Construction method of gastric mucosa intestinal epithelial metaplasm type organ model with goblet cell phenotype

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a construction method of a gastric mucosa intestinal epithelial metaplasm type organ model with goblet cell phenotype. The model is characterized by MUC2 + MUC6 + goblet cells, and histological characteristics of gastric mucosa intestinal metaplasia are more deeply simulated from the aspect of cell phenotype. The construction method comprises the following steps: (1) obtaining a human stomach gland to prepare an organoid; (2) subculturing the organoid; (3) construction of a pCDH-CMV-MCS-EF1-CDX2-Puro overexpression lentiviral vector, and construction of a pCDH- (4) virus packaging; (5) constructing CDX2 overexpression stably transfected cells and organoid; and (6) carrying out gradient differentiation on the CDX2 overexpression organoid to prepare the intestinal metaplasia organoid. According to the invention, the pathological transformation of the cytology level in the gastrointestinal mucosa intestinal epithelial metaplasia process is realized in the in-vitro model for the first time, and a new research model and theoretical guidance are provided for the diagnosis, prevention and individualized precise treatment of precancerous lesions of gastric cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for constructing a gastric mucosal intestinal metaplasia organoid model with a goblet cell phenotype, and in particular to a method for constructing an intestinal metaplasia organoid model using primary organoids derived from human gastric tissue. Background Art

[0002] Intestinal metaplasia (GIM) refers to the replacement of differentiated cells in the same tissue by another differentiated cell. It is a precursor to low-grade dysplasia and may further develop into cancer. Epidemiological studies have confirmed that intestinal metaplasia is one of the important risk factors for gastric cancer. Gastric intestinal metaplasia marks the transformation of gastric mucosa into an intestinal phenotype and can occur in all areas of the stomach, including the cardia, body, fundus and antrum. Intestinal metaplasia in the cardia is also known as Barret's esophagus / ulcer (BE). Long-term chronic inflammation can lead to mucosal atrophy, followed by intestinal metaplastic lesions, characterized by the appearance of intestinal-specific goblet cells, and the course of the disease often lasts for decades. These two types of metaplasia often coexist in patients with chronic atrophic gastritis and gastric cancer. At present, intestinal metaplasia can be divided into three types: complete (type I, small intestinal type) intestinal metaplasia, with increased expression of intestinal mucin 2 (MUC2) and loss of gastric mucin 1 (MUC1), MUC5AC and MUC6; incomplete (large intestinal type) intestinal metaplasia, which is structurally similar to colon glands, has no brush border, and expresses both gastric neutral mucin and intestinal acidic mucin. According to the type of acidic mucin, it can be further divided into type II intestinal metaplasia (expressing sialylated mucin) and type III intestinal metaplasia (expressing sulfated mucin), which can be distinguished by Alcian Blue Periodic Acid Schiff (AB-PAS) and High Iron Diamine / Alcian Blue (HID-AB). Among them, type III intestinal metaplasia is considered to be the type with the highest risk of gastric cancer.

[0003] Due to the lack of a suitable model to simulate the process of metaplasia in humans, the cellular origin of intestinal metaplasia and its exact molecular mechanism have not been fully elucidated. In the past 20 years, researchers have developed many mouse models of induced intestinal metaplasia to study the physiological and pathophysiological characteristics of this process; however, these models cannot truly reflect the course of intestinal metaplasia in humans. Therefore, there is an urgent need to develop a model that can better reflect the intestinal metaplasia lesions in humans.

[0004] Organoids are organ-specific stem cell clusters of a tissue that can maintain the function of the original organ and be induced to differentiate into most of the cells of its source tissue under specific conditions. This near-physiological 3D model helps to accurately study a series of in vivo biological processes, including tissue renewal, stem cell / niche function, and can be used for drug screening, transplantation, and combined with CRISPR-mediated editing for modeling of diseases including cancer. In particular, gastric organoids can differentiate into gastric epithelial progenitor cells and other mature epithelial cells under appropriate conditions.

[0005] Normal gastric and gastric cancer organoid models were established as early as 2016 and have been widely used to study host-microbe interactions. However, so far, no real "intestinal metaplasia" organoid has been established. In the existing technology, some studies have isolated and cultured organoids from tissues of patients with intestinal metaplasia. Although they have highly expressed intestinal metaplasia marker CDX2, they do not have other characteristic molecular markers of intestinal metaplasia; other studies have used normal gastric organoids to overexpress CDX2 to reprogram mouse organoids into a mixed gastrointestinal state, while expressing some gastric and intestinal molecular markers. Another study showed that using induced pluripotent stem cell-derived organoids, overexpression of CDX2 induced upregulation of the expression of some intestinal metaplasia-related genes, but no typical intestinal metaplastic cell phenotype appeared. These research results show that the establishment of the "intestinal metaplasia" organoid model is highly feasible, but its methods still need to be improved to better reflect the course of intestinal metaplasia in humans.

[0006] Some existing studies have attempted to construct an "intestinal metaplasia" organoid model, but in these studies, the models established are only based on changes in molecular markers, and there is no organoid model that simulates intestinal metaplasia at the cell phenotype level, so it cannot be called a true "intestinal metaplasia" model. Based on the existing model method, the present invention improves the organoid modeling method, aiming to construct an intestinal metaplasia organoid model with a characteristic goblet cell phenotype, and explore the molecular mechanism of intestinal metaplasia based on this model, as a new organoid model for screening therapeutic drugs that reverse intestinal metaplasia in the future. Summary of the invention

[0007] The purpose of the present invention is to construct a gastric mucosal intestinal metaplasia organoid model using primary organoids from human gastric tissue. + MUC6 +The goblet cells are characterized by more in-depth simulation of the histological characteristics of gastric mucosal intestinal metaplasia from the cell phenotype. The present invention realizes the pathological changes at the cytological level in the process of gastrointestinal mucosal intestinal metaplasia in an in vitro model for the first time, thereby providing a deeper understanding of the cytological and molecular mechanisms of the occurrence and development of gastric mucosal intestinal metaplasia, and providing a new research model and theoretical guidance for the diagnosis, prevention and individualized precision treatment of gastric cancer precancerous lesions.

[0008] Another object of the present invention is to provide the use of the gastric mucosal intestinal metaplasia organoid model in the preparation of intestinal metaplasia therapeutic products.

[0009] To achieve the above object, the technical solution created by the present invention is implemented as follows:

[0010] A method for constructing a gastric mucosal intestinal metaplasia organoid model with a goblet cell phenotype, comprising the following steps:

[0011] (i) Obtaining human gastric glands to prepare organoids;

[0012] (ii) Subculture of organoids;

[0013] (III) Construction of pCDH-CMV-MCS-EF1-CDX2-Puro overexpression lentiviral vector;

[0014] (iv) Virus packaging;

[0015] (V) Construction of CDX2-overexpressing stable cells and organoids;

[0016] (VI) CDX2 overexpression organoids were gradient differentiated to prepare gastric mucosal intestinal metaplasia organoids.

[0017] Specifically, in step (i), the gastric glands are isolated from the gastric mucosa of obese patients undergoing sleeve gastrectomy.

[0018] Specifically, the main operation of step (i) is to clean the gastric mucosal tissue of obese patients undergoing sleeve gastrectomy, remove fat, muscle layer and connective tissue, cut into pieces, collect tissue pieces by centrifugation, digest, incubate, terminate digestion, collect tissue fragments by centrifugation, separate gastric glands, add preheated 3D culture medium, and perform 3D organoid culture.

[0019] Specifically, in step (i), before adding the preheated 3D culture medium, Matrigel is first added to the isolated gastric glands and cultured for 15-20 minutes.

[0020] Specifically, in step (a), the main components of the preheated 3D culture medium are: Advanced DMEM / F12, 100 ng / mL Wnt3A, 500 ng / mL R-spondin1, 1% Glutamax, 2% B27, 1% N 2 ,20ng / mL human EGF, 150ng / mL human noggin, 150ng / mL human FGF-10, 1.25mM NacetylLcystein, 10mMnicotinamide, 10nM human gastrin, 2mM SB202190 and 1mM A83-01.

[0021] Specifically, in step (a), the 3D organoid culture conditions are 37°C, 5% CO 2 , 95% humidity.

[0022] Specifically, the main operation of step (ii) is to discard the old culture medium in the culture plate, add Advanced DMEM / F12 culture medium without fetal bovine serum FBS pre-cooled to 4°C, collect the organoids by centrifugation, digest, incubate, terminate digestion, collect the digested cells by centrifugation, add preheated 3D culture medium, and culture 3D organoids.

[0023] Specifically, in step (ii), before adding the preheated 3D culture medium, Matrigel matrix gel is first added to the digested cells and cultured for 15-20 minutes; the 3D organoid culture conditions in step (ii) are the same as those in step (i).

[0024] Specifically, the main operation of step (iii) is to first double-digest human-CDX2 CDS (i.e., CDX2 cDNAClone, NCBI No. NM_001265.6), and double-digest the pCDH-CMV-MCS-EF1-Puro cloning vector, and connect the double-digestion products to obtain the recombinant plasmid pCDH-CDX2; transform the recombinant plasmid into Escherichia coli DH10B competent cells, screen resistant single colonies, and extract the recombinant plasmid for later use.

[0025] Specifically, in step (iii), the human-CDX2 CDS double enzyme digestion system is:

[0026]

[0027] Specifically, in step (iii), the double restriction enzyme digestion system of the pCDH-CMV-MCS-EF1-Puro cloning vector is:

[0028]

[0029] Specifically, in step (iii), the ligation reaction system for the linearized vector pCDH-CMV-MCS-EF1-Puro and the enzyme-digested product human-CDX2CDS is:

[0030]

[0031] Specifically, the main operation of step (iv) is to digest the HEK293T cells in the logarithmic growth phase into a cell suspension and count them, spread them into a 100 mm culture dish, supplement DMEM high-glucose medium, and perform virus transfection packaging when the cells grow to a density of 70%, prepare a plasmid-PEI transfection complex, and add it to the cells in the culture dish, culture them in an incubator for 24-48 hours, collect the supernatant by centrifugation, and obtain a lentiviral suspension, determine the virus titer, aliquot, and store at low temperature.

[0032] Specifically, in step (iv), the preparation method of the plasmid-PEI transfection complex is: add 800 μL of Opti-MEM culture medium to a centrifuge tube, then add recombinant plasmid pCDH-CDX2, pCMV-psPAX2, and pMD2.G plasmid in a molar ratio of 1:1:1, with a total amount of 10 μg; mix and let stand for 3 minutes; add 30 μL of PEI transfection reagent, mix, and let stand for 20 minutes.

[0033] Specifically, the main operation of step (v) is to take out the lentivirus stored at low temperature (-80°C), melt it at 4°C, digest the organoids in step (i) into single cells, and then infect the digested organoid cells with the lentivirus, resuspend the cells, collect them by centrifugation, and resuspend the organoids with Matrigel gel. After an interval of 3-4 days, the infection efficiency is determined according to the growth of the organoids and the expression of green fluorescent protein (GFP), and subculture and screening are performed. After 10-15 days of screening, stably overexpressed organoids can be obtained.

[0034] Specifically, in step (v), when the lentivirus infects the digested cells, the specific operation is: add polybrene at a ratio of 8 μg / mL of the lentivirus, and evenly mix the lentivirus and the organoid cells at a multiplicity of infection MOI=1000.

[0035] Specifically, in step (v), during screening, a culture medium containing puromycin (1 μg / mL) is used for screening.

[0036] Specifically, the main operation of step (six) is to induce multi-stage differentiation of the organoid cells in step (fifth) after continuous in vitro passage, before sampling and detection, and harvest the organoids for phenotypic identification after 10-12 days of differentiation.

[0037] Specifically, in step (six), the multi-stage induced differentiation is as follows: 6 days before differentiation, EGF (epidermal growth factor), FGF-10 (recombinant human fibroblast growth factor-10) and CHIP99021 are supplemented; on the 7th and 8th days, PD0325901 and BMP4 are supplemented, and the EGF is reduced to 1 / 10 of the original amount; on the 9th and 10th days, PD0325901 and BMP4 are removed.

[0038] Furthermore, based on a general inventive concept, the present invention also provides a gastric mucosal intestinal metaplasia organoid model constructed by the above method. After single-cell sequencing identification, the model constructed by the present invention is a type III intestinal metaplasia organoid model.

[0039] Furthermore, based on a general inventive concept, the present invention also provides the use of the gastric mucosal intestinal metaplasia organoid model in the diagnosis, prevention or treatment of gastric cancer precancerous lesions.

[0040] Specifically, the present invention also provides the use of the gastric mucosal intestinal metaplasia organoid model in the development or screening of products for preventing and / or treating gastric mucosal metaplasia lesions and gastric cancer.

[0041] Specifically, the present invention also provides the use of the gastric mucosal intestinal metaplasia organoid model in screening intestinal metaplasia treatment products.

[0042] Furthermore, the product is a drug, a reagent or a kit.

[0043] Compared with the prior art, the beneficial effects of the present invention are:

[0044] 1. The present invention uses normal gastric organoids derived from human gastric mucosa, and constructs an intestinal metaplasia organoid model by overexpressing CDX2 in the organoids and inducing differentiation. Morphological identification shows that the control group (EV) maintains a spheroid morphology, while the intestinal metaplasia group (OV) organoid morphology is significantly changed, with the appearance of a large number of crypt structures.

[0045] 2. After further inducing cell differentiation, the appearance of goblet cells was confirmed by H&E staining, AB-PAS staining, and HID-AB staining. These cells expressed sulfated acidic mucins, which appeared brown in HID-AB staining. Further immunofluorescence staining results showed that the expression of gastric mucin MUC5AC in the "intestinalized" organoids was significantly reduced, while the expression of intestinal mucin MUC2 was increased.

[0046] 3. Single-cell sequencing results showed that the intestinal lineage cells in the intestinal metaplasia group organoids increased significantly, and two groups of goblet cells were identified. The C19 group of non-classical goblet cells were characterized by the expression of markers FCGBP and DMBT1, while the C24 classical goblet cells were characterized by the co-expression of markers MUC2, FCGBP and MUC6. Multiple identification results showed that the present invention successfully constructed a type III intestinal metaplasia organoid model.

[0047] 4. Based on the existing model method, this paper improves the organoid modeling method and constructs the first MUC2 + MUC6 + The intestinal metaplasia organoid model characterized by goblet cells achieved the histological transformation from stomach to intestine in vitro, which is of great significance for the subsequent study of the molecular mechanism and therapeutic drugs of intestinal metaplasia of human gastric mucosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Microscopic images of gastric organoids after overexpression of CDX2; A is the morphological changes of the control group (EV) and the intestinal metaplasia organoid group (OV) with increasing passage times, and B is the proportion of organoids with crypt structures in the OV group; (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001);

[0049] Figure 2 Immunofluorescence was used to identify the overexpression efficiency of CDX2 in the OV group; A is the immunofluorescence staining of CDX2 and the epithelial cell marker E-cadherin, and B is the statistical graph of the mean fluorescence intensity; (****: P < 0.0001);

[0050] Figure 3 Immunofluorescence identification of intestinal marker expression in organoids; A is the immunofluorescence staining of goblet cell marker MUC2 and intestinal stem cell marker OLFM4, and B is the statistical graph of mean fluorescence intensity; (****: P < 0.0001) (goblet cells are in the yellow box);

[0051] Figure 4 Immunofluorescence identification of gastric marker expression in organoids; A is the immunofluorescence staining of gastric mucin MUC5AC and cancer stem cell marker LGR5, and B is the statistical graph of mean fluorescence intensity; (****: P < 0.0001);

[0052] Figure 5 These are special stainings for the patient's intestinal metaplasia tissue and organoids; A is Alcian blue staining, B is H&E staining, and C is AB-PAS and HID-AB staining (positively stained goblet cells are shown in the yellow box);

[0053] Figure 6 is the organoid sc-RNA gene expression data; A is the intersection of differentially expressed genes and “intestinalization” genes between OV and EV groups at the meta-cell level; B is the heat map of the average expression values ​​of differentially expressed genes between OV and EV groups; C is the scatter plot of the expression of gastric marker MUC5AC and intestinal markers CDX2, MUC2, and FCGBP in organoids of EV and OV groups;

[0054] Figure 7 This is the heat map of the Top3 gene expressions in different cell clusters of scRNA-seq;

[0055] Figure 8 UPAM images of single-cell data; A is a UPAM image showing 25 cell clusters, and B is a distribution map that divides cells into gastric and intestinal lineages;

[0056] Fig. 9 Bubble plot of the average expression levels of gastric and intestinal marker genes in 25 cell clusters;

[0057] Fig.10 is the expression level of gastric and intestinal marker genes in gastric lineage cells;

[0058] Fig.11 is the expression level of gastric and intestinal marker genes in intestinal lineage cells;

[0059] Fig.12 is the expression level of gastric and intestinal marker genes in mixed lineage cells;

[0060] Fig.13 UPAM images of single cells of organoids in the EV and OV groups;

[0061] Fig.14 UPAM diagram and bubble diagram of the distribution of goblet cell markers MUC2, ZG16, SPINK4, CLCA1, ITLN1, etc. in single cell data;

[0062] Fig.15 This is the GO pathway enrichment analysis diagram of the core genes in different cell clusters;

[0063] Fig.16 Results of KEGG enrichment analysis of core genes in different cell clusters. DETAILED DESCRIPTION

[0064] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0065] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention.

[0066] Main materials and reagents

[0067] 1. Biomaterials

[0068] The human embryonic kidney cell line HEK293T was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. Patient specimens were collected from patients undergoing subtotal gastrectomy at the Fifth Affiliated Hospital of Zhengzhou University, and normal gastric tissue was collected for organoid culture. The following example protocol was approved by the Ethics Committee of the Fifth Affiliated Hospital of Zhengzhou University, and all patients signed informed consent.

[0069] 2. Experimental reagents

[0070] Table 1

[0071]

[0072]

[0073]

[0074] Example 1

[0075] Example 1 provides a method for constructing an intestinal metaplasia organoid model using primary organoids derived from human gastric tissue, aiming to construct a human intestinal metaplasia organoid model; and identifying various tissue and cytological characteristics of the constructed model to determine the successful construction and superiority of the model, specifically comprising the following steps:

[0076] (I) Isolation of gastric glands from gastric mucosa of obese patients undergoing sleeve gastrectomy and preparation of organoids

[0077] (1) Obtain the remaining samples from sleeve gastrectomy in obese patients in the operating room. Place the gastric mucosal tissue in a sterile culture dish and rinse with PBS to remove fat, muscle layer and connective tissue. Then place it in a 12-well plate, add 2 ml of PBS (containing 1% penicillin / streptomycin mixture) and place it on the operating table at room temperature for 30 minutes.

[0078] (2) Cut the tissue into pieces using sterile ophthalmic scissors and collect the pieces by centrifugation at 4°C and 1500 rpm for 5 min. Add chelating solution (distilled water, 5.6 mmol / L Na 2 HPO 4 , 8mmol / L KH 2 PO 4, 96.2mmol / L NaCl, 1.6mmol / L KCl, 43.4mmol / L sucrose, 54.9mmol / L D-sorbitol, 0.5mmol / L DL-dithiothreitol, 2mmol / L EDTA (pass through a 0.22μm filter after the above reagents are prepared) to digest the tissue, place it on a shaker, and incubate at 37℃ for 30min;

[0079] (3) The digestion was terminated with Advanced DMEM / F12 medium containing 10% fetal bovine serum (FBS), and the tissue fragments were collected by centrifugation at 1500 rpm for 5 min at 4°C. The fragments were placed in a culture dish and gently squeezed with a glass slide to separate the gastric glands.

[0080] (4) Filter through a 100 μm cell sieve to remove undigested tissue fragments, and then centrifuge at 1500 rpm for 5 min at 4°C;

[0081] (5) Discard the supernatant, add 40 μL of Matrigel (Corning 356231, growth factor-reduced matrix gel) to each well, mix gently on ice, and add to the preheated 24-well plate; place in the incubator for 15-20 min; preheat 3D culture medium (main components are Advanced DMEM / F12, 100 ng / mL Wnt3A, 500 ng / mL R-spondin1, 1% Glutamax, 2% B27, 1% N 2 ,20ng / mL human EGF, 150ng / mL human noggin, 150ng / mL human FGF-10, 1.25mMNacetylLcystein, 10mM nicotinamide, 10nM human gastrin, 2mM SB202190 and 1mM A83-01);

[0082] (6) After the Matrigel gel solidifies, add 500 μL of prepared and preheated 3D culture medium to each well; place the wells in a 37°C, 5% CO2, 95% humidity incubator for culture, and replace with fresh culture medium every 3 days.

[0083] 2. Subculture of organoids

[0084] (1) Discard the old culture medium in the culture plate, then add Advanced DMEM / F12 medium without fetal bovine serum (FBS) precooled to 4°C to each well, transfer to a 15 mL centrifuge tube, centrifuge at 4°C and 1200 rpm for 5 min, and discard the supernatant;

[0085] (2) Add 10 times the volume of Matrigel matrix gel to the centrifuge tube, blow evenly with a pipette, and digest in a 37°C water bath for 8 min;

[0086] (3) Add Advanced DMEM / F12 containing 10% fetal bovine serum (FBS) to terminate digestion, and then pipette up and down five times with a syringe;

[0087] (4) Centrifuge at 4°C and 1500 rpm for 5 min, discard the supernatant, add 40 μL of Matrigel to each well, mix gently on ice, add to the preheated 24-well plate, and place in the incubator for 15-20 min; preheat 3D culture medium (same as step (2));

[0088] (5) After the Matrigel gel solidifies, add 500 μL of pre-prepared 3D culture medium to each well and place at 37°C and 5% CO 2 , 95% Humidity incubator, and fresh culture medium was replaced every 3 days.

[0089] (III) Construction of pCDH-CMV-MCS-EF1-CDX2-Puro overexpression lentiviral vector

[0090] (1) human-CDX2 CDS (i.e., CDX2 cDNA Clone, NCBI number NM_001265.6) was synthesized by Changsha Youbao Biotechnology Co., Ltd. Since human-CDX2 CDS contains an EcoR1 restriction site at the 5' end and a Not1 restriction site at the 3' end, the enzyme digestion reaction system in Table 2 was used, and the synthesized product was placed in a PCR instrument and digested at 37°C for 2 h;

[0091] Table 2

[0092]

[0093] (2) pCDH-CMV-MCS-EF1-Puro cloning vector (purchased from Changsha Youbao Biotechnology) was digested with EocR1 and Not1 to linearize it, and then placed in a PCR instrument and incubated at 37°C for 2 h. The reaction system is shown in Table 3 below;

[0094] Table 3

[0095]

[0096]

[0097] (3) The products of step (1) and step (2) were subjected to DNA separation and purification by 1% agarose gel electrophoresis, and DNA was extracted and purified using a gel recovery kit. 2O elution and detection of product concentration in a micro-nucleic acid analyzer;

[0098] (4) Use a seamless cloning kit to connect the linearized vector of step (2) and the enzyme digestion product of step (1). Specifically, prepare the reaction system according to Table 4 below, place it in a PCR instrument, incubate it at 37° C. for 1 h to obtain the recombinant plasmid pCDH-CDX2, measure the concentration, and use it for subsequent transformation;

[0099] Table 4

[0100]

[0101] (5) agarose gel electrophoresis;

[0102] ① Add 100 mL of prepared 1×TAE buffer into a clean conical flask, weigh 1 g of agarose powder, and shake well to dissolve it;

[0103] ② Heat in a microwave oven at high heat to boil for 1 minute; then take out and cool naturally to about 55°C, add Gel-Green Plus nucleic acid dye at a ratio of 1 μL dye per 50 mL volume, shake well and pour into the mold to make gel;

[0104] ③Then let it stand at room temperature and cool naturally for 30 minutes. When the gel solidifies, add 600 mL of 1×TAE buffer to the electrophoresis tank, then put the solidified gel in, and add buffer until the gel surface is about 2 mm above the surface.

[0105] ④ Pull out the gel comb, mix the DNA sample with 5× DNA loading buffer at a ratio of 1:4, and add 8 μL to each spotting well;

[0106] ⑤ Cover the electrophoresis tank, set the voltage to 100V and the time to 30min for electrophoresis; then observe and take pictures under the exposure instrument, and cut out the gel of the specific band to recover the product;

[0107] (6) Plasmid transformation and screening;

[0108] ① Place the DH10B competent cells in an ice box to recover for 15 min, add 200 ng of the recombinant plasmid pCDH-CDX2 obtained in step (4), then gently pipette three times, insert the entire EP tube into ice, and let it stand for 30 min;

[0109] ②After completion, place the cells in a water bath and heat shock at 42°C for 90 seconds to promote the entry of the plasmid into the cells;

[0110] ③Then put the entire EP tube into ice for 5 minutes; after that, add 500 μL LB medium and place it in a 37°C incubator for 1 hour;

[0111] ④Take out the LB solid culture plate containing ampicillin, add an appropriate amount of transformed competent cells to the plate, spread them evenly with an inoculation loop, and place them in a 37°C incubator overnight;

[0112] ⑤ The next day, observe the colony formation on the plate; then add about 10 mL of LB liquid culture medium containing ampicillin to a sterile 15 mL centrifuge tube, then use a 10 μL sterile pipette tip to pick up a colony of appropriate size and put it into a centrifuge tube, place it in a constant temperature shaker, and culture it at 250 rpm and 37°C for 4 hours;

[0113] ⑥ When the bacterial solution becomes turbid, transfer it to a sterile 100mL conical flask and add culture medium to 100mL. Continue to culture overnight and then use it for plasmid extraction;

[0114] (7) Plasmid extraction

[0115] ① Add an equal volume of a portion of the cultured bacterial solution to 40% glycerol and freeze the glycerol bacteria in a -80℃ refrigerator; transfer the remaining bacterial solution to a 50mL sterile centrifuge tube and centrifuge at 12000rpm for 2min;

[0116] ② Discard the supernatant thoroughly, then extract the plasmid according to the instructions of the plasmid extraction kit, and add 8mL ZymoPURE TM P1, vortex to mix to fully lyse bacteria;

[0117] ③ Continue to add 8mL P2 lysis buffer and immediately invert the centrifuge tube 7-8 times; add 8mL P3 solution and gently invert to mix, avoiding violent movements to avoid DNA breakage;

[0118] ④ Then centrifuge at room temperature and 12000rpm for 2 minutes; transfer the supernatant to a new centrifuge tube and add 1 / 3 volume of ZymoPURE TM Buffer, vortex thoroughly to mix, connect the adsorption column to the syringe, and filter in batches;

[0119] ⑤ Add 5 mL of ZymoPURE cleaning solution 1 to the adsorption column and filter; repeat once;

[0120] ⑥ Use 5mL of ZymoPURE containing anhydrous ethanol TM Wash 2: clean the filter again;

[0121] ⑦ Place the adsorption column on the collection tube and spin dry the ethanol at 10,000 g for 2 min;

[0122] ⑧Place the adsorption column in a sterile 1.5mL EP tube, then add about 600μL ddH2O, let it stand for 2min to allow the plasmid to fully bind, then centrifuge at 12000g for 5min to elute, collect the plasmid, measure the purity and concentration with a micro-nucleic acid analyzer, and store it in a -20℃ refrigerator for future use.

[0123] (IV) Virus packaging

[0124] (1) HEK293T cells in the logarithmic growth phase were digested into cell suspension and counted; 3×10 6 The number of cells was plated into a 100 mm culture dish and supplemented with DMEM high glucose medium (containing 10% fetal bovine serum FBS) to 8 mL;

[0125] (2) When the cells grow to a density of 70%, perform virus transfection and packaging; replace with 7.2 mL of DMEM medium without antibiotics;

[0126] (3) Prepare plasmid-PEI transfection complex at a ratio of 800 μL per dish: add 800 μL Opti-MEM medium to a centrifuge tube, then add pCDH empty vector plasmid or pCDH-CDX2, pCMV-psPAX2, pMD2.G plasmid at a molar ratio of 1:1:1, with a total of 10 μg; gently pipette to mix and let stand for 3 minutes; add 30 μL PEI transfection reagent, pipette to mix and let stand for 20 minutes;

[0127] (4) Add the corresponding transfection complex to the corresponding cells, pay attention to slow and even dripping, try not to blow up the cells, shake gently to mix, and place in the incubator; collect the supernatant after 48 hours, centrifuge at 3000 rpm for 10 minutes to remove cell debris, and obtain a lentiviral suspension. The virus titer can be determined using a lentiviral titration kit; store in a -80°C refrigerator after aliquoting.

[0128] (V) CDX2 overexpression stable cells and organoid construction

[0129] (1) Take the aliquoted lentivirus out of the -80°C freezer in advance and thaw it in a 4°C refrigerator;

[0130] (2) Digest the organoids into a single cell suspension according to step (ii), count the cells and take an appropriate amount of cells for infection (generally one well is infected with one virus, and pCDH-co-GFP plasmid (Changsha Youbao Biotechnology) is used as a transfection control to observe the infection efficiency);

[0131] (3) Centrifuge at 1500 rpm for 5 min and discard the supernatant; add polybrene to the lentivirus at a ratio of 8 μg / mL, mix the virus and organoid cells evenly at a multiplicity of infection (MOI) of 1000, and resuspend the cells;

[0132] (4) Place in a horizontal centrifuge and centrifuge at 600 rpm at room temperature (generally set to 25°C) for 90 min;

[0133] (5) Discard the viral supernatant, resuspend the organoids with Matrigel, and follow the normal passaging steps;

[0134] (6) After an interval of 3-4 days, the expression of green fluorescent protein (GFP) was observed under a microscope based on the growth of the organoids to determine the infection efficiency;

[0135] (7) After the organoids are fully grown, they are passaged and screened with complete culture medium containing puromycin (1 μg / mL). Stably overexpressed organoids can be obtained after 15 days of screening. To ensure the infection efficiency, multiple infections and continuous passages can be performed. Organoids infected with the control plasmid are marked as the empty vector group (EV), and the CDX2 overexpression group is marked as OV (overexpress vector). Organoids infected with the GFP control virus are only used to observe the infection efficiency and no subsequent detection experiments are performed.

[0136] (VI) Gradient differentiation of CDX2-overexpressing organoids to generate intestinal metaplastic organoids

[0137] After continuous in vitro passage, the stably transfected organoids were induced to differentiate in multiple stages before sampling and testing. The specific steps are as follows:

[0138] (1) Differentiation medium mainly consists of Advanced DMEM-F12 with 1×N2, 1×B27 (without vitamin A), 2 mM L-glutamine, 1×pen / streptomycin, and 15 mM HEPES;

[0139] (2) Then, according to Table 5 below, growth factors and small molecules were added at different differentiation stages; 6 days before differentiation, EGF (epidermal growth factor) and FGF-10 (recombinant human fibroblast growth factor-10) were supplemented, 1 μg / mL CHIP99021; 1 μg / mL PD0325901 and 50 ng / mL BMP4 were supplemented on days 7-8, and EGF was reduced to 1 / 10 of the original amount; PD0325901 and BMP4 were removed on days 9-10; In addition, the differentiation status needs to be observed in time during the differentiation process. If obvious thick walls are found, the cells can be harvested as soon as possible during differentiation to avoid spheroid collapse;

[0140] Table 5

[0141]

[0142]

[0143] (3) After 10 days of differentiation, organoids were harvested according to the above steps for phenotypic characterization.

[0144] (VII) Phenotypic identification of intestinal metaplasia organoids

[0145] The tissue and cytological characteristics of intestinal metaplasia were identified by H&E staining, AB-PAS staining, HID-AB staining, immunofluorescence, immunohistochemistry and other techniques. The specific steps are as follows:

[0146] 1. H&E staining

[0147] (1) The embedded wax block was cut into 4 μm sections on a paraffin slicer and then baked in a drying oven at 65 °C for 1 h;

[0148] (2) The tissue sections were then dewaxed and immersed in xylene for 15 min, twice; then immersed in anhydrous ethanol (twice), 95%, 85%, and 75% alcohol for 10 min each to fully hydrate the tissue sections;

[0149] (3) Rinse in running tap water for 5 min; wash with PBS three times, 5 min each time;

[0150] (4) The sections were immersed in hematoxylin staining solution for 5 min and then rinsed in running water for 5 min;

[0151] (5) Add 1% concentrated hydrochloric acid to 75% alcohol and immerse the slices in it for 1 second;

[0152] (6) Rinse with running water for 5 minutes and observe the staining under a microscope. If the staining is light, re-stain it.

[0153] (7) Add 1% concentrated ammonia water to distilled water, immerse the sections in anti-blue for 3 seconds, and rinse with running water;

[0154] (8) Place the sections in eosin staining solution for 3 min and rinse with running water;

[0155] (9) Observe the staining condition under a microscope, then hydrate and transparentize the sections. The specific steps are: soak in 75%, 85%, and 95% water for 1 min, soak in anhydrous ethanol for 5 min (twice), and soak in xylene for 10 min (twice);

[0156] (10) Place a small drop of neutral gum on the tissue area and seal the slide with a coverslip; after fully drying in a fume hood, take a photo under a microscope.

[0157] 2. Mucin staining

[0158] (1) Alcian Blue Stain (AB): After the tissue sections were dewaxed and hydrated as described above, they were immersed in Alcian Blue acidification solution for 3 min, and then stained with Alcian Blue staining solution for 30 min. After rinsing with tap water, they were counterstained with Nuclear Fast Red reagent for 5 min. After rinsing, the sections were hydrated and mounted as described above.

[0159] (2) Alcian Blue-Periodic Acid Schiff (AB-PAS) staining: After the sections were dewaxed and hydrated in the same manner as above, they were immersed in AB staining solution for 20 min, washed with distilled water for 3 min, 3 times; oxidant was added for 6 min, washed with water 3 times; Schiff stain was added for 20 min, washed with distilled water for 5 min, 3 times; after completion, the sections were hydrated and sealed;

[0160] (3) High Iron Diamine / Alcian Blue (HID-AB) staining: The dewaxing and hydration steps of the sections were the same as above. The sections were placed in a humidified box, and HID staining solution A and B reagents were mixed in a ratio of 50:3 to prepare a working solution. After adding HID staining, the sections were left at room temperature overnight. The next day, the sections were washed with running water for 5 min, then immersed in Alcian Blue staining solution for 20 min, washed with water, and then stained with Nuclear Fast Red reagent for 5 min. After the completion, the sections were hydrated and sealed. Mucin staining was scored and statistically analyzed according to previously published methods (see the reference Vadivazhagan, Karthikeyan, Kalaivani Amitkumar, and Muthu Sudalaimuthu. "Quantitative Analysis of Mucin Expression Using Combined Alcian Blue-Periodic Acid Schiff (AB-PAS) Stain and Combined High Iron Diamine-Alcian Blue (HID-AB) Stain and the Correlation With Histomorphological Score in Chronic Calculous Cholecystitis." Cureus 14.11(2022)); the specific criteria are: 5 points if the percentage of positive cells in the visual field is 75-100%, 5 points if it is 50%-75%, 4 points if it is 25%-50%, 3 points if it is 5%-25%, 2 points if it is less than 5%, 1 point if it is negative.

[0161] 3. Immunofluorescence

[0162] (1) Slice and dewax using the same method as above;

[0163] (2) Rinse thoroughly with distilled water first, then rinse thoroughly with PBS solution for 3 min, 3 times;

[0164] (3) Heat the prepared sodium citrate antigen retrieval solution in a microwave oven at medium-high temperature for 5 min, place the slide in the oven, and retrieval at medium-high temperature for 20 min. Remove the slide and allow it to cool naturally at room temperature.

[0165] (4) Remove the slices and wash them thoroughly in PBS-T solution for 5 min, three times;

[0166] (5) After gently shaking off the water stains, use an immunohistochemistry pen to draw a circle along the edge of the tissue to mark the tissue position. Place the slice flat in a staining wet box, then add tissue autofluorescence quencher A solution, incubate at room temperature for 30 minutes, and wash with distilled water for 5 minutes;

[0167] (6) Add 50-100 μL of blocking solution (3% BSA dissolved in PBS-T) to each area of ​​the tissue to fully cover the tissue; block at room temperature for 1 hour;

[0168] (7) Dilute the corresponding primary antibodies (CDX2 antibody, E-cadherin antibody, MUC2 antibody, OLFM4 antibody, MUC5AC antibody, LGR5 antibody) directly with blocking solution in appropriate proportions. After blocking, carefully absorb the blocking solution with absorbent paper, then directly add 50-100 μL of the diluted primary antibody to fully cover the tissue and incubate at room temperature for 1 h.

[0169] (8) After the primary antibody incubation, the sections were washed three times with PBS-T for 5 min each time, and the fluorescent secondary antibody was diluted with blocking solution at an appropriate ratio; 50-100 μL of the diluted secondary antibody was added to the tissue area and incubated in the dark at room temperature for 1 h;

[0170] (9) Discard the secondary antibody and wash the cells with PBS-T three times for 5 min. Then, stain the cell nuclei with DAPI solution for 10 min.

[0171] (10) Add tissue autofluorescence quencher solution B, incubate at room temperature in the dark for 5 min, and rinse with running water for 5 min;

[0172] (11) Wash three times with PBS, seal with anti-fluorescence quenching sealing agent, dry at room temperature, and observe and photograph with a confocal microscope;

[0173] (12) Zeiss ZEN software was used to obtain the mean fluorescence intensity for semi-quantitative statistical analysis of protein expression; image J software was used for protein colocalization analysis.

[0174] (VIII) Further characterization of intestinal metaplasia organoid model by single-cell transcriptome sequencing

[0175] (1) Acquisition of “chemigenesis genes”

[0176] Based on previously published sequencing results based on a single intestinal metaplastic gland (see Lee, Hyuk-Joon, et al. "Gene expression profiling of metaplastic lineages identifies CDH17 as a prognostic marker in early stage gastric cancer." Gastroenterology 139.1 (2010): 23-225), we obtained genes differentially expressed in SPEM and intestinal metaplastic glands. These genes were then merged with genes from several reported intestinal metaplasia datasets (see Galamb, Orsolya, et al. "Helicobacter pylori and antrum erosion-specific gene expression patterns: the discriminative role of CXCL13 and VCAM1 transcripts." Helicobacter 13.2 (2008): 112-126; Tanaka S, Nagashima H, Cruz M, et al. Interleukin-17C in Human Helicobacter pylori). Gastritis[J].Infection and immunity,2017,85(10):e00389-17;Companioni O,Sanz-Anquela JM,Pardo ML,et al.Gene expression study and pathway analysis of histological subtypes of intestinal metaplasia thatprogress to gastric cancer[J].PloS one,2017,12(4):e0176043;Moore BD, Jin RU, Osaki L, et al. Identification of alanyl aminopeptidase(CD13) as asurface marker for isolation of mature gastric zymogenic chief cells[J].American journal of physiology Gastrointestinal and liver physiology, 2015, 309(12): G955-964; Singh H, Seruggia D, Madha S, et al. Transcription factor-mediated intestinal metaplasia and the role of a shadow enhancer [J]. Genes & development, 2022, 36(1-2): 38-52), and obtained a list of differentially expressed genes, which were named "metaplasia genes";.

[0177] (2) Single-cell transcriptome sequencing (scRNA-seq)

[0178] ① Single-cell sequencing was performed by Beijing Zebrafish Biology Co., Ltd. according to the 10×Genomics ScFlex protocol; a total of normal gastric organoids and stably transfected organoids from 3 independent individuals were tested;

[0179] ② Digest the organoids to prepare a single-cell suspension, count them, and centrifuge them at 4°C, 400g for 5 minutes;

[0180] ③ Remove the supernatant; add 1 mL 1% BSA-PBS solution and mix well by pipetting;

[0181] ④ Add AO / PI fluorescent dye and use a fluorescent cell counter to detect cell viability and concentration to ensure that the proportion of live cells is above 95% and the total number of cells is 3×10 5 above;

[0182] ⑤ Centrifuge at 400g for 5 min at 4°C in a horizontal refrigerated centrifuge to pellet the cells, add 1 mL of Fixation buffer, and mix gently; place at 4°C for 20 h;

[0183] ⑥ After fixation, centrifuge at 850g for 5 minutes at room temperature, then add 1mL Quenching buffer, mix gently, add 100μL Enhancer and 275μL 50% glycerol in sequence, mix well and store in a -80℃ refrigerator until sequencing library construction; the kit used in this step is the ScFlex single-cell fixation kit from 10×Genomics, USA.

[0184] (3) Single-cell sequencing data analysis

[0185] ① Raw data processing: 10× Genomics raw data were processed using Cell Ranger single-cell software (version 3.0), including converting raw base call files to FASTQ files using cellranger mkfastq, followed by pre-alignment, filtering, and UMI counting using cellranger counts; sequences were aligned to the hg19 reference genome using a prebuilt annotation package downloaded from the 10× Genomics website;

[0186] ②Cell clustering annotation: The samples were integrated into the R package “Seurat” using the anchoring method, and core cells were obtained by filtering scRNA-seq; unqualified cells included genes that could only be detected in 3 or fewer cells, and low-quality cells with less than 200 genes detected were excluded from subsequent analysis; the gene expression of core cells was normalized using a linear regression model, and then the top 2000 genes with highly variable characteristics were screened using analysis of variance; the UMAP algorithm was used to perform an overall dimensionality reduction analysis of the samples; the R package “singleR” was used to use the Human Primary Cell Atlas Data as reference data for auxiliary annotation, and then the CellMarker 2.0 database and previous single-cell sequencing studies on intestinal metaplasia and gastric cancer tissues were used to find marker genes for cells in order to manually annotate different clusters;

[0187] Screening of core cells and functional enrichment analysis of their marker genes: The FindAllMarkers function in the Seurat software package was used to find marker genes for each cluster by setting parameters min.pct=0.2 and only.pos=TRUE. Batch corrected differential analysis was performed using the "Harmony" package. The global characteristic genes of each cluster and comparative identity categories were identified by calculating the average normalized gene expression of individual cells. The threshold was set to P<0.01, |logFC|>2. Differentially expressed genes were identified by multiple comparisons using the Wilcoxon rank sum test and Bonferroni correction. Based on the significantly different marker genes of each cell type, the "clusterProfiler" package was used to perform GO and KEGG functional enrichment analysis on the significantly enriched differential genes in each cell to find the most significant features of each cell.

[0188] Experimental Results

[0189] (I) Identification of morphological changes in constructed intestinal metaplasia organoids

[0190] The present invention constructs an "intestinal metaplasia" organoid model through gastric organoids, overexpresses CDX2 in gastric organoids, and finds that the control group (EV group) maintains the normal round spherical morphology of gastric organoids; in the early stage of induction, gastric organoids overexpressing CDX2 do not show any morphological changes, that is, the intestinal metaplasia organoid group (OV group) has no obvious morphological changes in the first few passages, but after about 6-8 generations of continuous passages, morphological changes begin to appear, and CDX2-overexpressing organoids begin to "bud" and form obvious crypt-like structures, which are very obvious intestinal organoid characteristics, indicating that gastric organoids have undergone "intestinal metaplasia" changes. With continuous passage in vitro, the proportion of spheroids with crypts gradually increases ( Figure 1 ).

[0191] (II) Phenotypic identification of constructed intestinal metaplasia organoids

[0192] (1) Detection of intestinal metaplasia-related markers: Immunofluorescence revealed that there was almost no CDX2 protein in the EV group, while CDX2 was significantly upregulated in the OV group ( Figure 2 )(P<0.05); In addition, goblet cells with strong positive expression of MUC2 appeared in the OV group, and the intestinal progenitor cell marker OLFM4 was significantly higher than that in the EV group ( Figure 3 ); however, the gastric marker MUC5AC-positive cells were significantly reduced, accompanied by the upregulation of the stem cell marker LGR5 ( Figure 4 ).

[0193] (2) Identification by Alcian blue staining, H&E staining, AB-PAS staining, and HID-AB staining: The present invention then identified the organoid phenotype using a variety of methods. Using Alcian blue staining, a large number of goblet cells stained blue were observed using human intestinal metaplasia samples as a control. Cells with blue cytoplasm were also present in the OV group organoids ( Figure 5 A); H&E staining results showed that the OV group organoids had cells with goblet cell morphology, with a narrow nucleus at the bottom and a vacuolar shape at the top ( Figure 5 B); AB-PAS and HID-AB staining were then used to further identify the intestinal metaplasia subtype. AB-PAS staining is used to distinguish between gastric neutral mucin and intestinal acidic mucin. AB-PAS staining of intestinal metaplastic tissue showed both purple and red cells, of which gastric neutral mucin was stained red, while intestinal metaplastic glands secreted acidic mucus, which appeared blue; neutral and acidic mucus coexisted in gastrointestinal mixed glands, so they were stained brown-purple; brown-purple cells also appeared in the OV group organoids, but not in the EV group ( Figure 5 C), indicating that this is a mixed type of intestinal metaplasia (incomplete intestinal metaplasia).

[0194] HID-AB is mainly used to identify sulfated acidic mucus and sialic acid mucus. In the HID-AB staining of tissues, goblet cells are stained in two colors. Blue is the nitrogen-acetylated sialic acid mucus produced by the small intestinal epithelium, while cells expressing oxygen-acetylated sialic acid mucus and sulfated acidic mucus produced by the large intestinal epithelium are stained in brown ( Figure 5 C), The organoids in the EV group were negative for HID-AB staining, while the organoids in the OV group contained goblet cells stained brown, thus identifying them as type III incomplete intestinal metaplasia.

[0195] (III) Single-cell sequencing identifies the cellular composition of intestinal metaplastic organoids

[0196] (1) In order to verify the cell composition in intestinal metaplastic organoids, the present invention further performed scRNA sequencing on hGO samples from three different donors after induction of intestinal metaplasia. A total of 81,137 cells were detected. The global gene expression analysis based on the meta-cell level revealed that there were 5766 differentially expressed genes in the OV group and EV group organoids. After taking the intersection with the "metaplasia genes", 511 common up-regulated genes were obtained ( Figure 6 A), which shows that the organoid model largely simulates the molecular changes similar to those of human tissues (511 / 810). The average expression levels of the top 20 genes with significant differences were further displayed through the cluster heat map ( Figure 6 B), mainly intestinal stem / progenitor cell markers OLFM4, APOA1, APOC3, intestinal epithelial cell markers FABP1, FABP2, KRT20, ALPi and goblet cell marker MUC2 were upregulated, while gastric markers PGC, MUC5AC, GKN1, GKN2 were significantly downregulated. The scatter plot further showed that the CDX2 mRNA level in the OV group was significantly higher than that in the EV group, accompanied by a significant downregulation of the gastric mucosal marker MUC5AC, while the intestinal goblet cell markers MUC2 and FCGBP were significantly upregulated ( Figure 6 C).

[0197] (2) Seruat (single cell analysis) was used for cluster analysis, and the UMAP algorithm was used for dimensionality reduction to identify cell clusters. The present invention identified 25 cell clusters (Cluster, hereinafter referred to as C), which had different gene expression characteristics. The genes highly expressed in each cluster relative to other clusters were defined as the characteristic genes of the cluster. The top 3 genes with the highest expression in each cell cluster were plotted as a heat map for display ( Figure 7). Based on the prior knowledge of each cluster to various cell type marker genes, the cell types were annotated and 25 cell populations were identified, including stem cells (CDCA7, LGR5), pit mucous cells (GKN1, GKN2), glandular mucous cells (TFF2, MUC6), chief cells (PGC), multipotent progenitor cells (TCIM, CAPN8, TFF1), intestinal epithelial cells (FABP1, FABP2), intestinal epithelial progenitor cells (ADH1C, OLFM4), goblet cells (FCGBP, MUC2), and Paneth cells (PELI1). Figure 8 A), according to previously reported markers (see Aran D, Looney AP, Liu L, et al. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage [J]. Nature immunology, 2019, 20 (2): 163-172; Hu C, Li T, Xu Y, et al. CellMarker 2.0: an updated database of manually curated cell markers in human / mouse and web tools based on scRNA-seq data [J]. Nucleic acids research, 2023, 51(D1): D870-d876). The present invention divides these cells into three main lineages, including the gastric lineage (marked by MUC5AC, MUC6, TFF2, and ALDH1A3), the intestinal lineage (CDX2, CDX1, GPA33, OLFM4, FABP1 / 2, and REG4), and the mixed lineage (expressing both gastric and intestinal lineage genes). The latter two groups are collectively referred to as the metaplastic lineage ( Figure 8 B); the average expression levels of these marker genes are plotted as bubble charts ( Fig. 9 ).

[0198] Although some clusters have different expression of marker genes, these clusters are also identified as different subpopulations of the same type of cells based on cell markers (such as C2 (CEACAM7), C5 (TMEM37), C13 (ANPEP, PRAP1), C20 (APOA1, APOC3) are all annotated as Enterocytes). The highly enriched genes in these clusters were characterized using volcano plots. Cells of the gastric lineage have significantly high expression of gastric markers to varying degrees, such as C9 (foveolar mucous cells) with high expression of GKN1, GKN2 and MUC5AC, while intestinal gene expression is significantly lower than other cells ( Fig.10 ); In intestinal lineage and mixed lineage cells, the difference in gastrointestinal marker gene expression between different cells was lower than that in other cells ( Fig.11 , Fig.12 ); In intestinal lineage and mixed lineage cells, the expression of gastrointestinal marker genes differed greatly between cells ( Fig.11 , Fig.12 ).

[0199] The cell type composition of the EV group and the OV group showed significant differences ( Fig.13 ), the proportion of gastric lineage cells decreased significantly, C4 (6.45% vs 0.19%), C9 (5.49% vs 0.22%), C12 (37.11% vs 18.65%), C14 (1.74% vs 0.07%), C4 (2.09% vs 0.9%); while the proportion of intestinal lineage and mixed lineage cells increased significantly, C2 (1.48% vs 8.61%), C6 (0% vs 0.52%), C7 (1.57% vs 3.97%), C8 (0.35% vs 6.07%), C13 (11.85% vs 23.22%), C16 (4.62% vs 14.31%), C17 (0.26% vs 3%), C18 (0.09% vs 1.91%), C19 (0.52% vs 2.77%), C8 (0.35% vs 6.07%), C24 (0% vs 0.1%).

[0200] Importantly, the present invention identified two clusters of goblet cells, among which C24, although in a very small proportion, expressed the classical goblet cell markers MUC2 and FCGBP, and simultaneously expressed MUC2 and MUC6, as well as other classical goblet cell markers such as ZG16, SPINK4, CLCA1, ITLN1, and was named classical goblet cell ( Fig. 9 , Fig.12 , Fig.14 ); while C19 expressed FCGBP and the intestinal epithelial cell marker DMBT1 ( Fig.12), as reported in previous articles (see Goldenring JR, Mills J C. Cellular Plasticity, Reprogramming, and Regeneration: Metaplasia in the Stomach and Beyond [J]. Gastroenterology, 2022, 162 (2): 415-430; E EL,Martinez-Abad B,Arike L,et al.An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function[J].Science(New York,NY),2021,372(6539):eabb159). This group of cells is defined as atypical goblet cells for intestinal metaplasia classification based on gene expression. C24 expresses both MUC2 and MUC6, while C19 also expresses MUC6 ( Fig. 9 ), so that these two groups of cells were classified as incompletely metaplastic goblet cell lineages, which is consistent with the results of previous HID-AB mucus staining. These results together show that the present invention successfully constructed an organoid model of type III intestinal metaplasia.

[0201] (IV) Molecular characteristics of different cell populations in intestinal metaplastic organoids

[0202] In order to identify the characteristics of each cell, the present invention further performed enrichment analysis on the DEGs enriched in each cell cluster. The GO enrichment analysis results showed the five most significant pathways for each cell cluster ( Fig.15), in terms of biological processes, gastric cell cluster: C4 is associated with the establishment of protein localization to organelles, proteasome-mediated ubiquitin-dependent protein degradation, protein targeting, vesicle organization, the establishment of protein localization to membranes, etc.; C9: actin filament organization, positive regulation of protein localization, the establishment of protein localization to organelles, cell component decomposition, and the regulation of apoptosis signaling pathways; C12 and 14 are jointly enriched in actin synthesis and wound healing; C12, intrinsic apoptosis signaling pathway, cell junction assembly, and atypical NF-κB signaling transduction; C14 is also enriched in the positive regulation of cell adhesion; and C15 is characterized by pathways such as translation initiation, mitochondrial localization of proteins, and cytoplasmic translation initiation; C23 core pathways are cell-matrix adhesion, ribosome biogenesis, cell-matrix adhesion, cell-matrix adhesion regulation, and Rho protein signaling transduction regulation. In the intestinal lineage cell cluster, the three intestinal epithelial cell lineages, C5, 13, and 20, share the fatty acid metabolism process, and C5 and 20 are also characterized by the metabolism of purine compounds; nucleotide metabolism, ribonucleotide metabolism, and ribose phosphate metabolism; C13 is also enriched in the response to nutrient levels, the response to peptide hormones, and the negative regulation of protein modification processes such as fat and intestinal absorption; C20 is enriched in small molecule decomposition metabolism, purine nucleotide metabolism, and alcohol metabolism alone; in the progenitor cell group, the characteristic pathways shared by C8 and C18 are purine nucleotide metabolism and purine compounds metabolism; C8 is also enriched in organic compound oxidation to produce energy, cellular respiration, and ribonucleotide metabolism; and C18 is also characterized by chromosome separation, RNA splicing, and organelle fission pathways. The main enriched pathways of C6 are proteasome-mediated ubiquitin-dependent protein degradation, macroautophagy, positive regulation of cell decomposition, negative regulation of protein modification, and organic compound oxidation to produce energy. The most notable pathways in C19 non-classical goblet cells are small GTPase-mediated signal transduction, negative regulation of protein modification processes, RNA splicing, response to nutrient levels, and response to peptide hormones.

[0203] The results of KEGG enrichment analysis showed that ( Fig.16), the pathways enriched in the four gastric cell clusters (C4, 9, 14, and 15) were mainly regulating the actin cytoskeleton; in addition, the common pathways of the C4 and 9 clusters were protein processing in the endoplasmic reticulum. In the intestinal cell clusters, C5, 6, 8, 18, and 20 were enriched in the "thermogenesis" pathway; in addition, the oxidative phosphorylation pathway was significantly enriched in C5, 6, 8, and 20, while C13 and 19 shared tight junction and apoptosis pathways, indicating their potential function in forming the intestinal mucosal barrier. For the classical goblet cell cluster C24, only 9 pathways were enriched, namely thermogenesis, cell cycle, biosynthesis of O-glycosylated mucin, fatty acid degradation, vasopressin-regulated hydration absorption, biosynthesis of ubiquinone and other terpenoid quinones, glycolysis / glycogenogenesis, and glycine, serine, and threonine metabolic pathways. Based on these data, the "thermogenesis" pathway was enriched in most intestinal lineage cells, indicating an active state of energy metabolism in intestinal lineage cells.

Claims

1. A method for constructing a gastric mucosal intestinal metaplasia organoid model, characterized in that: The steps include: (i) Obtaining human gastric glands to prepare organoids; (ii) Subculture of organoids; (III) Construction of pCDH-CMV-MCS-EF1-CDX2-Puro overexpression lentiviral vector; (iv) Virus packaging; (V) Construction of CDX2-overexpressing stable cells and organoids; (VI) CDX2 overexpression organoids were gradient differentiated to prepare gastric mucosal intestinal metaplasia organoids.

2. The construction method according to claim 1, characterized in that: The main operation of step (i) is to clean the gastric mucosal tissue of obese patients undergoing sleeve gastrectomy, remove fat, muscle layer and connective tissue, cut into pieces, collect tissue pieces by centrifugation, digest, incubate, terminate digestion, collect tissue fragments by centrifugation, separate gastric glands, add preheated 3D culture medium, and perform 3D organoid culture.

3. The construction method according to claim 1, characterized in that: The main operation of step (ii) is to discard the old culture medium in the culture plate, add pre-cooled Advanced DMEM / F12 culture medium without fetal bovine serum (FBS), collect the organoids by centrifugation, digest, incubate, terminate digestion, collect the digested cells by centrifugation, add pre-warmed 3D culture medium, and culture 3D organoids.

4. The construction method according to claim 1, characterized in that: The main operation of step (iii) is to first double-digest human-CDX2 CDS, i.e., CDX2 cDNA Clone, and double-digest the pCDH-CMV-MCS-EF1-Puro cloning vector, and connect the double-digested products to obtain the recombinant plasmid pCDH-CDX2; transform the reorganized plasmid pCDH-CDX2 into Escherichia coli competent cells, screen resistant single colonies, and extract the recombinant plasmid for later use.

5. The construction method according to claim 4, characterized in that: In step (iii), the human-CDX2 CDS double enzyme digestion system is: In step (iii), the double restriction enzyme digestion system of pCDH-CMV-MCS-EF1-Puro cloning vector is: In step (iii), the ligation reaction system for the linearized vector pCDH-CMV-MCS-EF1-Puro and the enzyme digestion product human-CDX2 CDS is:

6. The construction method according to claim 1, characterized in that: The main operation of step (iv) is to digest the HEK293T cells in the logarithmic growth phase into a cell suspension and count them, spread them into a culture dish, supplement the culture medium, and perform virus transfection packaging when the cells grow to a density of 70%, prepare a plasmid-PEI transfection complex, and add it to the cells in the culture dish, culture them in an incubator for 24-48 hours, collect the supernatant by centrifugation, and obtain a lentiviral suspension, determine the virus titer, aliquot, and store at low temperature.

7. The construction method according to claim 1, characterized in that: The main operation of step (five) is to take out the cryopreserved lentivirus, melt it, digest the organoids in step (a) into single cells, and then infect the digested organoid cells with the lentivirus, resuspend the cells, collect them by centrifugation, and resuspend them with Matrigel. After an interval of 3-4 days, the infection efficiency is determined according to the growth of the organoids and the expression of green fluorescent protein, and subculture and screening are performed. After 10-15 days of screening, stably overexpressed organoids can be obtained.

8. The construction method according to claim 1, characterized in that: The main operation of step (six) is to induce multi-stage differentiation of the organoid cells in step (fifth) after continuous in vitro passage before sampling and detection. After 10-12 days of differentiation, the organoids are harvested for phenotypic identification.

9. A gastric mucosal intestinal metaplasia organoid model constructed by the method of any one of claims 1 to 8, characterized in that: The model is a type III intestinal metaplasia organoid model.

10. Use of the gastric mucosal intestinal metaplasia organoid model described in claim 9 in the development or screening of products for preventing and / or treating gastric mucosal metaplasia lesions and gastric cancer.

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