Mouse gastric adenocarcinoma cell line MPC as well as construction method and application thereof

Abstract

The invention discloses a mouse gastric adenocarcinoma cell line MPC as well as a construction method and application thereof, and belongs to the technical field of preparation of tumor cell lines and tumor models. The mouse gastric adenocarcinoma cell strain MPC is preserved in China Center for Type Culture Collection, has Cdh1 and Tp53 gene mutations at the same time, has stable cell biological characteristics and typical adenocarcinoma characteristics, can form tumors in immunodeficient and normal immune mice, can be used for preparing a mouse tumor model for occurrence, development and metastasis of gastric cancer, and has a good application prospect. The method is applied to important researches such as molecular biology, cytobiology, detection kit development, new drug research and development and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor cell lines and tumor model preparation, and particularly relates to a mouse gastric adenocarcinoma cell line MPC and a construction method and application thereof. Background Art

[0002] Gastric cancer is one of the most common malignant tumors in my country. It has become the second most common tumor in my country and the third most common tumor in terms of mortality. In recent years, the number of new cases of gastric cancer and gastric cancer-related deaths in my country has accounted for almost half of the world's total. Gastric adenocarcinoma accounts for more than 95% of gastric cancer. Patients with early-stage gastric adenocarcinoma can achieve a good prognosis through radical resection of gastric adenocarcinoma, but most patients have no obvious early symptoms and are already in the middle or late stages when diagnosed. Although there are currently a variety of treatments such as chemotherapy and radiotherapy that can benefit some patients, some patients are still insensitive to treatment and find it difficult to improve their survival from existing treatments. One of the important reasons is the lack of available mouse gastric adenocarcinoma cell lines.

[0003] A master craftsman must first sharpen his tools to achieve perfection. A deeper understanding of the pathogenesis of gastric adenocarcinoma and the development of more effective treatments are key to achieving breakthroughs in its treatment. Although patents exist for establishing mouse gastric cancer organoids and primary tumor models using transgenic mice, this approach suffers from long experimental cycles and high costs. The establishment of cell lines would greatly facilitate research, shorten the time required to establish gastric cancer models, reduce research costs, and enhance reproducibility. Mouse tumor cell lines are valuable tools for studying tumor development, progression, and metastasis, and are also commonly used in the development of tumor diagnostic and therapeutic methodologies. Currently, mouse gastric adenocarcinoma cell lines are rare. The only mouse gastric cancer cell line available from the ATCC is MFC, a squamous cell carcinoma that cannot form xenografts in immunocompetent mice, limiting its use in various research scenarios. Therefore, developing a gastric adenocarcinoma cell line that exhibits characteristics of adenocarcinoma and can form tumors in both immunodeficient and immunocompetent mice is crucial.

[0004] The tumor suppressor gene p53 (Tp53) plays a crucial role in cell cycle arrest, cellular senescence, apoptosis, differentiation, and metabolism. Alterations in the Tp53 gene are found in more than half of tumors, and mutations are associated with poor prognosis. Cadherin-1 (Cdh1) is a tumor suppressor gene that encodes epithelial cadherin (E-cadherin), which plays a crucial role in maintaining the structural and functional stability of epithelial tissues. Cdh1 gene deletion and decreased E-cadherin protein expression are found in gastric cancer tissues, closely associated with tumor cell invasion and metastasis. Cdh1 mutations are a single-gene predictor of poor prognosis in gastric cancer patients. Patients with Cdh1 mutations are more likely to develop peritoneal metastasis and have poorer overall survival than those without mutations. Whole-exome sequencing results indicate that peritoneal metastases are more likely to harbor mutations in Cdh1 and TP53 than primary lesions. Establishing mouse gastric cancer cell lines harboring Cdh1 and TP53 mutations is of great value for gastric cancer research.

[0005] Based on the above background, the present invention provides a mouse gastric adenocarcinoma cell line, which has double mutations in Cdh1 and Tp53, has stable cell biological characteristics and typical adenocarcinoma characteristics, can form tumors in immunodeficient and immune-normal mice, and can be directly applied to important research fields such as molecular biology, cell biology, construction of animal models, development of detection kits and new drug research and development. Summary of the Invention

[0006] The present invention aims to provide a mouse gastric adenocarcinoma cell line (MPCs); a second aim is to provide a method for constructing and using the MPCs. The mouse gastric adenocarcinoma cell line provided by the present invention can be stably passaged and can form tumors in both immunodeficient and immunocompetent mice. It can be used to study the mechanisms of gastric adenocarcinoma development, progression, and metastasis, as well as immune escape and drug resistance. It is an important tool for developing early screening methodologies for gastric adenocarcinoma and for drug screening for gastric adenocarcinoma.

[0007] The present invention includes the following technical solutions:

[0008] In a first aspect, the present invention provides a mouse gastric adenocarcinoma cell line MPC, wherein the mouse gastric adenocarcinoma cell line is deposited in the China Center for Type Culture Collection with a biological deposit number of CCTCC NO: C2024264.

[0009] Furthermore, the mouse gastric adenocarcinoma cell line provided by the present invention also includes daughter cells of the cell line contained therein.

[0010] In a second aspect, the present invention provides a method for constructing a mouse gastric adenocarcinoma cell line MPC, the method comprising the following steps:

[0011] 1) Cdh1 fl / fl Mist1-cre mice and Tp53 fl / fl Mist1-cre mice were mated to obtain Cdh1 fl / fl Tp53 fl / fl Mist1-cre mice;

[0012] 2) Isolation of Cdh1 from step 1) fl / fl Tp53 fl / fl Mist1-cre mouse stomach tissue was digested with tissue collagenase and cultured in complete organoid medium to obtain mouse gastric epithelial organoids Cdh1 fl / fl Tp53 fl / fl ;

[0013] 3) The mouse Cdh1 obtained in step 2) fl / fl Tp53 fl / fl Organoids were induced with tamoxifen to form mouse gastric cancer organoids Cdh1 - / - Tp53 - / - ;

[0014] 4) The mouse gastric cancer organoid Cdh1 obtained in step 3) - / - Tp53 - / - The mouse gastric adenocarcinoma cell line MPC was formed by acclimating the cells to the culture medium by gradually reducing the concentration of growth factors.

[0015] In a third aspect, the present invention provides a use of a mouse gastric adenocarcinoma cell line MPC in at least one of the following:

[0016] a) Application in preparing gastric adenocarcinoma cell model;

[0017] b) Application in preparing animal models of gastric adenocarcinoma;

[0018] c) use in the preparation and / or screening of drugs for preventing and treating gastric adenocarcinoma;

[0019] d) Application in establishing a research platform for drug resistance mechanisms in gastric adenocarcinoma;

[0020] e) Application in establishing a research platform for early screening and grading of gastric adenocarcinoma;

[0021] f) Application in establishing a research platform for the treatment of gastric adenocarcinoma.

[0022] The cell model and animal model of the present invention include cell or animal models of the occurrence, development and metastasis stages of gastric adenocarcinoma, and can be used to study the occurrence, development and metastasis mechanism of gastric adenocarcinoma.

[0023] In a fourth aspect, the present invention provides a method for constructing a gastric cancer mouse model, the method comprising: implanting the mouse gastric adenocarcinoma cell line MPC described in the first aspect of the present invention into a mouse, and feeding the mouse for 30-40 days to form a gastric cancer mouse model.

[0024] The gastric cancer mouse model includes a gastric cancer subcutaneous tumor mouse model, a gastric cancer in situ tumor mouse model and a gastric cancer metastasis tumor mouse model.

[0025] The gastric cancer described in the present invention is gastric adenocarcinoma.

[0026] In one embodiment of the present invention, the present invention provides a method for constructing a gastric adenocarcinoma subcutaneous tumor mouse model, comprising: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention was implanted in the armpit of mice at a volume of 0.1-0.5 mL per mouse and fed for 30-40 days to form a gastric adenocarcinoma subcutaneous tumor mouse model.

[0027] In one embodiment of the present invention, the present invention provides a method for constructing a gastric adenocarcinoma in situ tumor mouse model, comprising: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention was implanted into the gastric tissue of mice at a volume of 0.1-0.5 mL per mouse and fed for 60 days to form a gastric adenocarcinoma in situ tumor mouse model.

[0028] In one embodiment of the present invention, the present invention provides a method for constructing a gastric adenocarcinoma metastasis mouse model, comprising: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention is implanted into the abdominal cavity of mice at a volume of 0.1-0.5 mL per mouse and fed for 30-40 days to form a gastric adenocarcinoma metastasis mouse model.

[0029] In the present invention, the mice used to construct the mouse model are selected from immunodeficient mice or immune normal mice.

[0030] In some embodiments of the present invention, the immunodeficient mice include Balb / c nude mice, SCID mice, and severely immunodeficient NPG, NOG, and NSG mice.

[0031] In some embodiments of the present invention, the immune normal mice include C57BL / 6J mice.

[0032] In a fifth aspect, the present invention provides a gastric adenocarcinoma mouse model constructed according to the method of the fourth aspect of the present invention.

[0033] In a sixth aspect, the present invention provides a use of the gastric adenocarcinoma mouse model in at least one of the following:

[0034] a) use in the preparation and / or screening of drugs for preventing and treating gastric adenocarcinoma;

[0035] b) use as an animal model for preparing and / or screening drugs for preventing and treating gastric adenocarcinoma;

[0036] c) Application in establishing a research platform for drug resistance mechanisms in gastric adenocarcinoma;

[0037] d) Application in establishing a research platform for early screening and grading of gastric adenocarcinoma;

[0038] e) Application in establishing a research platform for the treatment of gastric adenocarcinoma.

[0039] In a seventh aspect, the present invention provides a method for preparing or screening a candidate drug for preventing and / or treating gastric adenocarcinoma, the method comprising at least one of the following:

[0040] m1) administering the test substance to a gastric adenocarcinoma cell model, wherein the test substance that inhibits cell proliferation or induces other programmed cell death is a candidate drug, wherein the gastric adenocarcinoma cell model is the gastric adenocarcinoma cell line MPC according to the first aspect of the present invention;

[0041] m2) administering the test substance to an animal model of gastric adenocarcinoma, monitoring the animal body weight and tumor growth, and the test substance that causes improvement or cure of gastric adenocarcinoma symptoms in the animal model is a candidate drug, wherein the gastric adenocarcinoma animal model is constructed by the method described in the fourth aspect of the present invention.

[0042] The method for administering the test substance described in method m2 is a conventional administration method in the art, including but not limited to tail vein injection, intraperitoneal injection, oral gavage, subcutaneous injection, intramuscular injection or local tumor administration, or a combination of two or more thereof.

[0043] The technical solution provided by the present invention has the following beneficial technical effects:

[0044] Currently, there are no relevant studies or reports on mouse gastric adenocarcinoma cell lines with double mutations of Tp53 and Chd1. The cell line provided by the present invention has both Tp53 and Chd1 mutations, filling the gap in this field. In addition, the mouse gastric adenocarcinoma cell line MPC provided by the present invention has stable cell biological characteristics and typical adenocarcinoma characteristics, with stable cloning, invasion and migration capabilities; it can form tumors in both immunodeficient mice and immune-normal mice, and can be successfully used to prepare a mouse tumor model of gastric cancer occurrence, development and metastasis. This model can be used to study the molecular mechanisms of gastric cancer occurrence and development, develop early screening methodologies for gastric cancer, screen drugs for the treatment of gastric cancer, and search for new biomarkers related to gastric cancer. It is an ideal tool for basic research and preclinical applications of gastric cancer.

[0045] Description of the deposit of biological materials involved in the present invention

[0046] Depository: China Center for Type Culture Collection (CCTCC);

[0047] Address: Wuhan University, Wuhan, China;

[0048] Deposit date: September 11, 2024;

[0049] Deposit number: CCTCC NO:C2024264;

[0050] Classification and nomenclature: Mouse gastric adenocarcinoma cell line MPC. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the process of establishing the mouse gastric adenocarcinoma cell line MPC.

[0052] Figure 2 These are cell morphology images of mouse normal gastric mucosal organoids (2a), mouse gastric cancer organoids (2b before low growth factor culture, 2c after low growth factor culture), and gastric adenocarcinoma cell line MPC (2d).

[0053] Figure 3 HE staining of the mouse gastric adenocarcinoma cell line MPC (3a) and the expression of the epithelial marker pan-CK (3b), as well as the expression of knockout genes encoding E-cadherin (3c) and p53 (3d).

[0054] Figure 4 This is a subcutaneous tumor of gastric adenocarcinoma MPC in C57BL / 6J mice.

[0055] Figure 5 HE staining of subcutaneous tumor tissue of gastric adenocarcinoma MPC in C57BL / 6J mice (5a), expression of adenocarcinoma epithelial marker pan-CK (5b), and expression of E-cadherin (5c) and p53 (5d).

[0056] Figure 6 This is the peritoneal tumor formation of gastric adenocarcinoma MPC cells in Balb / c nude mice. The green arrow points to the solid tumor formed in the peritoneal cavity.

[0057] Figure 7 HE staining of intraperitoneal tumor tissues of gastric adenocarcinoma MPC cells in Balb / c nude mice (7a and 7b) and the expression of adenocarcinoma biomarker pan-CK (7c).

[0058] Figure 8 Figure 8a shows the formation of tumors after orthotopic injection of gastric adenocarcinoma MPC cells into the stomach of Balb / c nude mice (8b and 8c). Figure 8b shows the formation of tumors after orthotopic injection of gastric adenocarcinoma MPC cells into the stomach of Balb / c nude mice (8a and 8b). DETAILED DESCRIPTION

[0059] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts are within the scope of protection of the present invention.

[0060] The compound names corresponding to the English abbreviations involved in the present invention are as follows:

[0061] Penicillin / Streptomycin Penicillin-streptomycin dual antibody <![CDATA[GlutaMAX TM Supplement]]> Glutamine Supplement N-Acetyl-L-cysteine N-Acetyl-L-Cysteine Nicotinamide Niacinamide R-Spondin-1 Wnt signaling pathway activators EGF Epidermal Growth Factor Thiazovivin Selective ROCK inhibitors B-27Supplement B-27 Serum-Free Supplement HEPES HEPES buffer N-2Supplement N-2 Additive Wnt-3a Wnt-3a growth differentiation factor Noggin Cell culture factors Gastrin I Gastric mucosal regeneration factor FGF-10 Fibroblast Growth Factor 10 MPC Mesenchymal progenitor cells

[0062] Example 1 Establishment of Mouse Gastric Adenocarcinoma Cell Line MPC

[0063] 1.Cdh1 fl / fl Tp53 fl / fl Mist1-cre mouse construct

[0064] Cdh1 fl / fl Mist1-cre C57BL / 6J mice and Tp53 fl / fl Cdh1 was obtained by mating Mist1-cre C57BL / 6J mice fl / fl Tp53 fl / fl Mist1-cre C57BL / 6J mice.

[0065] 2. Isolation of Cdh1 from step 1 fl / fl Tp53 fl / fl Mist1-cre mouse stomach tissue cultured into mouse gastric organoids

[0066] 2.1) 8-week-old Cdh1 fl / fl Tp53 fl / flMist1-cre mice were euthanized by CO2, and the stomach tissues were removed after dissection. The tissues were washed three times with ice-cold PBS.

[0067] 2.2) Use ophthalmic scissors to cut the tissue into 1-2 mm pieces. 3 The size of the cells was adjusted, and tissue digestion solution (Yeasen, #41423ES10) was added. The cells were digested in a 37°C water bath for 1.5 h. The cell-containing digestion suspension was filtered through a 70 μm pore size cell strainer (BIOFIL, #CSS-013-070), and the cell filtrate was collected and centrifuged at 350 g for 5 minutes to obtain a cell pellet. The cell pellet was resuspended in 200 μL of organoid complete medium and mixed with an equal volume of Matrigel (Corning, #356231) as the cell suspension. 50 μL of the system was seeded into a 6-well plate at 6 drops / well. After solidification at 37°C for 30 minutes, 2 mL of organoid complete medium was added for culture.

[0068] The organoid complete culture medium is AdDMEM / F-12 (Thermo, #12634010) culture medium containing the following specific components:

[0069]

[0070]

[0071] 2.3) Mouse normal gastric organoids cultured in step 2.2) Cdh1 fl / fl Tp53 fl / fl (Cell morphology as Figure 2 A final concentration of 20 nM Tamoxifen (Sigma-aldrich, #10540-29-1) was added to the mouse gastric cancer organoids (shown in a) and induced for 1 day to form mouse gastric cancer organoids Cdh1 - / - Tp53 - / - At the same time, in order to further purify mouse gastric cancer organoids, Cdh1 - / - Tp53 - / - Gastric cancer organoids need to add 20nM Nutlin-3 (a p53 protein degradation inhibitor; wild-type organoids cannot grow in organoids containing Nutlin-3) during the subsequent culture process, change the medium every 4 days, and subculture every 6-8 days.

[0072] The subculture method is as follows: When the gastric cancer organoids cultured above grow to a cell sphere diameter of 100 μm, the culture medium is aspirated and 500 μL of TrypLE is added. TMExpress enzyme (Thermo, #12604021), digest at 37℃ for 8-15min, add serum-containing medium to terminate digestion, mix thoroughly, centrifuge the cell suspension at 350g for 5min at room temperature, and remove the supernatant. Resuspend the cell pellet in organoid complete medium, mix the cell suspension with matrigel in a 1:1 ratio, and add 3×10 4 Cells / well, 50 μL / drop were inoculated into 6-well plates, 6 drops / well, and placed in a 37°C incubator for 30 min. At this time, the matrix gel has solidified and 2 mL of organoid complete culture medium was added.

[0073] 3. Domestication of Gastric Cancer Organoids into Cell Lines

[0074] For gastric cancer organoids cultured as in step 2, Cdh1 - / - Tp53 - / - Inject the cells into Balb / c nude mice (about 1*10^7 cells / mouse, containing 50% Matrigel) and observe the growth of the subcutaneous tumor. 3 , isolate the mouse subcutaneous tumor, and mince the tissue into 1-2 mm pieces using ophthalmic scissors. 3 The cells were sized and digested in tissue digestion buffer (Yeasen, #41423ES10) at 37°C for 0.5 h in a constant temperature water bath. The cell-containing digestion suspension was filtered through a 70 μm pore size cell strainer (BIOFIL, #CSS-013-070), and the cell filtrate was collected and centrifuged at 250 g for 5 minutes to obtain a cell pellet. The cell pellet was resuspended in 1640 medium containing 10% FBS (fetal bovine serum) and cultured in a 37°C, 5% CO2 incubator for 2D cell culture. If the cells are in poor condition during culture, 10% complete mouse gastric cancer organoid culture medium can be added. After 1 month of continuous culture, single clones were selected.

[0075] 4. Cell Monoclonal Selection

[0076] Collect the cells acclimated in step 3 and dilute them with ordinary culture medium to a suspension with a cell density of 1 cell / 0.4 mL. Add the cell suspension to a 96-well plate at 200 μL / well. Place the well plate in a cell culture incubator for culture. The cells in each well will gradually grow and divide to form a monoclonal clone. Then observe under a microscope and select the well where only one cell has grown. Further culture, expansion and seed preservation are performed, and this cell is named MPC. The cell morphology is as follows: Figure 2 As shown in d.

[0077] Example 2 Identification and Tumorigenicity Verification of Mouse Gastric Adenocarcinoma Cell Line MPC

[0078] 1. Culture of Mouse Gastric Adenocarcinoma Cell Line MPC

[0079] Mouse gastric adenocarcinoma cell line MPC was cultured at 4×10 5 Cells were plated at a density of 1000 cells / well in 6-well plates. Cell morphology and proliferation were observed. Cells were then cryopreserved and revived. Cell viability was observed after resuscitation to confirm cell passageability.

[0080] 2. Morphological Observation and Identification of Mouse Gastric Adenocarcinoma Cell Line MPC

[0081] 2.1) Observe the morphology of living cells using an inverted phase contrast microscope.

[0082] After changing the medium of the cells cultured in step 1, observe and photograph them under an inverted phase contrast microscope. It can be seen that the MPC cells are mostly irregular sheets, with uniform internal structure and a shape similar to "fried eggs" (such as Figure 2 d).

[0083] 2.2) HE staining of mouse gastric adenocarcinoma cell line MPC slides

[0084] In a 6-well plate containing a glass slide, after overnight culture, the supernatant was discarded, the cells were washed twice with PBS, and fixed with 4% paraformaldehyde (Biosharp, #BL539A) for half an hour. The MPC cell slides were washed three times with PBS, fixed with 95% ethanol for 20 minutes, and washed twice with PBS, each time for 1 minute. Nuclear staining: stain with hematoxylin solution for 2-3 minutes, and wash with tap water. Observe under a microscope. If the nucleus is stained too dark, decompose it with 1% hydrochloric acid alcohol solution for a few seconds and wash with tap water. Cytoplasm staining: immerse in eosin stain for 1 minute and wash with tap water. After blowing the stem cell slides, seal the slides with neutral gum, observe and photograph under an inverted phase contrast microscope. The results showed that the cell morphology was mostly flaky, some were long spindle-shaped, the cell nuclei were relatively uniformly round or oval, and the cell nuclei were large (such as Figure 3 a).

[0085] 2.3) Pan-CK, p53, and E-cadherin staining of mouse gastric adenocarcinoma cell lines

[0086] MPC cell slides were washed three times with PBS, fixed with 95% ethanol for 20 minutes, and washed twice with PBS for 1 minute each. The slides were incubated once with 0.5% Triton X-100 (in PBS) for 20 minutes, and washed three times with PBS for 2 minutes each. The slides were incubated with 3% H₂O₂ for 15 minutes, and washed three times with PBS for 2 minutes each. Blocking serum (5% normal secondary antibody serum in PBS) was used for 20 minutes. Primary antibody incubation (prepared with antibody diluent) was performed in a humidified chamber overnight at 4°C. The slides were washed three times with PBS for 5 minutes each, and then incubated with horseradish peroxidase-conjugated secondary antibody in a humidified chamber for 30 minutes at 37°C. The slides were washed three times with PBS for 5 minutes each. DAB staining was performed for 5 minutes in the dark, and the slides appeared brown under a microscope. The slides were washed twice with distilled water for 1 minute each. The slides were counterstained with hematoxylin for 1 minute, and washed with tap water for 20 minutes. Dehydrate with gradient ethanol concentrations: 80% for 2 minutes; 95% for 2 minutes; and 100% for 5 minutes. Clear the slides twice with xylene for 5 minutes each time, and then mount the slides with neutral gum. Observe and photograph the slides under an inverted phase-contrast microscope. The results showed that the mouse gastric adenocarcinoma cell line MPC widely expressed glandular epithelial markers pan-CK and E-cadherin, and lacked expression of p53 (see Table 1). Figure 3 b, 3c, and 3d).

[0087] 2.4) STR Identification of Mouse Gastric Adenocarcinoma Cell Line MPC

[0088] The cells were collected and subjected to STR analysis (Table 1). The results showed that the cell line was a mouse cell line. Comparison of the STR data for this cell line with those of cell lines included in the ATCC, DSMZ, JCRB, and RIKEN databases revealed no such cell line, indicating that this cell line was a novel mouse cell line.

[0089] Table 1 Genotyping results of MPC STR loci and Amelogenin loci in mouse gastric adenocarcinoma cell line

[0090]

[0091]

[0092] 3. Tumor formation of mouse gastric adenocarcinoma cell line MPC

[0093] 3.1) Subcutaneous tumor

[0094] When the mouse gastric adenocarcinoma cells (MPCs) were cultured to a confluence of 90%, the cells were collected and washed twice with PBS. The cell concentration was adjusted to 5 × 10 7The cells / mL were injected subcutaneously into the axilla of C57BL / 6 mice (8-week-old, female, Chengxi Biotechnology) at a volume of 0.1 mL / mouse. After 40 days, the mice were euthanized with CO2, and the tumors were dissected and photographed (e.g. Figure 4 The tumors were fixed in paraformaldehyde for subsequent pathological analysis.

[0095] 3.2) Abdominal tumor implantation

[0096] When the mouse gastric adenocarcinoma cells were cultured to a confluence of 90%, the cells were collected and washed twice with PBS. The cell concentration was adjusted to 10 × 10 7 The tumors were intraperitoneally injected into Balb / c nude mice (8-week-old, female, Chengxi Biotechnology) at a volume of 0.1 mL / mouse. After 40 days, the mice were euthanized with CO2, and the abdominal cavity of the mice was dissected to observe the tumor formation and take pictures (e.g. Figure 6 The intestinal tissues containing tumors of mice were fixed in paraformaldehyde for subsequent pathological analysis.

[0097] 3.3) Orthotopic gastric implantation

[0098] When the mouse gastric adenocarcinoma cells were cultured to a confluence of 90%, the cells were collected and washed twice with PBS. The cell concentration was adjusted to 10 × 10 7 The tumor was intraperitoneally injected into the serosa of the gastric antrum of Balb / c nude mice (8-week-old, female, Chengxi Biotechnology) at a volume of 0.05 mL per mouse. After 45 days, the mice were euthanized with CO2, and the abdominal cavity of the mice was dissected to observe the tumor formation and take pictures (such as Figure 8 The gastric tissue containing tumors of mice was fixed in paraformaldehyde for subsequent pathological analysis.

[0099] 4. Tumor Pathology Analysis

[0100] 4.1) Paraffin embedding of tissue

[0101] After trimming the fixed tissue, place it in an embedding cassette and rinse with running tap water for 30 minutes. Use a fully automatic dehydrator and set the program: soak in 75% ethanol for 30 minutes, soak in 80% ethanol for 30 minutes, soak in 90% ethanol for 30 minutes × 2 times, soak in 90% ethanol for 30 minutes × 2 times, soak in xylene for 20 minutes × 2 times, and soak in tissue paraffin for 90 minutes × 3 times. Paraffin embedding: Use hard wax (56-58°C), pour it into a metal frame, bury the tissue embedding cassette in the hot wax, place it on an ice table to cool for 4-6 hours, and remove it after the wax block is completely solidified. Fix the wax block on the microtome, hold the brush in your left hand, and turn the microtome with your right hand. First trim the tissue and make the cut surface smooth. Set the slice thickness to 8μm, and slice continuously to form a wax strip. Use a brush to gently lift the slice, spread the slice face up flat in warm water, and the slice will naturally unfold under the action of surface tension. Insert a glass slide vertically into the water, bring it close to the slice, and gently pull it out so that the slice adheres to the slide. Label the sample information. Place the slide in a 60°C constant temperature oven for half an hour to dry it for later use.

[0102] 4.2) HE staining

[0103] Dewax paraffin sections to dehydrate: Place sections in xylene I for 15 minutes, xylene II for 15 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes, and distilled water for 10 minutes. Stain with hematoxylin at room temperature for 1 minute, rinse in tap water for 8 minutes to blue, and rinse in distilled water for 3 minutes. Dehydrate and transparentize sections: Place sections in 70% ethanol for 3 minutes, 80% ethanol for 3 minutes, 90% ethanol for 3 minutes, stain with 1% eosin for 1 second, 95% ethanol for 3 minutes, 100% ethanol I for 3 minutes, 100% ethanol II for 3 minutes, xylene I for 5 minutes, xylene II for 5 minutes, and then mount with neutral resin. Observe and photograph under an inverted phase contrast microscope.

[0104] The results showed that in the subcutaneous tumor tissue of gastric adenocarcinoma MPC in C57BL / 6J mice, the nuclei of tumor cells were larger and there were obvious glandular duct structures (such as Figure 5 a) In the peritoneal implantation of gastric adenocarcinoma (MPC) in Balb / c nude mice, the tumor was mainly located in the intestinal mucosa, with no obvious intestinal infiltration. The nuclei of the tumor cells were enlarged, and the tissue had obvious glandular ductal structures (e.g. Figure 7 In the orthotopic gastric injection model, the tumor grew in the gastric serosa and infiltrated the muscularis, but did not break through the gastric mucosa (see Figures 8b and 8c).

[0105] 4.3) Immunohistochemical staining of pan-CK, E-cadherin, and p53

[0106] Dewax paraffin sections to dehydrate: Place sections in xylene I for 15 minutes, xylene II for 15 minutes, ethanol I for 5 minutes, ethanol II for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and finally wash with distilled water for 10 minutes. Antigen retrieval: Place tissue sections in a microwave oven filled with citric acid antigen retrieval buffer (pH 6.0). Incubate on medium heat for 8 minutes, then on medium-low heat for 8 minutes, then on medium-low heat for 7 minutes. During this process, prevent excessive evaporation of the buffer and avoid drying the slides. After cooling naturally, wash the slides three times (5 minutes each) in PBS (pH 7.4) on a destaining shaker. Blocking: Incubate sections in 3% hydrogen peroxide solution at room temperature in the dark for 25 minutes to block endogenous peroxidase. Wash the slides three times (5 minutes each) in PBS (pH 7.4) on a destaining shaker with shaking. Circle drawing: After briefly drying the sections, use a histochemical pen to draw a circle around the tissue to prevent antibody loss. Serum blocking: Add 3% BSA to the ring and incubate at room temperature for 30 minutes. Primary antibody application: Gently shake off the blocking solution and add the primary antibody prepared in the antibody diluent according to the specified ratio to the slide. Incubate the slides flat in a humidified chamber at 4°C overnight (add a small amount of water to the chamber to prevent antibody evaporation). Secondary antibody application: Wash slides in PBS (pH 7.4) three times for 5 minutes each on a shaker for destaining. After the slides are slightly dried, add a secondary antibody of the species corresponding to the primary antibody to the ring for covering the tissue and incubate at room temperature in the dark for 50 minutes. DAB color development and counterstaining of nuclei: Wash slides in PBS (pH 7.4) three times for 5 minutes each on a shaker for destaining. After the slides are slightly dried, add DAB color development reagent to the ring for monitoring color development under a microscope. After complete color development, rinse with distilled or tap water, counterstain with hematoxylin, differentiate with 1% hydrochloric acid and alcohol (approximately 1 second), rinse with tap water, blue with ammonia solution, and rinse with running water. Dehydration and mounting: Dehydrate and transparentize sections by placing them in 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, and xylene I for 5 minutes. Remove sections from xylene, allow to dry briefly, and mount with neutral gum. Observe and photograph under an inverted phase contrast microscope.

[0107] The results showed that in the subcutaneous tumor tissue of gastric adenocarcinoma MPC in C57BL / 6J mice, the nuclei of tumor cells were larger, mostly round or oval, with obvious glandular duct structure, and the adenocarcinoma biomarker pan-CK (such as Figure 5 b); Due to the knockout of CDH1 and TP53, the expression of E-cadherin and p53 in subcutaneous tumors is lost (such as Figure 5c and 5d). In the peritoneal implantation of gastric adenocarcinoma (MPC) tumor tissues in Balb / c nude mice, the tumors were mainly located in the intestinal mucosa, with no obvious intestinal infiltration. The nuclei of the tumor cells were large and mostly round or oval in shape. The tissues had obvious glandular ductal structures. The adenocarcinoma biomarker pan-CK (e.g., Figure 7 c).

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mouse gastric adenocarcinoma cell line MPC, which is deposited in China Center for Type Culture Collection with a biological deposit number of CCTCC NO: C2024264.

2. A method for constructing the mouse gastric adenocarcinoma cell line MPC according to claim 1, comprising the following steps: 1) Cdh1 fl / fl Mist1-cre mice and Tp53 fl / fl Mist1-cre mice were mated to obtain Cdh1 fl / fl Tp53 fl / fl Mist1-cre mice; 2) Isolation of Cdh1 from step 1) fl / fl Tp53 fl / fl Mist1-cre mouse stomach tissue was digested with tissue collagenase and cultured in complete organoid medium to obtain mouse gastric epithelial organoids Cdh1 fl / fl Tp53 fl / fl ; 3) The mouse Cdh1 obtained in step 2) fl / fl Tp53 fl / fl Organoids were induced with tamoxifen to form mouse gastric cancer organoids Cdh1 - / - Tp53 - / - ; 4) The mouse gastric cancer organoid Cdh1 obtained in step 3) - / - Tp53 - / - The mouse gastric adenocarcinoma cell line MPC was formed by acclimating the cells to the culture medium by gradually reducing the concentration of growth factors.

3. Use of the mouse gastric adenocarcinoma cell line MPC according to claim 1 in at least one of the following: a) Application in preparing gastric adenocarcinoma cell model; b) Application in preparing animal models of gastric adenocarcinoma; c) use in the preparation and / or screening of drugs for preventing and treating gastric adenocarcinoma; d) Application in establishing a research platform for drug resistance mechanisms in gastric adenocarcinoma; e) Application in establishing a research platform for early screening and grading of gastric adenocarcinoma; f) Application in establishing a research platform for the treatment of gastric adenocarcinoma.

4. A method for constructing a gastric cancer mouse model, comprising: The mouse gastric adenocarcinoma cell line MPC according to claim 1 is implanted into mice and fed for 30-40 days to form a gastric cancer mouse model.

5. The method according to claim 4, characterized in that The models include a gastric cancer subcutaneous tumor mouse model, a gastric cancer in situ tumor mouse model and a gastric cancer metastasis mouse model, and the gastric cancer is gastric adenocarcinoma.

6. The method according to claim 5, characterized in that The method for constructing a gastric adenocarcinoma subcutaneous tumor mouse model comprises: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention was implanted in the armpits of mice at an inoculation volume of 0.1-0.5 mL per mouse and fed for 30-40 days to form a gastric adenocarcinoma subcutaneous tumor mouse model. The method for constructing a gastric adenocarcinoma in situ tumor mouse model comprises: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention was implanted into the gastric tissue of mice at a volume of 0.1-0.5 mL per mouse and fed for 30-40 days to form a gastric adenocarcinoma in situ tumor mouse model; The method for constructing a gastric adenocarcinoma metastasis mouse model comprises: 7 The mouse gastric adenocarcinoma cell line MPC of the first aspect of the present invention is implanted into the abdominal cavity of mice at a volume of 0.1-0.5 mL per mouse and fed for 30-40 days to form a gastric adenocarcinoma metastasis mouse model.

7. The method according to claim 4, characterized in that The mice used to construct the mouse model are selected from immunodeficient mice or immune normal mice.

8. A gastric adenocarcinoma mouse model constructed according to the method of any one of claims 4 to 7.

9. Use of the gastric adenocarcinoma mouse model according to claim 8 in at least one of the following: a) use in the preparation and / or screening of drugs for preventing and treating gastric adenocarcinoma; b) use as an animal model for preparing and / or screening drugs for preventing and treating gastric adenocarcinoma; c) Application in establishing a research platform for drug resistance mechanisms in gastric adenocarcinoma; d) Application in establishing a research platform for early screening and grading of gastric adenocarcinoma; e) Application in establishing a research platform for the treatment of gastric adenocarcinoma.

10. A method for preparing or screening a candidate drug for preventing and / or treating gastric adenocarcinoma, the method comprising at least one of the following: m1) administering the test substance to a gastric adenocarcinoma cell model, wherein the test substance that inhibits cell proliferation or causes other programmed cell death is a candidate drug, wherein the gastric adenocarcinoma cell model is the gastric adenocarcinoma cell line MPC according to claim 1; m2) administering the test substance to an animal model of gastric adenocarcinoma, monitoring the animal body weight and tumor growth, and the test substance that causes improvement or cure of gastric adenocarcinoma symptoms in the animal model is a candidate drug, wherein the gastric adenocarcinoma animal model is constructed according to any one of the methods of claims 4 to 7.

Images