Mouse-derived gastric adenocarcinoma cell line MKC1 and application thereof

Abstract

The invention discloses a mouse-derived gastric adenocarcinoma cell line MKC1 and application thereof. The mouse gastric adenocarcinoma cell line MKC1 (Mus musculus) is preserved in the China Center for Type Culture Collection, the preservation date is June 15, 2024, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: C2024183. The cell line belongs to a murine gastric adenocarcinoma cell line, can be stably passaged, is high in growth speed, and can form primary tumors in a C57BL / 6 mouse body with a sound immune system. In addition, the cell line has the advantages of being high in repeatability, stable and single in property and the like, and is suitable for related research of gastric cancer immune microenvironment, tumor drug resistance, invasion and metastasis and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of gastric cancer cell lines, and particularly relates to a mouse-derived gastric adenocarcinoma cell line MKC1 and an application thereof. 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. Traditional treatments for gastric cancer are surgery, chemotherapy and radiotherapy, but 50% of treated patients will relapse, and the 5-year survival rate is only 20%. In particular, there is currently no effective treatment for locally advanced or metastatic gastric cancer, and the five-year survival rate is less than 10%. In order to develop more effective treatments, research on gastric cancer-specific molecular targets and the mechanisms of tumor occurrence and development has always been a hot topic in gastric cancer research.

[0003] In recent years, immunotherapy has made breakthrough progress in the treatment of various malignant tumors. Among them, the combination of nivolumab, a drug targeting programmed cell death protein-1 (PD-1), and chemotherapy has been approved as a first-line treatment for patients with locally advanced or metastatic gastric cancer. Despite the positive clinical progress, clinical application results show that only a small number of gastric cancer patients continue to benefit from immunotherapy, and most gastric cancer patients develop primary or secondary drug resistance, suggesting that there is still a need for in-depth research on the characteristics of the immune microenvironment of gastric cancer and specific immunosuppressive molecular targets.

[0004] In order to further study the immune microenvironment of gastric cancer, a tumor-bearing mouse model with a sound immune microenvironment is needed. Tumor-bearing mouse models are divided into spontaneous models and transplantation models. Among them, the cell-derived mouse tumor transplantation model (cell derived xenograft, CDX) is simple to operate and has stable and uniform tumor growth, which is a commonly used model for tumor research. To construct a mouse gastric cancer CDX model with a sound immune system, a mouse gastric cancer cell line is required. However, the currently available mouse gastric cancer cell lines have many defects: MFC cells were established by Qian Shusen et al. in 1985 and belong to squamous cell carcinoma cell lines; NCCG1 cells established by Wu Chen et al. also belong to squamous cell carcinoma cell lines, which are different from the common gastric adenocarcinoma in clinical practice. In 2018, the University of Tokyo Hospital constructed the gastric adenocarcinoma cell line YTN16 based on the separation of mouse gastric tumors induced by methylnitrosourea (MNU), but its successful construction was largely accidental. According to relevant literature descriptions, the research team only successfully established one gastric cancer cell line YTN16 among the 12 mouse tumors induced by MNU, and its construction process was difficult to be repeated by other laboratories. Summary of the invention

[0005] Purpose of the invention: In order to solve the problem of the lack of mouse-derived gastric adenocarcinoma cell lines, the present invention provides a mouse-derived gastric adenocarcinoma cell line MKC1 and its application. The gastric cancer cell line MKC1 established by the present invention grows adherently, has a fast growth rate, and has good in vitro amplification. In addition, the MKC1 cell line is isolated and cultured from MKC transgenic mice, has strong reproducibility, good passage stability, and is especially capable of forming tumors in C57BL / 6 mice with a sound immune system. The cell line can be used for research on the immune microenvironment, occurrence and development mechanism, distant metastasis mechanism, drug resistance mechanism, and new drug screening of gastric cancer.

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

[0007] In a first aspect, the present invention provides a mouse gastric adenocarcinoma cell line MKC1 (Mus musculus), wherein the mouse gastric adenocarcinoma cell line MKC1 is deposited in the China Center for Type Culture Collection on June 15, 2024, and the deposit number is CCTCC NO: C2024183.

[0008] In a second aspect, the present invention provides the use of the mouse gastric adenocarcinoma cell line MKC1 as an experimental tool in studying cancer.

[0009] As a specific embodiment, the cancer is gastric cancer.

[0010] As a specific embodiment, the cancer is gastric adenocarcinoma.

[0011] In a third aspect, the present invention provides the use of the mouse gastric adenocarcinoma cell line MKC1 in preparing a gastric cancer animal model.

[0012] As a specific embodiment, the gastric cancer is gastric adenocarcinoma.

[0013] As a specific implementation scheme, the gastric cancer animal model is a subcutaneous tumor formation animal model.

[0014] In a fourth aspect, the present invention provides the use of the mouse gastric adenocarcinoma cell line MKC1 cell line in screening drugs for treating gastric cancer.

[0015] As a specific embodiment, the gastric cancer is gastric adenocarcinoma.

[0016] The mouse gastric adenocarcinoma cell line MKC1 of the present invention is based on the Myc LSL / LSL ;Kras loxP / + ;

[0017] Anxa10-CreERT2 + / -(MKC) Gastric cancer spontaneous tumor transgenic mouse model, after the formation of gastric tumors, isolated and cultured, so named MKC1 cell line. This cell line belongs to the mouse gastric adenocarcinoma cell line, which can be stably propagated, grows fast, can form tumors in immunodeficient mice, especially in C57BL / 6 mice with a healthy immune system. In addition, this cell line has the advantages of strong reproducibility, stable and single properties, and is suitable for related research on gastric cancer immune microenvironment, tumor resistance, invasion and metastasis.

[0018] The method for constructing the transgenic mouse model comprises the following steps:

[0019] (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.

[0020] (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;

[0021] (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;

[0022] (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;

[0023] (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.

[0024] Beneficial effects: Compared with the prior art, the mouse gastric cancer cell line MKC1 established in the present invention is derived from the transgenic mouse Myc LSL / LSL ;Kras loxP / + ;Anxa10-CreERT2 + / - The construction process is highly reproducible and has good stability in passage. It can form tumors in immunodeficient mice, especially subcutaneously in C57BL / 6 mice with a healthy immune system. It is suitable for the study of the immune microenvironment and tumor occurrence and development mechanism of gastric cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 2 This is the electrophoresis diagram of the product amplified using MYC primers, where lanes 1, 2 and 3 represent three different samples.

[0027] Figure 3 The electrophoresis diagram of the product amplified using KRAS primers, where lanes 1, 2 and 3 represent three different samples.

[0028] Figure 4 This is a picture of tumor formation in transgenic MKC mice.

[0029] Figure 5 This is the microscopic morphology of MKC1 cells.

[0030] Figure 6 Results of MKC1 detection in clone formation experiment.

[0031] Figure 7This is the growth curve of CCK8 detecting MKC1, AGS, and HGC27.

[0032] Figure 8 This is a picture of subcutaneous tumor formation in C57BL / 6 mice injected with MKC1.

[0033] Fig. 9 This is the HE staining image of subcutaneous tumor tissue in C57BL / 6 mice. DETAILED DESCRIPTION

[0034] 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.

[0035] Example 1 Construction of MKC gastric cancer spontaneous tumorigenesis transgenic mouse model

[0036] 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.

[0037] The specific method for constructing the MKC mouse model is as follows:

[0038] 1. Gene sources and construction principles of each transgenic mouse

[0039] 1. Obtaining Anxa10-CreERT2 mice

[0040] 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.

[0041] Anxa10-2A-CreERT2 gene:

[0042] NCBI ID: 26359

[0043] MGI ID: 1347090

[0044] Ensembl ID: ENSMUSG00000031635

[0045] Pubmed: Anxa10

[0046] Anxa10 gene:

[0047] Anxa10 annexin A10[Mus musculus(house mouse)]

[0048] NCBI Gene ID:26359, updated on 11-Sep-2023

[0049] Examples of gene-marked cell types: gastric epithelial cells, skin hair follicle cells

[0050] 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.

[0051] 2. Obtaining MYC overexpressing mice

[0052] 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 / LSLHomozygous mice were purchased from Shanghai Model Organisms Technology Co., Ltd.

[0053] MYC gene:

[0054] MGI ID: 5461148

[0055] https: / / www.modelorg.com / index.php / portal / article / index / id / 858 / post_type / 3.html

[0056] 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.

[0057] 3.Kras gene G12D conditional point mutation mouse strain KRAS loxP / + Acquisition of mice

[0058] 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.

[0059] Kras gene:

[0060] Kras Kirsten rat sarcoma viral oncogene homolog[Mus musculus(housemouse)];

[0061] NCBI Gene ID:16653, updated on 11-Oct-2023

[0062] 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.

[0063] 2. Specific process of constructing gastric adenocarcinoma transgenic mouse model

[0064] (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.

[0065] (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.

[0066] (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;

[0067] (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

[0068] + / - ;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;

[0069] 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 G12D conditional point mutation of the Kras gene.

[0070] 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.

[0071] 3. Mouse genotype identification method

[0072] 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:

[0073] 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.

[0074] 1) The sequence of the wide type primer for identifying the Anxa10-CreERT2 recombinase gene is:

[0075] 5'-ATCTCCCATCCTCCCTAACTT-3' and 5'-ATCGCATCACCTTCAAACTCT-3'.

[0076] The mutant type primer sequences are:

[0077] 5'-CAGGGCACTATGAGAAAGCACTAC-3' and 5'-AGCCCGGACCGACGATGAAG C-3'.

[0078] 2) The Wide type primer sequence for identifying the MYC gene is:

[0079] 5'-ATAAGCCATTCTCCATTTCATAA-3' and 5'-CCCCTTGTTCCCTTTCTGC-3'.

[0080] The mutant type primer sequences are:

[0081] 5'-TTTGCCTTTGTTACCTGTTCCATC-3' and 5'-CTCCCCCGTGCCTTCCTTGAC-3'.

[0082] 3) The sequence of the wide type primer for identifying the KRAS point mutation gene is:

[0083] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-TGTCTTTCCCCAGCACAGT-3'.

[0084] The mutant type primer sequence is

[0085] 5'-CTGCATAGTACGCTATACCCTGT-3' and 5'-GCAGGTCGAGGGACCTAATA-3'.

[0086] Genotype identification system:

[0087] F (upstream primer): 0.25ul, R (downstream primer): 0.25ul, 2xTaq (buffer): 7.5ul, H2O: 5.5ul, mouse tail DNA: 1.5ul, Total: 15ul.

[0088] PCR instrument loading procedure:

[0089] 1) ANXA identification:

[0090] 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.

[0091] 2) Kras identification:

[0092] 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.

[0093] 3) MYC identification:

[0094] 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

[0095] The genotype identification results of mice are shown in Figure 1-3 , the three lanes in the figure represent three samples, Figure 1The 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.

[0096] Figure 2 For the electrophoresis of the product amplified with MYC primers, if a 434bp fragment can be specifically amplified, it is MYC LSL / LSL homozygous, if a 428bp fragment can be specifically amplified, it is MYC negative, and if a negative-positive band is amplified, it is heterozygous MYC LSL / +.

[0097] 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.

[0098] 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.

[0099] Example 2 Isolation and culture of gastric adenocarcinoma cell line MKC1

[0100] 1. The experimental steps for constructing primary cell lines are as follows:

[0101] (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;

[0102] (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;

[0103] (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;

[0104] (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.

[0105] (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.

[0106] (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 ).

[0107] (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).

[0108] 2. Primary cell line proliferation ability detection

[0109] (1) Colony formation assay: After trypsin digestion, cells were counted and 500 cells were added to each well of a 6-well plate. After 8-14 days of culture, cells were stained with 0.5% crystal violet (eg Figure 6 As shown, primary cells have the ability to form colonies).

[0110] (2) CCK-8 experiment: cells were seeded in 96-well plates (2-3×10 3 Cells / well) were added to each well and the plates were cultured in an incubator. After cell attachment, 10 μl of CCK-8 solution (Apexbio, TX, USA) was added to each well on days 1-4. After 2 h of incubation, the absorbance was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA). Figure 7 As shown, the proliferation capacity of primary cells MKC1 was significantly higher than that of gastric cancer cell lines HGC27 and AGS).

[0111] 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.

[0112] Example 3 Subcutaneous tumor formation and identification in C57BL / 6 mice

[0113] (1) Subcutaneous tumor formation in C57BL / 6 mice: After MKC1 was expanded and cultured, the cells were collected by trypsin digestion and suspended in physiological saline. The cell concentration was adjusted to 5×10 5 / 100μl. Six-week-old C57BL / 6 mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were used, and 100μl of cell suspension was injected subcutaneously on the back of each mouse. The mice were killed 4 weeks later and the tumors were removed (such as Figure 8 As shown, primary cells have the ability to form tumors subcutaneously in C57BL / 6 mice), were treated with formalin, embedded in paraffin and sectioned.

[0114] (2) Hematoxylin-eosin (HE) staining of subcutaneous tumor tissue: HE staining is one of the most commonly used staining methods in pathological sectioning technology. The steps are as follows: Place the mouse gastric cancer paraffin sections in a 60-degree oven for 90 minutes, quickly place the sections in xylene I for 15 minutes, and then transfer to xylene II for 15 minutes. Place the sections in absolute ethanol I, absolute ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol and 50% ethanol for 5 minutes each, and place the sections in deionized water for 5 minutes. Try to remove the moisture on the sections, then place the sections in hematoxylin for staining for 3-5 minutes, and rinse with deionized water. Place the sections in 1% hydrochloric acid ethanol for color separation for 30 seconds, and rinse with deionized water. Place the sections in tap water to turn blue for 3 minutes. Place the sections in 0.5% eosin solution for staining for 3 minutes, and rinse with deionized water. Place the slices in 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol II, and anhydrous ethanol I for 5 minutes each. Place the slices in xylene II and xylene I for 5 minutes each. Add a small amount of neutral gum to the sliced ​​tissue, gently cover with a coverslip, and air dry. Scan and photograph the sealed slices (such as Fig. 9 As shown, HE staining confirmed that the subcutaneous tumor in mice was gastric adenocarcinoma tissue).

[0115] 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 mouse gastric adenocarcinoma cell line MKC1, 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.

2. Use of the mouse gastric adenocarcinoma cell line MKC1 according to claim 1 as an experimental tool in cancer research.

3. The application according to claim 2, characterized in that: The cancer is gastric cancer.

4. The application according to claim 2, characterized in that: The cancer is gastric adenocarcinoma.

5. Use of the mouse gastric adenocarcinoma cell line MKC1 according to claim 1 in preparing a gastric cancer animal model.

6. The use according to claim 5, characterized in that: The gastric cancer is gastric adenocarcinoma.

7. The use according to claim 5, characterized in that: The gastric cancer animal model is a subcutaneous tumor formation animal model.

8. Use of the mouse gastric adenocarcinoma cell line MKC1 according to claim 1 in screening drugs for treating gastric cancer.

9. The use according to claim 8, characterized in that: The gastric cancer is gastric adenocarcinoma.

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