Mouse model for colitis-related intestinal cancer and construction method thereof

Abstract

The invention discloses a mouse model of intestinal cancer related to colitis and a construction method thereof, and belongs to the technical field of disease animal model construction. The construction method comprises the following steps: inserting loxP sites in the upstream of a No.2 exon and the downstream of a No.4 exon of a mouse GPX4 gene respectively to construct a parent mouse 1; hybridizing the parent mouse 1 and the parent mouse 2, and screening to obtain a filial generation mouse containing the GPX4 gene and the Cre gene; azomethane induction medicine and dextran sodium sulfate are adopted to jointly induce generation of intestinal cancer related to colitis of the filial generation rats; and monitoring the morbidity condition of the mouse colitis-related intestinal cancer to complete the construction of the mouse model. The finally obtained colitis-related intestinal cancer model has the characteristics that the body weight is reduced, the DAI score is higher, the intestinal permeability is increased more obviously, the intestinal tumor load is more, the mouse lifetime is shorter, and the reduction of the intestinal GPX4 of the mouse is similar to the intestinal expression of human colitis-related intestinal cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease animal model construction, and specifically relates to a colitis-related intestinal cancer mouse model and a construction method thereof. Background Art

[0002] Colitis-associated colorectal cancer (CAC) is a subtype of colorectal cancer closely related to inflammatory bowel disease (IBD). IBD primarily encompasses ulcerative colitis (UC) and Crohn's disease (CD). The incidence of IBD is increasing globally, and patients with long-term IBD have a significantly increased risk of developing CAC. Compared with sporadic colorectal cancer, CAC exhibits several unique clinicopathological features. For example, it typically presents with a more extensive lesion range, with multicentric origins being common. Histologically, it often presents as a mucinous adenocarcinoma or undifferentiated carcinoma, and its prognosis is relatively poor. This increasing disease burden poses a significant public health challenge, prompting researchers to intensify their research on CAC. Furthermore, due to the unique pathogenesis and clinical features of CAC, the efficacy of traditional colorectal cancer treatments, such as surgery, chemotherapy, and radiotherapy, may be limited in CAC. Furthermore, changes in the immune status and intestinal microecological environment of patients with inflammatory bowel disease also increase the complexity and difficulty of treatment. Therefore, in-depth research on the biological characteristics of colitis-related colorectal cancer is needed to explore new therapeutic targets and strategies to help improve the diagnosis and treatment of colitis-related colorectal cancer.

[0003] In biomedical research, animal models of human disease refer to experimentally established animal subjects and related materials that mimic the characteristics and manifestations of human diseases. These models provide important experimental platforms for studying the pathogenesis and progression of diseases, as well as for developing treatments. Currently, the most commonly used animal models of colitis-related colorectal cancer include chemically induced models, genetically engineered models, and cell transplantation models. Among these, the TNBS-induced model involves instilling a mixed solution of TNBS (trinitrobenzenesulfonic acid) and ethanol into the rectum of mice to induce colitis, which in turn triggers colorectal cancer. Mice are typically given a low dose initially, followed by repeated administration at varying time points, to induce chronic inflammation and tumorigenesis. TNBS-induced colitis is primarily characterized by a Th1 immune response, and the resulting tumors are often colon adenocarcinomas, similar to the immunopathological features of human Crohn's disease. However, this model is relatively complex to operate, and TNBS is known to be toxic, requiring high experimental staff expertise. Genetically engineered mice, ApcMin / + mice, are transgenic mice that carry a mutation in the Apc gene. The Apc gene is an important tumor suppressor gene. Its mutation causes abnormal proliferation of intestinal epithelial cells, forming multiple intestinal polyps. Over time, some polyps can develop into intestinal cancer. This model can spontaneously form intestinal tumors without the need for chemical induction. It is closer to the pathogenesis of human sporadic colorectal cancer and different from the pathogenesis of colitis-associated intestinal cancer. In addition, mouse colon cancer cell line CT26 cells are inoculated into immunodeficient mice (such as nude mice or SCID mice), and inflammation can be induced in the mice before or at the same time as inoculation, such as by giving DSS drinking water or rectal instillation of inflammatory inducers, and studying the interaction between tumor cells and the inflammatory microenvironment. However, since tumor cell lines are used, the initial stage of tumor development is different from that of real colitis-associated intestinal cancer, and it may not fully reflect the complex pathogenesis of colitis-associated intestinal cancer. The AOM+DSS-induced model can effectively simulate the development and progression of human colitis-associated colorectal cancer, including the onset of inflammation, disruption of the intestinal mucosal barrier, and tumor formation. It is relatively simple to operate and has good reproducibility, making it a commonly used animal model for studying colitis-associated colorectal cancer. However, the AOM+DSS model has random gene mutations that differ from those found in patients with colitis-associated colorectal cancer, requiring further optimization. Considering the pathological changes in patients, existing animal models, while inducing the onset of colitis-associated colorectal cancer through different mechanisms and with varying characteristics and symptoms, still cannot meet the needs of research on the mechanisms of inflammatory-cancer transformation. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a mouse model of colitis-related intestinal cancer and a method for constructing the same. The colitis-related intestinal cancer model finally obtained by the present invention can show a more reduced weight, a higher DAI score, a more obvious increase in intestinal permeability, a greater intestinal tumor load, a shorter survival period of mice, and the decrease in intestinal GPX4 in the mouse is similar to the intestinal expression characteristics of human colitis-related intestinal cancer.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides a method for constructing a mouse model of colitis-associated intestinal cancer, comprising the following steps:

[0007] S1. Insert loxP sites upstream of exon 2 and downstream of exon 4 of the mouse GPX4 gene to construct parental mouse 1. Simultaneously, select a mouse harboring intestinal epithelial cell-specific Cre gene as parental mouse 2.

[0008] S2. Cross parent mouse 1 with parent mouse 2 and screen for offspring containing the GPX4 gene and the Cre gene. fl / wt VilCre + ;

[0009] S3. Induction of GPX4 in offspring mice using azomethane-induced drugs and dextran sulfate sodium fl / wt VilCre + The development of colitis-related colorectal cancer;

[0010] S4. Monitor the incidence of colitis-related intestinal cancer in mice and complete the construction of the mouse model.

[0011] Preferably, the genetic background of the parent mice 1 and 2 in S1 is C57 / B6. The genetic background of the two parent mice of the present invention is C57 / B6. After hybridization and backcrossing, the GPX4 fl / wt VilCre + Mice developed more severe inflammatory and cancerous phenotypes after AOM+DSS induction.

[0012] Preferably, the methods used to construct the parent mouse 1 in S1 include but are not limited to CRISPR / Cas9 technology, NICER technology, and CHyMErA technology.

[0013] Preferably, the specific method for constructing the parent mouse 1 is:

[0014] The Gpx4-sgRNA DNA fragment was amplified by PCR using the sgRNA-Vector as a template. After gel recovery, it was used as a template for sgRNA in vitro transcription and purified.

[0015] Based on the selected sgRNA, a targeting vector is designed, containing homology arms and loxp sequences. After construction, it is enzymatically digested and purified, and then microinjected into C57 mouse embryos together with sgRNA and Cas9-mRNA. The injected embryos are then transplanted into the oviducts of surrogate recipient mice.

[0016] The present invention discovered that ferroptosis is a type of programmed cell death closely related to lipid peroxidation, and a growing number of studies have found that ferroptosis is involved in the pathogenesis of diseases related to intestinal involvement. Given the characteristics of intestinal epithelium during inflammatory-cancer transformation, such as cell damage, abnormal proliferation, and microenvironmental changes, and the key role that ferroptosis plays in regulating cell death, reshaping the inflammatory microenvironment, and influencing tumor development, it is reasonable and feasible to introduce the ferroptosis mechanism into animal models for research related to inflammatory-cancer transformation.

[0017] GPX4 encodes a member of the glutathione peroxidase family, whose function is to reduce hydroperoxides in membrane lipids and lipoproteins, thereby protecting mitochondrial function and inhibiting ferroptosis. Heterozygous and homozygous loxp mice are viable and fertile, and when bred with Cre recombinase mice, the offspring can produce inducible GPX4 gene knockout, with homozygous knockout being lethal and heterozygous knockdown being viable.

[0018] Preferably, the parent mouse 2 in S1 is a VilCre mouse, which has a nuclear-localized Cre recombinase on its Villin gene.

[0019] As an example, the induced progeny mouse GPX4 in S3 fl / wt VilCre + The specific steps of colitis-related colorectal cancer are:

[0020] Azomethane-induced drugs were introduced into the GPX4 offspring mice at a dose of 10 mg / kg. fl / wt VilCre + The offspring mice were then given water containing 2-3% dextran sulfate sodium for 5-7 consecutive days, after which they were switched to normal drinking water for two weeks, and this was repeated for three cycles.

[0021] As an example, the induced progeny mouse GPX4 fl / wt VilCre + Colitis-associated intestinal cancer develops when mice reach sexual maturity at eight weeks of age.

[0022] Descendant Mouse GPX4 fl / wt "fl" indicates that loxP sites are inserted on both sides of exons 2 and 4 of one allele (floxed), "wt" indicates that the other allele is unmodified, and "VilCre +” indicates carrying Villin-driven Cre recombinase.

[0023] Preferably, the items for monitoring colitis-related intestinal cancer in mice include: weight changes, blood in stool and stool characteristics, DAI score, inflammatory factors, intestinal permeability, colon damage, tumor load, and mouse survival time.

[0024] Preferably, the detection time is 1-3 months after administration. The present invention begins to show phenotypes 3 months after induction, including weight loss, increased DAI score, increased intestinal permeability, increased intestinal tumor load, and shortened mouse survival, which is consistent with the characteristics of human disease onset and more consistent with the characteristic of low GPX4 expression in the intestine of patients with inflammation and cancer.

[0025] In the second aspect, a mouse model of colitis-related intestinal cancer is constructed by the above-mentioned construction method.

[0026] Contains at least the following beneficial technical effects:

[0027] The present invention provides a method for constructing an animal model of colitis-related intestinal cancer. The method comprises first obtaining a mouse GPX4 containing a heterozygous GPX4 gene and a Cre gene. fl / wt VilCre + , and then use inflammation-cancer inducing drugs to further induce the disease in mice. Given that the present invention is a knockdown of specific genes in specific cells, the cause of the model's onset is clear, and it is caused by abnormal intestinal barrier system, increased intestinal damage, and increased tumor load, which ultimately lead to the onset of inflammation and cancer. At the same time, the colitis-related intestinal cancer model finally obtained by the present invention can show a greater weight loss, a higher DAI score, a more obvious increase in intestinal permeability, a greater intestinal tumor load, a shorter survival period of mice, and the decrease in intestinal GPX4 in the mice is similar to the intestinal expression characteristics of human colitis-related intestinal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Figure 3 is a comparison of GPX4 immunohistochemical staining of intestinal epithelial cells in healthy controls and patients; Figure A is the immunohistochemical staining of GPX4 in intestinal epithelial cells of healthy controls (HC) and patients with ulcerative colitis (UC); Figure B is the immunohistochemical staining of GPX4 in intestinal epithelial cells of healthy controls (HC) and patients with ulcerative colitis (UC), patients with inflammatory-cancer transformation (CAC), and common colon cancer (CRC).

[0029] Figure 2 GPX4 fl / wt VilCre +Schematic diagram of the construction steps; Figure A is a schematic diagram of gene targeting and conditional knockout of the Gpx4 gene intestinal epithelium conditional knockout mouse; Figure B is a hybridization strategy, which ultimately obtained the Gpx4 gene intestinal epithelium conditional knockdown mouse, namely GPX4 fl / wt VilCre + mouse.

[0030] Figure 3 Figure 1 is a phenotypic identification diagram of mice with conditional knockdown of intestinal epithelial cells; Figure A is GPX4 fl / wt VilCre + Western Blot images of intestinal epithelial cells of mice and control mice, Figure B is GPX4 fl / wt VilCre + In vivo imaging of mice and control mice after injection of L-012; Figure C is GPX4 fl / wt VilCre + Ultrasound images of the intestines of mice and control mice; Figure D is GPX4 fl / wt VilCre + HE staining of the intestine of mice and control mice;

[0031] Figure 4 For GPX4 fl / wt VilCre + Figure 3. Detection of AOM+DSS-induced inflammatory cancer model in control mice and control mice; Figure A is a representative picture of intestinal tumors, Figure B shows the changes in mouse survival, Figure C shows the weight of mice, and Figure D shows the number of left intestinal tumors; "*" means P < 0.05, "**" means P < 0.01, and "***" means P < 0.001. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] Unless otherwise specified, the "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C.

[0038] Example 1

[0039] Identification of GPX4 protein expression in intestinal epithelial cells of patients with inflammation and cancer

[0040] Patient specimens: This study was conducted in the Department of Gastroenterology at Beijing Friendship Hospital. Patients with intestinal polyps were selected as research subjects based on the principle of age and gender matching. Individuals without autoimmune diseases, inflammatory diseases, infectious diseases, and other underlying diseases served as healthy controls. The required clinical specimens were intestinal tissue wax blocks obtained from patients (UC patients, CAC patients, CRC patients) and controls during previous colonoscopy examinations at our hospital. The diagnosis of patients was based on the "Consensus on the Diagnosis and Treatment of Inflammatory Bowel Disease (2018, Beijing)" formulated by the Inflammatory Bowel Disease Group of the Gastroenterology Branch of the Chinese Medical Association, the "Guidelines for the Diagnosis and Treatment of Ulcerative Colitis in China (2023·Xi'an)", and the "Chinese Colorectal Cancer Diagnosis and Treatment Guidelines (2020 Edition)", issued by the National Health Commission; the above specimens were all collected from the archived data of the Pathology Department of our hospital. The clinical specimen collection plan involved in this study has been approved by the Ethics Committee of our hospital and was carried out in strict accordance with medical ethics standards.

[0041] The intestinal epithelial biopsy specimens were immediately fixed with formalin, embedded in paraffin, and subjected to immunohistochemical staining (see Figure 1A). The protocol was to incubate the anti-GPX4 antibody at 4°C overnight, then incubate with anti-rabbit IgG-HRP at room temperature for 1 hour. The immunohistochemical staining results were semi-quantitatively scored. Statistical scores indicated that the GPX4 content in the intestinal epithelium of UC patients was significantly lower than that of healthy controls ( Figure 1 A, B); In addition, the GPX4 content in the intestinal epithelium of patients with inflammatory cancer (CAC) was significantly lower than that of healthy controls, but the GPX4 content in the intestinal epithelium of patients with common colon cancer (CRC) was significantly increased ( Figure 1 B) This immunohistochemistry analysis showed that GPX4 levels were significantly reduced in the intestinal epithelium of patients with colitis-associated colorectal cancer.

[0042] Example 2

[0043] Experimental protocol for creating mice with GPX4 gene knockdown in intestinal epithelial cells

[0044] 1. Genetic information:

[0045] The GPX4 gene is located on mouse chromosome 10. The GPX4 gene encodes two main splice variants: GPX4-201 and GPX4-202. Both splice variants have seven exons, differing only in exon 1.

[0046] 2. Production principle:

[0047] The Cas9 / gRNA system was used to knock in two loxP elements upstream of exon 2 and downstream of exon 4 of the Gpx4 gene, i.e., two loxPs were used to anchor exons 2 to 4 of the Gpx4 gene. When these mice were hybridized with mice expressing Cre in tissues, exons 2 to 4 of the Gpx4 gene could be specifically deleted in tissues and organs expressing the Cre enzyme. These three exons are 392bp in length, which is not an integer multiple of 3. After deletion, both spliceosomes of the Gpx4 gene will undergo frameshift mutations, leading to Gpx4 gene knockout, thus obtaining mice with conditional Gpx4 gene knockout.

[0048] Gpx4 gene conditional knockout mouse gene targeting, and conditional knockout diagram as shown Figure 2 As shown in A.

[0049] 3. gRNA design, in vitro transcription and purification:

[0050] Select an sgRNA with a low off-target probability. Design two to three gRNAs near the pre-loxp target site through sequence alignment. After measuring the cleavage efficiency, select the gRNA with the highest cleavage activity for use in subsequent experiments. PCR amplify the Gpx4-sgRNA DNA fragment using the sgRNA-vector as a template, then recover the fragment on gel and use it as a template for in vitro transcription of the sgRNA. After in vitro transcription and purification of the sgRNA, aliquot and store in a -80°C freezer until ready for use.

[0051] 4. Design and construction of targeting vector:

[0052] Based on the selected sgRNA, a targeting vector is designed, containing homology arms and loxP sequences. After construction, the vector is digested and purified, and then microinjected into fertilized eggs along with the sgRNA and Cas9-mRNA.

[0053] 5. Microinjection:

[0054] The purified sgRNA and Cas9-mRNA were co-injected into C57 mouse embryos, and then the embryos were transplanted into the oviducts of surrogate recipient mice.

[0055] 6. Mouse identification:

[0056] Mice were born 21 days after embryo transfer, and genotype identification (mouse tail DNA identification) was completed about 2 weeks after birth, and the GPX4 mutant type was identified.

[0057] 7. Intestinal epithelial cell Cre:

[0058] This experiment used healthy 8-week-old VilCre mice purchased from Cytogenes. The Villin gene (Vil) in these mice has a nuclear-localized Cre recombinase. These VilCre mice can be used for Cre-lox studies of intestinal epithelial cells.

[0059] 8. The GPX4 mutant parent mouse 1 was hybridized with the intestinal epithelial cell-specific Cre mouse as the parent mouse 2, and the offspring mice containing the GPX4 gene and the Cre gene were screened to obtain GPX4fl / wt VilCre +

[0060] Hybridization strategies such as Figure 2 As shown in B.

[0061] Example 3

[0062] Phenotypic characterization of intestinal epithelial cell conditional knockdown mice

[0063] 1. Drug administration mutagenesis:

[0064] GPX4 in control mice without GPX4 knockdown fl / fl(purchased from Cytokine) and mice with conditional knockdown of GPX4 in intestinal epithelial cells fl / wt VilCre + The mice were treated with the inflammation-inducing drug AOM + DSS. Eight-week-old mice were first given a single intraperitoneal injection of AOM (10 mg / kg body weight). One week later, the mice were given a drinking water containing 2% DSS for one week, followed by regular drinking water for two weeks. This cycle lasted for a total of three cycles.

[0065] 2.Western blot:

[0066] At 3 months after AOM+DSS induction, the treated mice were sampled and intestinal epithelial cells were obtained from GPX4 fl / wt VilCre + and control mice (WT, GPX4 fl / fl ), and then western blot was performed after lysis, and GPX4 antibody was used for incubation. fl / wt VilCre + GPX4 expression in mouse intestinal epithelium was significantly decreased (see Figure 3 A), indicating that the model meets the design requirements.

[0067] 3. In vivo imaging:

[0068] At 3 months of AOM+DSS induction, mice were intraperitoneally injected with the chemiluminescent probe L-012 sodium to measure intestinal permeability and inflammation. L-012 sodium salt is a luminol-based chemiluminescent (CL) probe that is widely used in vivo to detect superoxide anions (O2·-) produced by NADPH oxidase (Nox). Mice were anesthetized with 2.0% isoflurane and then intraperitoneally injected with 25 mg / kg of L-012 solution. Subsequently, the mice were placed in an IVIS Spectrum CT bioluminescence imaging system (PerkinElmer, Waltham, MA, USA), and images were acquired using the automatic exposure option to optimize signal intensity. A region of interest was delineated on the abdomen of the mouse to measure the average radiant brightness of the chemiluminescence (p / s / cm 2 Semi-quantitative analysis was performed using the Living Image software.

[0069] GPX4 fl / wt VilCre + and control mice (GPX4 fl / fl WT) after AOM+DSS induction. Figure 3 As shown in B. Figure 3 B can be seen that GPX4 fl / wt VilCre +Intestinal permeability in mice was significantly increased after inflammation and cancer induction.

[0070] 4. Ultrasound imaging:

[0071] The experiment was conducted 3 months after AOM+DSS induction. Mice were fasted for 12 hours before the experiment, but water was not allowed to empty the intestinal contents and reduce intestinal gas interference; the abdominal hair of the mice was carefully removed using a shaver or depilatory cream, and the mice were fixed supinely on a heating pad on the ultrasound imaging platform. The mice were anesthetized with 2.0% isoflurane. An appropriate amount of ultrasound coupling agent was evenly applied to the abdomen of the mouse, and the ultrasound probe was gently placed on the abdomen of the mouse. Starting from the xiphoid process, the probe was slowly moved along the midline of the abdomen to gradually scan the entire intestinal area. For suspected tumor sites, the tumor growth was carefully observed, and the collected images and dynamic images were analyzed using the analysis software supporting the ultrasound imaging system.

[0072] GPX4 fl / wt VilCre + and control mice (GPX4 fl / fl WT) intestinal imaging after AOM+DSS induction Figure 3 As shown in C. Figure 3 C can be seen that GPX4 fl / wt VilCre + The increase in intestinal tumors in mice was more obvious after inflammation-induced cancer.

[0073] 5. HE staining:

[0074] Gpx4 was collected after inflammation and cancer stimulation fl / wt VilCre + mice and control mice (WT, GPX4 fl / fl ) intestinal tissue was fixed with formalin, and HE staining was used to detect intestinal damage and tumor growth in mice.

[0075] The results show that Gpx4 fl / wt VilCre + Mice had obvious tumor formation, more lymphatic infiltration (see Figure 3 D).

[0076] Example 4

[0077] Mouse tumor burden detection

[0078] 1. Mouse weight:

[0079] The initial body weight of the mice was weighed before drug induction, and the body weight of the mice was measured every other day after drug administration (3 times a week) and recorded; the changes in the body weight of the mice were statistically analyzed in % and a line graph was drawn.

[0080] 2. DAI scoring criteria are as follows

[0081]

[0082] 3. Counting Mouse Intestinal Tumors

[0083] The mouse colon was removed, the connective tissue was removed, and photographs were taken; the colon was then longitudinally dissected, the tumors were removed, and the tumors were counted and statistically analyzed.

[0084] 4. Mouse survival

[0085] The number of mice in each group was counted at the beginning. The death of mice during the whole experiment was recorded. The number of mice was also recorded on the last day of the experiment, and the survival period was finally counted.

[0086] In summary, see Figure 4 GPX4 fl / wt VilCre + Figure 4: Detection of AOM+DSS-induced inflammation and cancer model in control mice; Figure A is a representative image of intestinal tumors, Figure B shows the changes in mouse survival, Figure C shows the weight of mice, and Figure D shows the number of left intestinal tumors; "*" means P < 0.05, "**" means P < 0.01, and "***" means P < 0.001. fl / wt VilCre + Mice exhibited more pronounced intestinal inflammation and tumor burden after AOM+DSS induction. fl / wt VilCre + The mouse model of inflammation and cancer has a shorter survival period; at the same time, the decreased GPX4 expression in the intestine of these mice is similar to the intestinal expression characteristics of human colitis-associated intestinal cancer. Therefore, the mouse model obtained by this invention can be used as a new model for studying the pathogenesis of intestinal inflammation and cancer.

[0087] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for constructing a mouse model of colitis-associated intestinal cancer, characterized in that: The following steps are involved: S1. Insert loxP sites upstream of exon 2 and downstream of exon 4 of the mouse GPX4 gene to construct parental mouse 1. Simultaneously, select a mouse harboring intestinal epithelial cell-specific Cre gene as parental mouse 2. S2. Cross parent mouse 1 with parent mouse 2 and screen for offspring containing the GPX4 gene and the Cre gene. fl / wt VilCre + ; S3. Induction of GPX4 in offspring mice using azomethane-induced drugs and dextran sulfate sodium fl / wt VilCre + The development of colitis-related colorectal cancer; S4. Monitor the incidence of colitis-related intestinal cancer in mice and complete the construction of the mouse model.

2. The construction method according to claim 1, characterized in that The genetic backgrounds of parent mice 1 and 2 in S1 are both C57 / B6.

3. The construction method according to claim 1, characterized in that The methods used to construct the parent mouse 1 in S1 include but are not limited to CRISPR / Cas9 technology, NICER technology, and CHyMErA technology.

4. The construction method according to claim 3, characterized in that The specific method for constructing the parent mouse 1 is: The Gpx4-sgRNA DNA fragment was amplified by PCR using the sgRNA-Vector as a template. After gel recovery, it was used as a template for sgRNA in vitro transcription and purified. Based on the selected sgRNA, a targeting vector is designed, containing homology arms and loxp sequences. After construction, it is enzymatically digested and purified, and then microinjected into C57 mouse embryos together with sgRNA and Cas9-mRNA. The injected embryos are then transplanted into the oviducts of surrogate recipient mice.

5. The construction method according to claim 1, characterized in that The parent mouse 2 in S1 is specifically a VilCre mouse, which has a nuclear-localized Cre recombinase on its Villin gene.

6. The construction method according to claim 1, characterized in that The S3 induces GPX4 in progeny mice fl / wt VilCre + The specific steps of colitis-related colorectal cancer are: Azomethane-induced drugs were introduced into the GPX4 offspring mice at a dose of 10 mg / kg. fl / wt VilCre + The offspring mice were then given water containing 2-3% dextran sulfate sodium for 5-7 consecutive days, after which they were switched to normal drinking water for two weeks, and this was repeated for three cycles.

7. The construction method according to claim 6, characterized in that: The induced offspring mouse GPX4 fl / wt VilCre + Colitis-associated intestinal cancer develops when mice reach sexual maturity at eight weeks of age.

8. The construction method according to claim 1, wherein: The items for monitoring colitis-related intestinal cancer in mice include: weight changes, blood in stool and stool characteristics, DAI score, inflammatory factors, intestinal permeability, colon damage, tumor load, and mouse survival time.

9. The construction method according to claim 8, characterized in that: The detection time is 1-3 months after administration.

10. A mouse model of colitis-associated intestinal cancer, characterized in that: Constructed by the construction method according to any one of claims 1 to 9.

Images