JP2019041675A - Nash liver carcinogenic model nonhuman animal and its use - Google Patents

Abstract

To provide an NASH liver carcinogenic model nonhuman animal that develops a liver cancer from a background liver similar to human NASH, and an NASH liver carcinogenic model nonhuman animal manufacturing kit that enables an NASH liver carcinogenic model nonhuman animal that develops a liver cancer from a background liver similar to human NASH to be acquired easily.SOLUTION: An NASH liver carcinogenic model nonhuman animal is a nonhuman animal acquired by administering an LXRα agonist and a free radical generator to the nonhuman animal and breeding it by using high fat food. An NASH liver carcinogenic model nonhuman animal manufacturing kit includes an LXRα agonist, a free radical generator, and high fat food.SELECTED DRAWING: None

Description

  The present invention relates to a NASH liver carcinogenesis model non-human animal and its use. Specifically, the present invention relates to a NASH liver carcinogenesis model non-human animal, a kit for producing a NASH liver carcinogenesis model non-human animal, and a method for screening a compound for treatment or prevention of liver cancer.

  "Fatty liver" is classified into alcoholic fatty liver (AFL) that develops upon drinking and nonalcoholic fatty liver (NAFL) that develops without drinking. The state in which inflammation or fibrosis has progressed along with the fatification of the liver due to the onset of NAFL is nonalcoholic steatohepatitis (NASH). The NAFL and NASH together are called nonalcoholic fatty liver disease (NAFLD). In NAFLD, there are 10 million patients in Japan, of which 2 million are estimated to be NASH (see, for example, Non-Patent Document 1). NASH is known to shift to liver cancer via cirrhosis (see, for example, Non-patent Document 2). However, the mechanism by which NASH develops liver cancer is not yet clear.

  As experimental animals currently used for research as NASH models, genetically modified model animals (see, for example, Patent Document 1), genetically modified-meal-induced model animals (see, for example, Patent Document 2), diet-induced model Animals (see, for example, Non-Patent Document 3), diet-drug-induced model animals (see, for example, Patent Document 3), and the like can be mentioned (see, for example, Non-patent Document 4).

Patent No. 6020791 JP, 2017-006022, A JP, 2014-209849, A

Kojima S, et al., “Increase in the prevalence of fatty liver in Japan over the past 12 years: analysis of clinical background.”, J Gastroenterology, Vol. 38, p. 954-961, 2003. Cohen JC, et al., “Human Fatty Liver Disease: Old Questions and New Insights.”, Science, Vol. 332, No. 6037, p. 1519-1523, 2011. Leclercq IA, et al., “CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis.”, J Clin Invest., Vol. 105, No. 8, p. 1067-1075, 2000. Charrez B, et al., “Hepatocellular carcinoma and non-alcoholic steatohepatitis: The state of play.”, World J Gastroenterol., Vol. 22, No. 8, p. 2494-2502, 2016.

  Among the above-mentioned conventional NASH model animals, there is no model animal having both insulin resistance and histopathology according to human NASH, and furthermore, there is no model animal that develops hepatocellular carcinoma from NASH.

  The present invention has been made in view of the above circumstances, and provides a NASH liver carcinogenesis model non-human animal that develops liver cancer from a background liver similar to human NASH. In addition, the present invention provides a kit for producing a NASH liver carcinogenesis model non-human animal, which can be easily obtained from a NASH liver carcinogenesis model non-human animal that develops liver cancer from a background liver similar to human NASH. The present invention also provides a screening method for a compound for treating or preventing liver cancer, which can be conveniently obtained a compound for treating or preventing liver cancer.

That is, the present invention includes the following aspects.
The NASH liver carcinogenesis model non-human animal according to the first aspect of the present invention is a non-human animal obtained by administering a LXRα agonist and a free radical generator and rearing using a high-fat diet.

The LXRα agonist may be T0901317.
The free radical generator may be carbon tetrachloride.
The high fat diet may contain 40 kcal% or more and 80 kcal% or less of fat.

  In the NASH liver carcinogenesis model non-human animal according to the first aspect, the LXRα agonist is administered in a dose of 1.0 mg / kg to 3.0 mg / kg body weight at a time, 1 to 4 times a week as the number of doses It may be administered once or 10 weeks to 30 weeks as an administration period.

  In the NASH liver carcinogenesis model non-human animal according to the first aspect, the free radical generating agent is administered at a dose of 0.05 mL / kg body weight or more and 1.0 mL / kg body weight or less once, and once per week as the administration frequency. It may be administered up to 4 times, and 10 weeks or more and 30 weeks or less as an administration period.

The liver of the non-human animal may have the following pathological forms (a1) to (c1).
(A1) aggregation of chromatin in the nucleus of hepatocytes (b1) elimination of the structure of the nucleus of the nucleus of hepatocytes (c1)

The non-human animal may be a mammal.
The mammal may be a rodent.

  A kit for producing a NASH liver carcinogenesis model non-human animal according to a second aspect of the present invention comprises an LXRα agonist, a free radical generator, and a high fat diet.

The LXRα agonist may be T0901317.
The free radical generator may be carbon tetrachloride.
The high fat diet may contain 40 kcal% or more and 80 kcal% or less of fat.

  The kit for producing a NASH liver carcinogenesis model non-human animal according to the second aspect further comprises a NASH model non-human animal, wherein the NASH model non-human animal administers the LXRα agonist and the free radical generator, and It may be a non-human animal obtained by rearing using a fat diet.

  In the NASH model non-human animal, the LXRα agonist is administered at a dose of 1.0 mg / kg to 3.0 mg / kg body weight at a time, 1 to 4 times a week as an administration frequency, and as an administration period It may be administered for 1 week or more and 15 weeks or less.

  The NASH model non-human animal administers the free radical generator at a dose of 0.05 mL / kg body weight or more and 1.0 mL / kg body weight or less once, 1 to 4 times a week for administration, and It may be administered for 1 week to 15 weeks as a period.

The NASH model non-human animal exhibits insulin resistance, and the liver may have the pathological forms shown in (a2) to (d2) below.
(A2) macroscopic fat (b2) balloon-like degeneration of hepatocytes (c2) infiltration of inflammatory cells (d2) fibrosis

The screening method for a compound for treating or preventing liver cancer according to the third aspect of the present invention is a method comprising the following steps (A1) and (B1).
(A1) A step of administering a test substance to a NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 9 (B1) a step of evaluating the therapeutic effect on liver cancer

The screening method for a compound for treating or preventing liver cancer according to a fourth aspect of the present invention is a method comprising the following steps (A2) to (C2).
(A2) A step of producing a NASH model non-human animal by administering a LXRα agonist and a free radical generator and rearing using a high fat diet (B2) LXRα agonist and free radical generation of the NASH model non-human animal Of administering a drug and administering a test substance while rearing using a high-fat diet (C2) a step of evaluating the presence or absence of onset of liver cancer

  The NASH liver carcinogenesis model non-human animal of the above aspect is a non-human animal that develops liver cancer from a background liver similar to human NASH, and can be useful for elucidating the mechanism of developing liver cancer from NASH. Moreover, according to the production kit for NASH liver carcinogenesis model non-human animal of the above aspect, a NASH liver carcinogenesis model non-human animal that develops liver cancer from a background liver similar to human NASH can be obtained conveniently. According to the method of screening a compound for treatment or prevention of liver cancer of the above aspect, a compound effective for treatment or prevention of liver cancer can be conveniently screened.

The top is a diagram showing a protocol of a method for producing a NASH liver carcinogenesis model mouse in Example 1. Below is a diagram showing the flow of human normal liver developing at least one of NAFL and NASH and further developing at least one of cirrhosis and hepatocellular carcinoma. It is a Hematoxylin-Eosin (HE) stained image of a mouse liver 12 weeks after the start of breeding in Example 1. The scale bar represents 100 μm. FIG. 6 is a view of a silver stained liver of a mouse at 12 weeks after the start of breeding in Example 1. FIG. The scale bar represents 100 μm. It is an Oil-red stained image of the liver of the mouse of 12 weeks after the breeding start in Example 1. FIG. The scale bar represents 100 μm. FIG. 6 is a cutaway macroscopic view of the liver of a mouse at 24 weeks after the start of breeding in Example 1. FIG. FIG. 6 is an HE-stained image of the tumorous part of the liver of a mouse at 24 weeks after the start of breeding in Example 1. FIG. Scale bars in the left and right images indicate 500 μm and 100 μm, respectively. FIG. 6 is a view showing a stained silver image of a tumorous part of a liver of a mouse at 24 weeks after the start of breeding in Example 1. Scale bars in the left and right images indicate 500 μm and 100 μm, respectively. It is the image which detected the expression of each protein in the tumor part and non-tumor part of the liver of the mouse of 24 weeks after the breeding start in Example 1 by Western blotting. It is a graph which shows the ratio of phosphorylated Erk (p-Erk) with respect to the total Erk (t-Erk) in the tumor part and non-tumor part of the liver of the mouse in 24 weeks after the breeding start in Example 1. It is a graph which shows the ratio of phosphorylated Akt (p-Akt) to the total Akt (t-Akt) in the tumor part and non-tumor part of the liver of the mouse in 24 weeks after the breeding start in Example 1. FIG. It is a graph which shows the ratio of phosphorylated STAT3 (p-STAT3) to the total STAT3 (t-STAT3) in the tumor part and non-tumor part of the liver of the mouse in 24 weeks after the breeding start in Example 1. It is the image which detected the expression of each protein in the tumor part and non-tumor part of the liver of the mouse of 24 weeks after the breeding start in Example 1 by Western blotting. It is a graph which shows the expression level of Cyclin D1 in the tumor part and non-tumor part of the liver of the mouse in 24 weeks after the breeding start in Example 1. It is a graph which shows the ratio of phosphorylated JNK (p-JNK) to JNK total amount (t-JNK) in the tumor part and non-tumor part of the liver of the mouse in 24 weeks after the breeding start in Example 1.

«Nash liver carcinogenesis model non human animal»
A NASH liver carcinogenesis model non-human animal according to an embodiment of the present invention is a non-human animal obtained by administering a LXRα agonist and a free radical generator and rearing using a high-fat diet. In addition, the NASH liver carcinogenesis model non-human animal of the present embodiment is a non-human animal that has developed hepatocellular carcinoma from nonalcoholic steatohepatitis (NASH).

According to the conventional preparation method, it has not been possible to prepare a NASH liver carcinogenesis model non-human animal that develops hepatocellular carcinoma from a NASH model non-human animal having insulin resistance and human NASH pathological image.
On the other hand, in the NASH liver carcinogenesis model non-human animal of the present embodiment, accumulation of fat in the liver is induced by feeding a non-human animal with a high-fat diet and administering an LXRα agonist. In addition, administration of a free radical generator causes free radicals to be generated in liver cytochrome P4502E1 (CYP2E1) to cause oxidative stress and induce fibrosis and inflammation of the liver. Furthermore, this liver inflammation induces insulin resistance. From the above, non-human animals that exhibit insulin resistance and develop NASH having human NASH pathological image by showing accumulation of fat in the liver, fibrosis of the liver, inflammation and inflammation of the liver and induction of insulin resistance ( NASH model non-human animals are obtained. Furthermore, by continuously feeding a high-fat diet, an LXRα agonist and a free radical generator to this NASH model non-human animal, a NASH liver carcinogenic model non-human animal that has developed hepatocellular carcinoma from NASH can be obtained. In the obtained NASH liver carcinogenesis model non-human animal, as shown in the examples described later, the onset of hepatocellular carcinoma has been confirmed from its pathological form.

In the present specification, "human NASH pathological image" means the pathological form shown in the following (a2) to (d2) in the liver.
(A2) macroscopic fat (b2) balloon-like degeneration of hepatocytes (c2) infiltration of inflammatory cells (d2) fibrosis

Here, with regard to balloon-like degeneration of hepatocytes, the place where balloon-like swelling occurs is not particularly limited. Above all, it is preferable that the balloon-like swelling occurs around the central vein, from the viewpoint of being more similar to the symptoms of human NASH.
In addition, NASH model non-human animals causing liver fibrosis are in good agreement with NASH (Type 4) classified by Matteoni et al., Which is well-known as diagnostic criteria for NASH, and NASH with more advanced disease state. It can be a model non-human animal. Also, there is no particular limitation on the place where fibrosis occurs, but it is preferable that fibrosis occurs around the central vein from the point of being similar to the human NASH symptom.

In addition, insulin resistance in non-human animals can be determined by serum biochemical examination. Specifically, for example, serum is separated from blood collected from non-human animals, and glucose concentration in serum (especially fasting blood glucose), aspartate aminotransferase (AST (GOT)), alanine aminotransferase (ALT) Judgment as having insulin resistance when one or more selected from the group consisting of (GPT), free fatty acid (NEFA) and neutral fat (TG) is higher than non-human animals fed with normal feed Can.
More specifically, for example, the glucose concentration (fasting blood glucose) may be 200 mg / dL or more, 300 mg / dL or more, and 400 mg / dL or more. For example, the AST may be 200 U / L or more, 500 U / L or more, and 1000 U or more. For example, ALT may be 300 U / L or more, 800 U / L or more, and 1200 U / L or more. For example, free fatty acids (NEFA) may be 1000 μEq / L or more and may be 1200 μEq / L or more. The neutral fat may be at least 20 mg / dL and may be at least 30 mg / dL.
Also in human NASH patients, it is reported that the above-mentioned index is higher than that of healthy people, and the NASH liver carcinogenesis model non-human animal of this embodiment has a pathological condition similar to the human NASH symptoms in its breeding process. It is shown.

  The NASH liver carcinogenesis model non-human animal of the present embodiment has at least one (preferably all) of the pathological forms shown as the above-mentioned human NASH pathological image in its breeding process, and is insulin resistant Have. From this, it can be determined that the NASH liver carcinogenesis model non-human animal of the present embodiment is the one that developed liver cancer from a NASH model non-human animal having a background liver similar to human NASH.

Also, in general, "hepatic cancer" is roughly classified into "primary liver cancer" produced in the liver and "metastatic liver cancer" metastasized from another organ. As used herein, "liver cancer" refers to primary liver cancer.
In addition, in general, in "primary liver cancer", "hepatocellular carcinoma" in which the cells of the liver become cancer, and in the bile duct that plays a role of draining bile into the duodenum becomes "cancer. There are hepatocellular carcinoma which is liver cancer of children, mixed cancer of hepatocytes and cholangiocytes in adults, undifferentiated cancer, bile duct cystadenoma, neuroendocrine tumor and the like. The liver cancer developed by the NASH liver carcinogenesis model non-human animal of this embodiment particularly means hepatocellular carcinoma.

Moreover, the "pathological form of hepatocellular carcinoma" in the present specification means the forms shown in the following (a1) to (c1) in the liver.
(A1) aggregation of chromatin in the nucleus of hepatocytes (b1) elimination of the structure of the nucleus of the nucleus of hepatocytes (c1)

  The NASH liver carcinogenesis model non-human animal of the present embodiment has at least one (preferably all) of the forms shown as the pathological form of hepatocellular carcinoma. From this, it can be determined that the NASH liver carcinogenesis model non-human animal of the present embodiment has developed hepatocellular carcinoma. In addition, the NASH liver carcinogenesis model non-human animal of the present embodiment includes the NASH liver carcinogenesis model non-human animal obtained by the conventional preparation method (for example, administration of a carcinogen) and the pathological form of the above hepatocellular carcinoma. It is structurally distinguished in the points shown.

The animal used as the NASH liver carcinogenesis model non-human animal of the present embodiment is a non-human animal and is not particularly limited as long as it is generally used as an experimental animal, and it is a non-human vertebrate It is preferable that there be some, more preferably a non-human mammal, and even more preferably a rodent.
Specific examples of experimental animals include monkeys, dogs, cats, chickens, rabbits, pigs, cows, mice, rats, guinea pigs, hamsters and the like.
When a mouse is used as an experimental animal, it may be a normal mouse, and may be a hybrid or a strain, but a mouse used as an experimental animal is preferable. Specific examples of mice used as experimental animals include C57BL / 6 mice, C3H mice, ICR mice, and BALB / c mice.

<Method for producing NASH liver carcinogenesis model non-human animal>
The NASH liver carcinogenesis model non-human animal of the present embodiment can be prepared by administering an LXRα agonist and a free radical generator under the administration conditions shown below and rearing using a high fat diet. In other words, non-human animals obtained by administering a LXRα agonist and a free radical generator under the following administration conditions and rearing using a high-fat diet can be used as NASH liver carcinogenesis model non-human animals. .

[LXR alpha agonist]
The LXR alpha agonist is not particularly limited as long as it binds to the nuclear transcription factor liver X receptor alpha (Liver X receptor alpha; LXR alpha) and directly or indirectly induces fat accumulation in the liver . Specific examples of LXRα agonists include T0901317, GW3965 hydrochloride, 27-Hydroxycholesterol, WAY 252623 and the like. Among them, T0901317 is preferable as the LXRα agonist.

  The administration method of the LXRα agonist is not particularly limited, and examples thereof include intraperitoneal administration, intravenous administration, oral administration, subcutaneous administration and the like. Among them, as administration means, intraperitoneal administration is preferable.

The dose of the LXRα agonist is preferably 1.0 mg / kg or more and 3.0 mg / kg or less per dose, and 1.5 mg / kg or more per body weight or more per dose, for intraperitoneal administration. It is more preferable that it is not more than / kg body weight, and more preferably not less than 2.0 mg / kg body weight and not more than 2.5 mg / kg body weight per time.
When the dose is equal to or higher than the above lower limit, accumulation of fat in the liver of non-human animals can be induced, and hepatocellular carcinoma can be developed from background liver similar to human NASH with higher efficiency. On the other hand, the survival time of non-human animals can be kept longer by being below the above-mentioned upper limit.

  The number of administrations of the LXRα agonist may be appropriately adjusted according to the dosage in the above range. Specifically, the administration frequency of the LXR α agonist is preferably 1 to 4 times per week, more preferably 1 to 3 times per week, and still more preferably 1 to 2 times per week. preferable.

  The administration period of the LXRα agonist is not particularly limited as long as it is a period necessary for developing hepatocellular carcinoma from a background liver similar to human NASH, and is not particularly limited, and is appropriately adjusted according to the above dose and the above administration frequency. do it. Specifically, the administration period of the LXRα agonist is preferably 10 weeks to 30 weeks, more preferably 12 weeks to 28 weeks, and still more preferably 15 weeks to 25 weeks.

That is, as a preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, an LXRα agonist is administered in a dose of 1.0 mg / kg to 3.0 mg / kg body weight at a time per dose, and the number of doses per week It is administered 1 to 4 times, and 10 weeks to 30 weeks as an administration period.
In a more preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the LXR alpha agonist is administered at a dose of 1.5 mg / kg to 2.5 mg / kg body weight per dose, and the number of doses is 1 per week. It is administered three to three times and 12 weeks or more and 38 weeks or less as an administration period.
In a further preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the LXRα agonist is administered in a dose of 2.0 mg / kg to 2.5 mg / kg body weight at a time, 1 times a week as the number of doses. The administration is carried out twice to twice a week and 15 weeks or less as an administration period.

[Free radical generator]
The free radical generator is not particularly limited as long as it generates free radicals in the liver and directly or indirectly induces oxidative stress in the liver. Specific examples of free radical generators include carbon tetrachloride (arachidonic acid; AA), diethylnitrosamine (DEN), 2-acetylaminofluorene (2-Acetylaminofluorene: 2-AAF), and N-nitrosomorpholine. (N-Nitrosomopholine: NMOR), 1,2-dichloroethane (1,2-Dichloroethane; DCE), and the like. Among them, carbon tetrachloride is preferable as the free radical generator.

  The administration means of the free radical generating agent is not particularly limited, and examples thereof include intraperitoneal administration, intravenous administration, oral administration, subcutaneous administration and the like. Among them, as administration means, intraperitoneal administration is preferable.

The dose of the free radical generating agent is a dose lower than 1.0 ml / kg body weight /, which is the amount that causes liver damage due to normal oxidative stress. It is important for the onset of cancer. Specifically, in the case of intraperitoneal administration, the dose of the free radical generating agent is preferably 0.05 mL / kg body weight or more and 1.0 mL / kg body weight or less per dose, and 0.05 mL / kg per dose More preferably not less than body weight and not more than 0.5 mL / kg body weight, and still more preferably not less than 0.1 mL / kg and not more than 0.25 mL / kg body weight per dose.
When the dose is at least the above lower limit, oxidative stress in the liver of non-human animals can be induced, and hepatocellular carcinoma can be developed from a background liver similar to human NASH with higher efficiency. On the other hand, by setting the upper limit value or less, a background liver similar to human NASH can be obtained with higher efficiency before developing hepatocellular carcinoma in the liver of a non-human animal.

The number of times of administration of the free radical generating agent may be appropriately adjusted according to the dosage in the above range. Specifically, the number of times of administration of the free radical generating agent is preferably 1 to 4 times per week, more preferably 1 to 3 times per week, and 1 to 2 times per week Is more preferred.
The LXRα agonist and the free radical generator may be simultaneously administered to a non-human animal, or may be administered at different times on the same day or another day.

  The administration period of the free radical generating agent is not particularly limited as long as it is a period necessary to develop hepatocellular carcinoma from a background liver similar to human NASH, and is not limited, and it depends on the above dose and the above administration frequency. It may be adjusted appropriately. Specifically, the administration period of the free radical generating agent is preferably 10 weeks to 30 weeks, more preferably 12 weeks to 28 weeks, and still more preferably 15 weeks to 25 weeks. .

That is, as a preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the free radical generating agent is administered at a dose of 0.05 mL / kg body weight or more and less than 1.0 mL / kg body weight at a time, 1 times as the number of administrations. It is administered 1 to 4 times a week and 10 weeks or more and 30 weeks or less as an administration period.
In a more preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the free radical generating agent is administered at a dose of 0.05 mL / kg body weight or more and 0.5 mL / kg body weight or less once, and once a week as the administration frequency It is administered 1 to 3 times per dose, and 12 weeks to 38 weeks as a dosing period.
In a further preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the free radical generating agent is administered at a dose of 0.1 mL / kg body weight or more and 0.25 mL / kg body weight or less once, and once a week as the administration frequency It is administered once or twice, and as a administration period for 15 weeks or more and 25 weeks or less.

[High-fat diet]
The high-fat diet may be any diet containing 30 kcal% or more of fat, preferably 40 kcal% to 80 kcal%, and more preferably 50 kcal% to 70 kcal%. More preferably, the diet contains 60 kcal%. Here, as fats, all kinds of fats can be used regardless of animal fats and vegetable fats. Specific examples of the fat include corn oil, soybean oil, rapeseed oil, beef tallow, pork fat and the like. Among them, beef fat or pork fat is preferred as the fat. Further, the components other than fat in the high fat diet may be ordinary dietary components, and may contain, for example, proteins, carbohydrates, minerals and the like.
More specific examples of the high fat diet include High Fat Diet 32 (manufactured by CLEA Japan, Inc.) and D12492 (manufactured by Research Diet), which are commercially available as animal feed for research.

  The intake period of the high-fat diet is not particularly limited as long as it is necessary for obesity to occur. Specifically, one week or more is preferable, 4 weeks or more is more preferable, 10 weeks to 30 weeks is more preferable, and 12 weeks to 28 weeks is particularly preferable as the intake period of the high fat diet, and 15 weeks It is most preferable that it is 25 weeks or less. Also, the high-fat diet may be freely given to animals daily. In addition, it is preferable to continue to ingest the high fat diet even during the administration period of the LXRα agonist and the free radical generator.

  That is, as a preferred embodiment of the NASH liver carcinogenesis model non-human animal of the present invention, the high-fat diet is taken for a week or more, and then the above-mentioned LXRα agonist and the above-mentioned free radical generator are The administration is carried out under the above-mentioned administration conditions (dose amount, administration frequency and administration period).

  Moreover, as a timing which starts administration of said LXR (alpha) agonist and said free radical generating agent to a non-human animal, and intake of the said high fat diet, when a non-human animal is a mouse, 2 weeks or more and 10 weeks of age It is preferable that it is the following, It is more preferable that it is 4 weeks old or more and 8 weeks old or less, It is more preferable that it is 6 weeks old or more and 7 weeks old or less. Also, administration of the LXRα agonist and the free radical generator, and intake of the high-fat diet may be performed simultaneously. Alternatively, administration of the LXRα agonist and the free radical generator may be started after the intake of the high-fat diet is started a week or more ago and bred.

«Nash liver carcinogenesis model non-human animal production kit»
A kit for producing a NASH liver carcinogenesis model non-human animal according to an embodiment of the present invention comprises an LXRα agonist, a free radical generator, and a high fat diet.

  According to the production kit of the present embodiment, it is possible to easily obtain a NASH liver carcinogenesis model non-human animal that develops liver cancer from a background liver similar to human NASH.

Examples of the LXRα agonist, the free radical generator and the high fat diet provided in the production kit of the present embodiment include the same as those exemplified for the above-mentioned NASH liver carcinogenesis model non-human animal.
Among them, T0901317 is preferable as the LXRα agonist. The free radical generator is preferably carbon tetrachloride. The high-fat diet may be a diet containing 20 kcal% or more of fat, preferably a diet containing 40 kcal% or more and 80 kcal% or less, and more preferably a diet containing 50 kcal% or more and 70 kcal% or less More preferably, the diet contains 60 kcal%.

A non-human animal obtained by rearing a non-human animal under the administration conditions described in the above-mentioned NASH liver carcinogenesis model non-human animal using the production kit of the present embodiment is a NASH liver carcinogenesis model non-human animal. It can be used.
In addition, as a non-human animal to which the production kit of the present embodiment is applied, the same ones as exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal can be mentioned.

  By breeding a non-human animal under the administration conditions described in the method for producing a NASH liver carcinogenesis model non-human animal described above using the production kit of the present embodiment, NASH is caused to develop in the normal liver of the non-human animal, Furthermore, hepatocellular carcinoma can be developed to produce NASH liver carcinogenesis model non-human animals.

In addition, the production kit of the present embodiment may further include a NASH model non-human animal.
This NASH model non-human animal is a non-human animal obtained by administering the above-mentioned LXRα agonist and the above-mentioned free radical generator and rearing using the above-mentioned high fat diet.
In addition, a NASH model non-human animal included in the production kit of the present embodiment exhibits insulin resistance and has a human NASH pathological image. The "human NASH pathological image" referred to here is the same as that described in the above-mentioned NASH liver carcinogenesis model non-human animal.
The production kit of the present embodiment can further comprise a NASH model non-human animal, thereby producing a NASH liver carcinogenesis model non-human animal in a shorter period of time.

<Method of producing NASH model non-human animal>
A NASH model non-human animal included in the production kit of the present embodiment can be prepared by administering an LXRα agonist and a free radical generator under the administration conditions shown below, and rearing using a high fat diet. In other words, non-human animals obtained by administering the LXRα agonist and the free radical generator under the following administration conditions and rearing using a high-fat diet can be used as NASH model non-human animals.

  Examples of LXRα agonists include those similar to those exemplified above in the NASH liver carcinogenesis model non-human animal.

  As the administration means, dosage amount and administration frequency of the LXRα agonist, the same ones as exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal can be mentioned.

  The administration period of the LXRα agonist is not particularly limited as long as it is a period necessary for the appearance of a pathological image unique to human NASH, and may be appropriately adjusted according to the dose and the number of administrations. Specifically, the administration period of the LXRα agonist is preferably 1 week to 15 weeks, more preferably 5 weeks to 14 weeks, and still more preferably 10 weeks to 12 weeks.

That is, as a preferred embodiment of the NASH model non-human animal provided in the production kit of the present invention, the LXRα agonist is administered in a dose of 1.0 mg / kg to 3.0 mg / kg body weight at a time, 1 times as the number of administrations. It is administered 1 to 4 times a week and as a administration period for 1 week to 15 weeks.
In a more preferred embodiment of the NASH model non-human animal provided in the production kit of the present invention, the LXRα agonist is administered in a dose of 1.5 mg / kg to 2.5 mg / kg body weight at a time, for 1 week as the number of doses It is administered 1 to 3 times per, and 5 weeks or more and 14 weeks or less as an administration period.
In a further preferred embodiment of the NASH model non-human animal provided in the production kit of the present invention, the LXR alpha agonist is administered in a dose of 2.0 mg / kg to 2.5 mg / kg body weight at a time, for 1 week as the number of doses It is administered once or twice a day, and 10 weeks or more and 12 weeks or less as an administration period.

  Examples of the free radical generator include the same as those exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal.

As the administration means, dosage amount and administration frequency of the free radical generating agent, the same ones as exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal can be mentioned.
The LXRα agonist and the free radical generator may be simultaneously administered to a non-human animal, or may be administered at different times on the same day or another day.

  The administration period of the free radical generating agent is not particularly limited as long as it is a period necessary for the appearance of a pathological image unique to human NASH, and may be appropriately adjusted according to the dose and the administration frequency. Specifically, the administration period of the LXRα agonist is preferably 1 week to 15 weeks, more preferably 5 weeks to 14 weeks, and still more preferably 10 weeks to 12 weeks.

That is, as a preferred embodiment of the NASH model non-human animal provided in the production kit of the present invention, the free radical generating agent is administered in a dose of 0.05 mL / kg body weight or more and 1.0 mL / kg body weight or less, the number of times of administration It is administered as 1 to 4 times per week, and as a administration period for 1 week or more and 15 weeks or less.
In a more preferred embodiment of the NASH model non-human animal included in the production kit of the present invention, the free radical generating agent is used at a dose of 0.05 mL / kg body weight or more and 0.5 mL / kg body weight or less at one dose. It is administered 1 to 3 times a week and 5 weeks or more and 14 weeks or less as an administration period.
In a further preferred embodiment of the NASH model non-human animal provided in the production kit of the present invention, the free radical generating agent is administered in a dose of 0.1 mL / kg body weight or more and 0.25 mL / kg body weight or less per dose, as the administration frequency It is administered once or twice a week and as a administration period for 10 weeks or more and 12 weeks or less.

  Examples of the high fat diet include the same as those exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal.

  The intake period of the high fat diet includes the same ones as exemplified in the above-mentioned NASH liver carcinogenesis model non-human animal.

  That is, as a preferred embodiment of the NASH model non-human animal included in the production kit of the present invention, the above-mentioned LXRα agonist and the above-mentioned free radical generation are continued while taking the high fat diet after taking the high fat diet for one week or more. The agent is administered under the above-mentioned administration conditions (dose amount, administration frequency and administration period).

  Moreover, as a timing which starts administration of said LXR (alpha) agonist and said free radical generating agent to a non-human animal, and intake of the said high fat diet, when a non-human animal is a mouse, 2 weeks or more and 10 weeks of age It is preferable that it is the following, It is more preferable that it is 4 weeks old or more and 8 weeks old or less, It is more preferable that it is 6 weeks old or more and 7 weeks old or less. Also, administration of the LXRα agonist and the free radical generator, and intake of the high-fat diet may be performed simultaneously. Alternatively, administration of the LXRα agonist and the free radical generator may be started after the intake of the high-fat diet is started a week or more ago and bred.

«Methods of screening compounds for treatment or prevention of liver cancer»
First Embodiment
A method of screening a compound for treatment or prevention of liver cancer according to one embodiment of the present invention is a method comprising the following steps (A1) and (B1).
(A1) A step of administering a test substance to the above-mentioned NASH liver carcinogenesis model non-human animal (B1) a step of evaluating the therapeutic effect on liver cancer

  According to the screening method of the present embodiment, compounds effective for treatment or prevention of liver cancer can be screened. Furthermore, in particular, according to the screening method of the present embodiment, it is possible to screen a compound that is useful for suppressing the progression of the developed hepatocellular carcinoma or for killing and treating only the hepatocellular carcinoma. .

[Step (A1)]
Step (A1) is a step of administering a test substance to a NASH liver carcinogenesis model non-human animal.
A NASH liver carcinogenesis model non-human animal can be prepared by rearing a non-human animal under the administration conditions described in the method for preparing the NASH liver carcinogenesis model non-human animal above.

  There is no restriction | limiting in particular as a to-be-tested substance used for the screening method of this embodiment. Specific examples of the test substance include, for example, natural compounds, organic compounds, inorganic compounds, single compounds such as proteins and peptides, compound libraries, expression products of gene libraries, cell extracts, cell culture supernatants, fermentations Microbial products, marine organism extracts, plant extracts and the like can be mentioned.

  There are no particular limitations on the method of administering the test substance to the NASH liver carcinogenesis model non-human animal. Specific examples of the method of administering the test substance include, for example, intraperitoneal administration, intravenous administration, oral administration, subcutaneous administration and the like. When the test substance is a protein, for example, a viral vector having a gene encoding the protein may be constructed, and the infectivity may be used to introduce the gene into a NASH liver carcinogenesis model non-human animal. It is possible.

[Step (B1)]
Step (B1) is a step of evaluating the therapeutic effect of the test substance on liver cancer.
Specifically, it is possible to determine whether or not the hepatocellular carcinoma is improved by examining the pathological morphology of the NASH liver carcinogenesis model non-human animal. More specifically, for example, in a model animal to which a test substance is administered, when the hepatocellular carcinoma has disappeared or the sinusoidal structure is maintained, the test substance is a hepatocyte. It is judged to have a therapeutic effect on cancer.
Pathological forms of hepatocellular carcinoma include those described above for the NASH liver carcinogenesis model non-human animal.
In addition, "improvement of hepatocellular carcinoma" in the present specification means that the growth of hepatocellular carcinoma is suppressed, only hepatocellular carcinoma is killed, or the symptoms of hepatocellular carcinoma are alleviated. Point to what you are doing. One of ordinary skill in the art can use the pathological form of hepatocellular carcinoma described herein as an indicator to determine whether or not hepatocellular carcinoma is appropriately improved for a model non-human animal.

  In the step (B1), a test substance exhibiting an improvement effect can be selected as a therapeutic or prophylactic substance for hepatocellular carcinoma.

Second Embodiment
The screening method for a compound for treating or preventing liver cancer according to another embodiment of the present invention is a method for screening a compound for treating or preventing liver cancer comprising the following steps (A2) to (C2).
(A2) A step of preparing a non-alcoholic steatohepatitis (NASH) model non-human animal by administering a LXRα agonist and a free radical generator and rearing using a high-fat diet (B2) the aforementioned NASH model non-human A step of administering a test substance while administering an animal to a LXRα agonist and a free radical generator and rearing using a high-fat diet (C2) a step of evaluating the onset or absence of liver cancer

  According to the method of screening a compound for treatment or prevention of liver cancer of the present embodiment, a compound effective for treatment or prevention of liver cancer can be screened. Further, in particular, according to the screening method of the present embodiment, a compound useful for preventing the onset of hepatocellular carcinoma from NASH or preventing the onset of hepatocellular carcinoma by improving NASH It can be screened.

[Step (A2)]
Step (A2) is a step of producing a NASH model non-human animal.
As a specific production method, the same method as described in the above-mentioned production kit for NASH liver carcinogenesis model non-human animal can be mentioned.

[Step (B2)]
Step (B2) is a step of administering a test substance while administering the NASR model non-human animal obtained in (A2) with a LXRα agonist and a free radical generator and rearing using a high fat diet.
As described above in the NASH liver carcinogenesis model non-human animal, in the step (B2), the NASH model non-human animal is administered a LXRα agonist and a free radical generator without administering a test substance, and a high-fat diet Can be used to induce the onset of hepatocellular carcinoma from NASH.
As the test substance and the administration method thereof, the same ones as those exemplified in the first embodiment can be mentioned. In addition, the timing of administering the test substance may be the same as or different from the administration of the LXRα agonist and the free radical generator.

[Step (C2)]
Step (C2) is a step of evaluating the presence or absence of onset of liver cancer in a model non-human animal.
Specifically, the development of liver cancer can be determined by examining the pathological forms of model non-human animals. More specifically, for example, in a model animal to which a test substance is administered, (the pathological form of) NASH is improved or when it does not exhibit the pathological form of hepatocellular carcinoma. The test substance is determined to have a therapeutic effect on hepatocellular carcinoma.
Pathological forms of NASH and hepatocellular carcinoma include those described in the above NASH liver carcinogenesis model non-human animals.
Also, "amelioration of NASH" in the present specification refers to recovery of NASH symptoms to a normal state or relief of symptoms. One of ordinary skill in the art can use the pathological forms described herein as an indicator to determine whether the symptoms of NASH are appropriately improved for model non-human animals.

  In the step (C2), a test substance which does not exhibit the pathological form of hepatocellular carcinoma can be selected as a therapeutic or prophylactic substance for hepatocellular carcinoma. In addition, a test substance that exhibits ameliorating effect on the symptoms of NASH is also useful as a therapeutic or prophylactic substance for NASH.

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

Example 1
(1) Preparation of NASH model mouse A 12-week-old 7-week-old C57BL / 6 male mouse was allowed to freely ingest a high-fat diet (fat content: 60 kcal%, lard-containing, D12492, Research Diets) and Carbon chloride (0.1 mL / kg body weight, manufactured by Wako Pure Chemical Industries, Ltd.) was intraperitoneally administered twice a week. In addition, LXR agonist T0901317 (2.5 mg / kg body weight, Cayman Chemical Co. Ann Arbor, Michigan. USA) was intraperitoneally administered twice a week. Carbon tetrachloride and T0901317 were administered on the same day.

(2) Histopathological observation of NASH model mice (2-1) Hematoxylin-Eosin (HE) staining The liver is cut out from a part of mice (3 animals) at 12 weeks after the start of breeding by a normal method, and is wrapped in paraffin I buried it. Then, the paraffin block is sliced to a thickness of about 4 μm, and Hematoxylin-Eosin (HE) staining kit (Hematoxylin: manufactured by Merck, potassium aluminum sulfate: manufactured by Wako Pure Chemical Industries, Ltd., glycerin: manufactured by Wako Pure Chemical Industries, Ltd.) HE staining was performed using sodium iodate: Kosoh Chemical Co., Ltd.). Subsequently, it observed with the optical microscope (HS all-in-one fluorescence microscope BZ-9000, Keyence company make) (magnification: 400 times). The results are shown in FIG. 2A. In FIG. 2A, arrows indicate Mallory bodies and arrowheads indicate balloon-like cells. The term "Mallory body" as used herein refers to the accumulation of ubiquitinated proteins, keratins 8 and 18 as intermediate diameter filaments, p62, etc. appearing in the periphery of the nucleus of hepatocytes as an irregular structure. By acidic intracytoplasmic inclusion body is meant. It is known that the Mallory body is observed in fatty liver, liver cancer that has developed liver cancer and cirrhosis.

(2-2) Silver plated silver The paraffin block obtained in (2-1) is sliced to a thickness of about 4 μm, and a silver ammonia complex (silver nitrate, potassium hydroxide, aqueous ammonia: all manufactured by Wako Pure Chemical Industries, Ltd. Silver stain was applied using. Subsequently, it observed with the optical microscope (HS all-in-one fluorescence microscope BZ-9000, Keyence company make) (magnification: 100 times). The results are shown in FIG. 2B. In FIG. 2B, “CV” indicates central vein and “G” indicates Gleason sheath (interlobular connective tissue, Glisson sheath). In addition, arrows indicate fibrotic structures that bridge.

(2-3) Oil-red Stain The paraffin block obtained by (2-1) is sliced to a thickness of about 4 μm, and oil red (Oil-red) O staining kit (Oil red O: manufactured by KROMA, 2 -Propanol: Wako Pure Chemical Industries, Ltd.) was used to perform Oil-red staining. Subsequently, it observed with the optical microscope (HS all-in-one fluorescence microscope BZ-9000, Keyence company make) (magnification: 200 times). The results are shown in FIG. 2C.

(2-4) Observation results From FIG. 2A, Mallory bodies and balloon-like cells were observed. In addition, intrasinusoidal neutrophil infiltration was confirmed, and inflammatory cell infiltration was prominent.
From FIG. 2B, fibrosis was observed around the Grisson sheath, etc., and fibrosis was observed as bridging.
From FIG. 2C, large droplets of fat were observed. Fat droplets accounted for more than 30% of the leaflet area.
From the above, in the livers of mice at 12 weeks after the start of breeding, the pathological diagnostic criteria of NASH (Reference 1: Matteoni CA, et al., “Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. ", Gastroenterology, Vol. 116, Issue 6, p 1413-1419, 1999.), macroscopic fat, balloon-like degeneration of hepatocytes, inflammatory cell infiltration and fibrosis were all observed. That is, it was confirmed that the mouse liver at 12 weeks after the start of rearing had a pathological image of human NASH.

(3) Preparation of NASH Hepatic Carcinogenesis Model Mouse Further, mice were bred from the start of breeding until the 24th week under the same conditions as shown in (1). In addition, 4 individuals survived until the 24th week from the start of breeding.

(4) Histopathological observation of NASH liver carcinogenesis model mice (4-1) HE staining When the liver is cut out from mice (4 individuals) at 24 weeks after the start of breeding by a standard method, multiple masses in all individuals (See FIG. 3, arrows indicate the tumor site). Then, the tumor site of the excised liver was embedded in paraffin. Then, paraffin blocks in the tumor area are sliced to a thickness of about 4 μm, and Hematoxylin-Eosin (HE) staining kit (Hematoxylin: manufactured by Merck, potassium aluminum sulfate: manufactured by Wako Pure Chemical Industries, Ltd., glycerin manufactured by Wako Pure Chemical Industries, Ltd.) HE staining was performed using a sodium iodate manufactured by Kozo Chemical Co., Ltd.). Subsequently, it observed with the optical microscope (HS all-in-one fluorescence microscope BZ-9000, Keyence company make) (magnification: 100 times (left), 200 times (right)). The results are shown in FIG. 4A. In FIG. 4A, “CV” indicates central vein and “G” indicates Gleason sheath (interlobular connective tissue, Glisson sheath). Arrows indicate cell nuclei in which chromatin has aggregated, and arrowheads indicate balloon-like cells.

(4-2) Silver plated silver The paraffin block in the tumorous part obtained by (4-1) was sliced to a thickness of about 4 μm, and a silver ammonia complex (silver nitrate, potassium hydroxide, aqueous ammonia: all Wako Pure Chemical Industries, Ltd. Silver silver stain was applied using a product made in Japan. Subsequently, it observed with the optical microscope (HS all-in-one fluorescence microscope BZ-9000, Keyence company make) (magnification: 100 times (left), 400 times (right)). The results are shown in FIG. 4B. In FIG. 4B, "CV" indicates central vein (central vein) and "G" indicates Gleason sheath (interlobular connective tissue, Glisson sheath).

(4-3) Observation result From FIG. 4A, the normal structure was destroyed in the tumor part, and the portal vein area and the sinusoid structure became unclear. In addition, there were variations in cell size, and differences in nuclear size were observed. In addition, cell nuclei in which chromatin was aggregated were occasionally observed. In addition, fat vacuoles were noticeable.
From FIG. 4B, elimination of the sinusoid structure was observed in the tumorous part.
From the above, in the tumor area of the liver of mice at 24 weeks after the start of breeding, aggregation of chromatin in the cell nucleus, size unevenness of the cell nucleus and sinusoid structure, which are pathological findings of hepatocellular carcinoma developed from NASH The abolition of the That is, it was confirmed that the tumorous part of the liver of the mouse at 24 weeks after the start of breeding has a pathological image of hepatocellular carcinoma whose background is human NASH. In addition, tumors were formed in all four mice at 24 weeks after the start of breeding, and had a pathological image of hepatocellular carcinoma with human NASH as a background liver.

(5) Evaluation of protein expression in hepatocytes of NASH liver carcinogenesis model mice First, a cell lysate was prepared using the tumor area and non-tumor area of the mouse liver 24 weeks after the start of breeding. Next, proteins involved in the Jak / STAT3 system, PI3-K / Akt system and MAPK / Erk system (specifically, phosphorylated Erk, total Erk, total phosphorylated Akt, total Akt, total phosphorylated STAT3, STAT3 total, Cyclin D1, phosphorylated JNK and total amount of JNK) were detected by western blotting. In addition, GAPDH is a loading control. The results are shown in FIGS. 5A and 6A. Furthermore, from the detection brightness of each protein in Western blotting, the ratio of phosphorylated Erk to total Erk (p-Erk / t-Erk) (FIG. 5B), the ratio of phosphorylated Akt to total Akt (p-Akt / t-Akt) (FIG. 5C), Ratio of phosphorylated STAT3 to total STAT3 (p-STAT3 / t-STAT3) (FIG. 5D), expression level of Cyclin D1 (FIG. 6B) and ratio of phosphorylated JNK to total JNK (p-JNK) / T-JNK) (FIG. 6C) was calculated.

  From FIG. 5A to FIG. 5D and FIG. 6A to FIG. 6C, in the tumorous part, increased phosphorylation of proteins involved in each growth signal system was observed. In addition, the expression level of Cyclin D1 was significantly increased in the tumor site as compared to the non-tumor site.

In human hepatocellular carcinoma, increased phosphorylation of Erk, Akt, MEK, cmyc, BCL and STAT3 is known. In addition, it is known that the expression level of Cyclin D1 is increased.
From the above, it was confirmed that the tumorous part of the mouse liver at 24 weeks after the start of breeding reproduces the protein expression of hepatocellular carcinoma with human NASH as the background liver.

  The NASH liver carcinogenesis model non-human animal of the present embodiment is a non-human animal that develops liver cancer from a background liver similar to human NASH, and can be useful for elucidating the mechanism of developing liver cancer from NASH. In addition, according to the production kit for NASH liver carcinogenesis model non-human animal of the present embodiment, a NASH liver carcinogenesis model non-human animal that develops liver cancer can be easily obtained from a background liver similar to human NASH. According to the method of screening a compound for treatment or prevention of liver cancer of the present embodiment, a compound effective for treatment or prevention of liver cancer can be conveniently screened.

Claims (19)

NASH liver carcinogenesis model non-human animal,
NASH liver carcinogenesis model non-human animal obtained by administering a LXR alpha agonist and a free radical generator and rearing using a high fat diet.   The NASH liver carcinogenesis model non-human animal according to claim 1, wherein the LXRα agonist is T0901317.   The NASH liver carcinogenesis model non-human animal according to claim 1 or 2, wherein the free radical generator is carbon tetrachloride.   The NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 3, wherein the high fat diet contains 40 kcal% or more and 80 kcal% or less of fat.   The LXRα agonist is administered at a dose of 1.0 mg / kg body weight to 3.0 mg / kg body weight at a time, 1 to 4 times a week as administration times, and 10 weeks to 30 weeks as an administration period The NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 4.   The dose of the free radical generator is 0.05 mL / kg or more and 1.0 mL / kg or less per dose, 1 to 4 times a week for administration, and 10 weeks or more for 30 weeks or less for administration period. The NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 5, which is administered. The NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 6, wherein the liver of the non-human animal has the following pathological forms (a1) to (c1).
(A1) aggregation of chromatin in the nucleus of hepatocytes (b1) elimination of the structure of the nucleus of the nucleus of hepatocytes (c1)   The NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 7, wherein the non-human animal is a mammal.   The NASH liver carcinogenesis model non-human animal according to claim 8, wherein the mammal is a rodent. With an LXR alpha agonist
A free radical generator,
A kit for producing a NASH liver carcinogenesis model non-human animal comprising a high-fat diet.   The kit for producing a NASH liver carcinogenesis model non-human animal according to claim 10, wherein the LXRα agonist is T0901317.   The kit for producing a NASH liver carcinogenesis model non-human animal according to claim 10 or 11, wherein the free radical generator is carbon tetrachloride.   The kit for producing a NASH liver carcinogenesis model non-human animal according to any one of claims 10 to 12, wherein the high fat diet contains 40 kcal% or more and 80 kcal% or less of fat. Further equipped with NASH model non-human animals,
The NASH model non-human animal is a non-human animal obtained by administering the LXRα agonist and the free radical generator and rearing using the high-fat diet. Production kit for NASH liver carcinogenesis model non-human animal as described.   The NASH model non-human animal is a dose of 1.0 mg / kg or more and 3.0 mg / kg or less of body weight per dose, 1 to 4 times per week as dosing frequency, and a dose period of the LXRα agonist The kit for producing a NASH liver carcinogenesis model non-human animal according to claim 14, which is administered for 1 week to 15 weeks.   The NASH model non-human animal administers the free radical generator at a dose of 0.05 mL / kg body weight or more and 1.0 mL / kg body weight or less once, 1 to 4 times a week for administration, and The kit for producing a NASH liver carcinogenesis model non-human animal according to claim 14 or 15, which is administered for 1 week to 15 weeks as a period. The NASH model non-human animal exhibits insulin resistance, and the liver has a pathological form shown in (a2) to (d2) below: NASH according to any one of claims 14 to 16 Production kit for liver carcinogenesis model non-human animals.
(A2) macroscopic fat (b2) balloon-like degeneration of hepatocytes (c2) infiltration of inflammatory cells (d2) fibrosis A screening method for a compound for treating or preventing liver cancer, comprising the following steps (A1) and (B1):
(A1) A step of administering a test substance to a NASH liver carcinogenesis model non-human animal according to any one of claims 1 to 9 (B1) a step of evaluating the therapeutic effect on liver cancer A screening method for a compound for treating or preventing liver cancer, comprising the following steps (A2) to (C2).
(A2) A step of producing a NASH model non-human animal by administering a LXRα agonist and a free radical generator and rearing using a high fat diet (B2) LXRα agonist and free radical generation of the NASH model non-human animal Of administering a drug and administering a test substance while rearing using a high-fat diet (C2) a step of evaluating the presence or absence of onset of liver cancer