Stomach hypodifferentiation adenocarcinoma accompanied signet-ring cell carcinoma mouse model and construction method, cell strain and application thereof

Abstract

The invention provides a mouse model of gastric poorly differentiated adenocarcinoma accompanied with signet ring cell carcinoma as well as a construction method, a cell strain and application of the mouse model. The invention relates to a construction method of a mouse model of gastric hypodifferentiation adenocarcinoma accompanied with signet ring cell carcinoma. The construction method comprises the following steps: constructing a mouse M3 of which a Trp53 gene is conditionally knocked out; the method comprises the following steps: hybridizing a mouse M3 with a mouse M1 with a genotype of Anxa10CreERT2 / + to obtain a mouse M13, and carrying out interaction on the mouse M13 to obtain a mouse M4; the method comprises the following steps: hybridizing a mouse M4 with a mouse M2 of which the genotype is KRASLSL-G12D / + to obtain a mouse M42, and hybridizing the mouse M42 with the mouse M4 to obtain a mouse M5; chemical induction is carried out on the mouse M5 by administration of drugs, and the mouse model with hypogastric differentiation adenocarcinoma accompanied by signet-ring cell carcinoma is obtained; wherein the medicine is prepared from tamoxifen. By means of the construction method, the mouse model and the cell strain of gastric hypodifferentiation adenocarcinoma accompanied with signet-ring cell carcinoma can be obtained, the tumor formation rate of mouse gastric cancer can be increased, the tumor formation time can be shortened, and the tumor formation condition is stable.

Description

Technical Field

[0001] The present application relates to the technical field of animal model construction, and in particular to a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, a construction method thereof, a cell line, and applications thereof. Background Art

[0002] Gastric cancer is one of the most common malignant tumors worldwide, with high morbidity and mortality rates. In recent years, with the rise of tumor immunotherapy, especially the application of immune checkpoint inhibitors (such as PD-1 / PD-L1 antibodies), the treatment effect of gastric cancer patients has been improved to a certain extent. However, the overall efficacy of immunotherapy is still limited, and a considerable number of patients will develop primary or secondary drug resistance, leading to treatment failure. Therefore, in-depth exploration of the resistance mechanism of gastric cancer immunotherapy and the search for new therapeutic targets and strategies are of great significance for improving the prognosis of gastric cancer patients.

[0003] Ideal tumor models should mimic the development and progression of human tumors, as well as their response to treatment. These models primarily include animal models, organoid models, and cell models. Currently, genetically engineered mouse models (GEMMs) utilize gene editing techniques to induce oncogene activation or tumor suppressor gene inactivation in specific cells within the mouse body, leading to spontaneous tumor formation. These models, which possess a complete immune system, are powerful tools for studying tumor immunity.

[0004] The Cre-LoxP system is currently the most widely used conditional gene editing technology. It uses tissue-specific Cre recombinase to catalyze the recombination of DNA fragments between LoxP sites, achieving gene knockout, insertion, or inversion. In recent years, research using the Cre-LoxP system to construct gastric cancer models has steadily increased. In 2012, Japanese scientists reported a double-knockout mouse model in which Trp53 and CDH1 were conditionally deleted in gastric parietal cells using three genetic modifications: ATP4b-cre, TP53-flxo, and CDH1-flox. However, the number of gastric cancer GEMMs currently available is limited, and mouse models for poorly differentiated adenocarcinoma, the most common form of human gastric cancer, remain underdeveloped.

[0005] Tumor cell lines are important tools for in vitro research (such as cell biology, molecular biology, drug sensitivity testing, etc.). By performing various treatments and analyses on cell lines, we can gain a deeper understanding of the characteristics, signaling pathways, and drug resistance mechanisms of tumor cells, providing clues for finding new therapeutic targets and strategies. At present, the rumen-forming cancer cells of immunocompetent mice that have been reported and used in scientific research mainly include MFC gastric cancer cells and YTN3 gastric cancer cells. However, MFC gastric cancer cells mainly form tumors in 615 mice and the pathological type is squamous cell carcinoma, while YTN3 gastric cancer cells are difficult to progress to a larger size in C57BL / 6 mice. Therefore, the development of mouse gastric adenocarcinoma cell lines with new genetic configurations and pathological types is particularly important for gastric cancer research. Summary of the Invention

[0006] The purpose of this application is to provide a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, its construction method, cell line, and application, to improve the tumor formation rate of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma in mice, shorten the tumor formation time, and stabilize the tumor formation. The specific technical solution is as follows:

[0007] The first aspect of the present application provides a method for constructing a mouse model of gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma, comprising the steps of: constructing a mouse M3 with conditional knockout of the Trp53 gene; CreERT2 / + The mouse M1 was hybridized with the mouse M13, and the mouse M13 was intercrossed to obtain the mouse M4; the mouse M4 was hybridized with the mouse M13 with the genotype KRAS LSL-G12D / + The mouse M2 was hybridized to obtain the mouse M42, and the mouse M42 was hybridized with the mouse M4 to obtain the mouse M5; the mouse M5 was chemically induced by administering drugs to obtain a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma; wherein the drugs included tamoxifen.

[0008] In one embodiment of the present application, the method for constructing mouse M3 comprises: inserting flox into both sides of exon 5-7 of Trp53 gene through ES cell targeting to obtain mouse F1, mating the F1 mouse with the same genotype to obtain mouse M3 with conditional knockout of Trp53 gene, and the genotype of mouse M3 is Trp53 fl / fl .

[0009] In one embodiment of the present application, the genotype of mouse M13 is Anxa10 CreERT2 / + Trp53 fl / - The genotype of mouse M4 is Anxa10 CreERT2 / + Trp53 fl / fl ; The genotype of mouse M5 is Anxa10 CreERT2 / + Trp53 fl / fl ;KRAS LSL-G12D / + .

[0010] In one embodiment of the present application, the mouse M5 used for chemical induction is a C57BL / 6 mouse. When the drug is first administered, the age of the mouse M5 is 5 weeks to 7 weeks, and the weight is 18g to 20g; the drug concentration is 10mg / mL to 20mg / mL, the drug is administered by intraperitoneal injection, the drug dosage is 4.0mL / kg body weight to 6.0mL / kg body weight, the drug administration cycle is 1 week to 2 weeks, and the drug administration frequency is 2 times to 3 times a week; chemical induction can cause KRAS-G12D point mutation in the gastric epithelial cells of mouse M5 and knock out the Trp53 gene.

[0011] The second aspect of the present application provides a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma obtained according to the construction method described in the first aspect of the present application.

[0012] The third aspect of the present application provides a cell line constructed using the gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma mouse model described in the second aspect of the present application.

[0013] In one embodiment of the present application, the constructed method includes the steps of: obtaining gastric tumor tissue of a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, washing it, and then lysing it with an organoid lysis solution to obtain a tissue fragment solution; adding a stop solution to the tissue fragment solution to stop it, and then filtering and centrifuging it, adding red blood cell lysis solution to the collected precipitate, standing it and then centrifuging it to collect a mixed precipitate of tissue cells; resuspending the mixed precipitate of tissue cells using an organoid primary culture medium, adding Matrigel matrix glue for culturing, and obtaining contents containing primary cells; culturing the contents containing primary cells for 7-10 days, and then digesting them with trypsin digestion solution to separate the primary tumor cells and obtain a cell line.

[0014] In one embodiment of the present application, the volume ratio of gastric tumor tissue to organoid lysate is 1:(50-100);

[0015] The volume ratio of red blood cell lysate to sediment was 1:(3-5);

[0016] The primary culture medium of the organoids includes L-WRN cell culture supernatant, 50× B27 supplement without vitamin A 1vol%-3vol%, N-acetylcysteine ​​amide 1mM-1.5mM, niacinamide 8mM-12mM, FGF-1080ng / mL-120ng / mL, EGF 40ng / mL-60ng / mL, A83-01 TGFbeta inhibitor 0.5μM-1.5μM, Y-27632 dihydrochloride 8μM-12μM, Glutamax supplement 0.5vol%-1.5vol%, Matrigel 5vol%-10vol%, Hepes buffer 8mM-12mM, gastrin 0.5nM-2.0nM, Primocine 40 μg / mL-60 μg / mL, the volume ratio of the tissue cell mixed precipitate to the organoid primary culture medium is 1:(5-8), the concentration of Matrigel matrix gel is 8 mg / mL-12 mg / mL, and the volume ratio of the resuspended tissue cell mixed precipitate to Matrigel matrix gel is (10-20):1;

[0017] The concentration of the trypsin digestion solution is 0.05 w / v%-0.25 w / v%, the volume ratio of the contents containing primary cells to the trypsin digestion solution is 1:(10-20), and the digestion time is 3-6 minutes.

[0018] In one embodiment of the present application, the cell lines are deposited at the General Microbiology Center of China Culture Collection Administration of Microorganisms with the deposit numbers CGMCC No. 46347 and CGMCC No. 46348, respectively.

[0019] The fourth aspect of the present application provides a use of the mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma described in the second aspect of the present application or the cell line described in the third aspect of the present application in the preparation of a drug for treating poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma.

[0020] Beneficial effects of this application:

[0021] The present application provides a method for constructing a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma. The construction method of the present application can obtain a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma and a cell line. The construction method can increase the tumor formation rate of gastric cancer in mice and shorten the tumor formation time. At the same time, the tumor formation and tumor growth rate of the cell line are stable, and the difference between the groups is small, which solves the research problem of the lack of Trp53 gene knockout and KRAS-G12D point mutation mouse gastric cancer model and cell line. The cell line constructed in the present application will fill the gap in the pathological types of existing gastric cancer cell lines and provide a new and more representative research tool for basic research and clinical transformation of gastric cancer.

[0022] Of course, it is not necessary to achieve all the advantages described above at the same time when implementing any product or method of the present application.

[0023] Preservation Instructions

[0024] Classification and nomenclature: C57BL / 6 mouse gastric adenocarcinoma cell line BCH-812;

[0025] Deposit number: CGMCC No 46348;

[0026] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0027] Abbreviation of depository institution: CGMCC;

[0028] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0029] Date of preservation: April 22, 2025.

[0030] Classification and nomenclature: C57BL / 6 mouse gastric adenocarcinoma cell line BCH-912;

[0031] Deposit number: CGMCC No.46347;

[0032] Depository: General Microbiology Center of China Culture Collection Administration of Microorganisms;

[0033] Abbreviation of depository institution: CGMCC;

[0034] Storage address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

[0035] Date of preservation: April 22, 2025. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other embodiments can also be obtained based on these drawings.

[0037] Figure 1 The figures show the results of the mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma established in Example 1, wherein A is a cross-section of the abdominal cavity; B is the stomach tissue; C is the pathological staining result of the stomach tissue in B;

[0038] Figure 2 Graphs showing the in vitro proliferation curves of BCH-812 cells in Example 3, BCH-912 cells in Example 4, and YTN3 cells in Comparative Example 1;

[0039] Figure 3 Figure 3 shows the pathological staining results of tumor tissue obtained after the BCH-812 cell line in Example 3, the BCH-912 cell line in Example 4, and the YTN3 cell line in Comparative Example 1 were subcutaneously implanted in C57BL / 6 mice;

[0040] Figure 4 The graph is a volume growth curve of tumor tissue after the BCH-812 cell line in Example 3, the BCH-912 cell line in Example 4, and the YTN3 cell line in Comparative Example 1 were subcutaneously implanted in C57BL / 6 mice. DETAILED DESCRIPTION

[0041] The following will be combined with the embodiments of the present application and the accompanying drawings to clearly and completely describe the technical solutions in this application. Obviously, the embodiments described are only part of the embodiments of the present application, rather than all the embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0042] The first aspect of the present application provides a method for constructing a mouse model of gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma, comprising the steps of: constructing a mouse M3 with conditional knockout of the Trp53 gene; CreERT2 / + The mouse M1 was hybridized with the mouse M13, and the mouse M13 was intercrossed to obtain the mouse M4; the mouse M4 was hybridized with the mouse M13 with the genotype KRAS LSL-G12D / + The mouse M2 is hybridized to obtain the mouse M42, and the mouse M42 is hybridized with the mouse M4 to obtain the mouse M5; the mouse M5 is chemically induced by administering drugs to obtain a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma; wherein the drugs include tamoxifen. The construction method of the present application can obtain a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma and a cell line. The construction method can increase the tumor formation rate of gastric cancer in mice and shorten the tumor formation time. At the same time, the tumor formation and tumor growth rate of the cell line are stable, and the differences between groups are small, thus solving the research problem of the lack of gastric cancer models and cell lines in mice with Trp53 gene knockout and KRAS-G12D point mutation.

[0043] In this application, the genotype of mouse M1 is Anxa10 CreERT2 / + , indicating that a single allele at the Anxa10 locus of mouse M1 is an Anxa10-CreERT2 fusion gene, and even if the other allele of Anxa10 is not modified or is an Anxa10-CreERT2 fusion gene, it can induce a positive phenotype.

[0044] The genomic sequence of the mouse Anxa10 gene described in the present application is Chr8:62510076-62576184, which includes 31 exons, wherein the 2A-CreERT2-Wpre-pA element is inserted before the stop codon.

[0045] In this application, the genotype is Anxa10 CreERT2 / + Mouse M1 can be purchased. For example, the manufacturer and catalog number of mouse M1 can be Shanghai Model Organisms Technology Co., Ltd., NM-KI-200312.

[0046] Alternatively, mouse M1 can also be obtained by construction. This application does not particularly limit the construction method, as long as the purpose of this application can be achieved. For example, the construction method of mouse M1 may include: inserting 2A-CreERT2-Wpre-pA before the stop codon of the mouse Anxa10 gene to construct a mouse M1 that can inducibly express Cre recombinase, and the genotype of mouse M1 is Anxa10 CreERT2 / + .

[0047] In this application, the genotype of mouse M2 is KRAS LSL-G12D / + , indicating that a single allele at the KRAS locus in mouse M2 carries loxp-stop-loxp and exon2 containing the G12D point mutation, and can induce a positive phenotype even if the other allele is unmodified or carries loxp-stop-loxp and exon2 containing the G12D point mutation.

[0048] The mouse KRAS gene described in this application is located at Chr6:145162425-145195965, which includes 21 exons. Exon 2 is the second exon of the KRAS gene, and exon 2 containing a loxP-stop-loxP point mutation and a G12D point mutation replaces exon 2 of the KRAS gene.

[0049] In this application, the genotype is KRAS LSL-G12D / + Mouse M2 can be purchased. For example, the manufacturer and catalog number of mouse M2 can be Shanghai Model Organisms Science Co., Ltd., NM-KI-190003.

[0050] Alternatively, mouse M2 can also be obtained by construction. This application does not particularly limit the construction method, as long as the purpose of this application can be achieved. For example, the construction method of mouse M2 may include: replacing exon 2 of the KRAS gene with loxp-stop-loxp and exon 2 containing the G12D point mutation to construct mouse M2 with a KRAS gene G12D conditional point mutation. The genotype of mouse M2 is KRASLSL-G12D / + .

[0051] In one embodiment of the present application, the method for constructing mouse M3 comprises: inserting flox into both sides of exon 5-7 of Trp53 gene through ES cell targeting to obtain mouse F1, mating the F1 mouse with the same genotype to obtain mouse M3 with conditional knockout of Trp53 gene, and the genotype of mouse M3 is Trp53 fl / fl , indicating that mouse M3 is homozygous at the Trp53 locus, that is, both alleles of Trp53 are flox alleles.

[0052] The mouse Trp53 gene described in this application is located in the genomic sequence of GenBank Accession No. NC_000077.7 (Update Date 2024-02), nucleotide sequence 69471174-69482699, which includes 12 exons. Exon 5-7 is exon 5 to 7 of the gene. Flux was inserted on both sides of exon 5-7 of the Trp53 gene to obtain the mouse M3.

[0053] In one embodiment of the present application, the genotype of mouse M13 is Anxa10 CreERT2 / + Trp53 fl / - The genotype of mouse M4 is Anxa10 CreERT2 / + Trp53 fl / fl This indicates that one allele of Anxa10 in mouse M13 is an Anxa10-CreERT2 fusion gene, the other allele of Anxa10 is unmodified or is an Anxa10-CreERT2 fusion gene, one allele of Trp53 is a flox allele, and the other allele of Trp53 is unmodified; one allele of Anxa10 in mouse M4 is an Anxa10-CreERT2 fusion gene, the other allele of Anxa10 is unmodified or is an Anxa10-CreERT2 fusion gene, and both alleles of Trp53 are flox alleles.

[0054] In one embodiment of the present application, the genotype of mouse M5 is Anxa10 CreERT2 / + Trp53 fl / fl ;KRAS LSL-G12D / +This indicates that one allele of Anxa10 in mouse M5 is an Anxa10-CreERT2 fusion gene, the other allele of Anxa10 is unmodified or is an Anxa10-CreERT2 fusion gene, both alleles of Trp53 are flox alleles, one allele of KRAS carries loxp-stop-loxp and exon2 containing a G12D point mutation, and the other allele of KRAS is unmodified or carries loxp-stop-loxp and exon2 containing a G12D point mutation. Anxa10 expression is tissue- and cell-specific, primarily expressed in gastric epithelial cells. 2A-CreERT2-Wpre-pA was inserted before the stop codon of the mouse Anxa10 gene to generate the M1 mouse. M1 mice express the CreERT2 recombinase gene at the Anxa10 locus, specifically a fusion protein containing estrogen receptor mutant type 2 and Cre recombinase, forming a cytoplasmic-localized fusion protein, the Cre-ERT2 recombinase. The above steps were followed to generate the M5 mouse, which also expresses the CreERT2 recombinase gene at the Anxa10 locus. Upon drug administration and chemical induction, the Cre recombinase dissociates from the anchoring protein through a conformational change and enters the nucleus. Cre recombinase recognizes loxP sites in the KRAS and Trp53 genes in the M5 mouse, leading to genetic recombination. This results in the KRAS-G12D point mutation and Trp53 knockout in the gastric epithelial cells of the M5 mouse, creating a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma.

[0055] In one embodiment of the present application, the mouse M5 used for chemical induction is a C57BL / 6 mouse. When the drug is first administered, the age of the mouse M5 is 5 weeks to 7 weeks, and the weight is 18g to 20g; the drug concentration is 10mg / mL to 20mg / mL, the drug is administered by intraperitoneal injection, the drug dosage is 4.0mL / kg body weight to 6.0mL / kg body weight, the drug administration cycle is 1 week to 2 weeks, and the drug administration frequency is 2 times to 3 times a week; chemical induction can cause KRAS-G12D point mutation in the gastric epithelial cells of mouse M5 and knock out the Trp53 gene. For example, the age of the mouse when the drug is first administered can be 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks or 7 weeks; the weight of the mouse when the drug is first administered can be 18 g, 19 g, 20 g or a range consisting of any two values ​​therein; the drug administration concentration is 10 mg / mL, 12 mg / mL, 14 mg / mL, 16 mg / mL, 18 mg / mL, 20 mg / mL or a range consisting of any two values ​​therein; the drug dosage is 4.0 mL / kg body weight, 4.2 mL / kg body weight, 4.4 mL / kg body weight, 4.6 mL / kg body weight, 4.8 mL / kg body weight, 5.0 mL / kg body weight, 5.2 mL / kg body weight, 5.4 mL / kg body weight, 5.6 mL / kg body weight, 5.8 mL / kg body weight, 6.0 mL / kg body weight or a range consisting of any two values ​​therein; the drug administration cycle can be 1 week, 2 weeks or a range consisting of any two values ​​therein; and the drug administration frequency can be 2 or 3 times a week.

[0056] In one embodiment of the present application, administering a drug to mice M5 specifically comprises the following steps: administering the drug to the mice M5 via intraperitoneal injection; providing normal diet and water between administrations; and maintaining the mice conventionally for 8 to 16 weeks after administration. In the present application, the conventional maintenance period after administration can be 8 weeks, 10 weeks, 12 weeks, 14 weeks, 16 weeks, or a range consisting of any two of these values.

[0057] The second aspect of the present application provides a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma obtained by the construction method provided in the first aspect of the present application.

[0058] The third aspect of the present application provides a cell line, which is constructed using the gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma mouse model described in the second aspect of the present application. The cell line obtained by the gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma mouse model has stable tumor formation and tumor growth rate, small differences between groups, and is reproducible, solving the research problem of the lack of Trp53 gene knockout and KRAS-G12D point mutation C57BL / 6 mouse gastric cancer cell line. The cell line constructed in this application fills the gap in the pathological types of existing gastric cancer cell lines and provides a new and more representative research tool for basic research and clinical transformation of gastric cancer.

[0059] In one embodiment of the present application, the method of constructing comprises the steps of:

[0060] S1: Obtain gastric tumor tissue from a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, wash it, and lyse it with organoid lysis buffer to obtain tissue fragment solution;

[0061] S2: Add stop solution to the tissue fragment solution, filter and centrifuge, add red blood cell lysis solution to the collected precipitate, let it stand and then centrifuge to collect the tissue cell mixed precipitate;

[0062] S3: Resuspend the tissue cell mixture pellet in organoid primary culture medium and add Matrigel matrix gel for culture to obtain the contents containing primary cells;

[0063] S4: After culturing the contents containing the primary cells for 7-10 days, digesting them with trypsin to isolate the primary tumor cells and obtain a cell line. For example, the primary cells can be cultured for 7 days, 9 days, 10 days, or a range consisting of any two of these values.

[0064] In one embodiment of the present application, in step S1, the volume ratio of gastric tumor tissue to organoid lysate is 1:(50-100). For example, the volume ratio of gastric tumor tissue to organoid lysate can be 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or a range consisting of any two of these ratios.

[0065] In one embodiment of the present application, in step S2, the volume ratio of the precipitate to the red blood cell lysate is 1:(3-5). For example, the volume ratio of the precipitate to the red blood cell lysate is 1:3, 1:3.5, 1:4, 1:4.5, 1:5, or a range consisting of any two of these values.

[0066] In one embodiment of the present application, in step S3, the organoid primary culture medium includes L-WRN cell culture supernatant, 50× B27 supplement without vitamin A 1 vol%-3 vol%, N-acetylcysteine ​​amide (N-Acetyl-L-cysteine) 1 mM-1.5 mM, niacinamide 8 mM-12 mM, FGF-10 80 ng / mL-120 ng / mL, EGF 40 ng / mL-60 ng / mL, A83-01 TGFbeta inhibitor 0.5 μM-1.5 μM, Y-27632 dihydrochloride 8 μM-12 μM, Glutamax supplement 0.5 vol%-1.5 vol%, Matrigel 5 vol%-10 vol%, Hepes buffer 8 mM-12 mM, gastrin 0.5 nM-2.0 nM, Primocine 40μg / mL-60μg / mL, the volume ratio of the tissue cell mixed precipitate to the organoid primary culture medium is 1:(5-8), the concentration of Matrigel matrix gel is 8mg / mL-12mg / mL, and the volume ratio of the resuspended tissue cell mixed precipitate to Matrigel matrix gel is (10-20):1. For example, the volume ratio of the tissue cell mixed precipitate to the organoid primary culture medium can be 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8, or a range consisting of any two ratios therein; the concentration of Matrigel can be 8 mg / mL, 8.5 mg / mL, 9 mg / mL, 9.5 mg / mL, 10 mg / mL, 10.5 mg / mL, 11 mg / mL, 11.5 mg / mL, 12 mg / mL, or a range consisting of any two values ​​therein; the volume ratio of the resuspended tissue cell mixed precipitate to Matrigel can be 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, or a range consisting of any two ratios therein. Primocine refers to a primary cell antibiotic.

[0067] In one embodiment of the present application, in step S4, the concentration of the trypsin digestion solution is 0.05 w / v%-0.25 w / v%, the volume ratio of the contents containing primary cells to the trypsin digestion solution is 1:(10-20), and the digestion time is 3-6 minutes. For example, the concentration of the trypsin digestion solution can be 0.05 w / v%, 0.1 w / v%, 0.15 w / v%, 0.2 w / v%, 0.25 w / v%, or a range consisting of any two values ​​therein; the volume ratio of the contents containing primary cells to the trypsin digestion solution can be 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, or a range consisting of any two ratios therein; and the digestion time can be 3 minutes, 3.5 minutes, 4 minutes, 4.5 minutes, 5 minutes, 5.5 minutes, 6 minutes, or a range consisting of any two values ​​therein.

[0068] In the present application, in step S1, there is no particular limitation on the cleaning method, as long as the purpose of the present application can be achieved. For example, 1×PBS (PBS containing 1% (v / v) double antibody, that is, the working concentration of penicillin is 100U / mL, and the working concentration of streptomycin is 0.1mg / mL) can be used for washing 2-3 times; in the present application, the tissue fragments can be transferred to a shaker at 200rpm-300rpm, the shaking time is 100 minutes-110 minutes, and the shaking temperature is 37±0.5°C.

[0069] In this application, in step S2, the stop solution can be a complete culture medium containing 10% (v / v)-15% (v / v) fetal bovine serum (FBS) and 1% (v / v) double-antibody, i.e., the working concentration of penicillin is 100 U / mL and the working concentration of streptomycin is 0.1 mg / mL. For example, the components of the complete culture medium can be 500 mL of DMEM solution with 50 mL-75 mL of FBS and 5 mL of double-antibody added; in this application, the volume ratio of the tissue fragment solution to the stop solution is 1:(1-2).

[0070] In the present application, in step S2, filtration is performed through a filter membrane with a pore size of 22.0 μm-100.0 μm, and the tissue remaining on the filter membrane is ground during filtration. The grinding can be performed using a grinder, and the filtrate can be collected using a centrifuge tube such as a 10 mL-15 mL centrifuge tube; in the present application, centrifugation can be performed at 1200 rpm-1800 rpm for 2 min-8 min; in the present application, the standing temperature can be room temperature.

[0071] In the present application, in step S3, the culture conditions can be 5% CO2, 37±0.5℃; in the present application, the Matrigel matrix gelation method is a thin gel gelation method; rehydration is performed 20h-28h after obtaining the primary cells. Rehydration refers to the replenishment of organoid primary culture medium or complete culture medium. The present application does not specifically limit the amount of rehydration, as long as the purpose of the invention of the present application can be achieved. For example, the ratio of the supplemented volume to the original culture mixture volume is (1-2): 1. Some tissue fragments can be observed to adhere to the wall, and primary cells sprout and grow from the surrounding tissues usually after 72h.

[0072] In the present application, in step S4, after the primary cells are digested, most of the cells are poorly differentiated adenocarcinoma cells or signet ring cell cancer cells, and the cells contain only a small amount of tubular adenocarcinoma cells and fibroblasts, so the primary cells do not need to be separated.

[0073] In the present application, the cell line culture method is adherent culture.

[0074] In one embodiment of the present application, the cell lines are deposited at the China General Microbiology Center under the China Culture Collection Administration of Microorganisms with the deposit numbers CGMCC No. 46348 and CGMCC No. 46347, respectively.

[0075] The fourth aspect of the present application provides a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma provided in the second aspect of the present application or the use of the cell line provided in the third aspect of the present application in preparing a drug for treating poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma.

[0076] In the present application, the application includes screening and evaluating the efficacy of drugs for treating poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma.

[0077] Example

[0078] The following examples and comparative examples are provided to more specifically illustrate the embodiments of the present invention. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are based on mass.

[0079] Example 1

[0080] <Establishment of a Mouse Model of Poorly Differentiated Gastric Adenocarcinoma with Signet Ring Cell Carcinoma>

[0081] (1) M1 mice: C57BL / 6 M1 mice (6-8 weeks old, 18-20 g) were purchased from Shanghai Model Organisms Technology Co., Ltd. under the catalog name NM-KI-200312. The genotype of the mice was Anxa10. CreERT2 / -, that is, the single allele at the Anxa10 locus in mouse M1 is the Anxa10-CreERT2 fusion gene, and the other allele of Anxa10 is not modified;

[0082] (2) M2 mice: C57BL / 6 M2 mice (6-8 weeks old, 18-20 g) were purchased from Shanghai Model Organisms Technology Co., Ltd. under the catalog name NM-KI-190003. The genotype of M2 mice was KRAS LSL-G12D / - , that is, a single allele at the KRAS locus of mouse M2 carries loxp-stop-loxp and exon2 containing the G12D point mutation, while the other allele is unmodified;

[0083] (3) Construction of M3 mice: Using ES cell targeting, flox was inserted into both sides of exon 5-7 of the Trp53 gene of C57BL / 6 mice (6-8 weeks old, 18-20 g) to obtain F1 mice. The F1 mice were mated with the same genotype to obtain mice;

[0084] The mice were tested by tail identification and the genotype was Trp53. fl / fl The mice are M3 mice.

[0085] (4) Construction of mouse M4: Cross mouse M1 with mouse M3 to obtain mice. Take the mice and perform tail identification to test the genotype. The genotype selected is Anxa10 CreERT2 / - Trp53 fl / - The mice were M13 mice, and the mice were crossbred to obtain mice. The tails of the mice were taken to identify the genotype. The test showed that the genotype of the mice was Anxa10. CreERT2 / - Trp53 fl / fl or Anxa10 CreERT2 / CreERT2 Trp53 fl / fl The mice are mice M4;

[0086] (5) Construction of mouse M5: Mouse M4 and mouse M2 were hybridized to obtain mouse M42, and mouse M42 was hybridized with mouse M4 to obtain mice. The tails of the mice were taken to identify the genotype, and the genotype selected was Anxa10 CreERT2 / - Trp53 fl / fl ;KRAS LSL -G12D / - or Anxa10 CreERT2 / CreERT2 Trp53 fl / fl ;KRAS LSL-G12D / LSL-G12D or Anxa10 CreERT2 / CreERT2 Trp53 fl / fl ;KRAS LSL-G12D / - or Anxa10 CreERT2 / -Trp53 fl / fl ;KRAS LSL-G12D / LSL-G12D The mice were M5 mice;

[0087] (6) Chemical induction: M5 mice were 6 weeks old and weighed 18–20 g when the drug was first administered;

[0088] Mice M5 were intraperitoneally injected with drugs at a concentration of 20 mg / mL and a dosage of 5 mL / kg body weight.

[0089] The drug administration cycle was 1 week, and the drug administration frequency was 3 times a week; normal diet and water were given between administrations; after the end of administration, the mice were raised in a conventional manner until death.

[0090] A mouse model with 16 mice was established. Time t was recorded after the chemical induction was completed. The stomach tissues of 8 mice were taken for pathological staining at t = 3 weeks and 8 weeks respectively. The results of pathological staining showed that no cancer cells were found in the stomach tissues of the 8 mice at t = 3 weeks, indicating that the mice did not develop tumors; cancer cells were found in the stomach tissues of the 8 mice at t = 8 weeks, indicating that the mice developed tumors within 8 weeks, and the tumor formation rate of the mouse model was 8 / 8 (100%).

[0091] The mice died naturally about 12 weeks after the end of chemical induction, and the stomach tissues of the mice at the time of natural death were taken for pathological examination. Figure 1 As shown, from Figure 1 As shown in Figure A, when the mouse's abdominal cavity was opened, the stomach was significantly enlarged and hard to the touch. Figure 1 In Figure B, after the stomach cavity was opened along the greater curvature, it was seen that the gastric body and antrum of the mouse were significantly thickened, accompanied by white ulcers. Figure 1 In the C, Figure 1 The gastric tissue in Figure B was pathologically paraffin-embedded and sectioned. After H&E staining, it was observed that the main tissue type was poorly differentiated adenocarcinoma with signet ring cells.

[0092] Example 2

[0093] <Cell Line Construction>

[0094] (1) Obtain soybean-sized gastric tumor tissue (≈65 mm) of the mouse model of gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma 3 ), washed twice with 1× PBS, and then added to approximately 5.0 mL of organoid lysis buffer. 100 mL of OrganoPro tissue enzymatic solution (Beijing Ketu Medical Technology Co., Ltd.) was added to obtain a tissue fragment solution. The tissue fragments were transferred to a shaker at 250 rpm for 105 min at a shaking temperature of 37 ± 0.5°C.

[0095] (2) According to the volume ratio of tissue fragment solution to stop solution of 1:1, complete culture medium containing 10% (v / v) FBS and 1% (v / v) double antibody was added to the tissue fragment solution (50 mL of FBS and 5 mL of double antibody were added to 500 mL of DMEM solution, wherein the working concentration of penicillin was 100 U / mL and the working concentration of streptomycin was 0.1 mg / mL) to stop the reaction. Then, the solution was filtered through a filter membrane with a pore size of 100 μm. At the same time, the remaining tissue on the filter membrane was ground with a grinder, and the precipitate was collected by centrifugation at 1500 rpm for 5 min.

[0096] Red blood cell lysis buffer (Solarbio, R1010) was added to the collected sediment at a volume ratio of 1:3, and the mixture was allowed to stand at room temperature for 3 minutes and centrifuged at 1500 rpm for 5 minutes to collect the mixed tissue cell sediment.

[0097] (3) Resuspend the mixed tissue cell pellet in the organoid primary culture medium at a volume ratio of 1:5;

[0098] The resuspended tissue cell mixed precipitate and 12 mg / mL Matrigel matrix gel were added at a volume ratio of 16.7:1 to obtain a mixture;

[0099] The mixture was transferred to a 6-well plate, and approximately 1.0 mL of the mixture was added to each well. The plate was then cultured in a 5% CO2, 37°C constant temperature incubator to obtain contents containing primary cells. After 24 hours, 1.0 mL of complete culture medium was used to replenish the fluid. The complete culture medium was 500 mL of DMEM solution with 50 mL of FBS and 5 mL of double antibody added. After 72 hours, tissue fragments were observed attached to the 6-well plate, and tumor cells and fibroblasts were growing and sprouting from the surrounding tissue.

[0100] The primary organoid culture medium consists of L-WRN cell culture supernatant. Other components are shown in Table 1. The volume percentages of 50× B27 supplement without vitamin A, Glutamax supplement, and Matrigel are based on the volume of the primary organoid culture medium:

[0101] Table 1

[0102]

[0103]

[0104] (4) After culturing the contents containing primary cells for 7 days, the culture medium was aspirated and digested with 0.25 w / v% trypsin digestion solution (Gibco) at a volume ratio of 1:15 between the contents containing primary cells and trypsin digestion solution for 6 minutes. The cells were then centrifuged at 1200 rpm for 5 minutes and resuspended in culture medium to obtain cell lines. Among them, the BCH-812 cell line with a deposit number of CGMCC NO. 46348 and the BCH-912 cell line with a deposit number of CGMCC NO. 46347 were derived from different mouse models of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, respectively.

[0105] Example 3

[0106] The BCH-812 cell line with the deposit number of CGMCC NO.46348 was cultured in a 5% CO2, 37°C constant temperature incubator for adherent culture and the following tests were performed.

[0107] <In vitro proliferation ability test>

[0108] The in vitro proliferation capacity of the BCH-812 cell line, deposited with CGMCC NO. 46348, was tested and tested. Specifically, the cells were digested with 0.25 w / v% trypsin solution, centrifuged at 1200 rpm for 5 minutes, and then resuspended in complete culture medium (DMEM culture medium containing 10% (v / v) FBS and 1% (v / v) double-antibody), counted using a cell counter, and the cell concentration was adjusted to 20,000 cells / mL. The cells were then added to a 96-well plate at 2,000 cells / well and cultured in 5% CO2 and 37°C for 24 hours. The in vitro proliferation capacity of the cells was then analyzed using an IncuCyte device for cell counting.

[0109] <Establishment of tumor-bearing mouse model>

[0110] The BCH-812 cell line with the deposit number of CGMCC NO.46348 was obtained and digested with 0.25w / v% trypsin digestion solution. The cells were then centrifuged at a speed of 1200 rpm for 5 min. The cells were then resuspended in complete medium (DMEM medium containing 10% (v / v) FBS and 1% (v / v) double-antibody) and counted using a cell counter to adjust the cell concentration to 2×10 7 / mL; 100 μL of the adjusted BCH-812 cell suspension was injected subcutaneously into the back of C57BL / 6 mice (5-6 weeks old, weighing 16-18 g) using a 1.0 mL syringe. The tumor formation was observed every other day, and the length (mm) and width (mm) of the tumor were measured.

[0111] Example 4

[0112] The BCH-912 cell line with the deposit number CGMCC No. 46347 was cultured in a 5% CO2, 37°C constant temperature incubator for adherent culture and the following tests were performed.

[0113] <In vitro proliferation ability test>

[0114] The in vitro proliferation ability of the BCH-912 cell line, deposited with CGMCC No. 46347, was tested and tested. Specifically, the cells were digested with 0.25 w / v% trypsin solution, centrifuged at 1200 rpm for 5 minutes, and then resuspended in complete culture medium (DMEM culture medium containing 10% (v / v) FBS and 1% (v / v) double-antibody), counted using a cell counter, and the cell concentration was adjusted to 20,000 cells / mL. The cells were then added to a 96-well plate at 2,000 cells / well and cultured in 5% CO2 and 37°C for 24 hours. The in vitro proliferation ability of the cells was analyzed using an IncuCyte device for cell counting.

[0115] <Establishment of tumor-bearing mouse model>

[0116] The BCH-912 cell line with the deposit number of CGMCC No. 46347 was obtained and the cells were digested with 0.25 w / v% trypsin digestion solution, and then centrifuged at a centrifugal speed of 1200 rpm for 5 min. The cells were then resuspended in complete medium (DMEM medium containing 10% (v / v) FBS and 1% (v / v) double antibody) and counted using a cell counter to adjust the cell concentration to 2×10 7 / mL; 100 μL of the adjusted BCH-912 cell suspension was injected subcutaneously into the back of C57BL / 6 mice (5-6 weeks old, weighing 16-18 g) using a 1.0 mL syringe. The tumor formation was observed every other day, and the length (mm) and width (mm) of the tumor were measured.

[0117] Comparative Example 1

[0118] YTN3 gastric cancer cells were obtained from Professor Yukiyo Nomura of Japan.

[0119] In vitro proliferation ability test of YTN3 gastric cancer cell line: The obtained cell line was subjected to in vitro proliferation test. Specifically, the cells were digested with 0.25w / v% trypsin digestion solution, then centrifuged at a centrifugal speed of 1200rpm for 5min, and then resuspended in complete culture medium (DMEM culture medium containing 10% (v / v) FBS and 1% (v / v) double antibody), counted using a cell counter, and after adjusting the cell density, the cells were added to a 96-well plate at 2000 cells / well. After culturing for 24h under 5% CO2 and 37°C, the cell proliferation ability of the cells in vitro was analyzed by cell counting using an IncuCyte device.

[0120] <Establishment of tumor-bearing mouse model>

[0121] YTN3 cell line with the deposit number of CGMCC No.46347 was obtained and digested with 0.25w / v% trypsin digestion solution. The cells were then centrifuged at a speed of 1200rpm for 5min. The cells were then resuspended in complete medium (DMEM medium containing 10% (v / v) FBS and 1% (v / v) double-antibody) and counted using a cell counter to adjust the cell concentration to 2×10 7 / mL; 100 μL of the adjusted YTN3 cell suspension was injected subcutaneously into the back of C57BL / 6 mice (5-6 weeks old, weighing 16-18 g) using a 1.0 mL syringe. The tumor formation was observed every other day, and the length (mm) and width (mm) of the tumor were measured.

[0122] Comparative Example 2

[0123] The mouse M4 in Example 1 was chemically induced. When the drug was first administered, the mouse M4 was 6 weeks old and weighed 18-20 g.

[0124] Mice M4 were intraperitoneally injected with drugs at a concentration of 20 mg / mL and a dosage of 5 mL / kg body weight.

[0125] The drug administration cycle was 1 week, and the frequency of drug administration was 3 times a week; normal diet and water were given during the drug administration interval; conventional feeding was carried out after the end of drug administration. After 16 weeks of induction, no mouse model of gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma was obtained, and the tumor formation rate was 0 / 8.

[0126] Comparative Example 3

[0127] The mice M1 and M2 in Example 1 were hybridized to obtain offspring mice. The genotype of the offspring mice was tested by tail identification. The genotype was selected as Anxa10. CreERT2 / - ;KRAS LSL-G12D / - The mice were M12 mice.

[0128] The mice M12 in Example 1 were chemically induced. When the drug was first administered, the mice M12 were 6 weeks old and weighed 18-20 g.

[0129] M12 mice were intraperitoneally injected with drugs at a concentration of 20 mg / mL and a dosage of 5 mL / kg body weight.

[0130] The drug administration cycle was 1 week, and the frequency of drug administration was 3 times a week; normal diet and water were given during the drug administration interval; conventional feeding was carried out after the end of drug administration. After 16 weeks of induction, no mouse model of gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma was obtained, and the tumor formation rate was 0 / 8.

[0131] Experimental observations of tumor-bearing mice:

[0132] Each group had 4 mouse models. The BCH-812 cell line in Example 3, the BCH-912 cell line in Example 4, and the YTN3 cell line in Comparative Example 1 were all injected subcutaneously. The time t2 was recorded and the tumor volume (mm 3 )(Volume = length × width 2 / 2).

[0133] After 3-5 days, the formation of a palpable subcutaneous bulge that continued to grow was recorded as tumor formation, and the tumor formation time t1 was recorded. The tumor formation time was the average value of each parallel group. The absence of a palpable subcutaneous bulge was recorded as no tumor formation.

[0134] Tumor growth rate (mm 3 ) = tumor volume / (t2-t1), the average value of each parallel group was taken and recorded as the tumor growth rate.

[0135] The tumor volume of the mouse model at the time of mouse death was calculated, and the average value of each parallel group was taken as the maximum growth volume of the tumor;

[0136] The subcutaneous tumor tissue of the mouse model was obtained at the time of mouse death and subjected to pathological staining, and the pathological type was tested by H&E staining.

[0137] The mouse models and tissue samples obtained in Examples 3 and 4 and Comparative Example 1 were tested, and the results are shown in Table 2.

[0138] Table 2

[0139]

[0140] As can be seen from Table 2, in Example 3, Example 4 and Comparative Example 1, the tumor formation rate of the BCH-812 cell line in Example 3 and the BCH-912 cell line in Example 4 obtained by the construction method of the present application in the back of C57BL / 6 mice was 100%. The growth rate of the tumor tissue in the mouse models obtained in Example 3 and Example 4 was fast and stable, and the maximum growth volume was large; the tumor formation rate of the YTN3 cell line in Comparative Example 1 in the back of C57BL / 6 mice was 100%, but the tumor growth rate was slow and the maximum growth volume was small, which indicates that the cell line obtained by the construction method of the present application has a high tumor formation rate and a fast tumor growth rate.

[0141] from Figure 2 It can be seen that the in vitro proliferation abilities of the BCH-812 cells in Example 3 shown in Curve 1, the BCH-912 cells in Example 4 shown in Curve 2, and the YTN3 cells in Comparative Example 1 shown in Curve 3 are comparable.

[0142] The tumor tissues of the mouse models obtained in Example 3, Example 4, and Comparative Example 1 of the present application were subjected to H&E staining. The results are as follows: Figure 3 As shown, after the BCH-812 cell line in Example 3 and the BCH-912 cell line in Example 4 were implanted in the back of C57BL / 6 mice and formed tumors, the pathological type of the tumor tissue in the obtained mouse model was poorly differentiated adenocarcinoma with signet ring cell carcinoma, and the pathological type of the tumor tissue in the mouse model obtained in Comparative Example 1 was adenocarcinoma.

[0143] from Figure 4 It can be seen that the mouse gastric cancer cell lines BCH-812 and BCH-912 constructed by implanting the BCH-812 cell line in Example 3 and the BCH-912 cell line in Example 4 on the back of C57BL / 6 mice, respectively, can form tumors on the back of C57BL / 6 mice. The tumors in the mouse models constructed in Examples 3 and 4 grow steadily. In Comparative Example 1, the YTN3 gastric cancer cell line implanted on the back of C57BL / 6 mice showed a slower growth rate of the tumor.

[0144] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the scope of protection of the present application.

Claims

1. A method for constructing a mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, comprising the following steps: Construction of the Trp53 conditional knockout mouse M3; The mice M3 and Anxa10 CreERT2 / + The mouse M1 was hybridized to obtain the mouse M13, and the mouse M13 was intercrossed to obtain the mouse M4; The mice were M4 and KRAS LSL-G12D / + The mouse M2 was hybridized to obtain the mouse M42, and the mouse M42 was hybridized with the mouse M4 to obtain the mouse M5; The mouse M5 is given drugs for chemical induction to obtain the gastric poorly differentiated adenocarcinoma with signet ring cell carcinoma mouse model; wherein, The drug comprises tamoxifen.

2. The construction method according to claim 1, wherein The method for constructing the mouse M3 comprises: inserting flox into both sides of the Trp53 gene exon 5-7 through ES cell targeting to obtain the mouse F1, and mating the mouse F1 with the same genotype to obtain the mouse M3 with the conditional knockout of the Trp53 gene, wherein the genotype of the mouse M3 is Trp53 fl / fl .

3. The construction method according to claim 1, wherein: The genotype of the mouse M13 is Anxa10 CreERT2 / + Trp53 fl / - The genotype of the mouse M4 is Anxa10 CreERT2 / + Trp53 fl / fl ; The genotype of the mouse M5 is Anxa10 CreERT2 / + Trp53 fl / fl ;KRAS LSL-G12D / + .

4. The construction method according to any one of claims 1 to 3, wherein The mice M5 used in the chemical induction were C57BL / 6 mice. When the drug was first administered, the mice M5 were 5 to 7 weeks old and weighed 18 to 20 g. The drug has a concentration of 10 mg / mL to 20 mg / mL, is administered by intraperitoneal injection, has a dosage of 4.0 mL / kg to 6.0 mL / kg body weight, has an administration period of 1 to 2 weeks, and has an administration frequency of 2 to 3 times per week. The chemical induction can cause KRAS-G12D point mutation and knock out Trp53 gene in the gastric epithelial cells of the mouse M5.

5. The mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma obtained by the construction method according to any one of claims 1 to 4.

6. A cell line constructed using the mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma according to claim 5.

7. The cell line according to claim 6, wherein The method of constructing comprises the steps of: Obtaining gastric tumor tissue from the mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma, washing the tissue, and lysing the tissue with an organoid lysis buffer to obtain a tissue fragment solution; Adding a stop solution to the tissue fragment solution to stop the reaction, followed by filtering and centrifugation, adding a red blood cell lysis solution to the collected precipitate, allowing it to stand and then centrifuging to collect the tissue cell mixed precipitate; Resuspending the tissue cell mixed precipitate in an organoid primary culture medium, adding Matrigel matrix gel for culture, and obtaining a content containing primary cells; The contents containing primary cells are cultured for 7-10 days and then digested with trypsin digestion solution to separate the primary tumor cells to obtain the cell line.

8. The cell line according to claim 7, wherein The volume ratio of the gastric tumor tissue to the organoid lysate is 1:(50-100); The volume ratio of the precipitate to the red blood cell lysate is 1:(3-5); The organoid primary culture medium includes L-WRN cell culture supernatant, 50× B27 supplement without vitamin A 1vol%-3vol%, N-acetylcysteine ​​amide 1mM-1.5mM, niacinamide 8mM-12mM, FGF-1080ng / mL-120ng / mL, EGF 40ng / mL-60ng / mL, A83-01 TGFbeta inhibitor 0.5μM-1.5μM, Y-27632 dihydrochloride 8μM-12μM, Glutamax supplement 0.5vol%-1.5vol%, Matrigel 5vol%-10vol%, Hepes buffer 8mM-12mM, gastrin 0.5nM-2.0nM, Primocine 40 μg / mL-60 μg / mL, the volume ratio of the tissue cell mixed precipitate to the organoid primary culture medium is 1:(5-8), the concentration of the Matrigel matrix glue is 8 mg / mL-12 mg / mL, and the volume ratio of the resuspended tissue cell mixed precipitate to the Matrigel matrix glue is (10-20):1; The concentration of the trypsin digestion solution is 0.05 w / v%-0.25 w / v%, the volume ratio of the contents containing primary cells to the trypsin digestion solution is 1:(10-20), and the digestion time is 3-6 minutes.

9. The cell line according to claim 6, wherein The cell lines are deposited at the General Microbiology Center of China Culture Collection Administration CGMCC No. 46347 and CGMCC No. 46348 respectively.

10. Use of the mouse model of poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma according to claim 5 or the cell line according to any one of claims 6 to 9 in the preparation of a drug for treating poorly differentiated gastric adenocarcinoma with signet ring cell carcinoma.

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