Construction and identification method based on atrophic gastritis SPEM lesion model

Abstract

The invention relates to the technical field of life science, in particular to a method for constructing an atrophic gastritis-based SPEM lesion model, which comprises the following steps: S1, constructing an Slc7a11 conditional knockout mouse strain: carrying out cage hybridization on an Slc7a11 flox / flox mouse and an ATP4b-Cre transgenic mouse, and knocking out a gastric wall cell specific Slc7a11 gene; s2, carrying out genotyping identification; the identification method based on the atrophic gastritis SPEM lesion model and the construction method based on the atrophic gastritis SPEM lesion model comprise the following steps: S1, pathological model verification: taking a mouse gastric mucosa tissue slice, quantitatively analyzing the proportion of an SPEM region by adopting an immunofluorescence double-standard technology, and confirming Slc7a11 knockout induced SPEM pathological characteristics in combination with the existence of SPEM in the gastric mucosa; and S2, long-term pathology evolution monitoring. Through gastric wall cell specific Slc7a11 gene knockout and multiple PCR identification technologies, the initial response SPEM state after gastric mucosa damage is successfully simulated, and the kit is used for researching atrophic gastritis pathological mechanisms and evaluating drug intervention.

Description

Technical Field

[0001] The present invention relates to the field of life science technology, and in particular to a construction and identification method of an atrophic gastritis SPEM lesion model. Background Art

[0002] Chronic atrophic gastritis (CAG) is a precancerous lesion of the stomach characterized by a progression of inflammation, atrophy, metaplasia, and dysplasia. Metaplasia, a key node in the malignant progression of CAG, typically manifests as intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM). Previous studies have focused on the relationship between IM and gastric cancer. However, SPEM is actually the initial bioreactive response after gastric mucosal damage, meaning that IM originates from SPEM. Therefore, elucidating the pathological formation mechanism of SPEM is crucial for the early prevention and control of gastric cancer.

[0003] Although research on CAG and its precancerous lesions has made some progress, existing technologies have significant deficiencies in the following aspects: First, existing CAG models mostly rely on drug induction (such as MNNG, alcohol) or broad-spectrum gene knockout, which cannot achieve gastric parietal cell-specific genetic manipulation, resulting in poor model specificity and difficulty in accurately simulating the pathological characteristics of SPEM. For example, although drug-induced models can simulate gastric mucosal damage, they cannot precisely control gene expression, making it difficult to study the role of specific genes (such as Slc7a11) in SPEM. Second, existing models lack molecular markers and pathological identification criteria for SPEM, resulting in insufficient pathological verification. In addition, the pathological characteristics of existing drug-induced models vary significantly over time, with poor reproducibility and stability, making it difficult to observe the long-term transformation process of SPEM to IM. To address the problems of insufficient model specificity, incomplete pathological verification, and poor model stability in existing technologies, a method for constructing and identifying a SPEM lesion model based on atrophic gastritis is proposed. Summary of the Invention

[0004] To address the above problems, the present invention provides a method for constructing and identifying a SPEM lesion model based on atrophic gastritis. By using gastric parietal cell-specific Slc7a11 gene knockout and multiplex PCR identification technology, the initial innate response SPEM state after gastric mucosal damage is successfully simulated, which is used to study the pathological mechanism of atrophic gastritis and evaluate drug intervention.

[0005] In order to achieve the above object, the technical solution of the present invention is as follows: a method for constructing a SPEM lesion model of atrophic gastritis, comprising the following steps:

[0006] S1. Construction of Slc7a11 conditional knockout mouse strain: Slc7a11 flox / flox mice were co-bred with ATP4b-Cre transgenic mice to achieve gastric parietal cell-specific Slc7a11 gene knockout using the Cre-loxP system;

[0007] S2, genotyping identification: Multiplex PCR was performed using Slc7a11 flox locus primers F2 and R2, as well as Cre gene primers-1 and -2. Homozygous, heterozygous, and Cre-positive individuals were distinguished by PCR reaction. PCR detection using primers F2 and R1 confirmed that Cre-mediated Slc7a11 gene deletion manifested as a 235-bp band.

[0008] Furthermore, in S1, the Slc7a11 flox / flox mouse strain was C57BL / 6N-Slc7a11em1flox / Cya, and the ATP4b-Cre transgenic mouse strain was a gastric parietal cell-specific Cre-expressing mouse.

[0009] Furthermore, in S2, the sequence of primer F2 is 5′-AGACGTGATTCAGTTGTTGTTCCT-3′;

[0010] The sequence of primer R2 is 5′-AAGGGGATGCTTTGCTATTCTCTA-3′;

[0011] Primers F2 and R2 were used to amplify 216 bp and 155 bp bands;

[0012] The sequence of Cre gene primer-1 is 5′-GCAGATAGCAAGCAAGCTCCAACC-3′;

[0013] The sequence of Cre gene primer-2 is 5′-GGATTAACATTCTCCCACCGTCAG-3′;

[0014] Cre gene primer-1 and Cre gene primer-2 were used to amplify an 800 bp band.

[0015] Furthermore, in S2, the sequence of primer R1 is 5'-AGTAACCCTAAGGGTGGCAGCT-3'.

[0016] Furthermore, in S2, the PCR reaction system was performed under standard conditions in a volume of 25 μL for 35 cycles, and the PCR mixture contained 1.5 μL genomic DNA, 1.0 μL product primer F1, 1.0 μL product primer R1, 12.5 μL Premix Taq and 9.0 μL ddH2O, wherein the primer concentration was 10 μM.

[0017] Furthermore, the 35 cycles include the following steps:

[0018] S211, initial denaturation at 94°C for 3 min;

[0019] S212, denaturation was continued at 94°C for 30 seconds;

[0020] S213, annealing at 60 °C for 35 s and 35 cycles;

[0021] S214, extension at 72°C for 35 seconds;

[0022] S215, additional extension at 72°C for 5 minutes.

[0023] Furthermore, the extraction of genomic DNA contained in the PCR mixture comprises the following steps:

[0024] S221: After wearing sterile gloves, masks, caps, and sterile clothing, enter the SPF animal laboratory. Use sterile instruments to ear-tag the mice and place 3-5 mm of the mouse tail into the corresponding 1.5 ml Eppendorf tube. Add 200 μL of lysis buffer and 4 μL of proteinase K to obtain the treated mouse tail. The lysis buffer contains 10 μM Tris-HCl pH 8.0, 10 μM EDTA, 15 mM NaCl, and 0.5% SDS. Digest the treated mouse tail at 55°C overnight to obtain a lysis mixture.

[0025] S222, centrifuging the lysis mixture at 14000g for 10 min to obtain a layered lysis supernatant and impurity precipitate, taking 100uL of the lysis supernatant and mixing it with an equal volume of isopropanol, vortexing it for 10-20s to form an isopropanol-DNA mixture, centrifuging the isopropanol-DNA mixture at 14000g for 15 min to obtain a layered isopropanol supernatant and a DNA precipitate, discarding the isopropanol supernatant, adding 75% alcohol to the DNA precipitate for washing, and vortexing it for 10-20s to form an alcohol-DNA suspension, centrifuging the alcohol-DNA suspension at 14000g for 10 min to obtain an alcohol supernatant and a purified DNA precipitate, discarding the alcohol supernatant, and air-drying the purified DNA precipitate at room temperature for 30 min, adding 100uL ddH2O or TE water to dissolve it to obtain a DNA solution;

[0026] S223, according to the PCR reaction system, a corresponding program is selected and configured accordingly to obtain a PCR amplification product, and then band analysis of the PCR amplification product is performed by agarose gel electrophoresis.

[0027] Furthermore, the identification method based on the atrophic gastritis SPEM lesion model and the construction method based on the atrophic gastritis SPEM lesion model include the following steps:

[0028] S1. Pathological model validation: Mouse gastric mucosal tissue sections were obtained and immunofluorescence double-labeling was used to detect the co-localization of the gastric mucosal markers GIF and GSⅡ. The SPEM area ratio, i.e., the proportion of positive signals, was quantitatively analyzed. Mist1 and SPEM-related markers Clusterin-1 and HE4 were detected by immunoblotting. Immunoblotting was used to detect the expression levels of inflammatory factors, including downstream related factors such as IL-33 and NLRP3. Combined with the presence of SPEM in the gastric mucosa, the pathological characteristics of SPEM induced by Slc7a11 knockout were confirmed.

[0029] S2, long-term pathological evolution monitoring: after parietal cell-specific Slc7a11 knockout, the number of surface mucous cells, enteroendocrine cell chromogranin, as well as intestinal stem cells and crypt cells in gastric mucosal tissue were analyzed.

[0030] Furthermore, in S1, the double immunofluorescence staining method for detecting GIF and GSⅡ in gastric mucosal tissue comprises the following steps:

[0031] S11, paraffin-embedded mouse gastric mucosal tissue sections were obtained, dewaxed in xylene, hydrated with graded ethanol, and antigen heat retrieval was performed using pH 6.0 sodium citrate buffer;

[0032] S12, rinse three times with phosphate buffer for 5 min each time, then add serum homologous to the secondary antibody and incubate at room temperature for 30 min to block nonspecific binding sites;

[0033] S13, add diluted primary antibody solution, GIF and GSⅡ dropwise and incubate overnight at 4°C;

[0034] S14, rinse with phosphate buffer three times, 5 minutes each time, select the corresponding secondary antibody according to the species of the primary antibody, incubate for 2 hours in the dark, and then rinse again with phosphate buffer three times, 5 minutes each time;

[0035] S15, add 50 μl of DAPI staining solution, incubate for 10 min in the dark, and then stain the cell nuclei. Rinse with phosphate buffer three times, 5 min each time.

[0036] S16, the slides were mounted with anti-fluorescence quenching mounting medium and stored at 4°C in the dark until detection;

[0037] S17, take pictures by laser confocal microscopy, count the ratios of GIF, GSⅡ single-positive cells and GIF+GSⅡ+double-positive SPEM cells, and record the results.

[0038] Furthermore, in S2, when the numbers of surface mucous cells, enteroendocrine cell chromogranin, intestinal stem cells, and crypt cells in gastric mucosal tissue increased, it indicated that SLC7A11 knockout, which was specific to gastric mucosal parietal cells in mice, disrupted the differentiation of gastric mucosal epithelial glands in mice.

[0039] The above scheme has the following beneficial effects:

[0040] 1. This protocol improves the specificity of the model: By crossing Slc7a11 flox / flox mice with ATP4b-Cre transgenic mice, the Cre-loxP system is used to achieve gastric parietal cell-specific knockout of the Slc7a11 gene, avoiding the nonspecific effects of broad-spectrum gene knockout or drug-induced knockout. This model accurately simulates the pathological characteristics of human SPEM and provides a controllable genetic tool for studying the role of specific genes in the formation of SPEM.

[0041] 2. This protocol offers highly efficient genotyping and improved model stability: By designing a multiplex PCR primer set (F2 / R2, Cre-1 / Cre-2, F2 / R1), Slc7a11 flox homozygosity, Cre recombinase activity, and Slc7a11 knockout efficiency (verified by a 235bp band) can be simultaneously identified in a single reaction. Combined with an optimized PCR protocol, this significantly improves genotyping accuracy and ensures the stability and reproducibility of model construction.

[0042] 3. This protocol incorporates a comprehensive multi-dimensional pathological validation system: GIF / GSII dual immunofluorescence staining quantitatively analyzes the proportion of SPEM regions (percentage of double-positive signals), and molecular-level pathological validation criteria are established by detecting changes in the expression of Mist1 (a chief cell differentiation marker) and Clusterin-1 / HE4 (SPEM markers). Furthermore, IL-33 / NLRP3 inflammatory signaling assays are introduced to comprehensively assess the chronic inflammatory microenvironment induced by Slc7a11 knockout, addressing the limitations of traditional qPCR, which only detects IL-6 / TNF-α.

[0043] 4. This protocol, by tracking the dynamic changes of MUC5AC+ mucus cells, Chr-A+ enteroendocrine cells, GPR49 / LGR5+ intestinal stem cells, and Doublecortin (E-6)+ crypt cells, combined with β-catenin / CDX2 pathway analysis, achieved for the first time the long-term monitoring of the pathological evolution from SPEM to IM, maintaining a stable pathological phenotype over a longer period of time, and overcoming the problem of short-term pathological fluctuations caused by metabolic differences in drug-induced models.

[0044] 5. This protocol can not only be used to study the molecular mechanism of SPEM, but also serve as a screening platform for evaluating drugs targeting Slc7a11, providing a standardized tool for the early prevention and control of gastric cancer.

[0045] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the method steps of an embodiment of the method for constructing an atrophic gastritis SPEM lesion model according to the present invention;

[0047] Figure 2 Schematic diagram of the method steps of an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention;

[0048] Figure 3 Schematic diagram of the hybridization of Slc7a11 flox / flox and ATP4b-cre according to an embodiment of the method for constructing an atrophic gastritis SPEM lesion model of the present invention;

[0049] Figure 4 Identification of the Slc7a11flox / flox / ATP4b-cre mouse gene according to the embodiment of the method for constructing an atrophic gastritis SPEM lesion model of the present invention;

[0050] Figure 5 Schematic diagram of GRIM-19 protein expression analysis in gastric mucosal tissue of parietal cell-specific gene knockout mice according to an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention;

[0051] Figure 6 This is a schematic diagram of the expression level of GRIM-19 protein in gastric mucosal tissue detected by immunoblotting according to an embodiment of the identification method of the present invention based on the SPEM lesion model of atrophic gastritis;

[0052] Figure 7 Schematic diagram of HE staining of gastric mucosal tissue of parietal cell-specific SLC7A11 gene knockout mice according to an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention;

[0053] Figure 8 Schematic diagram of immunoblotting for detecting the expression of inflammatory factors in gastric mucosal tissue of parietal cell-specific SLC7A11 gene knockout mice according to an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention;

[0054] Figure 9Schematic diagram of SPEM marker analysis of gastric mucosal tissue in parietal cell-specific gene knockout mice according to an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention;

[0055] Figure 10 This is the GIF and GSⅡ immunofluorescence double-labeling analysis of gastric mucosal tissue of parietal cell-specific gene knockout mice in the identification method embodiment of the present invention based on the atrophic gastritis SPEM lesion model;

[0056] Figure 11 This is a schematic diagram of an embodiment of the identification method based on the atrophic gastritis SPEM lesion model of the present invention, further using immunofluorescence technology to analyze the differentiation of gastric mucosal glands. DETAILED DESCRIPTION

[0057] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0058] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0059] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0060] The following is further described in detail through specific implementation methods:

[0061] Example 1:

[0062] As attached Figures 1-11 As shown: The method for constructing the SPEM lesion model of atrophic gastritis includes the following steps:

[0063] S1. Construction of a Slc7a11 conditional knockout mouse strain: Slc7a11 flox / flox mice were co-bred with ATP4b-Cre transgenic mice. The Slc7a11 flox / flox mouse strain is C57BL / 6N-Slc7a11em1flox / Cya. The ATP4b-Cre transgenic mouse strain is a gastric parietal cell-specific Cre-expressing mouse. Gastric parietal cell-specific Slc7a11 gene knockout was achieved using the Cre-loxP system.

[0064] S2, Genotyping: Multiplex PCR was performed using primers F2 and R2 targeting the Slc7a11 flox locus, as well as Cre primers-1 and -2. The sequence of primer F2 is 5'-AGACGTGATTCAGTTGTTGTTCCT-3', and the sequence of primer R2 is 5'-AAGGGGATGCTTTGCTATTCTCTA-3'. Primers F2 and R2 amplify 216 and 155 bp bands, respectively. The sequence of Cre primer-1 is 5'-GCAGATAGCAAGCAAGCTCCAACC-3', and the sequence of Cre primer-2 is 5'-GGATTAACATTCTCCCACCGTCAG-3'. Cre primers-1 and -2 amplify an 800 bp band.

[0065] Homozygous, heterozygous, and Cre-positive individuals were distinguished by PCR reaction. The PCR reaction system was performed under standard conditions in a volume of 25 μL for 35 cycles. The 35 cycles included the following steps:

[0066] S211, initial denaturation at 94°C for 3 min;

[0067] S212, denaturation was continued at 94°C for 30 seconds;

[0068] S213, annealing at 60 °C for 35 s and 35 cycles;

[0069] S214, extension at 72°C for 35 seconds;

[0070] S215, additional extension at 72°C for 5 minutes.

[0071] The PCR mixture contains 1.5 μl of genomic DNA, 1.0 μl of product primer F1, 1.0 μl of product primer R1, 12.5 μl of Premix Taq, and 9.0 μl of ddH2O, wherein the primer concentration is 10 μM. The extraction of the genomic DNA contained in the PCR mixture includes the following steps:

[0072] S221: After wearing sterile gloves, masks, caps, and sterile clothing, enter the SPF animal laboratory. Use sterile instruments to ear-tag the mice and place 3-5 mm of the mouse tail into the corresponding 1.5 ml Eppendorf tube. Add 200 μL of lysis buffer and 4 μL of proteinase K to obtain the treated mouse tail. The lysis buffer contains 10 μM Tris-HCl pH 8.0, 10 μM EDTA, 15 mM NaCl, and 0.5% SDS. Digest the treated mouse tail at 55°C overnight to obtain a lysis mixture.

[0073] S222, centrifuging the lysis mixture at 14000g for 10 min to obtain a layered lysis supernatant and impurity precipitate, taking 100uL of the lysis supernatant and mixing it with an equal volume of isopropanol, vortexing it for 10-20s to form an isopropanol-DNA mixture, centrifuging the isopropanol-DNA mixture at 14000g for 15 min to obtain a layered isopropanol supernatant and a DNA precipitate, discarding the isopropanol supernatant, adding 75% alcohol to the DNA precipitate for washing, and vortexing it for 10-20s to form an alcohol-DNA suspension, centrifuging the alcohol-DNA suspension at 14000g for 10 min to obtain an alcohol supernatant and a purified DNA precipitate, discarding the alcohol supernatant, and air-drying the purified DNA precipitate at room temperature for 30 min, adding 100uL ddH2O or TE water to dissolve it to obtain a DNA solution;

[0074] S223, according to the PCR reaction system, a corresponding program is selected and configured accordingly to obtain a PCR amplification product, and then band analysis of the PCR amplification product is performed by agarose gel electrophoresis.

[0075] PCR detection was performed using primers F2 and R1, where the sequence of primer R1 was 5'-AGTAACCCTAAGGGTGGCAGCT-3', confirming that Cre-mediated Slc7a11 gene deletion was manifested as a 235 bp band.

[0076] The identification method of the atrophic gastritis SPEM lesion model, based on the method for constructing the atrophic gastritis SPEM lesion model, comprises the following steps: S1, pathological model verification: taking mouse gastric mucosal tissue sections, using immunofluorescence double labeling technology to detect the double-positive co-localization expression of mouse gastric mucosal markers GIF and GSⅡ, quantitatively analyzing the SPEM area ratio, that is, the positive signal ratio, detecting Mist1 and SPEM-related markers Clusterin-1 and HE4 by immunoblotting; using immunoblotting experiments to detect the expression level of inflammatory factors, inflammatory factors including downstream related factors such as IL-33 and NLRP3, wherein the double immunofluorescence staining detection method of gastric mucosal tissue GIF and GSⅡ comprises the following steps:

[0077] S11, paraffin-embedded mouse gastric mucosal tissue sections were obtained, dewaxed in xylene, hydrated with graded ethanol, and antigen heat retrieval was performed using pH 6.0 sodium citrate buffer;

[0078] S12, rinse three times with phosphate buffer for 5 min each time, then add serum homologous to the secondary antibody and incubate at room temperature for 30 min to block nonspecific binding sites;

[0079] S13, add diluted primary antibody solution, GIF and GSⅡ dropwise and incubate overnight at 4°C;

[0080] S14, rinse with phosphate buffer three times, 5 minutes each time, select the corresponding secondary antibody according to the species of the primary antibody, incubate for 2 hours in the dark, and then rinse again with phosphate buffer three times, 5 minutes each time;

[0081] S15, add 50 μl of DAPI staining solution, incubate for 10 min in the dark, and then stain the cell nuclei. Rinse with phosphate buffer three times, 5 min each time.

[0082] S16, the slides were mounted with anti-fluorescence quenching mounting medium and stored at 4°C in the dark until detection;

[0083] S17, take pictures by laser confocal microscopy, count the ratios of GIF, GSⅡ single-positive cells and GIF+GSⅡ+double-positive SPEM cells, and record the results.

[0084] Combined with the presence of SPEM in the gastric mucosa, Slc7a11 knockout induced SPEM pathological characteristics.

[0085] S2, long-term pathological evolution monitoring: After the parietal cell-specific Slc7a11 was knocked out, the changes in the number of surface mucus cells, enteroendocrine cell chromogranin, intestinal stem cells and crypt cells in gastric mucosal tissue were analyzed. When the parietal cell-specific SLC7A11 was knocked out, the number of surface mucus cells (MUC5AC+), enteroendocrine cell chromogranin A (Chr-A+), intestinal stem cells (GPR49 / LGR5+) and crypt cells in gastric mucosal tissue (Doublecortin (E-6)+) increased. This indicates that the knockout of SLC7A11-specific gastric mucosal parietal cells in mice disrupts the differentiation of gastric mucosal epithelial glands in mice. Among them, the transformation process of the gastric mucosa of the model mice from SPEM to IM was observed, including:

[0086] S21, observe the transformation process of gastric mucosa from SPEM to IM in model mice;

[0087] S22, analysis of changes in gene expression profiles during the transformation of SPEM to IM.

[0088] The specific implementation process is as follows: Construction and genotyping of the Slc7a11 conditional knockout mouse model based on this protocol:

[0089] 1.1 Experimental Purpose

[0090] A gastric parietal cell-specific Slc7a11 knockout mouse model was constructed by crossing Slc7a11 flox / flox mice with ATP4b-Cre mice, and genotyping and identification were performed.

[0091] 1.2 Experimental Materials

[0092] Mouse strains: C57BL / 6N-Slc7a11em1flox / Cya (Slc7a11 flox / flox mice), ATP4b-Cre transgenic mice.

[0093] Reagents: TaKaRa MiniBEST universal genomic DNA extraction kit (Ver.5.0), Premix Taq polymerase (Vazyme P222), primers F2, R2, primer-1, primer-2.

[0094] Instruments: PCR instrument, centrifuge, electrophoresis instrument.

[0095] 1.3 Experimental Procedure

[0096] 1.3.1 Mouse hybridization: Slc7a11 flox / flox mice were hybridized with ATP4b-Cre mice to obtain F1 mice. Slc7a11 flox / + / ATP4b-Cre individuals were selected from the F1 mice. These were then backcrossed with Slc7a11 flox / flox mice to obtain F2 mice. Slc7a11 flox / flox / ATP4b-Cre mice (i.e., Slc7a11- / - / ATP4b-Cre mice) were selected from the F2 generation.

[0097] 1.3.2 Genomic DNA Extraction: Take a 2-5 mm mouse tail segment, add 180 μL Buffer GL, 20 μL Proteinase K, and 10 μL RNase A, and incubate at 56°C overnight. Add 200 μL Buffer GB and 200 μL anhydrous ethanol, mix well, transfer to a spin column, and centrifuge at 12,000 rpm for 2 minutes. Wash the spin column with 500 μL Buffer WA and 700 μL Buffer WB, and finally elute the DNA with 50-200 μL sterile water.

[0098] 1.3.3 Genotyping PCR: Based on the S2 PCR reaction system, after an additional extension at 72°C for 5 minutes, 1.5% agarose gel electrophoresis was performed using the GeneRuler 100 bp DNA Ladder as a marker. Bands of 216 bp (Slc7a11flox), 155 bp (wild type), and 800 bp (Cre) were observed.

[0099] 1.4 Experimental Results

[0100] Genotyping results: Slc7a11 flox / flox mice: 216 bp band.

[0101] Slc7a11 flox / + / ATP4b-Cre mice: 216bp and 155bp bands, and an 800bp Cre band also appeared.

[0102] Slc7a11 flox / flox / ATP4b-Cre mice: 216 bp band, and 800 bp Cre band also appeared.

[0103] 1.5 Conclusion

[0104] A gastric parietal cell-specific Slc7a11 knockout mouse model was successfully constructed, and its genetic background was verified by genotyping.

[0105] Example 2:

[0106] The difference from Example 1 is that, as shown in the attached Figure 3-11 The specific implementation process is as follows: Based on this protocol, the SPEM pathological characteristics of Slc7a11 knockout mice were verified:

[0107] 2.1 Experimental Purpose

[0108] Verify whether Slc7a11- / - / ATP4b-Cre mice can simulate the pathological characteristics of SPEM, including changes in TFF2 expression, inflammatory factor levels and gastric acid secretion function.

[0109] 2.2 Experimental Materials

[0110] Mice: Slc7a11- / - / ATP4b-Cre mice (experimental group) and wild-type C57BL / 6N mice (control group).

[0111] Immunoblotting: Antibodies to SLC7A11, NLRP3, IL-33, ASC, Mist1, Clusterin-1, and HE4.

[0112] Immunofluorescence: GIF (gastric oxyntic cell marker), GSII (SPEM marker) and TFF2 antibodies.

[0113] Reagents: RIPA lysis buffer, SDS-PAGE gel precast, ECL chemiluminescent substrate, DAPI staining solution, anti-fluorescence quenching mounting medium.

[0114] Instruments: microscope (including fluorescence module), laser confocal microscope, microplate reader, pH meter, vertical electrophoresis tank, semi-dry transfer apparatus, chemiluminescence imaging system, slicer, antigen retrieval instrument and light-proof incubator.

[0115] 2.3 Experimental Procedure

[0116] 2.3.1 Tissue sample collection: After killing mice, gastric mucosal tissue was obtained, one part of which was used for paraffin embedding and sectioning, and the other part was used for RNA extraction.

[0117] 2.3.2 TFF2 immunohistochemical staining: TFF2 immunohistochemical staining method steps in S1 based on the identification method of the atrophic gastritis SPEM lesion model.

[0118] 2.3.3 Immunoblotting experiment: Gastric mucosal tissue was collected and added with RIPA lysis buffer (containing protease inhibitors). After homogenization, the cells were centrifuged at 4°C (12,000g, 15 min). The supernatant was collected to determine the protein concentration. 30 μg of protein was loaded and separated by SDS-PAGE gel electrophoresis. The membrane was then semi-dry transferred to a PVDF membrane and blocked with 5% skim milk for 1 h. The membrane was incubated with primary antibodies (such as SLC7A11, 1:1000; Mist1, 1:500) at 4°C overnight and HRP-conjugated secondary antibodies (1:5000) at room temperature for 1 h. The membrane was exposed to ECL chemiluminescent substrate, and the signals were captured using a chemiluminescent imaging system. The expression of the target protein was quantified using ImageJ.

[0119] 2.3.4 Immunofluorescence double labeling detection: The double immunofluorescence staining detection method of GIF and GSⅡ in gastric mucosal tissue in Example 1 was used for detection.

[0120] 2.3.5 Detection of inflammatory factors: RNA was extracted from gastric mucosal tissue and reverse transcribed into cDNA. The expression levels of IL-6 and TNF-α were detected by qPCR, with GAPDH as the internal reference; the expression of IL-33 and NLRP3 proteins were detected.

[0121] 2.3.6 Gastric juice pH determination: Take the gastric juice of mice and measure its pH value using a pH meter.

[0122] 2.4 Experimental Results

[0123] TFF2 expression: The proportion of TFF2-positive cells in the gastric mucosa of Slc7a11- / - / ATP4b-Cre mice was ≥30%, which was significantly higher than the proportion of TFF2-positive cells <5% in the wild type.

[0124] Immunofluorescence results: The proportion of GIF / GSII double-positive cells in the experimental group was ≥30%.

[0125] Immunoblotting results showed that SLC7A11 protein expression was absent in the experimental group, and Mist1 expression was downregulated by ≥50%. Clusterin-1 and HE4 protein expression were upregulated by ≥2 times. IL-33 and NLRP3 protein levels were increased by ≥1.5 times compared with the control group.

[0126] Inflammatory factor levels: IL-6 and TNF-α expression levels were ≥2 times higher than those of the wild type.

[0127] Gastric juice pH: The gastric juice pH of Slc7a11- / - / ATP4b-Cre mice was ≥4.0, which was significantly higher than the wild type pH of ≤2.5.

[0128] 2.5 Conclusion:

[0129] The Slc7a11- / - / ATP4b-Cre mouse model successfully mimics the pathological features of SPEM. At the molecular level, SLC7A11 deficiency leads to downregulation of Mist1 and upregulation of Clusterin-1 / HE4. Pathological hallmarks include a significant increase in TFF2 and GIF / GSII double-positive cells. Furthermore, within the inflammatory microenvironment, IL-33 / NLRP3 signaling is activated, IL-6 / TNF-α expression is elevated, and gastric acid secretion is impaired. This model demonstrates its potential for studying the mechanisms of SPEM and for drug screening.

[0130] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for constructing a SPEM lesion model of atrophic gastritis, characterized in that: The following steps are involved: S1. Construction of Slc7a11 conditional knockout mouse strain: Slc7a11 flox / flox mice were co-bred with ATP4b-Cre transgenic mice to achieve gastric parietal cell-specific Slc7a11 gene knockout using the Cre-loxP system; S2, genotyping identification: Multiplex PCR was performed using Slc7a11 flox locus primers F2 and R2, as well as Cre gene primers-1 and -2. Homozygous, heterozygous, and Cre-positive individuals were distinguished by PCR reaction. PCR detection using primers F2 and R1 confirmed that Cre-mediated Slc7a11 gene deletion manifested as a 235-bp band.

2. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 1, characterized in that: In S1, the Slc7a11 flox / flox mouse strain is C57BL / 6N-Slc7a11em1flox / Cya, and the ATP4b-Cre transgenic mouse strain is a gastric parietal cell-specific Cre-expressing mouse.

3. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 2, wherein: In S2, the sequence of primer F2 is 5′-AGACGTGATTCAGTTGTTGTTCCT-3′; The sequence of primer R2 is 5′-AAGGGGATGCTTTGCTATTCTCTA-3′; Primers F2 and R2 were used to amplify 216 bp and 155 bp bands; The sequence of Cre gene primer-1 is 5′-GCAGATAGCAAGCAAGCTCCAACC-3′; The sequence of Cre gene primer-2 is 5′-GGATTAACATTCTCCCACCGTCAG-3′; Cre gene primer-1 and Cre gene primer-2 were used to amplify an 800 bp band.

4. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 3, characterized in that: In S2, the sequence of primer R1 is 5′-AGTAACCCTAAGGGTGGCAGCT-3′.

5. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 4, characterized in that: In S2, the PCR reaction system was performed in a volume of 25 μL under standard conditions for 35 cycles. The PCR mixture contained 1.5 μL genomic DNA, 1.0 μL product primer F1, 1.0 μL product primer R1, 12.5 μL Premix Taq, and 9.0 μL ddH2O, wherein the primer concentration was 10 μM.

6. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 5, characterized in that: The 35 cycles include the following steps: S211, initial denaturation at 94°C for 3 min; S212, denaturation was continued at 94°C for 30 seconds; S213, annealing at 60 °C for 35 s and 35 cycles; S214, extension at 72°C for 35 seconds; S215, additional extension at 72°C for 5 minutes.

7. The method for constructing a SPEM lesion model of atrophic gastritis according to claim 6, characterized in that: Extraction of genomic DNA included in the PCR mixture involves the following steps: S221: After wearing sterile gloves, masks, caps, and sterile clothing, enter the SPF animal laboratory. Use sterile instruments to ear-tag mice and place 3-5 mm of mouse tails into corresponding 1.5 ml Eppendorf tubes. Add 200 μL of lysis buffer and 4 μL of proteinase K to obtain treated mouse tails. The lysis buffer contains 10 μM Tris-HCl pH 8.0, 10 μM EDTA, 15 mM NaCl, and 0.5% SDS. Digest the treated mouse tails at 55°C overnight to obtain a lysis mixture. S222, centrifuging the lysis mixture at 14000g for 10 min to obtain a layered lysis supernatant and impurity precipitate, taking 100uL of the lysis supernatant and mixing it with an equal volume of isopropanol, vortexing it for 10-20s to form an isopropanol-DNA mixture, centrifuging the isopropanol-DNA mixture at 14000g for 15 min to obtain a layered isopropanol supernatant and a DNA precipitate, discarding the isopropanol supernatant, adding 75% alcohol to the DNA precipitate for washing, and vortexing it for 10-20s to form an alcohol-DNA suspension, centrifuging the alcohol-DNA suspension at 14000g for 10 min to obtain an alcohol supernatant and a purified DNA precipitate, discarding the alcohol supernatant, and air-drying the purified DNA precipitate at room temperature for 30 min, adding 100uL ddH2O or TE water to dissolve it to obtain a DNA solution; S223, according to the PCR reaction system, a corresponding program is selected and configured accordingly to obtain a PCR amplification product, and then band analysis of the PCR amplification product is performed by agarose gel electrophoresis.

8. An identification method based on a SPEM lesion model of atrophic gastritis, based on the method for constructing a SPEM lesion model of atrophic gastritis according to claims 1-7, characterized in that: The following steps are involved: S1. Pathological model validation: Mouse gastric mucosal tissue sections were obtained and immunofluorescence double-labeling was used to detect the co-localization of the gastric mucosal markers GIF and GSⅡ. The SPEM area ratio, i.e., the proportion of positive signals, was quantitatively analyzed. Mist1 and SPEM-related markers Clusterin-1 and HE4 were detected by immunoblotting. Immunoblotting was used to detect the expression levels of inflammatory factors, including downstream related factors such as IL-33 and NLRP3. Combined with the presence of SPEM in the gastric mucosa, the pathological characteristics of SPEM induced by Slc7a11 knockout were confirmed. S2, long-term pathological evolution monitoring: after parietal cell-specific Slc7a11 knockout, the number of surface mucous cells, enteroendocrine cell chromogranin, as well as intestinal stem cells and crypt cells in gastric mucosal tissue were analyzed.

9. The identification method based on the atrophic gastritis SPEM lesion model according to claim 8, characterized in that: In S1, the double immunofluorescence staining method for GIF and GSⅡ in gastric mucosal tissue includes the following steps: S11, paraffin-embedded mouse gastric mucosal tissue sections were obtained, dewaxed in xylene, hydrated with graded ethanol, and antigen heat retrieval was performed using pH 6.0 sodium citrate buffer; S12, rinse three times with phosphate buffer for 5 min each time, then add serum homologous to the secondary antibody and incubate at room temperature for 30 min to block nonspecific binding sites; S13, add diluted primary antibody solution, GIF and GSⅡ dropwise and incubate overnight at 4°C; S14, rinse with phosphate buffer three times, 5 minutes each time, select the corresponding secondary antibody according to the species of the primary antibody, incubate for 2 hours in the dark, and then rinse again with phosphate buffer three times, 5 minutes each time; S15, add 50 μl of DAPI staining solution, incubate for 10 min in the dark, and then stain the cell nuclei. Rinse with phosphate buffer three times, 5 min each time. S16, the slides were mounted with anti-fluorescence quenching mounting medium and stored at 4°C in the dark until detection; S17, take pictures by laser confocal microscopy, count the ratios of GIF, GSⅡ single-positive cells and GIF+GSⅡ+double-positive SPEM cells, and record the results.

10. The identification method based on the atrophic gastritis SPEM lesion model according to claim 9, characterized in that: In S2, when the numbers of surface mucous cells, enteroendocrine cell chromogranin, intestinal stem cells, and crypt cells in gastric mucosal tissue increased, it indicated that gastric mucosal parietal cell-specific SLC7A11 knockout in mice disrupted the differentiation of gastric mucosal epithelial glands in mice.

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