WO2006093143A1 - Model for nash or liver tumor, method for production of the model, method for screening of therapeutic agent for nash or liver tumor, method for analysis of mechanism for development of nash or liver tumorm, and agent for prevention or treatment of nash - Google Patents

Abstract

[PROBLEMS] To provide a model animal which can present a symptom distinctive for non-alcoholic steatohepatitis (NASH) within a short period of time without artificially deleting a gene originally expressed in 'a hepatocyte' or a model animal for liver tumor caused by NASH; the elucidation of the mechanism for development of NASH or liver tumor; screening of an agent for the prevention or treatment of NASH or liver tumor; and an agent for the prevention or treatment of NASH. [MEANS FOR SOLVING PROBLEMS] A model animal for non-alcoholic steatohepatitis or liver tumor having a gene encoding a part or the entire of the amino acid sequence for SREBP1c having a transcriptional activity and a promoter gene capable of directing an adipose tissue-specific expression introduced therein; and an agent for preventing or treating NASH, comprising one or more substances selected from adiponectin or an analogue thereof and a gene encoding adiponectin or the analogue thereof as the active ingredient(s).

Description

 Specification

 NASH or liver tumor model and preparation method, NASH or liver tumor therapeutic agent screening method, NASH or liver tumor pathogenesis analysis method, and NASH prophylactic or therapeutic agent

 Technical field

 [0001] The present invention relates to analysis of the onset mechanism of non-alcoholic steatohepatitis or liver tumors resulting therefrom and screening for preventive or therapeutic agents, and to preventive or therapeutic agents for non-alcoholic steatohepatitis. .

 Background art

 [0002] In recent years, illness that exhibits symptoms similar to those of alcoholic liver diseases such as fatty liver, despite the fact that alcohol is not ingested, has become the focus of attention. Nonalcoholic fatty liver disease (NAFLD) It is called.) This NAFLD is a reversible benign disease and has been considered to be completely cured by improving lifestyle habits.

 [0003] However, even with NAFLD, despite the fact that alcohol is not ingested, it has been prominent that cirrhosis can lead to hepatocellular carcinoma and death, and this disease is non-alcoholic. It is named nonalcoholic steatohepatitis (hereinafter referred to as “NASH”), and is attracting attention as a lifestyle-related disease (metabolic syndrome).

 [0004] Regarding the pathogenesis of NASH, there are still many mysteries, and there is a need for elucidation and prevention or treatment agents.

 [0005] Recently, the power of mice that knocked out a tumor suppressor gene in the liver called PTEN has been proposed as a model of this disease state (Non-patent Document 1 below).

 [0006] However, this is a mouse in which a gene in a hepatocyte has been artificially deleted, and it is difficult to say that it is an accurate model of NASH.

[0007] This is because the pathology of NASH in humans is very unlikely to occur due to the deletion of certain genes, particularly the involvement of the PTEN gene, which is a tumor suppressor gene, and its related genes. Thus, it is considered to be a lifestyle-related disease (metabolic syndrome), and is caused by environmental factors such as overeating and lack of exercise in the absence of special disease gene abnormalities. This PTEN knockout mouse is considered to be one of the syndromes to be rubbed, because it is said that this PTEN knockout mouse does not necessarily develop in humans and shows the pathological condition of NASH. .

[0008] Furthermore, it is 40- to 78-week-old mice that have confirmed steatohepatitis-like symptoms in this PTEN knockout mouse, and a model that can be established more accurately and early is required. .

 [0009] On the other hand, it has been suggested that NASH can cause liver cancer and the like. It has not been confirmed that the NASH model actually develops a liver tumor.

[0010] Non-Patent Document 1: Horie et al. Volume 113, Number 12, June 2004, 1774-1783, Journal of Clin. Invest.

 Disclosure of the invention

 Problems to be solved by the invention

 [0011] The present inventors have found that the aP2_SREBPlc transgenic mouse, known as a model of lipotrophic diabetes, develops NASH symptoms at the age of 20 weeks. It was confirmed for the first time that he developed a hepatic neoplastic lesion, and the present invention has been achieved. The purpose of the present invention is to determine the genes that are naturally expressed in “hepatocytes” in a shorter period of time. An animal model that exhibits NASH symptoms without being artificially lost is provided, and a NASH-induced liver tumor model is provided for the first time as an animal model. Elucidation, to screen for NASH or liver tumor prevention or treatment.

 [0012] In addition, when the present inventors mated a separately developed high adiponectin-expressing model mouse with this fat-atrophic diabetes model mouse, the onset of NASH is suppressed even when reared up to 30 weeks of age. As a result, the second object of the present invention is to provide a preventive or therapeutic agent for NASH, which also has adiponectin or its related strength.

 Means for solving the problem

[0013] The above-mentioned object is to provide a non-alcoholic steatohepatitis animal model, transcriptional activity ability, characterized in that either (XI) or (X2) below is introduced and (Y) is introduced. The non-alcohol, wherein the amino acid sequence having the amino acid sequence is the N-terminal domain of the amino acid of SREBPlc Animal model of steatohepatitis or liver tumor, promoter with adipose tissue-specific expression One of the non-alcoholic steatohepatitis or liver tumor animal models, a following, (XI) or (X2) A non-alcoholic animal characterized in that an animal into which (Y) has been introduced is raised until at least one of the following findings (1) to (5) is observed together with fat deposition in hepatocytes. Non-alcoholic fat characterized in that a method for preparing an animal model for steatohepatitis, either (XI) or (X2) below, and an animal into which (Y) is introduced is raised to at least 20 weeks of age A method for preparing an animal model of hepatitis, preparation of an animal model of a liver tumor characterized in that either (XI) or (X2) below and an animal into which (Y) has been introduced are raised to at least 50 weeks of age Method, the non-alcoholic fatty liver Non-alcoholic steatohepatitis using non-alcoholic steatohepatitis or liver tumor prevention method using non-alcoholic steatohepatitis or liver tumor animal model using non-alcoholic steatohepatitis or liver tumor animal model A method for analyzing the onset mechanism of hepatitis or liver tumor, the prevention of non-alcoholic steatohepatitis characterized by containing as an active ingredient one or more selected from the following (A) to (D) force Achieved with therapeutic agents. (XI) A gene encoding an amino acid sequence having a transcription activity, which is part or all of the amino acid sequence of SREBPlc

 (X2) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in part or all of the amino acid sequence of SREBPlc and having transcriptional activity

 (Y) Promoter gene having adipose tissue-specific expression

 (1) Inflammatory cell infiltration into liver tissue

 (2) Focal necrosis of hepatocytes

 (3) Hepatocyte balloon

 (4) Mallory body expression in hepatocytes

 (5) Fibrosis in liver lobule

 (A) Adiponectin protein

(B) A protein comprising the amino acid sequence in which one or several amino acids of (A) are deleted, substituted or added and having the same activity as (A) (C) Gene encoding (A)

 (D) Gene encoding (B)

 The invention's effect

 [0015] The NASH or liver tumor model of the present invention (hereinafter sometimes collectively referred to as "the model of the present invention") can be obtained earlier by simply breeding a mouse that can be purchased as a known commercial product. It is more similar to the pathology of human NASH or the accompanying liver tumors. In addition, since the NASH or hepatic tumor preventive or therapeutic agent of the present invention is adiponectin or a similar compound originally present in the body of an organism, there is an advantage that there are few side effects. In particular, it is safer for human adiponectin and its analogs. BEST MODE FOR CARRYING OUT THE INVENTION

 [0016] SREBPlc used in the present invention is a subtype belonging to the SREBP (Sterol Regulatory Element Binding Protein) family, and is known to react with glucose and insulin and mainly control fatty acid synthesis. It is a protein that has been

 SREBPlc consists of about 1,140 amino acids and consists of the following three sites.

 [0017] (1) The transcription factor of the basic helix loop, one helix, single-hull zipper family present at the N-terminal site is composed of 480 amino acids.

 [0018] (2) The 80 amino acids present in the intermediate site are configured to surround two membrane-binding domains separated by 30 hydrophilic short-chain amino acids.

 [0019] (3) Long chain C-terminal region with a potential of 590 amino acids

 In the present invention, any or all of the amino acid sequences of SREBPlc, which are genes encoding an amino acid sequence having transcription activity, can be used. Only genes at amino acid sites having transcription activity For example, only the region encoding the transcription factor of (1) above can be used, and in particular, the gene encoding the amino acid terminal domain at positions 1-480 is preferred. It is preferable to use a gene encoding.

 Transcriptional activity refers to the function of SREBPlc as a natural transcription factor.

[0021] Mice overexpressing this gene are, for example, Volume 99, Number 5, March 1997, 846-854, Journal of Clin. Invest. And Volume 12, Number 20, October 1998, 3182-3194, It can be prepared by the method described in Genes Dev.

 This mouse overexpresses 480 transposers, consisting of a family of basic helix loops, helix bites, and isin zippers.

[0022] The gene used for introduction into the animal model in the present invention is thus a gene encoding an amino acid sequence having a transcriptional activity, which is a part or all of the amino acid sequence of SREBPlc.

 [0023] Further, in the present invention, as long as it has transcription activity, one or several amino acids of the above-mentioned "amino acid sequence having part or all of SREBPlc and having transcription activity" is present. It may be a deleted, substituted or added amino acid sequence.

[0024] Promoters having adipose tissue-specific expression of that used in the present invention, for example, a known aP2 (Adipocyte P2) promoter, Fabp4 (Fatty acid binding protein 4 ) Although promoter one coater and the like, _a P2 promoter Since it has a track record of being commercially available as aP2-SREBPlc transgenic mice, it is preferable because it is considered compatible with SREBPlc.

 [0025] As described above, in the animal model of the present invention, an artificial gene is generated in the hepatocyte gene by using a promoter that overexpresses part or all of SREBPlc in white fat related to obesity, which is not directly related to the liver. NASH can be developed without any significant improvement. This can be said to be closer to human spontaneous NASH.

 [0026] (type of animal)

 The animal used in the transgenic animal of the present invention is preferably a rodent animal, for example, a mouse, a rat, a hamster, etc. Among the particularly preferred mice, C57BL / 6N mice (B6 mice), Balb / c mice and the like are preferably used, and C5 7BL / 6N mice are particularly preferable. In the following description of the present invention, the case where the introduced rodent is a mouse may be described as an example, but the present invention is not limited to the mouse.

 <Transgenic animal of the present invention>

The method for introducing the above gene into the animal model of the present invention may be performed according to the usual method for introducing the above gene into the animal model. For example, an embryo obtained by the following method may be used. It can be produced by transplanting to a borrowed animal. However, methods that are generally used for gene transfer are not limited to these methods.

 [0028] (Method for producing embryo for production of transgenic animals)

 Examples include a method in which a gene fragment containing a specific promoter and part or all of the SREBPlc gene obtained as described above is injected into a pronuclear embryo of a mouse fertilized egg, for example, by microinjection or the like. . However, it is preferable to use a transgenic animal that stably expresses part or all of the SREBPlc gene. Therefore, the above-mentioned animal is founded as a founder and crossed with a wild-type mouse. It is preferable to select those that highly express part or all of SREBPlc. Examples of the screening method include PCR analysis using the genomic DNA of the tail and Southern blotting analysis. The amount of the gene fragment containing part or all of the promoter and the SREBPlc gene can be appropriately adjusted according to the degree to be developed, etc., and is not particularly limited.

 [0029] However, aP2-SREBPlc transgenic mice (mouse genetic background is C57BL / 6) can be purchased from the Jackson Laboratory in the United States.

 This known transgenic mouse did not develop NASH at the time of purchase, and because of the nature of its use as a disease state model, it has been used for experiments prior to the onset of NASH. That was completely unknown.

 [0030] The NASH animal model of the present invention is produced by breeding the above transgenic animal until at least one of the following findings (1) to (5) is observed together with fat deposition in hepatocytes. can do.

 [0031] (1) Inflammatory cell infiltration into hepatic thread and tissue

 (2) Focal necrosis of hepatocytes

 (3) Hepatocyte balloon

 (4) Mallory body expression in hepatocytes

 (5) Fibrosis in liver lobule

[0032] As a standard of a specific breeding period until the onset of NASH, the above-mentioned transgenic animal is preferably 20 weeks old, more preferably 25 weeks old, and particularly preferably 30 weeks old. 20 weeks NASH develops with a probability of approximately 50% at age, and almost all patients develop NASH at 30 weeks of age. Therefore, for example, if a 6-week-old commercially available transgenic mouse is used, the number of days for model creation is

Well known PTEN knockout mice (40-78 weeks old)

) Can be manufactured in a much shorter period of time.

[0033] In addition, the liver tumor model animal of the present invention can be prepared by continuing breeding under the same conditions after the onset of NASH. The specific breeding period until the onset of liver tumor is preferably 50 weeks old (after birth), more preferably 52 weeks old.

[0034] Rearing can be performed under the following conditions.

 Bait: Ordinary bait (manufactured by CLEA Japan, CE-2), free consumption

 Water: free intake

 Temperature: 22 degrees throughout the year

 Humidity: 40-60% throughout the year

 Light and dark: Lighting time is 12 hours (from 6 am)

[0035] In order to subculture the above-described model of the present invention, for example, a male SREBPlc transgenic mouse is crossed with a female of a wild-type mouse, and SDEBPlc is analyzed by genetic testing from its offspring. This is done by selecting the genes that have been incorporated. The wild-type mouse strain to be crossed is preferably the one having the same genetic background as the model of the present invention, such as C57BL / 6N described above, because the genetic background of the transgenic mouse does not change. Good.

 <Screening method of the present invention>

 By using the above-described model of the present invention, it is possible to screen for a preventive or therapeutic agent for NASH or liver tumor.

The screening referred to in the present invention includes so-called primary screening for selecting a desired preventive or therapeutic agent from a plurality of candidates, as well as secondary screening for confirming the prophylactic or therapeutic effect of a test substance ( Confirmation tests) are also included. Therefore, it can be used for confirming the effects of known NASH or therapeutic agents for liver tumors, or for confirming the therapeutic effects of NASH or therapeutic agents for liver tumors obtained by other primary screening methods. Because the animal model of the present invention is closer to human NASH than the traditional NASH model. Since the time required for screening (model preparation days) is as short as 14 weeks and 24 weeks from the purchase of commercially available transgenic mice, it is also useful for re-evaluation of known NASH therapeutic agents. It is. In addition, a gene capable of suppressing NASH or a liver tumor resulting therefrom can also be found by mating a transgenic mouse or a knockout mouse with the NASH or liver tumor model of the present invention.

<0037> <NASH or liver tumor pathogenesis analysis method of the present invention>

 By analyzing the NASH or liver tumor model of the present invention in detail, the pathogenesis mechanism of NASH can be analyzed. In particular, the model of the present invention is closer to the onset of symptoms because the genes of the hepatocytes themselves are not artificially manipulated compared to the conventionally known PTEN knockout mice! In addition, the pathogenesis of NASH and the pathogenesis of liver tumors caused by NASH can be confirmed. Examples of the analysis method include a method of exhaustively searching for genes and proteins specifically expressed in the animal model using cDNA microarray and proteome analysis.

<Prophylactic or therapeutic agent for NASH of the present invention>

 The NASH preventive or therapeutic agent of the present invention is characterized by containing, as an active ingredient, one or more selected from the following (A) to (D) as an active ingredient.

 (A) Adiponectin protein

 (B) A protein comprising the amino acid sequence in which one or several amino acids of (A) are deleted, substituted or added and having the same activity as (A)

 (C) Gene encoding (A)

 (D) Gene encoding (B)

[0039] As adiponectin used in the present invention, it is more preferable to use human adiponectin because it has fewer side effects. Human adiponectin is a well-known protein consisting of 244 amino acids, which is a cytodynamic force in which adipocytes are specifically expressed. Adiponectin is abundant in normal human blood. It is deficient in obese and type 2 diabetic patients, and this deficiency causes insulin resistance, not only obesity and type 2 diabetes, but also arteries. It is known to cause curing. [0040] The gene encoding adiponectin used in the present invention refers to a gene capable of expressing adiponectin, as long as the gene has substantially the same function as the polypeptide ability to be expressed, adiponectin, A part of the gene sequence may be deleted or replaced by another base, or another base sequence may be added in the structural gene, or at the 5 ′ end or 3, or the end.

 These genes can also be synthesized on the basis of cDNA prepared from adiponectin mRNA such as human adiponectin mRNA.

[0041] Examples of the protein having the same activity as (A) in (B) include a variant having an adiponectin active domain. There are various reports on the active domain of adiponectin. For example, in the case of human adiponectin, there is a report that the adiponectin is full-length (1-244 amino acids). Therefore, the C-terminal globular domain (107 to 244 amino acids), N-terminal fibrous dom ain (41 to 107 amino acids), and C-terminal + N-terminal combinations are considered active domains. Therefore, it is used as a preventive or therapeutic agent for NASH of the present invention (B) “(A) has one or more amino acids deleted, substituted or added, and has the same amino acid sequence as (A). Examples of the “protein having activity” include those in which one or several amino acids are deleted, substituted or added at a site excluding these active site candidates.

 [0042] In addition, as a gene used for the preventive or therapeutic agent in the present invention, RNA, plasmid, etc. can be used in addition to DNA, and it may be single-stranded or double-stranded. In addition, a therapeutic drug that has a genetic ability may be in the form of a viral vector or the like using, for example, adenovirus.

 [0043] The NASH prophylactic or therapeutic agent of the present invention can be appropriately administered to a patient in various preparation forms such as a preparation for oral administration, an injection, or an inhalant.

 [0044] The NASH preventive or therapeutic agent of the present invention can also be used in combination with other drugs such as steroids, immunosuppressants, anti-inflammatory drugs and the like as appropriate. Furthermore, two or more kinds of the active ingredients of the NASH prophylactic or therapeutic agent of the present invention can be used in combination as appropriate.

[0045] Various formulations of the preventive or therapeutic agent for NASH of the present invention can be produced by conventional methods. [0046] For example, dosage forms for oral administration include tablets, capsules, granules, fine granules, and powders. These preparations contain a small amount of active ingredients used in the pharmaceutical composition of the present invention. At least one type or two or more types and normal pharmaceutical additives such as lactose, corn starch, crystalline cellulose, magnesium stearate, carboxymethylcellulose calcium, hydroxypropylcellulose, and tar are mixed as appropriate, and are produced by conventional methods. The

 [0047] The injection can be produced by a conventional method, and isotonic agents such as mannitol, sodium chloride salt, gnolecose, sonorebit, glyceronole, xylitornole, funolectose, manoletos, mannose, etc. Stabilizers such as sodium and albumin and preservatives such as benzyl alcohol and methyl hydroxybenzoate can be added to the preparation.

 [0048] The injection may be a freeze-dried preparation for dissolution at the time of use. The lyophilized preparation can be produced by a conventional method, and the above-mentioned tonicity agent, stabilizer, preservative and the like can be appropriately added to the preparation.

 [0049] An inhalant can be produced by a conventional method. At least one or two of the active ingredients of the pharmaceutical composition used in the present invention are dissolved or suspended in a physiological saline solution, and a mantle is appropriately used. Sodium, sodium chloride, glucose, sorbit, glycerol, xylitol, fructose, maltose, mannose and other isotonic agents, sodium sulfite, albumin and other stabilizing agents, benzyl alcohol, norhydroxyhydroxybenzoate, etc. Prepared with the addition of preservatives.

 [0050] The dose of the NASH prophylactic or therapeutic agent of the present invention varies depending on the type of active ingredient used in the present invention, the route of administration, the patient's condition, age, body weight, etc. 0. Olmg strength 10, OOOmg, which should be administered at once or in 2 to 3 divided doses.

 Example 1

 [0051] About 6 weeks old aP2-SREBPlc transgenic mice were purchased from Jackson Laboratory. Three males were crossed with six female wild-type mice (C57BL / 6), and the offspring were screened for genetically tested SREBPlc genes and 20 transgenic mice. Got.

[0052] This transgenic mouse was bred under normal conditions and under the following conditions. There was a probability of 2 out of 4 animals, and at 30 weeks of age, 13 out of 14 had NASH. Therefore, the power of a transgenic mouse raised at 20 weeks of age or more can be used as the NASH animal model of the present invention.

[0053] Bait: Normal bait (manufactured by CLEA Japan, CE-2), free consumption

 Water: free intake

 Temperature: 18-22 degrees throughout the year

 Humidity: 40-60% throughout the year

 Light and dark: Lighting time is 12 hours (from 6 am)

[0054] HE staining of liver tissue of a 30-week-old transgenic mouse that developed NASH (Fig.

1) or Azan (AZAN) staining (Fig. 2) and X-magnified at 200x is shown.

[0055] In Fig. 1 (SREBPlc (HE)), lipid droplets (fat deposition in hepatocytes), ballooned hepatocytes, Mallory bodies, infiltration of inflammatory cells and focal necrosis in lobule were observed. (AZAN)

) However, central vein (C) fibrosis around hepatocytes was observed.

 Example 2

 [0056] In the same manner as in Example 1, when male transgenic mice were bred up to 52 weeks of age, it was confirmed that hepatic tumors had developed (arrows in Figs. 3 and 4). See section).

 FIG. 3 is a microscopic (X 20 times) histopathological photograph of HE-stained liver tissue of aP2- SREBPlc transgenic mouse 52 weeks old (male). In the liver, as indicated by the arrows, tumorous lesions that were macroscopically different from the surrounding tissues and developed to exclude the surrounding tissues were observed.

 Further, FIG. 4 is a microscopic (X 20 times) histopathological photograph of liver tissue stained with AZAN of aP2-SREBPlc transgenic mouse 52 weeks old (male). In the liver, as shown by the arrows, tumorous lesions that were visually different from the surrounding tissues and developed to exclude the surrounding tissues were observed.

 Within the neoplastic lesion, there was almost no hepatic interlobular portal vein region (asterisk) stained in blue with AZAN staining in the region considered to be non-neoplastic liver tissue.

This suggests that the model of the present invention can also be used as a liver tumor model. Example 3

 [0057] [Adiponectin power Proof that it is a preventive or therapeutic agent for NASH]

In order to prove that adiponectin or its related proteins and related genes are prophylactic or therapeutic agents for NASH, (1) an animal model that highly expresses adiponectin (see # 112004 -219279 ₀ WO 2006Z011491) was prepared. (2) The NASH animal model of the present invention was crossed.

 [0058] (1) Method for producing adiponectin transgenic animal

 The method for producing a transgenic mouse that highly expresses adiponectin is the force described in Japanese Patent Application No. 2004-219 279 (WO 2006/011491). First, the production method is briefly described below, and then the production method specifically produced Will be explained.

 [0059] <Plasmid vector>

 The plasmid vector contains a liver-specific expression promoter, adiponectin gene as its component.

 [0060] (Liver-specific expression promoter)

 Examples of the liver-specific expression promoter include a promoter derived from a gene of a protein specifically produced in the liver, such as an SAP promoter.

 [0061] The adiponectin gene refers to a gene capable of expressing adiponectin in an animal such as a human, and the gene has a gene sequence that has substantially the same function as that of the expressed polypeptide ability adiponectin. A part thereof may be deleted or substituted with another base, or another base sequence may be added in the structural gene, or added to the 5 ′ end or 3 ′ end. These genes can be artificially synthesized or can be prepared based on cDNA prepared from human adiponectin mRNA.

 [0062] The plasmid used in the plasmid vector is not particularly limited. For example, a gene having a large gene expression level in eukaryotes is preferred. It is preferable to contain poly A sequences that are useful in Specific examples include pSG5 plasmid vector (Stratagene).

[0063] (Production method of vector) A plasmid vector can be produced according to a conventional method.

 [0064] <Transgenic animal introduced with adiponectin>

 (Type of animal)

 As the animal to be used, as in the NASH animal model described above, a mouse is particularly preferred, for example, rodents such as mice, rats and hamsters are preferred. Among these, C57BL / 6N mice (B6 mice), Balb / c mice and the like are preferably used, and C57BL / 6N mice are particularly preferable.

 [0065] (Method for producing adiponectin transgenic animal)

 The adiponectin-introduced transgenic animal can be produced, for example, by transplanting an embryo obtained by the following method to a borrowed animal. However, it is not limited to these methods, and methods generally used for gene transfer can be used.

 [0066] (Method for producing embryo for production of transgenic animals)

 A method of injecting a gene fragment containing a specific promoter and adiponectin gene obtained as described above into a pronuclear embryo of a mouse fertilized egg, for example, by microinjection or the like

 [0067] However, it is preferable to use a transgenic animal that stably expresses the adiponectin gene. Therefore, the above-mentioned animal was founded as a founder and crossed with a wild-type mouse. It is preferable to select those that highly express adiponectin.

 [0068] Examples of the selection method include PCR analysis using tail genomic DNA, ELISA analysis of adiponectin in serum, and Western blotting analysis of adiponectin in liver tissue.

 [0069] The amount of the gene fragment containing the promoter and adiponectin gene can be appropriately adjusted according to the degree to which the gene fragment is desired to develop, and is not particularly limited.

 [0070] <Construction of specific high adiponectin transgenic mice>

SAP—promoter rabbit j8—globulin intron and human adiponectin cDNA—polyadrelate for expression of adiponectin in mouse liver at higher concentrations A vector containing Chillon signal was constructed. For this purpose, we used pSG5 vector (Stratagene), which is an expression vector with early SV40 promoter ^ —— rabbit 13-globulin intron polyadenylation signal. The pSG5 vector was transformed into SCSI 10 cells, and the pSG5 vector was increased using the Qiagen plasmid kit.

 The SAP promoter was used to selectively express adiponectin in the liver. Based on reports of overexpression of leptin and BNP in the liver using this promoter, PCR was performed by amplifying -677 bp to -9 bp upstream of the human SAP gene and adding Sal 1 and Cla 1 cleavage sites at both ends. The product was incorporated into the TA easy vector. By digesting this integrated TA easy vector with Sal 1 and Cla 1 restriction enzymes and ligating the excised PCR product to the Sal 1 and Cla 1 sites of the pSG5 vector, the early SV40 promoter in the pSG5 vector is obtained. Replaced with SAP promoter. In this way, an expression vector (hSAP_pSG5) containing a human SAP promoter rabbit j8-globulin intron-polyadenylation signal that can be expressed specifically in the liver was constructed. Using a cDNA library of mouse adipose tissue, primers were prepared to create BamHl and EcoRl sites at both ends, and the full length of human adiponectin cDNA was amplified by PCR. A PCR product with BamHl and EcoRl cleavage sites at both ends was incorporated into the TA easy vector. This integrated TA eas y vector was digested with BamHl and EcoRl restriction enzymes, and the excised product was ligated between BamHl and EcoRl present in the multicloning site of the hSAP-pSG5 expression vector.

[0071] As a result, a vector containing a human SAP promoter, one rabbit β globulin intron-human adiponectin cDNA polyaddition signal was constructed. The vector prepared above was cleaved with Sal 1, the vector-derived sequence was removed by agarose gel electrophoresis, and the DNA fragment (about 2 kb) was purified by Gene Clean II kit.

 [0072] Using the DNA fragment of the purified vector, 10 adiponectin transgenic mice were prepared according to a conventional method.

 [0073] (Confirmation of adiponectin expression)

When the concentration of adiponectin in the blood of this mouse was measured by ELISA, the amount of adiponectin was 5-10 times the normal amount. [0074] (2) Multiplication with NASH animal model of the present invention

 8-week-old aP2- SREBPlc transgenic mice were mated with 8-week-old high adiponectin transgenic mice obtained as described above, and both SREBPlc gene (SREBP lc) and adiponectin gene (ADIPO) were introduced. Selected double-transgenic mice.

 Fifteen animals were bred on normal diet, 8 of which were dissected at 30 weeks of age. In NASH model mice + high adiponectin transgenic mice, NASH was suppressed in all 8 mice (Figs. 5 and 6). This indicates that adiponectin suppresses NASH and, in turn, liver tumors caused by NASH.

[0075] Figure 5 (SREBPlc + ADIPO (HE)) (Here, HE means hematoxylin 'eosin staining.) Even in SREBP-lc transgenic mice, Infiltration of inflammatory cells with lighter degrees of fat deposition, Mallory bodies and hepatocyte ballooning were not observed.

 In addition, in FIG. 6 (SREBPlc + ADIPO (AZAN)) (where AZAN means Azan staining), the central vein (C) and portal vein (P) in the liver, the liver lobule, especially the liver It was strong with little fibrosis around the cells.

 Industrial applicability

[0076] It is possible to provide an animal model that exhibits symptoms of nonalcoholic steatohepatitis (NASH) in a shorter period of time without artificially deleting the gene originally expressed in “hepatocytes”. It is also possible to provide a model for liver tumors caused by NASH, and further to elucidate the pathogenesis of NASH or liver tumors using this model, and to develop a preventive or therapeutic agent for NASH or liver tumors. Screening became possible. In addition, specific NASH preventive or therapeutic agents such as adiponectin could be provided.

 Brief Description of Drawings

 [0077] [Fig. L] aP2- SREBPlc transgenic mouse 30 weeks of age (male) microscopic (X 200 times) histopathological image of HE-stained liver tissue.

FIG. 2 is a microscopic (X 200 times) histopathological photograph of an AZAN-stained liver tissue of aP2- SREBPlc transgenic mouse 30 weeks old (male). FIG. 3 is a microscopic (X 20 times) histopathological image of HE-stained liver tissue of aP2- SREBPlc transgenic mouse 52 weeks old (male).

 FIG. 4 is a microscopic (X 20 times) histopathological picture of liver tissue stained with AZAN of aP2- SREBPlc transgenic mouse 52 weeks old (male).

 [Fig. 5] Microscopic (200X magnification) histopathological image of liver tissue of a 30-week-old (male) double-transgenic mouse mated with aP2- SREBPlc transgenic mouse and adiponectin transgenic mouse It is a photograph of.

 [Fig. 6] Double-transgenic mouse mated with aP2- SREBPlc transgenic mouse and adiponectin transgenic mouse 30 weeks old (male), AZAN stained liver tissue microscope (X 200x) It is a picture of the statue.

Explanation of symbols

 C: Central vein

 P: Portal vein

Claims

Claims [1] A non-alcoholic steatohepatitis or liver tumor animal model, wherein (XI) or (X2) below is introduced and (Y) is introduced. (XI) A gene encoding an amino acid sequence that is part or all of SREBPlc and has transcriptional activity (X2) One or several amino acids are missing from part or all of the amino acid sequence of SREBPlc , A gene consisting of a substituted or added amino acid sequence and encoding a protein having transcriptional activity (Y) a promoter gene having adipose tissue-specific expression [2] The amino acid sequence having transcriptional activity is N of amino acids of SREBPlc The animal model for non-alcoholic steatohepatitis or liver tumor according to claim 1, wherein the animal model is a terminal domain. [3] The non-alcoholic steatohepatitis or liver tumor animal model according to claim 1 or 2, wherein the promoter is a P2 promoter having adipose tissue-specific expression. [4] Any of the following (XI) or (X2), and (Y) is introduced into the animal, along with fat deposition in hepatocytes, at least the findings (1) to (5) below: A method for producing an animal model of non-alcoholic steatohepatitis characterized by breeding until one or more species are recognized. (XI) A gene encoding an amino acid sequence that is part or all of SREBPlc and has transcriptional activity (X2) One or several amino acids are missing from part or all of the amino acid sequence of SREBPlc , A gene consisting of a substituted or added amino acid sequence and encoding a protein having transcriptional activity (Y) a promoter gene having adipose tissue-specific expression  (1) Inflammatory cell infiltration into liver tissue  (2) Focal necrosis of hepatocytes  (3) Hepatocyte balloon  (4) Mallory body expression in hepatocytes  (5) Fibrosis in liver lobule [5] At least 20 weeks after introducing either (XI) or (X2) below and (Y) Of non-alcoholic steatohepatitis animal model, characterized by breeding until age (XI) A gene encoding an amino acid sequence having a transcription activity, which is part or all of the amino acid sequence of SREBPlc  (X2) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in part or all of the amino acid sequence of SREBPlc and having transcriptional activity  (Y) Promoter gene having adipose tissue-specific expression [6] A method for producing a liver tumor animal model, characterized in that an animal introduced with either (XI) or (X2) below and (Y) is raised to at least 50 weeks of age.  (XI) A gene encoding an amino acid sequence having a transcription activity, which is part or all of the amino acid sequence of SREBPlc  (X2) A gene encoding a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added in part or all of the amino acid sequence of SREBPlc and having transcriptional activity  (Y) Promoter gene having adipose tissue-specific expression [7] A screening method for a preventive or therapeutic agent for nonalcoholic steatohepatitis or liver tumor using the nonalcoholic steatohepatitis or liver tumor animal model according to any one of claims 1 to 3. .  [8] A method for analyzing a non-alcoholic steatohepatitis or liver tumor pathogenesis using the non-alcoholic steatohepatitis animal model according to any one of claims 1 to 3.  [9] A prophylactic or therapeutic agent for nonalcoholic steatohepatitis comprising one or more of the following (A) to (D) forces selected as an active ingredient.  (A) Adiponectin protein  (B) A protein comprising the amino acid sequence in which one or several amino acids of (A) are deleted, substituted or added and having the same activity as (A)  (C) Gene encoding (A)  (D) Gene encoding (B)