Establishment method and application of mouse gastric cancer distant metastasis model

Abstract

The invention discloses an establishment method and application of a mouse gastric cancer remote metastasis model, and the establishment method comprises the following steps: injecting a mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) into a mouse body through intraperitoneal injection, portal intravenous injection, spleen injection or caudal intravenous injection, and feeding to obtain the mouse gastric cancer remote metastasis model. Compared with the prior art, the mouse gastric cancer distant metastasis model established in the invention has the advantages of strong repeatability in the construction process and good tumor formation stability, especially can form tumors in the C57BL / 6 mouse body with a sound immune system, and is suitable for research on gastric cancer distant metastasis molecular mechanism, immune microenvironment, chemotherapeutic drug resistance mechanism and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of gastric cancer animal models, and specifically relates to a method for establishing a mouse gastric cancer distant metastasis model and its application. Background Art

[0002] Gastric cancer is the fifth most common malignant tumor and the fourth leading cause of cancer-related death in the world. According to the Global Cancer Statistics Report and other relevant data, the number of new cases of gastric cancer in the world in 2020 will be nearly 1 million, of which 479,000 cases in China account for 43.9% of the new cases of gastric cancer in the world. Locally advanced or metastatic gastric cancer accounts for about 30% of the first cases of gastric cancer in China. There is currently no effective treatment, and the five-year survival rate is less than 10%. Despite progress in diagnostic and treatment methods, the prognosis of gastric cancer patients remains poor, especially for gastric cancer patients with distant metastasis. Common distant metastasis sites of gastric cancer include abdominal metastasis, liver metastasis, lung metastasis, etc., which are critical stages of disease progression and are closely related to prognosis. Understanding the underlying mechanisms driving distant metastasis of gastric cancer is crucial for developing targeted therapies and improving patient outcomes.

[0003] Immune surveillance plays a crucial role in identifying and eliminating cancer cells. However, as described by Stephen Paget's "seed and soil" theory, the tumor microenvironment has developed complex strategies to evade immune recognition and destruction, thereby promoting tumor growth and metastasis. In the context of distant metastasis, the interaction between tumor cells and the immune system is particularly critical due to the unique immune microenvironment of distant metastatic organs. Tumor-immune interactions and the immune landscape in the microenvironment are key determinants of gastric cancer progression and metastasis. The presence of distant metastasis is associated with systemic immune tolerance and a low response to immunotherapy. Currently, immunotherapy represented by targeting programmed cell death protein-1 (PD-1) has made progress in the treatment of a variety of malignant tumors, but its application in patients with distant metastasis of gastric cancer has not been effective. Therefore, elucidating the mechanism of immune escape in the context of distant metastasis will provide new ideas for the treatment of metastatic gastric cancer.

[0004] In order to further study the immune microenvironment mechanism of distant metastasis of gastric cancer, it is necessary to construct a distant metastasis model of gastric cancer in mice with a healthy immune system. The usual practice of establishing an animal model of distant metastasis in mice with a healthy immune system is to use mouse tumor cell lines for intraperitoneal injection (intraperitoneal metastasis model), portal vein or spleen injection (liver metastasis model), tail vein injection (lung metastasis model), etc. Through these methods, a series of distant metastasis mouse models such as pancreatic cancer distant metastasis model and colorectal cancer distant metastasis model have been successfully constructed. Specifically in the study of distant metastasis of gastric cancer, the mouse gastric cancer cell lines currently reported are not ideal, and it is difficult to construct a distant metastasis model of gastric cancer in mice with a healthy immune system. For example, the MFC cells constructed by Qian Shusen et al. are mouse gastric squamous cell carcinoma cell lines, while the vast majority of clinical gastric cancers are adenocarcinomas; the YTN16 cell line successfully constructed by Nomura et al. at the University of Tokyo in Japan is a chemically induced mouse tumor. Due to its low tumor formation rate and instability, the successful construction of the YTN16 cell line is very accidental, and the construction process is difficult to be replicated by other research groups, and there is no literature report on the successful construction of a distant metastasis model using the YTN16 cell line. Summary of the invention

[0005] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides a method for establishing a mouse gastric cancer distant metastasis model and its application. The gastric cancer distant metastasis model established by the present invention has strong repeatability and good tumor formation stability, and can form tumors in C57BL / 6 mice with a sound immune system. The model can be used to study the immune microenvironment, occurrence and development mechanism, and drug resistance mechanism of gastric cancer distant metastasis.

[0006] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for establishing a mouse gastric cancer distant metastasis model, comprising the following steps:

[0008] The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is injected into mice via intraperitoneal injection, portal vein injection, spleen injection or tail vein injection, and then the mice are fed to obtain the mouse gastric cancer distant metastasis model.

[0009] As a specific implementation scheme, the mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is deposited in the China Center for Type Culture Collection on June 15, 2024, with a deposit number of CCTCC NO: C2024183.

[0010] As a specific implementation scheme, the mouse gastric cancer distant metastasis model is a mouse gastric cancer abdominal metastasis model, and the establishment method includes the following steps:

[0011] The mouse gastric adenocarcinoma cell line MKC1 cell suspension is injected into the abdominal cavity of mice for feeding, thereby obtaining the mouse gastric cancer abdominal cavity metastasis model.

[0012] As a specific implementation scheme, the mouse gastric cancer distant metastasis model is a mouse gastric cancer liver metastasis model, and the establishment method includes the following steps:

[0013] The mouse spleen is exposed surgically, and a cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 is injected into the spleen. After a period of time, the mouse spleen is removed, the incision is sutured, and the mouse is fed to obtain the mouse gastric cancer liver metastasis model.

[0014] As a specific implementation scheme, the mouse gastric cancer distant metastasis model is a mouse gastric cancer lung metastasis model, and the establishment method includes the following steps:

[0015] The mouse gastric cancer lung metastasis model is obtained by injecting a cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 into the tail vein of the mouse for feeding.

[0016] As a specific embodiment, the feeding adopts a conventional diet for 25-30 days.

[0017] As a specific embodiment, the mouse is selected from 6-week-old C57 / 6J mice.

[0018] In a second aspect, the present invention provides the use of the mouse gastric cancer distant metastasis model obtained by the establishment method as an experimental tool in studying gastric cancer metastasis.

[0019] In a third aspect, the present invention provides an application of the mouse gastric cancer distant metastasis model obtained by the establishment method in screening drugs for treating gastric cancer metastasis.

[0020] In order to construct a stable and reproducible mouse gastric cancer orthotopic model, the present invention firstly constructed the Myc LSL / LSL ;Kras loxP / + ;Anxa10-CreER T2+ / -The mouse spontaneous tumor model of gastric cancer, i.e. the transgenic mouse model, is a model in which the gastric tumor tissue is removed and dissociated into a single cell suspension after the mouse spontaneously forms a primary gastric tumor. A homologous mouse gastric cancer cell line is constructed by culturing from the single cell clone. Experimental verification shows that this cell line grows rapidly and is stable in passage. Intraperitoneal injection, portal vein or spleen injection, tail vein injection, etc. can stably construct mouse gastric cancer abdominal metastasis, liver metastasis, lung metastasis and other models. The successful construction and application of this mouse gastric cancer distant metastasis model fills the gap in the lack of distant metastasis models in gastric cancer research. It can be used for the study of the immune microenvironment, occurrence and development mechanism, and drug resistance mechanism of distant metastasis of gastric cancer. In particular, it is helpful for the development and preclinical verification of immunotherapy drugs, and has good application prospects.

[0021] The method for constructing the transgenic mouse model comprises the following steps:

[0022] (1) Insert 2A-CreERT2-Wpre-pA into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + mice; the CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Mouse; loxp-stop-loxp and exon2 containing G12D replaced exon2 of Kras to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Mouse.

[0023] (2) MYC LSL / LSL Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + Mice;

[0024] (3) KRAS loxP / + Mice were mated with Anxa10-CreERT2 recombinase transgenic mice to obtain F1 generation mice, and heterozygous mice among the F1 generation mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mice;

[0025] (4) Transfect the Anxa10-CreERT2 in the above F1 generation + / - ;MYC LSL / +Mice with Anxa10-CreERT2 + / - ;Kras loxP / + The mice were mated to obtain F2 mice, and the genotype of the F2 mice was selected to be Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous mice, named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

[0026] (5) The Anxa10-CreERT2 with the same genotype in the F2 generation + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is the transgenic mouse model MKC.

[0027] Beneficial effects: Compared with the prior art, the mouse gastric cancer distant metastasis model established by the present invention has a strong repeatability in the construction process and good tumor stability, especially it can form tumors in C57BL / 6 mice with a sound immune system, and is suitable for the study of the molecular mechanism of gastric cancer distant metastasis, immune microenvironment, and chemotherapeutic drug resistance mechanism. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the electrophoresis diagram of the product amplified using the Anxa10-CreERT2 primer, where lanes 1, 2 and 3 represent three different samples.

[0029] Figure 2 This is the electrophoresis diagram of the product amplified using MYC primers, where lanes 1, 2 and 3 represent three different samples.

[0030] Figure 3 The electrophoresis diagram of the product amplified using KRAS primers, where lanes 1, 2 and 3 represent three different samples.

[0031] Figure 4 This is a picture of tumor formation in transgenic MKC mice.

[0032] Figure 5 This is the microscopic morphology of MKC1 cells.

[0033] Figure 6 Gross photos of abdominal metastasis in C57BL / 6 mice for the establishment of mouse gastric cancer cell line

[0034] Figure 7HE staining of mouse gastric cancer cell line for peritoneal metastasis in C57BL / 6 mice

[0035] Figure 8 Gross photo of liver metastasis in C57BL / 6 mice for establishment of mouse gastric cancer cell line

[0036] Fig. 9 HE staining of liver metastasis in C57BL / 6 mice for the establishment of mouse gastric cancer cell line

[0037] Fig.10 Gross photograph of lung metastasis in C57BL / 6 mice constructed for mouse gastric cancer cell line

[0038] Fig.11 HE staining of lung metastasis in C57BL / 6 mice for mouse gastric cancer cell line construction. DETAILED DESCRIPTION

[0039] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The experimental methods used in the examples are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0040] Example 1 Construction of MKC gastric cancer spontaneous tumorigenesis transgenic mouse model

[0041] The Anxa10-CreERT2 transgenic mice, which specifically expressed CreERT2 recombinase in gastric mucosal epithelial cells, were mixed with MYC overexpressing transgenic mice. LSL / LSL and mice with KRAS activating point mutations loxP / + The mice were crossed to obtain the F1 generation heterozygous mice, Anxa10-CreERT2 + / - ;MYC LSL / + Mice with Anxa10-CreERT2 + / - ;Kras loxP / + Mice. Two F1 generation mice were mated to obtain F2 mice. The genotype of the F2 generation mice was selected to be Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + The mice were mated and the F3 generation had the genotype of Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / + Mice (MKC mouse model), genotype identification as Figure 1-3 As shown. This genotype of mice was selected and injected with tamoxifen to induce Cre expression starting from 6 weeks of age. The vital signs and body weight of the mice were monitored. The transgenic mice formed primary gastric tumors after induction (as shown in Figure 2A). Figure 4 As shown in the figure, spontaneous tumors formed in the stomach of mice, and the mice were killed at week 8 of induction. The mice were dissected and the stomach was removed, the stomach wall was cut open, the stomach contents were taken out and the stomach cavity was flushed with PBS for subsequent processing.

[0042] The specific method for constructing the MKC mouse model is as follows:

[0043] 1. Gene sources and construction principles of each transgenic mouse

[0044] 1. Obtaining Anxa10-CreERT2 mice

[0045] 2A-CreERT2-Wpre-pA was inserted into the stop codon of mouse Anxa10 gene to obtain Anxa10-CreERT2 recombinase transgenic mouse Anxa10-CreERT2 + / + Mice. The mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0046] Anxa10-2A-CreERT2 gene:

[0047] NCBI ID: 26359

[0048] MGI ID: 1347090

[0049] Ensembl ID: ENSMUSG00000031635

[0050] Pubmed: Anxa10

[0051] Anxa10 gene:

[0052] Anxa10 annexin A10[Mus musculus(house mouse)]

[0053] NCBI Gene ID:26359, updated on 11-Sep-2023

[0054] Examples of gene-marked cell types: gastric epithelial cells, skin hair follicle cells

[0055] This mouse expresses CreERT2 recombinase at the Anxa10 locus. When Anxa10-2A-CreERT2 is bred with reporter mice, the reporter gene is expressed in the gastric epithelial cells of the adult after Tamoxifen induction; when bred with mice containing loxP flanking sequences, Tamoxifen-induced recombination will lead to the deletion of the floxed sequence in the gastric epithelial cells. It can be used for gastric cancer research.

[0056] 2. Obtaining MYC overexpressing mice

[0057] The CAG promoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 locus to establish the H11-LSL-Myc mouse model, which is a MYC overexpression gene mouse MYC LSL / LSL Homozygous mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0058] MYC gene:

[0059] MGI ID: 5461148

[0060] https: / / www.modelorg.com / index.php / portal / article / index / id / 858 / post_type / 3.html

[0061] The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis. The MYC proto-oncogene encodes a family of transcription factors and is one of the most common activated oncoproteins in human tumors. In fact, in most cancers, MYC aberrations or MYC-related pathways are upregulated through alternative mechanisms. MYC protein is a major regulator of cellular programs. The c-Myc gene (also called Myc) is abnormally expressed in many tumors, affecting cell proliferation, growth metabolism, gene instability, stimulating angiogenesis, cell malignant transformation, differentiation and apoptosis. The CAGpromoter-loxp-STOP-loxp-Myc-polyA conditional overexpression structure was inserted into the H11 site to establish the H11-LSL-Myc mouse model. The H11 site is located on mouse chromosome 11. This site has been shown to be similar to Rosa26 and can be used to express exogenous genes more widely. H11-LSL-Myc can highly express the Myc gene in Cre-expressing tissues after mating with Cre-expressing mice. It can be used to establish tumor models and conduct tumor research.

[0062] 3.Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Acquisition of mice

[0063] The loxp-stop-loxp and exon2 containing G12D replaced the exon2 of Kras to establish the Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Mice. The mice were purchased from Jackson lab.

[0064] Kras gene:

[0065] Kras Kirsten rat sarcoma viral oncogene homolog[Mus musculus(housemouse)];

[0066] NCBI Gene ID:16653, updated on 11-Oct-2023

[0067] KRAS mutations can lead to uncontrolled activation of RAS protein. RAS protein is located in the cell membrane and regulates cell growth, proliferation, movement, migration and angiogenesis. KRAS mutations will significantly increase the invasiveness of gastric cancer. KRAS expression is high in the Lauren classification of intestinal type. The most common model is to use K19-CreERT mice and Loxp-STOP-Loxp-K-Ras (G12D) mice to obtain mice that conditionally express K-Ras (G12D). These mice also have multiple dysplasia, metaplasia and adenocarcinoma in the stomach. The activation / inactivation effect of wild-type Kras is controlled, while the mutant Kras protein is dysfunctional and continuously in an activated state, leading to the continuous proliferation of tumor cells. The loxp-stop-loxp and exon2 containing G12D replace the Kras exon2 to establish a Kras gene G12D conditional point mutation mouse strain. After mating with Cre mice, G12D point mutation mice can be obtained.

[0068] 2. Specific process of constructing gastric adenocarcinoma transgenic mouse model

[0069] (1) MYC overexpression gene mice LSL / LSL The mice were mated with Anxa10-CreERT2 recombinase transgenic mice that specifically expressed CreERT2 recombinase in gastric epithelial cells to obtain F1 mice; heterozygous mice among the F1 mice were selected and named Anxa10-CreERT2 + / - ;MYC LSL / + Mouse.

[0070] (2) KRAS conditional point mutation activated mice - KRAS loxP / + The mice were mated with Anxa10-CreERT2 recombinase transgenic mice that specifically expressed CreERT2 recombinase in gastric epithelial cells to obtain F1 mice; heterozygous mice among the F1 mice were selected and named Anxa10-CreERT2 + / - ;Kras loxP / + Mouse.

[0071] (3) Transfecting the Anxa10-CreERT2 in the F1 generation + / - ;MYC LSL / + Anxa10-CreERT2 in mice and the F1 generation + / - ;KRAS loxP / + The mice were mated to obtain F2 mice; the F2 mice were selected to have the genotype of Anxa-CreERT2 heterozygous, MYC heterozygous, and KRAS heterozygous, and their genotype was named Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + Mice;

[0072] (4) Select the genotype of F2 mice to be Anxa10-CreERT2 + / - ;MYC LSL / + ;Kras loxP / + The mice were mated to obtain F3 mice, and the genotype of the F3 mice was selected to be Anxa10-CreERT2

[0073] + / - ;MYC LSL / LSL ;Kras loxP / + The mouse is a gastric epithelial cell-specific MYC overexpression and Kras activation mutation mouse, which is the gastric adenocarcinoma transgenic mouse model;

[0074] Anxa10-CreERT2 + / + The mice were homozygous for the Anxa10-CreERT2 recombinase transgenic mice. + / - The mice were Anxa10-CreERT2 recombinase transgenic heterozygous mice; MYC LSL / LSL Mice are homozygous for MYC overexpression. LSL / + MYC overexpression heterozygous mice; KRAS loxP / + The mice were heterozygous for the Kras gene G12D conditional point mutation.

[0075] The constructed Anxa10-CreERT2 + / - ;MYC LSL / LSL ;Kras loxP / +Mice, for adult mice, tamoxifen is injected intraperitoneally at 6 weeks of age. Dissolve tamoxifen in corn oil at a concentration of 20 mg / ml and shake overnight at 37 degrees Celsius. Store it at 4°C during injection. The injection dose is determined by weight, approximately 200 mg / kg body weight. Administer tamoxifen by intraperitoneal injection (using an ACUC-approved injection procedure) once every 24 hours for 3-5 consecutive days. For safety, disinfect the injection site with 70% ethanol before injection. After the last injection, mice should be isolated for 24 hours before returning to their normal animal room.

[0076] 3. Mouse genotype identification method

[0077] The genotype identification of gastric mucosal epithelial cell-specific Anxa10-CreERT2; MYC overexpression and Kras activation point mutation mice and the PCR identification method for obtaining heterozygous mice are as follows:

[0078] Take 2-3 mm of the tail of the offspring mouse and extract the genomic DNA of the tail using standard procedures. Use the genomic DNA of the tail as a template and use gene-specific primers to perform polymerase chain reaction (PCR) to identify the mouse genotype. Use the genomic DNA of the tail of wild-type mice C57 mice as a control.

[0079] 1) The sequence of the wide type primer for identifying the Anxa10-CreERT2 recombinase gene is:

[0080] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0081] The mutant type primer sequences are:

[0082] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and

[0083] 5'-AGCCCGGACCGACGATGAAGC-3'.

[0084] 2) The Wide type primer sequence for identifying the MYC gene is:

[0085] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0086] The mutant type primer sequences are:

[0087] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0088] 3) The sequence of the wide type primer for identifying the KRAS point mutation gene is:

[0089] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0090] The mutant type primer sequence is

[0091] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0092] Genotype identification system:

[0093] F (upstream primer): 0.25ul, R (downstream primer): 0.25ul, 2xTaq (buffer): 7.5ul, H2O: 5.5ul, mouse tail DNA: 1.5ul, Total: 15ul.

[0094] PCR instrument loading procedure:

[0095] 1) ANXA identification:

[0096] Volume: 15ul, Step1 94℃00:05:00(5min), Step2 94℃00:00:30(30s), Step3 60℃00:00:30(30s), Step4 72℃00:01:00(1min), Step 5Goto2 x35, Step6 72℃00:05:00(5min), Step7 12℃Forever.

[0097] 2) Kras identification:

[0098] Volume: 15ul, Step1 94℃00:03:00(3min), Step2 94℃00:00:15(15s), Step358'C00:00:15(15s), Step4 72℃00:00:40(40s), Step 5Goto2 x30, Step6 72℃00:05:00(5min), Step7 10'℃Forever.

[0099] 3) MYC identification:

[0100] Volume:15ul, Step1 94℃00:05:00(5min), Step2 94℃00:00:30(30s), Step3 65℃00:00:30.(30s), Step4 72℃00:01:00(1min), Step5 Goto2 x34, Step6 72℃00:05:00(5min), Step7 12℃Forever

[0101] The genotype identification results of mice are shown in Figure 1-3 , the three lanes in the figure represent three samples, Figure 1 The electrophoresis of the product amplified by Anxa10-CreERT2 primers was performed. If a 436bp fragment was specifically amplified, it was Anxa10-CreERT2 positive; if an 823bp fragment was specifically amplified, it was Anxa10-CreERT2 negative.

[0102] Figure 2 For the electrophoresis of the product amplified with MYC primers, if a 434bp fragment can be specifically amplified, it is MYC LSL / LSL homozygous, if a 428bp fragment can be specifically amplified, it is MYC negative, and if a negative-positive band is amplified, it is heterozygous MYC LSL / +.

[0103] Figure 3 The product amplified by KRAS primers is electrophoresed. If a 100bp fragment can be specifically amplified and a 200bp fragment can be amplified, it is a KRAS loxP / + heterozygote. If a 200bp fragment can be specifically amplified, it is KRAS negative and wild type.

[0104] Figure 1-3 The genotype of sample 1 is Anxa10-CreERT2+ / -; MYC LSL / +, the genotype of sample 2 is Anxa10-CreERT2+ / -; MYC LSL / LSL; KrasloxP / +, and the genotype of sample 3 is Anxa10-CreERT2+ / +; MYCLSL / +. Therefore, sample 2 is the required gastric epithelial cell-specific MYC overexpression and Kras activation point mutation mouse, that is, the MKC mouse model.

[0105] Example 2 Isolation and culture of gastric adenocarcinoma cell line MKC1

[0106] 1. The experimental steps for constructing primary cell lines are as follows:

[0107] (1) Place a clean cell culture dish on ice, and use sterile forceps to place the MKC mouse stomach tissue in Example 1 into the cell culture dish; rinse the stomach tissue three times with PBS containing penicillin-streptomycin;

[0108] (2) Use sterile surgical instruments to carefully remove non-target tissue parts such as blood vessels, fibers, mucosa, fat or necrotic tissue, and cut the target tissue into small pieces of about 2 mm;

[0109] (3) Transfer the tissue block to a 5 ml centrifuge tube, add RPMI 1640 digestion solution (1 mg / mL collagenase A, 0.1 mg / mL ribonuclease), and digest in a 37°C water bath for 20 min, pipetting every 5-10 min;

[0110] (4) Place a 70-micron cell sieve in a 15 ml centrifuge tube, rinse the cell sieve with RPMI 1640 + 2% FBS, pipette the sample through the sieve, add 2 ml RPMI 1640 + 2% FBS to rinse the cell sieve, and centrifuge at 4°C, 500 g for 5 min.

[0111] (5) Resuspend the cells in 1 ml RPMI 1640 + 2% FBS, add 3 ml red blood cell lysis buffer, lyse for 5 min, and centrifuge at 500 g at 4°C for 5 min.

[0112] (6) Resuspend the cells in RPMI 1640 medium containing 10% FBS and culture and subculture in a cell culture incubator (37° C., 5% CO 2 ).

[0113] (7) After 2-3 generations of cell culture, the cells were collected, counted, and added to a 96-well plate at 1 cell / 100 μl / well for monoclonal screening to obtain the mouse gastric cancer primary cell line MKC1 ( Figure 5 As shown, the primary cells are photographed under a microscope, and the cells are of uniform morphology and size).

[0114] The above-mentioned mouse gastric cancer cell line MKC1 (Mus musculus) was deposited in the China Center for Type Culture Collection (CCTCC) on June 15, 2024, with the deposit number CCTCC NO: C2024183, and the deposit address is Wuhan University, Wuhan City, Hubei Province, People's Republic of China.

[0115] Example 3: Injection of mouse gastric cancer cell line into the peritoneal cavity of C57 mice to establish a peritoneal metastasis model

[0116] 1) Collect mouse gastric cancer cell line MKC1, count and adjust the cell concentration to 5 million / 500ul.

[0117] 2) Select 6-week-old C57 / 6J mice and inject 500ul of cell suspension into the mouse peritoneal cavity using a syringe. Four weeks later, the mice were killed and laparotomy was performed to observe peritoneal metastasis lesions. Figure 6 As shown in the figure, the results showed that gastric cancer cells were widely implanted on the peritoneum of mice (red frame area). The peritoneal metastatic lesions were treated with formalin, the tissues were dehydrated, embedded and made into paraffin sections, and conventional HE staining was performed. The experimental results were observed under a microscope, as shown in the figure. Figure 7 As shown, the results showed that the peritoneal metastasis was gastric adenocarcinoma.

[0118] Example 4: Injection of mouse gastric cancer cell line into the spleen of C57 mice to establish a liver metastasis model

[0119] 1) Collect mouse gastric cancer cell line MKC1, count and adjust the cell concentration to 500,000 / 30ul.

[0120] 2) Select 6-week-old C57 / 6J mice, anesthetize them, make a left subcostal incision, cut open the mouse skin layer by layer, and expose the mouse spleen. Inject 30ul of cell suspension into the spleen, and press with a cotton swab to stop bleeding. After 10 minutes, remove the spleen of the mouse and suture the incision. After 4 weeks, kill the mouse, perform laparotomy on the mouse, and observe the liver metastasis of gastric cancer. Figure 8 As shown in the figure, the results showed that gastric cancer cells metastasized to the liver (white lesions). The liver metastatic lesions of gastric cancer were treated with formalin, the tissues were dehydrated, embedded and made into paraffin sections, and conventional HE staining was performed. The experimental results were observed under a microscope, as shown in the figure. Fig. 9 As shown, the results showed that the liver metastasis was gastric adenocarcinoma.

[0121] Example 5: Injection of mouse gastric cancer cell line into the tail vein of C57 mice to establish a lung metastasis model

[0122] 1) Collect mouse gastric cancer cell line MKC1, count and adjust the cell concentration to 1 million / 100ul.

[0123] 2) Select 6-week-old C57 / 6J mice, inject 100ul of cell suspension into the tail vein, and press with a cotton swab to stop bleeding. Four weeks later, the mice were killed and the thorax was cut open to observe the lung metastasis of gastric cancer. Fig.10 As shown in the figure, the results showed that gastric cancer cells metastasized to the lung tissue (white lesions). The gastric cancer lung metastasis lesions were treated with formalin, the tissues were dehydrated, embedded and made into paraffin sections, and conventional HE staining was performed. The experimental results were observed under a microscope, as shown in the figure. Fig.11 As shown, the results showed that the lung metastasis was gastric adenocarcinoma.

[0124] The above embodiments are the best implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for establishing a mouse gastric cancer distant metastasis model, characterized in that: The following steps are involved: The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is injected into mice via intraperitoneal injection, portal vein injection, spleen injection or tail vein injection, and then the mice are fed to obtain the mouse gastric cancer distant metastasis model.

2. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is deposited in the China Center for Type Culture Collection on June 15, 2024, with a deposit number of CCTCC NO: C2024183.

3. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The mouse gastric cancer distant metastasis model is a mouse gastric cancer peritoneal metastasis model, and the establishment method includes the following steps: The mouse gastric adenocarcinoma cell line MKC1 cell suspension is injected into the abdominal cavity of mice for feeding, thereby obtaining the mouse gastric cancer abdominal cavity metastasis model.

4. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The mouse gastric cancer distant metastasis model is a mouse gastric cancer liver metastasis model, and the establishment method includes the following steps: The mouse spleen is exposed surgically, and a cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 is injected into the spleen. After a period of time, the mouse spleen is removed, the incision is sutured, and the mouse is fed to obtain the mouse gastric cancer liver metastasis model.

5. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The mouse gastric cancer distant metastasis model is a mouse gastric cancer lung metastasis model, and the establishment method includes the following steps: The mouse gastric cancer lung metastasis model is obtained by injecting a cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 into the tail vein of the mouse for feeding.

6. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The feeding adopts a conventional diet and is fed for 25-30 days.

7. The method for establishing a mouse gastric cancer distant metastasis model according to claim 1, characterized in that: The mice were selected from 6-week-old C57 / 6J mice.

8. Use of the mouse gastric cancer distant metastasis model obtained by the establishment method according to any one of claims 1 to 7 as an experimental tool in studying gastric cancer metastasis.

9. Use of the mouse gastric cancer distant metastasis model obtained by the method according to any one of claims 1 to 7 in screening drugs for treating gastric cancer metastasis.

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