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 PDF

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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
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bladder
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江明
王海燕
曹靖晨
鄂群
张冲
张永辉
陈苗苗
吕秀芳
潘晓东
钱麟
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Nantong University
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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

Human Bladder Transitional Cell Carcinoma cell line and the method for building up of Bladder Cancer recombination function reconstruction model
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.
CN201610385304.3A 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 Pending CN106011072A (en)

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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

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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

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