JP6745450B2 - Colorectal cancer model animal, method for producing colorectal cancer model animal, anticancer agent, allergy model animal, method for producing allergy model animal, and screening method - Google Patents

Description

The present invention relates to a colorectal cancer model animal, a method for producing a colorectal cancer model animal, an anticancer agent, an allergy model animal, a method for producing an allergy model animal, and a screening method.

Colitis-associated colon cancer (CAC) is a subtype of colorectal cancer, and inflammation such as ulcerative colitis and Crohn's disease. It is believed to be due to sexual intestinal disease. Chronic inflammation is widely recognized as one of the major risk factors for developing cancer. However, the understanding of the mechanism of its occurrence is still insufficient.

Gasdermin D (Gsdmd) is a member of the Gasdermin (Gsdm) gene family. In mice, Gsdmd is the only gene belonging to the Gsdmd subfamily and is known to be specifically expressed in the intestinal epithelium such as the large intestine (Non-Patent Document 1). However, no abnormality was reported in the morphology of the intestinal tract of Gsdmd knockout (Gsdmd ^−/− ) mice (Non-Patent Document 2). In recent years, Gsdmd/GSDMD has also been reported to be a novel regulator of pyrotosis (Non-patent Documents 2 and 3).

Moreover, the onset of allergies has been increasing in recent years. In certain allergic diseases, an increase in immunoglobulin E (IgE) in the body causes an allergic reaction. IgE production is known to be promoted by specific cytokines such as interleukin-4 (IL-4).
When allergies become severe, rhinitis, urticaria, etc. occur and interfere with daily life, and in the case of shock symptoms, it may lead to a serious condition. Therefore, it is expected to elucidate the mechanism of allergies and to treat allergies.

Tamura, M. et al. Members of a novel gene family, Gsdm, are expressed exclusively in the epithelium of the skin and gastrointestinal tract in a highly tissue-specific manner.Genomics 89, 618-29 (2007). Fujii, T. et al. Gasdermin D (Gsdmd) is dispensable for mouse intestinal epithelium development. Genesis 46, 418-423 (2008). Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature (2015). Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signaling. Nature (2015).

The present invention has been made in view of the above circumstances, and provides a colorectal cancer model animal to be a colorectal cancer model, a method for producing the colorectal cancer model animal, a screening method using the colorectal cancer model animal, and an anticancer agent. Is an issue.

Another object of the present invention is to provide an allergy model animal, a method for producing the allergy model animal, and a screening method using the allergy model animal.

The present inventors have found that Gsdmd null (-/-) knockout mice produced by Cre-loxP site-specific recombination develop colon cancer, and completed the present invention.
In addition, the present inventors have found that in Gsdmd null (-/-) knockout mice produced by Cre-loxP site-specific recombination, the expression of interleukins associated with the development of allergy is enhanced, The present invention has been completed.
That is, the present invention is as follows.

(1) A colon cancer model animal that is a non-human animal in which the function of the Gasdermin D (Gsdmd) gene is deficient or reduced.
(2) The colorectal cancer model animal according to (1) above, which has a mutation in the Gsdmd gene or the expression control region of the Gsdmd gene.
(3) The colorectal cancer model animal according to (1) or (2) above, which contains a colorectal cancer-specific carcinogen and a colorectal inflammation substance in the body.
(4) The colorectal cancer model animal according to any one of (1) to (3), wherein the colorectal cancer is a colitis-related colorectal cancer (Coltis-associated colon cancer).
(5) The colorectal cancer model animal according to any one of (1) to (4) above, which is a rodent.
(6) A method for producing a colorectal cancer model animal, which comprises administering a colorectal cancer-specific carcinogen and a colon inflammatory substance to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.
(7) The method for producing a colorectal cancer model animal according to (6), wherein the colorectal cancer is a colitis-related colorectal cancer (Coltis-associated colon cancer).
(8) The test substance is administered to the colorectal cancer model animal according to any one of (1) to (5), and the test substance is administered to the colorectal cancer model animal, and the test substance is not administered. When compared with the colorectal cancer model animal, if the phenotype associated with colorectal cancer is suppressed in the colorectal cancer model animal administered the test substance, the test substance is effective in treating or preventing colorectal cancer A screening method comprising determining that the substance is a substance.
(9) An anticancer agent for colon cancer, which comprises any one or more of the following proteins (I) to (III) as an active ingredient.
(I) A protein having an amino acid sequence consisting of the 1st to 275th amino acids in the amino acid sequence represented by SEQ ID NO: 3 and having cytotoxic activity on colon cancer cells (II) represented by SEQ ID NO: 3 In the amino acid sequence consisting of the first to 275th amino acid sequences, it has an amino acid sequence in which one to several amino acids are deleted, substituted or added, and has cytotoxic activity on colon cancer cells. Protein (III) The amino acid sequence represented by SEQ ID NO: 3 has an amino acid sequence having 80% or more identity with the amino acid sequence consisting of the first amino acid to the 275th amino acid sequence, and has a cytotoxic activity on colon cancer cells. (10) An anticancer agent for colorectal cancer containing as an active ingredient an expression vector containing a gene encoding any one or more proteins of (I) to (III) below.
(I) A protein having an amino acid sequence consisting of the 1st to 275th amino acids in the amino acid sequence represented by SEQ ID NO: 3 and having cytotoxic activity on colon cancer cells (II) represented by SEQ ID NO: 3 In the amino acid sequence consisting of the first to 275th amino acid sequences, it has an amino acid sequence in which one to several amino acids are deleted, substituted or added, and has cytotoxic activity on colon cancer cells. Protein (III) The amino acid sequence represented by SEQ ID NO: 3 has an amino acid sequence having 80% or more identity with the amino acid sequence consisting of the first amino acid to the 275th amino acid sequence, and has a cytotoxic activity on colon cancer cells. An allergic model animal that is a non-human animal in which the function of the protein (11) Gasdermin D (Gsdmd) gene having the above is deficient or diminished, and is apt to develop an allergic disease.
(12) The allergy model animal according to (11) above, which has a mutation in the expression control region of the Gsdmd gene or the Gsdmd gene.
(13) The allergy model animal according to (11) or (12) above, which contains a carcinogen specific to colon cancer and a colon inflammatory substance in the body.
(14) The allergic model animal according to any one of (11) to (13), wherein the allergic disease is a type I allergic disease.
(15) The allergy model animal according to any one of (11) to (14) above, which is a rodent.
(16) A method for producing an allergic model animal, which comprises administering a colorectal cancer-specific carcinogen and a colon inflammatory substance to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.
(17) The method for producing an allergic model animal according to (16), wherein the colorectal cancer is colitis-associated colon cancer.
(18) The allergy model animal according to any one of (11) to (15) above, wherein the test substance is administered to the allergy model animal, and the test substance is administered to the allergy model animal and the allergy to which the test substance is not administered When the phenotype associated with allergy is suppressed in the allergy model animal to which the test substance is administered by comparison with a model animal, the test substance is considered to be an effective substance for treating or preventing allergic diseases. A screening method comprising determining.

According to the present invention, a colorectal cancer model animal serving as a colorectal cancer model can be provided. Further, it is possible to provide a method for producing the colorectal cancer model animal, and a screening method using the colorectal cancer model animal.
Further, according to the present invention, an allergy model animal can be provided. Further, it is possible to provide a method for producing the allergy model animal and a screening method using the allergy model animal.

It is a schematic diagram which shows typically the manufacturing procedure of the CAC(colitis-associated colon cancer) model animal in an Example. It is a graph which showed the result of the body weight change rate of the mouse during an AOM-DSS treatment period. It is a photograph showing the distal large intestine of a mouse after 12 weeks of AOM-DSS treatment. It is a dot plot which shows the number of tumors and tumor size in WT mouse and Gsdmd ^-/- mouse after 12 weeks AOM-DSS treatment. Fig. 6 is an image of a hematoxylin- and eosin-stained section of adenocarcinoma of the large intestine developed in a WT mouse and a Gsdmd ^−/− mouse. It is a figure which shows the uptake of BrdU in CAC of WT and Gsdmd ^-/- mouse, and the staining result of cleaved Caspase-3 (Casp3). It is an image which shows the result of the immunohistochemical test with respect to the colon adenocarcinoma obtained from WT mouse and Gsdmd ^-/- mouse, and the immunoblot. It is a schematic diagram which shows typically the procedure of the acute inflammation process in an Example. It is a graph which showed the weight change rate of the mouse during a DSS treatment period. It is an image which shows the result of the histological analysis after acute inflammation processing, and a graph which shows the histological score. It is a graph which shows the value of the intestinal length of WT mouse and Gsdmd ^-/- mouse after an acute inflammation process. It is an image which shows the result of the histological analysis of the intestine of WT mouse and Gsdmd ^-/- mouse after an acute inflammation process. It is an image which shows the result of the immunohistological analysis in the inflammation site|part of the large intestine of WT and Gsdmd ^-/- mouse. It is an image which shows the result of the immunohistological analysis in the inflammation site|part of the large intestine of WT and Gsdmd ^-/- mouse. It is a graph which shows the result of the real-time quantitative PCR obtained in the Example. It is a graph which shows the result of the real-time quantitative PCR obtained in the Example. It is a graph which shows the result of the real-time quantitative PCR obtained in the Example. 3 is an image showing the results of histological analysis of normal human large intestine and CAC specimens, and a graph showing the score of the IHC staining intensity. It is an image which shows the result of the histological analysis of the normal colonic epithelium of a mouse, and the colonic epithelium of the CAC model mouse which received the acute inflammation process. 3 is an image showing colo-320 cells into which a DNA sequence encoding GSDMD-N has been introduced, and control cells.

<< Colorectal cancer model animal >>
The colon cancer model animal of the present invention is a non-human animal in which the function of the Gsdmd gene is deficient or reduced.
Human (Homo sapiens) and mouse (Mus musculus) Gasdermin D (GSDMD and Gsdmd, respectively) are the only genes belonging to the Gsdmd subfamily. It has been clarified that mouse Gsdmd maps to chromosome 15 (15D3) (Non-patent document 1). The Gsdm gene family genes encode proteins consisting of 400 to 500 amino acids. It has been reported that the Gsdm family gene functions as a transcription factor, and Gsdmd may also function as a transcription factor (Lunny, DP et al. Mutations in gasdermin 3 cause aberrant differentiation of the hair follicle and sebaceous gland. J Invest Dermatol 124, 615-21 (2005).).

The amino acid sequences of the mouse Gsdmd gene and the protein encoded by it have been disclosed (Gene ID: 69146). The human GSDMD gene and the amino acid sequence of the protein encoded by it have been disclosed (Gene ID: 79792). The Gene ID represents the registration number of the Gene database (URL: ttp://www.ncbi.nlm.nih.gov/gene/).
The amino acid sequence of mouse GSDMD is shown in SEQ ID NO:1. The nucleotide sequence of DNA encoding mouse GSDMD is shown in SEQ ID NO:2. The amino acid sequence of human GSDMD is shown in SEQ ID NO:3. The nucleotide sequence of DNA encoding human GSDMD is shown in SEQ ID NO:4.

The non-human animal in the present specification is not particularly limited as long as it is a non-human animal, but is preferably a mammal, more preferably a rodent (Rodentia). Examples of animals classified as mammals include goats, pigs, dogs, cats, mice, rats, guinea pigs, hamsters and rabbits. Examples of animals classified into rodents include mice, rats, guinea pigs, hamsters and the like.

The colorectal cancer model animal of the present invention exhibits at least one phenotype associated with colorectal cancer.
Phenotypes associated with colorectal cancer include tumor development in the large intestine, tumor incidence, number of tumors, tumor size, degree of tumor invasion, colorectal cancer progression, colorectal cell differentiation abnormalities, Examples include morphological abnormalities of cells in the large intestine. These may be information obtained by confirming a tumor marker or other factors indicating the presence of a tumor in the large intestine. As the phenotype associated with colorectal cancer, those described in Examples of the present application can also be adopted.

The colorectal cancer may be colitis-related colorectal cancer.
Phenotypes associated with colitis-related colon cancer include inflammation, ulcers, erosions, etc. in the colonic mucosa. These may be information obtained by confirming inflammatory markers and other factors indicating the presence of colitis in the large intestine. As the phenotype associated with colitis-associated colorectal cancer, those described in Examples of the present application can also be adopted.

Examples of the Gsdmd gene in the present specification include a gene encoding a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3 above, and further include homologues, variants, and mutants of the gene.
When the Gsdmd gene in a model animal is known, it may be selected as the Gsdmd gene. Even if the Gsdmd gene in the model animal is not known, those skilled in the art can select the Gsdmd gene from the animal genome information of the model animal.

Examples of the Gsdmd gene or a homologue, variant, or mutant of the gene include a gene encoding a protein having any of the following amino acid sequences (a) to (c).
(A) a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3, which causes colorectal cancer development due to loss or deterioration of function;
(B) The amino acid sequence represented by SEQ ID NO: 1 or 3 has an amino acid sequence in which one to several amino acids have been deleted, substituted, or added, and causes the development of colorectal cancer due to loss or reduction in function. Protein,
(C) A protein having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 3 and causing a colorectal cancer due to loss or reduction in function

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 30, preferably 1 to 20, preferably 1 to 10, and more preferably 1 to 7, 1 to 5 is more preferable, 1 to 3 is particularly preferable, and 1 to 2 is most preferable.
Here, the identity with the amino acid sequence is preferably 80% or more, more preferably 85% or more, further preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more. The amino acid sequence identity can be determined by, for example, BLAST (reference URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi). For example, parameters are score = 100 and wordlength = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, parameters are score=50 and wordlength=3, for example. When using BLAST and Gapped BLAST programs, the default parameters for each program are used.

Here, the cause of colorectal cancer is defined as a model animal in which a function is deficient or diminished and an animal in which the function is deficient or diminished (animals that serve as controls such as wild type (WT) of model animals). It means that the phenotype associated with colorectal cancer is increased in the model animal in which the function is lost or reduced as compared with

The loss of Gsdmd gene function refers to a state in which the function originally possessed by the protein encoded by the Gsdmd gene is completely lost. The reduced function of the Gsdmd gene means a state in which the function originally possessed by the protein encoded by the Gsdmd gene is partially lost.

When the function originally possessed by the protein encoded by the Gsdmd gene is partially lost, the degree of the loss is compared between the model animal and a control animal such as a wild type animal of the model animal, It is sufficient that the condition recognized as a phenotype associated with colorectal cancer (difference from control) is more strongly manifested in the model animal.

As used herein, the large intestine includes the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum.

Examples of the colorectal cancer relating to the model animal of the present invention include Coltis-associated colon cancer. Examples of the colitis in the colitis-related colon cancer include ulcerative colitis and Crohn's disease.

The loss or reduction in the function of the Gsdmd gene can also be caused by the disappearance or reduction in the expression of the Gsdmd gene. Loss of Gsdmd gene expression means that the Gsdmd gene product is lost in a model animal. The expression of the Gsdmd gene being decreased means that the amount of the Gsdmd gene product is decreased as compared with a control animal such as a wild type animal of a model animal. When the expression of the Gsdmd gene is decreased, the degree of decrease is associated with colorectal cancer as described above in the model animal, as compared with a control animal such as a wild-type animal of the model animal. It is sufficient if the condition recognized as phenotype (difference from control) is more strongly manifested.

The disappearance or reduction of the expression of the Gsdmd gene can be caused by, for example, introducing a nucleic acid that causes RNAi for the Gsdmd gene or an antisense nucleic acid into a model animal and expressing it. Designing, introduction, and expression of these nucleic acids with respect to the target gene can be performed using known techniques.

The model animal of the present invention is preferably in a state in which a mutation has been introduced into the Gsdmd gene or the expression control region of the Gsdmd gene so that the function of the Gsdmd gene is deficient or decreased. Such model animals refer to genetically modified animals whose genetic information has been modified by a genetic engineering method, and examples thereof include transgenic mice and knockout mice.

The introduced mutation may be one that completely or partially loses the function originally possessed by the protein encoded by the Gsdmd gene, or one that eliminates or reduces the expression of the Gsdmd gene.

The Gsdmd gene includes an exon, which is a protein-coding region, and an intron when the expression of the Gsdmd gene can be eliminated or reduced, or the function of the protein encoded by the Gsdmd gene can be deleted or reduced. Be done.
The expression control region of the Gsdmd gene is a region on the genomic DNA that controls the expression of the Gsdmd gene, and is, for example, a promoter.

The introduced mutation includes deletion, substitution, or addition.
The loss or reduction in the function of the Gsdmd gene can be achieved by, for example, deleting all or part of the Gsdmd gene on the genomic DNA or the DNA corresponding to the expression control region of the Gsdmd gene. Further, the loss or reduction of the function of the Gsdmd gene can be achieved by, for example, substituting or adding a nucleic acid having a sequence different from the original one to the DNA corresponding to the Gsdmd gene or the expression control region of the Gsdmd gene on the genomic DNA. Is.

Techniques for introducing mutations into genes are known and can be performed by various genetic engineering techniques. For example, gene targeting by homologous recombination technology, gene modification, knockout, knockdown, knockin, site-specific recombination by Cre-loxP system and the like can be used. It can also be performed using a genome editing technique such as CRISPR/Cas or Transcription Activator-Like Effector Nucleases (TALEN).

The model animal according to the present invention shows a phenotype associated with colorectal cancer, although the frequency is low, only due to the loss or reduction of the function of the Gsdmd gene. However, the inventors have found that Gsdmd knockout (Gsdmd ^−/− ) mice administered with azoxymethane (AOM) and dextran sulfate sodium (DSS) develop colon cancer at a very high frequency.

In one embodiment, the model animal according to the present invention contains a carcinogen specific to colon cancer in its body. Examples of colorectal cancer-specific carcinogens include AOM, 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), and 1,2-Dimethylhydrazine (DMH).
In one embodiment, the model animal according to the present invention contains a colonic inflammatory substance in its body. Examples of the colon inflammatory substance include DSS, trinitrobenzene sulfonic acid (TNBS), and oxazolone (Oxazolone).

In one embodiment, the model animal of the present invention contains a colon cancer-specific carcinogen and/or a colon inflammatory substance in its body. The colorectal cancer-specific carcinogen and the colorectal inflammation substance may be used in combination. In one embodiment, the model animal of the present invention contains a colorectal cancer-specific carcinogen and a colon inflammatory substance in its body. A preferable combination of a carcinogen specific to colon cancer and a colon inflammatory substance includes a combination of AOM and DSS. In one aspect, the model animal according to the present invention contains AOM and DSS in its body.

A model animal containing a colon cancer-specific carcinogen and a colon inflammatory substance in the body is useful as a model animal for colitis-related colon cancer.

It was previously known that AOM and DSS induce colitis-related colorectal cancer. However, the fact that the loss or reduction of the Gsdmd gene function enhances the development of colitis-related colorectal cancer is a previously unknown new effect brought about by the loss or reduction of the Gsdmd gene function.

The model animal according to the present invention exhibits a phenotype associated with colon cancer. Therefore, it is very useful for elucidating the mechanism of the development of colorectal cancer, searching for substances useful for the treatment thereof, and developing treatment methods.

One aspect of the present invention is a method of using a non-human animal in which the function of the Gsdmd gene is deficient or reduced as a colorectal cancer model animal.

≪Allergy model animal≫
The allergy model animal of the present invention is a non-human animal in which the function of the Gsdmd gene is deleted or reduced.
Hereinafter, the allergy model animal of the present invention will be described, but the description of the same configuration as the colorectal cancer model animal will be omitted.

The allergy model animal of the present invention exhibits at least one phenotype associated with allergy.
Allergies are classified into types I to V depending on the mechanism of onset. Among them, type I allergy is also called immediate type allergy, which involves the production of IgE antibody in the animal body. Examples of the type I allergic disease include food allergy, hay fever, allergic rhinitis, bronchial asthma, atopic dermatitis and the like.
The allergy model animal of the present invention may exhibit at least one of the phenotypes associated with type I allergy. Phenotypes associated with type I allergies include rash, rhinitis, conjunctivitis, tear eyes, bronchitis, cough, dyspnea, and the like.

In addition, the presence of factors or cells related to the onset mechanism of type I allergy in the animal body may be analyzed as a phenotype. Examples of the factor or cells include IgE, type 2 helper T cells (Th2 cells), IL-4 that stimulates the production of IgE in antibody-producing cells, Th2 cytokines such as IL-5 and IL-13, and IL-25, IL-33, and the like that induce the production of Th2 cytokines are mentioned, and these factors or cells are known to be increased or activated by type I allergy. Therefore, the increase or activation of these factors or cells can be treated as a phenotype associated with type I allergy.
In addition to the case where the allergic disease is developed, when one or more of these factors or cells are increased, the allergic disease is easily developed. As shown in the examples, the expression levels of IL-4, IL-25, and IL-33 in the mouse lacking the function of the Gsdmd gene are significantly increased as compared with those in the wild-type mouse. It was

The animal model for allergy of the present invention may exhibit at least one of the phenotypes associated with colorectal cancer in addition to the phenotype associated with allergy.
Phenotypes associated with colorectal cancer include tumor development in the large intestine, tumor incidence, number of tumors, tumor size, degree of tumor invasion, colorectal cancer progression, colorectal cell differentiation abnormalities, Examples include morphological abnormalities of cells in the large intestine. These may be information obtained by confirming a tumor marker or other factors indicating the presence of a tumor in the large intestine. As the phenotype associated with colorectal cancer, those described in Examples of the present application can also be adopted.

Examples of the Gsdmd gene or a homologue, variant, or mutant of the gene include a gene encoding a protein having any of the following amino acid sequences (a) to (c).
(A) a protein having the amino acid sequence represented by SEQ ID NO: 1 or 3, which causes allergy due to loss or deterioration of function,
(B) In the amino acid sequence represented by SEQ ID NO: 1 or 3, it has an amino acid sequence in which one to several amino acids are deleted, substituted, or added, and causes a cause of allergy due to loss or reduction of function. Becomes a protein,
(C) A protein having an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1 or 3, and causing allergy due to loss or deterioration of function

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 30, preferably 1 to 20, preferably 1 to 10, and more preferably 1 to 7, 1 to 5 is more preferable, 1 to 3 is particularly preferable, and 1 to 2 is most preferable.
Here, the identity with the amino acid sequence is preferably 80% or more, more preferably 85% or more, further preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more. The amino acid sequence identity can be determined by, for example, BLAST (reference URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi). For example, parameters are score = 100 and wordlength = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, parameters are score=50 and wordlength=3, for example. When using BLAST and Gapped BLAST programs, the default parameters for each program are used.

Here, the cause of allergy is that a model animal having a loss or reduction in function and an animal not having a loss or reduction in function (animal to be a control of wild type (WT) etc. of model animal) It means that the model animal having the loss or reduction of the function has an increased phenotype associated with allergy, as compared with.

The loss of Gsdmd gene function refers to a state in which the function originally possessed by the protein encoded by the Gsdmd gene is completely lost. The reduced function of the Gsdmd gene means a state in which the function originally possessed by the protein encoded by the Gsdmd gene is partially lost.

When the function originally possessed by the protein encoded by the Gsdmd gene is partially lost, the degree of the loss is compared between the model animal and a control animal such as a wild type animal of the model animal, All that is necessary is that the condition recognized as a phenotype related to allergy (difference from the control) appears more strongly in the model animal.

The loss or reduction in the function of the Gsdmd gene can also be caused by the disappearance or reduction in the expression of the Gsdmd gene. Loss of Gsdmd gene expression means that the Gsdmd gene product is lost in a model animal. The expression of the Gsdmd gene being decreased means that the amount of the Gsdmd gene product is decreased as compared with a control animal such as a wild type animal of a model animal. When the expression of the Gsdmd gene is decreased, the degree of decrease is expressed in the model animal as compared with a control animal such as a wild-type animal of the model animal, and the expression related to allergy as described above. It only needs to be such that the condition recognized as a type (difference from the control) appears more strongly.

The model animal according to the present invention exhibits a phenotype associated with allergy only by the lack or reduction of the function of the Gsdmd gene. The present inventors have, Gsdmd knockout sodium dextran sulfate (DSS) was administered (Gsdmd ^{- / -)} mice, as well as azoxymethane (AOM) and dextran Gsdmd knockout administered sodium sulfate (DSS) (Gsdmd ^{- / -)} It was found that mice exhibit a phenotype significantly associated with allergy compared to wild type mice.

In one embodiment, the model animal according to the present invention contains a carcinogen specific to colon cancer in its body.
In one embodiment, the model animal according to the present invention contains a colonic inflammatory substance in its body.

In one embodiment, the model animal of the present invention contains a colon cancer-specific carcinogen and/or a colon inflammatory substance in its body. The colorectal cancer-specific carcinogen and the colorectal inflammation substance may be used in combination. In one embodiment, the model animal of the present invention contains a colorectal cancer-specific carcinogen and a colon inflammatory substance in its body. A preferable combination of a carcinogen specific to colon cancer and a colon inflammatory substance includes a combination of AOM and DSS. In one aspect, the model animal according to the present invention contains AOM and DSS in its body.

A model animal containing a colorectal cancer-specific carcinogen and a colon inflammatory substance in the body is useful as an allergy model animal.

It was previously known that AOM and DSS induce colitis-related colorectal cancer. However, the deficiency or reduction of Gsdmd gene function indicating a phenotype associated with allergy is a novel effect that has not been known so far, which is brought about by the loss or reduction of Gsdmd gene function.

The model animal according to the present invention exhibits a phenotype associated with allergy. Therefore, it is very useful for elucidating the mechanism of allergies, searching for substances useful for the treatment thereof, and developing treatment methods.

One aspect of the present invention is a method of using a non-human animal in which the function of the Gsdmd gene is deficient or reduced as an allergic model animal in which an allergic disease is likely to develop. The allergic model animal can be used, for example, after being treated with an allergen causing an allergic disease.

≪Method for producing model animals≫
The method for producing a colorectal cancer model animal of the present invention includes administering a colorectal cancer-specific carcinogen and/or a colon inflammatory substance to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.
The method for producing an allergic model animal of the present invention comprises administering a colorectal cancer-specific carcinogen and/or a colon inflammatory substance to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.
Hereinafter, the method for producing a colorectal cancer model animal of the present invention and the method for producing an allergy model animal of the present invention will be described as a method for producing a model animal of the present invention.

The colorectal cancer-specific carcinogen and the colorectal inflammation substance may be used in combination. In one aspect, the method for producing a model animal of the present invention comprises administering a colorectal cancer-specific carcinogen and a colon inflammatory substance to a non-human animal in which the function of the Gsdmd gene is deficient or reduced. A preferable combination of a carcinogen specific to colon cancer and a colon inflammatory substance includes a combination of AOM and DSS. In one aspect, the method for producing a model animal of the present invention comprises administering azoxymethane (AOM) and dextran sulfate sodium (DSS) to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.

Examples of the non-human animal in which the function of the Gsdmd gene is deficient or reduced include the model animals of the present invention, and examples thereof include those listed in <<Model animals>> above. Although the model animal of the present invention can be produced by the present production method, the model animal of the present invention is not limited to that obtained by the present production method.

Hereinafter, one embodiment of the method for producing a model animal according to the present invention will be described.
First, a non-human animal in which the function of the Gsdmd gene is deleted or reduced is prepared.
Next, a colorectal cancer-specific carcinogen and/or a colon inflammatory substance is administered to the non-human animal. Examples of colorectal cancer-specific carcinogens and colorectal inflammation substances include those listed in <<Model Animal>> above.
The form of administration of these substances to animals may be appropriately selected from various methods, and examples thereof include subcutaneous injection, intraperitoneal injection, and oral administration by addition of feed. For example, in the case of a mouse, 5 to 20 mg/kg (body weight) of a colon cancer-specific carcinogen and/or a colon inflammatory substance is administered to a mouse in which the function of the Gsdmd gene is deficient or reduced, about 3 to 3 times a week. It may be administered for 15 weeks for breeding.
By the above procedure, a model animal showing a phenotype associated with colitis-associated colorectal cancer can be produced.
Further, by the above procedure, a model animal showing a phenotype related to allergy can be produced.

In one aspect, the method for producing an allergic model animal of the present invention includes administering or exposing an allergen causing an allergic disease to a non-human animal in which the function of the Gsdmd gene is deficient or reduced.

<<Screening method>>
(Colorectal cancer)
The screening method of the present invention administers a test substance to the colorectal cancer model animal of the present invention, and compares the colorectal cancer model animal to which the test substance has been administered with the test substance to which the test substance has not been administered. In the case where the colorectal cancer model animal to which the test substance is administered has a suppressed phenotype related to colorectal cancer, it is determined that the test substance is a substance effective in treating or preventing colorectal cancer. ..
Examples of the model animal of the present invention include those listed in <<Model Animal>> above.

Examples of the method for administering the test substance to the colorectal cancer model animal include subcutaneous injection, intraperitoneal injection, oral administration and the like.
In the screening method, the above comparison shall be performed under the same conditions. That is, the comparison shall be made with respect to model animals bred under the same conditions under which the effectiveness of the test substance can be judged except for the presence or absence of administration of the test substance.

Examples of the test substance include peptides, proteins, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts and the like.

A substance determined to be a substance effective for treating or preventing colorectal cancer can be an active ingredient of a therapeutic or preventive agent for colorectal cancer.
Alternatively, instead of the test substance, a test sample containing the test substance may be administered, and the test sample may be determined to be a sample effective for treating or preventing colorectal cancer.

The screening method of the present invention is performed using a phenotype associated with colorectal cancer as an index. Examples of phenotypes associated with colorectal cancer include those listed in <<Colorectal Cancer Model Animal>> above. Examples of cases in which the phenotype is suppressed include cases in which the number of tumor occurrences has decreased, cases in which the values of tumor markers have decreased, and the like.

(Allergy)
The screening method of the present invention, a test substance is administered to the allergy model animal of the present invention, the allergy model animal to which the test substance was administered, and the allergy model animal to which the test substance is not administered are compared, When the phenotype associated with allergy is suppressed in the allergic model animal to which is administered, it is determined that the test substance is a substance effective for treating or preventing allergy.
Examples of the model animal of the present invention include those listed in <<Model Animal>> above.

Examples of the method for administering the test substance to the allergic model animal include subcutaneous injection, intraperitoneal injection, oral administration and the like.
In the screening method, the above comparison shall be performed under the same conditions. That is, the comparison shall be made with respect to model animals bred under the same conditions under which the effectiveness of the test substance can be judged except for the presence or absence of administration of the test substance.

Examples of the test substance include peptides, proteins, low molecular weight compounds, synthetic compounds, fermentation products, cell extracts and the like.

A substance determined to be a substance effective in treating or preventing allergy can be an active ingredient of a therapeutic or preventive agent for allergy.
Alternatively, a test sample containing the test substance may be administered instead of the test substance, and the test sample may be determined to be a sample effective for treating or preventing allergy.

The screening method of the present invention is carried out using an allergy-related phenotype as an index. Examples of phenotypes associated with allergies include those listed in <<Allergic Model Animals>> above. The case where the phenotype is suppressed includes, for example, the case where the expression level of IL-4 is decreased.

<<Anticancer agent for colorectal cancer>>
The anticancer agent for colon cancer of the present invention contains any one or more of the following proteins (I) to (III) as an active ingredient.
(I) A protein having an amino acid sequence consisting of the 1st to 275th amino acids in the amino acid sequence represented by SEQ ID NO: 3 and having cytotoxic activity on colon cancer cells (II) represented by SEQ ID NO: 3 In the amino acid sequence consisting of the first to 275th amino acid sequences, it has an amino acid sequence in which one to several amino acids are deleted, substituted or added, and has cytotoxic activity on colon cancer cells. Protein (III) The amino acid sequence represented by SEQ ID NO: 3 has an amino acid sequence having 80% or more identity with the amino acid sequence consisting of the first amino acid to the 275th amino acid sequence, and has a cytotoxic activity on colon cancer cells. Protein with

Here, the number of amino acids that may be deleted, substituted, or added is preferably 1 to 30, preferably 1 to 20, preferably 1 to 10, and more preferably 1 to 7, 1 to 5 is more preferable, 1 to 3 is particularly preferable, and 1 to 2 is most preferable.
Here, the identity with the amino acid sequence is preferably 80% or more, more preferably 85% or more, further preferably 90% or more, particularly preferably 95% or more, most preferably 98% or more. The amino acid sequence identity can be determined by, for example, BLAST (reference URL: http://blast.ncbi.nlm.nih.gov/Blast.cgi). For example, parameters are score = 100 and wordlength = 12. When the amino acid sequence is analyzed by BLASTX based on BLAST, parameters are score=50 and wordlength=3, for example. When using BLAST and Gapped BLAST programs, the default parameters for each program are used.

Here, whether the protein has the cytotoxic activity of colorectal cancer cells is determined by comparing the colorectal cancer cells to which the protein has been administered or expressed with the colorectal cancer cells to which the protein has not been administered or expressed. Or, in the expressed colon cancer cells, when the number of colon cancer cells decreases or does not increase, it can be said that the administered protein has cytotoxic activity. For example, even when colon cancer cells die, it can be said that the administered protein has cytotoxic activity.

The proteins (I) to (III) may consist of the proteins (I) to (III) as long as they have a cytotoxic activity on colon cancer cells, and the proteins (I) to It may be the protein of (III) to which another substance is added. Examples of other substances include amino acids, polypeptides, proteins, sugars, sugar chains, nucleic acids, polynucleotides and the like.

The anticancer agent for colon cancer of the present invention contains, as an active ingredient, an expression vector containing a gene encoding any one or more proteins of (I) to (III) above. Examples of the expression vector include gene therapy vectors such as adenovirus vector, adeno-associated virus (AAV) vector, and lentivirus vector.

The anticancer agent may be composed of only one or more proteins of any one of the above (I) to (III), or may be formulated in combination with other components. For example, various forms such as tablets, powders, granules, capsules, liquids and the like can be mentioned.

In these formulations, pharmacologically or pharmaceutically acceptable carriers, specifically, sterile water and physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, Flavoring agents, fragrances, excipients, vehicles, preservatives, binders, diluents, isotonic agents, bulking agents, disintegrating agents, buffering agents, coating agents, lubricants, coloring agents, sweetening agents, viscous agents. It can be appropriately combined with an agent, a flavoring agent, a solubilizing agent, or other additives. Further, other components having the cytotoxic activity of colon cancer cells may be further blended.

Examples of the administration method for administering the anticancer agent of the present invention to colorectal cancer patients include local administration such as injection to colorectal cancer, oral administration, and intravenous injection. In addition, it may be administered in combination with other components having the cytotoxic activity of colon cancer cells.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

The involvement of Gsdmd in the development of CAC was evaluated in an enhanced model of CAC development using azoxymethane (AOM) and dextran sulfate sodium (DSS).
FIG. 1 is a schematic diagram schematically showing the procedure for producing a model animal in the examples. In this model, mice were repeatedly injected with AOM as a mutagen and then repeatedly treated with 2.5% DSS.
FIG. 2 is a graph showing the weight change rate of mice during the AOM-DSS treatment period. Data are expressed as mean±SD [WT, n=18; Gsdmd ^−/− , n=14, **p<0.01]. After the first round of DSS treatment, Gsdmd ^−/− mice showed a reduced rate of weight loss compared to wild type (WT) mice.
FIG. 3 is a photograph showing the distal colon of a mouse after 12 weeks of AOM-DSS treatment. Twelve weeks after AOM injection, the number of colon cancers was dramatically increased in Gsdmd ^−/− mice compared to WT mice. A large number of colorectal cancers that were visually recognizable were also confirmed.
4A and 4B are dot plots showing the number of tumors (Tumor No./mouse) and the tumor size (mm ² ) in WT mice and Gsdmd ^−/− mice after 12 weeks of AOM-DSS treatment. Is. Bars represent mean values [WT, n=18; Gsdmd ^−/− , n=14; WT tumor, n=94; Gsdmd ^−/− tumor, n=130]. The tumor incidence and tumor size values of Gsdmd ^−/− mice were significantly higher than those of WT mice.
FIG. 5 is an image of hematoxylin- and eosin-stained sections of colorectal adenocarcinoma developed in WT and Gsdmd ^−/− mice. Histopathological analysis shows that AOM-DSS treatment induced adenocarcinoma in both WT and GSDMD ^−/− mice.
From these results, it was shown that Gsdmd deficiency increases the incidence of colitis-related colon cancer. Gsdmd has been shown to play a distinct role in tumor development and tumor growth in CAC.

Due to the difference in tumor growth between WT and Gsdmd ^−/− mice, colorectal cancer tumors in the size range of 10 to 20 mm ² were selected in each mouse to study tumor cell proliferation and apoptosis. Examined.
6(a) and 6(b) are diagrams showing the results of BrdU uptake and cleared caspase-3 (Casp3) in CAC of WT and Gsdmd ^−/− mice. Data are expressed as mean±SD [n=3, **p<0.01]. No difference in the ratio of BrdU-uptake cells was observed between WT and Gsdmd ^−/− mouse tumors. On the other hand, the number of apoptotic cells identified by the detection of cleared caspase-3 was significantly reduced in Gsdmd ^−/− mice compared to WT mice. These results indicate that Gsdmd specifically promotes apoptosis but not cell proliferation in tumor development.

It has been reported that the activation of two major signal cascades, NF-κB signaling and Signal transducer and activator 3 (Stat3) signaling pathway, is important for CAC development. Therefore, it was examined by immunohistochemical analysis and immunoblot whether their signaling was affected in tumors of Gsdmd ^−/− mice.
FIG. 7(a) shows phospho-Stat1 (p-Stat1), phospho-Stat3 (p-Stat3) and phospho-Ikkβ (p-IKKβ) for colorectal adenocarcinoma obtained from WT and Gsdmd ^−/− mice. 3 is an image showing the results of immunohistochemical examination of. FIG. 7( b) is an image showing the results of immunoblotting against lysates of colorectal adenocarcinoma obtained from WT and Gsdmd ^−/− mice against the described proteins. Each tumor (T) is a tumor of the same size obtained from a separate individual.
No significant differences in NF-κB and Stat3 signaling were observed between WT and Gsdmd ^−/− mice. On the other hand, in line with the decrease in apoptotic cells, the amount of phosphorylated Stat1 was also decreased in the tumor of Gsdmd ^−/− mouse. Thus, AOM-DSS-treated Gsdmd ^−/− mice had reduced CAC apoptosis and downregulated STAT1 signaling. This indicates that Gsdmd regulates apoptosis and Stat1 signaling in tumor cells.

The increased incidence of tumors was speculated to be due to susceptibility to acute inflammation induced by DSS treatment. To test this possibility, mice were treated with 2.5% DSS for 7 days and then fed H ₂ O for 3 days to induce acute inflammation. FIG. 8 is a schematic diagram schematically showing the procedure of acute inflammation treatment in Examples.
FIG. 9 is a graph showing the rate of change in body weight of mice during the DSS treatment period. Data are expressed as mean±SD [WT, n=5; Gsdmd ^−/− , n=5, **p<0.01]. Indeed, DSS-treated Gsdmd ^−/− mice showed a significant reduction in body weight loss compared to WT mice.

FIG. 10(a) is an image showing the result of histological analysis after acute inflammation treatment, and FIG. 10(b) is a graph showing the histological score. [WT, n=5; Gsdmd ^−/− , n=5, **p<0.01]. FIG. 11 is a graph showing the intestinal length values of WT mice and Gsdmd ^−/− mice after acute inflammation treatment. FIG. 12 is an image showing the results of histological analysis of the intestine of WT mice and Gsdmd ^−/− mice after acute inflammation treatment. The extent of intestinal length contraction due to inflammation was reduced in Gsdmd ^−/− mice. Histological analysis revealed that Gsdmd ^−/− mice had a moderate degree of destruction of colon tissue due to acute inflammation as compared to WT mice, as expected.

13 and 14(a) and (b) are images showing the results of immunohistological analysis for cleaved-Caspase3 (Casp-3) and p-Stat1 in the inflamed site of the large intestine of WT and Gsdmd ^−/− mice. Is. Similar to CAC in AOM-DSS-treated mice, a decrease in apoptotic cells and Stat1 activity was confirmed in DSS-treated Gsdmd ^−/− mice. This result supports that the inflammatory response was weakened by the loss of Gsdmd. This suggests that the function of Gsdmd is associated with the action of Stat1 in the response to inflammation and controls apoptosis.

Microarray analysis was performed on naive colon to identify pathways or genes that underlie the reduction of inflammatory responses in Gsdmd ^−/− mice. It was revealed that the expression of 13 genes increased 2-fold or more and the expression of 15 genes decreased 3-fold or more.
Using the large intestine in which acute inflammation occurred and CAC, the gene whose expression was significantly suppressed in the untreated large intestine and the expression level of inflammatory cytokine were examined by using real-time quantitative PCR.
15 and 16 are graphs showing the results of real-time quantitative PCR for the genes shown in the figures. The value is a relative value to the expression level of the Rps18 gene. 15 and 16, (D0) represents the result of the untreated large intestine, and (D7+3) represents the result of the acutely inflammatory large intestine of the mouse which was treated with DSS for 7 days and then fed with H ₂ O for 3 days. , (AOMDSS) represents the result of colorectal adenocarcinoma by AOM and DSS treatment according to the procedure shown in FIG. 1 [*p<0.05, **p<0.01].
Reduced expression of the Ly6 gene in Gsdmd ^−/− mice was consistently detected in both acute inflamed colon and CAC. These expression data at least suggested that Gsdmd is involved in the gene regulation of the Ly6 gene. The Ly6 gene encodes a GPI-anchored cell surface glycoprotein, and the expression of the Ly6 gene is induced by direct binding of Stat1 to the Ly6 promoter in response to stimulation of interferon γ (IFNγ). (Flanagan, K. et al. Intestinal epithelial cell up-regulation of LY6 molecules during colitis results in enhanced chemokine secretion. J Immunol 180, 3874-81 (2008), and Khan, KD et al. Induction. of the Ly-6A/E gene by interferon alpha/beta and gamma requires a DNA element to which a tyrosine-phosphorylated 91-kDa protein binds. Proc Natl Acad Sci USA 90, 6806-10 (1993)). Ifng expression was increased in Gsdmd ^−/− untreated colon, whereas Ly6 expression was decreased.

From the above results, it was hypothesized that Gsdmd deficiency causes an unresponsiveness to IFNγ stimulation and causes a decrease in the expression of the IFNγ responsive gene. It is well known that IFNγ directly induces the expression of Casp-1 gene through the activation of Stat1. To test this hypothesis, we compared Casp-1 gene expression in WT and Gsdmd ^−/− mouse splenocytes with and without IFNγ treatment.
17(a) and 17(b) are graphs showing the results of real-time quantitative PCR for the Gsdmd gene and the Casp1 gene. The value is a relative value to the expression level of the Rps18 gene [**p<0.01]. In WT mice, IFNγ treatment dramatically increased the expression of Casp-1. On the other hand, in Gsdmd ^−/− mice, such increased expression of Casp-1 was not observed. These data indicated that Gsdmd regulates IFNγ-Stat1 signaling.

As shown in FIG. 16, in Gsdmd ^−/− mice, the expression of IL-25, IL-33, and IL-4, which are known to be associated with allergies, were higher than those of (D0), (D7+3), and (AOMDSS), respectively. In the condition, there was a significant increase compared to WT mice.

Next, the expression of GSDMD was examined in the sample of colorectal cancer obtained from the patient.
18(a) and 18(b) are graphs showing images showing the results of histological analysis of specimens of the normal region and cancer region of the large intestine of a colorectal cancer patient, and the scores of the IHC staining intensity thereof [** p<0.01]. Interestingly, the expression of GMDMD protein was almost suppressed in human CAC.
FIG. 19 is an image showing the result of histological analysis of normal colon epithelium of a mouse and colon epithelium of a CAC model mouse subjected to acute inflammation treatment. In mice, Gsdmd was also expressed at the tip of the colon epithelium. As in the case of human colon cancer specimens, a decrease in Gsdmd protein was also observed in CAC model mice. These data indicate that GSDMD expression is closely associated with CAC progression.

Gsdmd is cleaved by caspase 1 (Casp-1), and it is known that the N-terminal portion of the Gsdmd protein induces cell death and pyrotosis associated with inflammation in macrophages and the like (Non-patent Document 3,). 4). Although Gsdmd functions as a substrate for caspase 1 (Casp-1), the data presented here suggest that Gsdmd also functions as a regulator of Casp-1.

A protein consisting of the 1st to 275th amino acids in the amino acid sequence represented by SEQ ID NO: 3 which is the N-terminal side portion of the human GSDMD protein was named human GSDMD-N.
A DNA sequence encoding human GSDMD-N was inserted into a pcDNA vector, the resulting expression vector was introduced into a human colon cancer-derived cell line (Colo-320), and GSDMD-N was tagged with Myc-His. A peptide linked protein was expressed.

The protein, which is the C-terminal portion of the human GSDMD protein and consists of the 276th to 484th amino acid sequences in the amino acid sequence represented by SEQ ID NO: 3, was named human GSDMD-C.
A DNA sequence encoding human GSDMD-C was inserted into a pcDNA vector, the obtained expression vector was introduced into a cell line (Colo-320) derived from human colon carcinoma, and GSDMD-C was tagged with Myc-His. A peptide linked protein was expressed.

As a control, a DNA sequence encoding GFP (Green Fluorescent Protein) was inserted into a pcDNA vector, the obtained expression vector was introduced into a human colon carcinoma-derived cell line (Colo-320), and Myc was added to GFP. -A protein linked with the His tag peptide was expressed.

The results are shown in Fig. 20. In the GSDMD-N-introduced colo-320 cells, swollen and ruptured cells were observed. No effect of transfection was observed in the GSDMD-C-introduced colo-320 cells and the GFP-introduced colo-320 cells.
From these results, it was revealed that the introduction of the human GSDMD-N protein induces cell death.

Hereinafter, the experimental methods and details of various materials used in the above-mentioned examples will be described.

(mouse)
The Gsdmd knockout (Gsdmd ^−/− ) mouse was produced as described in a previous report (Non-patent Document 2) and was backcrossed to the background of the C57BL/6 strain. C57BL/6 mice were purchased from CLEA Japan and all mice used were 6-8 week old males. All animal experiments were approved by the Laboratory Animal Committee of Juntendo University Shizuoka Hospital.
The targeting vector was constructed in the pLoxFNFDT plasmid. The pLoxFNFDT plasmid contains a long arm of 8.9 Kb consisting of a pgk-Neo ^r cassette containing a putative promoter region and one loxP site at the 3'end of Exon 1-2, and a 1.4 kb consisting of Exon 3-4. Of short arm and Diphtheriatoxin-A fragment cassette. Here, for the purpose of disrupting the conditional or conventional Gsdmd gene, an additional loxP site was introduced upstream of 1.95 kb of Exon 1 of the above long arm. The targeting vector was linearized with I-SceI and then electroporated into TT2 embryonic stem (ES) cells from (B6×CBA/J)F1 mice. For homologous recombination, ES colonies resistant to G418 were selected by PCR, and positive clones were aggregated with 8-cell stage embryos and transferred to a female substitute. Female chimeric mice confirmed to have germline transmission were crossed with B6 and CAG-Cre transgenic mice. Knockout allyl heterozygotes (Gsdmd ^+/- ) were obtained and crossed to produce homozygotes (Gsdmd ^-/- ).

(Biopsy organization)
Human colorectal cancer biopsies were obtained from patients treated at Juntendo University Shizuoka Hospital. The experiment was approved by the ethics committee of Juntendo University Shizuoka Hospital.

(Induction of colon cancer)
Induction of colorectal cancer was performed based on the method previously reported (Neufert, C. et al. An inducible mouse model of colon carcinogenesis for the analysis of sporadic and inflammation-driven tumor progression. Nat Protoc 2, 1998-2004 (2007)). It was Mice were injected intraperitoneally with 10 mg of azoxymethane (AOM) per kg body weight. Then, the mice were bred with drinking water containing 2.5% dextran sulfate sodium (DSS) for 1 week, and then with normal water for 3 weeks. The DSS treatment cycle was performed twice. Collection of large intestine tissue and evaluation of tumors were performed 12 weeks after AOM injection. Tumors of the large intestine were observed and evaluated under a microscope.

(Induction of acute inflammation)
The mice were bred with drinking water containing 2.5% DSS for 1 week, then switched to normal water and bred. The colon tissue was collected from the mouse 3 days after switching to normal water.

(Histological experiment)
The colon tissue was fixed with a fixative containing a phosphatase inhibitor and 4% paraformaldehyde at 4° C. overnight before embedding in paraffin. The deparaffinized section (5 μm) was subjected to hematoxylin/eosin staining or immunohistochemical treatment. In the immunohistochemical treatment, the sections were treated with 0.01 M citrate buffer (pH 6.0) at 105° C. for 15 minutes and then left at room temperature for 2 hours.
The primary antibodies used are shown below:
Anti-Cleaved Caspase-3 (Asp175) (1:800, #9664, Cell signaling technology),
anti-Phospho-Stat1 (Ser727) (1:800, #8826, Cell signaling technology),
anti-Phospho-Stat3 (Tyr705) (1:600, #9145, Cell signaling technology),
anti-Phospho-IKK a/b (Ser176/180) (1:150, #2697, Cell signaling technology),
anti-GSDMD (1:50, #3F12-1B2, Abnova),
anti-GSDMD (1:50, orb37999, biorbyt)
Detection of these antibodies was visualized using an appropriate secondary antibody using a VECTATAIN Elite ABC kit (Vector Laboratories) and DAB (Sigma) as a substrate for peroxidase.

(Quantification of cell proliferation)
The mice were intraperitoneally injected with 100 mg of BrdU (Sigma) per kg of body weight 2 hours before. Deparaffinized sections were treated with 2N HCl for 30 minutes at 37° C. to detect BrdU incorporated cells. Then, the section was washed with PBS and then twice with 0.1 M Tris-HCl (pH 7.5). Further, the section was treated with 0.1% trypsin at 37° C. for 3 minutes and then washed. Immunological detection of BrdU incorporated cells was performed using the anti-BrdU antibody (Sigma) described above.

(Immunoblotting)
Tissues were placed in a low temperature osmotic lysis buffer (10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.05% NP-40) with protease inhibitors (Complete mini, Roche) and phosphatase. It was dissolved in a solution containing inibitors (PhosSTOP, Roche) and centrifuged at 3000 rpm for 10 minutes at 4°C. The obtained supernatant was used as a cytoplasmic fraction. The resulting pellet was resuspended in extraction buffer (5 mM HEPES, pH 7.4, 1.5 mM MgCl ₂ , 300 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, 26% Glycerol), homogenized, and homogenized on ice 30 After allowing to stand for 1 minute, centrifugation was performed at 4° C. and 15000 rpm for 20 minutes, and the obtained supernatant was used as a nuclear fraction. The protein concentration was determined according to the instructions attached to the Bio-Rad protein assay (Bio Rad). An equal amount of each protein sample was added to each lane, separated by SDS-PAGE gel, transferred to nitrocellulose, and immunoblotted.
The antibodies used are shown below:
anti-Phospho-Stat1 (Ser727) (1:800),
Stat1(1:1000, #9172, Cell signaling technology),
anti-Phospho-Stat3 (Tyr705) (1:2000),
anti-Phospho-NF-κB p65 (Ser536) (1:1000,#3033, Cell signaling technology),
anti-NF-κB p65 (1:1000,#8242, Cell signaling technology),
anti-Caspase-1 (1:1000, #06-503, Millipore),
anti-b-Actin (1:1000, #8242, Cell signaling technology),
anti-Caspase-1 (1:1000, #06-503, Millipore)
The protein band was visualized by chemiluminescence using an ECL prime Western Blotting Detection System (GE Helthcare Japan).

(Scoring of colonic epithelial inflammation by morphological criteria)
The following four morphological characteristics were blindly subjected to morphological scoring using the total score. Inflammation (score 0: normal, score 1: inflammatory cells localized in lamina propria, score 2: inflammatory cells extending to submucosa, score 3: inflammatory cells with aggregation and expansion of submucosa), morphology ( Score 0: normal, score 1: goblet cell decrease, score 2: intestinal crypt dispersal, score 3: intestinal crypt disappearance), ulceration (score 0: absent, score 1: present), Lymphoid follicles (score 0: not present, score 1: present). For each morphological feature, the sum of the weighted scores was calculated by summing the field percentages (0%:0, 0.1-25%:1, 26-50%:2, 51-75%: 3, 76-100%: 4). The score range is 0-40.

(Scoring of colonic epithelial inflammation by morphological criteria)
Staining intensity and field ratios were blindly scored using the overall score. Strong intensity staining was scored 3, medium staining was scored 2, weak staining was scored 1 and negative staining was scored 0.
For each staining intensity, the sum of the weighted scores was calculated by summing the percentage of fields (0%:0, 0.1-25%:1, 26-50%:2, 51-75%:3, 76-100%: 4). The score range is 0-16.

(Gene expression analysis)
For microarray analysis, total RNA was purified from RNAlater (Qiagen)-treated colon tissue using RNAeasy mini kit (Qiagen). As the array, Affimetrix Mouse Genome 430 2.0 array was used. The amplification product of RNA was synthesized according to the protocol attached to the product. The data was analyzed by GeneSpring GX (Agilent). For Real-time PCR analysis, cDNA was synthesized from 500 ng of total RNA that had been Dnase-treated with PrimeScript II (TaKaRa). Real-time quantitative PCR was performed using TaKaRa Thermal Cycler Dice (TaKaTa) and SYBR premix Ex Taq II (TaKaRa).

(Isolation of intestinal epithelial cells and lymphocytes from the large intestine)
Intestinal epithelial cells (IECs) and lymphocytes have been reported previously (Weigmann, B. et al. Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue. Nat Protoc 2, 2307-11 (2007)). Isolated as described in. The large intestine was opened in the long axis direction and washed with cold PBS several times to remove adipose tissue and debris. The large intestine was cut into 5 mm pieces and incubated at 37° C. for 15 minutes in HBSS (with 5% FBS, 2 mM EDTA, 1 mM DTT, 10 mM HEPES added) to separate epithelial cells. After vortexing, tissue pieces containing medium were passed through a 100 μm cell strainer. The passed fraction was centrifuged to collect intestinal epithelial cells (IECs). The tissue piece remaining on the cell strainer was further incubated with PBS (5% FBS, 1.5 mg/ml Collagenase Type VIII, 0.1 mg/ml DnaseI added) at 37° C. for 45 minutes. After vortexing, the cells were filtered with a cell strainer, and the lamina propria mononuclear cells (LPMCs) were concentrated by density gradient centrifugation using Percoll (GE healthcare).

Each configuration and the combination thereof in each embodiment described above are examples, and addition, omission, replacement, and other changes of the configuration can be made without departing from the spirit of the present invention. The present invention is not limited to each embodiment, but is limited only by the scope of the claims.

Claims (6)

Gasdermin D (Gsdmd) mice der function is deficient or reduced gene is, including colon cancer-specific carcinogens and colon pathogenic substances in the body, colon cancer model animal. The colorectal cancer model animal according to claim 1, which has a mutation in the expression control region of the Gsdmd gene or the Gsdmd gene. The colon cancer, colitis associated colorectal cancer (Coltis-associated colon cancer) and colon cancer model animal according to claim 1 or 2. A method for producing a colorectal cancer model animal, which comprises administering a colorectal cancer-specific carcinogen and a colon inflammatory substance to a mouse in which the function of the Gsdmd gene is deficient or reduced. The method for producing a colorectal cancer model animal according to claim 4 , wherein the colorectal cancer is a colitis-related colorectal cancer (Coltis-associated colon cancer). Administering a test substance to the colon cancer model animal according to any one of claims 1 to 3, the said colon cancer animal model test substance has been administered, and the colon cancer animal model test substance is not administered If the phenotype associated with colon cancer is suppressed in the colorectal cancer model animal to which the test substance is administered, it is determined that the test substance is a substance effective for treating or preventing colorectal cancer. A screening method comprising: