CN109609460B - A kind of human glioma cell line and its method for building up and application - Google Patents
A kind of human glioma cell line and its method for building up and application Download PDFInfo
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Abstract
The invention discloses a kind of human glioma cell line, the human glioma cell line is CGMCC NO.16693, preservation date are as follows: on November 07th, 2018 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Human glioma cell line of the invention can stablize multiple passage, have Tumor formation, can prepare animal model for tumour, can be used for the research and development, the screening of human glioma drug and the in vitro study of human glioma of human glioma diagnostic reagent.Moreover, the present invention is divided into glioma cell using glioma stem cells, the quantity of heteroproteose cell can be reduced first, and after glioma stem cells are divided into glioma cell, the speed of growth is fast, and proliferative capacity is strong, remains the feature of glioma pathology tissue.
Description
Technical field
The invention belongs to medical domains, and in particular to a kind of human glioma cell line and its method for building up and application.
Background technique
Glioma is the common tumour of nervous centralis, and disease incidence is about 3-5/10 ten thousand people.Organize credit level Four: I grades, II
Grade is in low grade of malignancy, has benign organisms characteristic；III level and IV grades are High Grade Gliomas, especially glioblastoma,
Its median survival interval is only 12-18 months, and the median survival interval of change glioma (WHO III level) is 2-5, low level colloid
The median survival interval of tumor (WHO I, II grade) is also only 4-10.
Although current neurosurgery skill is being constantly progressive, operation instrument is increasingly advanced, also takes in conjunction with Radiotherapy chemotherapy
New progress was obtained, in addition there are also some new treatment methods, such as immunization therapy, using EGFR, IGFR, PDGFR as target spot
The treatment method of targeted therapy and anti-angiogenesis, these methods carry out use in conjunction generally according to the actual conditions of patient.But
5 years survival rates of glioma are not improved significantly, the main reason is that the high recurrent of glioma and it is heterogeneous with
And there is drug resistance phenomenon in chemotherapy process in Patients with gliomas, this serious therapeutic effect for affecting glioma.Therefore,
Novel treatment method is clinically not only needed, while being also required to develop effective therapeutic agent.
However, at present in worldwide, it is very few for the brain glioblastoma cell system of drug development and basic research,
Such as LN-229, U-87MG, U-118MG and U-138MG cell line derives from white people, with Asia ethnic group genetic background
Brain glioblastoma cell system is even more phoenix feathers and unicorn horns, compared with clinically large number of Patients with gliomas, brain glioblastoma cell system
Rare problem seriously constrains preclinical study.
Summary of the invention
For overcome the deficiencies in the prior art, one of the objects of the present invention is to provide a kind of human glioma cell lines.
The second object of the present invention is to provide a kind of progeny cell system of above-mentioned human glioma cell line.
The third object of the present invention is to provide a kind of genetically modified cell system based on above-mentioned human glioma cell line.
The fourth object of the present invention is to provide the method for building up of above-mentioned human glioma cell line.
The fifth object of the present invention is to provide the acquisition methods of above-mentioned progeny cell system.
The sixth object of the present invention is to provide a kind of animal model for tumour.
The seventh object of the present invention is to provide above-mentioned human glioma cell line, above-mentioned progeny cell system, above-mentioned tumour
The purposes of animal model, above-mentioned method for building up and acquisition methods.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of human glioma cell line, the human glioma cell line is in Chinese microorganism strain preservation conservator
The deposit number of meeting common micro-organisms center is CGMCC NO.16693, preservation date are as follows: on November 07th, 2018.
A kind of progeny cell system, the progeny cell system is passed on by above-mentioned human glioma cell line.
A kind of cell line of genetic modification, the cell line of the genetic modification be by above-mentioned human glioma cell line or on
Stating progeny cell is the cell line with oncogenicity obtained through genetic modification.
A kind of method for building up of above-mentioned human glioma cell line, in turn includes the following steps:
(a) clinical Human Brain Gliomas is obtained；
(b) humanized's tumor tissues heteroplastic transplantation model animal is constructed using the clinical Human Brain Gliomas；
(c) originally culture is carried out to the tumor tissues from humanized's tumor tissues heteroplastic transplantation model animal；
(d) secondary culture is carried out to the tumour cell of the originally culture, obtains the human glioma cell line.
In above-mentioned method for building up, humanized's tumor tissues heteroplastic transplantation model as a preferred implementation manner,
Animal used in the building process of animal is mammal；It is highly preferred that the mammal is nude mouse.
In above-mentioned method for building up, the originally culture uses as a preferred implementation manner, culture medium are as follows: contain
HEGF, human fibroblastic growth factor, B27 trophic factors and mycillin Neurobasal culture solution,
Wherein, the content of hEGF is 20ng/ml, and the content of human fibroblastic growth factor is 20ng/ml, B27 battalion
The content for supporting the factor is 2vol% (percent by volume), and the content of mycillin is 1vol% (percent by volume).
In above-mentioned method for building up, the secondary culture includes: glioma stem cells as a preferred implementation manner,
The secondary culture of (i.e. originally culture obtained cell) secondary culture and the glioma cell of stem cell differentiation.Preferably, described
Glioma stem cells secondary culture use culture medium are as follows: containing hEGF, human fibroblastic growth factor,
The Neurobasal culture solution of B27 trophic factors and mycillin, wherein the content of hEGF is 20ng/ml, people
The content of fibroblast growth factor is 20ng/ml, and the content of B27 trophic factors is 2vol%, and the content of mycillin is
1vol%；The culture medium that the secondary culture of the glioma cell of the stem cell differentiation uses are as follows: contain fetal calf serum and green chain
The DMEM culture solution of mycin, wherein in the DMEM culture solution, the volumn concentration of the fetal calf serum is 10%, green
The volumn concentration of streptomysin is 1%.
In above-mentioned method for building up, as a preferred implementation manner, in step (c), humanized's tumour is derived from
The tumor tissues of tissue heteroplastic transplantation model animal are repeatedly to be transplanted clinical Human Brain Gliomas in the mammalian body
It is obtained after culture；It is highly preferred that from humanized's tumor tissues heteroplastic transplantation model animal tumor tissues be by
Clinical Human Brain Gliomas is obtaining after 2-3 transplanting culture in the mammalian body.
A kind of acquisition methods of above-mentioned progeny cell system, using the DMEM culture solution pair containing fetal calf serum and mycillin
Above-mentioned human glioma cell line continues secondary culture, to obtain the progeny cell system, wherein train in the DMEM
In nutrient solution, the volumn concentration of the fetal calf serum is 10%, and the volumn concentration of mycillin is 1%.
A kind of animal model for tumour, the animal model for tumour are by by above-mentioned human glioma cell line, above-mentioned son
The cell line of continuous cell line or above-mentioned genetic modification is inoculated into be formed in the mammalian body, it is preferable that the animal is mouse.
Above-mentioned human glioma cell line, above-mentioned progeny cell system, the cell line of above-mentioned genetic modification, above-mentioned bearing animals
The acquisition methods of model, the method for building up of above-mentioned human glioma cell line or above-mentioned progeny cell system inhibit people in exploitation
Purposes in terms of glioma drug, the purposes in terms of preparing human glioma diagnostic reagent or be used for human brain colloid
Purposes in terms of tumor in vitro study.
Human glioma cell line of the invention, is named as BT-127, belongs to people's glioblastoma, in November, 2018
It is deposited within 07th China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: Chaoyang District, Beijing City
The institute 3 of North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101, deposit number is CGMCC NO.16693.
Compared with prior art, the invention has the following beneficial effects: human glioma cell lines of the invention to stablize
Repeatedly passage, have Tumor formation, animal model for tumour can be prepared, can be used for human glioma diagnostic reagent research and development,
The screening of human glioma drug and the in vitro study of human glioma.In addition, human glioma cell line of the invention
Method for building up is simple to operation, since tissue specimen is precious and few, can not only retain tissue specimen with PDX model, but also can be right
Tumour cell is screened in vivo, is proliferated active a part of tumour cell and is easier to grow, originally culture is easier to success, and tumour is thin
It is to be easier to success that born of the same parents, which build,.And it is traditional directly adopt Human Brain Gliomas carry out originally culture mode difficulty is big, cell not
Easily survival.Moreover, the present invention is divided into glioma cell using glioma stem cells, the quantity of heteroproteose cell can be reduced first,
After glioma stem cells are divided into glioma cell, the speed of growth is fast, and proliferative capacity is strong, remains glioma pathology tissue
Feature.
Detailed description of the invention
Fig. 1 is the form microphoto of BT-127 cell；Wherein, Fig. 1-A is that BT-127 cell G1 is commissioned to train feeding photo
(100×)；Fig. 1-B is that BT-127 cell G1 is commissioned to train feeding photo (100 ×) when cell density is larger；Fig. 1-C is that BT-127 is thin
Born of the same parents G20 is commissioned to train feeding photo；Fig. 1-D is that BT-127 cell G50 is commissioned to train feeding photo.
Fig. 2 is the chromosome parting microphoto of BT-127 cell；Wherein, (a) is to shoot photo under microscope；(b) it is
Photo after rearranging.
Fig. 3 is patient's glioma cells in tissue and nude mice model tumor tissue in pathomorphism photo, wherein (a) is patient's brain
Samples of human glioma；It (b) is F3 generation transplanting tumor tissue.
Fig. 4 is patient's glioma cells in tissue and transplanted tumor in nude mice histogenic immunity histochemical staining result photo；Wherein, (a-1)
For the cell proliferation markers Ki67 of patient's glioma cells in tissue；It (a-2) is the cell proliferation markers of F3 generation transplanting tumor tissue
Ki67；It (b-1) is the Deiter's cells marker neuroglial acidic protein GFAP of patient's glioma cells in tissue；(b-2) it is
F3 generation transplanting tumor tissue Deiter's cells marker neuroglial acidic protein GFAP；It (c-1) is patient's glioma cells in tissue
Nerve-specific Protein S 100；It (c-2) is the nerve-specific Protein S 100 of F3 generation transplanting tumor tissue.
Fig. 5 is that the GFAP immunofluorescence dyeing result of BT-127 cell shows photo, wherein (a) is intracellular GFAP dye
Color；It (b) is nuclear targeting；(c) picture is superimposed for GFAP and nuclear targeting.
Fig. 6 is tumor doubling time control curve figure.
Fig. 7 is cell tumor formation photo.
Fig. 8 is that oncolytic virus kills BT-127 tumour cell photo.
Fig. 9 be oncolytic virus kill F3 transplant tumor tissue in tumour cell result figure, wherein A be gross tumor volume at any time
Change curve, B is the photo of tumor size in different time points mouse after injecting oncolytic virus.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, below in conjunction with attached drawing to embodiment party of the present invention
Formula is described in further detail.
The present invention is studied to be ratified through Baijing Tiantan Hospital's Medical Ethics Committee.
The present embodiment can carry out originally culture and secondary culture using the transplanting tumor tissue of F1 generation, can also use F2 generation
Or the transplanting tumor tissue in F3 generation carries out originally culture and secondary culture, can adopt to the tumor tissues from PDX animal pattern
The foundation of cell line is carried out with the primary culture method of this field routine and secondary culture method, it is preferable to employ following embodiments
The method recorded in one.
The present invention is by establishing glioma PDX model supports Primary Tumor tissue growth, HE dyeing and immuning tissue
It learns dyeing and confirms that F3 generation transplanting tumor tissue remains the histological subtypes and phenotypic features of protocerebrum archicerebrum samples of human glioma；The present invention is excellent
It chooses the active transplantable tumor tissue block of F3 generation proliferation and carries out originally culture, it is primary thin using the purifying culture of Neurobasal culture solution
Born of the same parents, then use the DMEM culture solution culture containing fetal calf serum, and obtaining long term growth and to stablize the BT- of passage in vitro
127 cells.
Relevant biology, science of heredity and tissue-derived identification are carried out to BT-127 cell, until G50 generation all has phase
Same stable character.Through Germicidal efficacy and verifying, the BT-127 main body of growth in vitro is in shuttle-type, there are irregular branch or is divided
Fork, contact growth inhibition is unobvious when cell density is larger, in intersection accumulated growth state, after 50 passages, cell
Form still remains unchanged.Genetics research confirms that the cell is heteroploid, and Numerical and structural chromsomal aberrations are serious, meet pernicious
The genetics characteristics of tumour.The cell can form tumour in nude mouse, have oncogenicity.
The foundation of this cell line has following characteristics, and pathological tissue is derived from the pathological tissue of recurrent glioma patient, sharp first
PDX model is established with the pathological tissue, and passage can be stablized.Take the pathological tissue culture glioma stablized and passed on dry thin
Stem cell is divided into glioma cell after passing for 3 generations by born of the same parents, which can stablize passage 50 times or more.
Concrete operations see below with result.
The construction method of one human glioma cell line BT-127 of embodiment
1, tissue of patient sample process
1.1 patient profiles summarize:
The patient, male, 37 years old, is diagnosed as glioblastoma, undergoes before this glioma cells in tissue resection operation
2 glioma operations are crossed, and a Subcutaneous metastasis also has occurred in addition to encephalic primary tumor in the glioma this time recurred, it is seen that this example
The invasion of glioma is very strong, is transferred to that the case outside cranium is seldom for glioma, therefore transfer to the disease outside cranium
Tissue cultures are managed into PDX model, and then the quantity for being trained glioma cell line is less more, therefore human glioma of the invention
Cell line has important meaning for the invasion and transfer and drug test etc. of studying glioma.
In addition, this patient has carried out chemicotherapy, therefore human brain of the invention before the secondary glioma cells in tissue resection operation
Glioma cell line is very likely resistant to chemicotherapy, and the PDX model thus established may have the ability for resisting chemicotherapy, because
This cell line has great importance for research oncolytic virus to the therapeutic effect of glioma.
The processing of 1.2 tissue samples
The fresh resection of glioma sample (male, 37 years old, glioblastoma) that above-mentioned patients surgery is obtained is set immediately
In pre-cooling DMEM/F12 culture medium (mycillin containing 1vol%, mycillin of the invention be purchased from be purchased from Gibco, article No.
15140122, wherein every milliliter includes 10000 units of Penicillin (alkali) and 10000 μ g streptomysins (alkali), utilize 0.85wt% salt
The benzyl penicillin (sodium salt) and streptomycin sulphate of liquid form) in of short duration storage, to guarantee activity of the sample before reaching laboratory,
Pay attention to tissue sampling and keeps sterile during transporting.
Sample is taken out in aseptic superclean bench, is placed in 10cm sterile petri dish, and the PBS that 5ml pre-cooling is added is slow
Fliud flushing (pH=7.4) is cleaned 3 times, washes away blood and impurity as far as possible, then identifiable with the ophthalmic tweezers removal disinfected
After location of necrosis and blood vessel, samples of human glioma is moved into the tissue block for being cut into 2x2x3mm in new 10cm sterile petri dish, is added
The PBS buffer solution (pH=7.4) of 5ml pre-cooling is placed stand-by on ice.The sterile working of this process is completed within 30 minutes.
2, the building of PDX (heterograft of humanized's tumor tissues) animal pattern
6-8 week old male BALB/c-nu nude mice is chosen, under routine disinfection, anesthesia, cuts forelimb armpit on the right side of nude mice
Nest skin about 0.5cm long, tumor tissue is seeded to subcutaneously, is sewed up the incision.
To tumour growth to diameter about 1cm, routine puts to death mouse, aseptically tumor resection, the transplantable tumor of acquisition
Tissue is named as F1 generation, and a part of F1 generation transplanting tumor tissue is placed in STEM-CELL Banker frozen stock solution and is placed in -80 degree ice
Case freezes, and moves in liquid nitrogen container and saves after 3 days.
In 1 generation of residual F, transplants tumor tissue and continues in the above way to pass in nude mice by subcutaneous, and the transplanting tumor tissue of acquisition is named as
F2 generation, later every biography generation take a part transplanting tumor tissue to be transferred in liquid nitrogen according to the above method and save.
It repeats abovementioned steps and obtains F3 generation.
3, originally culture
In sterile super-clean bench, F3 is taken to be placed in 10cm for fresh tumor tissues (size is 4mm x 4mm x 4mm)
In sterile petri dish, the PBS buffer solution (pH=7.4) that 4 DEG C of 5ml pre-coolings are added is cleaned 3 times, is washed on tissue block as far as possible
Blood and impurity.Then after removing identifiable location of necrosis and blood vessel with the ophthalmic tweezers disinfected, tumor tissues are moved into
< 1mm is cut into new 10cm sterile petri dish3Fritter.The tissue block shredded is sucked into 15ml centrifuge tube simultaneously with dropper
The PBS buffer solution (pH=7.4) that 4 DEG C of 10ml pre-coolings are added mixes, and 1000 turns/min removes supernatant and boundary after being centrifuged 3 minutes
The impurity at place, the effect using pre-cooling PBS buffer solution are to reduce histocyte metabolic exhaustion.5ml erythrocyte cracked liquid is added
(Sigma), dropper blow it is even after centrifuge tube be put into ice chest stand 5-10 minutes.After 1000 turns/min is centrifuged 3 minutes in removal
Clear and intersection impurity.Then trypsin solution (Gibco, the article No. 1929961) digestion that 3ml0.05% is added into centrifuge tube is swollen
Tumor tissue block, dropper blow it is even after be fixed on uniform rotation device, be put into 37 DEG C of insulating boxs about 10 minutes.When seeing in centrifuge tube
When tissue block becomes cotton-shaped, DMEM culture solution (fetal calf serum containing 10vol%, 1vol% of twice of above-mentioned trypsin solution volume is added
Mycillin) terminate digestion, 1000 turns/min is centrifuged 3 minutes, removes supernatant and intersection impurity.10ml PBS buffering is added
Liquid (pH=7.4) is filtered after mixing is resuspended with screen, collects filtrate.After in 15ml centrifuge tube, 700 turns/min
Supernatant is sucked out in centrifugation 3 minutes, and 10ml Neurobasal culture solution (Gibco, article No. 21103049) is added into precipitating and (contains
20ng/ml hEGF, 20ng/ml human fibroblastic growth factor, 2vol%B27 trophic factors, 1vol%
Mycillin；), it is placed in 10cm sterile petri dish after mixing, in 37 DEG C of constant incubators 5%, CO2Under the conditions of cultivate 3 days.
4, secondary culture
After originally culture 3 days, it can be observed that the nerve ball (i.e. glioma stem cells) formed is suspended in culture solution, receive
Collect culture solution, 300g is centrifuged 3 minutes and removes supernatant, and adding the fresh Neurobasal culture solution of 10ml, (epidermis of people containing 20ng/ml is raw
The long factor, 20ng/ml human fibroblastic growth factor, 2vol%B27 trophic factors (be purchased from Gibco), 1vol% green chain
Mycin) nerve ball is resuspended in new Nerobasal culture solution and continues to cultivate in 10cm sterile petri dish, until nerve ball
It when diameter is greater than 3.0mm, is placed in 15ml centrifuge tube, is added 3ml PBS buffer solution (pH=7.4) with liquid-transfering gun picking nerve ball
Nerve ball is slowly rinsed, 300g is centrifuged 3 minutes abandoning supernatants, and cell precipitation uses 1ml1 × Accutase (Gibco, article No.
A1110501 digestion about 10 minutes) is resuspended, 300g is centrifuged 3 minutes after cell dispersion；Then the cell of collection is resuspended in
In 10ml Neurobasal culture solution (with corresponding culture solution used in originally culture), in new 10cm sterile petri dish
Continue to cultivate, when neural bulb diameter is greater than 3.0mm, passage.
Cell is continued to cultivate three generations by above-mentioned propagating method, and Neurobasal culture solution is then changed to DMEM culture solution
(mycillin of fetal calf serum containing 10vol%, 1vol%) is cultivated, and cell will be transformed into adherent growth by suspension growth,
And the cell of first adherent growth is named as BT-127 glioma cell, cell algebra is denoted as G1 generation.G1 is grown to for cell
Continue secondary culture when 80%-90% convergence degree, cultural method is as follows:
Outmoded culture solution in culture bottle is poured out, 2ml PBS buffer solution (pH=7.4) is added and slowly rocks flushing cell, and
Discard flushing liquor.0.05% trypsin solution room temperature of about 1ml is added into culture bottle to digest 1 minute or so, visible patch under mirror
Small circular and the travelling that suspends is presented in parietal cell, into culture bottle addition 2ml DMEM culture solution (fetal calf serum containing 10vol%,
The mycillin of 1vol%) digestion is terminated, dropper is blown and beaten repeatedly, and movement pays attention to softly, utmostly dispersing glioma cell
For individual cells, 15ml sterile centrifugation tube is then moved into, 800 turns/min removes supernatant after being centrifuged 3 minutes.Add into centrifuge tube
Enter the new DMEM culture solution of 6ml (mycillin of fetal calf serum containing 10vol%, 1vol%), dropper pressure-vaccum cell dispersion repeatedly,
Attention action is soft, which is divided into 3 culture bottles, then that 3ml is respectively added in 3 culture bottles is new
DMEM culture solution (mycillin of fetal calf serum containing 10vol%, 1vol%), the culture solution total volume in each culture bottle are
5ml, and the obtained passage cell after step culture is denoted as G2 generation, when G2 grows to 80%-90% convergence degree for cell
Continue to be passed on by above-mentioned secondary culture method, BT-127 growth of glioma cells is good, and form is more uniform, can be passaged to
50 is more than generation.
The morphological observation of two BT-127 cell of embodiment
The culture bottle for cultivating BT-127 cell is placed under inverted microscope, is observed under bright-field, Fig. 1 BT-
127 cell G1 are commissioned to train feeding photo, and as a result visible BT-127 cell bodies are in shuttle-type, and there are irregular branch or bifurcated (Fig. 1-
A, 100 ×)；It is unobvious that growth inhibition is contacted when cell density is larger, in intersection accumulated growth state, and by the outside spoke of habitat
Penetrate growth (Fig. 1-B, 100 ×).
Fig. 1-C is that BT-127 cell G20 is commissioned to train feeding photo.Fig. 1-D is that BT-127 cell G50 is commissioned to train feeding photo.By
The tetra- width figure of A-D of Fig. 1 compares it is found that after 50 passages, and the form of cell still remains unchanged, this illustrates prepared by the present invention
BT-127 cell can steadily pass on, facilitate pathophysiologic features, the hereditary feature of furtheing investigate the cell line for a long time,
Facilitate as the platforms such as drug test cell line or induce the cell line of animal model for tumour.
The chromosome karyotype analysis of three BT-127 cell of embodiment
The BT-127 cell for taking logarithmic phase to grow is added colchicine, makes its final concentration of 0.2 μ g/ml, in 37 DEG C of incubators
In continue culture 4 hours.Acquire metaphase cell, with fixer (methanol: glacial acetic acid=3:1 (V/V), matching while using) into
Row is about 1 hour fixed, then drips cell suspension on the micro slide of pre-cooling, is dyed with Giemas dyeing liquor, under microscope
Enumerating chromosomes number.As a result see Fig. 2, after BT-127 cell continuous passage, chromosome still keeps humanized's tumour cell chromosome
Feature, modal number (M) concentrates between 45~55, accounts for 70%, it is seen that the BT-127 cell is heteroploid, chromosome number
Mesh and structural aberration are serious, meet the genetics characteristics of malignant tumour.
Example IV BT-127 cell short-movie section repetitive sequence (STR) analysis
Short tandem repeat (short tandem repeat, STR) is also known as microsatellite DNA, refers on chromosome,
A kind of DNA sequence dna (the number of repetition 10 formed by several base-pairs as core unit (2~6 base-pairs), tandem sequence repeats
More than~60 times, genetic fragment is below 400 base-pairs)；Each duplicate number of core unit will appear individual difference, thus shape
At the different allele of fragment length.Therefore, the number of repetition of one group of STR sequence is almost unique in Different Individual,
It is the gene identities feature of individual and the main method that cell biology identifies cell identity and source.
It takes the BT-127 cell Microread Genomic DNA Kit of fresh cultured to extract DNA, uses
MicroreaderTM21ID System expands 20 STR bit points and sex identification site, uses ABI 3730xl type genetic analysis
Instrument carries out PCR product detection, is divided using GeneMapper3.2 software (Applied Biosystems) testing result
Analysis, and compared with American type culture collection (ATCC) and Germany Microbiological Culture Collection Center (DSMZ) database
It is right, identical STR testing result is not found, be the results are shown in Table 1, is not found human cell's cross contamination in cell.
1 STR situation summary sheet of table
The tissue-derived identification of embodiment five
Take F3 generation transplanting tumor tissue and protocerebrum archicerebrum samples of human glioma that rear paraffin embedding is fixed respectively, preparation is sliced and carries out
HE dyeing and immunohistochemical staining.HE coloration result shows that F3 generation transplanting tumor tissue and protocerebrum archicerebrum samples of human glioma are being organized
High consistency is presented in form and institutional framework, referring to Fig. 3, transplants the tissue that tumor tissue remains protocerebrum archicerebrum samples of human glioma
Learn hypotype；Immunohistochemical staining is the results show that referring to fig. 4, cell proliferation markers Ki67, nerve-specific Protein S 100 and mind
Table through spongiocyte marker neuroglial acidic protein (GFAP) in F3 generation transplanting tumor tissue and protocerebrum archicerebrum samples of human glioma
Up to no difference of science of statistics, it was confirmed that nude mice model tumor tissue remains the phenotypic features of protocerebrum archicerebrum samples of human glioma.
Further G42 is cultivated on the cover slip for BT-127 cell inoculation, after cytochrome oxidase isozymes, is fixed with 4% formaldehyde,
Carry out immunofluorescence dyeing.Referring to Fig. 5, as the result is shown Deiter's cells marker neuroglial acidic protein (GFAP) be in compared with
Strong positive expression, binding of pathological diagnosis, the cell are brain glioblastoma cell.
From the above results, BT-127 cell of the invention can express the typical marks object of glioma cell, in turn
Judge that the cell line has typical tumoral character.
Six cell Proliferation of embodiment and doubling time
The BT-127 cell of fresh cultured is taken to be laid in 96 orifice plates, after cell count, the cell concentration in every hole is 3000, often
100 μ l DMEM culture solutions (mycillin of fetal calf serum containing 10vol%, 1vol%) are added in hole, and 100 μ l are added in control wells
PBS buffer solution (pH=7.4), respectively spread 7 96 orifice plates, respectively at the 24th hour, 48 hours, 72 hours, 96 hours, 120 hours,
Corresponding 96 orifice plates are taken out after 144 hours, 168 hours, after exhausting the culture medium in hole, the fresh DMEM training of 100 μ l is added in every hole
Mixed solution (Dojindo, the article No. of nutrient solution (mycillin of fetal calf serum containing 10vol%, 1vol%) and 10 μ l CCK8
CK04 incubation 1 hour) is protected from light in 37 DEG C of insulating boxs.Microplate reader measures the absorbance value at 450nm, maps and uses formula
" doubling time=log2/ slope " calculates the doubling time.Cyto-dynamics result of study shows, the population doublings of BT-127 cell
Time is about 40 hours (referring to Fig. 6), shows that BT-127 cell viability is fine, and it is related to be applied to glioma as cell model
Research.
Seven heterograft of embodiment test
Take the BT-127 cell of fresh cultured, inoculate BALB/C immunodeficient mouse (tie up tonneau China in Beijing, male,
Age of mouse 6 weeks), every animal inoculation pvaccination 0.2ml contains 5 × 106A cell, daily observation and measurement the weight of animals and tumor size.It connects
Kind is after about 5 days, and tumour is initially formed and grown, tumor formation rate nearly 100%.As a result as shown in Figure 7, it is seen that the BT-127 cell can be
Tumour is formed in immunodeficient mouse body, and there is oncogenicity.
It follows that tumour can be formed in Mice Body by being inoculated with BT-127 cell, so that it is dynamic to form tumour
Object model.
Eight drug sensitivity testing in vitro of embodiment
External test glioma clinic commonly uses therapeutic agent: Temozolomide and lomustine, to BT-127 cell and brain
The antiproliferative effect of glioma cell line LN-229 (source ATCC, CRL-2611) cell.Logarithmic growth phase G46 is collected for BT-
127 cells and brain glioblastoma cell system LN-229 cell adjust concentration of cell suspension, the inoculating cell suspension (100 in 96 orifice plates
The hole μ l/), 10000, every hole cell is placed in 37 DEG C, 5%CO2In incubator after preculture 24 hours, it is separately added into Temozolomide
With lomustine and blank control, final concentration of 3 μM of drug, continue to be placed in 37 DEG C, 5%CO2It is cultivated in incubator.Drug is made
After 24 hours, cell viability is detected with CCK8 reagent.Specific step is as follows: exhausting cell culture fluid in orifice plate, then to every hole
100 microlitres of DMEM culture solutions (mycillin of fetal calf serum containing 10vol%, 1vol%) of middle addition and 10 microlitres of CCK8 solution,
Orifice plate is placed again into incubator and is incubated for 1 hour, using the absorbance at microplate reader measurement 450nm, the results are shown in Table 2,
We have found that the effect that each therapeutic agent compares LN-229 cell to the growth inhibition effect of BT-127 cell is weak, illustrate BT-127
Cell, can be as the cell model of screening new type antineoplastic medicine compared with LN-229 cell more drug resistance.
Table 2
The therapeutic evaluation of nine novel oncolytic virus of embodiment killing cell
Oncolytic virus, which refers to, infects cancer cell for antiviral selectivity natural or Jing Guo genetic recombination, certainly by virus
Cancer cell is killed and cracked to the function of body, at the same it can also challenge, it is residual to continue to kill to attract more immunocytes
Remaining cancer cell.Novel oncolytic virus is to carry out oncolytic virus made of genetic modification based on herpes simplex virus, and carry green
Color fluorescin (EGFP) reporter gene, the building of novel oncolytic virus are according to application number 2004100064921, and authorization is public
The method recorded in the patent application of announcement CN1283803C by wild type HSV-1 virus (No. GenBank of its gene order:
NC_001806 ICP34.5 gene and ICP47 gene knockout), and the position of the knockout ICP34.5 gene in HSV-1 virus is inserted
Enter the gene of the encoding green fluorescent protein guided by MMLV promoter.
By glioma cell U87MG (source ATCC, HTB-14), U251MG (source ATCC), BT-127 cell G1 and
The G50 of BT-127 is plated in 96 orifice plates respectively for cell, and 5000 cells are added in every hole and 100 μ l DMEM culture solutions (contain
The mycillin of 10vol% fetal calf serum, 1vol%) it is put into 37 DEG C of incubator 5%CO2Under the conditions of cultivate.It is believed that after 24 hours
Inoculating cell grows to 104Oncolytic virus is pressed MOI (i.e. infection multiplicity, the ratio of virus and cell quantity when referring to infection by a every hole
Value) it is 100、10-1、10-2、10-3With 10-4Five titres are added in corresponding orifice plate, and the DMEM training of same volume is added in control group
96 orifice plates, are then put into 37 DEG C of cell incubators and are incubated for by nutrient solution (mycillin of fetal calf serum containing 10vol%, 1vol%)
48 hours, fluorescence microscopy microscopic observation was simultaneously taken pictures.Visible novel oncolytic virus has BT-127 cell and kills under fluorescence microscope
Wound effect, and virus titer is got over High Fragmentation effect and is more obvious, as shown in figure 8, novel oncolytic virus is swollen as MOI value increases
Duplication increases (EGFP expression increases) in oncocyte, when MOI value reaches 100When, Partial tumors cell is dead, visible under mirror
EGFP expression decline.
Effect of the ten novel oncolytic virus of embodiment to Transplanted tumor model
It takes F3 generation transplanting tumor tissue to be inoculated in nude mice by method in above-mentioned PDX model construction and (chooses 6-8 week old male
BALB/c-nu nude mice) subcutaneous growth, nude mice is divided into control group and experimental group, is connect to Xenografts in nude mice tissue block diameter
When nearly 0.6cm, by the novel oncolytic virus injection in embodiment nine into transplanting tumor tissue, novel oncolytic virus is observed to transplanting
The effect of tumor.The physiological saline of the tumor tissue injection equivalent of control group, experimental group injection concentration is 3.75 × 108Pfu/ml's is new
Type oncolytic virus.Situations such as variation of every 2 days observation Xenografts in nude mice, such as whether there is or not hemorrhagic necrosis, and control is measured respectively
The length and width of group and experimental group transplantable tumor, make a record.
Referring to Fig. 9 A, visible experimental group transplantable tumor is obviously reduced after 2 weeks, and control group transplantable tumor still has becoming for continued growth
Nude mice is put to death after injecting virus 14 days, takes tumor tissue by gesture, obviously inhibits glioma after handling referring to Fig. 9 B oncolytic virus
Growth.
As known by the technical knowledge, the present invention can pass through the embodiment party of other essence without departing from its spirit or essential feature
Case is realized.Therefore, embodiment disclosed above, in all respects are merely illustrative, not the only.Institute
Have within the scope of the present invention or is included in the invention in the change being equal in the scope of the present invention.
Claims (4)
1. a kind of human glioma cell line, the human glioma cell line is in China Committee for Culture Collection of Microorganisms
The deposit number of common micro-organisms center is CGMCC NO.16693, preservation date are as follows: on November 07th, 2018.
2. a kind of construction method of animal model for tumour, which is characterized in that the step of the construction method of the animal model for tumour
Are as follows: human glioma cell line as described in claim 1 is inoculated into and forms the animal model for tumour in the mammalian body.
3. the construction method of animal model for tumour as claimed in claim 2, which is characterized in that the animal is mouse.
4. purposes of the human glioma cell line described in claim 1 in terms of preparing human glioma diagnostic reagent or
For the purposes in terms of the external non-treatment purpose research of human glioma.
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