A human osteosarcoma cell line amplified by myc and its application

Abstract

This invention discloses a myc-amplified human osteosarcoma cell line and its applications. The human osteosarcoma cell line, with accession number CCTCC NO:C2021308, was derived from clinical human osteosarcoma patients. It exhibits MYC amplification characteristics, stable in situ tumorigenesis in the tibia, and the ability to metastasize to the lungs. It can be transfected with lentiviruses and possesses gene modification capabilities. This human osteosarcoma cell line can be used to screen and develop drugs for treating osteosarcoma, construct drug-resistant osteosarcoma models, construct animal models of osteosarcoma, screen for osteosarcoma-related biomarkers, and study the mechanisms of osteosarcoma development and progression. This invention provides researchers with experimental materials for in-depth investigation of the mechanisms of human osteosarcoma development and progression, and for finding or evaluating treatment options.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a myc-amplified human osteosarcoma cell line and its applications. Background Technology

[0002] Osteosarcoma (OS) is a primary malignant bone tumor that commonly affects children and adolescents. It is highly malignant, has a high metastasis rate, and causes significant disability and mortality. It commonly occurs in the rapidly growing proximal tibia, proximal humerus, and distal femur, seriously endangering the health and lives of adolescents. With the widespread use of neoadjuvant chemotherapy combined with surgery, the five-year survival rate for osteosarcoma patients has increased to approximately 70%. However, osteosarcoma is prone to distant lung metastasis, and the five-year survival rate after lung metastasis is only 10%–20%. Osteosarcoma also easily develops chemotherapy resistance, so the development of drug treatment for it has been slow over the past two decades.

[0003] The etiology and pathogenesis of osteosarcoma are not fully understood, exhibiting complexity and heterogeneity. Genomic data from osteosarcoma patients, based on DNA copy number variation (CNV) analysis, have identified relatively common tumor-causing genes such as MYC and CDKN2A. Among these, MYC amplification occurs in approximately 50% of osteosarcoma patients and is closely related to drug resistance and metastasis. Overexpression of the MYC oncoprotein is considered a driver of poor prognosis in various human tumors, including osteosarcoma, with MYC amplification being one of the main factors contributing to this overexpression. Furthermore, MYC amplification leads to abnormal regulation of multiple signaling pathways in the tumor, thereby driving malignant tumor progression.

[0004] Therefore, identifying osteosarcoma cell lines with MYC amplification characteristics is crucial for the clinical differentiation, diagnosis, treatment selection, and follow-up of osteosarcoma. Currently, there are no osteosarcoma cell lines with MYC amplification. Therefore, isolating and culturing osteosarcoma cell lines from tumor tissues of clinically diagnosed osteosarcoma patients with MYC amplification is of significant value for studying the mechanisms of osteosarcoma development and progression.

[0005] Chinese patent document CN112080473A discloses a primary cell line for osteosarcoma lung metastases, its culture method, and its applications, used in research on the mechanism of osteosarcoma lung metastasis and anti-tumor drug research. Chinese patent document CN102154209A discloses a human osteosarcoma cell line and its applications; this cell line possesses the biological characteristics of clinical osteosarcoma and can be used in research on primary drug resistance and distant metastasis. Chinese patent document CN 102051344A discloses a group of human osteosarcoma cell lines and a mouse in vivo inhibition model, providing in vitro and in vivo experimental materials for clinical and basic research on osteosarcoma. The animal model is sensitive to commonly used chemotherapeutic drugs for osteosarcoma and can be used to simulate the outcome of osteosarcoma in clinical patients. However, no reports have been found regarding MYC-amplified osteosarcoma cell lines. Summary of the Invention

[0006] To address the aforementioned problems in the prior art, this invention provides a myc-amplified human osteosarcoma cell line and its applications.

[0007] The first aspect of the present invention is to provide a myc-amplified human osteosarcoma cell line, with accession number CCTCCNO:C2021308, classified and named human osteosarcoma cell line SGH-OS01, deposited on January 12, 2022, deposited at the China Center for Type Culture Collection, located at Wuhan University, China.

[0008] Furthermore, the aforementioned human osteosarcoma cell line exhibits MYC amplification characteristics.

[0009] A second aspect of the present invention is to provide the use of the above-described human osteosarcoma cell line, the use being selected from:

[0010] a) Screening and developing drugs for the treatment of osteosarcoma;

[0011] b) Construct a drug-resistant osteosarcoma cell tumor model;

[0012] c) Construct an animal model of osteosarcoma;

[0013] d) Constructing an animal model of osteosarcoma lung metastasis

[0014] d) Screening for osteosarcoma-related biomarkers; and

[0015] e) Study the mechanisms of osteosarcoma development and progression.

[0016] A third aspect of the present invention is to provide a method for constructing an animal model of osteosarcoma, comprising the step of injecting the above-described human osteosarcoma cell line into the tibial medullary cavity, subcutaneous tissue, or tail vein.

[0017] Furthermore, the aforementioned animals are non-human mammals.

[0018] Furthermore, the animals mentioned above were selected from rats, mice, dogs, rabbits, sheep, pigs, and monkeys.

[0019] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0020] 1. The cell line donor provided by this invention is a clinical osteosarcoma patient, and the cell line gene has been confirmed to have MYC amplification.

[0021] 2. The cell line provided by this invention has stable in situ tumorigenesis ability in the tibia, with a tumorigenesis rate of 100%.

[0022] 3. The cell line provided by this invention can be transfected with lentiviruses and has gene modification function.

[0023] 4. The cell line provided by this invention can be used to establish an animal model of osteosarcoma lung metastasis, providing experimental materials for researchers to conduct in-depth research on the pathogenesis and development mechanism of human osteosarcoma and to find or evaluate treatment options. Attached Figure Description

[0024] The myc-amplified human osteosarcoma cell line SGH-OS01 provided by this invention was deposited on January 12, 2022 at the China Center for Type Culture Collection (Wuhan University, 430072, China), with accession number CCTCCNO:C2021308 and culture name (classification name): human osteosarcoma cell line SGH-OS01.

[0025] Figure 1 It is a surgical specimen. Figure 1 A) and H&E histological staining images (Figures B and C);

[0026] Figure 2 These are microscopic images of human osteosarcoma cell lines SGH-OS01P0 generation (Figure A) and P11 generation (Figure B).

[0027] Figure 3 This is the identification result of the human osteosarcoma cell line SGH-OS01STR;

[0028] Figure 4 The figures show the growth curve (Figure A) and the colony formation results (Figure B) of the human osteosarcoma cell line SGH-OS01.

[0029] Figure 5 The images show the FISH results of the human osteosarcoma cell line SGH-OS01MYC amplification; Figure A shows the patient tissue, Figure B shows the cells from the patient's tumor tissue that have been passaged to P8, and Figure C shows the statistical graph of the FISH results.

[0030] Figure 6 The results of detecting MYC protein in the human osteosarcoma cell line SGH-OS01 are shown.

[0031] Figure 7 These are fluorescence images of human osteosarcoma cell line SGH-OS01 infected with lentivirus. In Figure A, cell morphology is shown under a regular microscope, and in Figure B, cell fluorescence is shown under a fluorescence microscope.

[0032] Figure 8 The images show the in situ tumor formation (Figure A), subcutaneous tumor formation (Figure B), and lung metastasis tumor formation (Figure C) of the human osteosarcoma cell line SGH-OS01 in the tibia of NCG mice, the MYC amplification results of in situ tumor formation (Figure D), the MYC amplification results of subcutaneous tumor formation (Figure E), and the statistical graph of FISH results (Figure F).

[0033] Figure 9 These are H&E histological staining images of the in situ tibial tumor and subcutaneous tumor models (Figures AB), and HE staining images of lung sections from the in situ tibial tumor, subcutaneous tumor, and lung metastasis tumor models (Figure CE). Detailed Implementation

[0034] This invention provides a myc-amplified human osteosarcoma cell line and its applications. The invention is described in detail below with specific examples to facilitate a better understanding; however, these examples do not limit the scope of the invention.

[0035] Example 1

[0036] In this embodiment, the human osteosarcoma cell line SGH-OS01 was constructed. The specific process is as follows:

[0037] The sample was obtained from a 15-year-old male patient with osteosarcoma. Surgical specimens and histological staining are shown in [reference needed]. Figure 1 Fresh surgical specimens were inoculated into culture dishes and primary cells were cultured using the tissue adherence method. When the cells reached approximately 80% confluence, they were passaged using trypsin digestion. The primary tumor cells were then inoculated into BNOD / SCID mice to establish a patient-derived xenograft (PDX) model. The primary tumor cells were then used for subsequent identification and other experiments.

[0038] Culture medium: DMEM high glucose medium (WISENT, 319-005-CL) containing 10% primary cell culture fetal bovine serum (WISENT, 086-550); Culture conditions: 5% CO2, saturated humidity, 37℃.

[0039] Example 2

[0040] This embodiment measures the performance of the above cell lines. The specific measurement process and results are as follows:

[0041] I. Cell line morphology observation and STR identification

[0042] Cell morphology was observed and photographed under a microscope at different growth densities in passaged cell lines. Cell morphology is as follows: Figure 2 As shown, the tumor cells are large and robust, spindle-shaped, containing a small number of irregular polygonal cells, and show no signs of aging. Human osteosarcoma cell lines passaged to P8 were compared with patient tumor tissue using STR identification (Genewiz Biotechnology, Suzhou, China, 200235). The results are as follows... Figure 3 As shown, STR analysis revealed that the cell line had a similarity of 94.4% to the patient's tumor tissue, indicating that the human osteosarcoma cell line originated from the patient's tumor tissue.

[0043] II. Cell growth curves and colony formation detection

[0044] (1) Cell growth curve

[0045] Cells from the established cell lines were passaged to P8 culture. After digestion and centrifugation to prepare a cell suspension, the cells were counted and cultured in 96-well plates at a density of 1000 cells per well. Cells were cultured overnight after adherence and monitored daily using CCK-8 reagent for 18 consecutive days to detect cell proliferation. Cell growth curves were plotted as shown below. Figure 4 As shown in Figure A, the cells grew well, and by day 17, the cell density in the culture wells reached saturation.

[0046] (2) Cell clone formation

[0047] Cells passaged to P8 were collected, digested, centrifuged, and resuspended. Cells were counted and cultured in 6-well plates at concentrations of 100, 200, and 500 cells per well for two weeks. After culture, the supernatant was discarded, and cells were washed twice with PBS. 1 ml of 4% paraformaldehyde was added to each well to fix the cells for 10 min. The fixative was removed, and cells were stained with crystal violet for 10 min. The staining solution was then slowly washed away with running water, and the cells were air-dried. Images were taken using an inverted microscope. The results are shown below. Figure 4 B. It has strong cell proliferation ability and good clonal formation ability.

[0048] III. MYC Amplification FISH Identification

[0049] Fluorescently labeled specific nucleic acid probes hybridize with intracellular target genes, and fluorescence signals are observed using fluorescence microscopy to determine the gene localization of the fluorescent probe in the sample, as well as to quantify the copy number of the target gene and the control gene on chromosome 8 (CEP8). Fluorescence in situ hybridization (FISH) is used to detect MYC amplification in passaged cell lines and patient tissue samples, employing a probe specifically targeting the target gene MYC(8q24) and a control probe targeting CEP8.

[0050] The FISH experiment steps are as follows:

[0051] (1) Dewaxing

[0052] ① Dewaxing with xylene three times, 5 minutes each time;

[0053] ② Wash three times with anhydrous ethanol, 2 minutes each time;

[0054] ③ Remove ethanol, then dry in a 60℃ oven.

[0055] (2) Protease treatment

[0056] ① Place in 2×SSC at 37℃ for 1 hour;

[0057] ② Place in freshly prepared protease digestion solution at 37°C for 20 minutes;

[0058] ③ Rinse the sections three times with 2×SSC at room temperature, 1 min each time;

[0059] ④ Gradient alcohol dehydration.

[0060] (3) Transformation

[0061] ① The specimen is placed in a denaturing solution at 75℃ for 2 minutes to denature;

[0062] ② Dehydrate the water sequentially in pre-cooled gradient alcohol solutions at 70%, 90%, and 100%, for 5 minutes each time;

[0063] ③ The air is dry.

[0064] (4) Hybridization

[0065] ① Prepare a two-color fluorescent probe;

[0066] ② Take the humidification box and place the slices inside;

[0067] ③ Place 10 μl of probe onto the tissue section and cover with a coverslip;

[0068] ④ Cover with a humidified container and incubate at 37°C for 16 hours.

[0069] (5) Washing

[0070] ① Carefully remove the coverslip with tweezers;

[0071] ② The slide specimen was washed three times with 50% formamide / 2×SSC preheated at 45℃, 5 min each time;

[0072] ③ Wash three times in 1×SSC preheated to 42-50℃, 5 minutes each time;

[0073] ④ At room temperature, gently wash the slide in 2×SSC, remove it, and let it air dry.

[0074] ⑤ Add 200 μl of DAPI staining solution to the slide specimen and cover with a slide.

[0075] (6) Mounting slides and observing FISH results under a fluorescence microscope

[0076] ① Seal the slide with mounting solution and store it in a dark box.

[0077] ② Observe the results under a fluorescence microscope and take pictures.

[0078] See results Figure 5 A MYC / CEP8 ratio ≥ 2.0 indicates MYC amplification, with the MYC to CEP8 copy number ratio significantly greater than 2. Therefore, consistent with the FISH test results of patient tissue, this human osteosarcoma cell line SGH-OS01 exhibits MYC amplification and retains the MYC amplification of the patient's tumor.

[0079] IV. Slow Viral Infection

[0080] Passaged cell lines were infected with lentivirus containing shRNA-MYC, and cellular susceptibility to the virus was assessed. The experimental procedures are as follows:

[0081] (1) Human osteosarcoma cell line SGH-OS01 cells were cultured in 6-well plates;

[0082] (2) When the cells are growing vigorously and reach 40% confluence, add the collected and filtered concentrated virus stock solution and infect at a ratio of 50 μl / well.

[0083] (3) Replace with fresh culture medium after 24 hours and continue culturing;

[0084] (4) Since the viral vector carrying the puromycin marker and the successfully infected cells carrying puromycin resistance, puromycin (1 μg / ml) was added for drug screening 48 hours later. After 48 hours, a large number of cells were observed to be alive under a microscope, while almost all the cells in the wells without the virus were dead, indicating that the virus transfection was successful;

[0085] (5) The above-mentioned recombinant cell line was passaged and cultured, and histones were extracted from the cells to identify whether MYC was successfully knocked out.

[0086] The results are as follows Figure 6 , Figure 7 As shown, compared with the Mock and shRNA-MYC controls, MYC expression was significantly reduced, indicating successful knockdown of MYC. This suggests that the human osteosarcoma cell line SGH-OS01 is easily transfected by lentiviruses and has good gene modification potential and applications.

[0087] Example 3

[0088] This embodiment constructs an NCG mouse tumor model and identifies its histological morphology. The specific construction process and identification results are as follows:

[0089] 1. Establishment of an in situ tumor model of NCG in rat tibia

[0090] The experimental steps are as follows:

[0091] (1) Human osteosarcoma cell line SGH-OS01 was cultured in vitro. After the cells reached the logarithmic growth phase, the cells were digested into a single-cell suspension, counted, and then prepared into 1×10⁶ cells with PBS. 8 cells / ml of cell suspension;

[0092] (2) Inoculate 10 μl of cell suspension into the tibial bone marrow cavity of mice using a microsyringe and record the weight of mice regularly;

[0093] (3) When the longest diameter of the tumor approaches 20 mm or the mouse dies, the experiment is terminated, the experimental mouse is euthanized, the thigh of the mouse bearing the tumor is removed, weighed, and the above tissue is fixed with formalin.

[0094] Please refer to the images of NCG mice and tumors. Figure 8 and Figure 9 , Figure 8 A is a photograph of NCG-carrying mice with tumors. 1×10 6 When the personal osteosarcoma cell line SGH-OS01 was inoculated into the tibial bone marrow cavity of NCG mice, a solid tumor grew at the inoculation site, and the tumor volume exceeded 1000 mm³ after 30 days. 3 This demonstrates that the human osteosarcoma cell line SGH-OS01 has excellent tumorigenic capacity in the tibia of NCG mice. Therefore, the human osteosarcoma cell line SGH-OS01 possesses excellent in situ tumorigenic capacity in the tibia of NCG mice, and its growth is relatively stable, making it suitable for in vivo studies in mice.

[0095] 2. Establishment of an NCG-induced subcutaneous tumor model in mice

[0096] The experimental steps are as follows:

[0097] (1) Human osteosarcoma cell line SGH-OS01 was cultured in vitro. After the cells reached the logarithmic growth phase, the cells were digested into a single-cell suspension, counted, and then prepared into 1×10⁶ cells with PBS. 7 cells / ml of cell suspension;

[0098] (2) Inoculate 100 μl of cell suspension into mice subcutaneously with a microsyringe and record the weight of the mice regularly;

[0099] (3) When the longest diameter of the tumor approaches 20 mm or the mouse dies, the experiment is terminated, the experimental mouse is euthanized, the subcutaneous tumor of the mouse is removed, weighed, and the above tissue is fixed with formalin.

[0100] Please refer to the images of NCG mice and tumors. Figure 8 AC. 1×10 6 When the personal osteosarcoma cell line SGH-OS01 was subcutaneously inoculated into NCG mice, solid tumors grew at the inoculation sites, and the tumor volume exceeded 1000 mm² after 30 days. 3 This demonstrates that the human osteosarcoma cell line SGH-OS01 has excellent tumorigenic ability in the subcutaneous tissue of NCG mice. Therefore, the human osteosarcoma cell line SGH-OS01 possesses excellent subcutaneous tumorigenic ability in NCG mice, and its growth is relatively stable, making it suitable for in vivo studies in mice.

[0101] 3. Establishment of an NCG-induced lung metastasis tumor model in mice

[0102] The experimental steps are as follows:

[0103] (1) Human osteosarcoma cell line SGH-OS01 was cultured in vitro. After the cells reached the logarithmic growth phase, the cells were digested into a single-cell suspension, counted, and then prepared into 1×10⁶ cells with PBS. 7 cells / ml of cell suspension;

[0104] (2) Inoculate 100 μl of cell suspension into the tail vein of mice using a microsyringe and record the weight of mice regularly;

[0105] (3) When mice show obvious emaciation or die, terminate the experiment, euthanize the mice, remove the lung tissue, weigh it, and fix the tissue with formalin.

[0106] Please refer to the images of NCG mice and tumors. Figure 8 , Figure 9 , Figure 8 C is a photograph of NCG-positive mice with tumors. 1×10 6 When the personal osteosarcoma cell line SGH-OS01 was subcutaneously inoculated into NCG mice, solid tumors grew at the inoculation sites, and the tumor volume exceeded 1000 mm² after 30 days. 3 This demonstrates that the human osteosarcoma cell line SGH-OS01 has excellent tumorigenic ability in the subcutaneous tissue of NCG mice. Therefore, the human osteosarcoma cell line SGH-OS01 possesses excellent subcutaneous tumorigenic ability in NCG mice, and its growth is relatively stable, making it suitable for in vivo studies in mice.

[0107] 4. Histological morphology and MYC amplification identification of NCG in situ tumorigenesis model in mice

[0108] Tumor tissue sections obtained from the in vitro cultured human osteosarcoma cell line SGH-OS01 and from NCG mouse tibial orthotopic transplantation and subcutaneous implantation models were subjected to HE staining and MYC amplification identification, respectively. These sections were then compared with clinical patient tumor tissue and PDX tumor tissue for histological morphology and MYC amplification identification using corresponding HE staining. The MYC amplification detection method (FISH detection experimental steps) is as described above, and the staining steps are as follows:

[0109] (1) HE staining

[0110] ① Digest adherent human osteosarcoma cell line SGH-OS01 cells with trypsin to adjust the cell concentration to approximately 1×10⁻⁶. 5 / ml was added to a 6-well plate, and after incubation for the corresponding time, the cell slides were removed and washed three times with PBS.

[0111] ② Fix with 95% ethanol for 20 min, then wash twice with PBS for 1 min each time.

[0112] ③ Stain with hematoxylin solution for 2-3 minutes, then wash with tap water.

[0113] ④ Observe under a microscope. If the cell nuclei are stained too darkly, separate the colors with 1% hydrochloric acid alcohol solution for a few seconds, and then wash with tap water.

[0114] ⑤ Immerse in eosin staining solution for 1 minute, then wash with tap water.

[0115] ⑥ Dry the stem cells by blowing or air-drying them naturally. After mounting the slides, mount them with neutral resin and take pictures with an inverted microscope.

[0116] (2) Immunohistochemical staining

[0117] ① Tissue fixation, dehydration, embedding, and sectioning

[0118] a. Fix the tumor tissue overnight at room temperature in 10% formalin solution, or store it at -4°C;

[0119] b. Dehydrate to wax using a gradient dehydration method: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 95% ethanol for 1 hour, 100% ethanol for 1 hour × 2 times, xylene for 1 hour × 2 times, wax impregnation > 2 hours;

[0120] c. Cut the tissue into a paraffin block as required for the experiment using a preheated embedding apparatus, and place it on ice to cool;

[0121] d. Pre-cool the paraffin block at -8℃, cut it into 4μm thick sections using a Leica tissue sectioner, spread the sections in 43℃ hot water until completely flat, remove the sections to prevent them from falling off, and then bake them in a 60℃ oven for >2h.

[0122] ②Section dewaxing and antibody repair

[0123] a. Preheat sections at 60℃ and dewax them in a gradient to water: xylene 10 min × 2 times, 100% ethanol 5 min × 2 times, 95% ethanol 5 min, 85% ethanol 5 min, 75% ethanol 5 min, PBS 5 min × 3 times.

[0124] b. Immerse the dehydrated sections in 0.01M citrate buffer, microwave on high until boiling, turn off the microwave, cool for 5 minutes, repeat this process 3 times for antigen retrieval, and finally cool naturally to room temperature along with the citrate buffer, and wash with PBS for 5 minutes × 3 times.

[0125] ③ Antibody incubation and DAB color development

[0126] a. Add 5% BSA to the tissue section and incubate at room temperature for 30 minutes;

[0127] b. Prepare primary antibody with 5% BSA, remove the blocking solution, and incubate the primary antibody at 4°C overnight;

[0128] c. Wash with PBS for 5 min × 3 times;

[0129] d. Incubate with the corresponding HRP-labeled secondary antibody at 37°C for 2 hours;

[0130] e. Wash with PBS for 5 min × 3 times;

[0131] f. Add the prepared DAB colorimetric solution, observe the colorimetric reaction under a microscope, and rinse with running water to stop the colorimetric reaction;

[0132] g. Remove residual liquid, add hematoxylin for counterstaining, differentiate with hydrochloric acid and alcohol, and soak in running water for 30 minutes for 8-inverse blue staining;

[0133] h. Dehydrate to xylene in a gradient: 75% ethanol for 5 min, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 5 min × 2 times, xylene for 10 min × 2 times;

[0134] i. Preserve by sealing with neutral resin.

[0135] ④ Slice photography and analysis

[0136] The slides were observed using a Leica inverted photographic microscope, and tumor tissue was photographed at 400×.

[0137] See relevant results Figure 8 DF and Figure 9 PDX tumor tissue is a patient-derived xenograft (PDX), which is obtained by transplanting a patient's tumor tissue into mice in tissue form, thus maintaining the heterogeneity of the tumor. Figure 8 D and Figure 8E represents the detection results of PDX in situ tumor tissue and lung metastasis tissue, respectively, and the statistical results are as follows ( Figure 8 F) shows that the copy number ratio of MYC to CEP8 is significantly greater than 2, indicating that both have MYC amplification ability, thus the result indicates that PDX has MYC amplification ability.

[0138] Depend on Figure 9 Immunohistochemical staining results showed that the tumor cells in both groups of sections were small and round, with a large nuclear-cytoplasmic ratio and obvious mitotic figures. Furthermore, there was no clear boundary between the tumor tissue and the stromal components, indicating that the tumor may be highly invasive.

[0139] In addition, we have established an orthotopic tumor model in 58 NCG mice using the human osteosarcoma cell line SGH-OS01, with a tumor formation rate of 100%.

[0140] In summary, the human osteosarcoma cell line SGH-OS01 provided by this invention is a clinical osteosarcoma patient as a donor. Moreover, this cell line has been shown to have MYC amplification characteristics in vitro, stable in situ tumor formation in the tibia of NCG mice, and the in situ tumor of the tibia of NCG mice has the ability to form plate cell clones. It can serve as experimental material for researchers to conduct in-depth research on the pathogenesis and development mechanism of human osteosarcoma and to find or evaluate treatment options.

[0141] The specific embodiments of the present invention have been described in detail above, but they are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A human osteosarcoma cell line amplified by myc, characterized in that, The accession number is CCTCC NO:C2021308.

2. The use of the human osteosarcoma cell line as described in claim 1, characterized in that, Selected from: a) Screening and developing drugs for the treatment of osteosarcoma; b) Construct a drug-resistant osteosarcoma cell model; c) Constructing an animal model of osteosarcoma; and d) Screening for osteosarcoma-related biomarkers.

3. A method for constructing an animal model of osteosarcoma, characterized in that, The procedure includes the step of injecting the human osteosarcoma cell line as described in claim 1 into the tibial medullary cavity, subcutaneous tissue, or tail vein.

4. The method according to claim 3, characterized in that The animal in question is an NCG mouse.

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