Human malignant bone giant cytoma cell line and application thereof

Abstract

The invention relates to the technical field of tumor biology, and particularly discloses a human malignant bone cytomegaloma cell line and application thereof.The human malignant bone cytomegaloma cell line is preserved on April 9, 2025, the preservation address is China Center for Type Culture Collection, the human malignant bone cytomegaloma cell line is located in Wuhan University, Wuhan, Hubei province, and the preservation number is CCTCC NO: C202539. The human malignant bone cytomegaloma cell line disclosed by the invention comes from tumor tissues of a Chinese malignant bone cytomegaloma patient after operation excision, and is suitable for being used as a cell material for research and development of malignant bone cytomegaloma related immunotherapy, diagnosis and medicine research and development. The cell line provided by the invention contains H3F3A G34W mutation, and has high sensitivity and specificity in diagnosis of malignant bone cytomegaloma. The cell line can be used as a tool cell for targeted drug research taking H3F3A as a target, a cell model is provided for research of malignant bone cytomegaloma of Chinese people, and an experimental platform is also provided for mechanism research of malignant bone cytomegaloma.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tumor biology, in particular, to a human malignant giant cell tumor of bone cell line and application thereof. BACKGROUND

[0002] Malignant giant cell tumor of bone is very rare, accounting for less than 10% of all giant cell tumors of bone. According to the difference of clinical background, malignant giant cell tumor of bone can be divided into primary and secondary two types, the former refers to the initial diagnosis of classic giant cell tumor and high-grade sarcoma component, the latter refers to the malignant transformation or recurrence of high-grade sarcoma at the primary site after surgical or radiotherapy intervention. The prognosis of malignant giant cell tumor of bone is similar to that of high-grade sarcoma, and the prognosis of secondary malignant subtype is usually worse than that of primary malignant subtype. At present, there is no standard treatment for malignant giant cell tumor of bone, and the treatment mode of high-grade sarcoma is usually adopted, that is, the comprehensive strategy of preoperative chemotherapy-surgery-postoperative chemotherapy. Especially for malignant giant cell tumor of bone with osteosarcoma phenotype, osteosarcoma chemotherapy regimen is usually adopted and extensive surgical resection is performed. However, because of low incidence, lack of effective comprehensive treatment means, the recurrence rate and mortality rate of the disease are very high. At the molecular diagnosis level, the monoclonal antibody targeting the histone H3.3 G34W epitope has high sensitivity and specificity in the diagnosis of giant cell tumor of bone and malignant giant cell tumor of bone. H3F3A gene G34W mutation accounts for 97.8% of GCTB cases. H3F3A gene mutation can be detected by immunohistochemistry or molecular biology methods, and is usually retained in malignant cell population. Therefore, in-depth study of the molecular pathogenesis of malignant giant cell tumor of bone and research of drugs targeting H3F3A may have great prospect for the treatment of malignant giant cell tumor of bone. At present, there is no widely used and standardized malignant giant cell tumor of bone cell line at home and abroad. This significantly limits the ability to study the malignant mechanism and develop targeted therapy for these patients.

[0003] Therefore, it is urgent to find a method for culturing malignant giant cell tumor of bone cells, establish a mature malignant giant cell tumor of bone cell line and corresponding drug research in current clinical research. This not only helps to fully reveal the biological diversity of the disease, but also provides more representative and widely used research resources for optimizing treatment plan and developing personalized treatment. SUMMARY

[0004] In view of this, the present application provides a human malignant giant cell tumor of bone cell line and application thereof, aiming at solving the problem that there is no widely used and standardized malignant giant cell tumor of bone cell line at home and abroad.

[0005] In one aspect, the present application provides a human malignant giant cell tumor of bone cell line, which is preserved on April 9, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, with a preservation number of CCTCC NO: C202539, and is named as human malignant giant cell tumor of bone cell line MGCTB-CZ1.

[0006] Further, the human malignant giant cell tumor of bone cell line MGCTB-CZ1 comprises a H3F3A G34W mutation, and can be continuously subcultured in vitro while maintaining the characteristics of malignant giant cell tumor of bone tumor cells unchanged.

[0007] Further, the construction method of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 is as follows: sampling the tumor tissue of the surgically resected malignant giant cell tumor of bone to obtain a tumor tissue sample; after pretreatment of the tumor tissue sample, adding a DMEM / F12 medium containing 5% fetal bovine serum and a tissue digestion solution, and digesting at 37°C for 1 hour to obtain a suspension; filtering the suspension through 70μm and 40μm cell screens in sequence, centrifuging the filtered suspension at 800 rpm for 5 minutes, collecting the precipitate to obtain tumor cells, resuspending the tumor cells with a tumor cell culture medium, and culturing at 37°C in a 5% CO2 atmosphere, during which the tumor cell culture medium is replaced every 2-3 days; when the cell density reaches between 80%-90%, the supernatant is aspirated, washed twice with DPBS, and digestion enzyme is added for digestion at 37°C for 3 minutes, then the tumor cell culture medium is added to terminate digestion, followed by centrifugation at 800 rpm for 5 min to aspirate the supernatant, resuspending the cells with the tumor culture medium, and then subculturing at 1:2.

[0008] Further, the pretreatment is to flush the tumor tissue sample twice with a tissue flushing solution, remove fibrous connective tissue, fat and necrotic tissue, and then cut it into granules.

[0009] Further, the tissue digestion solution is 1mg / ml type II collagenase.

[0010] Further, the configuration method of the tumor cell culture medium is as follows: in a DMEM / F12 basic medium system, 15% fetal bovine serum and 1% penicillin-streptomycin double antibody are added and mixed.

[0011] Further, the configuration method of the tissue flushing solution is as follows: adding 2% double antibody solution to HBSS buffer solution, mixing to obtain the tissue flushing solution.

[0012] Further, the configuration method of the digestive enzyme is: The trypsin containing 0.25% EDTA is added into PBS for dilution, to a final concentration of 0.05%.

[0013] In another aspect, the present application also provides a use of the human malignant giant cell tumor cell line in preparation of a cell model for studying the mechanism of malignant giant cell tumor.

[0014] In another aspect, the present application also provides a use of the human malignant giant cell tumor cell line in preparation of a malignant giant cell tumor diagnostic reagent or diagnostic kit.

[0015] Compared with the prior art, the present application has the beneficial effects that the human malignant giant cell tumor cell line MGCTB-CZ1 provided by the present application is a human single cell line, contains H3F3A G34W mutation, can be continuously subcultured in vitro while keeping the characteristics of malignant giant cell tumor tumor cells unchanged, and is suitable for being used as a cell material for researching and developing malignant giant cell tumor related immunotherapy, diagnosis and drug development. Moreover, the human malignant giant cell tumor cell line MGCTB-CZ1 provided by the present application contains H3F3A G34W mutation, and the mutation is considered to have the effect of driving the occurrence of malignant giant cell tumor and promoting tumor cell proliferation. Therefore, the cell line can be used as a tool cell for researching target drugs with H3F3A as a target. BRIEF DESCRIPTION OF DRAWINGS

[0016] Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The accompanying drawings are included to provide a description of preferred embodiments, and are not intended to limit the scope of the application. Furthermore, like reference numerals are intended to refer to like parts throughout the drawings. In the drawings: Figure 1 The morphological chart of different passages of the light microscope human malignant giant cell tumor cell line MGCTB-CZ1 cell provided by the embodiment of the present application is shown.

[0017] Figure 2 The HE staining chart of the human malignant giant cell tumor cell line MGCTB-CZ1 provided by the embodiment of the present application is shown.

[0018] Figure 3 The immunohistochemical H3.3G34W staining chart of the human malignant giant cell tumor cell line MGCTB-CZ1 provided by the embodiment of the present application is shown.

[0019] Figure 4 The cell growth curve chart of the human malignant giant cell tumor cell line MGCTB-CZ1 provided by the embodiment of the present application is shown. DETAILED DESCRIPTION

[0020] Exemplary embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it is understood that the present disclosure can be embodied in various forms and should not be limited by the embodiments set forth herein. Rather, these embodiments are provided so that the present disclosure can be thoroughly understood and fully conveyed to those skilled in the art. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the embodiments.

[0021] In some embodiments of the present application, a human malignant giant cell tumor cell line MGCTB-CZ Homosapiens is deposited on April 9, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, with the accession number CCTCC NO: C202539, and is classified and named as human malignant giant cell tumor cell line MGCTB-CZ1.

[0022] In some embodiments of the present application, the human malignant giant cell tumor cell line MGCTB-CZ1 contains a H3F3AG34W mutation and can be continuously subcultured in vitro while maintaining the characteristics of malignant giant cell tumor cells.

[0023] Specifically, the human malignant giant cell tumor cell line MGCTB-CZ1 of the present application is derived from a patient with malignant giant cell tumor (the patient is a 31-year-old male), and the tumor is located in the proximal right femur. Postoperative pathology shows that the malignant giant cell tumor has recurred. The tumor tissue sample after surgical resection is successfully cultured and established by primary culture.

[0024] Specifically, the human malignant giant cell tumor cell line MGCTB-CZ1 of the present application has vigorous cell growth, most of the cells are irregular polygons, a small part of the cells are spindle-shaped, round or oval, and the cell nucleus is obvious. Under high-density culture conditions, cell aggregation is observed, showing the malignant characteristics of losing contact inhibition. MGCTB-CZ1 cells grow in a typical epithelioid form. After adhering, the growth is fast, and has good in vitro culture amplification.

[0025] Specifically, the present inventors compared the STR sequencing results of the human malignant giant cell tumor cell line MGCTB-CZ1 with the databases of cell preservation libraries such as ATCC and DSMZ, and found no identical STR detection results, thereby confirming that MGCTB-CZ1 is a human single cell line from the same tissue or individual, and that no cross-contamination with other cell lines occurred during the primary culture process. Exon sequencing results show that the human malignant giant cell tumor cell line MGCTB-CZ1 contains the H3F3A G34W mutation.

[0026] The human malignant giant cell tumor cell line MGCTB-CZ1 of the present application is derived from the tumor tissue of a Chinese patient with malignant giant cell tumor after surgical resection. The STR detection results prove its uniqueness, and it can be continuously subcultured in vitro while maintaining the characteristics of malignant giant cell tumor cancer cells unchanged, and is suitable as a cell material for the research and development of malignant giant cell tumor related immunotherapy, diagnosis and drug development. The cell line of the present application contains the H3F3A G34W mutation, which is believed to have a role in driving the occurrence of malignant giant cell tumor and promoting tumor cell proliferation. Therefore, this cell line can be used as a tool cell for the research of targeted drugs targeting H3F3A, providing a new cell model for the study of Chinese malignant giant cell tumor, and providing a better platform for the mechanism research of human malignant giant cell tumor.

[0027] In some embodiments of the present application, a human malignant giant cell tumor cell line, the construction method of the human malignant giant cell tumor cell line MGCTB-CZ1 is: The tumor tissue of the malignant giant cell tumor after surgical resection is sampled to obtain a tumor tissue sample; After pretreatment of the tumor tissue sample, 5% fetal bovine serum-containing DMEM / F12 medium and tissue digestion solution are added, and digestion is carried out at 37°C for 1 hour to obtain a suspension; The suspension is filtered through 70μm and 40μm cell screens in sequence, the filtered suspension is centrifuged at 800 rpm for 5 minutes, the precipitate is collected, the tumor cells are resuspended using tumor cell culture medium, and the tumor cells are cultured at 37°C in a 5% CO2 atmosphere, during which the tumor cell culture medium is replaced every 2-3 days; When the cell density reaches between 80%-90%, the supernatant is aspirated, washed twice with DPBS, digestion enzymes are added, and digestion is carried out at 37°C for 3 minutes, then tumor cell culture medium is added to stop digestion, followed by centrifugation at 800 rpm for 5 min, aspiration of the supernatant, resuspension of the cells with tumor culture medium, and 1:2 subculture.

[0028] Specifically, the raw materials used in the construction method are all commercially available products.

[0029] Specifically, the tumor tissue of the malignant giant cell tumor of bone is derived from a patient with malignant giant cell tumor of bone (the patient is a 31-year-old male), and the tumor is located in the proximal femur. Postoperative pathology shows recurrence of malignant giant cell tumor of bone.

[0030] In some embodiments of the present application, the pretreatment is to rinse the tumor tissue sample twice with a tissue flushing solution, remove fibrous connective tissue, fat and necrotic tissue, and then cut it into granules.

[0031] Specifically, the tumor tissue sample is washed twice with a tissue flushing solution in a sterile clean bench, fibrous connective tissue, fat and necrotic tissue are removed, and the tumor tissue sample is cut into uniform granules close to 1 mm x 1 mm x 1 mm in a 6-well plate with a sterilized ophthalmic scissors.

[0032] In some embodiments of the present application, the tissue digestion solution is 1 mg / ml type II collagenase.

[0033] In some embodiments of the present application, the tumor cell culture medium is prepared by mixing 15% fetal bovine serum and 1% penicillin-streptomycin double antibody in a DMEM / F12 basic culture medium system.

[0034] In some embodiments of the present application, the tissue flushing solution is prepared by: Add 2% double antibody solution to HBSS buffer, mix, and obtain the tissue flushing solution.

[0035] In some embodiments of the present application, the digestion enzyme is prepared by: Add trypsin containing 0.25% EDTA to PBS for dilution to a final concentration of 0.05%.

[0036] On the other hand, in some embodiments of the present application, a human malignant giant cell tumor of bone cell line is also provided for use in preparing a cell model for studying the mechanism of malignant giant cell tumor of bone.

[0037] Specifically, the human malignant giant cell tumor of bone cell line MGCTB-CZ1 is compared with the peripheral blood from the same patient, and the differences in the transcriptome, epigenetic group and proteomics of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 are analyzed. The expression of genes and signal pathways in the human malignant giant cell tumor of bone cell line MGCTB-CZ1 is regulated by various technical means (such as CRISPR-Cas9, lentivirus transfection and inhibitor treatment), and the changes in the morphology and function of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 are detected. The tumor size, survival time and tumor metastasis of the two groups of animals are observed by inoculating cells in immunodeficient animals, and the molecular mechanisms regulating the occurrence and development of human malignant giant cell tumor of bone are found.

[0038] It can be understood that the human malignant giant cell tumor cell line MGCTB-CZ1 of the present application is established from a Chinese source, and has stable properties and a short establishment time. Using the malignant giant cell tumor cell line as a research model, by analyzing the cell morphology, function and genetic information of the cell line, it is very helpful to understand the mechanism of primary malignant giant cell tumor in Chinese people.

[0039] In another aspect, in some embodiments of the present application, the use of the human malignant giant cell tumor cell line in the preparation of a malignant giant cell tumor diagnostic reagent or diagnostic kit is also provided.

[0040] Specifically, since the human malignant giant cell tumor has specific tumor markers, a diagnostic reagent or diagnostic kit for detecting the occurrence or development of malignant giant cell tumor can be developed according to the tumor markers.

[0041] Specifically, by comparing the human malignant giant cell tumor cell line with normal osteoblasts and other types of cancer cells, analyzing the differences in transcriptome, epigenetic group and proteomics, etc., specific molecular markers of malignant giant cell tumor can be found, and the subsequent disease detection and drug development, etc. can be applied to the molecular markers.

[0042] Specifically, after finding the specific tumor markers of malignant giant cell tumor, a diagnostic reagent or diagnostic kit for detecting the occurrence or development of malignant giant cell tumor can be developed according to the tumor markers. By detecting the expression of the tumor markers in human peripheral blood, sputum or biopsy tissue, the occurrence and development of malignant giant cell tumor can be diagnosed, which can provide guidance and basis for clinical treatment.

[0043] Specifically, first, by adding different drugs to the culture medium of the human malignant giant cell tumor cell line, the cell state and functional changes are detected to obtain effective candidate drugs targeting human malignant giant cell tumor. The human malignant giant cell tumor cell line can be used as a tool for preclinical research of human malignant giant cell tumor drug treatment.

[0044] Example 1 S1, prepare tissue digestion solution: the tissue digestion solution is 1 mg / ml type II collagenase; S2, prepare tumor cell culture medium: in the DMEM / F12 basic culture medium system, supplement 15% fetal bovine serum and 1% penicillin-streptomycin double antibody, and mix thoroughly; S3, prepare tissue washing solution: add 2% double antibody solution to HBSS buffer, mix uniformly to prepare tissue washing solution; S4, preparation of digestive enzyme: 100 ml trypsin containing 0.25% EDTA was added to 20 ml PBS for dilution to obtain a final concentration of 0.05% digestive enzyme, and then was divided and stored in a refrigerator at -20°C for standby; S5, sample processing: tumor tissue after surgery from a case of malignant giant cell tumor of bone (the patient was a 31-year-old male, the tumor was located in the proximal femur, and postoperative pathology showed recurrence of malignant giant cell tumor of bone) was sampled, the sample was transferred to the laboratory from an ice bath box as soon as possible, the tumor tissue sample was washed twice with tissue flushing solution in a sterile clean bench, the fibrous connective tissue, fat and necrotic tissue were removed, the tumor tissue sample was cut into uniform granules close to 1 mm x 1 mm x 1 mm with a sterile ophthalmic scissors in a 6-well plate, and 9 mL of DMEM / F12 culture medium containing 5% fetal bovine serum and 1 mL of tissue digestion solution were added and placed in a 37°C incubator for digestion. After 1 hour, the suspension was filtered through 70 μm and 40 μm cell screens in turn, and the filtered suspension was centrifuged at 800 rpm for 5 minutes, the precipitate was collected, and the cells were resuspended with tumor cell culture medium and inoculated into a T25 culture flask, which was placed in a 37°C 5% CO2 incubator for culture.

[0045] S6, cell passage: tumor cell culture medium was changed every 2-3 days, when the cell density reached 80%-90%, the supernatant was aspirated, washed twice with DPBS, 1 mL of digestive enzyme was added, and the cells were digested at 37°C for 3 minutes, then the digestion was stopped by adding tumor cell culture medium, followed by centrifugation at 800 rpm for 5 min, then the supernatant was aspirated, the cells were resuspended with tumor culture medium, and then were passaged at a ratio of 1:2.

[0046] During the passage culture, the fibroblasts were continuously removed by differential digestion combined with differential adhesion method, taking advantage of the different sensitivity of fibroblasts and tumor cells to trypsin and the different adhesion ability of different types of cells to culture medium and solid surface. After multiple passages, fibroblasts could not be observed by naked eye in the culture dish, only single stromal cells remained and could continue to grow and pass. The in vitro culture time has exceeded half a year, and the cell number has exceeded 100 generations. After the cell line was successfully established, it was named human malignant giant cell tumor cell line MGCTB-CZ1.

[0047] Effect detection 1. The growth of the human malignant giant cell tumor cell line MGCTB-CZ1 cells in Example 1 was observed directly under an optical microscope.

[0048] As Figure 1 shown, Figure 1Figure 1 shows the morphological patterns of human malignant giant cell tumor of bone cell line MGCTB-CZ1 cells of different passages under light microscopy, wherein (a) p5; (b) p10; (c) p15; (d) p20; (e) p50; (f) p100, and the scale bars are all 100 μm.

[0049] It can be seen that the human malignant giant cell tumor of bone cell line MGCTB-CZ1 cells grow vigorously, and the MGCTB-CZ1 cells grow in a typical epithelioid form. Most of the cells are irregular polygons, and a small number of cells are spindle-shaped, round or oval, and the cell nuclei are obvious. Each generation of cells maintains vigorous metabolic activity, and the cell morphology is stable. Even when the cells are subcultured to the 20th passage, the morphology and growth rate of the cells remain unchanged. Under high-density culture conditions, cell aggregation is observed, showing the malignant characteristics of loss of contact inhibition.

[0050] Subsequently, HE staining was performed on the human malignant giant cell tumor of bone cells using an HE staining kit. After the cells adhered to the slides, the staining was performed strictly according to the standard operating parameters. First, the cell slides were washed with distilled water twice for 2 minutes each, and the samples were subjected to hematoxylin nuclear staining (5 minutes), hydrochloric acid ethanol rapid differentiation (30 seconds), and eosin cytoplasmic re-staining (120 seconds) in sequence, followed by gradient alcohol dehydration and xylene clearing treatment, and finally resin mounting and image acquisition. Figure 2 As shown in Figure 2, it is observed that the cells exhibit typical characteristics of malignant tumors, and the cell morphology is different, showing spindle-shaped, short spindle-shaped, and polygonal, and the cell nuclei are round or oval. The cell nuclei are round or oval, and part of the cell nuclei show a curved shape, and a small number of cell nuclei can be seen. In addition, the cell nuclei exhibit atypical proliferation characteristics, such as uneven aggregation of chromatin, roughness, thickening of the nuclear membrane, and the presence of large nucleoli.

[0051] 2. Detection of marker proteins of malignant giant cell tumor of bone cell line MGCTB-CZ1 Cell immunohistochemical staining was performed on the human malignant giant cell tumor of bone cell line MGCTB-CZ1 in Example 1. The MGCTB-CZ1 cells in the logarithmic growth phase were selected and plated on sterile slides 24 hours in advance, and then subjected to standard pretreatment such as PBS washing 3 times, fixation (4% paraformaldehyde room temperature immersion for 30 min), and membrane permeation treatment (0.5% Triton X-100 permeation for 20 min) in sequence. The cell slide samples were sent to the pathology department of Shanghai Changzheng Hospital for professional immunohistochemical staining treatment, and the results are shown in Figure 3. Figure 3 As shown in Figure 3, the H3.3 G34W protein in the tumor cell nucleus is diffusely positively expressed, and the staining is brownish.

[0052] The above results show that the human malignant giant cell tumor cell line MGCTB-CZ1 cell grows vigorously, based on the verification of histopathological characteristics and specific molecular markers, the MGCTB-CZ1 cell line is a cell line derived from malignant giant cell tumor, and retains the key molecules and malignant phenotype characteristics of the primary tumor.

[0053] 3. STR (Short Tandem Repeat) gene locus is composed of short tandem repeats of 3 to 7 base pairs. These sequences are ubiquitous in the human genome and are widely used in cell identification due to their high polymorphism. DNA fingerprinting is at the cellular level, and STR identification is the preferred method for human cell line authentication. Currently, STR genotyping has been recognized by authoritative organizations such as the International Cell Line Authentication Committee (ICLAC) and the American Type Culture Collection (ATCC), and has become the gold standard for cell identification. The human malignant giant cell tumor cell line MGCTB-CZ1 cells cultured in Example 1 were sent to a professional agency for STR detection, and the source of the cells was identified by STR analysis. Based on the STR typing technology, the present application analyzes the MGCTB-CZ1 cells and their original tumor tissue at 21 loci (see Table 1), and compares the above sequences with the cell line STR data collected in ATCC, DSMZ, JCRB and RIKEN databases. No identical STR detection results were found, which can prove its uniqueness, and no cross contamination with other cell lines occurred during primary culture.

[0054] Table 1 Comparison of STR analysis results of MGCTB-CZ1 and original tumor tissue

[0055] 4. Cell growth curve and cell population doubling time.

[0056] Cell Counting Kit-8, abbreviated as CCK-8 kit, is a widely used rapid high-sensitivity detection kit for cell proliferation and cytotoxicity based on WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt).

[0057] Take the logarithmic growth phase human malignant giant cell tumor of bone cell line MGCTB-CZ1 cells in Example 1, trypsin digestion of cells, centrifugation at 1000 rpm for 5 minutes to collect cell precipitate, using tumor cell culture medium resuspended and counting. Inoculated at a density of 3x10³ cells / well into 96-well plates set 5 time point groups (0, 24, 48, 72, 96 h), each group contains 9 technical repeat wells, a total of 45 wells, in a 37℃, 5% CO2 cell incubator. 2 hours before each time point, add 10% CK-8 reagent 100 μL to the corresponding wells, incubate in the dark, and then use a microplate reader to measure the absorbance at 450 nm. Using GraphPad Prism 9.5.1 software, plot the growth curve of the cells with culture time as the x-axis and OD value as the y-axis. Use the online doubling time calculation platform (https: / / doublingtime.com / ) to calculate the cell population doubling time. The results are shown in Figure 4 Figure 1, according to the growth curve, the doubling time of the cell line is about 40.36 hours.

[0058] 5、Human malignant giant cell tumor of bone cell line MGCTB-CZ1 exon sequencing.

[0059] Take the logarithmic growth phase human malignant giant cell tumor of bone cell line MGCTB-CZ1 cells in Example 1 and peripheral blood from the same patient, extract DNA, perform exon sequencing and analysis. 100% of the genomic sequence of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 in the present application is successfully aligned with the human reference genome (GRCh38 of NCBI), which proves that it is a human cell line. In addition, the human malignant giant cell tumor of bone cell line MGCTB-CZ1 contains the H3F3A G34W mutation.

[0060] It should be noted that: In the description provided herein, a large number of specific details are explained. However, it can be understood that the embodiments of the present application can be practiced without these specific details. In some examples, well-known structures and techniques are not shown in detail in order not to obscure understanding of the present description.

[0061] Similarly, it should be understood that, in order to simplify the present application and help understand one or more of the various inventive aspects, in the above description of the exemplary embodiments of the present application, various features of the present application are sometimes grouped together in a single embodiment, figure, or description thereof. However, this disclosure should not be interpreted as reflecting a schematic diagram that the claimed present application requires more features than those explicitly recited in each claim.

[0062] More specifically, as reflected by the claims below, the inventive aspects are in less than all of the features of the previously disclosed single embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, as part of this application, and each claim is hereby separately and independently incorporated into the detailed description as part of this application, with each claim standing on its own as a separate embodiment of this application.

[0063] Furthermore, to one of ordinary skill in the art, and as context can dictate, it is also noted that not all of the components, features, structures, characteristics, etc. described and depicted herein need be present in every embodiment of the application. In addition, those skilled in the art will recognize that elements from two or more of the figures can be combined into one embodiment.

[0064] For example, in the claims below, any of the claimed embodiments can be used in any combination. For example, in the claims below, any of the claimed embodiments can be used in any combination.

[0065] The foregoing is considered as illustrative only of the principles of the application. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the application to the exact construction and operation described. Accordingly, all such variations are intended to fall within the scope of the application. Accordingly, the scope of the application should be determined by the scope of the following claims and their equivalents.

Claims

1. A human malignant giant cell tumor of bone cell line, characterized in that, The human malignant giant cell tumor of bone cell line is preserved on April 9, 2025, at the China Center for Type Culture Collection, Wuhan University, Wuhan, Hubei, China, and the preservation number is CCTCC NO: C202539, and the classification and naming is human malignant giant cell tumor of bone cell line MGCTB-CZ1.

2. The human malignant giant cell tumor of bone cell line of claim 1, wherein, The human malignant giant cell tumor of bone cell line MGCTB-CZ1 contains H3F3A G34W mutation, and can be continuously subcultured in vitro without changing the characteristics of malignant giant cell tumor of bone tumor cells.

3. The human malignant giant cell tumor of bone cell line of claim 2, wherein, The construction method of the human malignant giant cell tumor of bone cell line MGCTB-CZ1 is as follows: The tumor tissue of the surgically resected malignant giant cell tumor of bone is sampled to obtain a tumor tissue sample; After pretreatment of the tumor tissue sample, 5% fetal bovine serum-containing DMEM / F12 medium and tissue digestion solution are added, and digestion is carried out at 37°C for 1 hour to obtain a suspension; The suspension is filtered through 70μm and 40μm cell screens in sequence, the filtered suspension is centrifuged at 800rpm for 5 minutes, the precipitate is collected, and the tumor cells are resuspended in tumor cell culture medium and cultured at 37°C in a 5% CO2 atmosphere. The tumor cell culture medium is replaced every 2-3 days during the culture; When the cell density reaches between 80%-90%, the supernatant is aspirated, washed twice with DPBS, and digestion enzyme is added for 3 minutes at 37°C, then tumor cell culture medium is added to terminate digestion, followed by centrifugation at 800rpm for 5min, aspiration of the supernatant, resuspension of the cells in tumor culture medium, and 1:2 passage.

4. The human malignant giant cell tumor of bone cell line of claim 3, wherein, The pretreatment is to rinse the tumor tissue sample twice with tissue rinse solution, remove fibrous connective tissue, fat and necrotic tissue, and then cut it into granules.

5. The human malignant giant cell tumor of bone cell line of claim 4, wherein, The tissue digestion solution is 1mg / ml type II collagenase.

6. The human malignant giant cell tumor of bone cell line of claim 5, wherein, The configuration method of the tumor cell culture medium is as follows: 15% fetal bovine serum and 1% penicillin-streptomycin double antibody are added to the DMEM / F12 basic medium system and mixed.

7. The human malignant giant cell tumor of bone cell line of claim 6, wherein, The configuration method of the tissue rinse solution is as follows: 2% double antibody solution is added to HBSS buffer solution, mixed to obtain the tissue rinse solution.

8. The human malignant giant cell tumor of bone cell line of claim 7, wherein, The configuration method of the digestion enzyme is as follows: Trypsin containing 0.25% EDTA is added to PBS for dilution to a final concentration of 0.05%.

9. Use of the human malignant giant cell tumor of bone cell line according to any one of claims 1-8 in the preparation of a cell model for the study of the mechanism of malignant giant cell tumor of bone.

10. Use of the human malignant giant cell tumor of bone cell line according to any one of claims 1-8 in the preparation of a malignant giant cell tumor of bone diagnostic reagent or diagnostic kit.

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