CN106834232A - Pituitary adenoma cell system and application thereof - Google Patents
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Abstract
The invention discloses one plant of Pituitary adenoma cell and application thereof, belong to oncobiology field.Pituitary adenoma cell strain (HPA1446), preserving number is CGMCC No.12672.It is an advantage of the invention that：Cell line of the present invention long-term cultivation and can keep cell characteristics constant in vitro, be the powerful of hypophysoma correlative study.It is the powerful studied tumor invasion and correlation molecule mechanism of action, prepare tumour relevant animal experimental model, research and development screening and evaluate anti-tumor medicine/method/diagnostic reagent, exploitation tumour medicine target spot, tumor biotherapy new technology and the engineering product research of detection associated biomolecule.
Description
Technical field
The present invention relates to one plant of Pituitary adenoma cell and application thereof, belong to oncobiology field.
Background technology
Pituitary adenoma accounts for the 10%-15% of Primary intracranial tumor, is one of most common nervous system neoplasm.Have been reported that
Pituitary adenoma postmortem discovery rate is claimed to be up to 14.4%, random crowd MRI recall rates are 22.5%.According to whether energy secreting hormone,
Hypophysoma can be divided into hormone secretion hypophysoma and non-functional adenoma.And according to the species of hormone secretion, hormone secretion hangs down
Body adenoma can be divided into prolactin, growth hormone, corticotropin, thyrotropic hormone and promoting sexual gland hormone point again
Secrete type hypophysoma.The clinical manifestation of hypophysoma be mainly tumour occupancy symptom and hormone secretion disorder caused by it is a series of
Clinical changes.The former is mainly shown as headache, visual impairment, defect of visual field etc., and the latter, may be for suffering from according to the species of hormone
The growth of person, development, labour capacity and reproductive function are produced and had a strong impact on.Cure rate is low, and treatment difficulty is big, and it is mesh that prognosis is not good
The problem of the preceding upper urgent need to resolve of clinically hypophysoma treatment.So far, although substantial amounts of research is expanded to hypophysoma, but
It is still clear far away for molecular mechanism that hypophysoma develops.Wherein, ripe efficient humanizing cells system is lacked, as mesh
The very big bottleneck of preceding restriction etiology of pituitary adenomas and biological characteristic research.
Pituitary adenoma cells culture is the Main Means that study of disease occurs and treats.People are different types of to mouse and people
Pituitary adenoma cells are studied for a long period of time, have been successfully established several muroid pituitary adenoma cells systems, obtain wide under study for action
General application.Be separately cultured at present successfully, can stablize and pass on and keep the having from Mouse Pituitary ACTH of good secreting function
The AtT-20 cell lines of adenoma cell, the GH3 cell lines from rat pituitary tumor, from rat pituitary prolactinoma
The cell line such as MMQ cell lines and α T3-1, GT1-1 from transgenic mice promoting sexual gland hormone pituitary adenoma.
Although carrying out the experiment porch of in vitro culture based on human pituitary tumor tissue, from drawing materials to the whole flow process side cultivated
Method, condition are after improvement, but the foundation of human pituitary adenomas cell line is difficult all the time.Because pituitary tumor cell growth is slow
Slowly, survival in vitro environment is difficult to maintain pituitary tumor cell secreting function and the problems such as fibroblast mixes, so far still nobody
Class immortalizes pituitary adenoma cells system and sets up.Therefore, it is based on mouse source pituitary tumor cell existing clinical and Basic Experiment Study more
It is to carry out on platform.But because there is larger thing in the origin of rodent pituitary tumor cell and people source pituitary tumor tissue
Interspecific difference, therefore the experimental data that obtains of mouse source pituitary tumor cell platform is still more difficult marries again in human experimentation, therefore the people of maturation
Source pituitary adenoma cells are urgently developed.
There was only mouse source pituitary tumor cell at present, not the pituitary tumor cell of humanization, people's hypophysoma of original cuiture is thin
Born of the same parents have many difficulty.
The content of the invention
The technical problem to be solved in the present invention is to provide one plant of people source Nonfunctional pituitary adenoma cell that can stablize passage
System.
To achieve the above object, the present invention uses following technical scheme：
Pituitary adenoma cell strain, the entitled HPA1446 of cell line is deposited in Chinese microorganism strain preservation conservator
Meeting common micro-organisms center, preserving number is CGMCC No.12672.
The cell has the biological characteristics of hypophysis derived cell：
Human pituitary's oncocyte system HPA1446 of the present invention is from the tumor tissues of Nonfunctional pituitary adenoma patient
Acquisition is separately cultured, the cell relies on method by cell factor and adherent method screens acquisition people's pituitary tumor cell repeatedly, by end
Granzyme is transfected, cell purification, and monoclonal filters out the cell line HPA1446 cells with stronger multiplication capacity, passes through in vitro
Secondary Culture more than 20 times, cell still keeps pituitary tumor characteristic.The cell has the ultra microstructure of pituitary tumor cell, and table
Up to the marker protein of various hypophysis derived tissues, while also having tumor characteristic and one-tenth knurl ability.
(1) partly it is triangle or polygonal under an optical microscope mostly in circle fusiformis, there is preferable refractivity, core
Film is complete, and kernel is clear；(2) stabilization passage, passes on 20 more than generation first；(3) chromosome is 100 different times of karyotypes；
(4) cell expressing protein sca-1 is positive, not express alpha-SMA；(5) visible shell membrane small circular promoting sexual gland hormone point under Electronic Speculum
Secretory granules；(6) cell has internal nude mice part one-tenth knurl ability.The cell is to enter growth stationary phase in growth 36h.
The culture of the immortalized cell line of Pituitary adenoma cell strain CGMCC No.12672 immortalizes monoclonal.
A kind of cell culture, comprising Pituitary adenoma cell strain CGMCC No.12672.
The purposes of cell line of the present invention includes but is not limited to following purposes：
Described Pituitary adenoma cell strain is used to prepare the purposes of tumor models.
Described Pituitary adenoma cell strain is used to prepare the purposes of animal model for tumour.
Described Pituitary adenoma cell strain is used to screen and/or evaluate/prepare the purposes of anti-tumor medicine.
Described Pituitary adenoma cell strain is for the purposes for developing tumour medicine target spot.
Described Pituitary adenoma cell strain is for the purposes for preparing tumour diagnostic reagent.
Purposes of the described Pituitary adenoma cell strain in screening tumor biotherapy medicine/reagent.
Purposes of the described Pituitary adenoma cell strain in oncotherapy new technology is developed.
Purposes of the described Pituitary adenoma cell strain in exploitation detection tumour associated biomolecule engineering product.
Cell line of the present invention possesses multiple use：Can be used for study Pituitaryadenoma morbidity and correlation molecule mechanism of action,
Research and development screening and evaluation Pituitaryadenoma medicine, the experimental evaluation of pituitary adenoma relevant animal, biological therapy new technology and inspection
Survey associated biomolecule engineering product etc. and necessary cell experiment instrument is provided.
It is an advantage of the invention that：Cell line of the present invention long-term cultivation and can keep cell characteristics constant in vitro, be hypophysis
The powerful of knurl related drugs research.
Biological deposits information：
Biomaterial title：HPA1446
Classification And Nomenclature：Pituitary adenoma cell strain
Preservation date：On June 24th, 2016
Preserving number：CGMCC No.12672
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center, address is exposed to the sun for Beijing
No. 3 Institute of Microorganism, Academia Sinica of institute of area North Star West Road 1, postcode 100101.
The present invention will be further described with reference to the accompanying drawings and detailed description, not limitation of the present invention.It is all
This area equivalent carried out according to the disclosure of invention, belongs to protection scope of the present invention.
Brief description of the drawings
Fig. 1 is the growth curve chart of primary cell and immortalized cells.
Fig. 2 is the 20th generation pituitary tumor cell of culture, is partly triangle or polygonal mostly in circle fusiformis.
Fig. 3 is the ultra microstructure of cell under transmission electron microscope.
Fig. 4 is the expression that immunofluorescence dyeing detects sca-1 albumen, and positive cell is the cell of red fluorescence, sca-1
Expression rate>80%.
Fig. 5 is the expression that immunofluorescence dyeing detects α-SMA albumen, and redfree fluorecyte, α-SMA are expressed as feminine gender,
Indicate without fibroblast pollution.
Fig. 6 is oil mirror observation and chromosome counting, sees that chromosome is 100 different times of karyotypes.
Fig. 7 is the Flow cytometry cell cycle.
Fig. 8-1 and Fig. 8-2 is tumor formation in nude mice result.
Specific embodiment
Embodiment 1：The acquisition of Pituitary adenoma cell strain and culture
First, HPA1446 primitive cell cultures
1st, biological material source
Cell line of the present invention is to be separately cultured to obtain from nonfunctioning pituitary adenoma operation patient's hypophysoma tissue tissue.
Tumor tissues take from a corrective surgery sample at Baijing Tiantan Hospital's neurosurgery center, and pathology is examined
It is nonfunctioning pituitary adenoma to break, and oncocyte dense distribution under microscope, after birth boundary is unclear, nucleus size and form not very
Cause, it is seen that kernel and double-core oncocyte.
2nd, isolated culture method
Tissue block is washed 2 times with containing the PBS solution (1: 1) for breaking red liquid (being purchased from Gibco companies) in aseptic super-clean bench, is removed
Blood vessel and slough, about 1mm is cut into tissue shear by tissue block3Size, adds 3ml 0.04%EDTA/0.5% tryptoses
Enzyme, 37 DEG C of water-baths digest 10 minutes, basis of microscopic observation, wait be digested to it is unicellular after, add completely trainings of the 3ml containing hyclone
Support base and terminate digestion.Filtered with the metallic sieve of 200 mesh, remove indigested tissue.Cell suspension after filtering is in PBS solution
In by centrifugation (1000rpm/ point, 10 minutes) washing 2 times, collect the sedimentation cell of centrifugation bottom of the tube, cell is suspended in and is contained
Have in the nutrient solution of growth factor, be inoculated in T25 blake bottles, be placed in 37 DEG C, 5%CO2Cultivated in incubator.
The preparation of the nutrient solution containing growth factor：90%DMEM/F12 (is purchased from Gibco companies), and 10%FBS (is purchased from
Gibco companies), basic fibroblast growth factor (the basic Fibroblast Growth of final concentration of 10ng/ml
Factor, bFGF, are purchased from inventrogen companies), the EGF (Epidermal of final concentration of 20ng/ml
Growth Factor, EGF, are purchased from inventrogen companies).
3rd, cell purification
Suspension cell is abandoned after cell culture 24h, attached cell changes the DMEM/F12 containing 20% hyclone and continues to cultivate, treats
Cell growth close to 90% converge when, PBS solution is washed 2 times, adds 3 points of 0.4ml 0.04%EDTA/0.5% Trypsin Induceds
Clock, is added and states nutrient solution piping and druming sub-bottle, and 37 DEG C are cultivated 30 minutes, collect suspension cell, abandons attached cell, 37 DEG C of trainings of suspension cell
Support 30 minutes, after cell is adherent again, collect suspension cell, abandon attached cell, suspension cell is cultivated 30 minutes for 37 DEG C, led to again
Adherent method repeatedly is crossed, fibroblast is finally removed.
4th, Secondary Culture
Cell growth after purification up to 90% converge when, add 0.4ml 0.04%EDTA/0.5% Trypsin Induceds 2
Minute, add and state nutrient solution piping and druming, sub-bottle, 37 DEG C, 5%CO2Secondary Culture in incubator.Said process is repeated, until passing 20
In generation, primary pituitary adenoma cell is obtained, carry out every detection.
2nd, the culture of the pituitary adenoma cells for immortalizing
1st, cell immortality processing method
Primary pituitary adenoma cell is filtered out with stronger multiplication capacity by Telomerase transfection, cell purification, monoclonal
Cell line be passaged to 20 generations through microsatellite analysis the peculiar site of cell without mutation and P53 and ras gene sequencing without site mutation
Being immortalized cell line after identification.
2nd, immortalized cells cultural method
(1) type of culture medium：DMEM- high glucose mediums (are purchased from Gibco companies)
(2) factor is added：10%FBS, 1%Penicillin, 1%Streptomycin.
(3) conditions of cryopreservation：90%FBS+10%DMSO.
(4) condition of culture：Reached when 70-80% converges whne cell and be ready for Secondary Culture；
(5) passage cultural method：
A) 25cm is suctioned out2Culture medium in blake bottle, with PBS cell once；
B) addition 0.25% tryptic digestive juice about 2ml is in blake bottle, 37 DEG C of warm bath 2-3min or so；It is inverted micro-
Microscopic observation, inhales after cell retraction is rounded and abandons digestive juice, adds complete culture solution and terminates digestion；
C) mixing is gently blown and beaten with suction pipe, by 1:2 appropriate ratios carry out inoculation passage, then supplement fresh training completely
Base is supported to 5ml, 37 DEG C, 5%CO are put into2Cultivated in cell culture incubator；
(6) pituitary tumor cell for being immortalized, and carry out biomaterial preservation, preserving number：CGMCC No.12672.
Embodiment 2：Cell growth curve is determined
First, method：
By the primary pituitary adenoma cell of embodiment 1 and the pituitary adenoma cells for immortalizing, adjust thin after cell is covered with
Born of the same parents' concentration is 5 × 104Cells/mL, the training of 96 holes is inoculated in by primary pituitary tumor cell and the pituitary tumor cell for immortalizing respectively
Plate is supported, per the μ l of hole 100,37 DEG C, 5%CO2Culture in incubator；Add in every hole after culture 12h, 24h, 36h, 48h, 72h respectively
Enter the MTT of 10 μ l, 37 DEG C, 5%CO2Culture lucifuge is incubated 3-4h in incubator；Liquid in exhaustion hole, adds 200 μ l's
DMSO, room temperature shakes 10min in shaking table；ELIASA 492nm wavelength measures same time point OD values, is divided with the OD values for measuring
Analysis.
2nd, result：
As shown in figure 1, the pituitary tumor cell for immortalizing enters growth stationary phase in 36h.Multiplication capacity is strong compared with primary cell.
Embodiment 3：Cellular morphology is detected
First, method：
1st, using phase contrast microscope observation of cell strain CGMCC No.12672.
2nd, using the ultra microstructure of transmission electron microscope observation cell line CGMCC No.12672：
When cell growth is converged up to 90%, nutrient solution is abandoned, add 3ml 0.1MPBS (pH value 7.4), scraped with rubber cell
Scraping cells, collect cell, and centrifugation (1000rpm/ points, 10 minutes) is abandoned supernatant, 2 hours are fixed before glutaraldehyde/osmium tetroxide,
0.1M PBS (pH value 7.4) are washed 3 times, 10 minutes/time.Then the rear fixation of rower sheet, be dehydrated, be impregnated with, embedding, cutting ultra-thin section
With heavy metal dyeing (Beijing Inst. of Neurosurgery's Electron Microscopy Room is completed).
2nd, result：
1st, cell line CGMCC No.12672 growth characteristics under phase contrast microscope：Be in mostly fusiformis, partly for triangle or
Polygonal, there is preferable refractivity, and nuclear membrane is complete, and kernel is clear.(see Fig. 2)
2nd, the ultra microstructure of transmission electron microscope observation cell line CGMCC No.12672：(see Fig. 3).
Tumour cell dense distribution, karyon form is irregular, and kernel is big, obvious, visible shell membrane roundlet in endochylema and projection
Shape gonadotrophin secretion particle.
Result is pointed out：The cell is originated for nonfunctional pituicyte.
Embodiment 4：The protein expression situation of Immunofluorescence test immortalized cells
First, method
Cell line CGMCC No.12672, by cell climbing sheet, PBS rinses 3 times afterwards, and 4% paraformaldehyde fixes 10min.
PBS rinses 5min, in triplicate.3%H2O2/ PBS is incubated at room temperature 10min, PBS rinsing 5min, in triplicate.5%FBS is closed,
Room temperature 15min.Sca-1 antibody (being purchased from santa cruz companies) and α-SMA antibody (being purchased from abcam companies), 4 DEG C are incubated respectively
Overnight incubation, PBS rinsing 5min, in triplicate.Secondary antibody (being purchased from inventrogen companies) is marked to drop in section cy3, often
Piece 1 drips, 37 DEG C of incubation 30min.PBS rinses 5min, in triplicate.Mounting, fluorescence microscopy Microscopic observation.
2nd, result
Fluorescence microscopy Microscopic observation,>80% cell shows red fluorescence, the sca-1 protein expressions positive (Fig. 4).α-
SMA antibody without cell present red fluorescence, α-SMA protein expressions feminine gender (Fig. 5).
Result is pointed out：The cell pollutes without fibroblast.
Embodiment 5：The tumor characteristic analysis of HPA1446 cells
First, method
1st, chromosome karyotype analysis：
Cell line CGMCC No.12672, after cell growth is converged up to 90%, change liquid and add final concentration of 0.5ug/
The colchicine working solution of ml makes cell be taken the logarithm growth period cell in stopping at division, plus colchicine is to the μ g/ of concentration 0.4
ML, cultivates 3 hours；Trypsin digestion and cell is added, with serum free medium washed cell once, 1500rpm centrifugation 5min,
Supernatant is abandoned, cell is collected；75mmol/L KCl hypotonic 30min, 37 DEG C；One is added to drip methyl alcohol/glacial acetic acid (3:1) fixer is mixed
4 DEG C of centrifugations, abandon supernatant afterwards；Plus 1mL fixers, 4 DEG C of placement 30min, 4 DEG C of centrifugations, abandon supernatant after mixing；Continuously add fixation
Liquid, mixes centrifugation；Drop piece, is air-dried, 80 DEG C of roasting piece 2h；Giemsa staining.Oily Microscopic observation.
2nd, the Flow cytometry cell cycle
Take the logarithm growth period cell (cell line CGMCC No.12672), abandon nutrient solution, trypsin digestion cell, plus culture
Liquid, centrifugation (1000rpm/ points, 14 minutes), removes supernatant.PBS is washed 2 times, 0.5mlPBS piping and druming, and cell is drawn with 5ml syringes,
Then firmly squeeze into 5ml 70% (precooling) ethanol, sealed membrane sealing, 4 DEG C of fixations are overnight.Centrifugation (800rpm/ points, 14 points
Clock), fixed cell is collected, PBS is washed 2 times, and 0.4ml PBS re-suspended cells simultaneously gently blow and beat (preventing clasmatosis).Plus RNase-
The μ l of A about 3 (final concentration of 50 μ g/ml), 37 DEG C of water-baths digest 30 minutes, plus 50 μ l PI (final concentration of 65 μ g/ml), in ice bath
Lucifuge is dyeed 30 minutes, 300 mesh (40~50 microns of aperture) nylon net filter, upper machine testing (Fig. 7).
3rd, one-tenth knurl ability detection
In order to detect the one-tenth knurl ability of cell, by 1.0*107Individual HPA1446 cells (cell line CGMCC No.12672) connect
Plant subcutaneous in NOD/SCID nude mices (being purchased from company of dimension tonneau China) oxter, observe the growing state of tumour after inoculation daily, record
Into knurl incubation period and tumor size.
2nd, result
1st, chromosome karyotype analysis：Oil mirror observation and chromosome counting, are shown in that chromosome is 100 different times of karyotypes
(Fig. 6).
2nd, the Flow cytometry cell cycle：The HPA1446 cell cycles cell percentage of each phase is：The G1 phases
66.6%th, S phases 24.4%, G2-M phases 9.02%.(Fig. 7)
3rd, one-tenth knurl ability detection：At the 14th day, nude mice oxter had tumour to occur, and tumor size (is schemed up to 1 centimetre at the 4th week
8-1, Fig. 8-2).
Claims (10)
1. Pituitary adenoma cell strain, the entitled HPA1446 of cell line is deposited in China Committee for Culture Collection of Microorganisms
Common micro-organisms center, preserving number is CGMCC No.12672.
2. Pituitary adenoma cell strain according to claim 1, it has following biological characteristics：(1) mostly in circle shuttle
Shape, is partly triangle or polygonal, there is preferable refractivity, and nuclear membrane is complete, and kernel is clear；(2) stabilization passage, passes on first
20 is more than generation；(3) chromosome is 100 different times of karyotypes；(4) cell expressing protein sca-1 is positive, not express alpha-
SMA；(5) visible shell membrane small circular gonadotrophin secretion particle under Electronic Speculum；(6) cell has internal nude mice part into knurl energy
Power.
3. the Pituitary adenoma cell strain described in claim 1 or 2 is used to prepare the purposes of tumor models.
4. the Pituitary adenoma cell strain described in claim 1 or 2 is used to prepare the purposes of animal model for tumour.
5. the Pituitary adenoma cell strain described in claim 1 or 2 for screen and/or evaluating/prepare anti-tumor medicine
Purposes.
6. the Pituitary adenoma cell strain described in claim 1 or 2 is for the purposes for developing tumour medicine target spot.
7. the Pituitary adenoma cell strain described in claim 1 or 2 is for the purposes for preparing tumour diagnostic reagent.
8. purposes of the Pituitary adenoma cell plant described in claim 1 or 2 in screening tumor biotherapy medicine/reagent.
9. purposes of the Pituitary adenoma cell plant described in claim 1 or 2 in oncotherapy new technology is developed.
10. the use in detecting tumour associated biomolecule engineering product is being developed in the Pituitary adenoma cell strain described in claim 1 or 2
On the way.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107937548A (en) * | 2018-01-04 | 2018-04-20 | 大连医科大学附属第二医院 | Application and expression of the OLFM3 genes in pituitary adenoma biomarker |
| CN114480639A (en) * | 2021-12-24 | 2022-05-13 | 中国医学科学院北京协和医院 | Novel targets for diagnosis and treatment of pituitary adenomas |
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| CN113025576B (en) * | 2021-03-12 | 2022-07-15 | 首都医科大学附属北京天坛医院 | Construction method and application of human adenohormone type pituitary adenoma stem cell line |
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| CN104212765A (en) * | 2013-05-30 | 2014-12-17 | 复旦大学附属华山医院 | Humanized drug-resistant GH-type pituitary tumor cell strain and establishment method and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107937548A (en) * | 2018-01-04 | 2018-04-20 | 大连医科大学附属第二医院 | Application and expression of the OLFM3 genes in pituitary adenoma biomarker |
| CN114480639A (en) * | 2021-12-24 | 2022-05-13 | 中国医学科学院北京协和医院 | Novel targets for diagnosis and treatment of pituitary adenomas |
| CN114480639B (en) * | 2021-12-24 | 2022-09-30 | 中国医学科学院北京协和医院 | Novel targets for diagnosis and treatment of pituitary adenomas |
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