Mutant spinal glioma cell line

Abstract

The invention provides a mutant spinal glioma cell line. The H3K27M mutant type glioma cell line is successfully constructed, the constructed cell line has more excellent multiplication capacity, migration capacity, tumor formation capacity and drug resistance, the curative effect and potential side effects of drugs can be more comprehensively evaluated, the cell line can be used for in-vitro research on the biological function of H3K27M glioma and response to drug treatment, and the cell line can be applied to in-vitro research of the H3K27M mutant type glioma. Wide application prospects are realized.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to a mutant spinal cord glioma cell line. Background Art

[0002] Spinal cord glioma is a spinal cord tumor disease with a low incidence but extremely high mortality and disability rates, accounting for approximately 2% to 4% of all primary nervous system tumors. Some evidence shows that spinal cord gliomas are involved in different tumor genetic induction factors compared to intracranial gliomas, and their molecular mechanisms are different from those of intracranial tumors. Based on the current situation, it is crucial to study the molecular mechanisms of the occurrence and development of spinal cord gliomas and to find molecular therapeutic targets for tumors. However, basic experiments at this stage still lack effective models of spinal cord tumors, which to some extent limits the research progress of basic experiments on spinal cord gliomas. Therefore, establishing a spinal cord glioma cell line is crucial to this field. Summary of the invention

[0003] To make up for the deficiencies of the prior art, the present invention provides a mutant spinal cord glioma cell line.

[0004] To achieve the above purpose, the present invention adopts the following technical solution.

[0005] The first aspect of the present invention provides an H3 K27M mutant spinal cord glioma cell line, which is deposited in the General Microbiology Center of China National Microorganism Culture Collection Administration, including SCA-S02 with a deposit number of CGMCC NO.45809, SCA-S09 with a deposit number of CGMCC NO.45810, SCA-S10 with a deposit number of CGMCC NO.45811, SCA-S12 with a deposit number of CGMCC NO.45812, SCA-S41 with a deposit number of CGMCC NO.45813, and SCA-S18 with a deposit number of CGMCC NO.45814.

[0006] The second aspect of the present invention provides a progeny cell line derived from the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present invention.

[0007] The third aspect of the present invention provides a reagent, which comprises the H3K27M mutant spinal cord glioma cell line described in the first aspect of the present invention or the progeny cell line described in the second aspect of the present invention.

[0008] The fourth aspect of the present invention provides a kit, which comprises the H3K27M mutant spinal cord glioma cell line described in the first aspect of the present invention, the progeny cell line described in the second aspect of the present invention, or the reagent described in the third aspect of the present invention.

[0009] The fifth aspect of the present invention provides a method for preparing an H3 K27M mutant spinal cord glioma model, the method comprising administering the H3 K27M mutant spinal cord glioma cell line described in the first aspect of the present invention, the progeny cell line described in the second aspect of the present invention, or the agent described in the third aspect of the present invention to an animal.

[0010] Furthermore, the animal is a mammal.

[0011] Furthermore, the mammal is a non-human mammal.

[0012] The sixth aspect of the present invention provides a method for screening drugs for treating H3 K27M mutant spinal glioma, the method comprising contacting the drug to be screened with the H3 K27M mutant spinal glioma cell line described in the first aspect of the present invention, the progeny cell line described in the second aspect of the present invention, or the reagent described in the third aspect of the present invention, and determining the inhibitory effect of the drug to be screened on the cells.

[0013] The seventh aspect of the present invention provides a method for evaluating the efficacy of a drug for H3 K27M mutant spinal glioma, the method comprising contacting the drug with the H3 K27M mutant spinal glioma cell line described in the first aspect of the present invention, the progeny cell line described in the second aspect of the present invention, or the reagent described in the third aspect of the present invention, and determining the inhibitory effect of the drug on the cells.

[0014] The eighth aspect of the present invention provides use of the H3 K27M mutant spinal cord glioma cell line described in the first aspect of the present invention or the progeny cell line described in the second aspect of the present invention in screening / evaluating / preparing drugs for treating spinal cord glioma.

[0015] Furthermore, evaluating a drug for spinal cord glioma includes evaluating the drug sensitivity of spinal cord glioma.

[0016] The ninth aspect of the present invention provides use of the H3 K27M mutant spinal cord glioma cell line described in the first aspect of the present invention or the progeny cell line described in the second aspect of the present invention in preparing a model.

[0017] Furthermore, the model includes a cell model and an animal model.

[0018] The tenth aspect of the present invention provides use of the H3 K27M mutant spinal cord glioma cell line described in the first aspect of the present invention or the progeny cell line described in the second aspect of the present invention in studying spinal cord glioma-related mechanisms.

[0019] Furthermore, the related mechanisms include the pathogenesis, development mechanism, and drug resistance mechanism of spinal cord glioma.

[0020] Advantages and beneficial effects of the present invention:

[0021] The present application successfully constructs a spinal cord glioma cell line carrying the H3K 27M mutation. The cell line constructed in the present application has more excellent proliferation ability, migration ability, tumor formation ability, and drug resistance, and can more comprehensively evaluate the efficacy and potential side effects of drugs. It can be used to study the biological functions of H3K27M spinal cord glioma and its response to drug treatment in vitro, and has broad application prospects.

[0022] Biomaterial Deposit Information

[0023] SCA-S02:

[0024] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45809.

[0025] SCA-S09:

[0026] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45810.

[0027] SCA-S10:

[0028] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45811.

[0029] SCA-S12:

[0030] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45812.

[0031] SCA-S41:

[0032] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45813.

[0033] SCA-S18:

[0034] Scientific description: human cells; depository: General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC); deposit address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; deposit date: January 22, 2024; deposit number: CGMCC NO.45814. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is a morphological picture of H3 K27M mutant spinal cord glioma cells;

[0036] Figure 2 This is a diagram of gene mutations in H3 K27M mutant spinal cord glioma cells;

[0037] Figure 3 is the proliferation curve of H3 K27M mutant spinal cord glioma cells;

[0038] Figure 4 This is a graph showing the radiosensitivity of H3 K27M mutant spinal cord glioma cells;

[0039] Figure 5 is the sensitivity curve of H3 K27M mutant spinal cord glioma cells to palbociclib;

[0040] Figure 6 is a curve diagram of the sensitivity of H3 K27M mutant spinal cord glioma cells to methotrexate;

[0041] Figure 7 is the sensitivity curve of H3 K27M mutant spinal cord glioma cells to tazostat;

[0042] Figure 8 is the sensitivity curve of H3 K27M mutant spinal cord glioma cells to temozolomide;

[0043] Fig. 9 This is a graph showing the sensitivity of H3 K27M mutant spinal cord glioma cells to panobinostat. DETAILED DESCRIPTION

[0044] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0045] The present invention provides a kit, which comprises the above-mentioned H3 K27M mutant spinal cord glioma cell line, the above-mentioned progeny cell line or the above-mentioned reagent.

[0046] In some embodiments, the kit may optionally include one or more components, such as instructions for use, devices, and additional reagents (e.g., sterile water or saline solution). In some embodiments, the kit may also contain reagents for sample collection, sample preparation and processing, and / or reagents for quantifying the amount of one or more surface markers in the sample (including but not limited to detection reagents, such as antibodies, buffers, enzyme staining substrates, chromogens, or other materials (e.g., slides, containers, microtiter plates)).

[0047] In some embodiments, test kit can be provided as an article comprising packaging materials for packaging cells or one or more other components. For example, the test kit can contain containers, bottles, tubes, vials and any packaging materials suitable for separating or organizing the components of the test kit. One or more containers can be formed by various materials (such as glass or plastic). In some embodiments, one or more containers hold cells or other compositions.

[0048] The present invention provides a method for preparing an H3 K27M mutant spinal cord glioma model, the method comprising administering the H3 K27M mutant spinal cord glioma cell line, the progeny cell line or the reagent to an animal.

[0049] In some embodiments, the model includes a cell model and an animal model. The animal model includes transplanting the above-mentioned H3 K27M mutant spinal cord glioma into a non-human animal to form an H3 K27M mutant spinal cord glioma animal model.

[0050] In some embodiments, the non-human animal refers to all animals, excluding humans, including but not limited to birds, farm animals (eg, cattle), sports animals (eg, horses), fish, reptiles, and non-human mammals.

[0051] In some embodiments, the non-human animal is preferably a non-human mammal.

[0052] In some embodiments, non-human mammals refer to all members of the class Mammalia, excluding humans, including but not limited to cats, dogs, and rodents, wherein rodents include but are not limited to rats, mice, rabbits, hamsters or guinea pigs.

[0053] In some embodiments, the H3 K27M mutant spinal cord glioma animal model can be transplanted and inoculated using any method disclosed in the prior art, such as subcutaneous injection of a single cell suspension.

[0054] The present invention provides a method for screening drugs for treating H3 K27M mutant spinal cord glioma, the method comprising contacting the drug to be screened with the above-mentioned H3 K27M mutant spinal cord glioma cell line, the above-mentioned progeny cell line or the above-mentioned reagent, and determining the inhibitory effect of the drug to be screened on the cells.

[0055] In some embodiments, screening for drugs for treating H3 K27M mutant spinal glioma comprises contacting the H3K27M mutant spinal glioma cell line with a drug to be tested, observing and comparing the tumor size, metastasis, survival period, etc. of the cells in the group contacted with the drug to be tested and the group not contacted with the drug to be tested, to screen for drugs that can treat H3 K27M mutant spinal glioma.

[0056] In some embodiments, contacting the H3 K27M mutant spinal glioma cell line with the drug to be tested refers to placing the H3 K27M mutant spinal glioma cell line and the drug to be tested in a contactable state. The contacting of the H3 K27M mutant spinal glioma cell line with the drug to be tested may be, for example, adding the drug to be tested to a solution containing the H3 K27M mutant spinal glioma cell line.

[0057] In some embodiments, the drug to be tested may be, for example, a low molecular weight compound, a protein (e.g., an antibody), DNA, RNA, a low molecular weight interfering RNA, or an antisense oligonucleotide. The drug to be tested may be, for example, a drug for treating a disease or cancer other than H3 K27M mutant spinal cord glioma. The drug to be tested may be, for example, a mixture of one or more than two. The drug to be tested is preferably a substance.

[0058] In some embodiments, the H3 K27M mutant spinal cord glioma cell line contacted with the above-mentioned drug to be tested is measured to include a decrease in cell viability, a decrease in cell proliferation, an increase in cell death, a change in cell size, the number of cells, and the expression of marker proteins constituting the cells. Among them, the change in cell size can be, for example, the size of the cell, which can be measured using a known device (such as a microscope or FACS). The size of the cell can be, for example, the circumference, diameter (such as the major diameter and the minor diameter) or area of ​​the cell.

[0059] In some embodiments, the measured value of a cell can be, for example, a measured value of a single measurement, or an average value of multiple measurements, or an average value of the measured values ​​of multiple cells. The measured value of a cell can be, for example, a change value (e.g., difference or multiple) before and after contact with the drug to be tested. The control value can be, for example, when the value after contact with the drug to be tested is used as the measured value of the cell, the measured value of the control cell before contact with the drug to be tested or not contacted with the drug to be tested can be used as the control value.

[0060] For example, when the measured value is the size of the cells, the determination of whether the drug to be tested is a substance capable of treating H3 K27M mutant spinal cord glioma may include the following steps: when the measured value of the size of the cells after contact with the drug to be tested is less than the control value (i.e., the degree of increase in the cell size decreases or the size is reduced), the drug to be tested is determined to be a drug capable of treating H3K27M mutant spinal cord glioma.

[0061] In some embodiments, treatment refers to any method used to partially or completely relieve, ameliorate, alleviate, inhibit, prevent, delay the onset of, reduce the severity and / or incidence of one or more symptoms or characteristics of a particular disease, disorder, and / or condition.

[0062] In some embodiments, prevention and grammatical variations thereof mean prevention and / or delay of the onset of a disease, illness and / or one or more clinical symptoms in an individual, and / or a reduction in the severity of the onset of a disease, illness and / or one or more clinical symptoms relative to what would occur in the absence of the method of the application. Prevention can be complete, such as the complete absence of a disease, illness and / or one or more clinical symptoms. Prevention can also be partial, such that the severity of the occurrence and / or onset of a disease, illness and / or one or more clinical symptoms in an individual is less than what would occur in the absence of the present invention.

[0063] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0064] Example

[0065] In this example, the patient-derived spinal cord H3 K27M variant cells were divided into two groups: "high proliferation and replication pressure type" and "high immune infiltration type" according to the patient's age and multi-omics information of the tumor. Among them, SCA-S10 and SCA-S12 are "high immune infiltration type" tumor patient-derived cell models, and SCA-S18, SCA-S41, SCA-S09, and SCA-S02 are "high proliferation and replication pressure type" cell models.

[0066] The process of cell suspension culture is as follows: place the glioma tissue block (the glioma tissue was surgically removed in Tiantan Hospital according to the operation procedures in line with medical ethics) in a beaker, rinse it with Hanks solution for 3 times to remove blood stains; then place it in a mixture containing penicillin and streptomycin for 60 minutes; use ophthalmic scissors to cut the tissue into 2-3 mm blocks for easy digestion; add trypsin solution equivalent to 50 times the volume of the total amount of tissue blocks, and then pour it into a conical flask, tie the bottle mouth or plug it with a rubber stopper; place it in a 37°C incubator for digestion, and shake it every 20 minutes during the digestion process; the digestion time is 60 minutes; then filter out the tissue blocks that have not been fully digested through a suitable stainless steel sieve; centrifuge the digestion solution at 800 rpm to obtain sample cells, aspirate the supernatant, add primary culture medium to the precipitate, and add the primary culture medium to the precipitate until the cell density is 10 5 / mL, and then placed in a constant temperature incubator at 37°C for culture. The basic culture medium of the primary culture medium is a suspension culture medium, which includes: 20ng / mL EGF cytokine, 20ng / mL bFGF cytokine, 20ng / mL streptomycin and 20ng / mL penicillin, B27 (50×) and 20ng / mL transferrin.

[0067] The single cell suspension obtained by the above suspension culture was inoculated into an ultra-low adsorption culture dish for culture, and then digested into single cells with digestive enzymes. When the operation was repeated and subcultured to the fourth generation, some primary cells died, stopped growing or grew slowly, and another part of the primary cells were able to show a good proliferation state in a serum-free suspension culture medium. These cell lines showing a good proliferation state were selected as further screening objects, and MTT experiments and Transwell experiments were performed to detect the proliferation and migration abilities of the cell lines. The six cell lines with the strongest proliferation and migration abilities were selected for preservation, namely, the H3 K27M mutant spinal cord glioma cells SCA-S02 (preservation number CGMCC NO.45809), SCA-S09 (preservation number CGMCC NO.45810), SCA-S10 (preservation number CGMCC NO.45811), SCA-S12 (preservation number CGMCC NO.45812), SCA-S41 (preservation number CGMCC NO.45813), and SCA-S42 (preservation number CGMCC NO.45814) disclosed in the present application. NO.45813), SCA-S18 (deposit number CGMCC NO.45814); the cell morphology of the above six cell lines is as follows Figure 1 As shown in Figure 2, different spinal glioma cells carry molecular mutations such as Figure 2 As shown, it can be seen that the above-mentioned multiple different spinal glioma cells all have H3 K27M mutations, while the accompanying other mutations are different.

[0068] Specifically, the H3 K27M mutant spinal cord glioma cell SCA-S02 has a deposit number of CGMCCNO.45809, tp53 has a missense mutation, myc has an amplification mutation, and pdgfra has an amplification mutation.

[0069] The deposit number of the H3 K27M mutant spinal cord glioma cell SCA-S09 is CGMCC NO.45810. The tp53 of the H3 K27M mutant spinal cord glioma cell has a frameshift mutation.

[0070] The deposit number of the H3 K27M mutant spinal cord glioma cell SCA-S10 is CGMCC NO.45811. Atrx of the H3 K27M mutant spinal cord glioma cell has a missense mutation, ppm1d has a stop mutation, and nf1 has a missense mutation.

[0071] The deposit number of H3 K27M mutant spinal cord glioma cell SCA-S12 is CGMCC NO.45812, ppm1d has a missense mutation, and nf1 has a frameshift mutation.

[0072] The deposit number of H3 K27M mutant spinal cord glioma cell SCA-S41 is CGMCC NO.45813, tp53 has a missense mutation, met has an amplification mutation, and myc has an amplification mutation.

[0073] The deposit number of H3 K27M mutant spinal cord glioma cell SCA-S18 is CGMCC NO.45814, tp53 has a missense mutation, met has an amplification mutation, and mycn has an amplification mutation.

[0074] In this example, whole exome sequencing was used to detect gene mutations and copy number variations in spinal cord glioma cells. The results are as follows: Figure 2 As shown, it can be seen that different spinal glioma cells all have H3 K27M mutation (H3F3A K27M), but there are differences in other accompanying mutations or copy numbers; the above cell lines can more objectively reflect the commonalities of this type of tumor cells and the inhibitory nature of tumors, and are suitable for evaluating the biological characteristics of cells carrying different molecular mutations and their response to drug treatment.

[0075] This example further uses the CCK-8 method to detect cell proliferation, and the specific process is as follows:

[0076] The above-mentioned different spinal astrocytoma cells in the logarithmic growth phase were taken and evenly plated in 96-well plates, with 2000 cells plated in each well. After culturing for 0, 2, 4, 6, and 8 days, CCK8 solution (purchased from Dojindo, Japan) was added to the cell culture medium (suspension culture medium) in a ratio of 1:10 to the cell culture medium of the 96-well plate. The culture was continued at 37°C for 2 hours, and the absorbance at a wavelength of 450nm was measured by an ELISA instrument. According to the OD results, GraphPad Prism 8 was used to fit the curve to determine the proliferation rate of different cell lines. In addition to the above-mentioned 6 spinal glioma cell lines, this example also measured the proliferation rate of SCA-S01 (deposit number CGMCC No. 23029) and SP0001 (deposit number CGMCC No. 23028) cells previously deposited by the inventors of the present disclosure. The results are as follows: Figure 3 As shown, the proliferation rates of the above 8 spinal cord glioma cells are significantly different. Specifically, SCA-S02 cells proliferate faster, SCA-S09 and SP0001 proliferate slower, and the other 6 spinal cord glioma cells proliferate more slowly. Therefore, it is further shown that the use of the above spinal cord glioma cells for determination can better reflect the commonalities, biological characteristics and response to drug treatment of spinal cord glioma cells.

[0077] Example 2

[0078] Radiotherapy is commonly used as a treatment for H3 K27M mutant spinal glioma in the prior art, but there is still great uncertainty as to whether patients can benefit from radiotherapy, and radiotherapy is usually not beneficial to patients' long-term survival. In order to detect the value of the 6 spinal glioma cells provided in Example 1 in evaluating H3 K27M mutant spinal glioma for radiotherapy and potential targeted therapy, this example measured the drug reactivity of the above 6 isolated H3K27M mutant spinal glioma cells and SCA-S01 and SP0001 cells previously deposited by the inventors of the present disclosure to different compounds, and the specific experiment is as follows.

[0079] In this example, the 8 spinal cord glioma cells were treated with radiotherapy doses of 0, 2, 4, 6, and 8 Gy, respectively. The number of surviving cells in different groups was measured 6 days after radiotherapy to evaluate their sensitivity to radiotherapy. Figure 4 As shown, it can be seen that SCA-S10, SCA-S12, SP0001, and SCA-SP01 are relatively resistant to radiotherapy, and the remaining SCA-S18, SCA-S41, SCA-S09, and SCA-S02 are relatively sensitive to radiotherapy, but still have the characteristics of resisting radiotherapy. When the radiotherapy dose is 8Gy, 50% of the cells still survive. It can be seen that this type of cell has a high resistance to radiotherapy as a whole, and corresponding drug screening is still needed according to its genetic background.

[0080] This example further measured the sensitivity of the above 8 cell lines to different chemotherapeutic drugs or different targeted drug treatments. The results are as follows: Figure 5-9 shown.

[0081] This example further determines the inhibitor concentration that can reduce cell viability by 50%, ie, the IC50 value, by calculation and fitting based on the data of spinal cord glioma cell viability at different inhibitor concentrations. The specific process is as follows.

[0082] Primary cultured spinal astrocytoma cells in the logarithmic growth phase were evenly plated in a 96-well plate, with 5000 cells plated in each well. After 24 hours, different concentrations of suspension culture medium containing inhibitors were added to culture the cells in the 96-well plate at a final concentration of 0-100 μM (specific concentrations were 0.00001; 0.00010; 0.00100; 0.01000; 0.10000; 1.00000; 10.00000; 100.00000 μM), with no less than 5 cells at each concentration, and continued to culture. After 6 days of culture, the cells were cultured using CellTiter-Lumi TM The cell viability was detected by luminescence 3D cell viability detection kit (Biyuntian C0061L).

[0083] The inhibitory rate of the inhibitor on spinal astrocytoma cells was calculated according to the following formula:

[0084] Inhibition rate (%) = (control group - drug-added group) / control group × 100%.

[0085] According to the calculation results, GraphPad Prism 8 was used to fit the curve and determine the half-maximal inhibitory concentration of various inhibitors on different spinal astrocytoma cells, as shown in Table 1.

[0086] Table 1 IC50 values ​​of different spinal glioma cells against different inhibitors

[0087]

[0088] The test results in Table 1 further showed that the sensitivity of the 8 spinal astrocytoma cells to various inhibitors varied, and 6 H3 K27M mutant spinal glioma cells were insensitive to temozolomide, a first-line chemotherapy drug for gliomas, which was consistent with the results of clinical cohort studies. They were more sensitive to the commonly used clinical targeted drug palbociclib (CDK6 inhibitor, FDA approved) and the targeted drug panobinostat for high-grade gliomas in children (HDAC inhibitor, in clinical trials); different spinal astrocytoma cell lines had different sensitivities to the clinical targeted drug tazemetostat (EZH2 inhibitor, in clinical trials) and methotrexate, which has the potential for intrathecal chemotherapy.

[0089] Specifically, the present invention successfully established 6 stem cell-like spinal cord glioma patient-derived cells carrying H3K27M mutation, among which the IC50 value of the SCA-S02 cell line for palbociclib was 4.439 μM, which was relatively low, indicating that SCA-S02 cells were relatively sensitive to palbociclib; the IC50 value for panobinostat was 0.01757 μM, indicating that SCA-S02 cells were highly sensitive to panobinostat; the IC50 values ​​for temozolomide and tazemetostat were 123.0 μM and 107.5 μM, respectively, which were relatively high, indicating that SCA-S02 cells were generally sensitive to these two drugs; the IC50 value for methotrexate was 662.5 μM, indicating that SCA-S02 cells were insensitive to methotrexate.

[0090] The IC50 value of SCA-S09 cell line for palbociclib is 4.533μM, indicating that SCA-S09 cells have a certain sensitivity to palbociclib; the IC50 value for panobinostat is 0.03588μM, which is at a relatively low level, indicating that SCA-S09 cells are relatively sensitive to panobinostat; the IC50 value for temozolomide is 164.2μM, which is relatively high, indicating that SCA-S09 cells are generally sensitive to temozolomide; the IC50 value for tazemetostat is 227.6μM, indicating that SCA-S09 cells are less sensitive to tazemetostat; the IC50 value for methotrexate is 28.94μM, which is at a relatively low level, indicating that SCA-S09 cells are highly sensitive to methotrexate.

[0091] The IC50 value of SCA-S41 cell line for panobinostat was 0.001297 μM, indicating that SCA-S41 cells were extremely sensitive to panobinostat; the IC50 value for palbociclib was 5.249 μM, indicating that SCA-S41 cells were somewhat sensitive to palbociclib; the IC50 value for temozolomide was 93.68 μM, which was relatively high, indicating that SCA-S41 cells were generally sensitive to temozolomide; the IC50 value for tazemetostat was 115.2 μM, which was relatively high, indicating that SCA-S41 cells were poorly sensitive to tazemetostat; the IC50 value for methotrexate was 117.4 μM, indicating that SCA-S41 cells were poorly sensitive to methotrexate.

[0092] The IC50 value of SCA-S10 cell line for palbociclib is 2.615 μM, indicating that the cell line has a certain sensitivity to palbociclib; the IC50 value for panobinostat is 0.004930 μM, indicating that the cell line is highly sensitive to panobinostat; the IC50 value for tazemetostat is 59.51 μM, indicating that the cell line is generally sensitive to tazemetostat; the IC50 value for methotrexate is 29714 μM, indicating that the cell line is extremely insensitive to methotrexate.

[0093] The IC50 value of SCA-S12 for palbociclib was 2.045 μM, indicating that the SCA-S12 cell line was relatively sensitive to palbociclib; the IC50 value for panobinostat was 0.03229 μM, indicating that the cell line was highly sensitive to panobinostat; the IC50 value for temozolomide was 16074 μM, indicating that the SCA-S12 cell line was extremely insensitive to temozolomide; the IC50 value for tazemetostat was 52.11 μM, indicating that the cell line was generally sensitive to tazemetostat; the IC50 value for methotrexate was 0.6891 μM, indicating that the cell line was relatively sensitive to methotrexate.

[0094] The IC50 value of SCA-S18 for palbociclib is 3.375 μM, indicating that the SCA-S18 cell line has a certain sensitivity to palbociclib; the IC50 value for panobinostat is 0.03960 μM, indicating that the cell line is relatively sensitive to panobinostat; the IC50 value for temozolomide is 1453 μM, indicating that the SCA-S18 cell line is poorly sensitive to temozolomide; the IC50 value for tazemetostat is 27.67 μM, indicating that the cell line is moderately sensitive to tazemetostat; the IC50 value for methotrexate is 0.01178 μM, indicating that the SCA-S18 cell line is extremely sensitive to methotrexate.

[0095] Through the above technical solution, the present disclosure successfully established 6 stem cell-like spinal cord glioma patient-derived cells carrying H3K 27M mutations. The above 6 cell lines have different sensitivities to different drugs, and their proliferation rate, migration rate and tumor formation ability are significantly better than other disclosed H3K 27M spinal cord glioma cell lines. The above 6 cell lines are combined to cover stem cell-like spinal cord gliomas with different mutations of H3K27M mutations and cell types with different drug sensitivities, so that the efficacy and potential side effects of drugs can be more comprehensively evaluated, and can be used to study the biological functions of H3K 27M spinal cord gliomas and their response to drug treatment in vitro.

[0096] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A H3 K27M mutant spinal cord glioma cell line, characterized in that: The cell lines are deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, including SCA-S02 with a deposit number of CGMCC NO.45809, SCA-S09 with a deposit number of CGMCC NO.45810, SCA-S10 with a deposit number of CGMCC NO.45811, SCA-S12 with a deposit number of CGMCC NO.45812, SCA-S41 with a deposit number of CGMCC NO.45813, and SCA-S18 with a deposit number of CGMCC NO.45814.

2. A progeny cell line derived from the H3 K27M mutant spinal cord glioma cell line according to claim 1.

3. A reagent, characterized in that The reagent comprises the H3 K27M mutant spinal cord glioma cell line of claim 1 or the progeny cell line of claim 2.

4. A kit, characterized in that: The kit comprises the H3 K27M mutant spinal cord glioma cell line of claim 1, the progeny cell line of claim 2 or the reagent of claim 3.

5. A method for preparing an H3 K27M mutant spinal cord glioma model, characterized in that: The method comprises administering the H3 K27M mutant spinal glioma cell line of claim 1, the progeny cell line of claim 2, or the agent of claim 3 to an animal; Preferably, the animal is a mammal.

6. A method for screening drugs for treating H3 K27M mutant spinal cord glioma, characterized in that: The method comprises contacting the drug to be screened with the H3 K27M mutant spinal cord glioma cell line of claim 1, the progeny cell line of claim 2 or the reagent of claim 3, and determining the inhibitory effect of the drug to be screened on the cells.

7. A method for evaluating the efficacy of a drug for H3 K27M mutant spinal cord glioma, characterized in that: The method comprises contacting the drug with the H3 K27M mutant spinal cord glioma cell line of claim 1, the progeny cell line of claim 2 or the reagent of claim 3, and measuring the inhibitory effect of the drug on the cells.

8. Use of the H3 K27M mutant spinal cord glioma cell line according to claim 1 or the progeny cell line according to claim 2 in screening / evaluating / preparing drugs for treating spinal cord glioma; Preferably, evaluating the drug of spinal cord glioma includes evaluating the drug sensitivity of spinal cord glioma.

9. Use of the H3 K27M mutant spinal cord glioma cell line of claim 1 or the progeny cell line of claim 2 in preparing a model; Preferably, the model includes a cell model and an animal model.

10. Use of the H3 K27M mutant spinal cord glioma cell line of claim 1 or the progeny cell line of claim 2 in studying spinal cord glioma-related mechanisms; Preferably, the related mechanisms include the pathogenesis, development mechanism, and drug resistance mechanism of spinal cord glioma.

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