Mouse head and neck squamous carcinoma cell line as well as construction method and application thereof

Abstract

The invention relates to the field of tumor cell lines, and provides a mouse head and neck squamous carcinoma cell line as well as a construction method and application thereof, the mouse head and neck squamous carcinoma cell line is named as 4N-MS1 and preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC NO: 65741. The construction method comprises the following steps: S1, inducing mouse oral mucosa canceration by using a chemical reagent; s2, cutting tumor tissues through an operation, cutting the tumor tissues into pieces, further cutting the tumor tissues into pieces through enzymatic dissociation, preparing a single-cell suspension, and transferring the cells into a pore plate for culture to obtain primary tumor cells; s3, purifying the primary tumor cells by an enzyme digestion method to obtain purified primary tumor cells; s4, transfecting the purified primary generation head and neck squamous cell carcinoma tumor cells with viruses to obtain the mouse head and neck squamous cell carcinoma cell line which grows stably. The cell line has the advantages that the cell line is high in purity, high in growth speed and high in tumor formation capacity in a mouse body, and a powerful tool is provided for later research on mechanisms of tumor immune response, escape and the like on an HNSCC mouse model and related treatment targets of an immune system.

Description

Technical Field

[0001] The present invention relates to the field of tumor cell lines, and more specifically, to a mouse head and neck squamous cell carcinoma cell line and a construction method and application thereof. Background Art

[0002] Head and neck cancer is one of the most common human malignant tumors in the world, and 90% of head and neck cancers are squamous cell carcinomas (HNSCC) involving the oral, pharyngeal and laryngeal mucosal surfaces. In recent years, despite the clinical adoption of a comprehensive treatment model that is mainly based on surgery and supplemented by radiotherapy, chemotherapy and other methods, the treatment effect is still not ideal, and the five-year overall survival rate is only maintained at around 50-60%. Therefore, it is very necessary to further study the pathogenesis of HNSCC and develop new treatment methods.

[0003] At present, the research paradigm for HNSCC is mainly based on in vivo and in vitro models. Compared with in vitro studies (mainly referring to studies on tumor cells), in vivo studies (animal experiments, mainly mouse models) can better reflect the complex microenvironment in the tumor and the biological process of tumor progression. The most ideal animal model is spontaneously generated HNSCC, which is very rare in experimental animals. The most commonly used models are human tumor cell line xenografts (Cell Derived Xenograft, CDX) and human tumor tissue derived transplant tumor models (Patient Derived Xenograft, PDX). The cell lines used in this model are derived from humans, and the main drawback is that the mice used for transplantation are naturally immune-deficient BALB / c-nu athymic nude mice, which cannot be used to study the tumor immune system and the role of immune checkpoint inhibitors.

[0004] In order to study the HNSCC tumor immune microenvironment in mice and discover new targets for tumor treatment, we need a reliable syngeneic transplant mouse model (Syngeneic model), which is to inoculate tumor cell lines from the same background into mice with sound immunity. The recipient mice have a complete mouse immune system with complete immune activity, and this immune system is compatible with the syngeneic transplant tumor tissue, which can maximize the simulation of the real situation of the tumor microenvironment. However, there are very few mouse-derived HNSCC cell lines at present, and the time and cost of obtaining them are high. The tumor formation effect on syngeneic mice cannot be verified, which restricts the use of syngeneic transplant mouse models. In addition, the failure rate of extracting and culturing cell lines from mouse HNSCC tumor models is very high, and it is difficult to obtain cell lines with stable growth rate, unlimited proliferation and tumor formation in syngeneic mice. Summary of the invention

[0005] The present invention aims to overcome at least one defect (shortcoming) of the above-mentioned prior art and provides a mouse head and neck squamous cell carcinoma cell line and a construction method and application. The cell line constructed by the present invention has a strong tumor-forming ability in mice, providing a good mouse in vivo model for research related to immunotherapy of head and neck squamous cell carcinoma.

[0006] One object of the present invention is to provide a mouse head and neck squamous cell carcinoma cell line, which is named 4N-MS1, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 10, 2025. Its preservation number is: GDMCCNO: 65741, and the address of the preservation unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] Furthermore, the strain of the mice is C57 mice.

[0008] Another object of the present invention is to provide a method for constructing the above-mentioned mouse head phosphoblastoma cell line, comprising the following steps:

[0009] S1. Use chemical reagents to induce oral mucosal cancer in mice;

[0010] S2, surgically remove the tumor tissue and cut it into pieces, use enzyme dissociation to further cut it into pieces and prepare it into a single cell suspension, transfer the single cell suspension to a well plate for culture, and obtain primary tumor cells;

[0011] S3, purifying primary tumor cells using an enzyme digestion method to obtain purified primary head and neck squamous cell carcinoma tumor cells;

[0012] S4. Virus transfection was used to purify primary head and neck squamous cell carcinoma tumor cells to obtain a stably growing mouse head and neck squamous cell carcinoma cell line.

[0013] Further, in step S2, the cells are further sheared and prepared into a single cell suspension using enzyme dissociation, and the single cell suspension is transferred to a well plate for culture, specifically:

[0014] The tumor tissue cut and shredded during surgery was transferred to a type I collagenase solution, incubated at a constant temperature with shaking, vortexed and shaken at intervals, centrifuged, the supernatant discarded, trypsin was added, oscillated at a constant temperature, DMEM culture medium containing 8-12% fetal bovine serum was added, the suspension containing tumor cells was filtered, the filtrate was taken, centrifuged, the supernatant discarded, red blood cell lysis solution was added, incubated, centrifuged, the supernatant discarded, and the cell suspension was resuspended in DMEM culture medium containing 8-12% fetal bovine serum to obtain a cell suspension. The cells were planted on a well plate, DMEM culture medium containing 10% fetal bovine serum, epidermal growth factor and penicillin-streptomycin were added, and the well plate was placed in an incubator for culture.

[0015] In more than one embodiment of the present invention, in step S2, the cells are further sheared and prepared into a single cell suspension using enzymatic dissociation, and the single cell suspension is transferred to a well plate for culture. The more specific steps are:

[0016] The tumor tissue was removed surgically and cut into pieces of 0.5-2 mm 3 Fragments, transfer the chopped tumor tissue to 300-600 μl of 1.5-2.5 mg / ml type I collagenase solution, place in a 36-38°C constant temperature shaker for 20-40 minutes, vortex and shake for 5-30 seconds every 5-15 minutes; then centrifuge at 300-400g for 3-10 minutes, and discard the supernatant. Add 400-600 μl of 1.5-2.5% trypsin solution, incubate in a 36-38°C constant temperature shaker for 3-8 minutes, and then add DMEM medium containing 8-12% fetal bovine serum (FBS) to neutralize trypsin. Filter the suspension containing tumor cells through a filter group with a pore size of 30-80 μm in sequence, collect the filtrate and centrifuge at 300-400g for 3-10 minutes, and discard the supernatant. Add 0.8-1.5ml red blood cell lysis buffer to resuspend the cells, incubate at room temperature for 8-15 minutes, centrifuge for 3-10 minutes, and discard the supernatant. Use 150-250μl of DMEM medium containing 8-12% FBS to resuspend the cells and prepare a cell suspension. Inoculate the cells in a culture plate, supplement with DMEM medium containing 8-12% FBS, 5-20ng / ml epidermal growth factor and 0.5-2% penicillin-streptomycin double antibody solution. Place the culture plate in an incubator and culture the culture system in a culture environment of 36-38°C, 4-6% CO2, pH 7.0-7.6 and 90-100% relative humidity.

[0017] Further, in step S3, the primary tumor cells are purified by enzyme digestion method as follows:

[0018] When the well-adherent cells grow to 85-90%, trypsin is added and incubated, and then DMEM medium containing 8-12% fetal bovine serum is added to allow some non-tumor cells to fall off, the liquid is aspirated, and trypsin solution is added again to digest until more than 90-95% of the cells fall off, centrifuge, discard the supernatant, and culture the cells in DMEM medium containing 4-6% FBS to obtain purified primary head and neck squamous cell carcinoma tumor cells.

[0019] In one or more embodiments of the present invention, in step S3, the enzymatic digestion method is used to purify primary tumor cells, and the more specific steps are:

[0020] When the density of cells with good adhesion reaches 85-95%, add 0.8-1.2 ml of 1.5-2.5% trypsin solution, incubate at 36-38°C for 0.5-2 minutes, and gently shake the culture bottle to evenly distribute the enzyme. Then add 0.8-1.2 ml of DMEM culture medium containing 8-12% fetal bovine serum (FBS) to neutralize trypsin, and observe under an optical microscope to confirm that some non-tumor cells have fallen off. Aspirate the liquid in the culture bottle, add 0.8-1.2 ml of 1.5-2.5% trypsin solution again, and continue digestion until more than 90-95% of the cells have fallen off. Centrifuge at 800-1200 rpm for 2-5 minutes, discard the supernatant, resuspend the cells in DMEM medium containing 4-6% FBS and continue to culture, add 5-20 ng / ml epidermal growth factor and 0.5-2% penicillin-streptomycin double antibody solution, maintain the culture system at 36-38°C, 4-6% CO2, pH 7.0-7.6 and 90-100% relative humidity, and obtain purified primary head and neck squamous cell carcinoma tumor cells after culture.

[0021] Further, in step S4, the virus is transfected into purified primary head and neck squamous cell carcinoma tumor cells, specifically:

[0022] For primary tumor cells, after changing to serum-free medium and culturing for a period of time, the pGLuc-Dura-SV40-N plasmid is transfected using liposome Lipofectin2000 as a vector to obtain a plasmid-Lipo2000 complex; the prepared plasmid-Lipo2000 complex is added to the cell culture medium, shaken to make it evenly distributed, and placed in an incubator for 24 to 48 hours; after being taken out from the incubator, the transfection-positive cells are screened with antibiotics, and the transfection is detected by luciferase expression. When the cell growth rate after successful transfection is stable, the DMEM medium containing 8-12% FBS is changed to continue culturing to obtain a stably growing head and neck squamous cell carcinoma cell line.

[0023] In one or more embodiments of the present invention, in step S4, the virus is transfected into purified primary head and neck squamous cell carcinoma tumor cells, and the more specific steps are:

[0024] For primary tumor cells with a growth density of 80-90%, replace the serum-free medium. After 0.5-1 hour, use liposome Lipofectin2000 as a carrier to transfect the pGLuc-Dura-SV40-N plasmid to obtain a plasmid-Lipo2000 complex. Add the prepared plasmid-Lipo2000 complex to the cell culture medium, shake it to make it evenly distributed, and place it in an incubator for 24-48 hours. The culture conditions are 36-38°C and 4-6% CO2. 24-48 hours after transfection, replace it with a complete medium containing 200-800μg / ml G418 (or other appropriate antibiotics); replace the fresh selection medium every 2-4 days, and continue to culture for 10-14 days; observe the cell survival under an optical microscope until the non-transfected cells are completely dead and only transfection-positive cells remain. The surviving cells are inoculated in 24-96-well plates and the density is adjusted to 1×10 4 -5×10 4 / well, culture until the cell density reaches 70-90%, discard the culture medium, and wash the cells 1-3 times with pre-cooled PBS. Add 50-100μl 1× luciferase lysis buffer to each well and incubate at room temperature for 10-20 minutes. Collect the lysate, centrifuge at 12000-15000g for 2-5 minutes, take 20-50μl cell lysis supernatant and mix it with an equal volume of luciferase substrate, use a luciferase detector to measure the relative light unit (RLU), use untransfected cells as negative controls, and when the cell growth rate after successful transfection is stable, culture in 8-12% FBS DMEM culture medium, 5-20ng / ml epidermal growth factor and 0.5-2% penicillin-streptomycin double antibody solution. The culture system is maintained in a culture environment of 36-38°C, 4-6% CO2, pH 7.0-7.6 and 90-100% relative humidity, and a head and neck squamous cell carcinoma cell line with stable growth and unlimited proliferation is obtained after culture.

[0025] Another object of the present invention is to provide a reagent for preparing a head and neck squamous cell carcinoma tumor model, wherein the reagent contains the above-mentioned mouse head and neck squamous cell carcinoma cell line.

[0026] Another object of the present invention is to provide a method for preparing a mouse head and neck squamous cell carcinoma transplanted tumor mouse model, wherein the mouse head and neck squamous cell carcinoma cell line or the above reagent is injected into the mouse body to induce tumor formation.

[0027] Another object of the present invention is to provide a use of the above-mentioned mouse head and neck squamous cell carcinoma cell line in preparing a head and neck squamous cell carcinoma mouse model.

[0028] Another object of the present invention is to provide a use of the above-mentioned mouse head and neck squamous cell carcinoma cell line in the preparation, screening or evaluation of drugs for treating head and neck squamous cell carcinoma.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention extracts, cultures and purifies tumor cells from a mouse HNSCC model induced by carcinogenic drugs, and can obtain a cell line that grows stably after transplantation in C57BL / 6 mice. The cell line has high purity, fast growth rate, strong tumor-forming ability in mice, and can be stably propagated, providing a powerful tool for subsequent research on tumor immune response, escape mechanisms and immune system-related therapeutic targets in HNSCC mouse models. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Shown: 4NQO-induced stable tumor formation pattern in C57BL / 6 mice.

[0032] Figure 2 Display: Schematic diagram of extraction, purification, culture and construction of cell lines from the mouse 4NQO-induced HNSCC tumor model.

[0035] Figure 3 Shown: cell growth pictures of the C57 mouse HNSCC cell line (4N-MS1) constructed by the method of the present invention under a light microscope, P0 refers to the primary cells without passage, P15 refers to passage 15 times, it can be seen that compared with the P0 cells, the P15 tumor cells have a more uniform morphology, with almost no fibroblasts, etc.;

[0036] Figure 4 Display: Somatic SNV statistics table, where the row names are explained as follows:

[0037] Exonic: The number of mutations occurring in the exon coding region

[0038] synonymous_SNV: number of synonymous mutations

[0039] nonsynonymous_SNV: number of nonsynonymous mutations

[0040] stopgain: the number of nonsense mutations, resulting in a new stop codon

[0041] stoploss: the number of mutations that lead to the loss of stop codons

[0042] startloss: the number of mutations that lead to loss of the start codon

[0043] unknown: The number of mutations with unknown functions due to incomplete gene structure annotation database

[0044] intronic: the number of mutations occurring in the introns of a gene

[0045] UTR3: Number of mutations occurring in the 3'UTR of the gene

[0046] UTR5: Number of mutations occurring in the 5'UTR of the gene

[0047] splicing: The number of mutations occurring in the 4bp region near the splicing site

[0048] ncRNA_exonic: the number of mutations occurring in the exon region of non-coding RNA

[0049] ncRNA_intronic: The number of mutations occurring in the intronic region of non-coding RNA

[0050] ncRNA_splicing: The number of mutations occurring in the non-coding RNA splicing site region

[0051] Upstream: The number of mutations occurring 1KB upstream of the gene transcription start site

[0052] Downstream: The number of mutations occurring 1KB downstream of the gene transcription termination site

[0053] intergenic: the number of mutations occurring in the intergenic region

[0054] total: total number of SNVs

[0055] Figure 5 Display: Copy number variation diagram of cell line 4N-MS1. Copy number variation (CNV) is manifested as an increase or decrease in the copy number of genomic fragments. It is an important component of genomic structural variation (SV). It can be divided into two types: deletion and duplication. It is an important molecular mechanism. HNSCC cell lines use Control-FREEC software to detect Somatic CNV, and paired normal samples are used for correction. If there are no normal samples, a single tumor sample is used for detection. The results are shown in the figure. The horizontal axis represents the position on the chromosome, and the vertical axis represents the standardized copy number. In the figure, red dots indicate an increase in copy number, blue dots indicate a decrease in copy number, and green dots indicate no significant change.

[0056] Figure 6 Shown: Pictures of 4N-MS1 cells forming subcutaneous tumors and in situ tumors on the dorsum of the tongue.

[0057] Figure 7Shown: Left: tumor growth curve of 4N-MS1 orthotopic tumor formed on the dorsum of tongue (n=11 mice); Right: tumor growth curve of 4N-MS1, MB49, B16F10 subcutaneous tumor formed (n=6 mice in each group). DETAILED DESCRIPTION

[0058] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is further described in detail below in conjunction with specific embodiments.

[0059] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0060] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0061] One object of the present invention is to provide a mouse head and neck squamous cell carcinoma cell line, which is named 4N-MS1, and was deposited in Guangdong Provincial Microbiological Culture Collection Center on January 10, 2025. Its preservation number is: GDMCCNO: 65741, and the address of the preservation unit is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0062] Furthermore, the strain of the mice is C57 mice.

[0063] Another object of the present invention is to provide a method for constructing the above-mentioned mouse head phosphoblastoma cell line, comprising the following steps:

[0064] S1. Use chemical reagents to induce oral mucosal cancer in mice;

[0065] S2, surgically remove the tumor tissue and cut it into pieces, use enzyme dissociation to further cut it into pieces and prepare it into a single cell suspension, transfer the single cell suspension to a well plate for culture, and obtain primary tumor cells;

[0066] S3, purifying primary tumor cells using an enzyme digestion method to obtain purified primary head and neck squamous cell carcinoma tumor cells;

[0067] S4. Virus transfection was used to purify primary head and neck squamous cell carcinoma tumor cells to obtain a stably growing mouse head and neck squamous cell carcinoma cell line.

[0068] Further, in step S2, the cells are further sheared and prepared into a single cell suspension using enzyme dissociation, and the single cell suspension is transferred to a well plate for culture, specifically:

[0069] The tumor tissue cut and shredded during surgery was transferred to a type I collagenase solution, incubated at a constant temperature with shaking, vortexed and shaken at intervals, centrifuged, the supernatant discarded, trypsin was added, oscillated at a constant temperature, DMEM culture medium containing 8-12% fetal bovine serum was added, the suspension containing tumor cells was filtered, the filtrate was taken, centrifuged, the supernatant discarded, red blood cell lysis solution was added, incubated, centrifuged, the supernatant discarded, and the cell suspension was resuspended in DMEM culture medium containing 8-12% fetal bovine serum to obtain a cell suspension. The cells were planted on a well plate, DMEM culture medium containing 10% fetal bovine serum, epidermal growth factor and penicillin-streptomycin were added, and the well plate was placed in an incubator for culture.

[0070] Further, in step S3, the primary tumor cells are purified by enzyme digestion method as follows:

[0071] When the well-adherent cells grow to 85-90%, trypsin is added and incubated, and then DMEM medium containing 8-12% fetal bovine serum is added to allow some non-tumor cells to fall off, the liquid is aspirated, and trypsin solution is added again to digest until more than 90-95% of the cells fall off, centrifuge, discard the supernatant, and culture the cells in DMEM medium containing 4-6% FBS to obtain purified primary head and neck squamous cell carcinoma tumor cells.

[0072] Further, in step S4, the virus is transfected into purified primary head and neck squamous cell carcinoma tumor cells, specifically:

[0073] For primary tumor cells, after changing to serum-free medium and culturing for a period of time, the pGLuc-Dura-SV40-N plasmid is transfected using liposome Lipofectin2000 as a vector to obtain a plasmid-Lipo2000 complex; the prepared plasmid-Lipo2000 complex is added to the cell culture medium, shaken to make it evenly distributed, and placed in an incubator for 24 to 48 hours; after being taken out from the incubator, the transfection-positive cells are screened with antibiotics, and the transfection is detected by luciferase expression. When the cell growth rate after successful transfection is stable, the DMEM medium containing 8-12% FBS is changed to continue culturing to obtain a stably growing head and neck squamous cell carcinoma cell line.

[0074] Another object of the present invention is to provide a reagent for preparing a head and neck squamous cell carcinoma tumor model, wherein the reagent contains the above-mentioned mouse head and neck squamous cell carcinoma cell line.

[0075] Another object of the present invention is to provide a method for preparing a mouse head and neck squamous cell carcinoma transplanted tumor mouse model, wherein the mouse head and neck squamous cell carcinoma cell line or the above reagent is injected into the mouse body to induce tumor formation.

[0076] Another object of the present invention is to provide a use of the above-mentioned mouse head and neck squamous cell carcinoma cell line in preparing a head and neck squamous cell carcinoma mouse model.

[0077] Another object of the present invention is to provide a use of the above-mentioned mouse head and neck squamous cell carcinoma cell line in the preparation, screening or evaluation of drugs for treating head and neck squamous cell carcinoma.

[0078] The present invention is now further described in conjunction with specific examples. The following examples are only for explaining the present invention, but do not constitute a limitation of the present invention. The test samples and test processes used in the following examples include the following (if the specific experimental conditions are not indicated in the examples, they are usually in accordance with conventional conditions or the conditions recommended by the reagent company; the reagents, consumables, etc. used in the following examples, if not otherwise specified, can all be obtained from commercial channels).

[0079] Example 1

[0080] 1. 4-Nitroquinoline-1-oxide (4NQO) induced stable tumor formation in C57BL / 6 mice (reference Figure 1 )

[0081] 80 6-8 week old C57BL / 6 female mice (Jiangsu Jicui Pharmaceutical) were used and fed with 4NQO (Sigma, N8141) in the drinking water at a concentration of 50 mg / kg. The water bottle was protected from light with tin foil. The water was changed once a week for 12 weeks, and then the mice were fed with purified water. Starting from the 16th week of medication, two mice were randomly selected each week and anesthetized and killed to obtain tongue tissues. The mice were fixed in formalin and embedded in paraffin, sliced, and stained with HE. The staining results were observed under a light microscope to determine whether a tumor occurred.

[0082] 2. Extraction, culture, and construction of cell lines from the mouse 4NQO-induced HNSCC tumor model (reference Figure 2 )

[0083] (1) At about 28 weeks of the 4NQO-induced model, the tumor size of the tumor-bearing mice was observed after isoflurane anesthesia. Mice with faster tumor growth were selected and intraperitoneally anesthetized with tribromoethanol (Avertin, 500 ul / mouse). After the mice were anesthetized, the mouse mouth was disinfected with type III iodine three times. Then, the mouse tumor tissue was removed along the junction of the tumor and the surrounding normal tissue with sterile surgical instruments. The excised tumor tissue was placed in PBS (Phosphate buffer saline) containing 0.5% FBS (fetal bovine serum);

[0084] (2) In order to obtain tumor cells with good activity, the tumor tissue was quickly cut into pieces with ophthalmic scissors to a size of 1 mm, and then the cut tissue was transferred to 500 ul of 2 mg / ml type I collagenase (Worthington Biochemical), incubated in a 37° constant temperature shaker for 30 minutes, shaken with a vortex oscillator for 10 seconds every 10 minutes, and then centrifuged at 300g for 5 minutes, the supernatant was discarded, and 500 ul of 2% trypsin was added and incubated in a 37°C constant temperature shaker for 5 minutes. The trypsin was then neutralized with DMEM culture medium containing 10% FBS, and the tumor cell suspension was then filtered using 70 μm and 40 μm pore size filters. The cell suspension obtained after filtration was centrifuged at 300g for 5 minutes, the supernatant was discarded, and about 1 ml of red blood cell lysis buffer (Biyuntian) was added to blow away the cells. After incubation at room temperature for 10 minutes, the cells were centrifuged at 300g for 5 minutes, the supernatant was discarded, and 10% The cells were resuspended in 200ul of FBS-containing DMEM medium and counted using a cell counting plate. Subsequently, the cells were planted at a density of 200,000 cells / well on a 12-well plate, and 10% FBS-containing DMEM medium was added, supplemented with 10ng / ml epidermal growth factor (Sigma) and 1% penicillin-streptomycin, and placed in a cell culture incubator to allow tumor cells to grow and proliferate rapidly. The incubator was maintained at 37°C, 5% CO2, pH 7.2-7.4, and 95% relative saturated humidity;

[0085] (3) In order to remove fibroblasts and the like in primary cells and obtain tumor cells with higher purity, for the above-mentioned cells with good adhesion, when the cells grow to about 90%, 1 ml of 2% trypsin is added and incubated at 37°C for 1 minute, the culture bottle is gently shaken, and then 1 ml of 10% FBS DMEM medium is added to neutralize the enzyme. After confirming that some non-tumor cells have fallen off under a light microscope, the liquid in the culture bottle is sucked away, and 1 ml of 2% trypsin is added again. Digest until more than 90% of the cells have fallen off, and the cells are collected by centrifugation at 1000 rpm for 3 minutes. The cells are placed in 5% FBS DMEM medium for culture, and 20 ng / ml epidermal growth factor and 2% penicillin-streptomycin double antibody solution are added. The culture system is maintained in a culture environment of 37°C, 5% CO2, pH 7.3 and 95% relative humidity. After culture, purified primary head and neck squamous cell carcinoma tumor cells are obtained. In some embodiments of the present invention, the primary tumor cells are then cultured for 3 generations according to the above steps to obtain HNSCC tumor cells with higher purity.

[0086] (4) In order to obtain stable growth and unlimited proliferation ability similar to that of commonly used cell lines in homologous mouse transplant tumor models of other cancer types (such as MB49 cells of bladder cancer, B16 cells of melanoma, and E0771 cells of breast cancer), and to prevent the gradual decrease in cell growth rate and division ability as the number of cell divisions increases, primary tumor cells with a growth density of 80-90% were transfected with pGLuc-Dura-SV40-N plasmid (Biyuntian) using liposome Lipofectin2000 (ThermoFish) as a vector, and the cells were replaced with serum-free culture medium 1 hour before transfection. Add the prepared plasmid-Lipo2000 complexes to the cell culture medium, shake gently to make it evenly distributed, place in a 37°C 5% CO2 incubator for 24-48 hours, and replace with a complete culture medium containing 200-800μg / ml G418 (or other appropriate antibiotics); replace the fresh selection medium every 3 days and continue culturing for 10 days; observe the cell survival under an optical microscope until the non-transfected cells die completely and only the transfected positive cells remain. The surviving cells are inoculated in a 96-well plate at a density of 3×10 4 / well, culture until the cell density reaches 80%, discard the culture medium, and wash the cells 3 times with pre-cooled PBS. Add 80μl 1× luciferase lysis buffer to each well and incubate at room temperature for 15 minutes. Collect the lysate, centrifuge at 12000g for 3 minutes, take 20μl cell lysis supernatant and mix it with an equal volume of luciferase substrate, use a luciferase detector to measure the relative light unit (RLU), and use untransfected cells as negative controls. When the cell growth rate after successful transfection is stable, culture in 10% FBS DMEM culture medium, 10ng / ml epidermal growth factor and 1% penicillin-streptomycin double antibody solution. The culture system is maintained in a culture environment of 37°C, 5% CO2, pH 7.2-7.4 and 95% relative humidity. After culture, a head and neck squamous cell carcinoma cell line with stable growth and unlimited proliferation is obtained, and the cell line is named 4N-MS1.

[0087] 3. Identification and testing of mouse HNSCC cell lines

[0088] (1) Perform whole exome sequencing (Shenzhen Chengqi Biotechnology) on the HNSCC cell line 4N-MS1, calculate the exome sequencing depth and coverage, and analyze the somatic mutations of the cell line;

[0089] The results of whole exome sequencing showed that the genome of cell line 4N-MS1 had a large number of different types of somatic single nucleotide mutations (somatic SNV), and the number of each type of mutation and the total number of mutations were similar, indicating that cell line 4N-MS1 met the genomic characteristics of malignant tumor cells (reference Figure 4); In addition, the 4N-MS1 genome copy number variation map showed that this cell line had a large number of gene fragments with increased copy numbers on chromosomes 1, 6, 12, and 15 (reference Figure 5 ).

[0090] (2) In vivo tumorigenesis experiments verified that 4N-MS1 has good in vivo tumorigenesis ability:

[0091] ① In situ tumor formation experiment on the tongue dorsum of C57 mice: C57 mice of the same origin as 4N-MS1 were used to prepare 4N-MS1 cells in the logarithmic growth phase, which were diluted to 10^7 cells / ml. 50ul / mouse was injected into the tongue dorsum of the mouse using a sterile syringe, and then the tumor growth was recorded. The tumor area was used to calculate the tumor size. The results showed that all the in situ tumor models transplanted with 4N-MS1 cells into the tongue dorsum of 11 C57 mice formed normal tumors ( Figure 6 ), indicating that 4N-MS1 has a faster growth rate in homologous mice and therefore has a stronger tumor-forming ability, which provides an effective tool for the study of animal models for the treatment of head and neck squamous cell carcinoma.

[0092] ②Subcutaneous tumor formation experiment: In order to compare the tumor formation ability of 4N-MS1 with other common tumors, three C57 mouse-derived cell lines, 4N-MS1, MB49, and B16F10, were used for subcutaneous tumor formation. The number of cells injected into each mouse was 10^6, and the tumor growth was recorded. Compared with two common bladder cancer cell lines (MB49) and melanoma cell lines (B16F10), 4N-MS1 grew slower ( Figure 7 ), in animal experiments to construct tumor models, 4N-MS1 leaves more time for operation and observation of drug treatment after tumor formation.

[0093] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to be exhaustive.

[0094] Limitation of the specific implementation methods of the present invention. Any modification, equivalent substitution and improvement made within the spirit and principle of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A mouse head and neck squamous cell carcinoma cell line, characterized in that The mouse head and neck squamous cell carcinoma cell line is named 4N-MS1 and is deposited in Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO: 65741.

2. The mouse head and neck squamous cell carcinoma cell line according to claim 1, characterized in that The strain of the mice is C57 mice.

3. A method for constructing a mouse head phosphocarcinoma cell line according to claim 1, characterized in that: The following steps are involved: S1. Use chemical reagents to induce oral mucosal cancer in mice; S2, surgically remove tumor tissue and cut it into pieces, use enzyme dissociation to further cut it into pieces and prepare it into a single cell suspension, transfer the single cell suspension to a well plate for culture, and obtain primary tumor cells; S3, purifying primary tumor cells using an enzyme digestion method to obtain purified primary head and neck squamous cell carcinoma tumor cells; S4. Virus transfection was used to purify primary head and neck squamous cell carcinoma tumor cells to obtain a stably growing mouse head and neck squamous cell carcinoma cell line.

4. The method for constructing a mouse head phosphoblastoma cell line according to claim 3, characterized in that: In step S2, the cells are further sheared and prepared into a single cell suspension using enzymatic dissociation, and the single cell suspension is transferred to a well plate for culture, specifically: The tumor tissue cut and shredded during surgery was transferred to a type I collagenase solution, incubated at a constant temperature with shaking, vortexed and shaken at intervals, centrifuged, the supernatant discarded, trypsin was added, oscillated at a constant temperature, DMEM culture medium containing 8-12% fetal bovine serum was added, the suspension containing tumor cells was filtered, the filtrate was taken, centrifuged, the supernatant discarded, red blood cell lysis solution was added, incubated, centrifuged, the supernatant discarded, and the cell suspension was resuspended in DMEM culture medium containing 8-12% fetal bovine serum to obtain a cell suspension. The cells were planted on a well plate, DMEM culture medium containing 10% fetal bovine serum, epidermal growth factor and penicillin-streptomycin were added, and the well plate was placed in an incubator for culture.

5. The method for constructing a mouse head phosphoblastoma cell line according to claim 3, characterized in that: In step S3, primary tumor cells are purified by enzymatic digestion as follows: When the well-adherent cells grow to 85-90%, trypsin is added and incubated, and then DMEM culture medium containing 8-12% fetal bovine serum is added to allow some non-tumor cells to fall off, the liquid is aspirated, and trypsin solution is re-added to digest until more than 90-95% of the cells fall off, the mixture is centrifuged, the supernatant is discarded, and the cells are cultured in DMEM culture medium containing 4-6% FBS to obtain purified primary head and neck squamous cell carcinoma tumor cells.

6. The method for constructing a mouse head phosphoblastoma cell line according to claim 3, characterized in that: In step S4, the virus is transfected into purified primary head and neck squamous cell carcinoma tumor cells, specifically: For primary tumor cells, after changing to serum-free medium and culturing for a period of time, use liposome Lipofectin2000 as a vector to transfect pGLuc-Dura-SV40-N plasmid to obtain a plasmid-Lipo2000 complex; add the prepared plasmid-Lipo2000 complex to the cell culture medium, shake it to make it evenly distributed, and culture it in an incubator for 24 to 48 hours; after taking it out of the incubator, screen the transfection-positive cells with antibiotics, and detect the transfection by luciferase expression. When the cell growth rate after successful transfection is stable, change to 8-12% FBS DMEM medium and continue to culture to obtain a stably growing head and neck squamous cell carcinoma cell line.

7. A reagent for preparing a head and neck squamous cell carcinoma tumor model, characterized in that: The reagent contains the mouse head and neck squamous cell carcinoma cell line according to claim 1.

8. A method for preparing a mouse head and neck squamous cell carcinoma transplanted tumor mouse model, characterized in that: The mouse head and neck squamous cell carcinoma cell line according to claim 1 or the reagent according to claim 7 is injected into mice to induce tumor formation.

9. Use of the mouse head and neck squamous cell carcinoma cell line according to claim 1 in preparing a head and neck squamous cell carcinoma mouse model.

10. Use of the mouse head and neck squamous cell carcinoma cell line according to claim 1 in the preparation, screening or evaluation of therapeutic drugs for head and neck squamous cell carcinoma.

Images