Establishment method and application of mouse gastric cancer orthotopic transplantation model

Abstract

The invention discloses an establishment method and application of a mouse gastric cancer orthotopic transplantation model, and the establishment method comprises the following steps: injecting a mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) into a mouse gastric tissue, and feeding to obtain the mouse gastric cancer orthotopic transplantation model. Compared with the prior art, the mouse gastric cancer in-situ model established in the invention has the advantages of strong repeatability in the construction process and good passage stability, especially can form tumor subcutaneously in a C57BL / 6 mouse with a sound immune system, has a therapeutic response to immunotherapeutic drugs such as anti-PD-1 monoclonal antibody, and is suitable for research on gastric cancer immune microenvironment and drug resistance mechanism of immunotherapeutic drugs.

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 orthotopic transplantation 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%. The key to improving prognosis lies in comprehensive treatment with targeted drugs as the core. However, the currently approved targeted therapeutic drugs for gastric cancer, such as trastuzumab and ramucirumab, have problems such as a small number of beneficiaries and limited extension of the median survival period.

[0003] In recent years, immunotherapy represented by targeting programmed cell death protein-1 (PD-1) has made breakthrough progress in the treatment of various malignant tumors. PD-1 is mainly expressed on the surface of immune cells such as activated T cells and natural killer (NK) cells, and is an important immunosuppressive molecule. The main mechanism of action of PD-1 (anti-PD-1) monoclonal antibody drugs is to block immunosuppressive pathways and reactivate immune cells. Among them, the combination of nivolumab, a PD-1 drug, and chemotherapy has been approved as a first-line treatment for patients with locally advanced or metastatic gastric cancer. Despite positive clinical progress, many clinical studies have shown that the objective response rate (ORR) of patients with advanced gastric cancer to immunotherapy is only 20% to 50%, indicating that up to 50% to 80% of patients with advanced gastric cancer have primary drug resistance, which seriously affects the application effect of immunotherapy drugs in gastric cancer.

[0004] The existing indicators for predicting the response to immunotherapy of gastric cancer mainly include microsatellite high instability (MSI-H), mismatch repair gene deficiency (dMMR), comprehensive immune positive score (CPS), Epstein-Barr virus (EBV) positive status, etc. Studies have shown that gastric cancer patients with MSI-H / dMMR, CPS ≥ 1 or EBV positive are usually accompanied by tumor interstitial immune cell infiltration and tumor gene mutation accumulation, and have a high response rate to immunotherapy drugs. However, these special cases account for less than 10% of the total gastric cancer cases. Studies have shown that among the remaining 90% of gastric cancer patients, some cases are also sensitive to immunotherapy, and there is currently no recognized tumor marker for predicting treatment response. In addition, although some research progress has been made in the mechanism of immunotherapy response of gastric cancer, the detailed mechanism of tumor microenvironment characteristics and cell-cell interactions during gastric cancer immunotherapy is still unclear. Therefore, it is necessary to further study the characteristics of the immune microenvironment of gastric cancer patients with resistance to immunotherapy drugs and explore tumor markers that can predict treatment response.

[0005] The tumor microenvironment is a tumor ecosystem interwoven by tumor cells, immune cells, stromal cells, and non-cellular components. Multiple cellular components together shape the organ-specific tumor environment and may lead to different responses of tumors to immunotherapy drugs. Therefore, systematically analyzing the dynamic changes of different cell subpopulations in the tumor microenvironment is the key to analyzing the mechanism of immunotherapy resistance and improving the treatment effect. In order to further study the mechanism of immunotherapy resistance, a gastric cancer mouse model with a sound immune microenvironment is needed. Mouse tumor models are divided into spontaneous tumor models and orthotopic transplantation models. Compared with spontaneous tumor models, orthotopic transplantation models have the advantages of simple construction methods, stable and rapid tumor formation, and can perform genetic intervention operations on transplants (cell lines, organoids, etc.), and are widely used in tumor immune microenvironment research. The ideal material for constructing mouse tumor orthotopic models is mouse tumor cell lines. At present, there are corresponding recognized orthotopic model construction methods for various digestive system tumors, such as the orthotopic model of mouse pancreatic cancer constructed using KPC cell lines and the orthotopic model of mouse colon cancer constructed using MC38 cell lines.

[0006] Specifically in gastric cancer research, the mouse gastric cancer cell lines that have been publicly reported are not ideal, and it is difficult to construct an in situ model of gastric cancer in mice with a sound 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 tumor in mice. Due to its low tumor formation rate and instability, the successful construction of the YTN16 cell line is largely accidental, and the construction process is difficult to be replicated by other research groups. Summary of the invention

[0007] Purpose of the invention: In order to solve the problems existing in the prior art, the present invention provides a method for establishing an orthotopic transplantation model of gastric cancer in mice and its application. The orthotopic transplantation model of gastric cancer established by the present invention has strong repeatability and good tumor stability, and can form tumors in C57BL / 6 mice with a sound immune system. The orthotopic transplantation model of gastric cancer can be used for the study of gastric cancer immune microenvironment, occurrence and development mechanism, distant metastasis mechanism, drug resistance mechanism, etc.

[0008] Technical solution: To achieve the above purpose, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a method for establishing an orthotopic transplantation model of gastric cancer in mice, comprising the following steps:

[0010] The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is injected into the gastric tissue of mice and fed, thereby obtaining the mouse gastric cancer orthotopic transplantation model.

[0011] 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.

[0012] As a specific embodiment, the mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is injected into the gastric tissue of the mouse, and the method includes the following steps:

[0013] The stomach of the mouse was exposed surgically, and the cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 was injected into the subserosa of the stomach, and the incision was sutured.

[0014] As a specific embodiment, the feeding adopts a regular diet for 25-30 days.

[0015] As a specific embodiment, the mouse is selected from 6-week-old C57 / 6J mice.

[0016] In a second aspect, the present invention provides the use of the mouse gastric cancer orthotopic transplantation model obtained by the establishment method as an experimental tool in studying cancer.

[0017] As a specific embodiment, the cancer is gastric cancer.

[0018] In a third aspect, the present invention provides the use of the mouse gastric cancer orthotopic transplantation model obtained by the establishment method in screening drugs for treating gastric cancer.

[0019] As a specific implementation scheme, the drug for treating gastric cancer is an anti-PD-1 monoclonal antibody drug.

[0020] As a specific implementation scheme, the drug for treating gastric cancer is a combination of 5-Fu and oxaliplatin.

[0021] 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 passages. When injected into the subgastric serosa of C57BL / 6 mice with a sound immune system, it can stably form an orthotopic tumor. The successful construction and application of this mouse orthotopic gastric cancer model fills the gap of the lack of an orthotopic transplantation model in gastric cancer research. It can be used for studies on the immune microenvironment, occurrence and development mechanism, distant metastasis mechanism, and drug resistance mechanism of gastric cancer, especially for the development and preclinical verification of immunotherapy drugs.

[0022] The method for constructing the transgenic mouse model comprises the following steps:

[0023] (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.

[0024] (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;

[0025] (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;

[0026] (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;

[0027] (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.

[0028] Beneficial effects: Compared with the prior art, the mouse in situ gastric cancer model established by the present invention has strong repeatability in the construction process and good stability in passage, especially it can form tumors subcutaneously in C57BL / 6 mice with a sound immune system and has a therapeutic response to immunotherapy drugs such as anti-PD-1 monoclonal antibody, and is suitable for the study of the immune microenvironment of gastric cancer and the resistance mechanism of immunotherapy drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

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

[0031] Figure 3 The electrophoresis diagram of the product amplified using KRAS primers, where lanes 1, 2 and 3 represent three different samples.

[0032] Figure 4 This is a picture of tumor formation in transgenic MKC mice.

[0033] Figure 5 This is the microscopic morphology of MKC1 cells.

[0034] Figure 6 This is a gross photograph of the tumor in the orthotopic model of gastric cancer in mice.

[0035] Figure 7 This is the HE staining image of tumor formation in the orthotopic model of gastric cancer in mice.

[0036] Figure 8 This is a Ki67 staining image of tumor formation in an orthotopic mouse gastric cancer model.

[0037] Fig. 9 To identify tumorigenic genotypes in an orthotopic mouse gastric cancer model.

[0038] Fig.10 Comparison of tumor size before and after using anti-PD-1 monoclonal antibody in the orthotopic model of gastric cancer in mice.

[0039] Fig.11 This is the IHC staining of CD8+ cells before and after using anti-PD-1 monoclonal antibody in the mouse gastric cancer orthotopic model.

[0040] Fig.12 Comparison of tumor formation before and after using 5-Fu and oxaliplatin in an orthotopic model of gastric cancer in mice. DETAILED DESCRIPTION

[0041] 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.

[0042] Example 1 Construction of MKC gastric cancer spontaneous tumorigenesis transgenic mouse model

[0043] 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.

[0044] The specific method for constructing the MKC mouse model is as follows:

[0045] 1. Gene sources and construction principles of each transgenic mouse

[0046] 1. Obtaining Anxa10-CreERT2 mice

[0047] 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.

[0048] Anxa10-2A-CreERT2 gene:

[0049] NCBI ID: 26359

[0050] MGI ID: 1347090

[0051] Ensembl ID: ENSMUSG00000031635

[0052] Pubmed: Anxa10

[0053] Anxa10 gene:

[0054] Anxa10 annexin A10[Mus musculus(house mouse)]

[0055] NCBI Gene ID:26359, updated on 11-Sep-2023

[0056] Examples of gene-marked cell types: gastric epithelial cells, skin hair follicle cells

[0057] 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.

[0058] 2. Obtaining MYC Overexpressing Mice

[0059] 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.

[0060] MYC gene:

[0061] MGI ID: 5461148

[0062] https: / / www.modelorg.com / index.php / portal / article / index / id / 858 / post_type / 3.html

[0063] 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.

[0064] 3.Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Acquisition of mice

[0065] 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.

[0066] Kras gene:

[0067] Kras Kirsten rat sarcoma viral oncogene homolog[Mus musculus(housemouse)];

[0068] NCBI Gene ID:16653, updated on 11-Oct-2023

[0069] 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.

[0070] 2. Specific process of constructing gastric adenocarcinoma transgenic mouse model

[0071] (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.

[0072] (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.

[0073] (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;

[0074] (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 + / - ;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;

[0075] 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.

[0076] 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.

[0077] 3. Mouse genotype identification method

[0078] 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:

[0079] 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.

[0080] 1) The sequence of the wide type primer for identifying the Anxa10-CreERT2 recombinase gene is:

[0081] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0082] The mutant type primer sequences are:

[0083] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and 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 2For 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 Mouse gastric cancer cell line in situ tumor formation experiment in mice

[0116] 1) Collect mouse gastric cancer cell line MKC1, count and adjust the cell concentration to 1 million / 10ul.

[0117] 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 stomach. Inject 10ul of cell suspension into the subserosa of the stomach. Suture the incision. After 4 weeks, kill the mice, perform laparotomy on the mice, and observe the gastric tumors. Figure 6 As shown, the results showed that a tumor had formed in the stomach. The tumor tissue was taken and treated with formalin. The tissue was dehydrated, embedded, and made into paraffin sections. Conventional HE staining and immunohistochemical staining (primary antibody was ki67, abcam, catalog number ab15580) were performed. The experimental results were observed and photographed under a microscope. The HE staining of the tumor section is shown in Figure 7 As shown, Ki67 staining Figure 8 shown.

[0118] Example 4 Identification of tumorigenic genotypes in an orthotopic model of gastric cancer in mice

[0119] Genomic DNA from tumor tissue was extracted using a genomic DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number DP304). DNA polymerase and corresponding primers were added for PCR amplification.

[0120] The sequence of the Wide type (WT) primer for identifying the Anxa10-CreERT2 recombinase gene is:

[0121] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0122] The sequence of the Mutant type (MUT) primer is:

[0123] 5′-CAGGGCACTATGAGAAAGCACTAC-3′ and 5′-

[0124] AGCCCGGACCGACGATGAAGC-3'.

[0125] The Wide type primer sequence for identifying the MYC gene is:

[0126] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0127] The mutant type primer sequences are:

[0128] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0129] ③The Wide type primer sequence for identifying KRAS point mutation gene is:

[0130] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0131] The mutant type primer sequences are:

[0132] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0133] The PCR reaction procedure is as follows:

[0134] ①Anxa10-CreERT2

[0135] Step1 94℃5min; Step2 94℃30s; Step3 60℃30s; Step4 72℃1min;

[0136] Step5 Go to 2×35; Step6 72℃5min; Step7 12℃∞.

[0137] ②KRAS

[0138] Step1 94℃3min; Step2 94℃15s; Step3 60℃15s; Step4 72℃40s;

[0139] Step5 Go to 2×30; Step6 72℃5min; Step7 10℃∞.

[0140] ③MYC

[0141] Step1 94℃5min; Step2 94℃30s; Step3 65℃30s; -0.5℃ / Cyc, 1Cyc-T;

[0142] Step4 72℃1min; Step5 Go to 2×34; Step6 72℃5min; Step7 12℃∞.

[0143] Prepare 2% agarose gel, add Ultra GelRed nucleic acid dye at 1:10000, mix well and pour into gel mold. Place agarose gel in electrophoresis tank, use 1×TAE as electrophoresis liquid, add DNA marker, test sample, negative control, set voltage to 135V, and run electrophoresis for 35 minutes. Finally, use gel imaging instrument to develop and take pictures, such as Fig. 9 As shown, the results showed that the genotype was correct.

[0144] Example 5 An anti-PD-1 monoclonal antibody drug application experiment was conducted in an in situ mouse gastric cancer model.

[0145] Six 6-week-old C57 / 6J mice were selected and randomly divided into two groups. A mouse gastric orthotopic tumor model was established according to the above method. The mice in the experimental group were intraperitoneally injected with anti-PD-1 monoclonal antibody three times a week, with an initial dose of 200ug and two subsequent doses of 100ug each. The mice in the control group were injected with the same dose of IgG control drug. The mice were killed after 4 weeks, and the mice were subjected to laparotomy. The stomach of the mice was removed and photographed. Fig.10 As shown in the figure, the results showed that after anti-PD-1 monoclonal antibody treatment, the gastric tumor shrank. The tumor tissue was taken and treated with formalin. The tissue was dehydrated, embedded, and made into paraffin sections. Conventional immunohistochemical staining was performed (the primary antibody was CD8, abcam, catalog number ab217344), and the experimental results were observed under a microscope and photographed, as shown in the figure. Fig.11 As shown, the results showed that T cell infiltration increased after anti-PD-1 monoclonal antibody treatment.

[0146] Example 6 Experimental study on mouse gastric cancer in situ model using 5-Fu and oxaliplatin

[0147] Six 6-week-old C57 / 6J mice were selected and randomly divided into two groups. The mouse gastric orthotopic tumor model was established according to the above method. The mice in the experimental group were intraperitoneally injected with 5-Fu (100 mg / kg) and oxaliplatin (5 mg / kg) once a week, and the mice in the control group were injected with the same dose of PBS. The mice were killed after 4 weeks, and the mice were processed by laparotomy. The stomach of the mice was removed and photographed. Fig.12 As shown, the results showed that the stomach tumor shrank after drug treatment.

[0148] 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 an orthotopic transplantation model of gastric cancer in mice, characterized in that: The following steps are involved: The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is injected into the gastric tissue of mice and fed, thereby obtaining the mouse gastric cancer orthotopic transplantation model.

2. The method for establishing an orthotopic transplantation model of gastric cancer in mice 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 an orthotopic transplantation model of gastric cancer in mice according to claim 1, characterized in that: The method of injecting mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) into mouse gastric tissue comprises the following steps: The stomach of the mouse was exposed surgically, and the cell suspension containing the mouse gastric adenocarcinoma cell line MKC1 was injected into the subserosa of the stomach, and the incision was sutured.

4. The method for establishing an orthotopic transplantation model of gastric cancer in mice according to claim 1, characterized in that: The feeding adopts a conventional diet and is fed for 25-30 days.

5. The method for establishing an orthotopic transplantation model of gastric cancer in mice according to claim 1, characterized in that: The mice were selected from 6-week-old C57 / 6J mice.

6. Use of the mouse gastric cancer orthotopic transplantation model obtained by the method according to any one of claims 1 to 5 as an experimental tool in cancer research.

7. The use according to claim 6, characterized in that: The cancer is gastric cancer.

8. Use of the mouse gastric cancer orthotopic transplantation model obtained by the method according to any one of claims 1 to 5 in screening drugs for treating gastric cancer.

9. The use according to claim 8, characterized in that: The drug for treating gastric cancer is an anti-PD-1 monoclonal antibody drug.

10. The use according to claim 8, characterized in that: The gastric cancer treatment drug is a combination of 5-Fu and oxaliplatin.

Images