A mutant spinal cord glioma cell line

Abstract

The application provides a mutant spinal cord glioma cell line. The application successfully constructs a H3K27M mutant spinal cord glioma cell line, the cell line constructed by the application has more excellent proliferation capacity, migration capacity, tumorigenic capacity and drug resistance, can more comprehensively evaluate the curative effect and potential side effects of drugs, can be used for in vitro research on the biological function of H3K27M spinal cord glioma and response to drug treatment, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a mutant spinal cord glioma cell line. BACKGROUND

[0002] Spinal cord glioma is a spinal cord tumor disease with low incidence but high mortality and disability rate, accounting for about 2% to 4% of all primary nervous system tumors. Some evidence shows that spinal cord glioma has different tumor genetic inducers than intracranial glioma, and the molecular mechanisms of development are different from intracranial tumors. Based on the current situation, it is crucial to study the molecular mechanisms of the development of spinal cord glioma and to find molecular therapeutic targets for tumors. However, the current basic experiment still lacks an effective model of spinal cord tumor, which to some extent limits the progress of basic experimental research on spinal cord glioma. Therefore, it is crucial to establish a spinal cord glioma cell line in the field. SUMMARY

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

[0004] To achieve the above-mentioned purpose, the present application adopts the following technical solutions.

[0005] The first aspect of the present application provides a H3 K27M mutant spinal cord glioma cell line, which is preserved in the China General Microbiological Culture Collection Center, and includes cell lines with preservation numbers of SCA-S02 (CGMCC NO.45809), SCA-S09 (CGMCC NO.45810), SCA-S10 (CGMCC NO.45811), SCA-S12 (CGMCC NO.45812), SCA-S41 (CGMCC NO.45813), and SCA-S18 (CGMCC NO.45814).

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

[0007] The third aspect of the present application provides a reagent, which includes the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application or the daughter cell line according to the second aspect of the present application.

[0008] The fourth aspect of the present application provides a kit, which includes the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application, the daughter cell line according to the second aspect of the present application, or the reagent according to the third aspect of the present application.

[0009] The fifth aspect of the present application provides a method for preparing a H3 K27M mutant spinal cord glioma model, which comprises administering the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application, the progeny cell line according to the second aspect of the present application or the reagent according to the third aspect of the present application into an animal.

[0010] Further, the animal is a mammal.

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

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

[0013] The seventh aspect of the present application provides a method for evaluating the efficacy of a drug for treating H3 K27M mutant spinal cord glioma, which comprises contacting the drug with the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application, the progeny cell line according to the second aspect of the present application or the reagent according to the third aspect of the present application, and determining the inhibitory effect of the drug on the cells.

[0014] The eighth aspect of the present application provides the use of the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application or the progeny cell line according to the second aspect of the present application in screening / evaluating / preparing a drug for treating spinal cord glioma.

[0015] Further, evaluating the drug for spinal cord glioma comprises evaluating the drug sensitivity for spinal cord glioma.

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

[0017] Further, the model comprises a cell model, an animal model.

[0018] The tenth aspect of the present application provides the use of the H3 K27M mutant spinal cord glioma cell line according to the first aspect of the present application or the progeny cell line according to the second aspect of the present application in researching a mechanism related to spinal cord glioma.

[0019] Further, the related mechanism comprises a pathogenesis, a development mechanism, a drug resistance mechanism of spinal cord glioma.

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

[0021] The application successfully constructs a H3K 27M mutant carrying spinal cord glioma cell line, the cell line constructed in the application has more excellent proliferation ability, migration ability, tumorigenicity and drug resistance, can more comprehensively evaluate the efficacy and potential side effects of drugs, can be used for in vitro research on the biological function of H3K27M spinal cord glioma and the response to drug treatment, and has wide application prospect.

[0022] Biological material preservation information

[0023] SCA-S02:

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

[0025] SCA-S09:

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

[0027] SCA-S10:

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

[0029] SCA-S12:

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

[0031] SCA-S41:

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

[0033] SCA-S18:

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

[0035] Figure 1 is a cell morphology diagram of H3 K27M mutant spinal cord glioma cells;

[0036] Figure 2 is a gene mutation condition diagram of H3 K27M mutant spinal cord glioma cells;

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

[0038] Figure 4 is a radiotherapy sensitivity curve diagram of H3 K27M mutant spinal cord glioma cells;

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

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

[0041] Figure 7 is a taselisib sensitivity curve diagram of H3 K27M mutant spinal cord glioma cells;

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

[0043] Figure 9 is a paribiclib sensitivity curve diagram of H3 K27M mutant spinal cord glioma cells. DETAILED DESCRIPTION

[0044] The following provides definitions of some terms used in the present specification. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0045] The present application provides a kit comprising 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 can 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 can also contain reagents for collection of the sample, preparation and processing of the sample, 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 stain substrates, chromogens, or other materials (such as slides, containers, microtiter plates)).

[0047] In some embodiments, the kit can be provided as an article of manufacture that includes packaging material for packaging the cells or one or more other components. For example, the kit can contain containers, bottles, tubes, vials, and any packaging material suitable for isolating or organizing the components of the kit. The one or more containers can be formed from a variety of materials such as glass or plastic. In some embodiments, the one or more containers hold a composition comprising the cells or other components.

[0048] The present application provides a method for preparing a H3 K27M mutant spinal cord glioma model, which comprises administering the above-mentioned H3 K27M mutant spinal cord glioma cell line, the above-mentioned progeny cell line or the above-mentioned reagent into an animal.

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

[0050] In some embodiments, the non-human animal refers to all animals, except humans, including but not limited to birds, farm animals (e.g., cows), sports animals (e.g., 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, the non-human mammal refers to all members of the mammalian class, except humans. It includes but is not limited to cats, dogs, and rodents. Among them, 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 by any method disclosed in the prior art, for example, it can be a single cell suspension subcutaneous injection, etc.

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

[0055] In some embodiments, the method for screening a drug for treating H3 K27M mutant spinal cord glioma comprises contacting the H3 K27M mutant spinal cord glioma cell line with a drug to be tested, and 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 a drug capable of treating H3 K27M mutant spinal cord glioma.

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

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

[0058] In some embodiments, the determination of the H3 K27M mutant spinal cord glioma cell line contacted with the drug to be tested includes 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. The change in cell size may, for example, be the determination of the size of the cells, which may, for example, be determined using a known device (e.g., a microscope or a FACS). The size of the cells may, for example, be the perimeter, diameter (e.g., major diameter and minor diameter), or area of the cells.

[0059] In some embodiments, the determined value of the cells may, for example, be one determined value, an average value of multiple determinations, or an average value of the determined values of multiple cells. The determined value of the cells may, for example, be a change value (e.g., a difference or a fold) before and after contacting the drug to be tested. The control value may, for example, be the determined value of a control cell before contacting the drug to be tested or not contacting the drug to be tested, when the value after contacting the drug to be tested is used as the determined value of the cells.

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

[0061] In some embodiments, treatment refers to any method for partial or complete alleviation, amelioration, relief, inhibition, prevention of one or more symptoms or features of a particular disease, disorder, and / or condition, delay of onset of one or more symptoms or features of a particular disease, disorder, and / or condition, reduction in severity and / or incidence of one or more symptoms or features of a particular disease, disorder, and / or condition.

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

[0063] The present application is further illustrated by the following specific examples. It is to be understood that the particular examples described herein are meant to be illustrative only and are not to be construed as limiting of the scope of the application. The main features of the application can be used in various embodiments without departing from the scope of the application.

[0064] Examples

[0065] In this embodiment, patient-derived spinal cord H3 K27M variant cells are divided into two groups of "high proliferation and replication pressure type" and "high immune infiltration type" according to the age of the patient and the multi-omics information of the tumor, wherein 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: the brain glioma tissue block (brain glioma tissue removed according to the operation process in accordance with the medical ethics in Tiantan Hospital) is placed in a beaker, rinsed with Hanks liquid for 3 times to remove blood stains; then placed in a mixed liquid containing penicillin for 60 minutes; the tissue is cut into 2-3 mm size pieces with ophthalmic scissors for easy digestion; add trypsin liquid equivalent to 50 times the volume of the total amount of tissue pieces, then pour into a flask, and the mouth is laced or plugged with a rubber plug; place in a 37℃ incubator for digestion, shake every 20 minutes during the digestion process; the digestion time is 60 minutes; then filter through a suitable stainless steel sieve to filter out the tissue pieces that have not been fully digested; centrifuge the digestion liquid at 800 rpm to obtain sample cells, aspirate the supernatant, and add primary culture medium to the sediment to a cell density of 10 5 individual / mL, and then placed in a 37℃ constant temperature incubator for culture. Among them, the basic medium of the primary culture medium is a suspension culture medium, which includes: 20 ng / mL of EGF cytokine, 20 ng / mL of bFGF cytokine, 20 ng / mL of streptomycin and 20 ng / mL of penicillin, B27 (50x) and 20 ng / mL of transferrin.

[0067] The single cell suspension obtained by the above suspension culture is inoculated in an ultra-low adsorption culture dish for culture, and then digested into single cells with digestive enzymes. When the repeated operation subculture reaches the 4th generation, part of the primary cells appear death, stop growing or grow slowly, and another part of the primary cells can present a good proliferation state in the serum-free suspension culture medium. These cell lines that present a good proliferation state are selected as further screening objects, MTT experiment and Transwell experiment are carried out to detect the proliferation and migration ability of the cell lines, and 6 cell lines with the strongest proliferation and migration ability are selected for preservation, which are 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-S18 (preservation number CGMCC NO. 45814) disclosed in the present application. The cell morphology of the above 6 cell strains is shown in Figure 1 , and the molecular mutations carried by different spinal cord glioma cells are shown in Figure 2 It can be seen that the above multiple different spinal cord glioma cells all have H3 K27M mutation, while the other mutations are different.

[0068] Specifically, the preservation number of the H3 K27M mutant spinal cord glioma cell SCA-S02 is CGMCC NO. 45809, the tp53 has a missense mutation, the myc has an amplification mutation, and the pdgfra has an amplification mutation.

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

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

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

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

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

[0074] In this embodiment, the gene mutations and copy number variations of the spinal cord glioma cells are detected by whole exome sequencing, and the results are shown in Table 1. Figure 2 As can be seen from Table 1, different spinal cord glioma cells all have H3 K27M mutations (H3F3A K27M), but the accompanying other mutations or copy numbers are different; the above cell strains can more objectively reflect the commonness of this type of tumor cells and the inhibition between tumors, and are suitable for evaluating the biological characteristics of cells carrying different molecular mutations and the response to drug treatment.

[0075] In this embodiment, the cell proliferation is further detected by the CCK-8 method, and the specific process is as follows:

[0076] The above different spinal cord astrocytoma cells in logarithmic growth phase were respectively uniformly plated into 96-well plates, 2000 cells were plated into each well, and after 0, 2, 4, 6, 8 days of culture, CCK8 liquid (purchased from Dojindo, Japan) was added to the 96-well plate cell culture medium at a ratio of 1:10 with the cell culture medium (suspension culture medium), and continued to be cultured at 37°C for 2h, and the absorbance value at 450nm was measured under the enzyme marker, and according to the OD result, the curve was fitted by GraphPad Prism 8 to determine the proliferation rate of different cell strains. In addition to the above 6 spinal cord glioma cells, the proliferation rate of SCA-S01 (preserved number CGMCC No. 23029) and SP0001 (preserved number CGMCC No. 23028) cells preserved by the present inventors in the previous stage was also determined, and the results are shown in Figure 3 The proliferation rates of the above 8 spinal cord glioma cells are obviously different, specifically, the SCA-S02 cell has a faster proliferation rate, the SCA-S09 and SP0001 have a slower proliferation rate, and the other 6 spinal cord glioma cells have a relatively moderate proliferation rate, thus further indicating that the use of the above spinal cord glioma cells for determination can better reflect the commonality, biological characteristics and response to drug treatment of spinal cord glioma cells.

[0077] Example 2

[0078] In the prior art, radiotherapy is commonly used as a treatment scheme for H3 K27M mutant spinal cord glioma, but there is still great uncertainty about whether the patient can benefit from radiotherapy, and the use of radiotherapy generally cannot make the patient benefit from long-term survival. In order to detect the value of the 6 spinal cord glioma cells provided in Example 1 in evaluating H3 K27M mutant spinal cord glioma for radiotherapy and potential targeted therapy, the drug responsiveness of the above 6 isolated H3K27M mutant spinal cord glioma cells and the SCA-S01 and SP0001 cells preserved by the present inventors in the previous stage to different compounds was determined in this embodiment. The specific experiment is as follows.

[0079] In this embodiment, the above 8 spinal cord glioma cells were treated with 0, 2, 4, 6, 8 Gy of radiotherapy dose, and the number of surviving cells in different groups was measured after 6 days of radiotherapy to evaluate their sensitivity to radiotherapy, as shown in Figure 4 As can be seen from the above table, SCA-S10, SCA-S12, SP0001, SCA-SP01 are relatively resistant to radiotherapy, the rest of SCA-S18, SCA-S41, SCA-S09, SCA-S02 are relatively sensitive to radiotherapy, but still have the characteristics of resisting radiotherapy, and still have 50% of the cells surviving when the radiotherapy dose is 8Gy, which shows that such cells have relatively high resistance to radiotherapy as a whole, and still need to be screened for corresponding drugs according to their genetic background.

[0080] The present embodiment further determines the sensitivity of the above-mentioned 8 cell lines to different chemotherapy drugs or different targeted drugs, and the results are shown in the following table. Figures 5-9

[0081] Based on the data of the viability of the spinal cord glioma cells under different inhibitor concentrations, the IC50 value of the inhibitor concentration that reduces the cell viability by 50% is determined by calculation and fitting, and the specific process is as follows.

[0082] The logarithmic growth phase of the spinal cord astrocytoma primary culture cells is uniformly spread in the 96-well plate, 5000 cells are spread in each well, and after 24 h, different concentrations of inhibitor-containing suspension medium are added, the cells in the 96-well plate are cultured in different inhibitors with a final concentration of 0-100 μM (specific concentrations are 0.00001; 0.00010; 0.00100; 0.01000; 0.10000; 1.00000; 10.00000; 100.00000 μM), and the cells under each concentration are not less than 5 wells, and the culture is continued; after 6 days of culture, the CellTiter-Lumi TM luminescence method 3D cell viability detection kit (Bi Yun Tian C0061L) is used to detect the cell viability.

[0083] The inhibition rate of the inhibitor on the spinal cord astrocytoma cells is calculated according to the following formula:

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

[0085] According to the calculation results, the half-inhibitory concentration of various inhibitors on different spinal cord astrocytoma cells is determined by using GraphPad Prism 8 to fit the curve, and the specific results are shown in Table 1.

[0086] Table 1 IC50 values of different spinal cord glioma cells to different inhibitors

[0087]

[0088] According to the detection results in Table 1, it is further indicated that the sensitivity of the 8 strains of spinal cord astrocytoma to various inhibitors is different, among which 6 strains of H3 K27M mutant spinal cord glioma cells are not sensitive to temozolomide, a first-line chemotherapy drug for glioma, which is consistent with the results of clinical cohort study. For the commonly used targeted drugs in clinical practice, such as palbociclib (CDK6 inhibitor, FDA approved) and pediatric high-grade glioma targeted drug paribedostat (HDAC inhibitor, in clinical trials), they are more sensitive; for the clinical targeted drugs such as tasidotin (EZH2 inhibitor, in clinical trials), and methotrexate with intrathecal chemotherapy potential, different spinal cord astrocytoma cell lines have different sensitivities.​

[0089] Specifically, the present disclosure successfully established 6 patient-derived cell lines carrying H3K27M mutation of stem cell-like spinal cord glioma, wherein the IC50 value of SCA-S02 cell line to palbociclib is 4.439 μM, which is relatively low, indicating that SCA-S02 cell is relatively sensitive to palbociclib; the IC50 value to paribranib is 0.01757 μM, indicating that SCA-S02 cell is highly sensitive to paribranib; the IC50 values to temozolomide and taselisib are 123.0 μM and 107.5 μM respectively, which are relatively high, indicating that SCA-S02 cell is generally sensitive to these two drugs; the IC50 value to methotrexate is 662.5 μM, indicating that SCA-S02 cell is not sensitive to methotrexate.

[0090] The IC50 value of SCA-S09 cell line to palbociclib is 4.533 μM, indicating that SCA-S09 cell has certain sensitivity to palbociclib; the IC50 value to paribranib is 0.03588 μM, which is at a relatively low level, indicating that SCA-S09 cell is relatively sensitive to paribranib; the IC50 value to temozolomide is 164.2 μM, which is relatively high, indicating that SCA-S09 cell is generally sensitive to temozolomide; the IC50 value to taselisib is 227.6 μM, indicating that SCA-S09 cell has poor sensitivity to taselisib; the IC50 value to methotrexate is 28.94 μM, which is at a relatively low level, indicating that SCA-S09 cell has high sensitivity to methotrexate.

[0091] The IC50 value of SCA-S41 cell line to paribranib is 0.001297 μM, indicating that SCA-S41 cell is extremely sensitive to paribranib; the IC50 value to palbociclib is 5.249 μM, indicating that SCA-S41 cell has certain sensitivity to palbociclib; the IC50 value to temozolomide is 93.68 μM, which is relatively high, indicating that SCA-S41 cell is generally sensitive to temozolomide; the IC50 value to taselisib is 115.2 μM, which is high, indicating that SCA-S41 cell has poor sensitivity to taselisib; the IC50 value to methotrexate is 117.4 μM, indicating that SCA-S41 cell has poor sensitivity to methotrexate.

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

[0093] The IC50 value of SCA-S12 for palbociclib is 2.045 μM, indicating that the SCA-S12 cell strain is sensitive to palbociclib; the IC50 value for paribromiclib is 0.03229 μM, indicating that the cell strain is highly sensitive to paribromiclib; the IC50 value for temozolomide is 16074 μM, indicating that the SCA-S12 cell strain is extremely insensitive to temozolomide; the IC50 value for thazovatin is 52.11 μM, indicating that the sensitivity to thazovatin is general; the IC50 value for methotrexate is 0.6891 μM, indicating that the cell strain is relatively sensitive to methotrexate.

[0094] The IC50 value of SCA-S18 for palbociclib is 3.375 μM, indicating that the SCA-S18 cell strain has certain sensitivity to palbociclib; the IC50 value for paribromiclib is 0.03960 μM, indicating that the cell strain is relatively sensitive to paribromiclib; the IC50 value for temozolomide is 1453 μM, indicating that the SCA-S18 cell strain has poor sensitivity to temozolomide; the IC50 value for thazovatin is 27.67 μM, indicating that the sensitivity to thazovatin is general; the IC50 value for methotrexate is 0.01178 μM, indicating that the SCA-S18 cell strain is extremely sensitive to methotrexate.

[0095] By the above technical solution, the present disclosure successfully establishes 6 patient-derived cells carrying H3K 27M mutation of stem cell-like spinal cord glioma, and the above 6 cells have different sensitivities to different drugs, and their proliferation speed, migration speed and tumorigenicity are significantly better than other disclosed H3K 27M spinal cord glioma cell lines. The combination of the above 6 cells covers H3K27M mutation of stem cell-like spinal cord glioma with different mutations and cell types with different drug sensitivities, so as to more comprehensively evaluate the efficacy and potential side effects of drugs, and can be used for in vitro research on the biological function of H3K 27M spinal cord glioma and the response to drug treatment.

[0096] The above description of the embodiments is only for understanding the method of the present disclosure and its core idea. It should be noted that for those skilled in the art, without departing from the principles of the present disclosure, some improvements and modifications can be made to the present disclosure, and these improvements and modifications will also fall within the protection scope of the claims of the present disclosure.

Claims

1. An H3 K27M mutant spinal cord glioma cell line, characterized in that, The cell line in question is the SCA-S02 cell line, deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.45809.

2. A reagent, characterized in that, The reagent includes the H3 K27M mutant glioma cell line of claim 1.

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

4. A method for preparing an H3 K27M mutant spinal cord glioma model, characterized in that, The method includes administering the H3 K27M mutant glioma cell line of claim 1 or the reagent of claim 2 to an animal.

5. The method according to claim 4, characterized in that, The animal in question is a mammal.

6. A method for screening drugs to treat H3 K27M mutant spinal cord gliomas, characterized in that, The method includes contacting the drug to be screened with the H3 K27M mutant spinal cord glioma cell line of claim 1 or the reagent of claim 2, and measuring the inhibitory effect of the drug to be screened on the cells.

7. A method for evaluating the efficacy of drugs for H3 K27M mutant spinal cord gliomas (not for therapeutic purposes), characterized in that, The method includes contacting the drug with the H3 K27M mutant spinal cord glioma cell line of claim 1 or the reagent of claim 2, and measuring the inhibitory effect of the drug on the cells.

8. The use of the H3K27M mutant glioma cell line of claim 1 in screening / evaluating drugs for the treatment of H3K27M mutant glioma for non-therapeutic purposes.

9. The application according to claim 8, characterized in that, Evaluation of drugs for spinal cord gliomas includes evaluating the drug sensitivity of spinal cord gliomas.

10. The application of the H3 K27M mutant glioma cell line of claim 1 in the preparation of an H3 K27M mutant glioma model.

11. The application according to claim 10, characterized in that, The models include cell models and animal models.

12. The application of the H3 K27M mutant glioma cell line of claim 1 in studying the mechanisms related to H3 K27M mutant glioma.

13. The application according to claim 12, characterized in that, The relevant mechanisms include the pathogenesis, development, and drug resistance mechanisms of H3 K27M mutant spinal cord glioma.

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