CN109749998A - A kind of primary bone giant cell tumor strain GCTB28-luc of people and its construction method and application - Google Patents
A kind of primary bone giant cell tumor strain GCTB28-luc of people and its construction method and application Download PDFInfo
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Abstract
A kind of primary bone giant cell tumor strain GCTB28-luc of people and its construction method and application, the primary bone giant cell tumor strain GCTB28 of people is preserved in China typical culture collection center, address: Wuhan University, Wuhan, China city, deposit number are CCTCC NO:C2018178.The cell strain GCTB28-luc that the present invention constructs has the characteristic of the primary giant cell tumor of bone tumour cell of people, meet establishment standard, in vitro culture can be carried out in the medium, and in vitro culture growth is stablized, energy continuous passage, and then it is readily available in establishing giant cell tumor of bone genesis mechanism model, the cell material that can be used as giant cell tumor of bone pathogenesis and individualized treatment in vitro study can be applied to prepare, screen, evaluate anti-giant cell tumor of bone drug.
Description
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of primary bone giant cell tumor strain GCTB28- of people
Luc and its construction method and application.
Background technique
Giant cell tumor of bone is one of most common primary bone tumour in Asia, accounts for the 13-15% of all primary bone tumour,
A kind of very special primary bone tumour, biological characteristics between it is good it is pernicious between, WHO (World Health Organization) is returned
In benign bone tumour, but compared with benign tumour, giant cell tumor of bone has higher invasion, is easy recurrence, can also turn
It moves, but transfer is very rare, metastasis site is mostly lung.Compared with malignant tumour, bone giant cell has relatively good pre-
Afterwards, it should be noted that seeming all not sensitive enough to chemotherapy and radiation.The even more noteworthy cytology of giant cell tumor of bone
Form it is sufficiently complex, be by three kinds of apocyte, stroma cell and monocyte cell compositions, and these three cells tumour send out
Role and mutual connection still fail to make known completely in life.
Summary of the invention
It is an object of that present invention to provide a kind of primary bone giant cell tumor strain GCTB28-luc of people and its construction method and
Using, the novel primary bone giant cell tumor strain GCTB28-luc of people can for research in patient body the generation of giant cell tumor of bone,
Resistance mechanism and treatment provide good external model, and then promote basic research, prevention and the clinical diagnosis and treatment of giant cell tumor of bone.
To achieve the goals above, the technical solution used in the present invention is:
The present invention has carried out multiple originally culture from a large amount of giant cell tumor of bone case, and to the method for animal-transplanted tumor
Explored, built up can stablize passage and the giant cell tumor of bone in the Asian source of tumorigenesis is thin in immunodeficient mouse
Born of the same parents' strain, it is believed that cell strain can provide good for the further mechanism of research giant cell tumor of bone and the opposite therapeutic strategy of formulation
Good basis.
A kind of primary bone giant cell tumor strain GCTB28-luc of people, the primary bone giant cell tumor strain GCTB28 of people
It is preserved in China typical culture collection center, address: Wuhan University, Wuhan, China city, preservation date: 2018.10.24, preservation
Number be CCTCC NO:C2018178.
The construction method of the primary bone giant cell tumor strain GCTB28-luc of people of the present invention comprising following step
It is rapid:
1) around by the tumor specimen after the excision of giant cell tumor of bone patients surgery using conventional trimming removal tumor tissues
Necrosis and nonneoplastic tissue, physiological saline repeated flushing, antiseptic sursery, which is cut, shreds tumor tissues, is placed in 50ml containing 10% tire ox
In the DMEM culture solution of serum, by nutrient solution volume be added II Collagenase Type 10mg/ml be placed in 37 DEG C of constant temperature oscillation casees with
150 revs/min of oscillation 2h；
2) postdigestive tissue specimen is taken out, is sieved with 300 mesh metallic sieves, the liquid for crossing sieve is slowly injected into pre-
In the centrifuge tube for having set Ficoll lymph separating liquid, after 2500 turns/min is centrifuged 30min, intermediate layer cell is collected, after PBS is resuspended
1000 turns/min is centrifuged 5min, abandons supernatant, DMEM adjusts cell density after being resuspended, with 1 × 106A/L concentration is inoculated in 25cm
In culture bottle, 37 DEG C are put into containing 5%CO2It is cultivated in the cell incubator of 95% air；With 0.25% pancreatin had digestive transfer culture；
3) by the cell inoculation after passage in new Tissue Culture Flask, DMEM culture medium is added and is cultivated；It is cultivating
During, the cell for selecting different algebra carries out freezing conservation, cell is resuspended with frozen stock solution after collecting cell, by cryopreservation tube
It is put into liquid nitrogen container and saves, which is named as GCTB28；
4) luciferase gene (Firefly luciferase gene) is integrated on cell GCTB28 chromosomal DNA,
Make cell expressing luciferase, constructs the cell strain of stable expressing luciferase.The present invention is by carrying out GCTB28
After luciferase gene modification, stable cell line is inoculated into animal body when carrying out animal experiment in vivo by this cell strain
It is interior, substrate luciferin (luciferin) is injected intraperitoneally after a certain period of time, can shine in a few minutes, i.e., available instrument detection, hair
Luminous intensity is related to the number of cell, keeps experiment in vivo more intuitive, facilitates and grasps experimental period and process etc..We will be this
The GCTB28 for carrying luciferase gene is further named as GCTB28-luc, and obtaining deposit number is CCTCC NO:C2018178
The primary bone giant cell tumor strain of people.
5) the primary bone giant cell tumor strain of people that the above-mentioned construction method of the present invention obtains is through G-band chromosome karyotyping table
It is bright its there are chromosome abnormalities.
Testing through nude mice tumorigenesis confirms: the primary bone giant cell tumor strain of the people of the invention can result in nude mice shin bone
Tumor formation has oncogenicity.
The present invention also provides the primary bone giant cell tumor strains of the people in establishing giant cell tumor of bone genesis mechanism model
Application.
The present invention also provides the primary bone giant cell tumor strains of the people to establish external people's bone giant-cell tumor animal mould
Application in type.
The primary bone giant cell tumor strain of people of the present invention can be applied to prepare, screen, evaluate the huge sarcoma drug of anti-bone.
Beneficial effects of the present invention:
The present invention constructs a kind of primary bone giant cell tumor strain GCTB28-luc of novel people, is one plant of newly-built people's bone
Giant-cell tumor tumor cell line can be used as the cell material of giant cell tumor of bone pathogenesis and individualized treatment in vitro study.
The primary bone giant cell tumor strain of the people that the present invention constructs can carry out in vitro culture, and in vitro culture in the medium
Stable, energy continuous passage is grown, and then is convenient to establish giant cell tumor of bone genesis mechanism model；It can be used as giant cell tumor of bone hair
Interpretation of the cause, onset and process of an illness system and the cell material of individualized treatment in vitro study, and then can be applied to prepare, screen, evaluate the huge sarcoma medicine of anti-bone
Object.
Detailed description of the invention
Fig. 1 is first generation bone giant cell tumor strain cell photo under inverted phase contrast microscope in the embodiment of the present invention 1.
Fig. 2 is third generation bone giant cell tumor strain cell photo under inverted phase contrast microscope in the embodiment of the present invention 1.
Fig. 3 is bone giant cell tumor strain cell transmission electron microscope picture in the embodiment of the present invention 2.
Fig. 4 is that bone giant cell tumor strain cell chromosome analyzes result in the embodiment of the present invention 3.
Fig. 5 is bone giant cell tumor strain cell growth curve in the embodiment of the present invention 4.
Fig. 6 is bone giant cell tumor strain cell growth cycle in the embodiment of the present invention 5.
Fig. 7 is that bone giant cell tumor strain cell and MG63 cell invasion ability compare in the embodiment of the present invention 6.
Fig. 8 is the shin bone tumor formation picture of bone giant cell tumor strain cellular immunity deficiency mouse in the embodiment of the present invention 7.
Fig. 9 is bone giant cell tumor strain cell mouse shin bone in the embodiment of the present invention 7 into tumor tissue HE coloration result.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be further described.
Embodiment 1GCT primary cell and osteoblasts cultivation
The tumor specimen cut off from giant cell tumor of bone patients surgery is put into 1000U/ml containing penicillin, 1000 μ of streptomysin
It in the Hanks liquid of g/ml, is placed in 50ml centrifuge tube, external ice bag is sent rapidly to laboratory.Tumor tissues are put into 10cm to put down
In ware, necrosis and nonneoplastic tissue around conventional trimming removal tumor tissues, physiological saline repeated flushing, antiseptic sursery cut by
Tissue shreds, and is placed in liquid containing 10%DMEM, and II Collagenase Type 10mg/ml is added by nutrient solution volume and is placed on 37 DEG C of constant temperature vibrations
It swings in case with 150 revs/min of oscillation 2h.Postdigestive tissue specimen is taken out, after the sieving of 300 mesh metallic sieves, will be sieved
The liquid of net is slowly injected into the centrifuge tube for preseting Ficoll lymph separating liquid, after 2500 turns of centrifugation 30min, collects middle layer
Cell, 1000 turns of centrifugation 5min after PBS is resuspended abandon supernatant, and DMEM adjusts cell density after being resuspended, with 106It is inoculated in 25cm
In culture bottle, 37 DEG C are put into containing 5%CO2It is cultivated in the cell incubator of 95% air.With 0.25% pancreatin had digestive transfer culture.
By the cell inoculation after passage in new 25cm2In Tissue Culture Flask, it is added and contains 10% fetal calf serum, penicillin
The DMEM culture medium of (100U/ml), streptomysin (100mg/ml) is cultivated；During culture, different algebra are selected
Cell carries out freezing conservation, and cell is resuspended with frozen stock solution after collecting cell, cryopreservation tube is put into liquid nitrogen container and is saved, this is thin
Born of the same parents system is named as GCTB28.
Experimental result: after cell suspension inoculation 72 hours, start it can be observed that attached cell, these cell sizes, shape
State is different, and the main stroma cell that can see form huge multinucleate giant cell and shuttle shape, wherein apocyte nucleus is arranged
Column are close, and spindle cell is not of uniform size, form of diverse, and Assessment of Nuclear Volume increases, and caryoplasm is out of proportion, and cell growth loses contact suppression
System, can interweave overlapping growth (as shown in Figure 1).It can be found that shuttle after the non-attached cell changed in liquid removal culture solution
Shape cell tight is centered around around multinucleate giant cell, quite similar with the feature on pathological section.It is worth noting that, by
After continuously passing on three times, multinucleate giant cell disappears in the visual field substantially, and the stroma cell of shuttle shape can only be observed (such as Fig. 2 institute
Show).
2 bone giant cell tumor strain cell transmission electron microscope of embodiment
Supernatant will be removed after the centrifugation of bone giant cell tumor strain cell, taking precipitate is fixed with 2.5% glutaraldehyde, then is passed through
L% osmic acid is fixed；The dehydration of gradient acetone, epoxy resin embedding, ultra-thin section after semithin section positioning, uranium-lead double staining, benefit
With transmission electron microscope observing, transmission electron microscope photo is as shown in Figure 3.
Experimental result: as shown in figure 3, scanning electron microscope shows that cell surface has gauffer, there are microvillus or digitation in periphery.
Transmission electron microscope shows that cell appearance is irregular, and core has greatly distortion, and nuclear proportion increases, and nuclear membrane has recess, there is multiple kernels, cytoplasm
Inside there are the organelles such as free ribosome abundant, mitochondria, endoplasmic reticulum, lysosome, high-visible, cell surface has microvillus,
It is closely connected between visible cell.
The analysis of 3 bone giant cell tumor strain cell chromosome of embodiment
It is (final concentration of that colchicine is added in the bone giant cell tumor strain cell for taking logarithmic growth in its culture solution
0.04g/m1), 4h is continuously cultivated；Training liquid is sucked out, is washed cell 1 time with Hank ' the s liquid of 37 DEG C of pre-temperatures, inhales and abandons cleaning solution；With
Simultaneously single cell suspension is made in 0.25% trypsin digestion and cell；1200r/min is centrifuged 10min, inhales and abandons supernatant；In respectively from
The mixing hypotonic medium 8ml (0.4% potassium chloride and 0.4% sodium citrate solution are mixed by 1:1) of 37 DEG C of pre-temperatures of addition in heart pipe, 37
DEG C water-bath 30-40min.The 2:1 methanol of 1ml Fresh, glacial acetic acid fixer is added in every pipe, mixes well, with 1200r/
Min is centrifuged l0min；Supernatant is abandoned, every pipe is added 2:1 methanol, the glacial acetic acid fixer of 8ml Fresh, mixes well, room temperature
Stand 30min；1200r/min is centrifuged l0min, is repeated twice；Abandoning supernatant, the 2:1 methanol of every pipe addition 1ml Fresh,
Glacial acetic acid fixer, mixes well, and single cell suspension is made；By above-mentioned suspension drop on the cleaning sheet glass for speckling with ice water, in
Room temperature is dried；With 1:l0Giemsa dye liquor (using 6.8 phosphate buffered saline of pH), 20-30min is dyed；It is rinsed with tap water
Completely, drying at room temperature, acetone are dehydrated 2 times (each 30s), and dimethylbenzene transparent 2 times (each 2min), neutral gum mounting is dry
Microscopy afterwards.30 well dispersed metaphase cells are selected at random and carry out chromosome analysis, and analysis result is referring to fig. 4.
Experimental result: select under the microscope it is well dispersed and than more complete 12nd generation metacinesis as being observed,
Count the chromosome number of 100 mitotic figures.As shown in Figure 4, observe that chromosome number is distributed mainly on 57-66, chromosome
Mode is 60-68, mostly hypo-triploid.
4 bone giant cell tumor strain cell proliferation experiment of embodiment
GCT cell in logarithmic growth phase is inoculated in 96 orifice plates by the concentration of 5000/100 μ l, every hole set 5 it is right
According to co-cultivation 7d；Respectively at the 1st day, the 2nd day, the 4th day and the 7th day, CCK810 μ l is added in every hole, 2h is incubated, with automatic
Microplate reader measures A (OD) value in each hole, its average value is taken to trace the growth curve of each cell at 450nm wavelength.This experiment weight
It is 3 times multiple.Growth curve chart is as shown in Figure 5.
Experimental result: when two points that the logarithmic growth phase of growth curve takes cell number to grow at double make vertical line measurement
Between, the cell population doublings time is 44.8h.Splitting index reaches top in culture 3d cell, is 49%, formation rate 42%.
5 bone giant cell tumor strain cell cycle analysis of embodiment
GCTB28 cell in logarithmic growth phase is cultivated into 48h under conditions of serum free medium, so that cell is big
Partial block is in G0/G1；Then give 1640 culture mediums containing 10%FBS and continue 12~16h of culture；After culture, with
The PBS of pre-cooling is washed cell 2 times, and with 0.25% trypsin digestion and cell, single cell suspension is made；It is added dropwise
In dehydrated alcohol, its final concentration is made to reach 75%；60min is fixed on ice, or is placed in -20 DEG C of refrigerators fixed, detection in 1 week.
Detection processing: first washing cell 2 times with the PBS of pre-cooling and is resuspended in 1 × PBS of 300-500 μ L (wherein containing 20 μ g/
MlRNaseA, 0.2%Triton X-100,0.2mmol/L EDTA and 20 μ g/ml Propidium Iodide), then at 37 DEG C
Water-bath in warm bath 15-30min；Thereafter, using the DNA content of flow cytomery cell, and with the progress of Modfit program
Manual synchronizing.
Experimental result: Fig. 6 is the growth cycle of bone giant cell tumor strain GCTB28 cell, it will be appreciated from fig. 6 that fluidic cell
Instrument measures cell G1=33.42%, G2=48.73%, S=17.88%.
The detection of 6 bone giant cell tumor strain cell invasion ability of embodiment
Transwell, 24 well culture plates, Matrigel, 1640 culture medium of serum-free and suction nozzle are placed in 4 DEG C of refrigerator overnights,
Matrigel and 1640 culture medium of serum-free are mixed in the ratio of 1:3 to prepare artificial basement membrane；Transwell is placed in 24 holes
In culture plate, the mixed liquor of 50 μ L is respectively added in upper chamber, 37 DEG C of incubation 2h are so that it is solidified；Upper and lower room be separately added into 100 μ L and
1640 culture medium of serum-free of 600 μ L, 37 DEG C of incubation 8h；By 1 × 106The concentration of/ml will be in the G1 and G2 of logarithmic growth phase
Cell is prepared into single cell suspension with 1640 culture medium of serum-free；The training liquid for discarding each room up and down is added 100 μ L's in upper chamber
Serum-free 1640 culture medium of the 600 μ L containing 5%fibronectin, every room is added in lower room in cell suspension (1 × 105 cell)
If 3 controls；In 37 DEG C, 5%CO2Condition in cultivate for 24 hours；Upper chamber is taken out, after the cell for failing invasion with cotton swab erasing,
30min, conventional H E dyeing are fixed with neutral formalin；Under 200 times of light microscopics, randomly selects 5 visuals field and count infiltrating cells, make even
Mean value is compared.
Experimental result: under phase contrast microscope, the GCTB28 cell number across Matrigel layers is counted.Each number of samples
10 visuals field, each field area are about 0.44mm2, calculate 1cm2The cell number passed through in area finds out the number with 10 the bottom of as
Logarithm.
Fig. 7 is the cell photo of GCTB28 cell, MG-63 cellular infiltration to the filter membrane back side under microscope.By being calculated
Logarithm and Fig. 7 it is found that the bone giant cell tumor strain cell that the present invention constructs has the close invasion energy of same MG-63 cell
Power.
7 bone giant cell tumor strain multicellular animal model foundation of embodiment
Nude mice is anaesthetized with 10g/L yellow Jackets (75mg/kg), dorsal position, the fixed forelimb of adhesive tape and left hind, after making the right side
Limb buckling, shin femur angle is at 45 °, then holds 1mlTB syringe -29G syringe needle and is pierced into shin femoral joint gap, closes from shin bone
Inserting needle at fossa point is saved, shin bone cancellous bone portion is slowly drilled through along shin bone backbone long axis direction and enters ossis, is had obvious
Breakthrough sense.Rotation slowly enters expansion marrow, exits after syringe needle and is inserted into ossis along former entry point microsyringe, slowly injects
GCTB28 cell suspension 20 μ l, about 5 × 106A cell, finally extracts syringe needle.It is steam again after nude mice revival.After modeling 2 weeks, often
Nude mice whole body bone x ray photograph of Zhou Jinhang.Prone position is fixed after nude mice is anaesthetized, and X-ray film is placed in below nude mice, is carried out
X line takes the photograph piece.Exposure factor: 40kV, 2mA, 3s, H 28cm.4 weeks execution nude mices after discovery tumour is formed are detected by iconography,
Suspicious tumor tissues are aseptically taken to fix with 10% formalin, paraffin embedding, slice, hematoxylin eosin staining,
Optical microphotograph microscopic observation tumour growth situation.
Experimental result: it is observed that shin bone changes by X-ray within 2 weeks after bone giant cell tumor strain cell marrow intraluminal grafting
Become, tumor formation rate 100%.X-ray check becomes apparent after 4 weeks, shows as eccentricity, osteolytic, dilatancy destruction of bone, and transverse diameter is normal
More than vertical diameter, destruction area is up to subchondral bone, and the different cortical bone continuity of degree, which occurs, in perilesional to interrupt, due to multiple bone
Ridge and formed soap bubble sample change.The reason of soap bubble sample changes is formed to intersect for bone interval, with the development of lesion,
Bone interval can be destroyed in succession again.Again because tumour is constantly expanded to surrounding, and there is new bone ridge in its marginal portion
And interval occurs；Vertebra shows as centrum osteolytic destruction of bone, inside sees separation of differing in size, eccentric growth, osteolytic sclerotin
It destroys, it is most without hardened edge and periosteal reaction with the characteristics of cortical bone expansion is thinning, it is more rare (as shown in Figure 8) to invade attachment.
Histopathology slide observation will be carried out after tumor resection it can be seen that tumour is mainly by Mononuclear stromal cell and more
Two kinds of cell compositions such as core giant cell, interstitial rich blood vessel.Stroma cell is shuttle shape, oval or circle, cell indefinite border
Chu, common endochylema protrusion.Nucleus is larger, and dyeing quality is medium, can have a kernel.Multinucleate giant cell often relatively evenly dissipates
Cloth is between stroma cell, the characteristics of being this tumor.The diameter of multinucleate giant cell is often 30~60 μm, and nucleus number is generally 15~20
It is a, at most up to 100 or more, often it is gathered in the center of cell.The form of core is similar to Mononuclear stromal cell.Cell boundaries are not
Rule, but it is clearer to demarcate, and endochylema is abundant, is in slightly basophilla, is also shown the foam cells containing a large amount of lipids sometimes (such as Fig. 9 institute
Show).This tumor interstitial rich blood vessel, the collagenous fibres how many is not waited.Tumour itself is shown in there is class bone group without skeletonization phenomenon sometimes
It knits and newborn bone trabecula, is common in around fibr tissue, it may be possible to shape after a kind of reactivity new bone formation or pathologic fracture
At poroma.
8 luciferase gene of embodiment is overexpressed stable cell line (GCTB28-luc) building
1, plasmid construction:
PCDH (catalog number CD510B-1, System Biosciences) plasmid EcoR I and BamH I
Two enzymes carry out digestion, and luciferase segment is obtained the segment (primers F: CGGAATTCCG of 1653bp or so by PCR amplification
ATGGAAGACGCCAAAAACAT；R:CGGGATCCCGTTACACGGCGATCTTTCCGC)；Digestion products carry out electrophoresis and glue returns
It receives；Luciferase segment and the carrier segments of linearisation are attached with ligase (TAKARA)；Conversion, picking Dan Ke
Grand, plasmid extraction；Sanger sequencing, correct recombinant plasmid are named as PCDH-luc.
2, virus packaging:
The day before transfection plants HEK-293T cell in 6cm culture dish, and cell density is about 80%-90% when transfection.It adopts
With three plasmid packaging systems: carrier PCDH-luc 4ug, Packaging plasmid (psPAX2) 3.6ug, Envelope
plasmid(PMD2.G)1.2ug.These plasmids are added in the Opti-MEM of 500uL, 20uL transfection reagent
Lipofectamine 2,000 is also added in the Opti-MEM of 500uL, and 5min is placed at room temperature for after mixing, then by plasmid with
Lipofectamine2,000 mixing, is placed at room temperature for 20min.It feeds the mixture into HEK-293T cell, after 6 hours, replacement
For fresh complete culture solution.After 48h, virus is collected and with 0.45um membrane filtration.
3, cell infection
The day before transfection is by GCTB28 cell inoculation in 6 orifice plates, and cell density is about 30%-40% before infecting.In every
2uL Polybrene (6ug/mL) is added in a hole, 37 DEG C, 5%CO230min is cultivated, GCTB28 cell then is added in virus
In.Fresh complete medium is replaced after infection 8h.Infect be added in each hole after 48h 2uL puromycin (4ug/mL) into
Row screening, improves efficiency of infection.
Experimental result
By above-mentioned cell expansion culture, as luciferase gene stablizes the cell strain (GCTB28-luc) being overexpressed, can
For the growing state of tracing in vivo and monitoring cell in vivo.
In conclusion the cell strain that the present invention constructs has the primary giant cell tumor of bone tumour cell of people according to establishment standard
Characteristic, meet establishment standard, be one plant of newly-built people's bone giant-cell tumor tumor cell line, can be used as giant cell tumor of bone morbidity machine
System and the cell material of individualized treatment in vitro study, so for establish giant cell tumor of bone genesis mechanism model and preparation, screening,
It evaluates anti-tumor drug and basis is provided.
The above is only the preferred embodiment of the present invention, protection scope of the present invention is not limited to reality shown in this article
Example is applied, all technical solutions belonged under thinking of the present invention all belong to the scope of protection of the present invention.It should be pointed out that being led for this technology
For the those of ordinary skill in domain, several modifications and retouching without departing from the principles of the present invention also should be regarded as of the invention
Protection scope.
Claims (8)
1. a kind of primary bone giant cell tumor strain GCTB28-luc of people, which is characterized in that the primary giant cell tumor of bone of people is thin
Born of the same parents' strain GCTB28-luc is preserved in China typical culture collection center, address: Wuhan University, Wuhan, China city, preservation date:
2018.10.24 deposit number: CCTCC NO:C2018178.
2. the construction method of the primary bone giant cell tumor strain GCTB28 of people as described in claim 1, includes the following steps:
1) by the tumor specimen after the excision of giant cell tumor of bone patients surgery using the necrosis around conventional trimming removal tumor tissues
And nonneoplastic tissue, physiological saline repeated flushing, antiseptic sursery, which is cut, shreds tumor tissues, is placed in 50ml containing 10% fetal calf serum
DMEM liquid in, by nutrient solution volume be added II Collagenase Type 10mg/ml be placed in 37 DEG C of constant temperature oscillation casees with 150 revs/min
Clock vibrates 2h；
2) postdigestive tissue specimen is taken out, is sieved with 300 mesh metallic sieves, the liquid for crossing sieve is slowly injected into and is preset
In the centrifuge tube of Ficoll lymph separating liquid, after 2500 turns/min is centrifuged 30min, intermediate layer cell is collected, 1000 after PBS resuspension
Turn/min centrifugation 5min, abandons supernatant, DMEM adjusts cell density after being resuspended, with 1 × 106A/L concentration is inoculated in 25cm culture
In bottle, 37 DEG C are put into containing 5%CO2It is cultivated in the cell incubator of 95% air；With 0.25% pancreatin had digestive transfer culture；
3) by the cell inoculation after passage in new Tissue Culture Flask, DMEM culture medium is added and is cultivated；In the mistake of culture
Cheng Zhong, the cell for selecting different algebra carry out freezing conservation, cell are resuspended with frozen stock solution after collecting cell, cryopreservation tube is put into
It is saved in liquid nitrogen container, which is named as GCTB28；
4) luciferase gene is integrated on cell GCTB28 chromosomal DNA, makes cell expressing luciferase, constructs stabilization
The cell strain of expressing luciferase, is named as GCTB28-luc, obtains the primary bone of people that deposit number is CCTCC NO:C2018178
Sarcoma cell strain.
3. construction method as claimed in claim 2, which is characterized in that in step 5), the DMEM culture medium contains 10% tire
Cow's serum, 100U/ml penicillin, 100mg/ml streptomysin.
4. the primary bone giant cell tumor strain GCTB28-luc of people as described in claim 1 is establishing external people's bone giant cell
Application in tumor immunodeficient animals model.
5. the primary bone giant cell tumor strain GCTB28-luc of people as described in claim 1 is establishing the generation of people's bone giant-cell tumor
Application in mechanism model.
6. the primary bone giant cell tumor strain GCTB28-luc of people as described in claim 1 is preparing anti-human giant cell tumor of bone medicine
Application in object.
7. the primary bone giant cell tumor strain GCTB28-luc of people as described in claim 1 is screening anti-human giant cell tumor of bone medicine
Application in object.
8. the primary bone giant cell tumor strain GCTB28-luc of people as described in claim 1 is evaluating anti-human giant cell tumor of bone medicine
Application in object.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116836934A (en) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477445A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院上海生命科学研究院 | Lentivirus vector expression system of luciferase and application thereof |
| CN102943065A (en) * | 2012-11-19 | 2013-02-27 | 上海市第十人民医院 | Cell strain of giant cell tumor of human bone and application thereof |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
| CN103911342A (en) * | 2013-01-08 | 2014-07-09 | 中国人民解放军第二军医大学 | Mice animal model of giant cell tumor of bone of human and construction method thereof |
-
2018
- 2018-12-26 CN CN201811600363.3A patent/CN109749998A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102477445A (en) * | 2010-11-23 | 2012-05-30 | 中国科学院上海生命科学研究院 | Lentivirus vector expression system of luciferase and application thereof |
| CN102943065A (en) * | 2012-11-19 | 2013-02-27 | 上海市第十人民医院 | Cell strain of giant cell tumor of human bone and application thereof |
| CN103911342A (en) * | 2013-01-08 | 2014-07-09 | 中国人民解放军第二军医大学 | Mice animal model of giant cell tumor of bone of human and construction method thereof |
| CN103865876A (en) * | 2014-03-26 | 2014-06-18 | 西北民族大学 | Method for primary culture of tumor cells |
Non-Patent Citations (5)
| Title |
|---|
| C.J.JOYNER，等: "Phenotypic characterisation of mononuclear and multinucleated cells of giant cell tumour of bone", 《BONE AND MINERAL》 * |
| ZHENHUA ZHOU，等: "ALCAM+ stromal cells: role in giant cell", 《CELL DEATH AND DISEASE》 * |
| ZHENHUA ZHOU，等: "Biological characteristics of a novel giant cell tumor cell line derived from spine", 《TUMOR BIOLOGY》 * |
| 谢丹，等: "骨巨细胞瘤细胞成分和细胞因子在溶骨中的作用", 《中山医科大学学报》 * |
| 赵宁侠，等: "骨巨细胞瘤中成纤维样基质细胞体外钙化能力的研究", 《北京医科大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116836934A (en) * | 2023-08-31 | 2023-10-03 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
| CN116836934B (en) * | 2023-08-31 | 2023-11-24 | 北京大橡科技有限公司 | Osteosarcoma organoid culture solution, culture reagent combination and culture method |
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