Construction method and application of GRIM-19 and IL-33 double-gene gastric wall cell specific knockout mouse model

Abstract

The invention 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 wall cell specific knockout mouse model. The construction method comprises the following steps: respectively carrying out flox modification on GRIM-19 and IL-33 genes of mice, and then hybridizing a GRIM-19 flox / flox mouse with an IL-33 flox / flox mouse, so as to obtain an F1-generation mouse; hybridizing the F1-generation mouse with an Atp4b-Cre mouse to obtain an F2-generation mouse; and hybridizing the F2-generation mouse with the F1-generation mouse to obtain the GRIM-19 and IL-33 double-gene gastric wall cell specific knockout mouse model. The model is helpful for exploring regulation and control mechanisms of GRIM-19 and IL-33 genes in generation and development of SPEM, and has important significance on treatment of SPEM and prevention and treatment of gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of animal models, and specifically relates to a method for constructing and applying a GRIM-19 and IL-33 double-gene gastric parietal cell specific knockout mouse model. Background Art

[0002] Gastric cancer is the fifth most common cancer and the third most common cause of cancer death worldwide, but the details of gastric cancer development remain 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 and the low diagnosis rate, the progression of gastric cancer greatly affects the treatment and survival of gastric cancer patients, and the recovery rate of patients with advanced gastric cancer is extremely low. Therefore, the discovery of molecular markers for early diagnosis and targeted treatment is crucial to improving the prognosis of gastric cancer.

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

[0004] Mitochondrial inner membrane protein GRIM-19 (Gene associated with retinoid interferon-induced mortality-19, also known as NDUFA13) is a functional component subunit of the mitochondrial respiratory chain MRC complex I. The gene of human GRIM-19 is located on chromosome 19p13.1, and both human and mouse GRIM-19 genes contain 5 exons. Compared with benign tumors, advanced tumors express very low levels of GRIM-19 protein, and the loss of GRIM-19 increases the susceptibility to tumor development. Specific knockout of GRIM-19 in gastric parietal cells can induce spontaneous gastric mucosal inflammation and SPEM pathology 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, IL-33 acts as a gene regulatory molecule in the nucleus in its full-length protein form (full-length IL-33 protein, flIL-33), while the mature form (mature IL-33, mIL-33) acts as an extracellular cytokine when released from damaged or necrotic cells, participating in the regulation of immune responses. IL-33 is a component of the tissue barrier in response to stimulation or cell damage, and is passively and rapidly released from damaged cells. 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] The latest research shows that IL-33 also plays an important role in the pathological formation of SPEM. Mice treated with IL33 develop SPEM and strong inflammatory responses in the lungs, intestines, and stomachs, indicating that IL33 is sufficient to recruit inflammatory cells and drive mucus production in the respiratory and intestinal epithelial tissues. IL-33 from damaged mucosal epithelial cells is a key initiator that induces the initial formation of SPEM. IL-33 is directly released into the extracellular space or enzymatically cleaved into active short peptides as an "Alarmin", which binds to its receptor ST2 (IL-1R4) to induce type II immune response, promote inflammation progression, and is also related to the pathogenesis of gastrointestinal-related cancers (including gastric cancer). Increased IL-33 is the cause of intestinal SPEM in mice and gastric cancer patients.

[0007] In order to further study the occurrence and development of SPEM, a good research model is crucial. However, the acute SPEM animal model constructed using chemical drugs can no longer meet current research needs, and the origin of SPEM is still inconclusive. Summary of the invention

[0008] In view of this, one of the objectives of the present invention is to provide a method for constructing a GRIM-19, IL-33 double-gene gastric parietal cell-specific knockout mouse model.

[0009] To achieve the above object, the present invention adopts the following technical solutions:

[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 knock out the gastric parietal cell GRIM-19 gene and IL-33 gene.

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

[0012] (1) The mouse GRIM-19 gene was floxed to obtain GRIM-19flox / flox mice;

[0013] (2) flox-modification of the mouse IL-33 gene to obtain IL-33flox / flox mice;

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

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

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

[0017] Furthermore, step (1) includes: inserting loxP sites on both sides of exon 3 of the mouse GRIM-19 gene to perform flox modification; the sequence of exon 3 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 exons 5 to 7 of the mouse IL-33 gene for flox modification; the sequence of exon 5 of the mouse IL-33 gene is shown in SEQ ID NO: 2, the sequence of exon 6 is shown in SEQ ID NO: 3, and the sequence of exon 7 is shown in SEQ ID NO: 4.

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

[0020] Furthermore, the method also includes performing PCR identification on the GRIM-19flox / flox mice, the IL-33flox / flox mice and the Atp4b-Cre mice, the PCR identification primers of the GRIM-19flox / flox mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 6; the PCR identification primers of the IL-33flox / flox mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 8; the PCR identification primers of the Atp4b-Cre mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 10.

[0021] Preferably, the GRIM-19-loxP PCR and 1133-loxP PCR systems are as follows:

[0022] rat tail DNA 1.5μl Forward primer (10 μM) 1μl Backward primer (10 μM) 1μl Taq Mix 12.5μl <![CDATA[ddH 2 The]]> 9μl Total 25μl .

[0023] Preferably, the GRIM-19-loxP PCR and Il33-loxP PCR procedures are as follows:

[0024]

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

[0026] <![CDATA[ddH 2 The]]> 6.0μl Forward primer (10 μM) 1.0μl Backward primer (10 μM) 1.0μl Taq Mix 10μl rat tail DNA 2μl Total 20μl .

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

[0028]

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

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

[0031] Preferably, in Atp4b-Cre PCR identification, if the PCR reaction product shows a band at 800 bp, the mouse is an Atp4b-Cre mouse; if the PCR reaction product shows no band, the mouse is a WT wild-type mouse.

[0032] Furthermore, the method also includes using reverse transcription polymerase chain reaction to identify the genotype of the F1 to F3 generations of mice, the GRIM-19 upstream primer sequence is shown in SEQ ID NO: 11, the GRIM-19 downstream primer sequence is shown in SEQ ID NO: 12; the IL-33 upstream primer sequence is shown in SEQ ID NO: 13, and the IL-33 downstream primer sequence is shown in SEQ ID NO: 14.

[0033] Preferably, the reaction system of the reverse transcription polymerase chain reaction 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 ddH 2 O was added to 10 μl.

[0034] Preferably, the reaction conditions of the reverse transcription polymerase chain reaction are: 95°C for 10 min; 95°C for 2 s, 60°C for 20 s, and 40 cycles.

[0035] Preferably, the mice are C57BL / 6J mice.

[0036] The second purpose of the present invention is to provide a GRIM-19, IL-33 double gene gastric parietal cell-specific knockout mouse model prepared by the aforementioned construction method for use in studying the occurrence and development mechanism of gastric precancerous lesions SPEM and screening candidate therapeutic drugs for SPEM.

[0037] Furthermore, the present invention focuses on the role of GRIM-19 and IL-33 in gastric diseases, and hybridization with other tool mice can also study their physiological and pathological effects on liver / kidney, colon, uterus and other related diseases.

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

[0039] The fourth object of the present invention is to provide an IL-33 expression inhibitor for use in the preparation of a product for treating gastric glandular disorders caused by GRIM-19 knockout and inhibiting the pathological progression of SPEM induced by GRIM-19 downregulation.

[0040] The beneficial effects of the present invention are:

[0041] 1. The present invention successfully constructed a GRIM-19 and IL-33 double-gene gastric parietal cell-specific knockout mouse model, which is helpful 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 study of human diseases and the development of new therapies.

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

[0043] Figure 1 The PCR identification results of different mice in Example 1 are shown;

[0044] Figure 2 This is a flow chart for constructing a GRIM-19 and IL-33 double-gene gastric parietal cell-specific knockout mouse model in Example 1 of the present invention;

[0045] Figure 3 HE staining picture;

[0046] Figure 4 The figure shows the results of double immunofluorescence staining. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be further described clearly and completely in conjunction with specific embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the protection scope of the present invention.

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

[0049] In the embodiments of the present invention, flox / flox indicates that the locus is double and both alleles carry flox sites, which enable tissues or cells expressing the Cre enzyme to specifically delete the floxed gene.

[0050] In the embodiment of the present invention, the mice are C57BL / 6J mice.

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

[0052] In the embodiments of the present invention, isopropanol was purchased from China Kangwei Century Company, ethanol was purchased from China Kangwei Century Company, DEPC water was purchased from Shanghai Biyuntian Biotechnology, QuantiNova Reverse Transcription Kit was purchased from Qiagen, Germany, SYBR Premix Ex Taq II was purchased from TAKARA Biotechnology (Dalian) Co., Ltd., agarose was purchased from Qingke Biotechnology, Goldview was purchased from Solebao Biological Reagents, and DNA Ladder was purchased from Mona Biotechnology Co., Ltd.; the manufacturer of the metal bath was Hangzhou Aosheng Instrument Co., Ltd., the manufacturer of the ordinary PCR instrument was Bio-Tek, USA, and the manufacturer of the fluorescent quantitative PCR instrument was Thermo Fisher Scientific, USA.

[0053] In the embodiment of the present invention, the mouse tail DNA lysis solution is prepared by mixing 4 ml of Tris-HCl, 0.4 ml of 1 mol EDTA, 2.668 ml of 3 mol NaCl, 0.8 ml of 10% SDS, and 32.132 ml of double distilled water, and the mixture is stored at room temperature.

[0054] In the embodiment of the present invention, 2% agarose gel was prepared by adding 2 g agarose powder to 0.1 L TBE buffer, heating to dissolve, adding 10 μl Goldview, mixing with a pipette tip, and quickly pouring into the gel plate to avoid generating bubbles.

[0055] Example 1. Construction of a GRIM-19, IL-33 dual-gene gastric parietal cell-specific knockout mouse model

[0056] The present invention uses the Cre-Lox system to specifically knock out the GRIM-19 gene and IL-33 gene of gastric parietal cells, thereby constructing a GRIM-19 and IL-33 double-gene gastric parietal cell specific knockout mouse model. Figure 2 As shown, the specific steps are as follows:

[0057] (1) loxP sites were inserted on both sides of exon 3 of the mouse GRIM-19 gene to perform flox modification to obtain GRIM-19 gene flox mice, which were named GRIM-19flox / flox mice;

[0058] (2) loxP sites were inserted on both sides of exons 5-7 of the mouse IL-33 gene to perform flox modification to obtain IL33 gene flox mice, which were named IL-33flox / flox mice;

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

[0060] (4) F1:GRIM-19flox / +,IL-33flox / + mice were crossed with Atp4b-Cre mice, and the exon sequences of the stomach-specific GRIM-19 and IL-33 genes were deleted through the action of ATP4B-Cre enzyme to obtain F2:GRIM-19flox / +,IL-33flox / +,Atp4b-Cre mice;

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

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

[0063] 1. Rat tail identification

[0064] (1) In an SPF animal laboratory, cut 3-5 mm mouse tails, add 200 uL tissue lysis buffer and 4 uL proteinase K, incubate at 56°C overnight for lysis, and extract DNA;

[0065] (2) The next day, the Eppendorf tube was centrifuged at 14,000 g for 10 min and DNA was extracted with an equal amount of isopropanol.

[0066] (3) After oscillation and mixing, centrifuge at 14000g / 10min;

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

[0068] (5) Add 30 μl 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] GRIM-19-loxPPCR, Il33-loxp PCR system is shown in Table 1; GRIM-19-loxPPCR, Il33-loxp PCR program is shown in Table 2; Atp4b-Cre PCR system is shown in Table 3; Atp4b-Cre PCR program is shown in Table 4; primer sequences are shown in Table 5;

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

[0072] rat tail DNA 1.5μl Forward primer (10 μM) 1μl Backward primer (10 μM) 1μl TaqMix 12.5μl <![CDATA[ddH 2 The]]> 9μl Total 25μl

[0073] Table 2. GRIM-19-loxP PCR, Il33-loxP PCR program

[0074]

[0075] Table 3. Atp4b-Cre PCR system

[0076]

[0077]

[0078] Table 4. Atp4b-Cre PCR program

[0079]

[0080] Table 5. Primer sequence list

[0081]

[0082] (7) Band analysis by agarose gel electrophoresis: Pour 2.0 g agarose into 100 ml 1X TBE buffer, heat in a microwave oven for 5 min, take out and cool at room temperature after the agarose is completely dissolved, add 10 ul developer and mix well, pour the gel solution into a gel mold with a fixed tooth comb, solidify at room temperature and place in an electrophoresis tank filled with 1X TBE buffer. Load 5 u1 of marker and 12 u1 of sample, perform electrophoresis at 120 V constant voltage for 30 min, take out the gel after the electrophoresis and place it in an imager for photo analysis.

[0083] (8) Identification method

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

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

[0086] Atp4b-Cre PCR: If the PCR reaction product shows a band at 800 bp, the mouse is an Atp4b-Cre mouse; if the PCR reaction product shows no band, the mouse is a WT wild-type mouse.

[0087] PCR identification results of different mice are as follows Figure 1 shown.

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

[0089] (1) Extraction of RNA from normal gastric mucosal cells

[0090] 1) Take 50 mg of mouse stomach tissue and put it into a pre-labeled enzyme-free Eppendorf tube. Add 1 ml of Tripure Isolation Reagent and homogenize it using a homogenizer until the tissue pieces are completely broken. Let it stand at room temperature for 5 minutes to ensure complete lysis.

[0091] 2) Add 0.2 ml of chloroform to the gastric tissue lysate, shake repeatedly for 15 seconds to mix thoroughly, let stand at room temperature for 5 minutes, and centrifuge at 12000g, 4°C for 15 minutes.

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

[0093] 4) Add 1 ml of 75% ethanol to the Eppendorf tube, gently invert several times, centrifuge at 7500 g, 4°C for 5 min, and 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, and measure the RNA concentration and OD260 / 280.

[0095] (2) Reverse transcription using the QuantiNova reverse transcription kit:

[0096] 1) Remove DNA from the sample, reaction system: 4×DN Master Mix 2μl, Total RNA 0.5μg, supplemented with RNase Free ddH 2 0 to 8 μl, incubate at 37°C for 5 min, and keep on ice until use.

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

[0098] 3) Add 40 μl RNase Free ddH 2 O, dilute to 50 μl for later use.

[0099] (3) Real-time PCR reaction

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

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

[0102] Results: After knockout, the transcription levels of GRIM-19 and IL-33 genes were significantly reduced.

[0103] Example 4. HE staining experiment

[0104] (1) After killing the mice, the stomach tissue was collected and placed in a 15 ml centrifuge tube containing 4% paraformaldehyde solution for fixation. After fixation at room temperature for 24 h, the sample was replaced with new 4% paraformaldehyde and placed in a 4°C refrigerator for slow fixation.

[0105] (2) After tissue dehydration, the tissue blocks were embedded in paraffin and solidified, 4 μm tissue sections were cut and baked in an oven;

[0106] (3) Dewaxing: Dewaxing in xylene I and xylene II for 20 min each;

[0107] (4) Hydration: Dehydrate with gradient ethanol and rinse with tap water according to the process;

[0108] (5) Stain with hematoxylin for 40 seconds and rinse with tap water for 3 minutes;

[0109] (6) Differentiate in 1% hydrochloric acid ethanol for 2 seconds and rinse in tap water for 1 minute;

[0110] (7) Blue color is reflected in saturated lithium carbonate;

[0111] (8) After cytoplasmic staining with eosin dye for a few seconds, dehydration was performed with graded ethanol;

[0112] (9) Xylene was used for transparency, and the slides were sealed and dried before scanning with an automatic slide scanner.

[0113] In this example, HE staining was used to evaluate the histological changes of embedded gastric tissue sections. The results are as follows: Figure 3 As shown in Figure 2, IL-33 knockout can improve gastric gland disorder caused by GRIM-19 knockout. Double immunofluorescence staining GIF / GSц co-labeled SPEM cells decreased, indicating that IL-33 knockout can alleviate SPEM pathological phenotypes on the basis of GRIM-19 knockout. For details, see Figure 4 This paper analyzes the key role of IL-33 in the formation of gastric mucosal SPEM induced by downregulation of GRIM-19 from the perspective of parietal cell origin, and expands the function of GRIM-19 from tumor suppression to precancerous inflammation regulation, which is expected to provide new ideas for the early prevention and treatment of intestinal 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 Cre-Lox system was used to specifically knock out the GRIM-19 and IL-33 genes in gastric parietal cells.

2. The construction method according to claim 1, characterized in that: The method comprises the following steps: (1) The mouse GRIM-19 gene was floxed to obtain GRIM-19flox / flox mice; (2) flox-modification of the mouse IL-33 gene to obtain IL-33flox / flox mice; (3) hybridizing the GRIM-19flox / flox mice obtained in step (1) with the IL-33flox / flox mice obtained in step (2) to obtain F1 mice, which were named GRIM-19flox / +, IL-33flox / + mice; (4) hybridizing the GRIM-19flox / +, IL-33flox / + mice obtained in step (3) with Atp4b-Cre mice to obtain F2 mice, which were named GRIM-19flox / +, IL-33flox / +, Atp4b-Cre mice; (5) The F2 mice obtained in step (4) were hybridized with the F1 mice obtained in step (3) to obtain F3 mice, which were named GRIM-19flox / flox, IL-33flox / flox, Atp4b-Cre mice.

3. The construction method according to claim 2, characterized in that: Step (1) comprises: inserting loxP sites on both sides of exon 3 of the mouse GRIM-19 gene to perform flox modification; the sequence of exon 3 of the mouse GRIM-19 gene is shown in SEQ ID NO:

1.

4. The construction method according to claim 2, characterized in that: In step (2), loxP sites are inserted on both sides of exons 5 to 7 of the mouse IL-33 gene for flox modification; the sequence of exon 5 of the mouse IL-33 gene is shown in SEQ ID NO: 2, the sequence of exon 6 is shown in SEQ ID NO: 3, and the sequence of exon 7 is shown in SEQ ID NO:

4.

5. The construction method according to claim 2, characterized in that: In step (4), the exon sequences of the stomach-specific GRIM-19 gene and IL-33 gene are deleted using the Atp4b-Cre enzyme.

6. The construction method according to claim 2, characterized in that: The method also includes performing PCR identification on the GRIM-19flox / flox mice, the IL-33flox / flox mice and the Atp4b-Cre mice, the PCR identification primers of the GRIM-19flox / flox mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 5 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 6; the PCR identification primers of the IL-33flox / flox mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 7 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO: 8; the PCR identification primers of the Atp4b-Cre mice include an upstream primer with a nucleotide sequence as shown in SEQ ID NO: 9 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO:

10.

7. The construction method according to claim 2, characterized in that: The method also includes using reverse transcription polymerase chain reaction to identify the genotype of the F1 to F3 generations of mice, the GRIM-19 upstream primer sequence is shown in SEQ ID NO: 11, the GRIM-19 downstream primer sequence is shown in SEQ ID NO: 12; the IL-33 upstream primer sequence is shown in SEQ ID NO: 13, and the IL-33 downstream primer sequence is shown in SEQ ID NO:

14.

8. Use of the GRIM-19, IL-33 double gene gastric parietal cell specific knockout mouse model prepared by the construction method according to any one of claims 1 to 7 in studying the occurrence and development mechanism of gastric precancerous lesions SPEM and screening candidate therapeutic drugs for SPEM.

9. Use of the GRIM-19, IL-33 double gene gastric parietal cell-specific knockout mouse model prepared by the construction method according to any one of claims 1 to 7 in screening products for treating gastric glandular disorders caused by GRIM-19 knockout and inhibiting the pathological progression of SPEM induced by GRIM-19 downregulation.

10. Use of IL-33 expression inhibitors in the preparation of products for treating gastric glandular disorders caused by GRIM-19 knockout and inhibiting the pathological progression of SPEM induced by GRIM-19 downregulation.

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