An organoid model of gastric mucosal IM with SPEM, its construction method and application

Abstract

This invention provides a gastric mucosal IM-SPEM organoid model, its construction method, and its applications, belonging to the field of biological technology. The construction method includes: polarizing mouse bone marrow-derived macrophages, and co-culturing the polarized macrophages with mouse gastric organoids. The gastric mucosal IM-SPEM organoid model constructed by this method can highly simulate the key pathological process of gastric "inflammation-cancer" transformation driven by a chronic inflammatory microenvironment in the human gastric mucosa, accurately reproducing the development path of normal gastric mucosa transforming from chronic non-atrophic gastritis to atrophic gastritis, SPEM, and IM lesions. This model integrates a multi-dimensional regulatory network including immune signaling, epithelial cell damage response, and stem cell fate reprogramming, and can dynamically monitor the interaction mechanism between macrophages and gastric epithelial cells, overcoming the technical deficiencies of traditional IM cell models in simulating complex in vivo microenvironments and analyzing intercellular communication mechanisms.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to a gastric mucosal IM-SPEM organoid model, its construction method, and its application. Background Technology

[0002] Intestinal metaplasia (IM), an important manifestation of gastric mucosal lesions, often occurs secondary to chronic atrophic gastritis. Its typical pathological feature is the abnormal replacement of gastric epithelial cells by intestinal-specific lineages; that is, the gastric mucosa contains epithelial cells similar to those in the intestinal mucosa, such as goblet cells and Paneth cells. IM is a recognized precancerous lesion (GPL), and its presence significantly increases the risk of gastric cancer (GC). According to the classic Correa cascade theory, IM serves as a bridge between gastric inflammation and cancer, representing a crucial stage in the malignant transformation of the gastric mucosa. The specific pathogenesis of IM is not fully understood. It is generally believed that the progression of IM is closely related to the continuous stimulation of the chronic inflammatory microenvironment, with chronic inflammatory stimuli such as Helicobacter pylori infection being the most significant driving factor inducing or aggravating IM. However, due to the lack of an in vitro model that accurately simulates the human pathological process, the cellular origin of IM and the molecular mechanisms of its malignant transformation driven by chronic inflammation remain unclear.

[0003] Spasmolytic polypeptide-expressing metaplasia (SPEM) refers to a metaplastic state in which SPEM cells appear at the base of gastric glands. These cells are characterized by the expression of trefoil factor 2 (TFF2) and mucinous cell-specific genes. SPEMs are also considered precursor cells of intraepithelial dysplasia (IM), and IM and gastric dysplasia (GC) may originate from SPEMs. This discovery challenges the classic Correa cascade theory, suggesting that the evolution of GC may have undergone a new cascade evolutionary pattern: chronic superficial gastritis – chronic atrophic gastritis – SPEM – IM – dysplasia – GC. Currently, there is still a lack of cell modeling methods for SPEM in clinical practice. Existing methods for constructing IM cell models, such as the traditional chenodeoxycholic acid-stimulated GES-1 IM cell model, the IM organoid model using N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-stimulated gastric organoids, and the organoid model overexpressing tailed homeobox transcription factor 2 (CDX2), all lack the participation of immune cells and cannot simulate the inflammatory microenvironment that is most critical to the "inflammation-cancer" transformation. Moreover, the above methods all ignore or skip the diagnosis of the pathological stage of SPEM, which is not conducive to studying the pathological mechanism of SPEM progressing to IM or direct carcinogenesis. There is an urgent need for a model that can simulate the clinical physiological and pathological microenvironment to break through the research bottleneck. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a gastric mucosal IM with SPEM organoid model, its construction method and application. The gastric mucosal IM with SPEM organoid model constructed by the method simulates the histological characteristics of gastric mucosal IM and SPEM and the evolution trajectory of metaplastic lesions under chronic inflammatory stimulation in a more in-depth manner from the cell phenotype.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:

[0006] This invention provides a method for constructing a gastric mucosal IM-SPEM organoid model, comprising the following steps: polarizing mouse bone marrow-derived macrophages, and co-culturing the polarized macrophages with mouse gastric organoids.

[0007] Preferably, polarization stimulation is performed on mouse bone marrow-derived macrophages cultured for 5-7 days.

[0008] Preferably, the polarization stimulation time is 40-56 hours.

[0009] Preferably, the culture medium formulation for polarization stimulation includes polarization factors and DMEM high-glucose medium containing 5-15% FBS;

[0010] Preferably, the polarization factor includes 15-25 ng / ml IL-4 and 15-25 ng / ml IL-13, or 95-105 ng / ml LPS.

[0011] Preferably, the polarized macrophages include M1 type bone marrow-derived macrophages or M2 type bone marrow-derived macrophages.

[0012] Preferably, the culture medium used for the polarized macrophages is DMEM high-glucose medium containing 5-15% FBS; and the culture medium used for the mouse gastric organoids is GO conditioned medium.

[0013] Preferably, the co-culture conditions include: culturing at 37-39℃ and 3-7% CO2 concentration for 2-9 days.

[0014] This invention provides a gastric mucosal IM-SPEM organoid model obtained by the construction method described above.

[0015] This invention provides the application of the gastric mucosal IM with SPEM organoid model obtained by the construction method, or the gastric mucosal IM with SPEM organoid model, in the development or screening of products for the prevention and / or treatment of gastric mucosal IM, SPEM lesions and gastric cancer.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention provides, for the first time, a method for constructing a gastric mucosal IM-SPEM organoid model. This method utilizes a dynamic interaction system established in a Transwell co-culture system between bone marrow-derived macrophages (BMDM) with tunable polarization and gastric organoids (GO). This method introduces physiologically and pathologically relevant macrophages as key microenvironmental regulators. Through the specific cytokine profile secreted by macrophages after polarization, GO can be effectively induced to differentiate into an IM phenotype with SPEM characteristics without any gene manipulation.

[0018] This invention abandons the static model that relies on cell passage selection after one-time gene manipulation (viral transfection). Instead, it constructs an experimental system for gastric mucosal IM with SPEM organoids that is closer to the physiological and pathological process and can simulate the effects of chronic inflammation. This system not only avoids the off-target effects, complexity, and risk of non-physiological overexpression associated with gene manipulation, but more importantly, it more realistically and progressively reproduces the complex pathophysiological nature of the continuous interaction between the immune microenvironment and epithelial cells in the development of gastric mucosal IM / SPEM through a time-controllable and continuous input of exogenous immune microenvironment.

[0019] The IM / SPEM organoid model co-cultured with M1 or M2 type BMDM and GO provided by this invention can effectively simulate the human chronic inflammatory microenvironment (M2 type BMDM infiltration is a SPEM marker event), integrate immune signals (cytokines), epithelial damage, and stem cell regulatory networks, and monitor macrophage-epithelial cell interactions in real time. It makes up for the technical deficiencies of traditional IM cell models in simulating complex in vivo microenvironments and analyzing intercellular communication mechanisms. It is more conducive to revealing the molecular changes in the gastric epithelial lineage during the development of IM / SPEM cells and the interaction between M1 or M2 type macrophages and gastric stem cells, SPEM cells, and intestinal epithelial cells, providing a new research model and theoretical guidance for the diagnosis, prevention, and personalized precision treatment of GPL.

[0020] The model constructed in this invention provides an unprecedentedly powerful tool capable of directly analyzing in vitro how M1 or M2 macrophages reshape the fate of gastric epithelial cells by secreting a series of cytokines, inducing GPL phenotypes such as SPEM and IM. Furthermore, the Transwell non-contact co-culture design combines separability and manipulability, allowing for the separate recovery of the two cell types for multi-omics analysis, or the convenient addition of neutralizing antibodies and inhibitors to target and validate specific signaling pathways, thereby elucidating their molecular mechanisms in depth. Therefore, this model is not only an important complement to existing technologies, but also a novel research platform for discovering new mechanisms driving carcinogenesis through the inflammatory microenvironment and for screening intervention strategies, providing an irreplaceable and powerful tool for in-depth research into the immune-epithelial dialogue mechanisms in gastric cancer development. Attached Figure Description

[0021] Figure 1 The changes in macrophage mRNA expression in each group after LPS stimulation are shown. "#" indicates a difference compared to the control group. P <0.05; "##" indicates that compared with the control group, P <0.01; BMDM represents the control group; BMDM+LPS represents the M1 polarization group.

[0022] Figure 2 The changes in mRNA and protein levels in macrophages after stimulation with IL-4 and IL-13 are shown. Specifically: A. mRNA expression levels in macrophages of different groups under combined IL-4 and IL-13 stimulation; B. changes in CD163 protein expression levels in macrophages of different groups after stimulation with IL-4 and IL-13; C. relative CD163 protein expression levels in macrophages of different groups under combined IL-4 and IL-13 stimulation. "##" indicates the difference compared to the control group. P <0.01; "###" indicates that compared with the control group, P <0.001; BMDM represents the control group; BMDM+IL-4+IL13 represents the M2 polarization group.

[0023] Figure 3 Changes in mRNA expression related to different subtypes of BMDM co-cultured with GO for 3 days were observed. "#" indicates a difference compared to the control group. P <0.05; "##" indicates that compared with the control group, P <0.01; "###" indicates that compared with the control group, P <0.001; M0+GO represents the control group; M1+GO represents the M1 polarization co-culture group; M2+GO represents the M2 polarization co-culture group.

[0024] Figure 4 The changes in mRNA expression related to different subtypes of BMDM co-cultured with GO for 6 days were analyzed. "#" indicates a difference compared to the control group.P <0.05; "##" indicates that compared with the control group, P <0.01; "###" indicates that compared with the control group, P <0.001; M0+GO represents the control group; M1+GO represents the M1 polarization co-culture group; M2+GO represents the M2 polarization co-culture group.

[0025] Figure 5 The changes in mRNA expression related to different subtypes of BMDM co-cultured with GO for 9 days were analyzed. "#" indicates the expression level compared to the control group. P <0.05; "##" indicates that compared with the control group, P <0.01; M0+GO represents the control group; M1+GO represents the M1 polarization co-culture group; M2+GO represents the M2 polarization co-culture group.

[0026] Figure 6 The changes in mRNA expression related to different subtypes of BMDM co-cultured with GO for 12 days were analyzed. Note: "#" indicates a difference compared to the control group. P <0.05; "##" indicates that compared with the control group, P <0.01; "###" indicates that compared with the control group, P <0.001; "####" indicates that compared with the control group, P <0.0001; M0+GO represents the control group; M1+GO represents the M1 polarization co-culture group; M2+GO represents the M2 polarization co-culture group.

[0027] Figure 7 After co-culturing for 12 days and changing the medium, we observed significant collapse and floating loss of the matrix gel.

[0028] Figure 8 Bright-field microscopic observation and HE staining were performed on BMDM and GO co-cultured for 3 days. Specifically: A. Bright-field microscopic observation of BMDM and GO co-cultured for 3 days; B. HE staining of BMDM and GO co-cultured for 3 days.

[0029] Figure 9 The expression levels of AQP5, MUC2, and GSII were detected by IF assay after 3 days of co-culturing BMDM and GO. Specifically, A. AQP5 expression level after 3 days of co-culturing BMDM and GO; B. MUC2 expression level after 3 days of co-culturing BMDM and GO; C. GSII expression level after 3 days of co-culturing BMDM and GO.

[0030] Figure 10 The effect of serum containing Weiwei Decoction on GO in M2 type BMDM co-cultured with GO for 3 days was investigated. Where n=6; "#" indicates the difference compared to the control group. P <0.05; "##" indicates that compared with the control group,P <0.01; "###" indicates that compared with the control group, P <0.001; "Indicates a comparison with the model group, P <0.05; "Indicates a comparison with the model group, P <0.01; "Indicates a comparison with the model group, P <0.001; M0+GO represents the control group; M2+GO represents the model group; M2+GO-L represents the low-dose Weiwei Decoction group; M2+GO-M represents the medium-dose Weiwei Decoction group; M2+GO-H represents the high-dose Weiwei Decoction group.

[0031] Figure 11 The effect of serum containing Weiwei Decoction on M2 type BMDM co-cultured with GO for 3 days was investigated. Where n=6; "#" indicates the difference compared to the control group. P <0.05; "###" indicates that compared with the control group, P <0.001; "Indicates a comparison with the model group, P <0.05; "Indicates a comparison with the model group, P <0.01; "Indicates a comparison with the model group, P <0.001; M0+GO represents the control group; M2+GO represents the model group; M2+GO-L represents the low-dose Weiwei Decoction group; M2+GO-M represents the medium-dose Weiwei Decoction group; M2+GO-H represents the high-dose Weiwei Decoction group.

[0032] Figure 12 The effect of serum containing Weiwei Decoction on M2 type BMDM co-cultured with GO for 6 days was investigated. Where n=6; "#" indicates the difference compared to the control group. P <0.05; "##" indicates that compared with the control group, P <0.01; "###" indicates that compared with the control group, P <0.001; "Indicates a comparison with the model group, P <0.05; "Indicates a comparison with the model group, P <0.01; "Indicates a comparison with the model group, P <0.001; M0+GO represents the control group; M2+GO represents the model group; M2+GO-L represents the low-dose Weiwei Decoction group; M2+GO-M represents the medium-dose Weiwei Decoction group; M2+GO-H represents the high-dose Weiwei Decoction group.

[0033] Figure 13 Changes in the expression of IM and SPEM-related markers after co-culturing THP-1 and GES-1 for 72 h.

[0034] Figure 14 The results show the changes in the expression of IM and SPEM-related markers after THP-1 was co-cultured with primary gastric mucosal epithelial cells for 72 h.

[0035] Figure 15 For GO seeding in the lower chamber, a small-volume, multi-point seeding method is adopted. Detailed Implementation

[0036] This invention provides a method for constructing a gastric mucosal IM-SPEM organoid model, comprising the following steps: polarizing mouse BMDM, and co-culturing polarized macrophages with mouse GO. The co-culture uses Transwell 6-well plates. Polarized macrophages are placed in the upper chamber of the Transwell, and mouse GO is placed in the lower chamber. The upper chamber of the Transwell is embedded in the culture plate containing the lower chamber of the mouse GO for co-culture. The material used for co-culture is a Transwell nested with a 6-well plate (PET membrane, 24 mm, 0.4 μm), purchased from Corning Incorporated, USA, catalog number 3450.

[0037] In this invention, mice are cultured separately from mice with GO before the completion of BMDM polarization stimulation.

[0038] In this invention, mouse bone marrow-derived mononuclear cells (BMDMs) are induced to adhere to the culture medium by adding macrophage colony-stimulating factor (M-CSF) to DMEM high-glucose medium containing 5-15% FBS to obtain mouse BMDMs. Specific steps include: centrifuging freshly extracted mouse bone marrow-derived mononuclear cells to obtain cell pellet, resuspending the cells in DMEM high-glucose medium containing 5-15% FBS, 10-30 ng / mL M-CSF, and 0.5-1.5% penicillin-antibody solution, and then transferring the cell suspension to the upper chamber of a Transwell 6-well plate; installing the Transwell upper chamber system in a sterile 6-well culture plate, and culturing the cells in an incubator.

[0039] In this invention, mouse gastric stem cells are extracted and cultured to obtain mouse GO. The preparation of mouse GO according to this invention includes the following steps: After the mouse is euthanized by cervical dislocation, the mouse is immersed in alcohol, and then the abdominal cavity of the mouse is quickly cut open to separate the entire stomach. The operation is then transferred to a clean bench, and the fundus (foregut), antrum and pylorus of the stomach are cut off, leaving only the stomach body. The stomach body is repeatedly washed with sterile PBS to ensure that visible residue is removed. The stomach body is cut into small pieces the size of rice grains and washed repeatedly with D-PBS at least 20 times. Then EDTA is added and the tissue is gradually lysed by slow shaking at room temperature. Then the lysis buffer containing EDTA is removed, and the stomach body is gently pressed with a sterile glass slide to squeeze out the stem cells. The cells are filtered through a 60-80 μm filter and centrifuged to separate the mouse GO. Then, GO is cultured in GO conditioned medium containing key factors (8-12 µM Y-27632, 8-12 µM SB431542, 8-12 µg / ml gentamicin). In this invention, mouse GO cells are transferred to the lower chamber of a Transwell 6-well plate and cultured in an incubator. The mouse GO cells cultured in this invention are replaced with fresh GO conditioned medium every 40-56 hours during days 1-6 of culture.

[0040] In this invention, polarization stimulation is performed on BMDM mice cultured for 5-7 days; preferably, polarization stimulation is performed on BMDM mice cultured for 6 days. The duration of polarization stimulation is 40-56 hours, more preferably 48 hours. The culture medium formulation used for polarization stimulation in this invention includes a polarization factor and DMEM high-glucose medium containing 5-15% FBS; preferably, it includes a polarization factor and DMEM high-glucose medium containing 10% FBS. The polarization factor in this invention includes 15-25 ng / ml IL-4 and 15-25 ng / ml IL-13, or 95-105 ng / ml lipopolysaccharide (LPS); preferably, it includes 20 ng / ml IL-4 and 20 ng / ml IL-13, or 100 ng / ml LPS. After polarization stimulation, the old culture medium is discarded, and DMEM high-glucose medium containing 5-15% FBS is added; preferably, the culture medium is DMEM high-glucose medium containing 10% FBS.

[0041] In this invention, GO is cultured for 5-6 days and then passaged for the first time. The specific process of the first passage is as follows: GO is broken into smaller clumps or single-cell clumps, centrifuged, and then resuspended in the matrix gel at a ratio of 1:1-2. The cells are then seeded into a new Transwell lower chamber at a passage ratio of 1:1-3 and cultured for further culture or used directly for co-culture.

[0042] In this invention, BMDM and GO are completely separated and cultured independently after initial extraction. BMDM differentiates into unpolarized macrophages (M0) under the action of M-CSF, while GO proliferates in GO conditioned medium to form primary organoid structures.

[0043] In this invention, BMDM cells are cultured in DMEM high-glucose medium containing 5-15% FBS and 10-30 ng / mL M-CSF for approximately 5-6 days, with the medium being replaced regularly (usually every 2-3 days) to allow them to fully differentiate into mature, unpolarized M0 macrophages. In this invention, the old medium in the BMDM cells is discarded, and 95-105 ng / mL LPS is added to DMEM high-glucose medium containing 5-15% FBS for 40-56 hours to construct M1-type BMDM cells. In this invention, the old medium in the BMDM cells is discarded, and 15-25 ng / mL IL-4 and 15-25 ng / mL IL-13 are added to DMEM high-glucose medium containing 5-15% FBS for 40-56 hours to construct M2-type BMDM cells. The FBS used in this invention is preferably Australian fetal FBS.

[0044] In this invention, polarization stimulation and GO passage are performed simultaneously. This synchronization ensures that when polarization is complete, GO is also in a suitable stage of active proliferation, in good condition, and has undergone amplification, preparing for co-culture. In this invention, the BMDM polarization process (days 6-8) and GO culture (days 6-8) remain physically separated. Polarization is carried out in a BMDM-only culture system, while GO grows and is passaged in its own culture medium. Contact between the two is strictly avoided before polarization is complete. Preferably, BMDM is inoculated into the upper chamber of a Transwell 6-well plate, and the upper chamber containing BMDM is placed on an empty bottom plate for culture. Preferably, GO is inoculated into the lower chamber of a Transwell 6-well plate and cultured separately.

[0045] In this invention, the co-culture conditions include: culturing at 35-39℃ and 3-7% CO2 concentration for 2-9 days; preferably at 37℃ and 5% CO2 concentration for 3-6 days. In this invention, during co-culture, factors secreted by BMDM diffuse through the 0.4μm PET membrane pores to the lower chamber, acting on GO, without direct cell migration. During co-culture, every 2-4 days, the old BMDM upper chamber (along with its culture medium and cells) is discarded and replaced with a freshly prepared BMDM upper chamber that has undergone the same polarization stimulation (M1 or M2 type) for 40-56 hours (obtained by repeating the polarization process on days 6-8); the culture medium in the lower chamber GO is replaced with fresh GO conditioned medium as usual (every 48 hours). Because the activity of macrophages and the secretion profile of key factors after polarization are maintained for a limited time (usually about 3 days), a strict dynamic replacement procedure must be implemented to achieve long-term stable microenvironment stimulation to induce chronic IM / SPEM phenotype formation. This systematic and timed replacement of the upper chamber of polarized macrophages is the core mechanism for ensuring a continuous and effective supply of macrophage-derived active factors within the co-culture system, precisely simulating the continuous infiltration and activation of immune cells during chronic pathological processes. At the start of co-culture, this invention thoroughly removes the old BMDM medium containing polarizing factors, then adds new DMEM high-glucose medium containing 5-15% FBS without polarizing factors before co-culturing, preventing the polarizing factors in the old medium from affecting GO.

[0046] In this invention, the preparation of GO conditioned medium (L-WRN conditioned medium) is based on the Murine L-WRN Conditioned Medium Protocol from the MD Anderson Cancer Center in the United States. The steps include: resuscitating and culturing L-WRN cells, and passaged to 5 cells of 25 cm². 2 After the cells were cultured in the flask, they were transferred to a 180cm³ culture medium. 2 Cells were cultured in culture flasks; cells were screened once with G-418 and hygromycin according to experimental requirements to remove non-resistant cells; then the cells were passaged into five 180 cm⁻¹ culture flasks. 2 Once the cells have grown to fill the entire bottom of the culture flask, add DMEM-F12 medium containing 1% penicillin-dextrose, 20% FBS and 200mM L-glutamate. Collect the culture medium every 24 hours, mix all the collected culture medium, shake well and freeze.

[0047] The dynamic replacement procedure described in this invention can also be used to first co-culture M1 macrophages for 3 days, and then replace them with M2 macrophages after 3-6 days. Alternatively, the entire process can be standardized by replacing either M2 or M1 macrophages.

[0048] The present invention also provides a gastric mucosa IM with SPEM organoid model obtained by the above construction method. The gastric mucosa IM with SPEM organoid model of the present invention has IM and SPEM phenotypes, and can effectively simulate the dynamic evolution process of gastric "inflammation-cancer" transformation under chronic inflammatory stimulation in the human body.

[0049] The present invention also provides the use of the gastric mucosa IM with SPEM organoid model obtained by the above construction method or the gastric mucosa IM with SPEM organoid model in the development or screening of products for preventing and / or treating gastric mucosa IM, SPEM lesions and gastric cancer.

[0050] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well-known to those skilled in the art.

[0051] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the embodiments in the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0052] The reagents or materials involved in the following examples are as follows:

[0053] For the experiment, 30 4-week-old SPF-grade male C57BL / 6J mice were selected and provided by Zhuhai Besttone Biotechnology Co., Ltd. (Animal Production License: SCXK (Guangdong) 2020-0051). L-WRN cells were purchased from MEISEN CTCC / Meisen Cells (product number: CTCC-001-0253).

[0054] DMEM high-glucose medium was purchased from Gibco (C11995500BT), fetal bovine serum (FBS) from Thermo Fisher Scientific (Australia, C10099141C), penicillin antibody from Dalian Meilun Pharmaceutical Co., Ltd. (MA0110), M-CSF protein (HY-P7085), IL-4 protein (Mouse, HY-P70644), IL-13 protein (Mouse, HY-P70460), and lipopolysaccharide (LPS, HY-D1056) from MCE (USA), Transwell nested 6-well plates (PET membrane, 24 mm, 0.4 μm) from Corning (C3450), Matrigel (LDEV-free) from Corning (C356234), and TGF-β type I receptor inhibitors SB431542 (HY-10431) and Y-27632. ROCK inhibitor (HY-10071) and gentamicin (HY-A0276) were purchased from MCE (USA). DMEM-F12 culture medium (containing L-glutamine and HEPES, catalog number C11330500BT-500ml) was purchased from Gibco (USA). G-418 Geneticin (BS150-1g) and hygromycin B (BS204-1g) were purchased from Beijing Lanjieke Company. Related PCR detection kits were purchased from Beijing Jinsha Company.

[0055] Example 1: Construction of a gastric mucosal IM-SPEM organoid model

[0056] I. Obtaining and Basic Culture of Mouse BMDM (Day 1): On the first day of the experiment (Day 1), femurs and tibias of C57BL / 6J mice were aseptically isolated, bone marrow cells were flushed out, and after erythrocyte lysis, the cells were resuspended in DMEM high-glucose medium containing 20 ng / ml M-CSF, 10% FBS and 1% penicillin antibiotics, and seeded in the upper chamber of a Transwell plate (the upper chamber was placed on an empty 6-well plate) and cultured in a 37℃ 5% CO2 incubator to induce differentiation into mature, unpolarized M0 macrophages.

[0057] II. GO Extraction and Primary Culture in Mice: On day 1, the gastric body mucosa tissue of mice was obtained by dissection. After EDTA digestion, mechanical dissociation, and filtration, the obtained gastric glandular epithelial cells or cell clumps were embedded in matrix gel and seeded into the lower chamber of a Transwell co-cultured plate (a 6-well plate without an upper chamber). GO conditioned medium containing key factors (10 µM Y-27632, 10 µMSB431542, 10 µg / ml gentamicin) was added, and the cells were cultured in a 37°C, 5% CO2 incubator.

[0058] GO routine culture: During the first 1-6 days of GO culture, the entire GO conditioned medium should be replaced with fresh GO conditioned medium every 48 hours.

[0059] III. Co-culture of GO and M2-type BMDM

[0060] ① Steps one and two above were cultured separately and simultaneously. On day 6, BMDM polarization stimulation and GO passage culture were performed simultaneously, and cultured for another 48 hours.

[0061] M2-type BMDM polarization stimulation: Discard the old culture medium in the BMDM chamber, add DMEM high-glucose medium containing 10% FBS with 20 ng / ml IL-4 and 20 ng / ml IL-13, and stimulate for 48 h to construct M2-type BMDM. GO passage culture: When GO culture reaches day 6 (usually when sufficient number and size of organoids have formed), passage at a 1:2 ratio in separate flasks. Specific passage procedure: Dissociate the matrix gel on ice, mechanically break up the organoids into smaller clumps or single-cell clusters, centrifuge, and resuspend the cells in fresh matrix gel at a 1:1 ratio. Re-seed directly into the prepared Transwell lower chamber. Replace with fresh GO conditioned medium and continue culturing in a 37°C, 5% CO2 incubator.

[0062] ② Dynamic co-culture initiation and maintenance (starting from day 8)

[0063] Establishment of the co-culture system (day 8): After 48 hours of M2 polarization stimulation of the BMDM (i.e., day 8), discard the old culture medium in the lower chamber of the GO Transwell (culture well) and the upper chamber (or culture vessel) of the polarized BMDM Transwell. Add fresh GO conditioned medium to the lower chamber well of the Transwell containing GO. Embed the upper chamber of the Transwell containing the polarized M2 BMDM into the culture plate with the lower chamber of GO already placed. Add DMEM high-glucose medium containing 10% FBS but without polarization factors (IL-4 and IL-13) to the upper chamber (BMDM side). Place the entire Transwell device in a 37°C incubator with 5% CO2 concentration to begin co-culture. At this time, the factors secreted by the M2 BMDM can diffuse through the 0.4μm PET membrane pores to the lower chamber and act on GO, while the cells themselves do not migrate directly. After 3 days of culture, the gastric mucosal IM organoid model with SPEM is obtained.

[0064] Dynamic Maintenance Mechanism: Due to the limited duration (3 days) of polarized macrophage activity and key factor secretion profile maintenance, a strict dynamic replacement procedure must be implemented to achieve long-term stable microenvironment stimulation to induce progressive IM / SPEM phenotype formation and promote IM / SPEM progression. Specific Procedure: After co-culture begins, every 72 hours (3 days) (days 3, 6, 9, and 12, depending on the total culture time), carefully discard the entire old BMDM upper chamber (along with its culture medium and cells). Replace it with a freshly prepared M2-type BMDM upper chamber that has undergone the same polarization stimulation for 48 hours (i.e., obtained by repeating the polarization process from days 6 to 8). The GO medium in the lower chamber is replaced with fresh GO conditioned medium as usual (every 48 hours). This systematic, timed replacement of the polarized macrophage upper chamber is the core mechanism to ensure a continuous and effective supply of macrophage-derived active factors within the co-culture system, precisely mimicking the continuous infiltration and activation of immune cells during chronic pathological processes.

[0065] Example 2

[0066] In Example 1, step three, the M2-type BMDM polarization stimulation was replaced with M1-type BMDM polarization stimulation. The specific steps for M1-type BMDM polarization stimulation are as follows: Discard the old culture medium in the BMDM chamber, add DMEM high-glucose culture medium containing 100 ng / ml LPS and 10% FBS, and culture for 48 hours to construct the M1-type BMDM. In subsequent experimental operations in Example 1, the M2-type BMDM was replaced with the M1-type BMDM.

[0067] Example 3

[0068] I. BMDM Acquisition and Basic Culture (Day 1), the specific steps are as follows:

[0069] ① After euthanizing 4-6 week old C57 mice by cervical spine, they were disinfected by immersing them in 75% alcohol for 2 minutes. The legs were then cut off from the thigh to the ankle, and the femurs were removed in a laminar flow hood and transferred to a new culture dish containing 10% FBS in DMEM high-glucose medium.

[0070] ② Use sterilized scissors to cut off both ends of the femur, cut off the bone and muscle, and then use sterile gauze to carefully remove the remaining muscle tissue, being careful not to break the bone.

[0071] ③ Use pre-cooled DMEM high-glucose medium containing 10% FBS as the rinsing solution to process bone samples using a 1ml syringe. Insert the syringe needle into both ends of the femur and apply pressure to expel bone marrow cells from the bone cavity. Transfer the cells to a pre-prepared fresh culture dish and rinse until the bone marrow turns white (2-3ml). Discard the bone.

[0072] ④ Repeatedly blow and agitate the red blood cell clumps in the dish to disperse them, then transfer the cell suspension into a 50ml centrifuge tube with a 70μm cell filter membrane (pre-wetted) to remove excess impurities.

[0073] ⑤ After the filtered single-cell suspension is sealed, it is centrifuged at 1500 rpm for 5 minutes at room temperature, and then the supernatant is removed.

[0074] ⑥ Rinse the precipitate with 2-3 ml PBS, resuspend it, transfer it to a 15 ml centrifuge tube, centrifuge at 1500 rpm for 3 min, and discard the supernatant.

[0075] ⑦ Add 3 times the cell volume of red blood cell lysis buffer, resuspend, and lyse on ice for 5-10 minutes.

[0076] ⑧ Add an equal volume of DMEM high-glucose medium containing 10% FBS to terminate the lysis. Centrifuge the cell suspension after lysing the red blood cells at 1500 rpm for 5 min.

[0077] ⑨ After centrifugation, discard the supernatant, add 2-3 ml of PBS to wash, resuspend, centrifuge at 1000 rpm for 2 min, discard the supernatant, and ensure complete lysis.

[0078] ⑩ Cells were resuspended in DMEM high-glucose medium containing 10% FBS and 20 ng / mL M-CSF, and then the cell suspension was transferred to the upper chamber of a Transwell 6-well plate. The Transwell chamber system was installed in a sterilized 6-well culture plate and cultured in an incubator at 37°C with 5% CO2 concentration.

[0079] II. GO Extraction and Initial Culture: Synchronized on Day 1, the specific steps are as follows:

[0080] 1. GO extraction

[0081] ① Four- to six-week-old C57BL / 6 mice were euthanized by cervical dislocation, and the stomach tissue was quickly dissected and placed in pre-cooled (4°C) D-PBS buffer (containing 1% penicillin antibody).

[0082] ② The stomach was dissected longitudinally along the greater curvature of the mouse stomach, and then the gastric body mucosa was separated under low temperature: the muscle layer was peeled off with microforceps, the mucosal epithelial tissue was preserved, and the tissue was cut into 1-2 mm pieces. 3 Fragments.

[0083] ③ Transfer tissue fragments to a 15mL centrifuge tube, add 10mL of pre-cooled D-PBS and wash repeatedly by pipetting (about 10 times), let stand for 1 minute to allow the tissue to settle and then discard the supernatant. Repeat until the supernatant is clear (about 5-8 times) to remove mucus and blood residue.

[0084] ④ After the final rinse, remove most of the supernatant, add 25 mL of D-PBS (pH 7.4) containing 10 mM EDTA to the precipitate, and digest at 37°C with shaking for 60 min before discarding the supernatant.

[0085] ⑤ After digestion is terminated, let stand on ice for 5 minutes, discard the supernatant, add 10 mL of pre-cooled D-PBS and gently pipette, discard the supernatant, cover the tissue block with a sterile glass slide, put on sterile gloves, press the glass slide with both thumbs to squeeze out the cells from the glands of the tissue block; filter through a 70 μm cell sieve.

[0086] ⑥ Centrifuge at 200×g for 5 min (4℃), discard the supernatant, and resuspend the precipitate in 1 mL of pre-cooled mixture of matrix gel and GO conditioned medium until the cell volume is 0.2-2×10⁻⁶ cells / mL. 5 Count / mL, operate on ice to avoid colloid solidification and bubble formation.

[0087] ⑦ Using the microdroplet embedding method, 50 μL of the mixture was inoculated into the center of a preheated 6-well plate and incubated at 37°C for 30 min to form a dome structure.

[0088] ⑧ Slowly add 500 μL of GO conditioned medium (containing 10 µM SB431542, 10 µM Y-27632 and 10 µM gentamicin) along the well wall.

[0089] ⑨ Change the entire medium every 48 hours. When aspirating the old medium, keep the pipette tip close to the well wall and avoid touching the matrix gel dome.

[0090] ⑩ Passage the cells on day 6 of culture based on the number of organoids.

[0091] 2. GO Generation

[0092] ① Matrix gel dissociation and organoid collection: Place the 6-well plate containing organoids on ice, aspirate the culture medium, add about 1 mL of pre-cooled GO conditioned medium, mechanically pipette several times to break up the matrix gel, and collect the suspension.

[0093] ②Incubate on ice for 10 minutes to soften the matrix gel, and then blow and agitate several times until more than 80% of the organoid structures dissociate into single-cell clumps.

[0094] ③ Organoid reseeding: Centrifuge at 1500 rpm for 5 min (4℃), discard the supernatant, passage at a ratio of 1:2, resuspend the cells in fresh matrix gel at a ratio of 1:1, seed into a preheated 6-well plate, and continue routine culture procedures.

[0095] 3. Preparation of GO conditioned medium

[0096] ① Cell resuscitation: Remove the L-WRN cell cryopreservation tube from liquid nitrogen, thaw rapidly in a 37°C water bath, transfer to a 15mL centrifuge tube containing 10mL of pre-warmed DMEM-F12 medium containing 10% FBS, centrifuge at 1000rpm for 5min (room temperature), and discard the supernatant.

[0097] ② Primary expansion: After resuspending the cell pellet, the cells were seeded into T25 culture flasks, and 5 mL of DMEM-F12 medium containing 10% FBS was added. The cells were cultured at 37℃ and 5% CO2 for 24 h. The cell adhesion rate was confirmed to be >80% by using an inverted microscope.

[0098] ③ Gradual passage: Pass the cells to T175 culture flasks at a ratio of 1:3 to maintain cell confluence at 80-90% and avoid excessive growth that could lead to a decline in secretory function.

[0099] ④ Drug screening: Add 25ml of DMEM-F12 medium containing 500μg / mL G418, 500μg / mL hygromycin and 10% FBS for cell culture. Through a continuous screening process of 72h, untransfected cell populations are effectively removed.

[0100] ⑤ Conditioned medium induction: Discard the antibiotic-containing DMEM-F12 medium, gently wash twice with pre-cooled PBS-EDTA buffer (0.5mM, pH 7.4), add 0.25% trypsin-EDTA (containing 0.02% EDTA) and digest at 37℃ for 3-5 min, resuspend at a ratio of 1:5 and inoculate into 5 T175 culture flasks, and replace with antibiotic-free DMEM-F12 medium containing 10% FBS.

[0101] ⑥ Supernatant collection: Replace with primary culture medium (DMEM-F12 medium containing 20% ​​FBS and 1% L-glutamine 200mM (100x) + 1% double antibiotics) and continue culturing. Every other day, filter the culture supernatant through a 0.22μm pore size filter membrane. Repeat this process five times before using it for subsequent experiments.

[0102] ⑦ Concentration and preparation: Mix the supernatant from 5 batches with freshly prepared primary culture medium at a 1:1 ratio, and supplement with 10 μM Y-27632, 10 μM SB431542 and 10 μM gentamicin. Collect the final volume through a filter and dispense into 50 mL centrifuge tubes to obtain GO conditioned medium (40 mL per tube). Store at -20℃ for ≤3 months.

[0103] III. Co-cultivation of GO and BMDM: The steps are the same as in Example 1.

[0104] Example 4

[0105] In Example 3, step three of the M2-type BMDM polarization stimulation was replaced with M1-type BMDM polarization stimulation. The specific steps for M1-type BMDM polarization stimulation are as follows: Discard the old culture medium in the BMDM chamber, add DMEM high-glucose culture medium containing 100 ng / ml LPS and 10% FBS, and stimulate for 48 h to construct the M1-type BMDM. In subsequent experimental operations in Example 3, the M2-type BMDM was replaced with the M1-type BMDM.

[0106] Comparative Example 1

[0107] Replace the M2 type BMDM polarization stimulation in step three of Example 3 with the M0 type BMDM polarization stimulation. The specific steps for M0 type BMDM polarization stimulation are as follows: Discard the old culture medium in the BMDM chamber, add freshly prepared DMEM high-glucose culture medium containing 10% FBS and continue culturing for 48 hours to construct the M0 type BMDM.

[0108] Experimental Example 1

[0109] 1. Construction and validation of the M1 macrophage model

[0110] The M1 type BMDM (BMDM+LPS group) and the unpolarized BMDM (BMDM group) obtained in Example 4 were subjected to PCR detection. Figure 1 It can be seen that after LPS stimulation, compared with the BMDM group, the M1 polarization group showed significantly higher expression of M1 polarization marker-induced nitric oxide synthase (INOS). P <0.05), while the M1 polarization marker, mannose receptor C-type 1 (C-type 1 or CD206) ( P <0.05), peroxisome proliferator-activated receptor γ (PPARG) ( P<0.01), chitinase-like protein 3 (YM1) P <0.01) and resistance-like molecule α (FIZZ1) P The values ​​of <0.01) all decreased significantly, indicating that M1 polarization induction was successful.

[0111] 2. Construction and validation of the M2 macrophage model

[0112] The M2 type BMDM (BMDM+IL-4+IL-13 group) and the unpolarized BMDM (BMDM group) obtained in Example 3 were subjected to PCR detection. Figure 2 Compared with the BMDM group, the M2 polarization group showed significantly higher expression of CD206 ( P <0.01), PPARG ( P <0.001), Arginase 1 (ARG1) P <0.001), YM1 ( P <0.01), FIZZ1 ( P <0.001), monocyte chemoattractant protein 1 (MCP1) P M2 polarization markers such as <0.001 were observed, while the M2 polarization marker leukocyte differentiation antigen 86 (CD86) was significantly reduced. P <0.01), indicating that M2 polarization induction was successful.

[0113] Experimental Example 2: Validation of the Construction of the BMDM and GO Co-cultivation IM System

[0114] Based on the method described in Example 1, M0 type BMDM (Comparative Example 1), M1 type BMDM (Example 4), and M2 type BMDM (Example 3) were co-cultured with GO for 3 days, 6 days, 9 days, and 12 days, respectively, to explore the optimal induction conditions for IM organoid phenotypes.

[0115] 1. Construction of a 3-day co-training system for BMDM and GO

[0116] M0, M1, and M2 BMDMs were co-cultured with GO for 3 days, and RNA was extracted for PCR detection. Figure 3 The results showed that the mRNA expression of CDX2, mucin 2 (MUC2), and SRY-associated high-mobility cassette transcription factor 9 (SOX9) in the M2 polarization co-culture group was significantly higher than that in the control group. P <0.05 indicates that GO has IM lesions. The mRNA expression of malignant brain tumor deletion protein 1 (DMBT1) was significantly increased compared with the control group. P<0.05), the mRNA expression of WAP tetradisulfide core domain protein 2 (WFDC2) also showed an upward trend, and the mRNA expression of muscle, intestinal and gastric transcription factor 1 (MIST1) was significantly reduced compared with the control group. P <0.05 indicates that GO also exhibits SPEM lesions. Furthermore, the mRNA expression of the α subunit of potassium hydrogen ATPase (ATP4A) and trefoil factor 1 (TFF1) was significantly lower in the M2 polarization co-culture group compared to the control group. P <0.05 indicates that GO has undergone gastric mucosal atrophy, characterized by parietal cell loss, which is consistent with the clinicopathological features of IM / SPEM often secondary to chronic atrophic gastritis. The expression levels of MUC2 and interleukin-33 (IL-33) mRNA in the M1 polarization co-culture group were significantly increased compared to the control group. P <0.05) indicates that GO has IM and SPEM lesions, and the modeling effect of the M2 polarization co-culture group is better than that of the M1 polarization co-culture group.

[0117] 2. Construction of a 6-day co-training system for BMDM and GO

[0118] M0, M1, and M2 BMDMs were co-cultured with GO for 6 days before PCR detection. Figure 4 The results showed that the mRNA expression of CDX2, MUC2, and SOX9 in the M2 polarization co-culture group was significantly higher than that in the control group. P <0.05 indicates that GO has IM lesions. The mRNA expression of IL-33 and leukocyte differentiation antigen 44 variant 9 (CD44V9) was significantly increased compared with the control group. P <0.05), MIST1 mRNA expression was significantly reduced in both the M1 and M2 polarization co-culture groups. P <0.05 indicates that GO also exhibits SPEM lesions. Compared to the control group, ATP4A mRNA expression was significantly reduced in both the M1 and M2 polarization co-culture groups. P <0.05), and TFF1 mRNA expression also showed a decreasing trend, indicating that GO underwent gastric mucosal atrophy represented by parietal cell loss. MUC2, IL-33, and WFDC2 were significantly increased in the M1 polarization co-culture group ( P <0.05) indicates that GO has IM accompanied by SPEM lesions, and the modeling effect of the M2 polarization co-culture group is better than that of the M1 polarization co-culture group.

[0119] 3. Construction of a 9-day co-training system for BMDM and GO

[0120] M0, M1, and M2 BMDMs were co-cultured with GO for 9 days before PCR detection. Figure 5The results showed that the mRNA expression of tail-type homeobox transcription factor 1 (CDX1) and SOX9 was significantly increased in the M2 polarization co-culture group. P <0.05 indicates that GO has IM lesions. The mRNA expression of SOX9 and signal transducer and activator of transcription 3 (STAT3) was significantly increased in the M1 polarization co-culture group ( P <0.05 indicates that GO also showed IM lesions. The mRNA expression of DMBT1, aquaporin 5 (AQP5), and mucin 6 (MUC6) was significantly increased in both the M1 and M2 polarization co-culture groups. P <0.05), IL-33 and TFF2 mRNA expression were significantly increased in the M1 polarization co-culture group ( P <0.05), indicating that GO also showed SPEM lesions, and the modeling effect of the M2 polarization co-culture group was better than that of the M1 polarization co-culture group.

[0121] 4. Construction of a 12-day co-training system for BMDM and GO

[0122] M0, M1, and M2 BMDMs were co-cultured with GO for 12 days before PCR detection. Figure 6 The results showed that the expression of ATP4A and MIST1 mRNA was significantly reduced in both the M1 and M2 polarization co-culture groups. P <0.05 indicates that GO has undergone gastric mucosal atrophy and SPEM lesions, represented by parietal cell loss. However, the mRNA expression of IM markers such as CDX2, trefoil factor 3 (TFF3), SOX9, and olfurcin-4 (OLFM4) and SPEM markers such as IL-33, DMBT1, and MUC6 was significantly lower in the M1 and M2 polarized co-culture groups than in the control group, indicating that mature IM cells and SPEM cells died. The expression of cell proliferation marker KI67 mRNA was significantly decreased in both the M1 and M2 polarized co-culture groups. P <0.05 indicates weakened GO stemness and reduced proliferation activity. Furthermore, during medium change on day 12 of co-culture, matrix gel collapse and floating loss were observed (…). Figure 7 The message indicates that GO needs to be propagated, and a new substrate glue should be used to replant the board.

[0123] In summary, with the extension of co-culture time, the expression of IM and SPEM-related markers mRNA in GO showed a dynamic trend of first increasing and then decreasing. Among them, co-culturing GO with M1 and M2 type BMDM for 3-9 days can induce IM with SPEM lesions, and the induction effect is better when co-culturing with M2 type BMDM for 3-6 days. This indicates that the immunosuppressive microenvironment induced by macrophage M2 polarization is more conducive to the formation of IM / SPEM.

[0124] 5. Pathological morphological observation and HE staining, IF detection of IM and SPEM-related markers after 3 days of co-culture of BMDM and GO in Example 3 (M2+GO+3DAY group) and Comparative Example 1 (M0+GO+3DAY group)

[0125] like Figure 8 Microscopic observation under bright field microscopy revealed significant phenotypic differences between IM-like gastric GO co-cultured with M2-type BMDM and normal gastric GO. The main differences were: normal GO exhibited a regular "gastric unit-like" three-dimensional structure with a smooth surface and clear polarity; while IM-like GO showed multicystic changes, disordered polarity, and cobblestone-like protrusions. HE staining showed that normal GO was predominantly composed of mucinous neck cells with basal nuclei, maintaining gastric glandular characteristics; IM-like GO, however, showed typical IM features such as cytoplasmic vacuoles (suggesting goblet cell formation), glandular lumen dilation, and increased nuclear atypia.

[0126] like Figure 9 IF staining revealed that SPEM markers AQP5 and genus lectin II (GSII) showed focal weak positive expression on the cell membranes of gastric gland neck mucus cells in the M0+GO+3DAY group, and continuous linear strong positive expression on the extracellular membrane of IM-like GO cells. The IM-specific marker MUC2 was almost not expressed in GO cells of the M0+GO+3DAY group, but showed significantly high expression in IM-like GO cells, exhibiting granular aggregation in both the cytoplasm and extracellular space of metaplastic cells.

[0127] The above experimental results further confirm that co-culturing GO with M2 macrophages for 3 days can induce IM and SPEM lesions in GO.

[0128] Experimental Example 3: The intervention effect of Weiwei Decoction on the IM-SPEM organoid models co-cultured for 3 and 6 days in Example 3.

[0129] 1. Weiwei Decoction is a clinically proven formula of Shenzhen Traditional Chinese Medicine Hospital and an in-house preparation of medical institutions in Guangdong Province (Guangdong Pharmaceutical Preparation Permit: Z20070548). The formula consists of: Astragalus membranaceus 30g, Codonopsis pilosula 30g, Sparganium stoloniferum (processed with vinegar) 15g, Curcuma zedoaria (processed with vinegar) 15g, Panax notoginseng 10g, Lycium barbarum 20g, Ophiopogon japonicus 15g, Dendrobium nobile 15g, Hedyotis diffusa 20g, Scutellaria barbata 20g, Atractylodes macrocephala 15g, and Glycyrrhiza uralensis (processed with honey) 5g. Serum concentrations of Weiwei Decoction at 5%, 7.5%, and 10% were used as low, medium, and high doses in a co-culture system for subsequent experiments.

[0130] 2. Experimental procedures for the intervention of serum containing Weiwei Decoction on M2 type BMDM co-culture with GO.

[0131] Based on the co-culture experiment in Example 3, for the system co-cultured for 3 days, at the beginning of co-culture, culture medium containing the corresponding concentration and volume of Weiweitang drug-containing serum was added to the upper chamber containing M2 type BMDM and the lower chamber containing GO, respectively. RNA was extracted after co-culture for 3 days. For the system co-cultured for 6 days, M2 type BMDM and GO were co-cultured for 3 days to induce phenotypic changes of IM and SPEM in GO. Then, the old culture medium was discarded, the medium was changed, and the corresponding concentration of Weiweitang drug-containing serum was added to the newly prepared culture medium and cultured for 3 days before RNA was extracted.

[0132] 3. Results of the intervention effect of serum containing Weiwei Decoction on IM phenotype formation in the co-culture system of GO and M2 type BMDM.

[0133] like Figure 10 To investigate the effect of serum containing Weiwei Decoction on IM organoid formation, low, medium, and high doses of serum containing Weiwei Decoction were added simultaneously at the beginning of the 3-day co-culture system of GO and M2 type BMDM. The results showed that Weiwei Decoction could not only significantly reduce the mRNA expression levels of IM markers such as tailed homeobox transcription factor 2 (CDX2), MUC2, and Kruppel-like factor 4 (KLF4) in GO, but also downregulate the expression of SPEM markers such as WFDC2, AQP5, MUC6, and IL-33 (P<0.05), indicating that Weiwei Decoction has a therapeutic effect in inhibiting IM and SPEM formation.

[0134] 4. Results of the intervention effect of serum containing Weiwei Decoction on BMDM in GO and M2 type BMDM co-cultured for 3 days.

[0135] like Figure 11 After intervention with Weiwei Decoction, the expression levels of polarization-related genes (CD206, PPARG, FIZZ1) and anti-inflammatory factors (IL-10, IL-4, IL-6) in M2 type BMDM were significantly reduced in the co-culture system. At the same time, it also restored the expression level of M1 polarization marker CD86 to a certain extent (P<0.05). This suggests that Weiwei Decoction may exert its therapeutic effect on IM through the molecular mechanism of dynamically regulating macrophage polarization homeostasis.

[0136] 5. Results of the intervention effect of serum containing Weiwei Decoction on GO and M2 type BMDM co-culture for 6 days

[0137] like Figure 12 Serum intervention with Weiwei Decoction significantly reduced the expression levels of IM markers such as CDX2, MUC2, KLF4, chorionic villi 1 (VIL1), and OLFM4, and SPEM markers such as DMBT1, WFDC2, TFF2, MUC6, and IL-33 in the model group GO (P<0.05), and also inhibited the expression level of KI67 (P<0.05).

[0138] The above results indicate that Weiwei Decoction not only inhibits IM formation but also has a therapeutic effect in inhibiting IM progression. Furthermore, Weiwei Decoction can inhibit the expression of SPEM-specific molecular markers such as AQP5, WFDC2, TFF2, and IL-33 in IM organoid models, suggesting that Weiwei Decoction can inhibit SPEM formation and may inhibit the biological process of SPEM transforming into IM, thereby blocking the IM formation pathway. In addition, it was observed that after intervention with Weiwei Decoction, M2 polarization-related genes (CD206, PPARG, FIZZ1) and immunosuppression-related inflammatory molecules (IL-10, IL-4, IL-6) in M2-type BMDM were significantly reduced, indicating that Weiwei Decoction may exert its therapeutic effect on IM by inhibiting M2 polarization of macrophages and reducing anti-inflammatory cytokines such as IL-10 and IL-4.

[0139] Cell screening and validation during the construction process in Experiment Example 4

[0140] 1. Co-culture was performed using classic human gastric mucosal epithelial cells (GES-1) and human leukemia mononuclear macrophages (THP-1).

[0141] Experimental protocol: THP-1 cells were revived and cultured in THP-1-specific medium (purchased from Wuhan Pronosai Life Science Technology Co., Ltd., catalog number CM-0233). After treatment with 100 ng / ml PMA (purchased from MCE, catalog number: HY-18739) for 24 h, they were induced to differentiate into macrophages and adhere to the culture medium. Then, 20 ng / ml IL-4 (purchased from MCE, catalog number: HY-P70445) and 20 ng / ml IL-13 (purchased from MCE, catalog number: HY-P72795) were added to stimulate THP-1 cells to polarize into the M2 phenotype for 48 h. Subsequently, GES-1 cells were passaged and seeded in DMEM high-glucose medium (Gibco, C11995500BT) containing 10% Australian fetal bovine serum (Thermo Fisher Scientific, USA; 10099141C). The resulting GES-1 cells were then co-cultured for 72 h in nested Transwell 6-well plates (containing 6-well plates, PET membrane, 24 mm, 0.4 μm, Corning, USA; catalog number: 3450). The upper chamber contained THP-1 cells, and the lower chamber contained GES-1 cells. The respective cell culture media required for each cell type were added to both chambers. The control group was co-cultured with unpolarized THP-1 cells at the same time.

[0142] After culture, corresponding RNA was extracted for PCR detection. The results showed that, except for a slight upregulation trend of TFF3, MUC6, and CD44 after co-culture with M2 macrophages, no significant changes in the expression of IM (CDX2, CDX1, MUC2, VIL1, TFF3, KLF4) and SPEM (WFDC2, MUC6, AQP5, DMBT1, CLU, CD44) related marker genes were observed. Figure 13 This indicates that the co-culture method using the above cell lines cannot construct cell models with IM or SPEM phenotypic changes.

[0143] 2. Co-culture using primary human gastric mucosal epithelial cells extracted clinically in a timely manner.

[0144] Primary gastric mucosal epithelial cells were co-cultured for 72 hours using a special culture medium for primary gastric mucosal epithelial cells (purchased from Wuhan Pronosei Life Sciences Co., Ltd., catalog number CM-H048) in step 1 of this experiment, replacing the GES-1 in the lower chamber with primary human gastric mucosal epithelial cells. The control group also used THP-1 without inflammatory factor stimulation; the model group used polarized THP-1 with added IL-4-IL-13 for co-culture. RNA was extracted from the primary gastric mucosal epithelial cells in the lower chamber for PCR detection. The results showed a significant upward trend in more IM and SPEM indicators, but these were not statistically significant, such as IM indicators like CDX1, MUC2, and VIL1. For SPEM indicators such as AQP5, TFF2, WFDC2, and MUC6, the differences between groups were relatively large. Figure 14 .

[0145] Based on the above experimental findings, it is believed that selecting gastric mucosal epithelial cells and macrophages that are closer to the primary cells for co-culture experiments has greater phenotypic plasticity, is closer to clinical practice, and is more likely to induce IM and SPEM lesions. Therefore, primary BMDM extracted from mouse bone marrow and mouse GO cells rich in stem cells and proliferative capacity were finally selected for co-culture experiments.

[0146] Experiment 5: Placement and observation of co-cultured cells in upper and lower chambers

[0147] After identifying the cells in Experiment 4, GO was initially seeded in the upper chamber and BMDM in the lower chamber for co-culture for 6 days. However, due to the requirements of the plate material for the MIG plate to solidify, when the MIG plate containing organoids was seeded in the upper chamber of the Transwell plate (which has a PET diaphragm at the bottom to allow small molecules to move), slight collapse of the MIG plate was observed, and the shape was inconsistent with the normal columnar three-dimensional structure. Furthermore, when the medium was changed on the third day of the experiment, further collapse of the MIG plate was observed. This situation was not conducive to the 3D culture conditions of GO. Therefore, the placement of the upper and lower chambers was changed in subsequent experiments, with BMDM seeded in the upper chamber and GO seeded in the lower chamber.

[0148] After determining that GOs would be planted in the lower chamber, the volume of the substrate gel used for planting could not be too large. Otherwise, during co-culture, the Transwell chambers would be added and press down on the top of the substrate gel, potentially affecting its curing morphology. Therefore, a "small volume, multiple points" planting method was chosen in a 6-well plate. This method ensured the number and density of organoids to meet experimental needs without compressing the substrate gel by the chambers. Figure 15 .

[0149] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an organoid model of gastric mucosal intestinal metaplasia (IM) accompanied by spasmolytic polypeptide expression metaplasia (SPEM), characterized in that, Includes the following steps: Step 1: Obtaining and basic culture of mouse bone marrow-derived macrophages: On day 1 of the experiment, femurs and tibias of C57BL / 6J mice were aseptically isolated, bone marrow cells were flushed out, and after erythrocyte lysis, the cells were resuspended in DMEM high-glucose medium containing 10-30 ng / ml M-CSF, 5-15% FBS and 0.5-1.5% penicillin-dextrose antibodies. The cells were seeded in the upper chamber of a Transwell plate, which was then placed on an empty 6-well plate and cultured in an incubator to induce differentiation into mature, unpolarized M0 macrophages. Step 2, Extraction and Primary Culture of Mouse Gastric Organoids: On day 1, the mucosal tissue of the mouse gastric body was obtained by dissection. After EDTA digestion, mechanical dissociation, and filtration, the obtained gastric glandular epithelial cells or cell clumps were embedded in matrix gel and seeded into the lower chamber of a 6-well Transwell plate without an upper chamber for co-culture. Gastric organoid conditioned medium containing key factors 8-12µM Y-27632, 8-12µM SB431542, and 8-12µg / ml gentamicin was added and cultured in an incubator. Routine Culture of Mouse Gastric Organoids: During the first 6 days of culture, the conditioned medium for mouse gastric organoids was completely replaced with fresh gastric organoid conditioned medium every 48 hours. Step 3: Co-culture of mouse gastric organoids and M2 type bone marrow-derived macrophages ① Steps one and two above are cultured separately and simultaneously; on day 6, polarization stimulation of mouse bone marrow-derived macrophages and passage culture of mouse gastric organoids are performed simultaneously and cultured for another 48 hours; the polarization stimulation is achieved by discarding the old culture medium and adding DMEM high glucose medium containing 5-15% FBS containing 15-25 ng / ml IL-4 and 15-25 ng / ml IL-13 for 48 hours to construct M2 type bone marrow-derived macrophages; ② Start dynamic co-culture initiation and maintenance on day 8 On day 8, the co-culture system was established: After 48 hours of polarization stimulation of mouse bone marrow-derived macrophages, the old culture medium in the lower chamber of the Transwell for mouse gastric organoids and the upper chamber of the Transwell for M2 bone marrow-derived macrophages was discarded; fresh gastric organoid conditioned medium was added to the lower chamber well of the Transwell containing the mouse gastric organoids; the upper chamber of the Transwell containing M2 bone marrow-derived macrophages was embedded in the culture plate in which the lower chamber of the gastric organoids had been placed, and DMEM high-glucose medium containing 5-15% FBS but without polarization factors IL-4 and IL-13 was added to the upper chamber; the culture was carried out at 37-39℃ and 3-7% CO2 concentration for 3 days to obtain the gastric mucosal IM with SPEM organoid model; Dynamic maintenance mechanism: After the start of co-culture, the old upper chamber of M2 bone marrow-derived macrophages, along with the culture medium and cells therein, is discarded every 3 days and replaced with a freshly prepared upper chamber of M2 bone marrow-derived macrophages that has undergone the same polarization stimulation for 48 hours. The culture medium of the gastric organoids in the lower chamber is replaced with fresh gastric organoid conditioned medium every 48 hours. The co-culture conditions include: incubation at 37-39℃ and 3-7% CO2 concentration for 3-9 days; The conditioned medium for the gastric organoids is L-WRN conditioned medium.

2. A method for constructing a gastric mucosal IM-SPEM organoid model, characterized in that, Includes the following steps: Step 1: Obtaining and basic culture of mouse bone marrow-derived macrophages: On day 1 of the experiment, femurs and tibias of C57BL / 6J mice were aseptically isolated, bone marrow cells were flushed out, and after erythrocyte lysis, the cells were resuspended in DMEM high-glucose medium containing 10-30 ng / ml M-CSF, 5-15% FBS and 0.5-1.5% penicillin-dextrose antibodies. The cells were seeded in the upper chamber of a Transwell plate, which was then placed on an empty 6-well plate and cultured in an incubator to induce differentiation into mature, unpolarized M0 macrophages. Step 2, Extraction and Primary Culture of Mouse Gastric Organoids: On day 1, the mucosal tissue of the mouse gastric body was obtained by dissection. After EDTA digestion, mechanical dissociation, and filtration, the obtained gastric glandular epithelial cells or cell clumps were embedded in matrix gel and seeded into the lower chamber of a 6-well Transwell plate without an upper chamber for co-culture. Gastric organoid conditioned medium containing key factors 8-12µM Y-27632, 8-12µM SB431542, and 8-12µg / ml gentamicin was added and cultured in an incubator. Routine Culture of Mouse Gastric Organoids: During the first 6 days of culture, the conditioned medium for mouse gastric organoids was completely replaced with fresh gastric organoid conditioned medium every 48 hours. Step 3: Co-culture of mouse gastric organoids and M1 type bone marrow-derived macrophages ① Steps one and two above are cultured separately and simultaneously; on day 6, polarization stimulation of mouse bone marrow-derived macrophages and passage culture of mouse gastric organoids are performed simultaneously and cultured for another 48 hours; the polarization stimulation is achieved by discarding the old culture medium and adding DMEM high-glucose culture medium containing 95-105 ng / ml LPS and 5-15% FBS for 48 hours to construct M1 type bone marrow-derived macrophages; ② Start dynamic co-culture initiation and maintenance on day 8 Establishment of the co-culture system on day 8: After 48 hours of polarization stimulation of mouse bone marrow-derived macrophages, the old culture medium in the lower chamber of the Transwell for mouse gastric organoids and the upper chamber of the Transwell for M1 bone marrow-derived macrophages was discarded; fresh gastric organoid conditioned medium was added to the lower chamber well of the Transwell containing the mouse gastric organoids; the upper chamber of the Transwell containing M1 bone marrow-derived macrophages was embedded in the culture plate in which the lower chamber of the gastric organoids had been placed, and DMEM high-glucose medium containing 5-15% FBS but without polarization factor LPS was added to the upper chamber; the culture was carried out at 37-39℃ and 3-7% CO2 concentration for 3 days to obtain the gastric mucosal IM with SPEM organoid model; Dynamic maintenance mechanism: After the start of co-culture, the old upper chamber of M1 bone marrow-derived macrophages, along with the culture medium and cells therein, is discarded every 3 days and replaced with a freshly prepared upper chamber of M1 bone marrow-derived macrophages that has undergone the same polarization stimulation for 48 hours. The culture medium of the gastric organoids in the lower chamber is replaced with fresh gastric organoid conditioned medium every 48 hours. The co-culture conditions include: incubation at 37-39℃ and 3-7% CO2 concentration for 3-9 days; The conditioned medium for the gastric organoids is L-WRN conditioned medium.

3. The construction method as described in claim 1, characterized in that, The culture medium used for the polarization-stimulated bone marrow-derived macrophages was DMEM high-glucose medium containing 5-15% FBS.

4. The application of the gastric mucosal IM-SPEM organoid model obtained by the construction method according to any one of claims 1-3 in screening products for the prevention and / or treatment of gastric mucosal IM, SPEM lesions and gastric cancer.

Images