CN117106724A - H3K27M mutant spinal glioma cell and culture method thereof - Google Patents
H3K27M mutant spinal glioma cell and culture method thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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Abstract
The application provides an isolated H3K27M mutant spinal glioma cell, wherein the preservation number of the H3K27M mutant spinal glioma cell is CGMCC No.23028. The spinal glioma cells have stem cell characteristics, can grow in a balling mode, highly express GFAP and Ki-67, can form tumors under the skin of an immunodeficiency mouse, have the same natural drug resistance to Temozolomide (TMZ) as spinal glioma, and can serve for in vitro research on biological functions and response to drug treatment of H3K27M spinal glioma.
Description
Technical Field
The application relates to the field of biomedicine, in particular to an H3K27M mutant spinal glioma cell and a culture method thereof.
Background
In recent years, the theory of tumor stem cells has been paid attention to, and researches show that the growth of tumors is the result of the continuous proliferation of a very small amount of tumor stem cells in the tumors, and only the small group of tumor stem cells can be killed to cure cancer better. Therefore, the potential biological functions of stem cells and the differentiation and proliferation conditions of the stem cells are known, and the method has important clinical significance for early detection, treatment and diagnosis of tumors. However, currently the ratio of tumour stem cells in tumour tissue is very low. In recent years, along with the gradual elucidation of genetic basis of tumorigenesis and the rapid development of molecular diagnosis, molecular pathological diagnosis is gradually widely applied in clinic.
The unique expression of the H3K27M mutation in diffuse midline gliomas among spinal gliomas has become a new class in the central nervous system tumor classification. Therefore, the finding of the glioma cells of the H3K27M mutant type with specific tumor stem property has very important significance for the drug screening of glioma.
Disclosure of Invention
The present disclosure is directed to the development of an isolated H3K27M mutant spinal glioma cell for drug screening of spinal gliomas.
To achieve the above object, a first aspect of the present disclosure provides an isolated H3K27M mutant spinal glioma cell, wherein the H3K27M mutant spinal glioma cell has a preservation number of CGMCC No.23028.
According to the present disclosure, the H3K27M mutant spinal glioma cells have tumor stem properties and can be obtained by suspension culture.
The second aspect of the present disclosure also provides the culture method of glioma cells according to the first aspect, wherein the method comprises the steps of: s1, performing enzymolysis digestion and centrifugation on the glioma cells in the first aspect, and collecting sample cells; s2, re-suspending and centrifuging sample cells by using PBS, and inoculating the sample cells into a suspension culture medium for suspension culture to obtain single cell suspension; s3, inoculating the single cell suspension into an ultralow adsorption culture dish for culture, and then digesting the single cell suspension into single cells by using digestive enzymes.
According to the present disclosure, in step S2, the suspension culture conditions include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
According to the present disclosure, in step S3, the conditions of the subculture include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
According to the present disclosure, wherein the suspension medium contains bFGF cytokine, EGF cytokine, streptomycin, penicillin, B27 supplement, and transferrin.
According to the present disclosure, wherein the concentration of bFGF cytokine is 15-30ng/mL, the concentration of EGF cytokine is 15-30ng/mL, the concentration of B27 supplement is B27 (50X), the concentration of streptomycin is 15-30ng/mL, and the concentration of penicillin is 15-30ng/mL.
According to the present disclosure, wherein the cell density inoculated into the suspension medium is 10 4 -10 6 And each mL.
According to the present disclosure, wherein the conditions of PBS resuspension centrifugation include: the centrifugation speed is 800-1200rpm/min, the centrifugation temperature is 4-35 ℃, and the centrifugation time is 1-10min.
The disclosure also provides the use of the H3K27M mutant spinal glioma cells of the first aspect in screening for a medicament for treating H3K27M mutant spinal glioma.
Through the technical scheme, the spinal glioma cells with stem cell characteristics can be cultured in a suspending mode, carry H3K27M mutation, grow in a balling mode, express GFAP and Ki-67 in a high degree, can form tumors under the skin of an immunodeficiency mouse, have the same natural drug resistance to Temozolomide (TMZ) as spinal glioma, and can serve in-vitro study of biological functions, response to drug treatment and the like of the H3K27M spinal glioma.
Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.
Biological material preservation information
The biological preservation information to which the present disclosure relates includes: the classification is named: a human cell; the preservation units are as follows: china general microbiological culture Collection center (China Committee for culture Collection); the addresses are: beijing, chaoyang area, north Chenxi Lu No. 1, 3; the preservation date is 2021-09-17; the preservation number is: CGMCC No.23028.
Drawings
The accompanying drawings are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:
FIG. 1 is a cell morphology of the H3K27M mutant spinal glioma cells SCA-S01 cultured according to the present disclosure.
FIG. 2 is the second generation sequencing results of the cultured H3K27M mutant spinal glioma cells SCA-S01 of the present disclosure.
FIG. 3 is a chart of immunohistochemical staining of the cultured H3K27M mutant spinal glioma cells SCA-S01 of the present disclosure.
FIG. 4 is a response of the cultured H3K27M mutant spinal glioma cells SCA-S01 of the present disclosure at different concentrations of TMZ.
Figure 5 is a response of control brain glioma stem cells at different concentrations of TMZ.
Detailed Description
The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.
The first aspect of the present disclosure provides an isolated H3K27M mutant spinal glioma cell with a preservation number of CGMCC No.23028.
According to the disclosure, wherein the H3K27M mutant spinal glioma cells have tumor stem properties, can be obtained by suspension culture.
The H3K27M mutation is a high grade glioma of the infiltrating midline predominantly astrocyte differentiation accompanied by H3K27M mutation, wherein the lysine at position 27 in the H3 histone is mutated to an egg amino acid.
A second aspect of the present disclosure provides a method for culturing the glioma cells of the first aspect, wherein the method comprises the steps of:
s1, performing enzymolysis digestion and centrifugation on the glioma cells of the first aspect, and collecting sample cells;
s2, re-suspending and centrifuging sample cells by using PBS, and inoculating the sample cells into a suspension culture medium for suspension culture to obtain single cell suspension;
s3, inoculating the single cell suspension into an ultralow adsorption culture dish for culture, and then digesting the single cell suspension into single cells by using digestive enzymes.
According to the present disclosure, spinal glioma cells are seeded at a lower density in a liquid or semi-solid serum-free medium, and an ultra-low adsorption culture vessel is used to reduce cell attachment during culture, and the serum-free medium and the ultra-low adsorption culture vessel can maintain the cells in an undifferentiated state, and only cells with stem cell potential can survive and proliferate for a long period of time in a serum-free environment.
According to the present disclosure, in step S2, the suspension culture conditions include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
According to the present disclosure, in step S3, the conditions of the subculture include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
According to the present disclosure, wherein the suspension medium contains bFGF cytokine, EGF cytokine, streptomycin, penicillin, B27 supplement, and transferrin.
According to the present disclosure, wherein the concentration of bFGF cytokine is 15-30ng/mL, the concentration of EGF cytokine is 15-30ng/mL, the concentration of B27 supplement is B27 (50X), the concentration of streptomycin is 15-30ng/mL, and the concentration of penicillin is 15-30ng/mL.
According to the present disclosure, wherein the cell density inoculated into the suspension medium is 10 4 -10 6 And each mL.
According to the present disclosure, wherein the conditions of PBS resuspension centrifugation include: the centrifugation speed is 800-1200rpm/min, the centrifugation temperature is 4-35 ℃, and the centrifugation time is 1-10min.
The disclosure also provides the use of the H3K27M mutant spinal glioma of the first aspect in screening a medicament for the treatment of H3K27M mutant spinal glioma.
The present disclosure is further illustrated by the following examples, but the present disclosure is not limited thereby.
Example 1
Cell suspension culture process
Placing the glioma tissue blocks (glioma tissues which are extracted by operation in the Tiantan hospital according to the operation flow conforming to medical ethics) in a beaker, and rinsing for 3 times by Hanks liquid to remove blood stains; then placing the mixture in a mixed solution containing the green streptomycin for 60 minutes; cutting the tissue into pieces of 2-3 mm size with an ophthalmic scissors to facilitate digestion; adding trypsin liquid which is 50 times of the total tissue mass, and then pouring the trypsin liquid into the triangular flask, ligating a bottle mouth or plugging a rubber plug; placing the mixture in a 37 ℃ incubator for digestion, and shaking the mixture once every 20 minutes in the digestion process; digestion time was 60 minutes; filtering out tissue blocks which are not fully digested by a proper stainless steel screen; separating heart digestive juice at 800 rpm to obtain sample cells, sucking out supernatant, adding primary culture medium into the precipitate until cell density is 10 5 Each mL was then incubated in an incubator at 37 ℃. Wherein, the basic culture medium of the primary culture medium is a suspension culture medium, and the suspension culture medium comprises: EGF cytokine 20ng/mL, bFGF cytokine 20ng/mL, streptomycin 20ng/mL and penicillin 20ng/mL, B27 (50X) and transferrin 20 ng/mL.
Inoculating the single cell suspension obtained by suspension culture into an ultralow adsorption culture dish for culture, then digesting the single cell by using digestive enzyme, repeating the operation and subculturing until the 4 th generation, wherein part of primary cells die, stop growing or grow slowly, the other part of primary cells can be in a good proliferation state in a serum-free suspension culture medium, selecting cell lines in the good proliferation state as further screening objects, carrying out MTT experiments and Transwell experiments, detecting proliferation and migration capacities of the cell lines, selecting one cell line with the strongest proliferation and migration capacities for preservation, namely the glioma cell SCA-S01 carrying the H3K27M mutation, which is cultured by the disclosure, with the preservation number of CGMCC No.23028, wherein the cell morphology picture is shown in figure 1, the left side is the glioma stem cell established by the disclosure, and the right side is the glioma stem cell (cultured by a laboratory).
The H3K27M mutation carried by established glioma stem cells was determined by a second generation sequencing technique and the results showed that the cells carried the H3K27M mutation, as shown in figure 2.
The cells are injected subcutaneously into an immunodeficient mouse, and a spinal glioma tumor can be formed in the tumor of the immunodeficient mouse. After the tumor was removed, paraffin-embedded sections were prepared and immunohistochemical staining was performed, and it was observed that the tumor carried the H3K27M mutation, and Ki-67 was highly expressed, as shown in FIG. 3.
Example 2
Temozolomide drug is known to be effective in some glioma patients, but not in another; wherein temozolomide is not effective against spinal gliomas of H3K27M, the test examples separately determine drug responsiveness of spinal glioma cells SCA-S01 of the present disclosure and control cells (brain glioma stem cells cultured in the laboratory) to temozolomide.
Temozolomide drug was administered to control brain glioma stem cells and spinal glioma cells SCA-S01 of the present disclosure at final drug concentrations of 100, 200, 400, 500, 800, 1000, and 2000 μm, respectively, and after 72 hours of drug action, cell viability was measured using CCK8 kit (available from merck corporation) and the results are shown in fig. 4 and 5.
Fig. 4 is a graph showing the inhibition of cells and TMZ IC50 values of spinal glioma cells SCA-S01 cultured according to the present disclosure under different concentrations of TMZ treatment, and fig. 5 is a graph showing the inhibition of cells and TMZ IC50 values of control brain glioma stem cells under different concentrations of TMZ treatment. The result shows that the difference of the response of the brain glioma stem cells to TMZ compared with the control shows that the spinal glioma cells SCA-S01 prepared by the method can resist TMZ, further proves that the spinal glioma cells SCA-S01 provided by the method has tumor stem property, carries H3K27M mutation and can feed back the characteristic that the spinal glioma resists temozolomide.
Through the technical scheme, the application successfully establishes a stem cell-like spinal glioma patient-derived cell (SCA-S01 stem cell-like cell) carrying the H3K27M mutation, the cell grows in a balling mode, is naturally resistant to TMZ as parent spinal glioma, can form tumors under the skin of an immunodeficient mouse, and the spinal glioma cell SCA-S01 carries the H3K27M mutation and highly expresses Ki-67, and can be used for in vitro research on biological functions of the H3K27M spinal glioma, response to drug treatment and the like.
The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.
In addition, the specific features described in the above embodiments may be combined in any suitable manner without contradiction. The various possible combinations are not described further in this disclosure in order to avoid unnecessary repetition.
Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.
Claims (10)
1. An isolated H3K27M mutant spinal glioma cell, which is characterized in that the preservation number of the H3K27M mutant spinal glioma cell is CGMCC No.23028.
2. The spinal glioma cell according to claim 1, wherein the H3K27M mutant spinal glioma cell has tumor stem property, and can be obtained by suspension culture.
3. The method of culturing spinal glioma cells according to claim 1 or 2, wherein the method comprises the steps of:
s1, performing enzymolysis digestion and centrifugation on the glioma cells in claim 1 or 2, and collecting sample cells;
s2, re-suspending and centrifuging sample cells by using PBS, and inoculating the sample cells into a suspension culture medium for suspension culture to obtain single cell suspension;
s3, inoculating the single cell suspension into an ultralow adsorption culture dish for culture, and then digesting the single cell suspension into single cells by using digestive enzymes.
4. A culture method according to claim 3, wherein in step S2, the conditions of the suspension culture include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
5. A culture method according to claim 3, wherein in step S3, the conditions of the subculture include: the culture time is 3-10 days, and the culture temperature is 35-45 ℃.
6. The culture method according to claim 3, wherein the suspension medium contains bFGF cytokine, EGF cytokine, streptomycin, penicillin, B27 supplement and transferrin.
7. The culture method according to claim 6, wherein the bFGF cytokine is at a concentration of 15-30ng/mL, the EGF cytokine is at a concentration of 15-30ng/mL, the B27 supplement is at a concentration of B27 (50X), the streptomycin is at a concentration of 15-30ng/mL, and the penicillin is at a concentration of 15-30ng/mL.
8. The culture method according to claim 3, wherein the cell density inoculated into the suspension medium is 10 4 -10 6 And each mL.
9. A culture method according to claim 3, wherein the conditions of PBS resuspension centrifugation include: the centrifugation speed is 800-1200rpm/min, the centrifugation temperature is 4-35 ℃, and the centrifugation time is 1-10min.
10. Use of the H3K27M mutant spinal glioma cells of claim 1 or 2 in the screening of a medicament for the treatment of H3K27M mutant spinal glioma.
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