Human brain glioma primary cell, progeny cell and application thereof

Abstract

The invention discloses a primary cell and a progeny cell of human brain glioma and application of the primary cell and the progeny cell. The invention provides a human brain glioma primary cell which is a human brain glioma primary cell Glioma-2, and the preservation number of the human brain glioma primary cell Glioma-2 in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: C202420. The human brain glioma primary cell Glioma-2 provided by the invention can form clone in soft agar, and shows different drug toxic reactions to common chemotherapeutic drugs (cis-platinum, carboplatin, etoposide, carmostine and irinotecan) for brain glioma. The human brain glioma primary cell Glioma-2 provided by the invention can be used for analyzing the pathogenesis of brain glioma in vitro and in vivo and detecting related characteristics such as drug sensitivity in vitro, and an experimental material closer to clinical tumor biological characteristics is provided for research of human brain glioma.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology, and particularly relates to a primary human glioma cell, a progeny cell thereof, and their applications. Background Art

[0002] Glioma refers to a tumor originating from glial cells of the brain nerve, and is the most common primary intracranial tumor. The diagnosis of glioma requires obtaining specimens through tumor resection surgery or biopsy surgery, and performing integrated diagnosis of tissue pathology and molecular pathology to determine the pathological grade and molecular subtype. Molecular markers are of great significance for the individualized treatment of glioma and the judgment of clinical prognosis. The treatment of glioma mainly relies on surgical resection, combined with comprehensive treatment methods such as radiotherapy and chemotherapy. The treatment of glioma requires multi-disciplinary cooperation (MDT) among departments such as neurosurgery, neuroimaging, radiotherapy, neuro-oncology, pathology, and neuro-rehabilitation. It is necessary to follow the principles of evidence-based medicine, adopt individualized comprehensive treatment, optimize and standardize the treatment plan, in order to achieve the maximum treatment benefit, as much as possible to extend the progression-free survival time and overall survival time of patients, improve the quality of life, and enable patients to obtain the most optimized comprehensive treatment.

[0003] However, currently, only a few cell lines are mainly used for basic research and drug sensitivity testing of glioma globally, and these cell lines lack tumor heterogeneity and biological characteristics in vivo. There is an urgent clinical need for more and better models to study new treatment methods and develop new therapeutic drugs. Summary of the Invention

[0004] The purpose of the present invention is to provide a primary human glioma cell, a progeny cell thereof, and their applications.

[0005] In the first aspect, the present invention claims to protect a primary human glioma cell.

[0006] The primary human glioma cell claimed by the present invention is the primary human glioma cell Glioma-2, and its preservation number at the China Center for Type Culture Collection is CCTCC NO: C202420.

[0007] The STR data of the primary human glioma cells (i.e., the primary human glioma cells Glioma-2) is the same as that of the tissue of the patient from whom the primary cells were derived, and is as follows: Amelogen in-X / X, D3S1358-17 / 17, vWA-14 / 16, D7S820-8 / 11, CSF1PO-10 / 12, PentaE-12 / 15, D8S1179-15 / 16, D21S11-32.2 / 32.2, D16S539-11 / 11, D2S1338-23 / 24, PentaD-9 / 12, D19S433-13 / 13.2, TH01-7 / 9, D13S317-8 / 10, TPOX-8 / 9, D18S51-18 / 20, D6S1043-11 / 14, D1S1656-14 / 17, D5S818-12 / 12, D12S391-18 / 22, FGA-22.2 / 25, covering 21 loci commonly detected.

[0008] In a second aspect, the present invention claims the progeny cells of the primary human glioma cells described in the first aspect above.

[0009] The progeny cells have the same characteristics as the primary cells (i.e., the primary human glioma cells Glioma-2).

[0010] In a third aspect, the present invention claims the use of the primary human glioma cells described in the first aspect above or the progeny cells described in the second aspect above in any of the following:

[0011] (A1) Preparing a product for studying the pathogenesis of human glioma;

[0012] (A2) Studying the pathogenesis of human glioma.

[0013] In a fourth aspect, the present invention claims the use of the primary human glioma cells described in the first aspect above or the progeny cells described in the second aspect above in any of the following:

[0014] (B1) Preparing a product for in vitro screening of therapeutic drugs for human glioma;

[0015] (B2) In vitro screening of therapeutic drugs for human glioma.

[0016] In a fifth aspect, the present invention claims the use of the primary human glioma cells described in the first aspect above or the progeny cells described in the second aspect above in any of the following:

[0017] (C1) Preparing a product for studying the action targets and / or pharmacological mechanisms of therapeutic drugs for human glioma;

[0018] (C2) Study the action targets and / or pharmacological mechanisms of drugs for the treatment of human glioma.

[0019] In the sixth aspect, the present invention claims the use of the primary human glioma cells described in the first aspect above or the progeny cells described in the second aspect above in constructing a tumor model of human glioma.

[0020] Beneficial effects of the present invention: The primary human glioma cell Glioma-2 provided by the present invention, identified by STR gene typing, is a primary human glioma cell that has never been registered at home and abroad. Karyotype analysis shows that the chromosome number is 47-98, most of the chromosome numbers are above 69, and the abnormal ratio is 100%. This cell can form clones in soft agar and shows different drug toxicity responses to common chemotherapy drugs for glioma (cisplatin, carboplatin, etoposide, carmustine, irinotecan). This cell has a stable state and provides experimental materials and models closer to the clinical tumor biological characteristics for the study of human glioma. The primary cells obtained in the present invention can be directly used as a basic research model for studying the pathogenesis of human glioma, and can also be used as a screening model for precision drug use plans for human glioma in vitro before clinical trials.

[0021] Depository Instructions

[0022] Classification and naming: Primary human glioma cell Glioma-2;

[0023] Depository institution: China Center for Type Culture Collection;

[0024] Abbreviation of depository institution: CCTCC;

[0025] Address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province;

[0026] Date of deposit: January 5, 2024;

[0027] Registration number in the depository center: CCTCC NO: C202420. Description of the drawings

[0028] Figure 1 It is the cell morphology of the primary human glioma cell Glioma-2 under an optical microscope.

[0029] Figure 2 It is the immunofluorescence identification of the primary human glioma cell Glioma-2. The left column is DAPI staining, the middle is antibody staining, and the right column is the staining of DAPI and antibody merged together.

[0030] Figure 3STR data atlas of human glioma tissue and primary human glioma cells Glioma-2. A is the STR data atlas of human glioma tissue; B is the STR data atlas of primary human glioma cells Glioma-2.

[0031] Figure 4 Karyotype analysis chart of primary human glioma cells Glioma-2.

[0032] Figure 5 Soft agar colony formation assay in vitro of primary human glioma cells Glioma-2.

[0033] Figure 6 Drug sensitivity screening results chart of primary human glioma cells Glioma-2. Specific implementation manners

[0034] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0035] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0036] Example 1: Obtaining, identification and application of primary human glioma cells Glioma-2

[0037] I. Experimental methods

[0038] 1. Isolation, culture and passage of primary cells

[0039] A. Isolation and culture of primary cells

[0040] (1) Quickly rinse the retrieved surgical specimen (the patient's glioma tissue specimen) with 95%-100% ethanol, and then transfer it to a new culture dish after rinsing with PBS at 4°C.

[0041] (2) Cut the tissue into small tissue pieces (<10 mm) with sterilized surgical scissors.

[0042] (3) Take a 15 mL centrifuge tube, add enzyme A, H and R in the Miltenyi human tissue dissociation kit to 4.7 mL of DMEM or RPMI1640 medium, and mix to prepare a digestive enzyme solution.

[0043] (4) Transfer the cut tissue fragments to a 15 mL centrifuge tube with digestive enzymes using a Pasteur pipette, seal with parafilm and label the information.

[0044] (5) Transfer to an incubator at 37 °C and culture on a rotary shaker for 0.5 h - 3 h. Observe the digestion of the tissue blocks every half hour during this period.

[0045] (6) Centrifuge at 500 g, 4 °C for 5 min and discard the supernatant.

[0046] (7) Resuspend the precipitate in 10 mL of complete DMEM medium, filter through a 100 μm filter membrane, and then centrifuge at 300 g, 4 °C for 5 min.

[0047] (8) Resuspend the precipitate in the primary medium and plate it in a culture plate. Culture in an incubator at 37 °C, 5% CO₂. Refer to 201910212202.5 for completion.

[0048] (9) Observe the cell growth under a microscope every two days and change the medium as needed.

[0049] B. Digestive subculture

[0050] When the cells grow to 80% - 90% confluence, wash the remaining liquid with PBS, then digest with 0.25% trypsin / EDTA for 1 - 3 min. Stop the digestion after all epithelial cells are digested, resuspend the cells in 5 - 10 mL of complete medium containing 10% FBS, and centrifuge at 500 g for 30 min to collect the cells.

[0051] 2. STR identification (sent to an identification company for identification)

[0052] 3. Immunofluorescence identification of primary human glioma cells

[0053] Seed 10 5 of the cells in a 24-well plate containing cell slides. After the cells adhere the next day, rinse twice with PBS and fix with 4% paraformaldehyde for 30 min. Identify the primary cells using immunofluorescence. The antibodies used for immunofluorescence are Pan-CK (ab195203), P53 (ab26), and GFAP (ab7260).

[0054] 4. Karyotype analysis

[0055] Take approximately 10 6For the cell count of the cells to be tested, take the cells out of the 37°C, 5% CO₂ incubator, add colchicine (final concentration 0.2 μg / ml), shake well and incubate at 37°C for 1 - 2 hours. While waiting, prepare the hypotonic solution 0.075 M KCl (preparation: 8 ml of sterile water + 44.7 mg of KCl), and preheat it in a 37°C water bath. Digest the incubated cells with trypsin, centrifuge at 1200 rpm for 5 min, collect in a centrifuge tube, remove the supernatant, resuspend with 8 ml of hypotonic solution, mix well and place in a 37°C water bath for 40 min. Add 2 ml of freshly prepared fixative (methanol / glacial acetic acid = 2 / 1 to 3 / 1, volume ratio), mix well, let stand at room temperature for 10 min, then centrifuge at 1000 rpm for 10 min, remove the supernatant (leave 500 μl, first blow the cells at the bottom evenly), add 8 ml of fresh fixative, centrifuge at 1200 rpm for 10 min, repeat three times, then drop the cells onto a slide and age in an 80°C oven for 3 hours. Then place the dried slide in freshly prepared trypsin (0.25%) and digest for 1 min (strictly control the time), then place it in Giemsa stain and stain for 5 - 10 minutes (first stain 1 slide for 5 min, adjust the subsequent staining time according to the color), take out, rinse off the stain with running water, and observe the results under a microscope.

[0056] 5. Soft agar colony formation assay

[0057] (1) Take cells in the logarithmic growth phase, digest with 0.25% trypsin and gently pipette to make single cells, perform viable cell counting, and adjust the cell density to 1×10 6 / L with DMEM medium containing 20% fetal bovine serum. Then make gradient dilutions according to the experimental requirements.

[0058] (2) Prepare low melting point agarose solutions at two concentrations of 1.2% and 0.7% respectively with distilled water, autoclave, and maintain them at 40°C without solidifying.

[0059] (3) Mix 1.2% agarose and 2×DMEM medium (containing 2× antibiotics and 20% calf serum) in a 1:1 ratio, take 3 mL of the mixture and inject it into a 6 cm diameter petri dish (add 7 - 10 mL to a 10 cm petri dish), cool and solidify to make the bottom agar, and place it in a CO₂ incubator for later use.

[0060] (4) Mix 0.7% agarose and 2×DMEM medium in a 1:1 ratio in a sterile test tube, then add 0.2 mL of the cell suspension to the tube, mix well, and inject it into the petri dish with the 1.2% agarose bottom layer to form a double agar layer. After the upper agar solidifies, place it in a 37°C, 5% CO₂ incubator and culture for 10 - 14 days.

[0061] (5) Place the petri dish under an inverted microscope and observe the number of cell colonies.

[0062] 6. Drug sensitivity screening

[0063] The primary cells were seeded in a 96-well plate at a density of 10 4 / well. After the cells adhered to the plate the next day, different drugs were added and the cells were treated for 48 h. The OD value of the cells was detected by the CCK8 method, and the inhibition rate of the drugs was calculated.

[0064] II. Results and analysis

[0065] 1. Culture and passage of primary human glioma cells

[0066] After culturing the isolated primary human glioma cells in a culture plate for one week, cell clones could be clearly observed. As Figure 1 shown, the cells were closely arranged, connected like cobblestones paving a road, with different sizes and various shapes, including triangle, spindle, polygon and other shapes. The results indicated that the primary human glioma cells were successfully cultured.

[0067] 2. Immunofluorescence identification of primary human glioma cells

[0068] The cultured primary human glioma cells were identified by immunofluorescence. As Figure 2 shown, the immunofluorescence results showed that Pan-CK was negative, and both P53 and GFAP were positively expressed. The immunofluorescence antibody expression results were consistent with the clinical pathological results of the patient's tumor, suggesting that the cultured primary cells were human glioma tumor cells.

[0069] 3. STR gene typing of primary human glioma cells

[0070] As Figure 3As shown in the figure. The STR data of the primary human glioma cells obtained in the present invention are the same as those of the tissue of the patient from whom the primary cells were derived, and are: Amelogenin-X / X, D3S1358-17 / 17, vWA-14 / 16, D7S820-8 / 11, CSF1PO-10 / 12, PentaE-12 / 15, D8S1179-15 / 16, D21S11-32.2 / 32.2, D16S539-11 / 11, D2S1338-23 / 24, PentaD-9 / 12, D19S433-13 / 13.2, TH01-7 / 9, D13S317-8 / 10, TPOX-8 / 9, D18S51-18 / 20, D6S1043-11 / 14, D1S1656-14 / 17, D5S818-12 / 12, D12S391-18 / 22, FGA-22.2 / 25, covering 21 loci commonly detected. The STR genotyping identification results show that the primary human glioma cells are primary human glioma cells that have never been registered at home and abroad, and are named primary human glioma cells Glioma-2.

[0071] 4. Karyotype analysis of primary human glioma cells

[0072] The results show that: The karyotype analysis and identification of the primary human glioma cells Glioma-2 show that the chromosomes are 47-98, most of the chromosome numbers are above 69, and the abnormal ratio is 100%. As Figure 4 shown in the figure. Karyotype analysis shows that the chromosome number of the primary human glioma cells Glioma-2 is polyploid, and each chromosome has copy number variations in different situations, and there is a chromosome A that cannot be distinguished, revealing that the primary cells are cancer cells.

[0073] 5. In vitro soft agar colony formation of primary human glioma cells

[0074] As Figure 5 shown in the figure. The primary human glioma cells Glioma-2 can form obvious cell colonies in soft agar, indicating that the primary human glioma cells Glioma-2 have tumorigenicity in vitro.

[0075] 6. Drug sensitivity screening of primary human glioma cells

[0076] As Figure 6 shown in the figure. The commonly used clinical chemotherapy drugs for glioma include cisplatin, carboplatin, etoposide, carmustine, irinotecan, etc. The in vitro drug sensitivity test results show that: The primary human glioma cells Glioma-2 have different sensitivities to different chemotherapy drugs, and the sensitivities from high to low are irinotecan > carboplatin > carmustine > cisplatin > etoposide.

[0077] The primary glioma cells of human brain Glioma-2 were deposited at the China Center for Type Culture Collection on January 5, 2024, with the deposit number of CCTCC NO:C202420.

[0078] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art.

Claims

1. A primary human glioma cell, characterized in that: The primary human glioma cells are primary human glioma cells Glioma-2, and its deposit number in the China Center for Type Culture Collection is CCTCC NO: C202420.

2. The progeny cells of the primary human glioma cells described in claim 1.

3. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the preparation of products for studying the pathogenesis of human glioma.

4. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the study of the pathogenesis of human glioma.

5. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the preparation of products for in vitro screening of therapeutic drugs for human glioma.

6. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the in vitro screening of therapeutic drugs for human glioma.

7. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the preparation of products for studying the action targets and / or pharmacological mechanisms of therapeutic drugs for human glioma.

8. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the study of the action targets and / or pharmacological mechanisms of therapeutic drugs for human glioma.

9. The application of the primary human glioma cells described in claim 1 or the progeny cells described in claim 2 in the construction of a tumor model of human glioma.

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