A medulloblastoma cell line

Abstract

The application discloses a medulloblastoma cell line. The medulloblastoma cell line provided by the application is identified by STR and karyotype analysis, no human cell cross contamination is found in the cell, and no cell with more than 80% matching degree of STR typing data is found in the Cellosaurus database. The cell line is also proved to be a neural tumor by HE staining, kki67 staining and synaptophysin staining. The cell can be stably passaged and can be used for drug sensitivity test. The cell line provided by the application provides a new direction for the research on the related mechanism and treatment method of medulloblastoma.

Description

TECHNICAL FIELD

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

[0002] Medulloblastoma (MB) is a common intracranial malignant tumor in childhood, which is an embryonic tumor. Most MBs occur in children under the age of 10, and adult MBs are rare, accounting for about 1% of the incidence of MB. MB is highly malignant, and when the patient is diagnosed, the patient has often had cerebrospinal fluid dissemination metastasis, and the incidence rate is as high as 30%. The growth site of MB is often in the midline of the posterior fossa, often grows rapidly to occupy the fourth ventricle, and invades the cerebellum and forms adhesion to the brain stem. The clinical manifestations of MB patients are mostly related to increased intracranial pressure, because the rapid growth of the tumor hinders the normal circulation of cerebrospinal fluid, and some patients also show corresponding symptoms due to cerebellar and local nerve compression. The current treatment for MB is a comprehensive scheme combining surgery and radiotherapy and chemotherapy. The size and location of the tumor may affect the total resection rate of surgery, which may directly affect the prognosis of the patient.

[0003] In order to further study the pathogenesis and treatment method of medulloblastoma, it is crucial to establish a suitable research model. SUMMARY

[0004] In order to make up for the deficiencies of the prior art, the present application provides a medulloblastoma cell line.

[0005] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The first aspect of the present application provides a medulloblastoma cell line, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202469.

[0007] Further, the medulloblastoma cell line also includes a daughter cell line thereof.

[0008] The daughter cell line is obtained after the medulloblastoma cell line is cultured. The daughter cell line basically or entirely retains the characteristics of the parent cell.

[0009] The second aspect of the present application provides a cell model, which includes the medulloblastoma cell line of the first aspect of the present application.

[0010] The third aspect of the present application provides a reagent, which includes the medulloblastoma cell line of the first aspect of the present application.

[0011] The fourth aspect of the present application provides a kit, which includes the medulloblastoma cell line of the first aspect of the present application or the reagent of the third aspect of the present application.

[0012] Further, the kit further comprises an instruction.

[0013] Further, the kit further comprises a reagent for processing the sample.

[0014] The fifth aspect of the present application provides any one of the following uses:

[0015] (1) the medulloblastoma cell line of the first aspect of the present application in the preparation of a model;

[0016] (2) the medulloblastoma cell line of the first aspect of the present application in the screening / evaluation / preparation of a drug for treating medulloblastoma;

[0017] (3) the medulloblastoma cell line of the first aspect of the present application in the study of medulloblastoma-related mechanisms;

[0018] (4) the medulloblastoma cell line of the first aspect of the present application in the development of a drug target for medulloblastoma;

[0019] (5) the medulloblastoma cell line of the first aspect of the present application in the preparation of a diagnostic reagent for medulloblastoma.

[0020] Further, the model comprises a cell model, an animal model.

[0021] Further, the drug for evaluating medulloblastoma comprises evaluating the drug sensitivity of medulloblastoma.

[0022] Further, the drug comprises one or more of cisplatin, cyclophosphamide or vincristine.

[0023] Further, the medulloblastoma-related mechanism comprises the pathogenesis, development mechanism, and drug resistance mechanism of medulloblastoma.

[0024] The sixth aspect of the present application provides a method for preparing a medulloblastoma model, the method comprising administering the medulloblastoma cell line of the first aspect of the present application to an animal.

[0025] Further, the animal is a mammal.

[0026] The seventh aspect of the present application provides a method for screening a drug for treating medulloblastoma, the method comprising contacting the drug to be screened with the medulloblastoma cell line of the first aspect of the present application, and determining the inhibitory effect of the drug to be tested on the cells. The drug that inhibits the growth of medulloblastoma cells is selected.

[0027] The eighth aspect of the present application provides a method for testing the sensitivity of medulloblastoma drugs, which comprises contacting a drug to be tested with the medulloblastoma cell line of the first aspect of the present application, and further determining the IC50.

[0028] The ninth aspect of the present application provides a method for constructing the medulloblastoma cell line of the first aspect of the present application, which comprises culturing after tumor tissue digestion, and using semi-replacement principle for subculture.

[0029] Further, the tumor tissue is digested by enzyme digestion method.

[0030] Further, the enzyme comprises collagenase.

[0031] Further, the collagenase is selected from type IV collagenase.

[0032] Further, the components of the culture medium after tumor tissue digestion comprise FBS, basal medium, double antibodies, serum-free additives, ITS, and FGF.

[0033] Further, the basal medium is selected from DMEM.

[0034] Further, the FGF is selected from bFGF.

[0035] Further, the double antibodies comprise penicillin and streptomycin.

[0036] Further, the serum-free additives comprise B-27 and N-2.

[0037] Preservation information of the biological material:

[0038] Scientific description: human child medulloblastoma primary cell line CMPC Homo sapiens

[0039] Preservation unit: China Center for Type Culture Collection (CCTCC)

[0040] Preservation address: Wuhan University, Wuhan, China

[0041] Preservation date: May 11, 2024

[0042] Preservation number: CCTCC NO: C202469

[0043] Advantages and beneficial effects of the present application:

[0044] The medulloblastoma cell line provided by the application is identified by STR and karyotype analysis, no human cell cross contamination is found in the cell, and no cell with more than 80% matching STR typing data is found in the Cellosaurus database, cell line HE staining, ki67 staining and synaptophysin staining also prove that the cell is a neural tumor, and the cell can be stably passaged and used for drug sensitivity test. The cell line provided by the application provides a new direction for the research on the related mechanism and treatment method of medulloblastoma. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 is a drug sensitivity diagram of cisplatin, cyclophosphamide and vincristine;

[0046] Figure 2 is a cell passage diagram;

[0047] Figure 3 is a karyotype analysis diagram of the cell;

[0048] Figure 4 is a cell line HE staining diagram;

[0049] Figure 5 is a ki67 staining diagram;

[0050] Figure 6 is a synaptophysin staining diagram;

[0051] Figure 7 is a cell passage result diagram. DETAILED DESCRIPTION

[0052] The following provides definitions of some terms used in the specification. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the application belongs.

[0053] The application provides a kit, which comprises the above-mentioned medulloblastoma cell line or the above-mentioned reagent.

[0054] In one embodiment, 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 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 (such as slides, containers, microtiter plates).

[0055] In one embodiment, the kit can be provided as an article of manufacture including 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 separating 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.

[0056] The present application provides any one of the following uses:

[0057] (1) use of the above medulloblastoma cell line in preparing a model;

[0058] (2) use of the above medulloblastoma cell line in screening / evaluating / preparing a drug for treating medulloblastoma;

[0059] (3) use of the above medulloblastoma cell line in studying the mechanism related to medulloblastoma;

[0060] (4) use of the above medulloblastoma cell line in developing a drug target for medulloblastoma;

[0061] (5) use of the above medulloblastoma cell line in preparing a diagnostic reagent for medulloblastoma.

[0062] In one embodiment, the model described in (1) includes a cell model, an animal model. The animal model includes transplanting the above medulloblastoma into a non-human animal to form a medulloblastoma animal model.

[0063] In one embodiment, the non-human animal refers to all animals, except humans, and includes but is not limited to birds, farm animals (e.g., cows), sports animals (e.g., horses), fish, reptiles, and non-human mammals.

[0064] In one embodiment, the non-human animal is preferably a non-human mammal.

[0065] In one embodiment, 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 and guinea pigs.

[0066] In one embodiment, the medulloblastoma animal model can be transplanted by any method disclosed in the prior art, for example, it can be subcutaneous injection of single cell suspension, etc.

[0067] In one embodiment, the screening of a drug for treating medulloblastoma comprises contacting the medulloblastoma 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, and screening a drug capable of treating medulloblastoma.

[0068] In one embodiment, the contacting of the medulloblastoma cell line with the drug to be tested means placing the above-mentioned medulloblastoma cell line and the drug to be tested in a state where they can contact each other. The contacting of the above-mentioned medulloblastoma cell line with the drug to be tested can be, for example, the addition of the drug to be tested to a solution containing the above-mentioned medulloblastoma cell line.

[0069] In one embodiment, the drug to be tested can be, for example, 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 can be, for example, a drug for treating a disease or cancer other than medulloblastoma. The drug to be tested can be, for example, one or a mixture of two or more. The drug to be tested is preferably one substance.

[0070] In one embodiment, the measurement of the medulloblastoma cell contacted with the above-mentioned 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 a marker protein constituting a cell. Among them, the change in cell size can be, for example, the measurement of the size of a cell, and the size of a cell can be measured using a publicly known device (e.g., a microscope or a FACS). The size of a cell can be, for example, the circumference, the diameter (e.g., the major diameter and the minor diameter), or the area of a cell.

[0071] In one embodiment, the measurement value of a cell can be, for example, one measurement value measured at a time, an average value of a plurality of measurement values measured at a plurality of times, or an average value of a plurality of measurement values of a plurality of cells. The measurement value of a cell can be, for example, a change value (e.g., a difference or a fold) before and after the contact with the drug to be tested. The control value can be, for example, a measurement value of a control cell before the contact with the above-mentioned drug to be tested or a measurement value of a control cell not contacted with the above-mentioned drug to be tested, when the value after the contact with the drug to be tested is used as the measurement value of a cell.

[0072] For example, in the case where the measurement value is the size of a cell, the determination of whether or not the drug to be tested is a substance capable of treating medulloblastoma can include the step of determining the above-mentioned drug to be tested as a drug capable of treating medulloblastoma when the measurement value of the size of a cell after the contact with the drug to be tested is smaller than the control value (i.e., the degree of increase in the size of a cell is decreased or the size of a cell is decreased).

[0073] In one embodiment, developing a medulloblastoma drug target includes studying molecular markers of medulloblastoma by comparing normal cells with medulloblastoma cells, and developing drugs based on the molecular markers.

[0074] In one embodiment, preparing a medulloblastoma diagnostic reagent includes studying tumor molecular markers specific to medulloblastoma by comparing normal cells with medulloblastoma cells, and developing detection reagents and kits for detecting the occurrence or development of medulloblastoma based on the tumor molecular markers.

[0075] In one embodiment, treatment refers to any method for partially or completely relieving, ameliorating, reducing, inhibiting, preventing one or more symptoms or characteristics of a particular disease, disorder and / or condition, delaying the onset of one or more symptoms or characteristics of a particular disease, disorder and / or condition, and / or reducing the severity and / or incidence of one or more symptoms or characteristics of a particular disease, disorder and / or condition.

[0076] The present application provides a method for constructing the above-mentioned medulloblastoma cell line, which comprises culturing after tumor tissue digestion and using semi-replacement principle for subculture.

[0077] In one embodiment, the tumor tissue is digested by using enzyme digestion method. The enzyme can be any enzyme capable of achieving tumor tissue digestion, including but not limited to trypsin, collagenase, hyaluronidase, deoxyribonuclease I (DNase I), dispase, and elastase.

[0078] Among them, collagenase can be subdivided into type I collagenase (type I collagenase), type II collagenase (type II collagenase), type III collagenase (type III collagenase), type IV collagenase (type IV collagenase), and type V collagenase (type V collagenase).

[0079] In a specific embodiment, the collagenase is selected from type IV collagenase.

[0080] In one embodiment, the culture medium after tumor tissue digestion comprises FBS, basal medium, double antibody, serum-free additive, ITS, and FGF.

[0081] In an embodiment, a culture medium is recognized in the art and generally refers to any substance or preparation used to cultivate living cells. As used in cell culture, a culture medium includes the components of the environment surrounding the cells. A culture medium can be a mixture of solids, liquids, gases, and materials. Culture media include liquid growth media. Culture media also include gelatinous media, such as agar, agarose, gelatin, and collagen matrices. Exemplary gaseous culture media include a gas phase to which cells growing on a petri dish or other solid or semi-solid support are exposed. Culture medium also refers to materials intended for use in cell culture, even if the materials have not yet been brought into contact with cells. In other words, a nutrient-rich liquid prepared for use in cultivation is a culture medium. A basal medium refers to a medium that promotes the growth of many types of microorganisms without the need for any special nutritional supplements. Basal media are generally used as a base for more complex media, to which supplements such as serum or serum substitutes, buffers, growth-promoting factors, antibiotics, and the like are added.

[0082] In an embodiment, a culture medium includes a basal medium, including but not limited to Dulbecco's Modified Eagle Medium (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Glasgow Minimal Essential Medium (G-MEM), Eagle's Basal Medium (BME), DMEM / Ham's F12, DMEM / F12, Iscove's Modified Dulbecco's Medium, Ham's F-10, Ham's F-12, 199 Medium, RPMI 1640 Medium.

[0083] In a specific embodiment, the basal medium is selected from DMEM.

[0084] In an embodiment, examples of fibroblast growth factor (FGF) in the culture medium include, but are not limited to, one or several of FGF-1, FGF-2 (bFGF), FGF-3, FGF-4, FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-15, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20, FGF-21, FGF-22, FGF-23.

[0085] In a specific embodiment, the FGF is selected from bFGF.

[0086] In one embodiment, the culture medium can further comprise amino acids, including L-alanine, L-arginine, L-asparagine, L-aspartate, L-cysteine, L-cystine, L-glutamic acid, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

[0087] In one embodiment, the culture medium can further comprise vitamins, including but not limited to thiamine (vitamin Bl), riboflavin (vitamin B2), niacin (vitamin B3), D-calcium pantothenate (vitamin B5), pyridoxal / pyridoxal phosphate / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), ergocalciferol (vitamin D2), DL-alpha-tocopherol (vitamin E), biotin (vitamin H), menadione (vitamin K).

[0088] In one embodiment, the culture medium can further comprise inorganic salts, including but not limited to salts of calcium, copper, iron, magnesium, potassium, sodium, zinc. The salts are generally used in the form of chlorides, phosphates, sulfates, nitrates, and bicarbonates. More specifically, the salts include but are not limited to CaCl2, CuSO4-5H2O, Fe(NO3)9H2O, FeSO4-7H2O, MgCl, MgSO4, KCl, NaHCO3, NaCl, Na2HPO4, Na2HPO4-H2O, ZnSO4-7H2O.

[0089] In one embodiment, the culture medium can further comprise sugars that can be a carbon energy source. The sugars include but are not limited to glucose, galactose, mannose, fructose. Among them, as the sugar, glucose is preferred, and D-glucose (dextrose) is more preferred.

[0090] In one embodiment, the culture medium can further comprise trace elements. The trace elements include barium, bromine, cobalt, iodine, manganese, chromium, copper, nickel, selenium, vanadium, titanium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, tin, zirconium, cadmium, zinc, aluminum, or ions thereof.

[0091] In one embodiment, passaging refers to the division of cells in a culture vessel into 2 or more culture vessels, typically including the addition of fresh culture medium. Passaging is typically performed when the cells reach a certain density in the culture.

[0092] The present application is further illustrated by the following specific examples. It is to be understood that the specific embodiments described herein are presented by way of example only and are not intended to be limiting. The essential features of the present application can be used in various embodiments without departing from the scope of the present application.

[0093] Examples

[0094] 1. Primary cell culture, passaging and preservation

[0095] The core part of medulloblastoma tumor tissue is obtained aseptically during surgery, and after the tumor tissue is removed from the body, it is placed in a centrifuge tube containing DMEM (the liquid level is higher than the tumor tissue), and it is placed in an ice box for half an hour and then transferred to the laboratory for primary cell culture operation. Wash twice with phosphate buffered saline (PBS) to wash away residual red blood cells, and mechanically dissociate (1-2 mm in diameter) with sterile scissors to prepare a single cell suspension, then centrifuge (1200 rpm, 3 min, 4°C). After centrifugation, add 5 ml of DMEM and 100 μl of type IV collagenase, mix well, and place in a 37°C humidified incubator containing 5% CO2 for 15 minutes of complete digestion, blowing every five minutes to ensure full contact. After complete digestion, the suspension is filtered through a sterile filter with a pore size of 200 um and centrifuged (1200 rpm, 3 min, 4°C). After centrifugation, the cell pellet is resuspended in 5 ml of pre-prepared special culture medium containing 10% serum [for example, 500 ml of culture medium: fetal bovine serum (FBS) 50 ml + DMEM 450 ml + double-antibiotic 10 ml (100 U / ml penicillin and 0.1 mg / ml streptomycin) + B-27 serum-free supplement (50X) 10 ml + N-2 supplement (100X) 5 ml + ITS solution 5 ml + bFGF 100 μl], and cultured in a T25 culture dish (2 ml of Matrigel is added and coated overnight at 37°C in a humidified incubator containing 5% CO2).

[0096] In the first few passages, the semi-replacement principle is adopted. When the primary tumor cells in the culture dish reach 90% confluence, they are digested with 0.25% trypsin and passaged at a ratio of 1:3, or all stable passaged cells are centrifuged and resuspended in 1 ml of culture medium (containing 10% dimethyl sulfoxide), then transferred to liquid nitrogen for long-term preservation after gradient cooling at -80°C overnight.

[0097] 2. Drug sensitivity test

[0098] The primary cell line of medulloblastoma was subjected to drug sensitivity test of conventional chemotherapeutic drugs, and the corresponding concentration gradient was set for the killing experiment of cisplatin, cyclophosphamide and vincristine (cisplatin concentration gradient: 0 μM; 3.906 μM; 7.813 μM; 15.625 μM; 31.25 μM; 62.5 μM; 125 μM; 250 μM cyclophosphamide concentration gradient: 0 μM; 3.906 μM; 7.813 μM; 15.625 μM; 31.25 μM; 62.5 μM; 125 μM; 250 μM; 500 μM; vincristine concentration gradient: 0 μM; 3.906 μM; 7.813 μM; 15.625 μM; 31.25 μM; 62.5 μM; 125 μM; 250 μM; 500 μM;), and the IC50 of the three conventional chemotherapeutic drugs was determined (cisplatin IC50: 155.5 μM; cyclophosphamide IC50: 376.8 μM; vincristine: 65.08 μM), proving that the cell line can be applied to drug sensitivity test Figure 1 ).

[0099] 3. Cell proliferation

[0100] Three kinds of cells of different generations (P10; P20; P40) of the tumor primary cells were selected for proliferation experiment to detect the proliferation of different generations of the primary cell strain. The cells were planted in 96-well plates at 3000 / well, the natural growth of the cells was monitored for seven days, the cell growth on the same day was detected by celltiter every day, and the growth curve for seven days was recorded, proving that the cells can be stably passaged to 40 generations and maintain a certain growth rate Figure 2 ).

[0101] 4. Cell STR identification

[0102] 20 STR loci and 1 gender locus were amplified by multiplex PCR complex amplification system (CELL STR ); the PCR amplification products were analyzed by ABI 3130xl DNA Analyzer (Applied ); and the detection results were analyzed by GeneMapper ID-X v1.5 (Applied ) software.

[0103] The results proved that no human cell cross contamination was found in the cells, and no cell with STR typing data matching degree greater than 80% (8 STR loci matched) was found in the Cellosaurus database; the STR typing results showed that no multiple alleles were found at each locus. No human cell cross contamination was found in the cells (Table 1, Table 2).

[0104] Table 1 sample control results

[0105]

[0106]

[0107] Table 2 Genotyping results of STR loci and Amelogenin loci of cell TTST0815

[0108]

[0109] 5. Karyotype analysis

[0110] The cell was subjected to chromosome karyotype analysis by G banding, and 130 were scanned, and 14 were polyploid. 20 were analyzed for diploidy, and the chromosome number was 46, the number was normal, the sex chromosome was composed of XY, and it was a male karyotype; no obvious abnormality was found in the chromosome structure. Figure 3 ).

[0111] 6. Staining

[0112] HE staining:

[0113] 1) Paraffin section dewaxing to water: sequentially place the section into environment-friendly dewaxing solution I for 20 min - environment-friendly dewaxing solution II for 20 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - 75% alcohol for 5 min, and wash with water. Rewarming fixation of frozen section: take the frozen section out of the -20°C refrigerator to room temperature, fix with tissue fixing solution for 15 min, and then wash with running water.

[0114] 2) Pretreatment: place the section into high-definition constant dyeing pretreatment solution for 1 min.

[0115] 3) Hematoxylin staining: place the section into hematoxylin dyeing solution for 3-5 min, wash with water, differentiate with differentiation solution, wash with water, return to blue with return blue solution, and wash with running water.

[0116] 4) Eosin staining: dehydrate the section in 95% alcohol for 1 min, and then dye in eosin dyeing solution for 15 s.

[0117] 5) Dehydration and mounting: sequentially place the section into anhydrous ethanol I for 2 min - anhydrous ethanol II for 2 min - anhydrous ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min, and then transparentize and mount with neutral balsam.

[0118] 6) Microscope examination, image collection and analysis.

[0119] Cell climbing sheet immunohistochemistry:

[0120] 1) Cell membrane breaking: After the slide is slightly dried, draw a circle in the middle of the cover glass where the cells are evenly distributed to prevent the antibody from flowing away. Add 50-100 μL of membrane breaking solution and incubate at room temperature for 20 min. Wash with PBS for 3 times, 5 min each time.

[0121] 2) Serum blocking: Add 3% BSA evenly to the tissue in the circle and block at room temperature for 30 min.

[0122] 3) First antibody: Gently shake off the blocking solution and add the first antibody in the cell well plate. Incubate at 4°C overnight.

[0123] 4) Second antibody: Place the cell well plate on the decolorizing shaker and shake for 3 times, 5 min each time. After slightly shaking, add the second antibody (HRP labeled) corresponding to the first antibody in the kit to cover the tissue and incubate at room temperature for 50 min.

[0124] 5) DAB color development: Place the cell well plate in PBS (pH 7.4) on the decolorizing shaker and shake for 3 times, 5 min each time. After slightly shaking, add freshly prepared DAB color developing solution to the circle. Control the color development time under a microscope. The positive result is brownish yellow. Wash the slice to stop the color development.

[0125] 6) Cell nucleus restaining: Add hematoxylin for 3 min or so, wash with water, add hematoxylin differentiation solution for a few seconds, wash with water, add hematoxylin blue solution to return to blue, and wash with water.

[0126] 7) Dehydration and mounting: Add 75% alcohol for 5 min, then 85% alcohol for 5 min, then anhydrous ethanol I for 5 min, and then anhydrous ethanol II for 5 min. Take the cover glass out of the alcohol and dry it with a hair dryer. Then mount the cell side down on a glass slide with neutral balsam.

[0127] 8) Microscope examination, image collection and analysis.

[0128] Cell line HE staining ( Figure 4 ), ki67 ( Figure 5 ) and synaptophysin ( Figure 6 ) staining pictures prove that the cells are neural tumors.

[0129] In addition, different generation characteristic cell photos show that the representative cells of each generation are visible, and the cell morphology observed under a microscope does not change significantly ( Figure 7 ).

[0130] The above description of the embodiments is only for understanding the method of the present application and its core idea. It should be noted that, for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications will also fall within the protection scope of the claims of the present application.

Claims

1. A medulloblastoma cell line, characterized in that, It is preserved in China Center for Type Culture Collection, and the preservation number is CCTCC NO: C202469.

2. The medulloblastoma cell line of claim 1, wherein, The medulloblastoma cell line also includes its daughter cell line.

3. A cellular model, characterized in that, The cell model includes the medulloblastoma cell line of claim 1 or 2.

4. An agent, characterized in that, The reagent includes the medulloblastoma cell line of claim 1 or 2.

5. A kit characterized in that, The kit includes the medulloblastoma cell line of claim 1 or 2 or the reagent of claim 4.

6. The kit of claim 5, wherein The kit also includes instructions.

7. The kit of claim 5, wherein The kit also includes reagents for processing samples.

8. Any of the following applications: (1) the medulloblastoma cell line of claim 1 or 2 for use in preparing a cell model or an animal model; (2) the medulloblastoma cell line of claim 1 or 2 for use in screening / evaluating / preparing a drug for treating medulloblastoma; (3) the medulloblastoma cell line of claim 1 or 2 for use in studying the pathogenesis, development mechanism or drug resistance mechanism of medulloblastoma; (4) the medulloblastoma cell line of claim 1 or 2 for use in developing a drug target for medulloblastoma; (5) the medulloblastoma cell line of claim 1 or 2 for use in preparing a diagnostic reagent for medulloblastoma.

9. Use according to claim 8, characterized in that, The drug for evaluating medulloblastoma includes a drug for evaluating the sensitivity of medulloblastoma.

10. Use according to claim 9, characterized in that, The drug includes one or more of cisplatin, cyclophosphamide or vincristine.

11. A method of preparing a medulloblastoma model, characterized by, The method includes administering the medulloblastoma cell line of claim 1 or 2 to a non-human animal.

12. The method of claim 11, wherein, The animal is a non-human mammal.

13. A method of screening for a drug for treating medulloblastoma, characterized by, The method includes contacting the drug to be screened with the medulloblastoma cell line of claim 1 or 2, and determining the inhibitory effect of the drug to be tested on the cells.

14. A method of testing medulloblastoma drug sensitivity, characterized by, The method includes contacting the drug to be tested with the medulloblastoma cell line of claim 1 or 2.

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