CN106011072A - Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction - Google Patents
Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction Download PDFInfo
- Publication number
- CN106011072A CN106011072A CN201610385304.3A CN201610385304A CN106011072A CN 106011072 A CN106011072 A CN 106011072A CN 201610385304 A CN201610385304 A CN 201610385304A CN 106011072 A CN106011072 A CN 106011072A
- Authority
- CN
- China
- Prior art keywords
- cell
- bladder
- carcinoma
- cell line
- pbs
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004027 cell Anatomy 0.000 title claims abstract description 91
- 206010005003 Bladder cancer Diseases 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 15
- 201000001531 bladder carcinoma Diseases 0.000 title claims abstract description 10
- 206010005084 bladder transitional cell carcinoma Diseases 0.000 title claims abstract description 10
- 201000001528 bladder urothelial carcinoma Diseases 0.000 title claims abstract description 10
- 238000005215 recombination Methods 0.000 title claims abstract description 10
- 230000006798 recombination Effects 0.000 title claims abstract description 10
- 208000010570 urinary bladder carcinoma Diseases 0.000 title claims abstract description 10
- 238000010172 mouse model Methods 0.000 title claims abstract description 7
- 241000699670 Mus sp. Species 0.000 claims abstract description 8
- 238000004113 cell culture Methods 0.000 claims abstract description 7
- 238000003364 immunohistochemistry Methods 0.000 claims abstract description 4
- 230000001413 cellular effect Effects 0.000 claims description 20
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 18
- 206010028980 Neoplasm Diseases 0.000 claims description 18
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 18
- 239000012531 culture fluid Substances 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 18
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 18
- 239000000499 gel Substances 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 13
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 210000003734 kidney Anatomy 0.000 claims description 11
- 239000000463 material Substances 0.000 claims description 10
- 108091009167 Bloom syndrome protein Proteins 0.000 claims description 9
- 230000012010 growth Effects 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 108090000623 proteins and genes Proteins 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000004043 dyeing Methods 0.000 claims description 8
- 230000007935 neutral effect Effects 0.000 claims description 7
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 claims description 6
- 229930040373 Paraformaldehyde Natural products 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 6
- 230000018044 dehydration Effects 0.000 claims description 6
- 238000006297 dehydration reaction Methods 0.000 claims description 6
- 238000001962 electrophoresis Methods 0.000 claims description 6
- 238000011010 flushing procedure Methods 0.000 claims description 6
- 238000011534 incubation Methods 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 239000012188 paraffin wax Substances 0.000 claims description 6
- 229920002866 paraformaldehyde Polymers 0.000 claims description 6
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 claims description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 claims description 6
- 102000008186 Collagen Human genes 0.000 claims description 5
- 108010035532 Collagen Proteins 0.000 claims description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 238000010166 immunofluorescence Methods 0.000 claims description 5
- 230000000877 morphologic effect Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 claims description 4
- 238000011729 BALB/c nude mouse Methods 0.000 claims description 4
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 claims description 4
- 102000004142 Trypsin Human genes 0.000 claims description 4
- 108090000631 Trypsin Proteins 0.000 claims description 4
- 239000012160 loading buffer Substances 0.000 claims description 4
- 230000008520 organization Effects 0.000 claims description 4
- 239000003531 protein hydrolysate Substances 0.000 claims description 4
- 239000012588 trypsin Substances 0.000 claims description 4
- 239000012224 working solution Substances 0.000 claims description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 claims description 3
- 101100298998 Caenorhabditis elegans pbs-3 gene Proteins 0.000 claims description 3
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 claims description 3
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 claims description 3
- 241000446313 Lamella Species 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 240000003186 Stachytarpheta cayennensis Species 0.000 claims description 3
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 230000001464 adherent effect Effects 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 239000000427 antigen Substances 0.000 claims description 3
- 102000036639 antigens Human genes 0.000 claims description 3
- 108091007433 antigens Proteins 0.000 claims description 3
- 238000009835 boiling Methods 0.000 claims description 3
- 244000309466 calf Species 0.000 claims description 3
- 239000002775 capsule Substances 0.000 claims description 3
- 239000012141 concentrate Substances 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 238000010494 dissociation reaction Methods 0.000 claims description 3
- 230000005593 dissociations Effects 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000000975 dye Substances 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 238000000799 fluorescence microscopy Methods 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000007654 immersion Methods 0.000 claims description 3
- 238000003119 immunoblot Methods 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 239000006166 lysate Substances 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- 238000001556 precipitation Methods 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 238000010814 radioimmunoprecipitation assay Methods 0.000 claims description 3
- 238000000926 separation method Methods 0.000 claims description 3
- 235000020183 skimmed milk Nutrition 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 2
- 239000000512 collagen gel Substances 0.000 claims description 2
- 230000001605 fetal effect Effects 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 238000010827 pathological analysis Methods 0.000 claims description 2
- 238000011160 research Methods 0.000 abstract description 3
- 238000010171 animal model Methods 0.000 abstract description 2
- 230000004791 biological behavior Effects 0.000 abstract description 2
- 239000000090 biomarker Substances 0.000 abstract description 2
- 238000011161 development Methods 0.000 abstract description 2
- 230000001225 therapeutic effect Effects 0.000 abstract description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 abstract 1
- 238000000338 in vitro Methods 0.000 abstract 1
- 238000001727 in vivo Methods 0.000 abstract 1
- 230000008521 reorganization Effects 0.000 abstract 1
- 210000001519 tissue Anatomy 0.000 description 24
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 9
- 210000004379 membrane Anatomy 0.000 description 6
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 3
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 3
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 3
- 206010044412 transitional cell carcinoma Diseases 0.000 description 3
- 102100032912 CD44 antigen Human genes 0.000 description 2
- 201000009030 Carcinoma Diseases 0.000 description 2
- 101001066288 Gallus gallus GATA-binding factor 3 Proteins 0.000 description 2
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 description 2
- 101000994460 Homo sapiens Keratin, type I cytoskeletal 20 Proteins 0.000 description 2
- 101000763314 Homo sapiens Thrombomodulin Proteins 0.000 description 2
- 102100032700 Keratin, type I cytoskeletal 20 Human genes 0.000 description 2
- 102100026966 Thrombomodulin Human genes 0.000 description 2
- -1 UP III Proteins 0.000 description 2
- 230000003187 abdominal effect Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108090000386 Fibroblast Growth Factor 1 Proteins 0.000 description 1
- 102100031706 Fibroblast growth factor 1 Human genes 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241001416149 Ovis ammon Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000000270 basal cell Anatomy 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000002771 cell marker Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
- C12N5/0695—Stem cells; Progenitor cells; Precursor cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0331—Animal model for proliferative diseases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Oncology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for establishing a human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction. The method includes the primary cell culture, immortalized cell line identification, tissue reorganization and planting, HE staining, and immunohistochemistry. The invention establishes a novel stable human bladder transitional cell carcinoma cell line, and uses the cell line to construct the mouse model of bladder carcinoma tissue recombination functional reconstruction. The occurrence and development of bladder carcinoma are simulated in mice. The method researches on the biological behavior of bladder carcinoma from in vitro molecular and cell biology and in vivo animal models, and provides an effective tool for the discovery of new biomarkers and therapeutic measures.
Description
Technical field
The present invention relates to a kind of Transitional cell carcinoma cell line and the method for building up of Bladder Cancer recombination function reconstruction model thereof.
Background technology
Prior art lacks stable Human Bladder Transitional Cell Carcinoma cell line and bladder cancer functional reconstruction model thereof.Translational medicine is studied in the urgent need to setting up such model.
Summary of the invention
It is an object of the invention to provide a kind of Transitional cell carcinoma cell line and the method for building up of Bladder Cancer recombination function reconstruction mouse model thereof.
Summary of the invention
The technical solution of the present invention is:
The method for building up of a kind of Human Bladder Transitional Cell Carcinoma cell line, is characterized in that: comprise the following steps:
(1) primitive cell culture
By drawing materials under aseptic for Freshman bladder cancer specimens from pri and being placed on 15mL sterile centrifugation tube, add 10ml primitive cell culture liquid, 4 DEG C of preservations；Specimen is divided into two parts, a part fix with 4% paraformaldehyde, gives over to HE dyeing and definitive pathological diagnosis；Another part shears shreds, 1 × PBS 3 times, transfers in centrifuge tube by the piece of tissue shredded, after being centrifuged, resuspended with original cuiture liquid, puts in T-25 original cuiture bottle, is placed on 5%CO2, 37 DEG C of constant incubators are cultivated；Every day is the growth conditions of tissues observed block under inverted microscope；After cultivating 1-2 days, at the bottom of piece of tissue sticks to bottle after, change liquid, continue to cultivate with original cuiture liquid；After primary cell grows around piece of tissue, with trypsin by cell dissociation, examine under a microscope, when cellular morphology becomes transparent spherical shape, add serum, terminate digestion, blow and beat cell with pipettor gently so that it is suspend；Liquid in culture bottle is moved into centrifuge tube, centrifugal, collect cell precipitation, remove supernatant, add original cuiture liquid, put in new culture bottle and cultivate, obtain the primary Urothelial cell of human bladder carcinoma tissue；Fully digest the cell grown with pancreatin, continue cultivate cell and pass on 10-20 time, put into T-25 Tissue Culture Flask, change regular growth culture fluid and cultivate, it is achieved cellular immortalization；
(2) cell line is identified
Immunoblotting analysis: with protein lysate: RIPA lysate: PMSF=100:1, collection cell protein sample, for guaranteeing that albumen applied sample amount is consistent, adjusting protein concentration through BCA albuminimetry is 2ug/ul；The SDS-PAGE albumen sample-loading buffer of concentration, boiling water bath 5min is added in protein sample；Preparation 10%SDS-PAGE gel, by every hole 25ul loading, carries out albumen constant voltage electrophoresis, concentrates glue voltage 80V, separation gel voltage 100V；After electrophoresis terminates, pvdf membrane constant current transferring film, 260mA, 2h；Taking out pvdf membrane, 5% skim milk room temperature closes 2h；Hatch one to resist, 4 DEG C, overnight；Two anti-HRP labellings are added after washing film；ECL developer；Scanning;
Immunofluorescence: trophophase cell of taking the logarithm, after trypsinization, is inoculated in poly-D-lysine is coated 24 hole breezing plates of coverslip, puts into coverslip in hole, be placed at the bottom of hole, make cell climb on the cover slip；Cell density about 10^4/ hole；Stand overnight, treat that cell is the most adherent, when cell covers with to 60%, after cellular morphology is normal, abandoning culture fluid, 4% paraformaldehyde fixes 30min, 0.2%Triton X-100 rupture of membranes 5min, and 5% calf serum closes 30min, with pipettor sucking-off serum, dropping one resists, 4 DEG C of overnight incubation；5min × 3 time are rinsed with 1 × PBS；Dropping fluorescein labelling two resists, and incubated at room 2h, PBS rinses 5min × 3 time, and DAPI redyes cover plate；Fluorescence microscopy Microscopic observation is also taken pictures.
The method for building up of a kind of Bladder Cancer reconstructed models, is characterized in that:
Take primary cell, after trypsinization, centrifugal, use culture fluid re-suspended cell, counting, it is divided into two parts, mixing by 1:2 with a copy of it cell after being shredded by the rat fetal bladder interstitial lamella cell rat-BLM of conceived 18.5 days, 2000rpm is centrifuged 3 minutes, discards culture fluid；The collagen gel that concentration is 10 g/L big rat-tail extracted is mixed by 2:1 with working solution, is placed on ice, is separately added into cell or the cell mixed with BLM, makes in final each Cellular gels block containing 2 × 105Individual cell, draws 50ul collagen protein mixed liquor and drops in sterile petri dish, be put in 37 DEG C of incubators and solidify 30 min, after Cellular gels solidifies, adds culture fluid, makes Cellular gels be immersed in culture fluid, be placed in 37 DEG C, 5% CO2Incubator is cultivated, uses the most afterwards.
A kind of Bladder Cancer recombination function rebuilds the method for building up of mouse model, it is characterized in that: aseptically, organization restructuring block is inoculated in the subrenal capsule of 8 week old BALB/C nude mices；Treating 1-2 month, tumor is formed, and puts to death mice, takes out the mouse kidney with tumor tissues, is soaked in 4% formaldehyde fixing, standby；
HE dyeing, immunohistochemistry
Taking Freshman bladder cancer specimens from pri and the mouse kidney with tumor tissues, be placed in 4% formaldehyde the fixing 24h of immersion, gradient alcohol dehydration, paraffin embedding, preparation section, conventional H E dyes, and neutral gum mounting carries out morphological observation under light microscopic；Taking paraffin section, dewaxing, aquation, 1 × PBS rinses, 0.3% H2O2Hatching 5min, 1 × PBS rinses, antigen retrieval, 1 × PBS rinses, and dropping lowlenthal serum is closed 10min, 1 × PBS and rinsed, one resists 4 DEG C of overnight incubation, and 1 × PBS rinses, two anti-incubated at room 1h, 1 × PBS rinses, and DAB develops the color, and after flushing, haematoxylin is redyed, hydrochloride alcohol breaks up, tap water rinses, and distilled water flushing, dehydration of alcohol, dimethylbenzene are transparent, neutral gum mounting；Light Microscopic observation.
The present invention establishes the Human Bladder Transitional Cell Carcinoma cell line (NTU1) that a strain is stable, utilize this cell line to construct Mice Body inner tissue recombination function and rebuild translational medicine study model, simulation tumor occurs and development pathobiology process, from external molecular biology and cytobiology angle and internal Research of Animal Model for Study tumor biological behavior and mechanism, early examine for bladder cancer and early control, find that new biomarker and therapeutic strategy provide effective instrument and means.
Below in conjunction with embodiment, the invention will be further described.
Detailed description of the invention
Experiment 1: primitive cell culture
Major experimental reagent and material:
Tissue-derived: to be provided fresh clinical bladder cancer specimens from pri by The First People's Hospital of Nantong's Urology Surgery
Reagent: DMEM/F12 basal medium, RPMI1640 basal medium,
Vascular endothelial cell growth factor (BPE), epithelical cell growth factor (EGF), Insulin-Transferrin-selenium
(ITS), dual anti-, hyclone, 0.25% trypsin, PBS
Material: operating theater instruments (all processes through autoclave sterilization), pipettor
Original cuiture bottle, Tissue Culture Flask, 15ml centrifuge tube
CO2Incubator, inverted phase contrast microscope, centrifuge
Experimental technique:
The fresh bladder cancer specimens from pri taken out from clinic, is immediately placed in primitive cell culture liquid, 4 degrees Celsius of preservations.Specimen is divided into two parts, a part fix with 4% paraformaldehyde, gives over to HE dyeing and case is made a definite diagnosis；Another part shears shreds, the smaller the better, PBS 3 times, transfers in centrifuge tube by the tissue shredded, after being centrifuged, resuspended with original cuiture liquid, puts in T-25 original cuiture bottle, is placed on 5%CO2, 37 DEG C of constant incubators are cultivated.Every day is the growth conditions of tissues observed block under inverted microscope.After cultivating 1-2 days, at the bottom of piece of tissue sticks to bottle after, change liquid, continue to cultivate with original cuiture liquid.After primary cell grows around piece of tissue, with trypsin by cell dissociation, examine under a microscope, when cellular morphology becomes transparent spherical shape, add serum, terminate digestion, blow and beat cell with pipettor gently so that it is suspend, be careful not to blow afloat piece of tissue.Liquid in culture bottle is moved into centrifuge tube, centrifugal, collect cell precipitation, remove supernatant, add appropriate original cuiture liquid, put in new culture bottle and cultivate, obtain the primary Urothelial cell of human bladder carcinoma tissue.Fully digest the cell grown with pancreatin, continue cultivate cell and pass on 10-20 time, put into general T-25 Tissue Culture Flask, change regular growth culture fluid and cultivate, it is achieved cellular immortalization.
Experimental result:
Cell has just started poor growth, and in spherical shape, after 7 generations, growth is stable, within 2-3 days, can pass on.Cellular morphology is spindle shape, has multiple elongated feeler, has epithelial cell speciality.Growth quickly, can be covered with and pass on (1:2) for general 3-5 days.The most stably pass on half a year more than, become immortalized cell line.
Experiment 2: cell line is identified
Major experimental reagent and material:
Reagent:
Protein lysate, sample-loading buffer, defatted milk powder, ECL chemiluminescence developing agent,
One resists: pan-CK, EGFR, UP III, GATA-3, TM (THBD), EGFR, CD44 and CK20 etc.
Two resist: mountain sheep anti mouse, goat antirabbit
Material:
Just putting fluorescence microscope, chemiluminescence imaging system, electrophresis apparatus
Experimental technique:
Immunoblotting analysis (western blot): collect cell protein sample with protein lysate (RIPA lysate: PMSF=100:1), for guaranteeing that albumen applied sample amount is consistent, adjusting protein concentration through BCA albuminimetry is 2ug/ul.The appropriate SDS-PAGE albumen sample-loading buffer concentrated, boiling water bath 5min is added in protein sample.Preparation 10%SDS-PAGE gel, by every hole 25ul loading, carries out albumen constant voltage electrophoresis, concentrates glue voltage 80V, separation gel voltage 100V.After electrophoresis terminates, pvdf membrane constant current transferring film, 260mA, 2h.Taking out pvdf membrane, 5% skim milk room temperature closes 2h.Hatch one to resist, 4 DEG C, overnight.Two anti-(HRP labellings) are added after washing film.ECL developer.
Immunofluorescence (immunofluorescence): trophophase cell of taking the logarithm, after trypsinization, is inoculated in poly-D-lysine is coated 24 hole breezing plates of coverslip, puts into coverslip in hole, be placed at the bottom of hole, make cell climb on the cover slip.Cell density about 10^4/ hole.Stand overnight, treat that cell is the most adherent, when cell covers with to 60%, after cellular morphology is normal, abandon culture fluid, 4% paraformaldehyde fixes 30min, 0.2%Triton X-100 rupture of membranes 5min, and 5% calf serum closes 30min, with pipettor sucking-off serum, do not wash, drip diluted in proportion and resist, 4 DEG C of overnight incubation.5min × 3 time are rinsed with 1 × PBS.The fluorescein labelling two anti-(1%BSA-PBS dilution) of dropping proper proportion dilution, incubated at room 2h, 1 × PBS rinses 5min × 3 time, and DAPI redyes cover plate.Fluorescence microscopy Microscopic observation is also taken pictures.
Experimental result:
Clinical operation specimen HE stained is diagnosed as differentiated transitional cell carcinoma of bladder.The immunofluorescence dyeing qualification result of primary cell NTU0 and cell line NTU1 shows, bladder cancer specific labelled antibody UP III, GATA-3, THBD are high expressed, it was demonstrated that cell line NTU1 built up is transitional cell bladder carcinoma cell line.Epithelial cell marker albumen EGFR, CK20 also have expression.CD44 is strong positive, illustrates that this cell line NTU1 is based on bladder cancer basal cell (dry, CFU-GM) sample.
Experiment 3: organization restructuring and plantation mouse model
Major experimental reagent and material:
Reagent: rat tail collagen albumen, working solution
Material: operating theater instruments (through autoclave sterilization)
Stereomicroscope, Tissue Culture Dish
Mice (innate immunity defect BALB/C nude mice, SPF level, 8 week old)；Pregnant rat
Experimental technique:
Taking the rat of conceived 18.5 days, after anesthesia, cut abdominal part open, take out children Mus from embryo gently, abdominal part upward, opens extremity, operates, mention and from section bladder, isolating bladder interstitial lamella (rat-BLM), totally remove epithelial layer under stereomicroscope.Take human bladder cancer's primary cell or cell line, after trypsinization, centrifugal, use culture fluid re-suspended cell, counting, it is divided into two parts, mixes by 1:2 with a copy of it cell after rat-BLM is shredded, 2000rpm is centrifuged 3 minutes, discards culture fluid.The collagen protein (concentration is 10 g/L) rat-tail extracted is mixed by 2:1 with working solution (setting solution), is placed on ice, is separately added into cell or the cell mixed with rat-BLM, makes in final each Cellular gels block containing 2 × 105Individual cell, draws 50ul collagen protein mixed liquor and drops in sterile petri dish, be put in 37 DEG C of incubators and solidify 30 min, after Cellular gels solidifies, add a small amount of culture fluid, make Cellular gels be immersed in culture fluid, be placed in 37 DEG C, 5% CO2Incubator is cultivated, uses the most afterwards.Aseptically, organization restructuring block is inoculated in the subrenal capsule of BALB/C nude mice (Nude), after stitching, treats that mouse revives completely and be placed in barrier system raising.Treating 1-2 month, tumor is formed, and puts to death mice, takes out the mouse kidney with tumor tissues, is soaked in 4% formaldehyde fixing, standby.
Experimental result:
Cellular gels block becomes tumor very fast, within one month, can become tumor, tumor formation rate 100%.After becoming tumor, mice form is substantially become thin, and both sides kidney highlights.Putting to death mice, take out its kidney and observe, tumor tissues substantially highlights, and is creamy white.Part cell is had to invade inside kidney.Have no neoplasm metastasis.
Experiment 4:HE dyeing, immunohistochemistry
Major experimental reagent and material:
Reagent: DAB colour reagent box, formaldehyde, haematoxylin, hydrochloric acid, ethanol, lowlenthal serum, H2O2, neutral gum
Material: embedded box, microtome, microscope slide, coverslip
Experimental technique:
Treating excess syndrome tests the specimen in 1 and the mouse kidney with tumor tissues, is placed in 4% formaldehyde the fixing 24h of immersion, gradient alcohol dehydration, paraffin embedding film-making, and conventional H E dyes, and neutral gum mounting carries out morphological observation under light microscopic.Taking paraffin section, dewaxing, aquation, 1 × PBS rinses, H2O2Hatching 5min, 1 × PBS rinses, antigen retrieval, 1 × PBS rinses, and dropping lowlenthal serum is closed 10min, 1 × PBS and rinsed, one resists 4 DEG C of overnight incubation, and 1 × PBS rinses, two anti-incubated at room 1h, 1 × PBS rinses, and DAB develops the color, and after flushing, haematoxylin is redyed, hydrochloride alcohol breaks up, tap water rinses, and distilled water flushing, dehydration of alcohol, dimethylbenzene are transparent, neutral gum mounting.Light Microscopic observation.
Experimental result:
Observing after basic stitch block HE dyeing, its tissue morphology is well differentiated transitional cell carcinoma.The mouse kidney HE coloration result not adding rat-BLM shows, the lump of formation is solid tumor, has transitional cell Morphological Features and histological characteristic, but differentiation degree is relatively low.The mouse kidney HE coloration result adding rat-BLM shows, the tumor of formation is the shifting shape cell carcinoma of differentiated, and relevant with implantation time length, pathomorphology is consistent with basic stitch block form.
Three, conclusion
Cultivate and external identification experiment through original cuiture, spontaneous immortalization, successfully build up Human Bladder Transitional Cell Carcinoma cell line NTU1 that a strain is stable；Utilizing this cell line NTU1, be successfully set up human bladder carcinoma tissue recombination function and rebuild mouse transforming medical research model, its this cell line of implantation tumor model validation keeps consistent morphological characteristic with primary carcinoma.
Claims (3)
1. a method for building up for Human Bladder Transitional Cell Carcinoma cell line, is characterized in that: comprise the following steps:
(1) primitive cell culture
By drawing materials under aseptic for Freshman bladder cancer specimens from pri and being placed on 15mL sterile centrifugation tube, add 10ml primitive cell culture liquid, 4 DEG C of preservations；Specimen is divided into two parts, a part fix with 4% paraformaldehyde, gives over to HE dyeing and definitive pathological diagnosis；Another part shears shreds, 1 × PBS 3 times, transfers in centrifuge tube by the piece of tissue shredded, after being centrifuged, resuspended with original cuiture liquid, puts in T-25 original cuiture bottle, is placed on 5%CO2, 37 DEG C of constant incubators are cultivated；Every day is the growth conditions of tissues observed block under inverted microscope；After cultivating 1-2 days, at the bottom of piece of tissue sticks to bottle after, change liquid, continue to cultivate with original cuiture liquid；After primary cell grows around piece of tissue, with trypsin by cell dissociation, examine under a microscope, when cellular morphology becomes transparent spherical shape, add serum, terminate digestion, blow and beat cell with pipettor gently so that it is suspend；Liquid in culture bottle is moved into centrifuge tube, centrifugal, collect cell precipitation, remove supernatant, add original cuiture liquid, put in new culture bottle and cultivate, obtain the primary Urothelial cell of human bladder carcinoma tissue；Fully digest the cell grown with pancreatin, continue cultivate cell and pass on 10-20 time, put into T-25 Tissue Culture Flask, change regular growth culture fluid and cultivate, it is achieved cellular immortalization；
(2) cell line is identified
Immunoblotting analysis: with protein lysate: RIPA lysate: PMSF=100:1, collection cell protein sample, for guaranteeing that albumen applied sample amount is consistent, adjusting protein concentration through BCA albuminimetry is 2ug/ul；The SDS-PAGE albumen sample-loading buffer of concentration, boiling water bath 5min is added in protein sample；Preparation 10%SDS-PAGE gel, by every hole 25ul loading, carries out albumen constant voltage electrophoresis, concentrates glue voltage 80V, separation gel voltage 100V；After electrophoresis terminates, pvdf membrane constant current transferring film, 260mA, 2h；Taking out pvdf membrane, 5% skim milk room temperature closes 2h；Hatch one to resist, 4 DEG C, overnight；Two anti-HRP labellings are added after washing film；ECL developer；Scanning;
Immunofluorescence: trophophase cell of taking the logarithm, after trypsinization, is inoculated in poly-D-lysine is coated 24 hole breezing plates of coverslip, puts into coverslip in hole, be placed at the bottom of hole, make cell climb on the cover slip；Cell density about 10^4/ hole；Stand overnight, treat that cell is the most adherent, when cell covers with to 60%, after cellular morphology is normal, abandoning culture fluid, 4% paraformaldehyde fixes 30min, 0.2%Triton X-100 rupture of membranes 5min, and 5% calf serum closes 30min, with pipettor sucking-off serum, dropping one resists, 4 DEG C of overnight incubation；5min × 3 time are rinsed with 1 × PBS；Dropping fluorescein labelling two resists, and incubated at room 2h, PBS rinses 5min × 3 time, and DAPI redyes cover plate；Fluorescence microscopy Microscopic observation is also taken pictures.
2. a method for building up for Bladder Cancer reconstructed models, is characterized in that:
Take primary cell, after trypsinization, centrifugal, use culture fluid re-suspended cell, counting, it is divided into two parts, mixing by 1:2 with a copy of it cell after being shredded by the rat fetal bladder interstitial lamella cell rat-BLM of conceived 18.5 days, 2000rpm is centrifuged 3 minutes, discards culture fluid；The collagen gel that concentration is 10 g/L big rat-tail extracted is mixed by 2:1 with working solution, is placed on ice, is separately added into cell or the cell mixed with BLM, makes in final each Cellular gels block containing 2 × 105Individual cell, draws 50ul collagen protein mixed liquor and drops in sterile petri dish, be put in 37 DEG C of incubators and solidify 30 min, after Cellular gels solidifies, adds culture fluid, makes Cellular gels be immersed in culture fluid, be placed in 37 DEG C, 5% CO2Incubator is cultivated, uses the most afterwards.
3. Bladder Cancer recombination function rebuilds a method for building up for mouse model, it is characterized in that: aseptically, organization restructuring block is inoculated in the subrenal capsule of 8 week old BALB/C nude mices；Treating 1-2 month, tumor is formed, and puts to death mice, takes out the mouse kidney with tumor tissues, is soaked in 4% formaldehyde fixing, standby；
HE dyeing, immunohistochemistry
Taking Freshman bladder cancer specimens from pri and the mouse kidney with tumor tissues, be placed in 4% formaldehyde the fixing 24h of immersion, gradient alcohol dehydration, paraffin embedding, preparation section, conventional H E dyes, and neutral gum mounting carries out morphological observation under light microscopic；Taking paraffin section, dewaxing, aquation, 1 × PBS rinses, 0.3% H2O2Hatching 5min, 1 × PBS rinses, antigen retrieval, 1 × PBS rinses, and dropping lowlenthal serum is closed 10min, 1 × PBS and rinsed, one resists 4 DEG C of overnight incubation, and 1 × PBS rinses, two anti-incubated at room 1h, 1 × PBS rinses, and DAB develops the color, and after flushing, haematoxylin is redyed, hydrochloride alcohol breaks up, tap water rinses, and distilled water flushing, dehydration of alcohol, dimethylbenzene are transparent, neutral gum mounting；Light Microscopic observation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610385304.3A CN106011072A (en) | 2016-06-03 | 2016-06-03 | Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610385304.3A CN106011072A (en) | 2016-06-03 | 2016-06-03 | Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106011072A true CN106011072A (en) | 2016-10-12 |
Family
ID=57089409
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610385304.3A Pending CN106011072A (en) | 2016-06-03 | 2016-06-03 | Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106011072A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107361024A (en) * | 2017-07-18 | 2017-11-21 | 郑州大学 | A kind of method for building up of esophageal squamous cell carcinoma early stage metastasis model |
| CN107523543A (en) * | 2017-09-12 | 2017-12-29 | 哈尔滨医科大学 | A kind of construction method of the small micella of biological microemulsion, transitional cell bladder carcinoma cell line suspension and mouse air bag bladder cancer models |
| CN108624561A (en) * | 2018-05-26 | 2018-10-09 | 复旦大学 | Primary tumor cell culture medium, cultural method and application |
| CN110251164A (en) * | 2019-06-14 | 2019-09-20 | 上海交通大学医学院附属仁济医院 | A kind of dyeing interpretation method of cell capture device, cystoscope and sample cell |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
| CN111197030A (en) * | 2020-02-17 | 2020-05-26 | 上海嗣新生物科技有限公司 | Method for culturing bladder cancer organoid in vitro |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344910A (en) * | 2011-10-18 | 2012-02-08 | 许传亮 | Human bladder cancer cell line |
| CN103911343A (en) * | 2014-03-07 | 2014-07-09 | 许传亮 | Human bladder cancer cells capable of realizing multi-organ metastasis |
-
2016
- 2016-06-03 CN CN201610385304.3A patent/CN106011072A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102344910A (en) * | 2011-10-18 | 2012-02-08 | 许传亮 | Human bladder cancer cell line |
| CN103911343A (en) * | 2014-03-07 | 2014-07-09 | 许传亮 | Human bladder cancer cells capable of realizing multi-organ metastasis |
Non-Patent Citations (2)
| Title |
|---|
| KEITH SYSON CHANA等: "identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells", 《PNAS》 * |
| 徐伟东: "中国人膀胱癌细胞系及其多器官转移模型建立、鉴定及转移机制研究", 《中国优秀硕士学位论文全文数据库，医学卫生科技辑，第二军医大学硕士学位论文》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107361024A (en) * | 2017-07-18 | 2017-11-21 | 郑州大学 | A kind of method for building up of esophageal squamous cell carcinoma early stage metastasis model |
| CN107523543A (en) * | 2017-09-12 | 2017-12-29 | 哈尔滨医科大学 | A kind of construction method of the small micella of biological microemulsion, transitional cell bladder carcinoma cell line suspension and mouse air bag bladder cancer models |
| CN107523543B (en) * | 2017-09-12 | 2020-07-14 | 哈尔滨医科大学 | Biological microemulsion small micelle, bladder cancer cell suspension and construction method of mouse air sac bladder cancer model |
| CN108624561A (en) * | 2018-05-26 | 2018-10-09 | 复旦大学 | Primary tumor cell culture medium, cultural method and application |
| CN108624561B (en) * | 2018-05-26 | 2021-09-17 | 复旦大学 | Primary tumor cell culture medium, culture method and application |
| CN110251164A (en) * | 2019-06-14 | 2019-09-20 | 上海交通大学医学院附属仁济医院 | A kind of dyeing interpretation method of cell capture device, cystoscope and sample cell |
| CN111110385A (en) * | 2019-12-31 | 2020-05-08 | 南京普恩瑞生物科技有限公司 | Construction method of human tumor xenograft model |
| CN111197030A (en) * | 2020-02-17 | 2020-05-26 | 上海嗣新生物科技有限公司 | Method for culturing bladder cancer organoid in vitro |
| CN111197030B (en) * | 2020-02-17 | 2023-08-22 | 上海嗣新医药科技有限公司 | Method for in vitro culture of bladder cancer organoids |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106011072A (en) | Method for establishing human bladder transitional cell carcinoma cell line and a mouse model of bladder carcinoma tissue recombination functional reconstruction | |
| Keller et al. | Consensus recommendations for trabecular meshwork cell isolation, characterization and culture | |
| Nguyen-Ngoc et al. | 3D culture assays of murine mammary branching morphogenesis and epithelial invasion | |
| Parekh et al. | Descemet membrane endothelial keratoplasty tissue preparation from donor corneas using a standardized submerged hydro-separation method | |
| CN104411816B (en) | The allogeneic blood vessel transformed through bioengineering | |
| CN107937341A (en) | The production method of retinal pigment epithelium lamella | |
| Mi et al. | The formation of a tissue-engineered cornea using plastically compressed collagen scaffolds and limbal stem cells | |
| CN108977407A (en) | A kind of preparation and application for the cadherins fusion protein functional modification tissue engineering bracket promoting vascularization | |
| CN114854671A (en) | Preparation method and application of endometriosis invading vascularization 3D cell model | |
| CN108774629A (en) | A kind of primary culture method of Microhyla ornata alveolar epithelial cells | |
| Pang et al. | Neuron/oligodendrocyte myelination coculture | |
| Mehta et al. | Ex vivo mouse aortic ring angiogenesis assay | |
| JP3723204B1 (en) | Impermeable tissue quick fixative | |
| CN112608384A (en) | Preparation method and application of echinococcus granulosus adult epitopic antigen monoclonal antibody | |
| CN106676074A (en) | Method for inducing liver cell cells to be transformed into liver cancer stem cells | |
| CN105087466B (en) | The culture medium and method that inducing umbilical cord mesenchymal stem breaks up to corneal epithelial cell | |
| Mirando et al. | Assessing the effects of threonyl-tRNA synthetase on angiogenesis-related responses | |
| Nicosia et al. | Rat aortic ring assay of angiogenesis | |
| US20220317129A1 (en) | Method for preparing biological material for microscopy analysis | |
| Figueiredo et al. | Two-step Approach to Explore Early-and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm | |
| US20210123015A1 (en) | Methods of isolating cells from placental tissue | |
| Carabaña et al. | Multidimensional fluorescence imaging of embryonic and postnatal mammary gland development | |
| Vertrees et al. | Cellular differentiation in three-dimensional lung cell cultures | |
| Poulaki | Angiogenesis assays | |
| CN104807788B (en) | A kind of method and kit for monitoring vascular remodeling phenomenon |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161012 |