symptoms, a method for preparing this model, a method for using a rodent animal that stably exhibits human NASH symptoms as a human NASH model, and such rodents. An object of the present invention is to provide a method for screening human NASH therapeutic agents using dental animals.

In order to solve the above problems, the present inventors have conducted extensive research and found that chimeric rodents in which some or all of the hepatocytes have been replaced with human hepatocytes have a content of choline or a salt thereof of 0.01% by weight. Hereinafter, by breeding with a mixed feed adjusted to a methionine content of 0.5% by weight or less and a fat content of 25 kcal% or more, macrovesicular fat deposition, We found that NASH-characteristic lesions such as fibrosis, inflammatory cell infiltration, hepatocyte ballooning, and Mallory bodies were observed. In particular, ballooning and mallory bodies are difficult to appear in experimental animals, and there are reports that they are lesions specific to human NASH (PLoS One. 2014 Dec 23; 9(12): e115922. doi: 10.1371 /journal.pone.0115922. eCollection 2014.), this rodent was found to serve as a model that recapitulated human NASH.

In addition, the present inventors have found that raising human hepatocyte chimeric rodents with the above-mentioned adjusted feed increases the human ALT1 concentration without changing the alanine transaminase (ALT) concentration of the rodents. rice field. When hepatocytes are damaged, cytoplasmic enzymes leak out of the cells and enter the blood. Since ALT is contained most abundantly in the liver, ALT activity in the blood is an indicator of liver damage. Therefore, it was found that the above-mentioned conditioned feed can specifically induce liver injury in human hepatocytes without inducing injury in the recipient animal's hepatocytes remaining in the human hepatocyte chimeric rodent. In this respect as well, it can be seen that the conditioned feed described above can induce symptoms characteristic of human NASH.

In addition, it was confirmed that NASH symptoms could be reproducibly introduced by breeding human hepatocyte chimeric rodents on the above-mentioned adjusted diet. Therefore, it was found that human hepatocyte chimeric rodents fed with the above-mentioned modified diet can be used as an animal model for study of NASH pathology and drug efficacy screening.

In human hepatocyte chimeric rodents, human hepatocytes engrafted in the liver function by interacting with rodent cells in other tissues or organs and with rodent hepatocytes remaining in the liver. do. This raises the possibility that mouse hepatocytes and human hepatocytes have different sensitivities to various disorders, and it is often difficult to specifically damage human hepatocytes. For example, as shown in the Examples section, inflammation and fibrosis produced by administration of carbon tetrachloride (CCl ₄ ) to normal mice is commonly used as a hepatitis or cirrhosis model, but human liver It is difficult to obtain a hepatitis or cirrhosis model with human hepatocyte injury because administration of CCl ₄ to cell chimeric mice induces much stronger injury and necrosis in mouse hepatocytes than in human hepatocytes.
Under these circumstances, it is not surprising that feeding human hepatocyte chimeric rodents with the above-mentioned adjusted diet specifically damaged human hepatocytes and strongly expressed lesions characteristic of human NASH. It is amazing.

In addition, as described in Non-Patent Document 1, there are species differences in the induction of lesions by modified feeds among rodents. In this respect as well, it is surprising that NASH could be induced in rodent livers replaced with human hepatocytes by administration of a conditioned diet.

The present invention has been completed based on the above findings, and provides the following [1] to [14].
[1] A chimeric rodent animal in which human hepatocytes have been partially or entirely replaced with human hepatocytes is fed with a modified feed having one or more of the following characteristics (a), (b), and (c): A human non-alcoholic steatohepatitis model, including an animal obtained by
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate, and fat contained in the adjusted feed [2] Chimeric rodents are primary chimeric rodents, or subcultured The human nonalcoholic steatohepatitis model according to [1], which is a chimeric rodent animal.
[3] The human non-alcoholic steatohepatitis model of [1] or [2], which is fed with a modified diet for 1 week or more.
[4] Raising a chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes, on a modified feed having one or more of the following characteristics (a), (b), and (c): as a human non-alcoholic steatohepatitis model.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate, and fat contained in the adjusted feed [5] Chimeric rodents are primary chimeric rodents, or subcultured The method of [4], which is a chimeric rodent.
[6] The method of [4] or [5], wherein the animal is fed with a modified feed for one week or longer.
[7] A chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes is fed with a modified feed having one or more of the following characteristics (a), (b), and (c): A method for producing a human non-alcoholic steatohepatitis model, comprising steps.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate, and fat contained in the adjusted feed [8] Chimeric rodents are primary chimeric rodents, or subcultured The method of [7], which is a chimeric rodent.
[9] The method of [7] or [8], wherein the animal is fed with a modified feed for 1 week or more.
[10] A chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes is fed with a modified feed having one or more of the following characteristics (a), (b), and (c): The step of administering a test substance to an animal obtained by the method comprising the step and the degree of symptoms of non-alcoholic steatohepatitis before and after administration are compared, or the chimeric rodent animals administered the test substance and the test substance A method of screening for a therapeutic agent for human non-alcoholic steatohepatitis, comprising the step of comparing the degree of symptoms of non-alcoholic steatohepatitis between untreated chimeric rodent animals.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more relative to the total calorific value of protein, carbohydrate, and fat contained in the adjusted feed [11] Chimeric rodents are primary chimeric rodents, or subcultured The method of [10], which is a chimeric rodent.
[12] The method of [10] or [11], wherein the animal is fed with a modified feed for 1 week or more.
[13] A chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes is fed with a modified feed having one or more of the following characteristics (a), (b), and (c): as a human non-alcoholic steatohepatitis model.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more relative to the total calorific value of protein, carbohydrate, and fat contained in the adjusted feed [14] Passage in which part or all of the hepatocytes are replaced with human hepatocytes A human simple fatty liver model, including transplanted chimeric rodents.

The human NASH animal model of the present invention has all or part of the liver replaced with human hepatocytes, so unlike conventional rodent NASH models, it exhibits various symptoms characteristic of human NASH. It is a model that accurately reproduces NASH. In addition, in the human NASH animal model of the present invention, the transplanted human hepatocytes are specifically damaged, which also supports the presentation of lesions characteristic of human NASH. Moreover, the NASH symptoms of the human NASH animal model of the present invention are reproducible, whereas the NASH symptoms of the conventional rodent animal model having human hepatocytes were not reproducible.
In addition, human hepatocyte chimeric rodents fed a normal diet have simple fatty liver but do not progress to NASH. Chimeric rodent animals can be used as control animals. The inventors of the present invention found that primary chimeric rodents exhibited symptoms of simple fatty liver, and reported in Patent Document 1. It was discovered in the present invention that the symptoms of
Thus, the human NASH animal model of the present invention forms human NASH pathology with high reproducibility and can be compared with control animals. It is a better model than
Based on these facts, the model of the present invention can be suitably used as a pathological model animal that accurately reflects the pathological condition of human NASH for research on the onset mechanism of NASH, screening of agents for its prevention or treatment, and the like.

Fig. 10 is a graph showing changes in blood human albumin concentration when serially transplanted chimeric mice (human hepatocyte Lot No. BD195) were fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 4 is a graph showing changes in plasma human ALT activity and mouse ALT activity when serially transplanted chimeric mice (human hepatocyte lot No. BD195) were fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. . Fig. 2 shows hematoxylin and eosin stained images (magnification: 40) of liver sections of serially transplanted chimeric mice (human hepatocyte lot No. BD195) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 2 shows hematoxylin and eosin staining images (magnification: 400) of liver sections of serially transplanted chimeric mice (human hepatocyte lot No. BD195) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 2 shows hematoxylin and eosin stained images (magnification: 400) of liver sections of serially transplanted chimeric mice (human hepatocyte Lot No. IVTJFC) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. FIG. 10 shows Sirius red-stained liver sections of serially transplanted chimeric mice (human liver cell lot No. BD195) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. FIG. 2 shows Sirius red-stained liver sections of serially transplanted chimeric mice (human hepatocyte Lot No. IVTJFC) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 2 shows the ratio of fibrosis area calculated from Sirius red-stained images of liver sections of serially transplanted chimeric mice (human hepatocyte lot No. BD195) fed with ultra-high-fat choline-deficient methionine-reduced diet or normal diet. graph. Fig. 10 shows anti-F4/80 antibody immunostaining images of liver sections after serially transplanted chimeric mice (human hepatocyte Lot No. BD195) were bred with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Macrophages/Kupffer calculated from anti-F4/80 antibody immunostaining images of liver sections after serially transplanted chimeric mice (human hepatocyte lot No. BD195) were bred with ultra-high-fat choline-deficient methionine-reduced diet or normal diet. It is a graph which shows the ratio of a cell-positive area. Fig. 10 shows anti-αSMA antibody immunostaining images of liver sections of serially transplanted chimeric mice (human hepatocyte Lot No. BD195) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. It is a TUNEL-stained image of a liver section after serially transplanted chimeric mice (human hepatocyte Lot No. BD195) were fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 2 shows hematoxylin and eosin stained images (400x magnification) of liver sections of primary chimeric mice (human hepatocyte Lot No. IVTJFC) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. It is Sirius red-stained images of liver sections of primary chimeric mice (human hepatocyte Lot No. IVTJFC) fed with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. It is an anti-F4/80 antibody immunostaining image of a liver section after breeding primary chimeric mice (human hepatocyte Lot No. IVTJFC) with an ultra-high-fat choline-deficient methionine-reduced diet or a normal diet. Fig. 3 is a graph showing plasma total ALT activity and human ALT activity in human hepatocyte chimeric mice (human hepatocyte Lot No. BD195) or SCID mice in CCl _4- administered or non-administered groups. Fig. 10 is a hematoxylin/eosin-stained image of a liver section after administration of CCl ₄ to a human hepatocyte chimeric mouse (human hepatocyte Lot No. BD195).

(1) Human NASH rodent model and its production method The method for producing the human non-alcoholic steatohepatitis (NASH) model of the present invention is a chimeric rodent model in which some or all of the hepatocytes are replaced with human hepatocytes. A group consisting of (a) 0.01% by weight or less of choline or a salt thereof, (b) 0.5% by weight or less of methionine, and (c) 25 kcal% or more of fat. A method comprising feeding a conditioned feed having at least one selected characteristic.

Human hepatocyte chimeric rodents Chimeric rodents in which some or all of the hepatocytes are replaced with human hepatocytes can be produced by transplanting human hepatocytes into immunodeficient liver-damaged rodents ( primary chimeric animals). In addition, serially transplanted chimeric rodents are obtained by transplanting human hepatocytes proliferated in the body of primary chimeric rodents into immunodeficient liver-damaged rodents of the same type as the immunodeficient liver-damaged rodents described above. can be obtained by Transplantation of human hepatocytes grown in chimeric rodents can be performed once or multiple times.

Rodents Examples of rodents include rodents such as mice and rats, guinea pigs, squirrels and hamsters, and rodents such as mice and rats, which are widely used as experimental animals, are easy to use.
They may be male or female, but males are preferred.

Immunodeficiency Liver Injury Rodent Immunodeficiency Liver Injury Rodents are immunodeficient in that they do not reject cells derived from different species, and the cells of the rodent's native liver are damaged. animals that are receiving If human hepatocytes are transplanted due to damage to the animal's original cells, the liver function will be maintained by the transplanted human hepatocytes, and the animal will be an animal that accurately reflects the individual functions of human hepatocytes. Become. It also facilitates the proliferation of transplanted human hepatocytes.

Immunodeficiency hepatopathy animals can be prepared by subjecting the same individual to liver injury-inducing treatment and immunodeficiency-inducing treatment. Liver injury-inducing treatments include administration of liver injury-inducing substances such as carbon tetrachloride, yellow phosphorus, D-galactosamine, 2-acetylaminofluorene, and pyrrolizidine alkaloids, irradiation, and surgical partial liver resection. mentioned. Immunodeficiency-inducing treatments include administration of immunosuppressants and thymectomy.

An immunodeficient liver-damaged animal can also be produced by subjecting a genetically immunodeficient animal to liver damage-inducing treatment. Genetic immunodeficiency animals include animals with severe combined immunodeficiency (SCID) showing T cell lineage deficiency, animals with loss of T cell function due to hereditary thymic deficiency, and animals with known RAG2 gene. Examples include gene targeting (Science, 244:1288-1292, 1989) and animals knocked out by genome editing technology. Specifically, SCID mice, RAG2 knockout mice, IL2Rgc/Rag2 knockout mice, NOD mice, NOG mice, nude mice, nude rats, immunodeficient rats obtained by transplanting SCID mouse bone marrow into X-ray irradiated nude rats. (JP-A-2007-228962, Transplantation. 60(7):740-7, 1995).

An immunodeficient liver-damaged animal can also be produced by subjecting a genetic liver-damaged animal to immunodeficiency-inducing treatment. As a genetic liver-damaged animal, a known transgenic method (Proc. Natl. Acad. USA 77;7380-7384, 1980). In such animals, the hepatopathy-inducing protein is expressed in a liver-specific manner, resulting in hepatopathy. Proteins that are specifically expressed in the liver include serum albumin, cholinesterase, Hagemann factor, and the like. Liver injury-inducing proteins include urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and the like. Animals with hereditary liver damage can also be obtained by knocking out genes responsible for liver function, such as the fumarylacetoacetate hydrolase gene. In addition, administration of ganciclovir to mice transfected with a thymidine kinase gene under the albumin enhancer promoter can also induce liver injury.

Furthermore, an immunodeficient liver-damaged animal can also be produced by mating a genetically immunodeficient animal with a genetic liver-damaged animal of the same species.
As the genetic immunodeficiency hepatopathy animal, an animal that is homozygous or heterozygous for each of the hepatopathy gene and the immunodeficiency gene can be used.

Human hepatocytes used for transplantation can be isolated from human liver tissue by a conventional method such as the collagenase perfusion method. For example, by using human hepatocytes from children under the age of 14, a high replacement rate with human hepatocytes is achieved. In addition, by using proliferative hepatocytes with the ability to proliferate in vivo, human hepatocyte populations that can rapidly proliferate in the body of recipient rodents and exhibit normal liver function can be generated in a short period of time. can be formed. Such proliferative human hepatocytes include small human hepatocytes invented by the present inventors (JP-A-8-112092, etc.). In addition, human hepatocytes obtained from hepatic progenitor cells such as Clip cells and pluripotent stem cells such as iPS cells and ES cells can also be used.

Human hepatocytes can be transplanted into the liver via the spleen of immunodeficient hepatopathic animals. It can also be transplanted directly from the portal vein. The number of human hepatocytes to be transplanted can be about 1 to 2 million.
The sex of the immunodeficient liver-damaged animal is not particularly limited. In addition, the age of immunodeficient liver-damaged animals at the time of transplantation is not particularly limited. As far as possible, it is preferable to use animals that are about 6 weeks old immediately after birth.
Animals after transplantation may be bred by a conventional method. For example, by rearing for about 3 to 30 weeks after transplantation, a primary chimeric animal in which some or all of the hepatocytes have been replaced with human hepatocytes can be obtained.

Next, a method for producing a serially transplanted chimeric animal will be described. Human hepatocytes proliferated in the chimeric animal can be recovered, for example, by treating the liver tissue of the chimeric animal with collagenase. Since the cytotoxicity of collagenase to rodent hepatocytes is higher than that to human hepatocytes, by adjusting the collagenase treatment time, chimeric hepatocytes were damaged and almost exclusively human hepatocytes were isolated. can do.
The collected hepatocytes include not only human hepatocytes proliferated in the chimeric animal body, but also a small amount of hepatocyte non-parenchymal liver cells and recipient animal hepatocytes. Therefore, although the collected hepatocytes may be used for transplantation as they are, the purity of human hepatocytes can be increased using a monoclonal antibody that specifically recognizes human hepatocytes or recipient animal hepatocytes.
The method of transplanting human hepatocytes isolated from the primary chimeric animal into the liver of a rodent and proliferating the cells is the same as that for producing the primary chimeric animal.

Both primary chimeric rodents and passage-transplanted chimeric rodents can be used in the present invention. Subcultured chimeric rodents may be those that have been subcultured once, or those that have been subcultured twice or more. For example, 2-4 passaged chimeric rodents can be used. In both cases, human NASH symptoms can be sufficiently induced by feeding them with a modified diet.

で示される第４級アンモニウムカチオンである。
コリン塩としては、それには限定されないが、塩化物、水酸化物、リン酸塩、リン酸一水素塩、リン酸二水素塩、炭酸塩、炭酸水素塩、硫酸塩のような無機塩；酒石酸塩、酒石酸水素塩（重酒石酸塩）、クエン酸塩、酢酸塩、シュウ酸塩、乳酸塩、リンゴ酸塩、フマル酸塩、マロン酸塩、コハク酸塩のような有機酸塩に代表される有機塩が挙げられる。調整飼料中では、コリンは、通常、塩として存在する。 Adjusted feed When the adjusted feed used in the present invention has a reduced amount of choline or its salt, the amount of choline or its salt is 0.01 relative to the total amount of the adjusted feed (that is, the final concentration). It is preferably 0.001% by weight or less, more preferably 0.001% by weight or less, and most preferably does not contain or substantially does not contain choline or a salt thereof. This is sufficient to induce NASH symptoms. In addition, even if choline or its salt is not added, there is no hindrance to growth.
The compounding amount of choline or its salt referred to here is the amount of choline or its salt added to the feed. Materials in feeds for feeding rodents may inherently contain trace amounts of choline or its salts, but the content is usually negligible.
Choline has the following formula (1)

is a quaternary ammonium cation represented by
Choline salts include, but are not limited to inorganic salts such as chlorides, hydroxides, phosphates, monohydrogen phosphates, dihydrogen phosphates, carbonates, hydrogen carbonates, sulfates; tartaric acid; salts, hydrogen tartrate (bitartrate), citrate, acetate, oxalate, lactate, malate, fumarate, malonate, organic acid salts such as succinate Organic salts are mentioned. In formulated feeds, choline is normally present as a salt.

When the adjusted feed used in the present invention has a reduced methionine content, the methionine content is preferably 0.5% by weight or less, and 0.2% by weight or less, relative to the total amount of the adjusted sample (that is, the final concentration). is more preferred, and 0.1% by weight or less is even more preferred. Moreover, the amount of methionine can be 0.03% by weight or more, 0.05% by weight or more, or 0.1% by weight or more. Within this range, NASH symptoms can be induced while weight loss due to loss of muscle mass is suppressed, making it practical as an experimental animal.
The amount of methionine added here is the amount of methionine added to the feed. Materials in feeds intended for feeding rodents may naturally contain trace amounts of methionine, but the content is usually negligible. Methionine, which constitutes the peptide, is not included in the amount of methionine blended here.

When the adjusted feed used in the present invention has an increased fat content, the fat content is 25 kcal% or more with respect to the total calorie (100 kcal%) of protein, carbohydrate, and fat in the feed , preferably 40 kcal% or more, especially 50 kcal% or more, especially 60 kcal% or more. This is sufficient to induce NASH symptoms. In addition, the fat content should be 120 kcal% or less, especially 90 kcal% or less, especially 70 kcal% or less, especially 60 kcal% or less, relative to the total calorific value (100 kcal%) of protein, carbohydrate, and fat in the feed. can be Within this range, NASH symptoms can be induced and practical use as experimental animals can be achieved.

In addition, when the adjusted feed used in the present invention has an increased fat content, the fat content is 10% by weight or more, especially 20% by weight or more, especially 30% by weight, based on the total amount of the adjusted feed. Above all, 35% by weight or more is preferable. This is sufficient to induce NASH symptoms. Also, the fat content can be 70% by weight or less, especially 60% by weight or less, especially 50% by weight or less, relative to the total amount of the prepared feed. Within this range, NASH symptoms can be induced and practical use as experimental animals can be achieved.

In the present invention, the "fat" in the conditioned feed includes any of vegetable oils, animal oils and mineral oils.
Vegetable oils include, but are not limited to, corn oil, soybean oil, sesame oil, rapeseed oil, rice oil, bran oil, camellia oil, safflower oil, coconut oil, cottonseed oil, sunflower oil, perilla oil, linseed oil, olive oil, and peanut oil. , almond oil, avocado oil, hazelnut oil, walnut oil, grapeseed oil, cocoa butter, peanut butter, and the like. Animal fats and oils include whale oil, shark oil, liver oil, horse oil, lard, beef tallow, horse fat, milk fat, hydrogenated oils and fats thereof, and the like.

The prepared feed used in the present invention has (a) a choline or its salt content of 0.01% by weight or less, (b) a methionine content of 0.5% by weight or less, and (c) a fat content of 25 kcal% or more. Any one or two or more of the characteristics may be provided. Specifically, (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), and (a) and (b) Any of (c) is acceptable. Among them, a combination of (a), (b) and (c) is preferable.

The prepared feed used in the present invention contains nutrients such as proteins, carbohydrates, minerals, and vitamins normally contained in animal feed within the range satisfying (a), (b), and/or (c) above. can be done.

For chimeric rodents, in both primary chimeric animals and serially transplanted chimeric animals, the adjusted feed is administered when the human hepatocyte replacement rate is 30% or more, especially 50% or more, especially 70% or more. It is preferable to start In addition, when the human hepatocyte replacement rate reaches 80% or more or 90% or more, or when all the hepatocytes of the rodent are replaced with human hepatocytes, administration of the modified feed may be started. .
The human hepatocyte replacement rate is measured, for example, by preparing a liver section of a chimeric animal and staining (e.g., hematoxylin and eosin staining) to measure the area ratio of human hepatocytes, or by using a human hepatocyte-specific antibody (e.g., , cytokeratin 8/18 antibody or STEM121 antibody), and the human hepatocyte area ratio (human cytokeratin 8/18 or STEM121 positive area ratio) is measured. By collecting liver sections from all seven lobes of the chimeric animal and calculating the average value of the human hepatocyte area ratio, the human hepatocyte replacement rate can be accurately determined. Since the human hepatocyte replacement rate of the right lateral lobe section is highly correlated with the average value of the human hepatocyte replacement rate of all seven lobe sections, the human hepatocyte replacement rate can also be obtained using the right lateral lobe section. .
The human hepatocyte replacement rate can also be estimated by measuring the blood human albumin concentration of the chimeric animal and applying it to a previously prepared calibration curve.

When the chimeric rodent is reared on the modified diet, the modified diet may be fed ad libitum or forced-fed.
In addition, the administration period of the adjusted feed can be 1 week or longer, 2 weeks or longer, 3 weeks or longer, or 4 weeks or longer. It can also be 40 weeks or less, 30 weeks or less, 20 weeks or less, or 10 weeks or less. This range can sufficiently induce NASH symptoms.

Nonalcoholic steatohepatitis (NASH)
A human hepatocyte chimeric rodent exhibiting human NASH symptoms is obtained as described above, and this animal can be used as a human NASH model (human NASH rodent model).
Human NASH is associated with at least lipid droplet deposition, inflammatory cell infiltration, and fibrosis in the liver, among fat-accumulating liver diseases other than alcoholic liver disease in humans (non-alcoholic fatty liver disease (NAFLD)). A disease that presents the symptoms of

Deposition of lipid droplets in the liver may be observed even a little, but the area ratio of lipid droplets observed by staining the liver section (e.g., Oil Red O staining) is preferably 5% or more, 33% and even more preferably greater than 66%. The NAFLD activity score (NAS score) determined by the Nonalcoholic Steatohepatitis Clinical Research Network (Hepatology. 2005 Jun;41(6):1313-21.) is "1" when the area ratio of lipid droplets is 5% or more and 33% or less. "2" when more than 33% and 66% or less, and "3" when more than 66%.

Inflammatory cell infiltration in the liver should be observed even if only a little, but when liver sections are stained (e.g., hematoxylin and eosin staining, Masson's trichrome staining), inflammatory foci are found within the liver lobules per x200 field of view. is preferably one or more, more preferably two or more, more preferably four or more. In the above NAS score, 1 or less than 2 inflammatory foci per 200x field of view of the liver lobule is '1', 2 or more and 4 or less is '2', and more than 4 is '3'. be.

Even a little liver fibrosis should be observed, but it should be observed in the perisinusoidal and central hepatic lobules or periportal vein when the liver section is stained (e.g., Sirius red staining, Masson's trichrome staining). is preferred (NAS score Fibrosis stage “1”), more preferably both perisinusoidal and periportal veins (NAS score Fibrosis stage “2”), and bridging fibrosis is observed Even more preferably (NAS score Fibrosis stage '3'), even more preferably liver cirrhosis is observed (NAS score Fibrosis stage '4').

In addition to the lesions described above, iron deposition and hepatocyte apoptosis can also be observed in the liver of chimeric rodents exhibiting NASH symptoms obtained by the above method. Iron deposition can be confirmed, for example, by iron staining or hematoxylin-eosin staining of a liver section. Apoptosis of hepatocytes is confirmed, for example, as apoptotic bodies in hematoxylin and eosin staining. It can also be confirmed by detecting DNA fragments generated by apoptosis by the TUNEL (TdT-mediated dUTP nick end labeling) method or by immunostaining for activated caspase 3.

Oxidative stress is also considered to be one of the causes of progression of symptoms from non-alcoholic fatty liver disease (NAFLD) to NASH. CYP2E1 and 4-hydroxy-2-nonenal (4-HNE) are known proteins that cause oxidative stress. CYP2E1 is one of the enzymes that generate free radicals, and 4-HNE is a lipid peroxidase. In the liver of chimeric rodents exhibiting NASH symptoms, expression or enhanced expression of proteins that cause oxidative stress, such as CYP2E1 and 4-HNE, can be observed. Expression of CYP2E1 and 4-HNE can be confirmed by immunostaining of liver sections using human CYP2E1 and human 4-HNE antibodies, respectively.

In addition, lesions characteristic of human NASH, such as ballooning of hepatocytes and Mallory bodies, can be observed in the livers of chimeric rodents exhibiting NASH symptoms. Ballooning of hepatocytes and Mallory bodies can be confirmed by staining liver sections (eg, hematoxylin-eosin staining, Azan-Mallory staining, Masson's trichrome staining). The NAS score is "1" when a small number of ballooning degeneration of hepatocytes in the liver is observed, and "2" when many are observed.

In addition, enhanced liver-enriched human alanine transaminase (ALT) activity may be seen in blood, plasma, or serum. ALT catalyzes the transfer reaction between the α-keto group of α-ketoglutarate and the amino group of L-alanine to produce pyruvate and glutamate, which is coupled to lactate dehydrogenase (LDH) to convert β- Since nicotinamide adenine dinucleotide reduced form (NADH) is converted to β-nicotinamide adenine dinucleotide oxidized form (NAD), the ALT activity value can be obtained by measuring the rate of NADH decrease using absorbance as an index. .
Although mouse hepatocyte-derived ALT and human hepatocyte-derived ALT cannot be distinguished by ALT activity measurement, if human ALT1 concentration is measured by ELISA in addition to ALT activity measurement, it accounts for ALT. It is possible to grasp the ratio of ALT derived from human hepatocytes.

(2) Screening method for agent for prevention, treatment, or improvement of human NASH The method for screening for agent for prevention, treatment, or improvement of human NASH of the present invention is performed by adding a test substance to the rodent animal model of human NASH of the present invention described above. and comparing the degree of NASH symptoms before and after administration of the test substance, or comparing the degree of NASH symptoms between a test substance-administered group and a non-administered group.

A comparison of the degree of NASH symptoms before and after administration of the test substance may be made for any of the above symptoms observed in human NASH. That is, lipid droplet deposition, inflammatory cell infiltration, fibrosis, iron deposition, hepatocyte apoptosis, expression of proteins that cause oxidative stress, ballooning of hepatocytes, Mallory bodies, and ALT activity in the liver were investigated. good too. Among them, it is preferable to perform lipid droplet deposition, inflammatory cell infiltration, and/or fibrosis. In addition to one or more of lipid droplet deposition, inflammatory cell infiltration, and fibrosis, one of iron deposition, hepatocyte apoptosis, expression of proteins leading to oxidative stress, ballooning of hepatocytes, Mallory bodies, and ALT activity By comparing the above, more accurate screening can be performed.
If these symptoms are relieved or improved, the test substance can be determined to be effective in treating or improving human NASH. If any NASH symptom is significantly alleviated or improved, it can be determined that the test substance is effective for treating or improving human NASH, but the It is also possible to use the mitigation or improvement of In addition, since a drug that is effective in treating or improving a disease is usually also effective in preventing the disease, such a test drug can be determined to be also effective in preventing human NASH.

Test substances are not particularly limited, and include low-molecular-weight compounds, amino acids, nucleic acids, lipids, sugars, extracts of natural products, and the like.
The administration route of the test substance is not particularly limited and includes oral administration, intraperitoneal administration, intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, transdermal administration and the like.
The dosage of the test substance and the dosing schedule, such as the frequency of administration, can be determined for each test substance so that the presence or absence of effects can be determined.
For example, 1 to 8 weeks after administration of the test substance, the degree of NASH symptoms can be confirmed and compared with the degree of NASH symptoms in the non-administration group. Alternatively, the degree of NASH symptoms can be compared before and after administration of the test substance.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these.
(1) Generation of primary human hepatocyte chimeric mice
Generation of immunodeficient liver-damaged mice
uPA-Tg(+/+)/SCID(+/+) mice used as recipient animals were bred by PhoenixBio. This mouse was produced by the following method.

First, uPA-Tg mice (hemizygote, +/-) were produced by the method described in Examples 1 and 2 of JP-A-2013-230093.
For the uPA gene, total RNA was extracted from mouse liver by the AGPC method (acid-guanidinium-isothiocyanate-phenol-chloroform) and dissolved in RNase-free water. Using the total RNA obtained above, the sequence of the uPA gene registered in a public database (accession number: uPA gene-specific primer created from NM008873 (1341st to 1360th base antisense sequence) and Reverse transcription reaction with LongRange Reverse Transcriptase (manufactured by Qiagen) was performed at 25°C for 10 minutes, then at 42°C for 90 minutes, and reverse transcriptase inactivation treatment was performed at 85°C for 5 minutes. Invitrogen) was added and treated at 37° C. for 20 minutes to digest the mRNA and leave only cDNA.PCR was performed using the synthesized cDNA as a template for PCR reaction. Phusion DNA polymerase (manufactured by Fynnzymes) was used as the enzyme, and the PCR primer (sense sequence from base 39 to base 61) was prepared from the uPA gene sequence (accession number: NM008873). The amplified fragment in the PCR reaction has a length of base numbers 39 to 1360. The obtained DNA fragment was introduced into an expression plasmid having a mouse albumin promoter/enhancer described below to construct "mAlb uPAInt2". mAlb uPAInt2" is a mouse albumin enhancer/promoter downstream of rabbit β-globin exon 2, intron, 3rd exon, mouse uPA ORF, and rabbit β-globin 3rd exon polyA signal. be.

Plasmid "mAlb uPAInt2" was introduced into ES cells obtained from 129SvEv mice by electroporation, and then selective culture was performed with G418. The resulting G418-resistant colonies were assayed for gene-introduced ES cells by PCR as follows.

25-30 μg of vector for homologous recombination (uPA) DNA was linearized by restriction enzyme digestion and purified. This DNA was suspended in an electroporation buffer (20 mM HEPES pH 7.0, 137 mM NaCl, 5 mM KCl, 6 mM D-glucose, 0.7 mM Na ₂ HPO ₄ ) containing 3×10 ⁶ mouse ES cells. Gene transfer was performed under the conditions of turbidity, Field Strength of 185 V/cm, and Capacitance of 500 μF. After 24 hours from the introduction, selective culture was performed with G418 (Geniticin) (SIGMA G-9516) at a final concentration of 200 μg/mL. Dulbecco's Modified Eagle's Medium (DMEM) (Gibco/BRL 11965-084) culture medium containing fetal bovine serum (Hyclone SH30071) at a final concentration of 15% and L-glutamine at a final concentration of 2 mM (Gibco /BRL 25030-081), non-essential amino acids (Gibco/BRL 11140-050) at a final concentration of 100 μM each, HEPES at a final concentration of 10 mM (Gibco/BRL 15630-080), a final concentration of 100 U each. /mL penicillin/streptomycin (Gibco/BRL 15140-122), β-mercaptoethanol (SIGMA M-7522) at a final concentration of 100 μM, and ESGRO(LIF) (Gibco/BRL) at a final concentration of 1000 U/mL. 13275-029) was used (hereinafter referred to as ES medium).

MEF (Mouse Embryonic Fibroblast) cells isolated from E14.5 embryos were used as feeder cells for ES cells, and the culture medium was DMEM (Gibco/BRL 11965-084) with a final concentration of 10%. fetal bovine serum (Hyclone SH30071), L-glutamine (Gibco/BRL 25030-081) at a final concentration of 2 mM, non-essential amino acids (Gibco/BRL 11140-050) at a final concentration of 100 μM each, final concentration was added with 100 U/mL of penicillin/streptomycin (Gibco/BRL 15140-122) (hereinafter referred to as MEF medium). MEF cells cultured to confluence in a 150 cm ² flask were detached with trypsin/EDTA (0.05%/1 mM, Gibco/BRL 25300-047) and plated in four 10 cm dishes, two 24-well plates, Two 6-well plates, six 25 cm ² flasks and two 75 cm ² flasks were each re-plated at the optimal concentration.

ES cells for genotyping were prepared as follows.
From day 5 after gene introduction, the G418-resistant colonies that emerged were subcultured to a 24-well plate as follows. That is, using Pipetman (Gilson), G418-resistant colonies were transferred to a 96-well microplate containing 150 µL of trypsin/EDTA solution, treated in an incubator at 37°C for 20 minutes, and then pipetted with Pipetman. made into single cells. This cell suspension was transferred to a 24-well plate and culture continued. After 2 days, the cells on the 24-well plate were split into two, one for cryopreservation and one for DNA extraction. Briefly, the cells were treated with 500 μL of trypsin/EDTA for 20 minutes in a 37° C. incubator, then 500 μL of ES medium was added and gently pipetted with a Pipetman to make single cells. Half of the cell suspension was then transferred to a 24-well plate containing 1 mL of ES medium, and 1 mL of ES medium was also added to the original 24-well plate. Two days later, the medium was removed from one of the 24-well plates, and fetal bovine serum at a final concentration of 10% and dimethylsulfoxide (DMSO) at a final concentration of 10% (Sigma D-5879) were added to the ES medium. 1 mL of freezing medium was added, and after sealing, the tube was cryopreserved at -70°C.

Assays for gene-introduced ES cells were performed by PCR as follows. That is, the medium was removed from each well of the 24-well plate in which the cells had grown to confluence, washed with PBS, and then added with 250 μL of lysis buffer (1% SDS, 20 mM EDTA, 20 mM Tris pH 7.5) and proteinase K. (20 mg/mL) was added, shaken well, and dissolved by heating at 52°C. DNA was extracted from the dissolved samples by phenol/chloroform extraction and used as template DNA for PCR.

uPA gene-introduced ES cells were selected by the following procedure.
The PCR primers used were set in rabbit β-globin. The sequences are sense primer: GGGCGGCGGTACCGATCCTGAGAACTTCAGGGTGAG (SEQ ID NO: 1) and antisense primer: GGGCGGCGGTACCAATTCTTTGCCAAAATGATGAGA (SEQ ID NO: 2). The reaction was carried out according to the attached method of AmpliTaqGold (ABI). After activating the enzyme at 95°C for 9 minutes, the reaction was repeated 40 times at 94°C for 30 seconds (denaturation), 63°C for 30 seconds (annealing), and 72°C for 1 minute (elongation). After completion, the reaction solution was electrophoresed on a 2% agarose gel to confirm the PCR product.

Clones for which gene transfer was confirmed by PCR analysis were thawed by warming the frozen 96-well plate to 37° C. and subcultured to a 24-well plate. After culturing this 24-well plate at 37° C. for 24 hours, the medium was changed to remove DMSO and liquid paraffin. When each clone reached 75-90% confluence, it was subcultured from 24-well to 6-well plate. Furthermore, when 2 wells were obtained that had grown to 75-90% confluence in a 6-well plate, 1 well was cryopreserved, and the remaining 1 well was used for injection into blastocysts and DNA extraction. .

Cryopreservation was performed as follows. That is, after rinsing the cells twice with PBS, add 0.5 mL of Trypsin, incubate at 37°C for 15-20 minutes to treat with trypsin, add 0.5 mL of ES cell medium, and repeat 35-40 times. Pelling was performed to completely dissociate the ES cell clusters. This cell suspension was transferred to a 15 mL centrifugation tube, and the wells were washed with 1 mL of ES cell medium and collected in the tube. The tube was centrifuged at 1,000 rpm for 7 minutes, the medium was removed and resuspended in 0.25 mL ES cell medium and 0.25 mL of 2x freezing medium was added. The contents of the wells were transferred to cryogenic vials, frozen at -80°C and stored in liquid nitrogen.

Cells for injection into blastocysts and DNA extraction were used for injection into blastocysts after completely dissociating the ES cell clumps, one fourth of which was used for injection into blastocysts, and one third of the remaining cells, and two-thirds were subcultured onto gelatin-coated 60 mm dishes, respectively. For the former, when the cells grew to confluency, genomic DNA for PCR analysis was extracted, and for the latter, when the cells grew to confluency, they were divided into three and frozen.

Using ES cells having the uPA gene, chimeric mice were produced as follows.
For the ES cell clones in which the gene transfer was confirmed, chimeric embryos were produced using C57BL/6J mouse blastocysts as host embryos, which were transplanted into the uterine horns of pseudopregnant mice to obtain offspring. Host embryos were harvested on day 3 of pregnancy by perfusing the oviduct and uterus with Whitten's medium supplemented with 100 μM trypsin/EDTA. Eight-cell stage embryos or morulae were cultured in Whitten's medium for 24 hours and the resulting blastocysts were used for injection. The ES cells used for injection were dispersed by TE treatment 2 or 3 days after passage, and allowed to stand at 4°C until subjected to micromanipulation. As a pipette for injection of ES cells, Sutter's glass capillary tubing (inner diameter: about 20 μm) was used. As a pipette for holding embryos, a micro glass tube (NARISHIGE) with an outer diameter of 1 mm was stretched thinly using a microelectrode fabricator (Sutter P-97/IVF), and then the outer diameter was measured using a microforge (De Fonburun). A piece cut at a portion of 50 to 100 μm and further processed to have a diameter of 10 to 20 μm was used. The injection and retention pipettes were connected to a micromanipulator (Leica) with a piezo system (Primetech PAMS-CT150). The chamber used for micromanipulation consisted of a perforated glass slide with a coverslip attached with beeswax. The top surface was covered with mineral oil (Sigma). About 100 ES cells were placed in one drop, and about 20 expanded blastocysts were placed in the other drop, and about 15 ES cells were injected per embryo. All micromanipulations were performed under an inverted microscope. The manipulated embryos were implanted into the uterine horns of day 2 pseudopregnant ICR recipient females. Recipient females who did not deliver offspring by the expected delivery date underwent caesarean section and were nursed by foster parents. As a result of injecting 45 clones of ES cells into C57BL/6J mouse blastocysts, male chimeric mice were obtained in 39 clones.

The uPA-Tg mice (hemizygote, +/-) thus obtained were back-crossed to SCID-bg mice twice to generate mice with the uPA-Tg(+/-)SCID(+/+) genotype. Obtained. Sperm were collected from the male mice, unfertilized eggs of SCID mice (homozygote, +/+), and after in vitro fertilization, they were returned to surrogate mothers. Mice with Tg gene were selected among the born offspring, and uPA-Tg(+/-)/SCID (+/+) mice having both traits were obtained by natural mating. Distinction between uPA-Tg(+/-) and uPA-Tg(-/-) was performed by genome PCR method using transgene-specific sequences as primers.
forward primer
5'-GGGCGGCGGTACCGATCCTGAGAACTTCAGGGTGAG-3' (SEQ ID NO: 3)
reverse primer
5′-GGGCGGCGTACCAATTCTTTGCCAAAATGATGAGA-3′ (SEQ ID NO: 4)
SCID (+/+), SCID (+/-) and SCID (-/-) were identified by PCR-RFLP method.

Next, the obtained uPA-Tg(+/-)/SCID(+/+) were mated to obtain uPA-Tg(+/+)/SCID(+/+) and uPA-Tg(+/-). I got /SCID(+/+). Identification of uPA-Tg(+/+) and uPA-Tg(+/-) was performed by Southern blotting. The tails of 8- to 10-day-old mice were cut about 5 mm, solubilized with SDS and proteinase K solution, and mixed protein components were removed by phenol and chloroform extraction. After degrading contaminating RNA with DNase-free RNase A, high-molecular-weight genomic DNA was precipitated by isopropanol precipitation. The above genomic DNA was washed with 70% ethanol, air-dried, and redissolved in TE. Genomic DNA extracted from the sample, positive and negative control genomic DNA, 5 μg each, were completely digested with EcoR1, the resulting DNA fragments were separated by agarose electrophoresis, and transferred to a nylon membrane. A DNA fragment suitable for Southern hybridization probe was purified from the uPA cDNA probe/TA using the restriction enzyme EcoR1 (379 bp). The above DNA fragment was [32P]-labeled by the random prime method. DNA fragments transferred to nylon membranes were hybridized with RI-labeled uPA cDNA probes. Non-specifically bound probes were removed by washing, and radioactive signals originating from foreign genes introduced into mAb-uPA-Int2 Tg mouse candidates were detected by exposure to X-ray film. Genotype mAlb-uPA-Int2 Tg mice by detecting a specific signal of 1.5 kb from the wild-type locus and a specific signal of 0.4 kb (wt:1.5 kb) from the mutant locus. was judged.
uPA-Tg(+/+)/SCID(+/+) mice and SCID/cb-17 mice (Charles River Japan) were crossed to obtain uPA-Tg(+/-)/SCID(+/+) mice. rice field. uPA-Tg(+/+)/SCID(+/+) mice were used for primary transplantation, and uPA-Tg(+/-)/SCID(+/+) mice were used as host mice for passage.

Human hepatocyte transplantation As human hepatocytes, hepatocytes purchased from BD Gentest (Lot No. BD195, girl, 2 years old) and hepatocytes purchased from BioIVT (Lot No. IVTJFC, boy, 1 year old), each used. These frozen hepatocytes were described by Chise Tateno, Yasumi Yoshizane, Naomi Saito, Miho Kataoka, Rie Utoh, Chihiro Yamasaki, Asato Tachibana, Yoshinori Soeno, Kinji Asahina, Hiroshi Hino, Toshimasa Asahara, Tsuyoshi Yokoi, Toshinori Furukawa, Katsutoshi Yoshizato: Near -completely humanized liver in mice shows human-type metabolic responses to drugs.

A 3- to 5-week-old uPA-Tg(+/+)/SCID(+/+) mouse was anesthetized with isoflurane, an approximately 5-mm incision was made in the left flank, and ^10.0x105 human hepatocytes were removed from the splenic head. After injection, the spleen was sutured back into the abdominal cavity.
After the transplantation, the mice were reared for about 100 days by ad libitum intake of normal feed CRF-1 (Oriental Yeast Co., Ltd.) and tap water containing 0.0125% sodium hypochlorite solution.

SCID/c.b-17 mice used for mating do not have T cells or B cells, but are known to have NK cells. To prevent the transplanted human hepatocytes from being attacked by mouse NK cells, an antibody that inhibits NK activity was administered intraperitoneally the day before transplantation.

The replacement rate of human hepatocytes in the obtained primary chimeric mouse liver was 90-95%. This substitution rate was determined by measuring the human albumin concentration in mouse blood. That is, blood was collected from the tail vein of the chimeric mouse, 2 μl of the collected blood was added to 200 μL of LX-Buffer, and human albumin in mouse blood was analyzed by immunoturbidimetric analysis using an automatic analyzer JEOL BM6050 (JEOL). Concentration was measured. The human hepatocyte replacement rate was estimated by applying this human albumin concentration to a calibration curve prepared in advance. For the standard curve, frozen sections of chimeric mouse liver were prepared and immunostained using a human hepatocyte-specific cytokeratin 8/18 antibody (ICN Pharmaceuticals, Inc.). It was created between the actual replacement rate of human hepatocytes and the human albumin concentration in blood of chimeric mice.

(2) Production of chimeric mouse with serial transplantation of human hepatocytes Liver tissue of the primary chimeric mouse was treated with collagenase to collect cells rich in human hepatocytes. 3-5 week old uPA-Tg(+/-)/SCID(+/+) mice were transplanted from the spleen with 1×10 ⁶ hepatocytes isolated from primary chimeric mice. The transplantation method is the same as that of human hepatocyte transplantation into primary chimeric mice.
After transplantation, the mice were reared for about 100 days by ad libitum access to tap water containing CRF-1 (Oriental Yeast Co., Ltd.) and 0.0125% sodium hypochlorite solution.
The replacement rate of human hepatocytes in the livers of the obtained serially transplanted chimeric mice was 90-97%.

(3) Induction of NASH symptoms by administration of modified feed
(3-1) Subcultured human hepatocyte chimeric mice
Blood human albumin concentration/serum ALT activity As mentioned above, when hepatocytes are damaged, cytoplasmic enzymes leak out of the cells and enter the blood. Since ALT is contained most abundantly in the liver, an increase in ALT activity in the blood is an indicator of liver damage. Also, since albumin is produced in the liver and is present in the blood, when the liver is damaged, the albumin concentration in the blood decreases. Therefore, a decrease in blood albumin concentration is an index of liver damage.

The serially transplanted human hepatocyte chimeric mice obtained in the item "(2) Preparation of serially transplanted human hepatocyte chimeric mice" were fed with an ultra-high-fat choline-deficient methionine-reduced diet (A06071302; Research Diets, Inc) or a normal diet. CRF-1 (Oriental Yeast Co., Ltd.) and 0.0125% sodium hypochlorite solution added tap water were fed ad libitum for 12 or 14 weeks. The 2-week feeding group and the 4-week feeding group consisted of 3 rats in each group. In the 8-week feeding group, the ultra-high-fat choline-deficient methionine-reduced diet feeding group consisted of 4 rats, and the normal feed feeding group consisted of 3 rats. In the 12-week feeding group and the 14-week feeding group, 3 rats were fed to the ultra-high-fat choline-deficient methionine-reduced diet, and 3 or 4 rats were fed to the normal diet.
Before the start of the test, 2 weeks, 4 weeks, 8 weeks, and 12 weeks later, blood was collected from the tail vein of the serially transplanted chimeric mice fed with adjusted diet and normal diet, respectively, and 2 µl of the collected blood was physiologically administered. It was added to 200 μL of saline solution and analyzed using an automatic analyzer BioMajesty ^TM (JCA-BM6050, JEOL, Tokyo) using latex agglutination immunoturbidimetry (LZ test 'Eiken' U-ALB, Eiken Chemical Co., Ltd., Tokyo). ) to measure the blood human albumin concentration (mg/ml).
FIG. 1 shows the results of serially transplanted chimeric mice transplanted with human hepatocyte Lot No.BD195. It can be seen that the feeding of the modified feed markedly decreased the blood human albumin concentration and induced functional deterioration of human hepatocytes.

The ultra-high fat choline-deficient methionine diet (A06071302; Research Diets, Inc) does not contain added choline or its salts, and contains L-methionine at a final concentration of 0.1% by weight. Also, the ratio of the calorie of fat to the total calorie of protein, carbohydrate and fat is 62 kcal%. Also, the fat content is 35.7% by weight relative to the total weight of the feed.

In addition, before the start of the test, 2 weeks, 4 weeks, 8 weeks, and 12 weeks later, blood was collected from the tail vein of the serially transplanted chimeric mice fed with adjusted diet and normal diet, respectively. was measured using Fuji Dry Chem 7000 (Fuji Film) and Fuji Dry Chem Slide GTP/ALT-PIII (Fuji Film), and human ALT1 concentration (ng/mL) was measured using an ELISA kit (human ALT1 ELISA kit, Phoenix Bio Co., Ltd.). was measured using Furthermore, human ALT activity (U/L) was determined from human ALT1 concentration (ng/mL). Mouse ALT activity was calculated by subtracting human ALT1 activity from total ALT activity.
FIG. 2 shows the time course of ALT activity in the serially transplanted chimeric mice transplanted with human hepatocyte Lot No. BD195. It was found that mouse ALT activity was not increased by feeding the modified diet, but human ALT activity was increased after 2 and 4 weeks, and changed in parallel with total ALT activity. It can be seen that feeding with the modified feed specifically induced liver injury in human hepatocytes.

The passage-transplanted human hepatocyte chimeric mice obtained in the item "(2) Preparation of serial-transplanted human hepatocyte chimeric mice" were fed with a choline-deficient methionine-depleted high-fat diet (CDAHFD) (super high-fat The rats were reared for 12 weeks with choline-deficient methionine-reduced diet A06071302 (Research Diets, Inc) or normal diet CRF-1 (Oriental Yeast Co., Ltd.) and tap water added with 0.0125% sodium hypochlorite solution ad libitum.
2 weeks, 4 weeks, 8 weeks, and 12 weeks after the initiation of the test, paraffin sections of the livers of the subcultured chimeric mice were prepared and stained with hematoxylin and eosin (HE). FIG. 3 shows histological images at 40x magnification of the serially transplanted chimeric mouse transplanted with human hepatocyte Lot No. BD195. Lipid droplets, especially macrovesicular lipid droplets, increased remarkably after 2 and 4 weeks in the modified diet compared to the normal diet.

In addition, FIG. 4 shows histological images of the serially transplanted chimeric mice transplanted with human hepatocyte Lot No. BD195 at a magnification of 400 after 12 weeks. In addition, FIG. 5 shows a histological image at 400 times magnification of the serially transplanted chimeric mouse transplanted with human hepatocyte Lot No.IVTJFC after 14 weeks. When reared on the modified diet, unlike when reared on the normal diet, infiltration of inflammatory cells such as macrophages (arrows) was observed, and ballooning cells with Mallory body-like structures in the cytoplasm (hepatocyte ballooning cells) were observed. large) (arrowheads) were observed. It can be seen that long-term administration of the modified diet induced lesions characteristic of human NASH.

In addition, a paraffin section of the liver of the serially transplanted chimeric mouse transplanted with human hepatocyte Lot No. BD195 was prepared 12 weeks after the start of the test and stained with Sirius red. Histological images at 40x and 400x magnification are shown in FIG. In addition, 14 weeks after the start of the test, paraffin sections of the liver of the serially transplanted chimeric mice transplanted with human hepatocyte Lot No.IVTJFC were prepared and stained with Sirius red. Histological images at 400x magnification are shown in FIG. When animals were fed the modified diet, the fibrosis area (red-stained area) increased, and pericellular or perisinusoidal fibrosis extending from the pericentral vein or portal vein area was observed compared to the normal diet. observed.
The Sirius Red-positive area (fibrotic area) per total area on the Sirius Red-stained tissue section of the serially transplanted chimeric mouse transplanted with human hepatocyte Lot No. BD195 was calculated. The results are shown in FIG. It can be seen that the modified diet increased the fibrosis area by about 1.6 times after 8 weeks and about 2.4 times after 12 weeks compared to the normal diet. .

In addition, serially transplanted chimeric mice transplanted with human hepatocyte Lot No. BD195 were immunized with anti-F4/80 antibody (clone BM8, BMA Biomedicals) on paraffin sections of the liver 8 weeks and 12 weeks after the start of the test. dyed. F4/80 is one of the antigens expressed in intrahepatic Kupffer cells, macrophages, etc. Immunostaining with an anti-F4/80 antibody can stain Kupffer cells and macrophages brown. FIG. 9 shows an immunostaining image of the anti-F4/80 antibody. When the mice were fed the modified feed, there were clearly more brown-stained areas than when the mice were fed the normal feed, indicating an increase in Kupffer cells and macrophages.

For serially transplanted chimeric mice transplanted with human hepatocyte Lot No. BD195, 8 weeks and 12 weeks after the start of the test, paraffin sections of the liver were stained with anti-F4/80 antibody, and 10 sections were randomly determined, Photographs were taken at a magnification of 100 times, and the ratio of F4/80-positive area (Kupffer cell/macrophage-positive area) per photographed area was calculated. The results are shown in FIG. It can be seen that the modified feed increases the F4/80-positive area by about 2.7 times after 8 weeks and about 2.1 times after 12 weeks, compared to when the animals are fed with the normal feed.

In addition, with respect to chimeric mice transplanted with human hepatocytes Lot No. BD195, paraffin sections of the liver 2 weeks, 4 weeks, 8 weeks, and 12 weeks after the start of the test were treated with anti-α-smooth muscle actin (α-smooth muscle actin). Immunostaining was performed using an αSMA) antibody (clone 1A4, Sigma). αSMA is a marker for activated hepatic stellate cells, and activated hepatic stellate cells are thought to produce collagen fibers and play an important role in liver fibrosis. Activated astrocytes can be stained brown by immunostaining with an αSMA antibody. FIG. 11 shows an immunostained image of the αSMA antibody (100x magnification). From FIG. 11, it can be seen that the brown-stained αSMA-positive regions clearly increased during any period of feeding on the modified feed compared to the case of feeding on the normal feed.

FIG. 12 shows a TUNEL-stained image (magnification: 100) of the serially transplanted chimeric mouse transplanted with human hepatocyte Lot No. BD195. In addition, paraffin sections of the liver 8 weeks and 12 weeks after the initiation of the test were stained by the TUNEL method. The TUNEL method is a staining method that specifically stains apoptotic cells. From FIG. 12, it can be seen that brown-stained TUNEL-positive cells clearly increased in the animals fed the modified feed compared to those fed the normal feed. Since apoptosis is a mode of cell death that is characteristic of NASH, NASH symptoms were induced by feeding the modified diet.

(3-2) Primary human hepatocyte chimeric mice The primary human hepatocyte chimeric mice transplanted with human hepatocyte Lot No. IVTJFC obtained in the item "(1) Production of primary human hepatocyte chimeric mice" were choline-deficient. Methionine-reduced high-fat diet (CDAHFD) (super-high-fat choline-deficient methionine-reduced diet, A06071302) (Research Diets, Inc) or normal diet CRF-1 (Oriental Yeast Co., Ltd.) and sodium hypochlorite solution The rats were fed with 0.0125% added tap water ad libitum for 12 weeks.
Twelve weeks after the start of the test, paraffin sections of the liver of the primary transplanted chimeric mouse were prepared and stained with hematoxylin and eosin (HE). Histological images at 400x magnification are shown in FIG. When reared on the modified diet, unlike when reared on the normal diet, infiltration of inflammatory cells such as macrophages (arrows) was observed, and ballooning cells with Mallory body-like structures in the cytoplasm (hepatocyte ballooning cells) were observed. large) (arrowheads) were observed. It can be seen that long-term administration of the modified diet induced lesions characteristic of human NASH.

In addition, about primary human hepatocyte chimeric mice transplanted with human hepatocyte Lot No.IVTJFC, liver paraffin sections were prepared 12 weeks after the start of the test, and Sirius red staining was performed. A 400-fold histological image is shown in FIG. When animals were fed the modified diet, the fibrosis area (red-stained area) increased, and pericellular or perisinusoidal fibrosis extending from the pericentral vein or portal vein area was observed compared to the normal diet. observed.

In addition, 12 weeks after the start of the test on primary human hepatocyte chimeric mice implanted with human hepatocyte Lot No.IVTJFC, paraffin sections of the liver were immunostained using an anti-F4/80 antibody (clone BM8, BMA Biomedicals). rice field. FIG. 15 shows an immunostaining image of the anti-F4/80 antibody. When the mice were fed the modified feed, there were clearly more brown-stained areas than when the mice were fed the normal feed, indicating an increase in Kupffer cells and macrophages.

(4) Carbon tetrachloride (CCl ₄ ) administration test to human hepatocyte chimeric mice
_CCl4 is known to induce damage to the liver. The effects of the same dose of CCl ₄ on the liver were compared between passage-transplanted human hepatocyte chimeric mice and normal mice. As an index of liver injury, the elevation of ALT activity in plasma was measured.

Three serially transplanted human hepatocyte chimeric mice transplanted with human hepatocyte Lot No. BD195 obtained in the item "(2) Preparation of serially transplanted human hepatocyte chimeric mice" (human hepatocyte replacement rate 75-86 %) were orally administered CCl ₄ dissolved in corn oil at a dose of 50 mg/kg. It was administered once per day for 7 days, and each mouse was dissected on the day after the last administration. In addition, before the start of the test, 2 days, 4 days, and 8 days later, blood was collected from each mouse, and total ALT activity in plasma was measured using Fuji Dry Chem 7000 (Fuji Film) and Fuji Dry Chem Slide GTP/ALT-PIII (Fuji Film). and the human ALT1 concentration (ng/mL) was measured using an ELISA kit (human ALT1 ELISA kit, PhoenixBio Co., Ltd.). Furthermore, human ALT activity (U/L) was determined from human ALT1 concentration (ng/mL). As a control, 3 chimeric mice with serially transplanted human hepatocytes were bred for 8 days without administration of CCl ₄ , and after 2, 4, and 8 days, blood was collected and total ALT activity was measured in the same manner. , measured human ALT activity.
In addition, 3 male SCID mice aged 10-12 weeks were prepared, and oral administration of CCl ₄ , blood sampling, and measurement of total ALT activity were performed in the same manner. As a control, 3 SCID mice were bred for 8 days without CCl ₄ administration, and after 2, 4, and 8 days, blood was collected and total ALT activity was measured in the same manner.

The upper part of FIG. 16 shows changes over time in total ALT activity in the serially transplanted chimeric mice and SCID mice. Administration of CCl ₄ markedly increased total ALT activity in SCID mice, and all animals became unwell on day 2 and were euthanized. In contrast, the total ALT activity during CCl ₄ administration was about one-tenth that of SCID mice in serially transplanted chimeric mice. In addition, as shown in the lower part of FIG. 16, human ALT activity in human hepatocyte chimeric mice was only slightly increased in the CCl ₄ administration group. The human ALT activity of the human hepatocyte chimeric mouse was about 1/10 of the total ALT activity. Considering the high human hepatocyte replacement rate of 75-86% in human hepatocyte chimeric mice, mouse hepatocytes are much more sensitive than human hepatocytes to damage by _CCl4 . I understand.

Human hepatocyte chimeric mice were euthanized 7 days after CCl ₄ administration, and SCID mice were euthanized 2 days after CCl ₄ administration due to poor prognosis. Paraffin sections of the liver were prepared and HE stained. Histological images at 40x magnification are shown in FIG. Hepatocyte necrosis was observed specifically in the mouse region in the liver tissue of the human hepatocyte chimera mouse (arrow), and extensive hepatocyte necrosis was observed in the liver tissue of the SCID mouse (arrow). This also indicates that CCl ₄ administration is difficult to cause specific damage to human hepatocytes.

(5) Control animals of the model of the present invention As shown on the left side of Figs. 3 and 4, the serially transplanted chimeric mice fed with a normal diet show the pathology of simple fatty liver. In addition, as shown in this study, the subcultured chimeric mice do not progress from simple steatosis to NASH even after being fed a normal diet for a long period of time. Therefore, the passage-transplanted chimeric rodent can be used as a model of human simple fatty liver and can be used as a control animal for the NASH model of the present invention.

The human non-alcoholic steatohepatitis rodent model of the present invention accurately reflects human NASH because the liver is replaced with human hepatocytes. Since the NASH symptoms can be obtained stably, it can be suitably used as a rodent animal model having human hepatocytes for studying the pathology of NASH, and screening agents for the prevention, treatment, or improvement of NASH.

Claims (13)

A human obtained by feeding a chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes on a modified diet having the following characteristics (a), (b), and (c ) : A human non-alcoholic steatohepatitis model consisting of animals in which liver injury is specifically induced in hepatocytes.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate and fat contained in the formulated feed. 2. The human non-alcoholic steatohepatitis model according to claim 1, wherein the chimeric rodent is a primary chimeric rodent or a subcultured chimeric rodent. 3. The human non-alcoholic steatohepatitis model according to claim 1 or 2, which is fed with a modified diet for 1 week or longer. A human obtained by feeding a chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes on a modified diet having the following characteristics (a), (b), and (c ) : A method of using an animal in which liver injury is specifically induced in hepatocytes as a human non-alcoholic steatohepatitis model.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate and fat contained in the formulated feed. 5. The method of claim 4, wherein the chimeric rodent is a primary chimeric rodent or a subcultured chimeric rodent. 6. The method according to claim 4 or 5, wherein the animal is fed with the modified feed for 1 week or more. human liver, comprising the step of feeding a chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes, on a modified diet having the following characteristics (a), (b), and (c ) : A method for preparing a human non-alcoholic steatohepatitis model comprising an animal in which cell-specific liver injury is induced.
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate and fat contained in the formulated feed. 8. The method of claim 7, wherein the chimeric rodent is a primary chimeric rodent or a subcultured chimeric rodent. 9. The method according to claim 7 or 8, wherein the animals are fed with the modified feed for one week or more. Obtained by a method comprising the step of feeding a chimeric rodent animal in which some or all of the hepatocytes have been replaced with human hepatocytes, on a modified diet having the characteristics of (a), (b) and (c) below. A step of administering a test substance to an animal in which hepatic injury is specifically induced in human hepatocytes, and the degree of symptoms of non-alcoholic steatohepatitis before and after administration are compared, or chimeric rodents administered with the test substance are compared. A screening method for a therapeutic agent for human non-alcoholic steatohepatitis, comprising the step of comparing the degree of symptoms of non-alcoholic steatohepatitis between a dental animal and a chimeric rodent to which no test substance is administered. .
(a) The content of choline or its salt is 0.01% by weight or less of the total weight of the prepared feed.
(b) The content of methionine is 0.5% by weight or less based on the total amount of the prepared feed.
(c) The fat content is 25 kcal% or more of the total calorific value of protein, carbohydrate and fat contained in the formulated feed. 11. The method of claim 10, wherein the chimeric rodent is a primary chimeric rodent or a subcultured chimeric rodent. 12. The method according to claim 10 or 11, wherein the conditioned feed is fed for one week or more. A human simple fatty liver model consisting of serially transplanted chimeric rodents in which some or all of the hepatocytes have been replaced with human hepatocytes.

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