Construction method and application of grimm19-il-33 double gene gastric parietal cell-specific knockout mouse model

Abstract

The application belongs to the technical field of animal models, and particularly relates to a construction method and application of a GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model. The construction method comprises the following steps: respectively modifying mouse GRIM-19 and IL-33 genes by flox, then crossing the GRIM-19flox / flox mouse with the IL-33flox / flox mouse to obtain F1 generation mice; crossing the F1 generation mice with Atp4b-Cre mice to obtain F2 generation mice; crossing the F2 generation mice with the F1 generation mice to obtain a GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model. The model is helpful for exploring the regulation mechanism of the GRIM-19 and IL-33 genes in SPEM occurrence and progression, and is of great significance for the treatment of SPEM and the prevention and treatment of gastric cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal models, and particularly relates to a construction method and application of a GRIM-19 and IL-33 double-gene gastric parietal cell-specific knockout mouse model. BACKGROUND

[0002] Gastric cancer is the fifth most common cancer worldwide and the third most common cause of cancer death, but the details of gastric cancer development are still unclear. In recent years, the cure rate of early gastric cancer can reach more than 90%, but due to the atypical symptoms of early gastric cancer, the diagnosis rate is low, and after progression, it greatly affects the treatment and survival of gastric cancer patients, and the recovery rate of patients with advanced gastric cancer is extremely low. Therefore, finding molecular markers for early diagnosis and targeted therapy is crucial for improving the prognosis of gastric cancer.

[0003] Two precancerous metaplasias are associated with gastric precancerous lesions: intestinal metaplasia (IM) and spasmolytic polypeptide expressing metaplasia (SPEM). SPEM is considered to be a potential cell source of intestinal epithelial metaplasia and eventually adenocarcinoma, and persistent stimulation of chronic inflammation can promote SPEM to progress to dysplasia and even cancer. Many mouse models have led to the evolution of SPEM. At present, the mechanism of SPEM has not been fully elucidated, and the research progress is relatively slow.

[0004] Mitochondrial inner membrane protein GRIM-19 (Gene associated with retinoid interferon-induced mortality-19, also known as NDUFA13), as a functional component of MRC complex I of mitochondrial respiratory chain, the gene of human GRIM-19 is located in chromosome 19p13.1, and the human and mouse GRIM-19 genes both contain 5 exons. Compared with benign tumors, the expression level of GRIM-19 protein in advanced tumors is extremely low, and the deletion of GRIM-19 increases the susceptibility to tumor development. Gastric parietal cell-specific knockout of GRIM-19 can induce spontaneous gastric mucosal inflammation and SPEM pathological formation in mice.

[0005] Interleukin-33 (IL-33) is a member of the IL-1 cytokine family and plays an important regulatory role in the immune system. As an immune system regulator, the full-length protein form of IL-33 (full-length IL-33 protein, flIL-33) acts as a gene regulator in the nucleus, while the mature form (mature IL-33, mIL-33) is used as an extracellular cytokine when released from damaged or necrotic cells, and is involved in the regulation of immune response. IL-33 is released from damaged cells as a passive and rapid response to stimulation or cell damage. However, it can also be actively secreted by immune cells. During inflammation, the abundant basal IL-33 expression in tissues can be further increased.

[0006] Recent studies have shown that IL-33 also plays an important role in the formation of SPEM pathology. Mice treated with IL33 developed SPEM and strong inflammatory responses in the lungs, intestines and stomach, indicating that IL33 was sufficient to recruit inflammatory cells and drive mucus production in the epithelial tissues of the respiratory tract and intestines. IL-33 from damaged mucosal epithelial cells is a key initiator of the initial formation of SPEM. IL-33 is released directly into the extracellular space or cleaved into an active short peptide by enzymes, binds to its receptor ST2 (IL-1R4), and induces a type II immune response, promoting inflammation progression, and is also related to the pathogenesis of gastrointestinal-related cancers, including gastric cancer. IL-33 increase is the cause of intestinalization of SPEM in mice and gastric cancer patients.

[0007] In order to further study the occurrence and development of SPEM, a good research model is essential. However, the acute SPEM animal model constructed by applying chemical drugs cannot meet the current research needs, and the origin of SPEM is still not determined. SUMMARY

[0008] Therefore, one of the purposes of the present application is to provide a method for constructing a GRIM-19 and IL-33 double-gene gastric parietal cell-specific knockout mouse model.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0010] The method for constructing a GRIM-19 and IL-33 double-gene gastric parietal cell-specific knockout mouse model uses the Cre-Lox system to specifically knockout the GRIM-19 gene and the IL-33 gene of the gastric parietal cells.

[0011] Further, the method comprises the following steps:

[0012] (1) The mouse GRIM-19 gene is modified by flox to obtain GRIM-19flox / flox mice;

[0013] (2) The IL-33 gene of the mouse is modified by flox to obtain IL-33flox / flox mice;

[0014] (3) The GRIM-19flox / flox mice obtained in step (1) are crossed with the IL-33flox / flox mice obtained in step (2) to obtain F1 generation mice, which are named GRIM-19flox / +, IL-33flox / + mice;

[0015] (4) The GRIM-19flox / +, IL-33flox / + mice obtained in step (3) are crossed with Atp4b-Cre mice to obtain F2 generation mice, which are named GRIM-19flox / +, IL-33flox / +, Atp4b-Cre mice;

[0016] (5) The F2 generation mice obtained in step (4) are crossed with the F1 generation mice obtained in step (3) to obtain F3 generation mice, which are named GRIM-19flox / flox, IL-33flox / flox, Atp4b-Cre mice.

[0017] Further, step (1) comprises: inserting loxP sites on both sides of the 3rd exon of the mouse GRIM-19 gene for flox modification; the sequence of the 3rd exon of the mouse GRIM-19 gene is shown in SEQ ID NO: 1.

[0018] Further, in step (2), loxP sites are inserted on both sides of the 5th-7th exons of the mouse IL-33 gene for flox modification; the sequence of the 5th exon of the mouse IL-33 gene is shown in SEQ ID NO: 2, the sequence of the 6th exon is shown in SEQ ID NO: 3, and the sequence of the 7th exon is shown in SEQ ID NO: 4.

[0019] Further, in step (4), the exon sequences of the stomach-specific GRIM-19 gene and IL-33 gene are deleted by the action of ATP4B-Cre enzyme.

[0020] Further, the method further comprises PCR identification of the GRIM-19flox / flox mouse, the IL-33flox / flox mouse and the Atp4b-Cre mouse, wherein the PCR identification primer of the GRIM-19flox / flox mouse comprises an upstream primer with a nucleotide sequence as set forth in SEQ ID NO: 5 and a downstream primer with a nucleotide sequence as set forth in SEQ ID NO: 6; the PCR identification primer of the IL-33flox / flox mouse comprises an upstream primer with a nucleotide sequence as set forth in SEQ ID NO: 7 and a downstream primer with a nucleotide sequence as set forth in SEQ ID NO: 8; and the PCR identification primer of the Atp4b-Cre mouse comprises an upstream primer with a nucleotide sequence as set forth in SEQ ID NO: 9 and a downstream primer with a nucleotide sequence as set forth in SEQ ID NO: 10.

[0021] As preferred, the GRIM-19-loxP PCR, 1133-loxp PCR system is as follows:

[0022] Rat tail DNA 1.5 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl Taq Mix 12.5 μl ddH2O 9 μl Total 25 μl .

[0023] As preferred, the GRIM-19-loxP PCR, 1133-loxp PCR procedure is as follows:

[0024]

[0025] As preferred, the Atp4b-Cre PCR system is as follows:

[0026] ddH2O 6.0 μl Forward primer (10 μM) 1.0 μl Reverse primer (10 μM) 1.0 μl Taq Mix 10 μl Rat tail DNA 2 μl Total 20 μl .

[0027] As preferred, the Atp4b-Cre PCR procedure is as follows:

[0028]

[0029] As preferred, in the GRIM-19-loxP PCR identification, if the PCR reaction product shows one band and the position is 382bp, then the mouse is a GRIM-19flox / flox homozygous mouse; if the PCR reaction product shows two bands and the positions are 382bp and 302bp, then the mouse is a GRIM-19flox / + heterozygous mouse; if the PCR reaction product shows one band and the position is 302bp, then the mouse is a WT wild type mouse.

[0030] As preferred, in the IL-33-loxp PCR identification, if the PCR reaction product shows one band and the position is 355bp, the mouse is IL-33flox / flox homozygous mouse; if the PCR reaction product shows two bands and the positions are 355bp and 297bp, the mouse is IL-33flox / + heterozygous mouse; if the PCR reaction product shows one band and the position is 297bp, the mouse is WT wild type mouse.

[0031] As preferred, in the Atp4b-Cre PCR identification, if the PCR reaction product shows one band and the position is 800bp, the mouse is Atp4b-Cre mouse; if the PCR reaction product shows no band, the mouse is WT wild type mouse.

[0032] Further, the method further comprises genotype identification of the F1-F3 generation mice by reverse transcription polymerase chain reaction, the GRIM-19 upstream primer sequence is shown as SEQ ID NO: 11, the GRIM-19 downstream primer sequence is shown as SEQ ID NO: 12; the IL-33 upstream primer sequence is shown as SEQ ID NO: 13, and the IL-33 downstream primer sequence is shown as SEQ ID NO: 14.

[0033] As preferred, the reverse transcription polymerase chain reaction system is: 2×SYBR Green Mix 5 μl, RT product 1 μl, Primer F (10 μM) 0.4 μl, Primer R (10 μM) 0.4 μl, RNase Free ddH2O is added to 10 μl.

[0034] As preferred, the reverse transcription polymerase chain reaction condition is: 95℃ 10min; 95℃ 2s, 60℃ 20s, 40 cycles.

[0035] As preferred, the mouse is C57BL / 6J mouse.

[0036] The second purpose of the present application is to provide an application of the GRIM-19 and IL-33 double gene parietal cell-specific knockout mouse model prepared by the foregoing construction method in the research of the occurrence and development mechanism of precancerous lesion SPEM of gastric cancer and the screening of candidate therapeutic drugs for SPEM.

[0037] Further, the present application focuses on the role of GRIM-19 and IL-33 in gastric diseases, and the crossbreeding with other tool mice can also study the physiological and pathological role in related diseases of liver / kidney, colon, uterus, etc.

[0038] The third object of the present application is to provide an application of the GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model prepared by the aforementioned construction method in screening products for treating gastric gland disorders caused by GRIM-19 knockout and inhibiting pathological progression of SPEM induced by down-regulation of GRIM-19.

[0039] The fourth object of the present application is to provide an application of an expression inhibitor of IL-33 in preparing products for treating gastric gland disorders caused by GRIM-19 knockout and inhibiting pathological progression of SPEM induced by down-regulation of GRIM-19.

[0040] The present application has the following advantages:

[0041] 1. The present application successfully constructs a GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model, which helps to explore the regulatory mechanism of GRIM-19 and IL-33 genes in the occurrence and progression of SPEM, and is of great significance for the research of human diseases and the development of new therapies.

[0042] 2. Lack of early clinical indicators will hinder early diagnosis and treatment of diseases. The present application reveals the key role of parietal cell-derived IL-33 and GRIM-19 double genes in the pathological process of SPEM from the animal level: parietal cell IL-33 knockout can reduce spontaneous gastric mucosal inflammation induced by GRIM-19 knockout in mice and inhibit the formation of SPEM pathology. This finding will greatly help the early detection and even reversal of gastric cancer, and help to promote the process of prevention and treatment of gastric cancer and provide new diagnostic and therapeutic targets for the treatment of SPEM. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Fig. 1 is a diagram of PCR identification results of different mice in Example 1;

[0044] Figure 2 Fig. 3 is a flow chart of construction of the GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model in Example 1 of the present application;

[0045] Figure 3 Fig. 5 is a HE staining chart;

[0046] Figure 4 Fig. 7 is a double immunofluorescence staining result chart. DETAILED DESCRIPTION

[0047] The technical solutions of the present application will be described further clearly and completely in combination with specific embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Therefore, all the other embodiments obtained by those skilled in the art based on the embodiments in the present application without creative labor are within the protection scope of the present application.

[0048] The present application constructs a GRIM-19flox / flox; IL-33flox / flox, Atp4b-Cre double-gene gastric wall cell-specific knockout mouse model by specifically knocking out the GRIM-19 gene and the IL-33 gene of the gastric wall cell through the Cre-Lox system. The principle of the Cre-Lox system is that: Cre recombinase is originally found in P1 phage, can specifically recognize the loxP site, and mediate the DNA sequence between the two loxP sites to be deleted or recombined. The sequence of the coding region of the Cre recombinase gene is 1029 bp in length (EMBL database accession number X03453), and encodes a 38 kDa monomer protein composed of 343 amino acids. In the Cre-loxP system, after the mice carrying the loxP site (i.e. Floxed mice) are mated with the Cre mice, gene knockout or expression can be achieved in specific tissues or cells. The LoxP sequence is derived from P1 phage, and is composed of two 13 bp inverted repeat sequences and an 8 bp sequence in the middle interval. The 8 bp interval sequence also determines the direction of LoxP. Cre is covalently bound to DNA in the process of catalyzing DNA strand exchange, and the 13 bp inverted repeat sequence is the binding domain of Cre enzyme.

[0049] In the embodiments of the present application, flox / flox indicates that the gene locus is double two alleles with flox sites, and these sites enable the tissue or cell expressing Cre enzyme to specifically delete the floxed gene.

[0050] In the embodiments of the present application, C57BL / 6J mice are selected.

[0051] In the embodiments of the present application, the Gene ID of GRIM-19 in NCBI is 67184, the sequence of the 3rd exon is shown as SEQ ID NO: 1; the Gene ID of IL-33 in NCBI is 77125, the sequence of the 5th exon is shown as SEQ ID NO: 2, the sequence of the 6th exon is shown as SEQ ID NO: 3, and the sequence of the 7th exon is shown as SEQ ID NO: 4.

[0052] In the embodiment of the present application, isopropyl alcohol is purchased from China Kangwei Century Company, ethanol is purchased from China Kangwei Century Company, DEPC water is purchased from Shanghai Biyun Tian Biology, QuantiNova Reverse Transcription Kit is purchased from Germany Qiagen, SYBR Premix Ex Taq II is purchased from TAKARA Baosheng Engineering (Dalian) Co., Ltd., agarose is purchased from Qikeng Biology, Goldview is purchased from Solaybao Biological Reagent, DNA Ladder is purchased from Mona Biological Technology Co., Ltd.; the manufacturer of the metal bath pot is Hangzhou Aosheng Instrument Co., Ltd., the manufacturer of the general PCR instrument is the United States Bio-Tek Company, and the manufacturer of the fluorescent quantitative PCR instrument is the United States Thermo Fisher Scientific Company.

[0053] In the embodiment of the present application, the preparation of the mouse tail DNA lysis solution is as follows: Tris-HCl 4ml, 1mol EDTA 0.4ml, 3mol NaCl 2.668ml, 10% SDS 0.8ml, and 32.132ml of double-distilled water, which are uniformly mixed and stored at room temperature.

[0054] In the embodiment of the present application, the preparation of 2% agarose gel is as follows: 2g of agarose powder is added to 0.1L of TBE buffer solution, heated and dissolved, 10ul of Goldview is added, the tip is uniformly mixed, and quickly poured into the gel plate to avoid air bubbles.

[0055] Example 1. Method for constructing a GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model

[0056] The present application constructs a GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model by specifically knocking out the GRIM-19 gene and the IL-33 gene of parietal cells through the Cre-Lox system. The operation process is as shown in Figure 2 , and the specific steps are as follows:

[0057] (1) The loxP site is inserted on both sides of the 3rd exon of the mouse GRIM-19 gene, the flox modification is performed, the flox mouse of the GRIM-19 gene is obtained, and the mouse is named GRIM-19flox / flox mouse;

[0058] (2) The loxP site is inserted on both sides of the 5th-7th exon of the mouse IL-33 gene, the flox modification is performed, the flox mouse of the IL33 gene is obtained, and the mouse is named IL-33flox / flox mouse;

[0059] (3) Cross GRIM-19flox / flox mice with IL-33flox / flox mice to obtain F1: GRIM-19flox / +, IL-33flox / + mice;

[0060] (4) Cross F1: GRIM-19flox / +, IL-33flox / + mice with Atp4b-Cre mice to delete the exon sequence of stomach-specific GRIM-19 and IL-33 genes by ATP4B-Cre enzyme to obtain F2: GRIM-19flox / +, IL-33flox / +, Atp4b-Cre mice;

[0061] (5) Cross F2: GRIM-19flox / +, IL-33flox / +, Atp4b-Cre mice with GRIM-19flox / +, IL-33flox / + mice to obtain F3: GRIM-19flox / flox, IL-33flox / flox, Atp4b-Cre mice.

[0062] Example 2. Identification of GRIM-19 and IL-33 double-gene stomach parietal cell-specific knockout mouse model

[0063] 1. Mouse tail identification

[0064] (1) In the SPF animal laboratory, cut 3-5 mm mouse tail and add 200 uL tissue lysis solution and 4 uL protease k, and lyse overnight at 56°C constant temperature to extract DNA;

[0065] (2) The next day, centrifuge the Ep tube at 14000g / 10min, and extract DNA with an equal amount of isopropanol;

[0066] (3) Shake well and centrifuge at 14000g / 10min;

[0067] (4) Washing: pour off the supernatant, wash the DNA with 70% anhydrous ethanol at 14000g / 15min, pour off the anhydrous ethanol, and air dry;

[0068] (5) Add 30μl of enzyme-free water to the filtered EP tube to dissolve the DNA;

[0069] (6) Prepare the corresponding PCR reaction system and select the corresponding program for reaction;

[0070] Table 1. GRIM-19-loxp PCR, Il33-loxp PCR system table

[0071] Table 1. GRIM-19-loxp PCR, Il33-loxp PCR system table

[0072] Rat tail DNA 1.5 μl Forward primer (10 μM) 1 μl Reverse primer (10 μM) 1 μl Taq Mix 12.5 μl ddH2O 9 μl Total 25 μl

[0073] Table 2. GRIM-19-loxp PCR, Il33-loxp PCR procedure table

[0074]

[0075] Table 3. Atp4b-Cre PCR system table

[0076]

[0077]

[0078] Table 4. Atp4b-Cre PCR procedure table

[0079]

[0080] Table 5. Primer sequence table

[0081]

[0082] (7) Agarose gel electrophoresis for band analysis: 2.0 g of agarose was poured into 100 ml of 1X TBE buffer, heated in a microwave oven for 5 min, and after the agarose was completely dissolved, 10 ul of developing solution was added and mixed, the gel solution was poured into the gel mold fixed with the comb, and the gel was cooled at room temperature and placed in an electrophoresis tank containing 1X TBE buffer. Marker loading amount 5ul, sample loading amount 12ul, 120V constant voltage electrophoresis 30min, after electrophoresis, the gel was taken out and placed in an imaging instrument for photographing and analysis.

[0083] (8) Identification method

[0084] GRIM-19-loxP PCR: If the PCR reaction product shows one band and the position is 382bp, then the mouse is GRIM-19flox / flox homozygous mouse; if the PCR reaction product shows two bands and the position is 382bp and 302bp, then the mouse is GRIM-19flox / + heterozygous mouse; if the PCR reaction product shows one band and the position is 302bp, then the mouse is WT wild type mouse.

[0085] IL-33-loxp PCR: If the PCR reaction product shows one band and the position is 355bp, then the mouse is IL-33flox / flox homozygous mouse; if the PCR reaction product shows two bands and the position is 355bp and 297bp, then the mouse is IL-33flox / + heterozygous mouse; if the PCR reaction product shows one band and the position is 297bp, then the mouse is WT wild type mouse.

[0086] Atp4b-Cre PCR: If the PCR reaction product shows one band and the position is 800bp, then the mouse is Atp4b-Cre mouse; if the PCR reaction product shows no band, then the mouse is WT wild type mouse.

[0087] The PCR identification results of different mice are shown in Table 1. Figure 1

[0088] 2. Identification of cDNA level GRIM-19flox / flox, IL-33flox / flox, Atp4b-Cre mouse model

[0089] (1) Extract normal gastric mucosa cell RNA

[0090] 1) Take 50mg of mouse gastric tissue into a pre-labeled de-enzyme Ep tube, add 1ml of Tripure Isolation Reagent, use a homogenizer to homogenize until the tissue block is completely broken, stand at room temperature for 5 minutes to ensure complete lysis.

[0091] 2) Add 0.2ml of chloroform to the gastric tissue lysis solution, shake thoroughly for 15s, stand at room temperature for 5min, centrifuge at 12000g, 4℃ for 15min.

[0092] 3) Absorb the upper colorless liquid into a new de-enzyme Ep tube, add 0.5ml of isopropanol, invert several times to mix thoroughly, stand at room temperature for 10min to precipitate the RNA, centrifuge at 12000g, 4℃ for 10min, discard the supernatant.

[0093] 4) Add 1ml of 75% ethanol to the Ep tube, gently invert several times, centrifuge at 7500g, 4℃ for 5min, discard the supernatant.​

[0094] 5) Dry at room temperature for 5 min to remove excess ethanol, add 100 μl RNase-free water to dissolve the precipitate, measure the RNA concentration and OD260 / 280.

[0095] (2) Reverse transcription was performed using QuantiNova Reverse Transcription Kit:

[0096] 1) Remove DNA from the sample, reaction system: 4 x DN Master Mix 2 μl, Total RNA 0.5 μg, add RNase Free ddH2O to 8 μl, incubate at 37°C for 5 min, and store on ice.

[0097] 2) Perform reverse transcription, reaction system: 8 μl of the first step mixture, 5 x RT Master Mix II 2 μl, a total of 10 μl of reaction system; reaction conditions: incubate at 37°C for 15 min, heat at 98°C for 5 min, and store on ice for standby.

[0098] 3) Add 40 μl of RNase Free ddH2O to the product, dilute to 50 μl for standby.

[0099] (3) Real-time PCR reaction

[0100] The primers for reverse transcription polymerase chain reaction (RT-PCR) were designed according to the reference sequence of GRIM-19 cDNA 3 exon, and the sequence of the upstream primer of GRIM-19 is shown in SEQ ID NO: 11, and the sequence of the downstream primer of GRIM-19 is shown in SEQ ID NO: 12. The primers for reverse transcription polymerase chain reaction (RT-PCR) were designed according to the reference sequence of IL-33 cDNA 5-7 exon, and the sequence of the upstream primer of IL-33 is shown in SEQ ID NO: 13, and the sequence of the downstream primer of IL-33 is shown in SEQ ID NO: 14.

[0101] Reaction system: 2 x SYBR Green Mix 5 μl, RT product 1 μl, Primer F (10 μM) 0.4 μl, Primer R (10 μM) 0.4 μl, RNase Free ddH2O added to 10 μl. Reaction conditions: 95°C for 10 min; 95°C for 2 s, 60°C for 20 s, 40 cycles, and measure the fluorescence value at the end of each cycle. Three replicate wells were set for all detection indicators, and mouse β-actin was used as an internal control for detection, and relative quantitative analysis was performed by comparing CT values (2-△△CT).

[0102] Results: The transcriptional levels of GRIM-19 and IL-33 genes were significantly reduced after knockout.

[0103] Example 4. HE staining experiment

[0104] (1) After the mice were sacrificed, the stomach tissue was taken into a 15ml centrifuge tube containing 4% paraformaldehyde solution, and the tissue sample was fixed. After 24h of room temperature fixation, the new 4% paraformaldehyde was replaced and placed in a 4°C refrigerator for slow fixation;

[0105] (2) After dehydration of the tissue, the paraffin-embedded tissue block was solidified, and after solidification, 4μm tissue sections were prepared and baked in an oven;

[0106] (3) De-paraffinization: sequentially de-paraffinized in xylene I and xylene II for 20min each;

[0107] (4) Hydration: sequentially perform gradient ethanol dehydration and tap water washing according to the process;

[0108] (5) Stain with hematoxylin dye for 40s and wash with tap water for 3min;

[0109] (6) Differentiate in 1% hydrochloric acid ethanol for 2s and wash with tap water for 1min;

[0110] (7) Counterstain with saturated lithium carbonate;

[0111] (8) After staining the cytoplasm with eosin dye for a few seconds, perform gradient ethanol dehydration;

[0112] (9) Xylene transparency, mounting, drying, and scanning with a full-automatic slide scanning instrument.

[0113] This example evaluates the histological changes of the embedded gastric body tissue sections by HE staining, and the results are shown in Figure 3 , and the knockout of IL-33 can improve the gastric gland disorder caused by the knockout of GRIM-19. Double immunofluorescence staining GIF / GSц co-labels SPEM cells, indicating that the knockout of IL-33 on the basis of the knockout of GRIM-19 can alleviate the SPEM pathological phenotype, as described in detail in Figure 4 . The present application analyzes the key role of IL-33 in the formation of gastric mucosal SPEM induced by the down-regulation of GRIM-19 from the parietal cell source, expands the function of GRIM-19 from tumor suppression to precancerous inflammation regulation, and is expected to provide a new idea for early prevention and treatment of intestinal-type gastric cancer.

Claims

1. A method for constructing a GRIM-19 and IL-33 double gene gastric parietal cell-specific knockout mouse model, characterized in that, The method comprises the following steps of: (1) inserting loxP sites at both sides of the 3rd exon of a mouse GRIM-19 gene to perform flox modification, and obtaining a GRIM-19 flox / flox mouse; the sequence of the 3rd exon of the mouse GRIM-19 gene is shown as SEQ ID NO: 1; (2) inserting loxP sites at both sides of the 5th-7th exons of a mouse IL-33 gene to perform flox modification, and obtaining an IL-33 flox / flox mouse; the sequence of the 5th exon of the mouse IL-33 gene is shown as SEQ ID NO: 2, the sequence of the 6th exon is shown as SEQ ID NO: 3, and the sequence of the 7th exon is shown as SEQ ID NO: 4; (3) crossing the GRIM-19 flox / flox mouse obtained in step (1) with the IL-33 flox / flox mouse obtained in step (2) to obtain an F1 generation mouse, which is named as a GRIM-19 flox / +, IL-33 flox / + mouse; (4) crossing the GRIM-19 flox / +, IL-33 flox / + mouse obtained in step (3) with an Atp4b-Cre mouse to obtain an F2 generation mouse, which is named as a GRIM-19 flox / +, IL-33 flox / +, Atp4b-Cre mouse; (5) crossing the F2 generation mouse obtained in step (4) with the F1 generation mouse obtained in step (3) to obtain an F3 generation mouse, which is named as a GRIM-19 flox / flox, IL-33 flox / flox, Atp4b-Cre mouse.

2. The construction method of claim 1, wherein, In step (4), the Atp4b-Cre enzyme is used to delete the exon sequences of the stomach-specific GRIM-19 gene and IL-33 gene.

3. The construction method of claim 1, wherein, The method further comprises performing PCR identification on the GRIM-19 flox / flox mouse, the IL-33 flox / flox mouse and the Atp4b-Cre mouse; the PCR identification primers of the GRIM-19 flox / flox mouse comprise an upstream primer with the nucleotide sequence shown as SEQ ID NO: 5 and a downstream primer with the nucleotide sequence shown as SEQ ID NO: 6; the PCR identification primers of the IL-33 flox / flox mouse comprise an upstream primer with the nucleotide sequence shown as SEQ ID NO: 7 and a downstream primer with the nucleotide sequence shown as SEQ ID NO: 8; and the PCR identification primers of the Atp4b-Cre mouse comprise an upstream primer with the nucleotide sequence shown as SEQ ID NO: 9 and a downstream primer with the nucleotide sequence shown as SEQ ID NO:

10.

4. The construction method of claim 1, wherein, The method further comprises genotype identification of the F1-F3 generation mice by reverse transcription polymerase chain reaction, wherein the sequence of the upstream primer of GRIM-19 is shown as SEQ ID NO: 11, the sequence of the downstream primer of GRIM-19 is shown as SEQ ID NO: 12; the sequence of the upstream primer of IL-33 is shown as SEQ ID NO: 13, and the sequence of the downstream primer of IL-33 is shown as SEQ ID NO:

14.

5. Application of the GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model prepared by the construction method of any one of claims 1-4 in the study of the mechanism of the occurrence and development of precancerous lesions SPEM and the screening of candidate therapeutic drugs for SPEM.

6. Application of the GRIM-19 and IL-33 double-gene parietal cell-specific knockout mouse model prepared by the construction method of any one of claims 1-4 in the screening of products for treating gastric gland disorders caused by GRIM-19 knockout and inhibiting the pathological progression of SPEM induced by the down-regulation of GRIM-19.

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