CN105483086B - A kind of brain glioblastoma cell and application thereof - Google Patents
A kind of brain glioblastoma cell and application thereof Download PDFInfo
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- CN105483086B CN105483086B CN201511005867.7A CN201511005867A CN105483086B CN 105483086 B CN105483086 B CN 105483086B CN 201511005867 A CN201511005867 A CN 201511005867A CN 105483086 B CN105483086 B CN 105483086B
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Abstract
The present invention provides a kind of brain glioblastoma cells, wherein the deposit number of the brain glioblastoma cell is CGMCC NO.11100.The present invention also provides purposes of the brain glioblastoma cell as described above in screening drug.Through the above technical solutions, the present invention can simplify and accelerate the drug screening to brain glioblastoma cell hypotype.
Description
Technical field
The present invention relates to technical field of cell biology, and in particular, to a kind of brain glioblastoma cell and the glioma
The purposes of cell.
Background technique
Glioblastoma is the highest glioma of grade malignancy in astrocytic tumor.The tumour is located under cortex, more
Number is grown on curtain everywhere in cerebral hemisphere.In infiltrative growth, several cerebral lobes are often invaded, and invade DEEP STRUCTURE, it can also be through
Corpus callosum involves contralateral hemispheres.Happening part is most common with frontal lobe, other are followed successively by temporal lobe, top, and minority is found in pillow
Leaf/thalamus and Basal ganglia etc..
Glioblastoma growth speed is fast, the course of disease is short, and for 70-80% patient's course of disease at 3-6 months, the course of disease was more than 1 year person
Only 10%.Single cases can fall ill because of tumour bleeding in stroke sample, and since tumour growth is rapid, the extensive intracranial pressure of brain edema increases
High symptom is obvious, and almost all patient has headache, vomiting, papilledema to have headache, spiritual change, limb adynamia, vomiting, meaning
Know obstacle and disfluency.Tumor infiltrating destroys brain tissue, causes a series of focal symptom, and patient has different degrees of inclined
Paralysis, hemidysesthesia aphasia and hemianopsia etc..Neurological examination can find hemiplegia, cranial nerve lesions, hemidysesthesia with
Hemianopsia.About 33% patient has epileptic attack, and about 20% patient shows the mental symptoms such as indifferent, dull-witted, hypophrenia.
Glioblastoma can be divided into the secondary glioblastoma developed by low level astrocytoma
(secondary glioblastoma) and the primary glioblastoma (primary for not showing low lesion pre-malignant
Glioblastoma) two class, and be histologically difficult to distinguish secondary glioblastoma and primary glioblastoma.
At present, it has been found that the mutation of isocitric dehydrogenase (Isocitrate Dehydrogenase, IDH) is only in secondary colloid
The mutation of isocitric dehydrogenase does not occur in blastoma, but still in some secondary glioblastoma.
It is level Four glioblastoma that primary glioblastoma, which is from first time clinical diagnosis, and most apparent point
Subcharacter is EGFR amplification, mutation or overexpression (40%), and P53 is mutated (30%), and CDKN2A/B lacks (30-40%), and RB1 is prominent
Become or missing, No. 10 chromosome diminutions (70%) and PTEN are mutated (30%) etc..Secondary glioblastoma is by rudimentary
The not level Four glioblastoma of (II grades or III level) progress.Its clinical diagnosis for the first time is Low grade glioma,
The general tumour after operation or chemicotherapy occurs again, and rank proceeds to level Four glioblastoma.The study found that
The molecular marker and gene cell access of secondary glioblastoma are different from primary glioblastoma.Research at present
Most hot is that isocitric dehydrogenase 1 is mutated (70%), additionally includes P53 mutation (65%), and PDGFA and PDGFRA are overexpressed
(60%), the missing (50%) of No. 19 chromosome long arms and the mutation of RB1 or missing (25%).
The targeted therapy for being found to be glioblastoma of these molecular markers provides important target spot.However, being directed to
The targeted drug of these molecular markers is very much, but does not all actually enter the clinical application stage, is that targeting is closed after all
The specificity of system is lower, thus leads to that curative effect is lower and side effect is larger, is unsuitable for clinical application.
The present inventor has found in research before, in glioma, there is the patient-specific of a monoid
A kind of fusion protein is expressed on ground, and the patient's monoid for expressing the fusion protein belongs to a new hypotype of glioma, is shown
The prognosis and drug susceptibility different from other Patients with gliomas.But due to lacking suitable cell model, at present to this
There is screening difficulty in the drug development of glioma.
Summary of the invention
In order to overcome the defect for lacking suitable cell model, the present invention provides a kind of brain glioblastoma cell, the brain glue
Drug screening of the matter oncocyte particularly suitable for above-mentioned glioma hypotype.
The present inventor carries out primitive cell culture from glioma cells in tissue and passage cell culture, screening obtain
The brain glioblastoma cell of one plant of steady and continuous passage, which, which stablizes, expresses above-mentioned fusion protein, and shows
Special drug susceptibility, results in the present invention.
The present invention provides a kind of brain glioblastoma cells, wherein the deposit number of the brain glioblastoma cell is CGMCC
NO.11100。
The present invention also provides purposes of the brain glioblastoma cell as described above in screening drug.
Through the above technical solutions, the present invention can simplify and accelerate the drug sieve to above-mentioned brain glioblastoma cell hypotype
Choosing.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Biomaterial preservation
Brain glioblastoma cell of the invention is that the present inventor carries out primitive cell culture from glioma cells in tissue
With passage cell culture, screening has obtained the brain glioblastoma cell of one plant of steady and continuous passage, deposit number CGMCC
No.11100, the deposit date is on August 31st, 2015, depositary institution was that China Committee for Culture Collection of Microorganisms is commonly micro-
Bio-Centers, address are located at Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, classification naming
For Genus Homo brain glioblastoma cell system.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is the photo for the cell of the invention that deposit number is CGMCC No.11100.
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The present invention provides a kind of brain glioblastoma cells, wherein the deposit number of the brain glioblastoma cell is CGMCC
NO.11100。
The present invention also provides purposes of the brain glioblastoma cell as described above in screening drug.
Wherein, brain glioblastoma cell as described above can be used for the screening of a variety of drugs, it is preferable that the drug can be with
For the drug for treating tumour；It is highly preferred that the tumour is glioblastoma.
Wherein, a kind of particularly preferred embodiment, the glioblastoma are expressed fusion protein according to the present invention
Glioblastoma hypotype, the fusion protein is on N-terminal to C-terminal direction by the first protein segment and the second albumen flakes
Section is formed by connecting, and the first protein segment is as shown in SEQ ID NO:1,2,3 or 4, second protein fragments such as SEQ ID
Shown in NO:5 or 6.
Wherein, the sequence of SEQ ID NO:1 is outside first from people PTPRZ1 gene (NCBI Gene ID is 5803)
The protein sequence of aobvious son and intron sequences coding.
Wherein, the sequence of SEQ ID NO:2 is Second Exon and intron sequences coding from people's PTPRZ1 gene
Protein sequence.
Wherein, the sequence of SEQ ID NO:3 is third exon and intron sequences coding from people's PTPRZ1 gene
Protein sequence.
Wherein, the sequence of SEQ ID NO:4 is the 8th exon and intron sequences coding from people's PTPRZ1 gene
Protein sequence.
Wherein, the sequence of SEQ ID NO:5 is from the homologous different of people MET gene (NCBI Gene ID is 4233) coding
The segment of 1 protein sequence of structure body.
Wherein, the sequence of SEQ ID NO:6 is the segment of 2 protein sequence of homology isomer from people's MET gene coding.
In the present invention, above-mentioned fusion protein can be encoded by integrative nucleic acid, wherein encode fusion protein as described above
The sequence of integrative nucleic acid can decode to obtain according to the amino acid codes table of comparisons, due to the presence of Codon degeneracy, coding
Sequence with multiple integrative nucleic acids of the fusion protein of amino acid sequence can be different from each other.
Preferably, the glioblastoma hypotype is the glioblastoma hypotype that also transcription has fusion mRNA, described
The cDNA of fusion mRNA is formed by connecting on 5 ' to 3 ' directions by the first nucleic acid fragment and the second nucleic acid fragment, first nucleic acid
Segment is as shown in SEQ ID NO:7,8,9 or 10, and second nucleic acid fragment is as shown in SEQ ID NO:11 or 12.
Wherein, First Exon and intron sequences of the sequence of SEQ ID NO:7 from people's PTPRZ1 gene, coding is such as
Albumen shown in SEQ ID NO:1.
Wherein, Second Exon and intron sequences of the sequence of SEQ ID NO:8 from people's PTPRZ1 gene, coding is such as
Albumen shown in SEQ ID NO:2.
Wherein, third exon and intron sequences of the sequence of SEQ ID NO:9 from people's PTPRZ1 gene, coding is such as
Albumen shown in SEQ ID NO:3.
Wherein, the sequence of SEQ ID NO:10 is the 8th exon and intron sequences from people's PTPRZ1 gene, is compiled
Code albumen as shown in SEQ ID NO:4.
Wherein, the sequence of SEQ ID NO:11 is the segment from people's MET gene, is encoded as shown in SEQ ID NO:5
Albumen.
Wherein, the sequence of SEQ ID NO:12 is the segment from people's MET gene, is encoded as shown in SEQ ID NO:6
Albumen.
Hereinafter, present invention will be further described in detail through examples.
Embodiment 1:
Glioma cells in tissue block (in the brain colloid that Tiantan Hospital is won according to the operating process operation for meeting Medical Ethics
Tumor tissue) it is placed in a beaker, it is rinsed 3 times with Hanks liquid, removes blood stains；60 are subsequently placed in the mixed liquor containing mycillin
Minute；Tissue is cut into eye scissors the block of 2-3 mm in size, in order to digest；Addition is equivalent to 50 times of bodies of tissue block total amount
Long-pending trypsin solution, is then poured into conical flask together, ligatures bottleneck or plug with rubber plug；37 DEG C of incubator digestion are placed in, are disappeared
It was shaken once in change every 20 minutes；Digestion time is 60 minutes；Then by being suitable for stainless steel sieve, not yet abundant digestion is filtered
The tissue block opened；800 revs/min of centrifugation digestive juices；Supernatant is sucked out, primary culture medium is added into precipitating, until cell density is 105
A/mL；It is placed in 37 DEG C of incubator cultures.Wherein, the basal medium of primary culture medium is DMEM culture medium；Also contain 10 volume %
Fetal calf serum, the FGF cell factor of EGF, 0.1ng/ml of 0.1ng/ml, streptomysin and penicillin is dual anti-, B27 and turns iron egg
It is white.
When subculture to 7 generation, primary culture medium is replaced with to the DMEM culture medium without serum；Secondary culture 7 again
Generation, during 7 generation secondary culture, primary cell occur all it is dead, stop growing or the phenomenon that slow growth；By accident,
There are individual cell lines the state being well proliferated can be presented in the DMEM culture medium without serum, these can be well proliferated
Cell line as further screening object.
In further screening process, the verifying of expressing fusion protein is carried out by PCR.It extracts thin to what is further screened
The cDNA that the RNA of born of the same parents is obtained carries out the PCR verifying of fusion protein.PCR verifying primer used is as shown in SEQ ID NO:17
The first primer and the composition of the second primer as shown in SEQ ID NO:18.The operation of PCR is according to synthetic primer and PCR kit
Specification carries out.The product of PCR shows the presence or absence of amplified band, the amplified band occurred by Ago-Gel nucleic acid electrophoresis
It is recycled using DNA gel QIAquick Gel Extraction Kit (QIAquick PCR purification kit is purchased from Qiagen), then
It is cloned into carrier T (pGEM-T easy vector, be purchased from Promega), and with DNA sequencer (3730 × l of ABI Prism
DNA Sequencer is purchased from Applied Biosystems) it is sequenced.It to sequencing result, tests, expands according to table 1
The cell line for occurring any one in SEQ ID NO:13-16 in product is to meet the desired cell line of screening.
Table 1
In meeting the desired cell line of screening, further progress MTT experiment and Transwell experiment detection are proliferated and move
Shifting ability, chooses proliferation and the strongest cell line of transfer ability carries out preservation, and deposit number is CGMCC No.11100,
Brain glioblastoma cell as of the invention, cellular morphology picture is as shown in Figure 1, fragment sequence such as SEQ in its amplified production
Shown in ID NO:14.
Comparative example 1
It is operated first with slow virus carrier PCDH (SBI company article No. CD510B-1) by its specification, preparation expression fusion
The carrier for expression of eukaryon of gene (the first nucleic acid fragment is SEQ ID NO:8 and the first nucleic acid fragment is SEQ ID NO:12) is used in combination
Packaging plasmid (SBI company article No. LV500A-1) cotransfection 293T cell of virus is to prepare slow virus.
Human glioblastoma Cell line U87 is purchased from Chinese Academy of Medical Sciences's cell bank, does not express the above-mentioned fusion of the present invention originally
Albumen.With the slow-virus infection U87 cell for having packed above-mentioned carrier, puromycin screening (0.5 μ g/mL screening, 0.2 μ g/ are carried out
ML is maintained), the cell model for stablizing expressed fusion protein is established, and verify above-mentioned cell membrane with immuning hybridization and reverse transcription PCR
Type has the expression of fusion protein and the transcription of fusion.The U87 cell model for stablizing expressed fusion protein is this comparison
The cell model of example.
Testing example 1
Known Temozolomide is effective to part Patients with gliomas, but to the above-mentioned fusion protein of expression of the present invention
Patients with gliomas is invalid；And gram azoles has for Patients with gliomas of the Buddhist nun to the above-mentioned fusion protein of expression of the present invention
Effect, and it is invalid to the other Patients with gliomas in part.This testing example to original U87 cell, stablize expressed fusion protein
The cell of the invention that U87 cell and deposit number are CGMCC No.11100 surveys gram azoles for the drug responsiveness of Buddhist nun
Examination.
It is to original U87 cell, transfection blank control cell, the U87 cell of stable expressed fusion protein and deposit number
The cell of the invention of CGMCC No.11100 is separately added into Temozolomide, gram azoles for Buddhist nun and blank control, drug final concentration
It is 3 μM.After drug effect 24 hours, cell viability is detected with thiazole bromide blue tetrazolium (MTT), the results are shown in Table 2.
Table 6
According to the result of table 2, the cell of the invention of the U87 cell with track fusion and CGMCC No.11100
Proliferation obviously accelerate compared to original U87 cell, Temozolomide is able to suppress the proliferation of original U87 cell, to having fusion base
Because the inhibited proliferation of the U87 cell of expression is weaker；And it cannot substantially inhibit that CGMCC No.11100's is of the invention thin
The proliferation of born of the same parents, gram azoles is able to suppress the proliferation of original U87 cell for Buddhist nun, to the proliferation of the U87 cell with track fusion
Also there is stronger inhibiting effect；Also it can inhibit the proliferation of the cell of the invention of CGMCC No.11100.Thus illustrate, CGMCC
The cell of the invention of No.11100 can be as the cell model of the glioma hypotype of screening expressed fusion protein.
Claims (6)
1. a kind of brain glioblastoma cell, which is characterized in that the deposit number of the brain glioblastoma cell is CGMCC NO.11100.
2. purposes of the brain glioblastoma cell described in claim 1 in screening drug.
3. purposes according to claim 2, wherein the drug is the drug for treating tumour.
4. purposes according to claim 3, wherein the tumour is glioblastoma.
5. purposes according to claim 4, wherein the glioblastoma is the glioblast of expressed fusion protein
Tumor hypotype, the fusion protein are formed by connecting on N-terminal to C-terminal direction by the first protein segment and the second protein fragments,
The first protein segment is as shown in SEQ ID NO:1,2,3 or 4, the second protein fragments such as institute of SEQ ID NO:5 or 6
Show.
6. purposes according to claim 5, wherein the glioblastoma hypotype is the glue that also transcription has fusion mRNA
Matter blastoma hypotype, the cDNA of the fusion mRNA are connected on 5 ' to 3 ' directions by the first nucleic acid fragment and the second nucleic acid fragment
It connects, first nucleic acid fragment is as shown in SEQID NO:7,8,9 or 10, second nucleic acid fragment such as SEQ ID NO:
Shown in 11 or 12.
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Citations (4)
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|---|---|---|---|---|
| CN103627673A (en) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | Human brain glioma cell line, and establishing method and application thereof |
| CN103525765B (en) * | 2013-10-18 | 2014-12-17 | 李文斌 | Drug-resistant glioblastoma cell line of primary temozolomide and bevacizumab, construction method and application of cell line |
| CN104946593A (en) * | 2015-04-30 | 2015-09-30 | 复旦大学附属金山医院 | Adriamycin-resistant U87 cell model and application thereof |
| CN104974986A (en) * | 2015-05-29 | 2015-10-14 | 苏州大学 | Human-derived stem cell-like brain glioma cell line and uses thereof |
-
2015
- 2015-12-29 CN CN201511005867.7A patent/CN105483086B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103627673A (en) * | 2012-08-21 | 2014-03-12 | 上海睿智化学研究有限公司 | Human brain glioma cell line, and establishing method and application thereof |
| CN103525765B (en) * | 2013-10-18 | 2014-12-17 | 李文斌 | Drug-resistant glioblastoma cell line of primary temozolomide and bevacizumab, construction method and application of cell line |
| CN104946593A (en) * | 2015-04-30 | 2015-09-30 | 复旦大学附属金山医院 | Adriamycin-resistant U87 cell model and application thereof |
| CN104974986A (en) * | 2015-05-29 | 2015-10-14 | 苏州大学 | Human-derived stem cell-like brain glioma cell line and uses thereof |
Non-Patent Citations (4)
| Title |
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| Brainstem Glioma: A Review;Sean A. Grimm et al.;《Curr Neurol Neurosci Rep》;20131231;第13卷;全文 * |
| deFuse: An Algorithm for Gene Fusion Discovery in Tumor RNA-Seq Data;Andrew McPherson et al.;《PLoS Computational Biology》;20110531;第7卷(第5期);全文 * |
| Identification of human brain tumour initiating cells;Sheila K. Singh et al.;《NATURE》;20041118;第432卷;全文 * |
| Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line;Toru Kondo et al.;《PNAS》;20040120;第101卷(第3期);全文 * |
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