CN103756966A - Chinese tongue squamous epithelial cell carcinoma cell line CTSC-2 and establishment method thereof - Google Patents
Chinese tongue squamous epithelial cell carcinoma cell line CTSC-2 and establishment method thereof Download PDFInfo
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Abstract
The invention provides a method for establishing a Chinese tongue squamous epithelial cell carcinoma cell line CTSC-2, and provides a material basis for researching the characteristics of human tongue squamous epithelial cell carcinoma and therapeutic drugs thereof. The method comprises the following specific steps: carrying out primary culture on tongue squamous epithelial cell carcinoma cells by adopting a tissue mass adherence method; subculture was carried out when the cell density reached about 80%; RT-PCR detection of the passage stable cells shows that the cells express human tongue squamous epithelial cell carcinoma cell marker molecules CK8, 18 and 19; the western blot experiment detects that the adherent cells express the CK8 and 18 proteins of the marker molecules of human tongue squamous epithelial cell carcinoma cells, so that the successful establishment of the Chinese tongue squamous epithelial cell carcinoma cell line CTSC-2 is proved on the molecular level.
Description
Technical field
The present invention relates to people's tongue squamous cell cancer cell technology field, particularly, relate to a kind of Chinese's tongue squamous cell cancer clone CTSC-2 and establishment method thereof.
Background technology
The morbidity of 1.1 tongue squamous cell cancers and prognosis
Tongue cancer is modal oral squamous cell carcinoma, often occurs in front 2/3 place of tongue.Tongue cancer is occurred frequently in South East Asia, and with India, for the highest, Annual occurence rate reaches 9.4/100 ten thousand.Recent study demonstration, in world wide, the total incidence of tongue cancer progressively improves, and has the trend of rejuvenation.Due to the histological characteristic of its happening part and the biological characteristics of tongue cancer, tongue cancer makes patient's tongue limitation of movement, and difficulty causes swallowing, speaks, takes food.In addition, tongue cancer easily shifts, and prognosis is poor.
1.2 tumor stem cell progress of research
1.2.1 the proposition of tumor stem cell theory and development
Traditional concept thinks, tumour be a group by somatic mutation, the cell that obtains unlimited multiplication capacity forms, but the experiment of mouse tumorigenesis and body outer clone formation experimental result show, be not that all tumour cells can be bred.Illustrate that tumour is not the individuality of a homogeneous, it has heterogeneity, has different cell subsets in tissue.In tumor tissues, may some have the cell of stem cell characteristic, this cell type is called as tumor stem cell (cancer stem cell, CSC).
Tumor stem cell theory is proved in leukemia, Lapidot in 1994 etc. are utilized and in cell surface marker CD34+CD38-and mouse tumorigenesis model, are isolated people's acute myeloblastic leukemia (Acutemyeloid leukemia by FACS, AML) stem cell, subsequently, the discoveries such as Bonnet only have CD34+/CD38-leukemia cell can be in non-diabetic severe severe combined immunodeficiency (nonobese diabetic/severe combined immunodeflcient, NOD/SCID) Mice Body tumorigenesis.2003, Clarke etc. applied to tumor stem cell theory in solid tumor for the first time, and they have confirmed this viewpoint in mammary cancer, CD44+CD24-/low cell only need 100 just can tumorigenesis.Afterwards, research is found also can isolate tumor stem cell in the cancers such as cerebral tumor, liver cancer, lung cancer such as glioblastoma multiforme, has supported this theory.2006, AACR (American As sociation for Cancer Research) made definition to tumor stem cell: in tumour, have self-renewal capacity, and can produce the cell of heterogeneous tumour cell.It has three large characteristics: 1, differentiation capability: tumor stem cell is served as the role who forms and break up all types of tumour cells in tumour, in Tumor Growth, constantly supplements required all kinds of tumour cells.2, self-renewal capacity: generate a part with own identical, there is the cell of propagation, expansion and differentiation potential, thereby maintain the homeostasis of stem cell.3, Steady-State Control ability: the differentiation of adjusting and balance tumour, according to the stimulation of environment, tumour is made to regulation and control.
1.2.2 research and the application thereof of tumor stem cell in oral carcinoma
The cancer cells that includes different differentiation degrees, different steps in the same cancer kitchen range of oral carcinoma tissue.Studies confirm that, in incidence squamous cell cancer, exist a group to there is self, multi-lineage potential and have the cancer cells of tumorigenesis ability.Tumor stem cell is mostly in dormant state, insensitive to Radiotherapy chemotherapy, it is the root that after causing performing the operation, tumour easily recurs, resistance is strong, refractory is healed, the proposition of tumor stem cell theory can make the target for the treatment of from tumour global transfer to tumor stem cell, proposes the comprehensive therapy strategy combining for cancer cells and tumor stem cell.The research of tumor stem cell is also for the research of tumour has brought a new thinking.
The foundation of 1.3 clones
Setting up tumor cell line is that research tumour causes the important link in pass.Clone is widely used in each research field of biology.It refers to that directly separation and Culture goes out cell from the tissue of organism, for follow-up study provides a kind of method of stable cell lines.Clone is growth, metabolism, the modulating properties of research specific cells, cell and cell-cell interaction, and the interactions of cell and matrix etc. are research material effectively.The first strain continuous cell line of the mankind is the cervical cancer cell system (Hela) being set up by George Otto Gey laboratory nineteen fifty-one, the foundation of this strain clone is the milestone of human body cell vitro culture, Hela cell has advanced the process of many research fields such as cell, gene, virus, vaccine, has also deepened the understanding of people to tumour: find the various characteristics of tumour, reason and process, the screening cancer therapy drug etc. of research tumor development.
Except the developing of the research characteristic of cell itself and cancer, be also applied to the research field of heredity, immunity, differentiation, growth.In addition, many enterprises can also utilize cell to produce the various factors, albumen etc. both at home and abroad.The widespread use of cell has promoted the flourish of cytobiology.
Former culture mainly contains mechanical process and enzyme digestion.Mechanical process is mainly to utilize physics to shear, and directly tissue is shredded, and then these is organized to the method for fritter adherent culture.Thereby enzyme digestion is the suitable enzymatic lysis tissue of application isolate unicellular, directly suspension or the method for adherent culture.For the enzyme digesting, mainly contain: trypsinase, collagenase, pronase, Unidasa etc.
The evaluation of squamous cell cancer clone has the following aspects: 1, pathological section is identified: the World Health Organization (WHO, 2005) is divided into high, medium and low three grades according to cell and nuclear polymorphism, cell fission state, angling degree etc.Differentiated squama cancer is similar with normal tesselated epithelium, angling obviously, nuclear fission mutually less, cell and nucleus polymorphism not obvious; Middle differentiated squamous-cell carcinomas angling is more rare, nuclear fission mutually many, cell polymorphism is obvious; Low differentiated squamous-cell carcinomas has a large amount of nuclear fission phases, cell polymorphism is obvious, angling is very rare; 2, cellular form: be generally polygon Epithelial, nuclear-cytoplasmic ratio is high, the clear number of kernel is more, after cell arrangement is tight, is paving stone sample; 3, iuntercellular desmosome connects: on the film between flanking cell, have a border circular areas, responsible intercellular connection, communication, opposing external force are pullled, desmosome is more common in epithelium, the stratified squamous epithelium iuntercellular that skin, oral cavity, oesophagus etc. are located is more, can be destroyed by trypsinase, collagenase and Unidasa, under Electronic Speculum, mostly visible macula densa and Intermediate Filaments, be keratin filament in epithelial cell; 4, molecular marker: be mainly cytokeratin (cytokeratin, CK), be expressed in specifically epithelial cell, it is the Intermediate Filaments proteinoid that forms epithelial cell inner cell skeleton, have 20 kinds, CK1-9 is meta-alkalescence or neutral II type Keratin sulfate, and CK10-20 is the I type Keratin sulfate of slant acidity, these Keratin sulfate General Expressions are in specific organ or tissue, and CK8, CK18, CK19 are often expressed in simple epithelium.The above-mentioned biological characteristics that is characterized as the newly-established tongue squamous cell cancer clone of analysis of squamous cell cancer provides theoretical foundation.
The first strain tongue squamous cell cancer Establishment of Cell Line was in 1981, and by Japan Report, this strain clone fails to make mouse tumorigenesis.Then nineteen eighty-three has been set up a strain tongue cancer Tca8113 at China's Shanghai report, and 1995 have set up a strain tongue cancer cells PWH-S1 at China's Hong Kong report.Existing at American type culture collection(ATCC) in tongue cancer cells have 5 strains, be respectively SCC-15, SCC-4, SCC-25, SCC-9, CAL27, are all eighties in 20th century, rarely have afterwards report.
The biological characteristics of different Human Tongue Carcinoma Lines can there are differences, the method that this species diversity comes from patient itself and receives treatment.The degree of differentiation of some tongue cancer is low, and grade malignancy is high; The degree of differentiation of some tongue cancer is high, and grade malignancy is low.Some patient accepts chemotherapy in the preoperative, and therefore postoperative resulting cell has higher resistance.The existence of this species diversity just, makes us to the diversity of tongue cancer and complicacy, have more understanding, and the tongue cancer cells there are differences just becomes effective external model that development occurs research tongue cancer.
Summary of the invention
For this reason, the invention provides a kind of Chinese's tongue squamous cell cancer clone CTSC-2 and establishment method thereof.
Technical scheme of the present invention is as follows:
The establishment method of Chinese's tongue squamous cell cancer clone CTSC-2, comprises following concrete steps:
A) adopt tissue block adherent method to carry out the former culture of tongue squamous cell cancer cell;
B) cultivation of going down to posterity when cell density reaches approximately 80%;
C) the stable cell that goes down to posterity is detected and shows cell expressing people tongue squamous cell cancer cell sign molecule CK8,18,19 through RT-PCR;
D) through immunoblot experiment, detect attached cell and express people's tongue squamous cell cancer cell sign molecule CK8,18 albumen, from molecular level, prove and successfully set up Chinese's tongue squamous cell cancer clone CTSC-2 thus; The CTSC-2 obtaining is preserved in Chinese Typical Representative culture collection center, address: Wuhan, China city Wuhan University; Preserving number CCTCC NO C2012177, preservation date is on November 14th, 2012.
The establishment method of described Chinese's tongue squamous cell cancer clone CTSC-2, the concrete grammar of steps A is:
1) the coated culture dish of type i collagen, room temperature is static more than 1 hour, and 1 * PBS washes ware once, stand-by;
2) prepare nutrient solution, be put in 37 ℃ of thermostat water baths incubation 10 minutes;
3) in super clean bench, take out tongue squamous cell cancer tissue, in alcohol, sterilization is no more than 10 seconds, soaks in 1 * PBS, removes unwanted position, is put in nutrient solution;
4) tissue is cut into fine grained chippings, evenly divides and plant in culture dish, add appropriate nutrient solution, make to organize half to be dipped in nutrient solution;
5) be placed in 37 ℃, 5%CO
2incubator in cultivate;
6) second day supplements appropriate nutrient solution to submergence tissue block slightly;
7) visual cell's growing state suitably changes liquid.
The establishment method of described Chinese's tongue squamous cell cancer clone CTSC-2, the concrete grammar of step B is:
1) abandon nutrient solution, with 1 * PBS, wash once, add tryptic digestion;
2) add serum to stop digestion;
3) piping and druming cell, is placed in centrifuge tube, adds appropriate nutrient solution piping and druming;
4) centrifugal 1000rpm, 5 minutes;
5) abandon supernatant, add appropriate nutrient solution piping and druming loose;
6) cell suspension is added in new culture dish, obtain described Chinese's tongue squamous cell cancer clone CTSC-2.
The purposes of described Chinese's tongue squamous cell cancer clone CTSC-2 in the medicine of research treatment people tongue squamous cell cancer.
The purposes of described Chinese's tongue squamous cell cancer clone CTSC-2 in the medicine for the treatment of people tongue squamous cell cancer.
Described Chinese's tongue squamous cell cancer clone CTSC-2 at Chinese Typical Representative culture collection center (Wuhan University's preservation center) carry out preservation.
Beneficial effect of the present invention is: the present invention has set up Chinese's tongue squamous cell cancer clone CTSC-2, for studying characteristic and the medicine thereof of people's tongue squamous cell cancer, provides basic substance.
Accompanying drawing explanation
Fig. 1, cell derived patient tissue section H & E dyeing, the cellular form of light Microscopic observation primary cell and CTSC-2 the 16th generation continuous cell line.
Under Fig. 2, transmission electron microscope, observe the ultrastructure figure of CTSC-2 cell.
Fig. 3, sepharose detect the CK8,18 of normal buccal mucosa cell, CAL27 tongue cancer cells, CTSC-2 cell, the expression of 19 mRNA;
Fig. 4, Western western blotting method detect the CK8,18 and the protein expression situation of E-cadherin of CAL27 tongue cancer cells, CTSC-2 cell.
Fig. 5, CTSC-2 cell chromosome show;
Fig. 6, random selection are added up acquired results with SSCP after having calculated 100 finely dispersed CTSC-2 cell chromosomes.
The cell growth curve of Fig. 7, CTSC-2 cell;
The clone of Fig. 8, CTSC-2 cell forms;
Fig. 9, CTSC-2CTSC-2 cell mouse transplanted tumor tissue slice H & E dyeing;
Figure 10, immunofluorescence detect CTSC-2 clone to CD133, Sox2, Nanog, Oct4, SSEA-1, SSEA-4, TRA-1-60, the expression of TRA-1-81;
Figure 11, serum-free suspension culture enrichment CTSC-2 clone tumor stem cell and morphological observation thereof;
Figure 12, H & E dyeing CTSC-2 sacculus cell;
Figure 13, CTSC-2 sacculus cell and the expression of attached cell to CK8, CK18, CK19; Figure 14, CTSC-2 attached cell and the clone of sacculus cell in BME form statistics;
Figure 15, flow cytometer detect the contrast of the stem cell molecular marker expression of CTSC-2 sacculus cell and attached cell;
Figure 16, by the method for fatty inducing culture, detect the differentiation capability result of CTSC-2 sacculus cell.
Embodiment
Below with reference to specific embodiment, the establishment method of Chinese's tongue cancer cells CTSC-2 of the present invention is further illustrated.
One, foundation and the CHARACTERISTICS IDENTIFICATION of tongue squamous cell cancer clone CTSC-2
Material explanation:
1 clinical samples
This is tested oral cavity tissue used and all takes from Zhongshan University's attached brilliance stomatological hospital excision sample.
2 clones
This tests tongue squamous cell cancer CAL27 cell strain used purchased from ATCC.
3 key instruments
4, main agents
5 main solution and formulas
5.1 cell cultures
1) 10 * PBS Buffer:NaCl80g, KCl2g, Na
2hPO
411.55g, KH
2pO
42g, adds the ultrapure water of about 800ml, and the abundant stirring and dissolving of magnetic stirring apparatus regulates pH value to 7.4, is settled to 1L, after autoclave sterilization, and room temperature preservation.
2) foetal calf serum (FBS): purchased from Hyclone company, 4 ℃ of preservations, and substratum is made into suitable proportion.
3) 10 * Trypsin-EDTA (TE): take 1.25g trypsin, 1.00g EDTA is dissolved in 1 * PBS of 250ml, filtration sterilization ,-20 ℃ of preservations.Before using, with 1 * PBS buffer, be diluted to proper concn peptic cell.
4) DMEM/F12(DF) substratum: be dissolved in the ultrapure water of 1L, regulate pH value to 7.2, utilize peristaltic pump and 0.22 μ m membrane filtration degerming.4 ℃ of preservations.2 * DF component doubles, and the water yield is constant.DF(-) represent not add Ampicillin and Kanamycin.
5) mouse tail NTx: during former culture, be diluted to 1% concentration with 1 * PBS, at the bottom of adding 1.5mL to cover ware on each ware of six centimetres, room temperature is coated more than 1 hour.
6) soft agar: soft agar adds ultrapure water, autoclave sterilization is made into 0.66% and 1.33%.
7) colchicine: 40 μ g/mL mother liquors, final working concentration is 0.2 μ g/mL.
8) the primary nutrient solution of tongue cancer: DF+10%FBS.
5.2 nucleic acid electrophoresis
1) 50 * TAE electrophoresis Buffer(pH8.5): in the vial of 1L, add 242g Tris, 18.6gNa
2eDTA2H
2o, then adds the secondary water of about 800ml, abundant stirring and dissolving, then add the acetic acid of 57.1ml, and fully stir, add secondary water and be settled to 1L, room temperature preservation.During use, with 1:50, dilute.
2) ethidium bromide (EB): storage liquid concentration is 10mg/ml, working concentration is 0.5 μ g/ml.
3) 1% Normal Agarose Gel: take 1g agarose (agarose), add 100ml1 * TAE buffer, heated and boiled to agarose dissolves completely, be chilled to 50 ℃ of left and right, add 5 μ l EB storage liquid, making final concentration is 0.5 μ g/ml, records gel flat board after shaking up.
5.3Western immunoblotting
1) protein lysate (RIPA):
2) proteinase inhibitor: EDTA-free Protease Inhibitor Cocktail tablet (Roche, Cat.No.04693159001); In cocktail, include Pancreas-extract, Thermolysin (Metalloprotease), Chymotrypsin, Trypsin, the proteinase inhibitor such as Papain.A slice tablet adds 1ml1 * PBS and is made into 10 * cocktail storage liquid, during use, be diluted to 1 *.
3) protein quantification: carry out according to GE2-D Quant Kit specification sheets.
4) 1M Tris-HCl (pH6.8): weigh 121.1g Tris and be placed in 1L bottle, add the secondary water of about 800ml, fully stirring and dissolving, with dissolved salt acid for adjusting pH value to 6.8, is settled to 1L by solution, after autoclave sterilization, room temperature preservation.
5) 1.5M Tris-HCl (pH8.8): weigh 181.7g Tris and be placed in 1L bottle, add the secondary water of about 800ml, fully stirring and dissolving, with dissolved salt acid for adjusting pH value to 8.8, is settled to 1L by solution.After autoclave sterilization, room temperature preservation.
6) 10% (W/V) ammonium persulphate claims 1.0g ammonium persulphate, adds 10ml secondary water fully to dissolve, 4 ℃ of temporary transient preservations.Or be distributed into 100 μ l aliquots, seal-20 ℃ of preservations.
7) 5 * Tris-Glycine Buffer (SDS-PAGE electrophoretic buffer) takes 15.1g Tris, 94g glycine, and 5.0g SDS, adds secondary water and is settled to 1L, room temperature preservation.During use, with secondary water, be diluted to 1 * damping fluid.
8) 5 * SDS-PAGE Loading Buffer: the 1M Tris-HCl (pH6.8) that adds 1.25ml in the plastic centrifuge tube of 50ml, 0.5g SDS, 25mg tetrabromophenol sulfonphthalein, 2.5ml glycerine, then add secondary water and be settled to 50ml, a little after heat of solution, after aliquot (1ml/ pipe) packing, room temperature preservation.Before using, every 1ml Loading Buffer adds the 1M DTT of 50 μ l2-mercaptoethanols or 0.25ml, fully mixes room temperature preservation.
9) 5% concentrated glue
10) 10% separation gel
11) transferring film liquid: 2.9g Glycine, 5.8g Tris, 0.37g SDS, the methyl alcohol of 200ml, is settled to 1L with secondary water, room temperature preservation.
12) 1 * TBST buffer:8.8g NaCl, the 1M Tris-HCl (pH8.0) of 20ml, adds the secondary water of about 800ml, and fully stirring and dissolving, is settled to 1L with secondary water, fully mixes room temperature preservation after finally adding 1.0ml Tween20.
13) confining liquid: take 0.5g skim-milk and add in 10ml TBST buffer, fully mix dissolving, now with the current.
14) ECL luminescent solution: pierce ECL enhance chemiluminescent westhern blotting substrate, purchased from Thermo, before using, 1:1 mixes two kinds of liquid, now with the current.
5.4 histological chemistry
1) 10% formalin solution: 50mL formaldehyde solution (37%-40%) is added in 450mL1 * PBS, room temperature preservation.
2) 4% paraformaldehyde (4%PFA): the paraformaldehyde of 20g is poured in 1 * PBS of 400mL, 65 ℃ of dissolvings, during dissolving, every 10-20 minute, shake up, be bordering on while dissolving completely, the 1M NaOH that adds 40 μ l, 1 * PBS is settled to 500mL, after mixing, is cooled to room temperature, 4 ℃ of preservations.
5.5 antibody
5.6 primer
Human keratin8（CK8）
Forward TTC CTG GAG CAG CAG AAC AA
Reverse GAG GAC AAA TTC GTT CTC CAT
Human keratin18（CK18）
Forward TCA GCA GAT TGA GGA GAG CA
Reverse TCT GAC TCA AGG TGC AGC AG
Human keratin19（CK19）
Forward ATT CTT GGT GCC ACC ATT GA
Reverse TCC TCA TGG TTC TTC TTC AGG
6 methods and result:
Foundation and the morphological observation of 6.1 tongue squamous cell cancer clone CTSC-2
6.1.1 the former culture of cell and the cultivation of going down to posterity
Adopt tissue block adherent method to carry out the former culture of tongue squamous cell cancer cell:
1) more than the coated static 1h of culture dish room temperature of type i collagen, 1 * PBS washes ware once, stand-by;
2) prepare nutrient solution, be put in 37 ℃ of thermostat water baths incubation 10 minutes;
3) in super clean bench, take out tissue (Zhongshan University's attached brilliance stomatological hospital excision sample), in alcohol, sterilization is no more than 10 seconds, soaks in 1 * PBS, removes unwanted position, is put in nutrient solution;
4) tissue is cut into fine grained chippings, evenly divides and plant in culture dish, add appropriate nutrient solution, make to organize half to be dipped in nutrient solution;
5) be placed in 37 ℃, 5%CO
2incubator in cultivate;
6) second day supplements appropriate nutrient solution submergence oral cavity tissue piece extremely slightly;
7) visual cell's growing state suitably changes liquid.
When reaching approximately 80%, cell density goes down to posterity:
1) abandon nutrient solution, with 1 * PBS, wash once, add trysinization;
2) add serum to stop digestion;
3) piping and druming cell, is placed in centrifuge tube, adds appropriate nutrient solution piping and druming;
4) centrifugal 1000rpm, 5min;
5) abandon supernatant, add appropriate nutrient solution piping and druming loose;
6) cell suspension is added in new culture dish.
Interpretation of result:
By after above-mentioned primary culture method the 5th day, start to occur epithelioid cell and inoblast around in tissue block.By taking gradient digestion and scraping eliminating method and remove inoblast.Because epithelioid cell breeds comparatively fast, within 17 days, just can go down to posterity afterwards, live through and go down to posterity for 4 times and remove after inoblast simultaneously, inoblast fades away, and obtains epithelioid clone, called after CTSC-2 cell.So far, CTSC-2 cell has gone down to posterity more than 50 times, and cell state is stable.
Under light microscopic, the form of CTSC-2 passage cell as shown in Figure 1.Light Microscopic observation, CTSC-2 passage cell is all typical Epithelial form, flat, polygon, cell presents paving stone shape after covering with culture dish.The feeler of CTSC-2 passage cell is more, and free property is stronger, has certain distance after usually can having observed a lot of cell fission with archeocyte.Nuclear-cytoplasmic ratio is high, and kernel is clear, and quantity differs, and can reach at most 5-6.
6.1.2 fixing, the dehydration of oral cavity tissue, embedding, H & E dyeing procedure
1) 10% formalin or 4%PFA fixing port cavity tissue spend the night;
2) on automatic dehydration instrument, dewater: 70% ethanol 2 hours, 80% ethanol 2 hours, 95% ethanol 30 minutes, 95% ethanol 30 minutes, dehydrated alcohol 30 minutes, dehydrated alcohol 2 hours, dehydrated alcohol+TO(1:1) 30 minutes, TO30 minute, TO2 hour, TO+ paraffin (1:1) 30 minutes, paraffin 3 hours, paraffin 2 hours.Totally 12 barrels, 16 hours;
3) on embedding machine, with paraffin, carry out embedding, on slicing machine, cut into slices;
4) 60 ℃, tissue is de-cured, dimethylbenzene 5 minutes, dehydrated alcohol 1 minute, 90% ethanol 1 minute, 80% ethanol 1 minute, 70% ethanol 1 minute, distillation washing 4 minutes;
5) Hematorylin liquid dyeing 1-2 minute, tap water rinses 10 minutes, water-soluble Yihong dyeing 2-3 minute;
6) dehydration, transparent and mounting: section is added to 70% ethanol 30 seconds, 80% ethanol 30 seconds, 90% ethanol 1 minute, dehydrated alcohol 1 minute, dimethylbenzene 5 minutes, finally uses resinene mounting.
Under light microscopic, CTSC-2 patient tissue form as shown in Figure 1, light Microscopic observation, the section of oral cavity tissue patient tissue, is visible as squamous cell cancer, and cell is the polygon differing in size, there is polymorphism, be arranged in nido, have matrix to hold around, nuclear fission is many mutually, nuclear-cytoplasmic ratio is high, multinuclear benevolence or lobulated, there is keratin pearl in the patient tissue visible tumor center of cutting into slices, tumor invasion muscle layer, visible lymphocytic infiltration in tissue, in tissue slice, keratin pearl is more obvious, more and area is large, therefore CTSC-2 is compared with differentiated.
6.1.3CTSC-2 cell Ulntrastructure
As the Ulntrastructure of observing CTSC-2 cell under Fig. 2 transmission electron microscope as shown in Figure 2, visible cell has first antenna shape projection, and cell pimple is more.In tenuigenin, can be observed golgi body, rrna, endoplasmic reticulum distribution, also had a small amount of cavity, nucleus is irregular, has 1-2 kernel obvious.Cell is not observed contacting between obvious desmosome cell and cell with intercellular connection.
6.1.4CTSC-2 the evaluation of the significant molecule of cell
6.1.4.1 the extraction of normal buccal mucosa cell, CAL27 tongue cancer cells, CTSC-2 attached cell RNA (pressing the operation of Invitrogen Trizol test kit specification sheets)
1) cell in vegetative period of taking the logarithm, abandons nutrient solution, adds the cell concentration of 1ml Trizol(6cm ware more suitable in culture dish), with pipettor, blow and beat to lysis completely, liquid is drawn to 1.5ml without in enzyme centrifuge tube; 2) room temperature is placed and within 5 minutes, is made nucleic acid-protein mixture completely separated, and 4 ℃, centrifugal 10 minutes of 12000xg, gets supernatant;
3) every use 1ml Trizol adds chloroform 0.2ml, violent jolting 15s, and the standing 3min of room temperature makes it layering, and 4 ℃, centrifugal 15 minutes of 12000xg;
4)≤50% upper strata water is carefully transferred to another 1.5ml without in enzyme centrifuge tube;
5) every use 1ml Trizol adds 0.5ml Virahol, mixes gently standing 10 minutes of room temperature, 4 ℃, centrifugal 10 minutes of 12000xg;
6) abandon supernatant, along wall, add without enzyme 75% ethanol 1ml, mix gently;
7) 4 ℃, centrifugal 5 minutes of 7500xg, abandons supernatant, and 1.5ml centrifuge tube is inverted, with the micropipet of the 100 μ l remaining ethanol that exhausts, dry, precipitation be dissolved in 20～40 μ l without in RNase water;
8) get 2 μ l, with ultraviolet spectrophotometer (Nanodrop), measure A260nm and A280nm, RNA concentration (μ g/ml)=A260nm * 40 * extension rate; Purity is with A260/A280 than value representation, and reaching 1.8-2.0 can be for experiment, and-80 ℃ save backup.
6.1.4.1.2 extract oral cavity tissue RNA
1) claim oral cavity tissue 50-100mg to 1.5ml without in enzyme centrifuge tube, add 1ml Trizol, homogenizer is homogenate on ice; Or add again Trizol cracking with liquid nitrogen grinding is broken;
Subsequent step is with 2 of 6.1.4.1)-8).
6.1.4.1.3RT-PCR reverse transcription reaction (by TOYOBO ReverTra Ace-α-test kit specification sheets operation) 1) in PCR reaction tubes, be sequentially added into:
10pmol/L Oigo(dT) 5μl
RNase Free H
2O 6μl
2) ice bath 1min after 65 ℃ of effect 5min, puts on ice and adds:
3) PCR instrument is set, 42 ℃, 60min, 99 ℃, 5min, 4 ℃, hold, put-20 ℃ and save backup.
6.1.4.1.4PCR(press the operation of TOYOBO KOD-Plus-test kit specification sheets)
1) in PCR pipe, be sequentially added into:
2) PCR instrument is set, different primers is by characteristic settings such as various annealing temperatures, and PCR product first detects with agarose gel electrophoresis.
Sepharose detects the CK8,18 of normal buccal mucosa cell, CAL27 tongue cancer cells, CTSC-2 cell, the expression of 19 mRNA, and result as shown in Figure 3.
6.1.4.1.5Western immunoblotting detects CAL27 tongue cancer cells, the CK8 of CTSC-2 cell, 18 protein expression situation.
Protein extracting method is as follows: by cell volume, add 5 times of cell volume protein lysates (RAPI), standing cracking on ice 60 minutes, ultrasonic disruption 10 minutes; 4 ℃, centrifugal 8 minutes of 12000xg; Supernatant is shifted and to enter the 1.5mL centrifuge tube without enzyme, add 10 * proteinase inhibitor to 1 * ,-80 ℃ save backup; With conventional Western western blotting method, detect CAL27 tongue cancer cells, the CK8 of CTSC-2 cell, 18 protein expression situation.Result as shown in Figure 4.
Result shows, normal buccal mucosa epithelial cell, CAL27 tongue cancer cells, these three kinds of cells of CTSC-2 cell are all expressed CK8,18,19 mRNA, expression amount no significant difference.Because normal buccal mucosa epithelial cell is less, fail to propose albumen, therefore only detected CAL27 tongue cancer cells, the CK8 of CTSC-2 attached cell, 18 protein expression situation, result shows that the two all expresses, and expression amount is basically identical.This result has confirmed that from molecular level CTSC-2 cell is typical squamous cell.
6.1.5CTSC-1 identify with the DNA fingerprint of CTSC-2
6.1.5.1 STR (STR) is analyzed: STR (STR) is extensively to exist the strand of dna connection in genome to read sequence again, the core sequence of 2-6bp, consists of, and conventionally repeats 15-30 time.STR sequence in organism genome is consistent and can entails filial generation, therefore can experimental results show that whether this research cultured cells is originated by its patient with STR.The cell that oral cavity tissue is respectively got to a fritter and the whole ware of CTSC-2 (1st generation) is delivered to Zhongshan University's verification centre of forensic medicine and is carried out Analysis and Identification.Result is as shown in table 1.
Table 1CTSC-2 cell and oral cavity tissue STR result
| Oral cavity tissue | CTSC-2 | |
| D3S1358 | ||
| 15，16 | 15，16 | |
| |
7 | 7 |
| D21S11 | 32.2,33.2 | 32.2,33.2 |
| D18S51 | 12,15 | 15 |
| |
19,20 | 19,20 |
| D5S818 | 13,14 | 13,14 |
| |
8,9 | 8,9 |
| D7S820 | 11,13 | 11,13 |
| D16S539 | 9 | 9 |
| CSF1PO | 12 | 12 |
| |
7,9 | 7,9 |
| |
14,18 | 14,18 |
| D8S1179 | 12,15 | 12,15 |
| |
8,9 | 8,9 |
| FGA | 20,25 | 20,25 |
| Aml | | X |
| D18S1364 | ||
| 16，18 | 16 | |
| D12S391 | 18,19 | 18,19 |
| |
19,20 | 19,20 |
| |
14,18 | 14,18 |
| D2S1772 | 21,24 | 21,24 |
| |
18,19 | 18,19 |
| D22-GATA198BO5 | 17,21 | 17,21 |
| D8S1132 | 20,21 | 20,21 |
| D7S048 | 20 | 20 |
* light grey lattice represent the vicissitudinous gene of number
This experimental analysis has detected 25 sites, comprises an individual character chromosomal foci and 24 non-sex chromosome sites.CTSC-2 is at D18S51, and on D18S1364 site, cell is compared with tissue, has lacked a kind of tumor-necrosis factor glycoproteins, and all the other all mate completely.The process of considering cell cultures has the process of a screening to tissue, tissue has more diversity compared with cell, cell chromosome may produce variation in various degree, so or can judge, CTSC-2 clone is to cultivate out from this patient's oral cavity tissue.
6.1.5.2, the aobvious band of Chromosome G is analyzed:
In the CTSC-2 of logarithmic phase cell, through colchicine (0.2 μ g/mL), process after 2 hours, peptic cell, after 1 * PBS is resuspended, deliver to Zhongshan University's genetic research center and carry out film-making, the aobvious band of G is analyzed karyomit(e) situation, then fetches after 100 finely dispersed CTSC-2 cell chromosomes are calculated in random selection and add up acquired results with SSCP.Shown in result Fig. 5.
6.1.5.3, chromosome karyotype analysis:
1) in logarithmic phase CTSC-2, change to the nutrient solution containing colchicine (0.2 μ g/mL), cultivate 2 hours;
2) discard supernatant, 1 * PBS washes ware once, discards 1XPBS, trypsin digestion and cell;
3) after FBS stops digestion, centrifugal 5 minutes of 1000rpm, removes supernatant;
4) add the KCL solution (0.075M) of 37 ℃ of preheatings to process 30 minutes (different cell asynchronism(-nization));
5) add 1mL stationary liquid (methyl alcohol: acetic acid=3:1, now with the current), dispel and beat, centrifugal 5 minutes of 1000rpm, discard supernatant, add fresh stationary liquid, dispel, effect 20-30 minute, 1000rpm is centrifugal 5 minutes again, abandons supernatant, adds 0.5mL stationary liquid, makes cell suspension;
6) by cell suspension at the slide (bubble in 30% ethanol, 4 ℃ of refrigerator precoolings) that drips at place of 40cm above slide glass, dispel immediately, spirit lamp is dried;
7) Gimsa stoste: 1XPBS=1:9 dyeing is 20 minutes, and tap water rinses.Result as shown in Figure 5 and Figure 6.
The biological characteristics of 6.2CTSC-2 cell
6.2.2.1 cell counting detects CAL27 cell, the cell growth curve of CTSC-2 cell and doubling time.
1) the CAL27 cell of taking the logarithm vegetative period and CTSC-2 cell, be inoculated in 24 orifice plates with the cell concn in 3000/ml/ hole;
2) every 24h counting once, carries out cell counting, continuous counter 6 days after 1% trysinization;
3) take every day cell density average is ordinate zou, and incubation time is X-coordinate, draws cell growth curve.Result as shown in Figure 7.
4) by following formula, calculate the doubling time.
TD=t log2/（logNt-logN0）
(TD: doubling time; T: incubation time: Nt: cultivate the cell count after t hour; N0: postvaccinal cell count)
6.2.2.2CTSC-2 cell is cloned formation in BME
1) BME is placed on ice, puts into 4 ℃ of refrigerator overnight and dissolve;
2) digestion logarithmic phase CTSC-2 cell, counting, a small amount of DF is resuspended;
3) the CTSC-2 cell of some amount is added in BME, notice that whole process all will be on ice, when piping and druming mixes, avoid producing bubble;
4) plant in a hole of 96 orifice plates, after solidifying, upper strata adds 100 μ lDF+10%FBS nutrient solutions;
5) within 3 days, change a not good liquor, Continuous Observation two weeks.
BME clone forms result as shown in Figure 8, is having matrix to exist, and in the situation of DF+10%FBS culture condition, CTSC-2 cell clonal formation rate is 1.01 ± 0.23%.
6.2.2.3CTSC-2 the tumorigenicity of cell
In BALb/c nude mouse, become knurl experiment
1) the adherent CTSC-2 cell of digestion in logarithmic phase, is resuspended in 1 * PBS;
2) be subcutaneously injected into the back, both sides of female BalB/C nude mice of 6-8 week, be divided into 1 * 10
7, 2 * 10
6two order of magnitude groups;
3) observe once weekly, measure size, after 3 months, put to death and take pictures.
Above experimentation on animals meets the requirement of Declaration of Helsinki (The Declaration of Helsinki), and obtains the approval of Ethics Committee of Zhongshan University and the approval of Experimental Animal Center.
Inject altogether 4 nude mices, every kind of clone is injected two, left side injection 1 * 10
7individual cell/100 microlitre, the right injection 2 * 10
6individual cell/100 microlitre.Tumorigenesis mouse starts Continuous Observation growing state from cell injection, measures diameter of tumor, takes pictures, puts to death nude mice, tumour is separated after 3 months.
1 * 10
7the mouse of CTSC-2 cell injection, the about 0.7cm of its tumor size, all without being connected, coating is complete with skin, muscle, peritonaeum, is that tumour leans on skin side to have below extravasated blood, internal organ are intact does not find macroscopic metastasis.
As shown in Figure 9, the tumor biopsy H & E demonstration of dyeing, tumour is squamous cell cancer, can observe keratin pearl.Get part cell cultures, observe with epithelioid cell's form was consistent originally, can stablize propagation, frozen standby after amplification.
Two, enrichment and the CHARACTERISTICS IDENTIFICATION of tumor stem cell in tongue squamous cell cancer cell
1 material explanation
Clone and tissue mainly contain CTSC-2 clone and cell derived patient's tissue slice, and CTSC-2 clone is cultivated gained voluntarily by this laboratory, organizes and all takes from Zhongshan University's attached brilliance stomatological hospital excision sample.
2 key instruments
| Instrument title | Producer | Instrument title | Producer |
| Laser co-focusing | Leica | Magnetic stirring apparatus | IKA |
| Transmission electron microscope | NEC JEM | Quantitative PCR instrument | Roche LightCycler480 |
| Microplate reader | Bio-ted | Ultracentrifuge | BeckMan |
| Albumen bilateral system | GE | The |
3 main agents
All the other reagent are same, foundation and the CHARACTERISTICS IDENTIFICATION of tongue squamous cell cancer clone CTSC-2.
4 main solution and formulas
4.1 cell cultures
1) 0.7% agarose (agarose): 0.7g agarose adds 100ml ultrapure water, uses after autoclave sterilization.
2) serum-free tumor stem cell is cultivated the factor (8F): patent is maintained secrecy.
3) Trypsin inhibitor (TI): take 1.25g trypsin inhibitor and be dissolved in 1 * PBS of 250ml, be made into 10 * TI ,-20 ℃ of preservations.Before using, with 1 * PBS buffer, be diluted to 1 * TI and stop peptic cell.
4) Poly-2-hydroxyethyl methacrylate (polyHEMA): 95% alcohol is made into 36mg/mL, 4 ℃ of refrigerator overnight are dissolved, very thickness.
Other solution is same, foundation and the CHARACTERISTICS IDENTIFICATION of tongue squamous cell cancer clone CTSC-2.
4.2 histogenic immunity chemistry
1) 3% H
2o
2the H of/methanol solution: 20ml30%
2o
2with 180ml methyl alcohol
2) citrate buffer solution: 0.01M, contains trisodium citrate 3g, citric acid 0.4g, 1L secondary water in pH6.0:1L
5 antibody
6 primers
Nanog Forward：CAAAGGCAAACAACCCACTT
Reverse：TCTGCTGGAGGCTGAGGTAT
Oct4 Forward：CGACCATCTGCCGCTTTGAG
Reverse：CCCCCTGTCCCCCATTCCTA
Sox2 Forward：ATGCACCGCTACGACGTGA
Reverse：CTTTTGCACCCCTCCCATTT
c-kit Forward：ATGTGGGCAAGACTTCTGCCTA
Reverse：ATCATGCCAGCTACGATTA
ABCA1 Forward：TCTGGAAAGCTCTGAAGCCG
Reverse：TGAGTTCCTCCCACATGCCT
ABCB1 Forward：CCCATCATTGCAATAGCAGG
Reverse：GTTCAAACTTCTGCTCCTGA
ABCC1 Forward：CTTCTGGAGGAATTGGTTGTATAGAAG
Reverse：GGTAGACCCAGACAAGGATGTTAGA
ABCG1 Forward：ACCGGGGAAAAGTCTGCAAT
Reverse：TCACCAGCCGACTGTTCTGA
ABCG2 Forward：AGTTCCATGGCACTGGCCATA
Reverse：TCAGGTAGGCAATTGTGAGG
7, method and result
7.1CTSC-2 there is parts of fine cellular expression stem cell molecular marked compound in clone
7.1.1RT-PCR reverse transcription method detects the Sox2 in CTSC-2 cell, Nanog, the expression of the mRNA of Oct4
Adopt the method identical with 6.1.4.1 to extract total RNA of CTSC-2 cell, and employing and the 6.1.4.1.3 reverse transcription method cDNA that is CTSC-2 cell by total RNA reverse transcription of CTSC-2 cell.Detected the Sox2 in CTSC-2 cell, Nanog, the expression of the mRNA of Oct4, result shows CTSC-2 cell expressing Sox2, Nanog, the mRNA of Oct4.Subsequently, with the expression that Western immunoblotting has detected Sox2, also confirmed that its protein level is consistent with mrna expression level.7.1.2 cell immunofluorescent staining method detects CTSC-2 clone to CD133, Sox2, Nanog, Oct4, SSEA-1, SSEA-4, TRA-1-60, the expression of TRA-1-81
Cell immunofluorescent staining method is as follows:
1) by clean aseptic cover glass in each hole as for six orifice plates, the coated slide of I type mouse tail collagen more than 1 hour, is drawn supernatant, and 1 * PBS washes 2 times, stand-by;
2) the CTSC-2 cell of digestion logarithmic phase, resuspended rear kind enters to have in six orifice plates of cover glass;
3) be placed in CO2gas incubator and be cultured to the about 70%-80% of cell density;
4) 1 * PBS washes slide 2 times, by the 4%PFA room temperature of precooling, fixes 10 minutes;
5) abandon stationary liquid, 1 * PBS washes 3 times, 10 minutes/time;
6) 1 * PBS dripping containing 5%BSA mixes (1:1) with lowlenthal serum confining liquid, seals 1 hour;
7) inhale and abandon confining liquid, drip the suitably primary antibodie of dilution, 4 ℃ of refrigerator overnight;
8) inhale and abandon primary antibodie, 1 * PBS washes 3 times, 10 minutes/time;
9) drip fluorescein-labelled two of suitable same kind of diluting and resist, incubated at room 1-2 hour;
10) inhale and to abandon two anti-ly, 1 * PBS washes 3 times, 10 minutes/time;
11) with the mountant mounting containing DAPI;
12) under laser confocal microscope, observe, choose at random 5 visuals field (10 * 10 times) and take pictures and add up, the result of taking pictures is as Figure 10, and statistical result showed is at table 2.
Table 2 immunofluorescence detects the statistics of cell expressing stem cell markers
| % | CD133 | Sox2 | Nanog | Oct4 | SSEA-1 | SSEA-4 | TRA-1-60 | TRA-1-81 |
| CTSC-2 | 99.0±0.5 | 4.1±1.2 | 3.0±1.0 | 22.3±2.0 | 25.8±1.6 | 27.5±1.8 | <1 | 5.7±1.3 |
The result of two experiments has all been pointed out and in cell, has been had tumor stem cell, but results suggest CD133 all cells in CTSC-2 clone is all expressed, therefore be not suitable in this experiment for doing the screening of the marker of tongue cancer stem cell, remaining stem cell marker only has part cell to present positive expression.
Tumor stem cell in 7.2 serum-free suspension culture enrichment CTSC-2 clones
The CTSC-2 attached cell of digestion logarithmic phase, counting, is resuspended in DF+8F nutrient solution; Plant to enter to use in advance in the coated culture dish of aseptic 0.7%agarose.Found that, in the coated ware of agarose, by after serum-free for attached cell (DF+8F) nutrient solution suspension culture, can observe sacculus fine and close, neat in edge and form, result is as Figure 11.Then, we determine the optimum cell thickness of sowing of enrichment tumor stem cell, observe 1 * 10
5, 5 * 10
5, 1 * 10
6, 2 * 10
6, 5 * 10
6the impact of five cell count magnitudes of/10cm on bioaccumulation efficiency.Found that at 2X10
6under the quantity of/10cm, without adding the somatomedins such as FGF2 or TGF-β, the plastidogenetic Balloon size of CTSC-2 is more even, and edge is more neat, cell state is good, therefore the condition unification of follow-up cultivation sacculus is: DF+8F, 0.7%agarose are coated with 10cm ware, 2X10
6individual cell/ware, the culture condition while going down to posterity is also like this.
As shown in figure 11, light Microscopic observation, balloon structure is compacted, whole bright, in sacculus, can not clearly distinguish cell and iuntercellular boundary.In the same time of cultivating, size the heterogeneity of sacculus, more than large diameter can reach 100 μ m, little diameter only has 30 μ m left and right.As shown in figure 12, H & E dyeing sacculus shows that spheroid is fine and close, between cell and cell be connected closely and mutually chimeric.After counting, plant lower 2 * 10
6individual cell, sacculus formed rear cultivation after 7 days, calculated sacculus more than 50 μ m under light microscopic, and result shows that CTSC-2 cell can form approximately 600 cells, and what be about kind of lower cell count is about 0.03 ± 0.01%.
Figure 13 demonstration, sacculus cell and attached cell do not have notable difference to the expression level of CK8, CK18, CK19, and prompting tumor stem cell does not obviously change on cell type.
The biological characteristics of 7.3CTSC-2 sacculus
By the experimental result of this research, and in conjunction with bibliographical information, think in the attached cell that DF+10%FBS cultivates and contain a small amount of tumor stem cell, and in the sacculus cell of DF+8F suspension culture, contain more tumor stem cell, therefore, the experiment of this research is usingd attached cell as reference, and tumor stem cell is carried out to a series of analysis and research.
7.3.1CTSC-2 the comparative analysis of the cell cycle of sacculus cell and attached cell
Utilize the cell cycle that flow cytometer carries out to detect, cell is divided into 3 classes, the first kind is the cell in the G0/G1 phase, and the cell of G0 phase stops division, and the cell of G1 phase is just being prepared division; Equations of The Second Kind is the cell of S phase, and the cell of this period is carrying out chromosomal copying; The 3rd class is the G2/M phase, and the cell of this period has completed chromosomal doubling.The CTSC-2 attached cell G0/G1 phase is 87.24 ± 7.95%, the CTSC-2 sacculus cell G0/G1 phase is 96.28 ± 3.11%, and both compare, and has more cell in G0 in sacculus, the G1 phase G0/G1 phase, difference has statistical significance (P < 0.05).The Leukopenia explanation of division, the culture condition of suspension serum-free, can make tumor stem cell well maintain dryness.
7.3.2CTSC-2 the contrast of the clonality of sacculus cell and attached cell
Attached cell and sacculus cell are digested respectively for unicellular, unicellular kind is entered in BME, with the culture condition of DF+8F, observe the situation that clone forms in BME.And clone's formation result is added up, as shown in figure 14.
7.3.3CTSC-2 the contrast of the resistance of sacculus cell and attached cell
This experiment utilizes the method for CCK8 to detect CTSC-2 sacculus and the tolerance of attached cell to 5-Fu and two kinds of anticarcinogens of DDP.Experiment is divided into 5-Fu and two experimental group of DDP, and in 5-Fu group, when the concentration of 100 μ g/mL, CTSC-2 attached cell median lethal concentration is 30 μ g/mL, and CTSC-2 sacculus cell median lethal concentration is 75 μ g/mL; In DDP group, CTSC-2 attached cell median lethal concentration is 4 μ g/mL, and CTSC-2 sacculus cell median lethal concentration is 14 μ g/mL, and the tolerance difference of chemotherapeutics is had to statistical significance (P<0.01).Result shows the rising along with chemotherapeutics concentration, and cell survival rate all reduces, but under same drug level effect, the survival rate of sacculus is higher than attached cell.In proof sacculus, mdr cell content is higher, may be because the high institute of tumor stem cell content causes.By detecting the several main genes relevant to resistance of ABC family, as ABCA1, ABCB1, ABCC1, ABCG1, finds after the expression of ABCG2, and the ABCG1 of sacculus is than the up-regulated of adherent cell, and prompting may be relevant to ABCG1 path.
7.3.4CTSC-2 the contrast that the stem cell molecular marker of sacculus cell and attached cell is expressed
The expression of the significant molecule protein of this research application flow cytometer detection stem cell, result is as Figure 15, and flow cytometer detected result shows, and the Sox2 expression of CTSC-2 sacculus cell does not obviously raise than CTSC-2 cell, may be that CTSC-2 expression amount is all very low.And the expression of SSEA-1 is CTSC-2 attached cell, be 28.79% positive, CTSC-2 sacculus cell is 44.89%, and CTSC-2 sacculus cell is compared up-regulated with attached cell, and difference has statistical significance.Many than in attached cell of the cell of high expression level Sox2 and SSEA-1 in results suggest sacculus cell.
7.3.5CTSC-2 sacculus has certain differentiation potential
Having differentiation potential is a large feature for stem cell, so this experiment has detected the differentiation potential of CTSC-2 sacculus cell by two kinds of methods, and contrasts with CTSC-2 attached cell.First method is, sacculus kind, in culture dish that can be adherent, is added to DF+10%FBS nutrient solution, continues to cultivate, and observes that sacculus can differentiate gradually and the similar epithelioid cell of attached cell originally, as Figure 16.At the 2nd day that cultivates, just having more than 50% CTSC-2 sacculus cytodifferentiation was flat adherent squamous cell, within the 4th day, was just all divided into squamous cell.
Second method is the method by above-mentioned fatty inducing culture, detects the differentiation capability of CTSC-2 sacculus cell, and CTSC-2 attached cell is as contrast, as Figure 16.Inducing culture carries out fat stains after 30 days, attached cell does not have obvious form to change, more but cavity appears in tenuigenin.CTSC-2 sacculus, after induction, has part cell in tenuigenin, to occur fat particle.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, its framework form can be flexible and changeable, can subseries product.Just make some simple deduction or replace, all should be considered as belonging to the present invention by the definite scope of patent protection of submitted to claims.
Claims (5)
1. the establishment method of Chinese's tongue squamous cell cancer clone CTSC-2, is characterized in that, comprises following concrete steps:
A) adopt tissue block adherent method to carry out the former culture of tongue squamous cell cancer cell;
B) cultivation of going down to posterity when cell density reaches approximately 80%;
C) the stable cell that goes down to posterity is detected and shows cell expressing people tongue squamous cell cancer cell sign molecule CK8,18,19 through RT-PCR;
D) through immunoblot experiment, detecting attached cell expression people tongue squamous cell cancer cell sign divides
soncK8,18 albumen, prove and successfully set up Chinese's tongue squamous cell cancer clone CTSC-2, the CTSC-2 of acquisition is preserved in Chinese Typical Representative culture collection center, preserving number CCTCC NO:C2012177 thus from molecular level.
2. the establishment method of Chinese's tongue squamous cell cancer clone CTSC-2 as claimed in claim 1, is characterized in that, the concrete grammar of steps A is:
1) the coated culture dish of type i collagen, more than the static 1h of room temperature, 1 * PBS washes ware once, stand-by;
2) prepare nutrient solution, be put in 37 ℃ of thermostat water baths incubation 10 minutes;
3) in super clean bench, take out tongue squamous cell cancer tissue, in alcohol, sterilization is no more than 10 seconds, soaks in 1 * PBS, removes unwanted position, is put in nutrient solution;
4) oral cavity tissue is cut into fine grained chippings, evenly divides and plant in culture dish, add appropriate nutrient solution, make to organize half to be dipped in nutrient solution;
5) be placed in 37 ℃, 5%CO
2incubator in cultivate;
6) second day supplements appropriate nutrient solution to submergence tissue block slightly;
7) visual cell's growing state suitably changes liquid.
3. the establishment method of Chinese's tongue squamous cell cancer clone CTSC-2 as claimed in claim 1, is characterized in that, the concrete grammar of step B is:
1) abandon nutrient solution, with 1 * PBS, wash once, add tryptic digestion;
2) add serum to stop digestion;
3) piping and druming cell, is placed in centrifuge tube, adds appropriate nutrient solution piping and druming;
4) centrifugal 1000rpm, 5min;
5) abandon supernatant, add appropriate nutrient solution piping and druming loose;
6) cell suspension is added in new culture dish, obtain described Chinese's tongue squamous cell cancer clone CTSC-2.
4. the purposes of Chinese's tongue squamous cell cancer clone CTSC-2 as claimed in claim 1 in the medicine of research treatment people tongue squamous cell cancer.
5. the purposes of Chinese's tongue squamous cell cancer clone CTSC-2 as claimed in claim 1 in the medicine for the treatment of people tongue squamous cell cancer.
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| CN102911916A (en) * | 2012-08-24 | 2013-02-06 | 东莞中山大学研究院 | A Chinese tongue squamous cell carcinoma cell line CTSC-2 and its establishment method |
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| CN1861786A (en) * | 2005-05-11 | 2006-11-15 | 郭伟 | Establishing and application of scale cancer cell line of purify species New Zealand mice oral cavaty and palatal surface parts |
| US20070232668A1 (en) * | 2006-03-31 | 2007-10-04 | The Board Of Regents Of The University Of Texas System | Orally Bioavailable Caffeic Acid Related Anticancer Drugs |
| CN101240261A (en) * | 2007-02-06 | 2008-08-13 | 中山大学 | A kind of human breast cancer cell line SK-3rd and its establishment method |
| CN102433305A (en) * | 2011-12-31 | 2012-05-02 | 广州呼吸疾病研究所 | Cell strain derived from human lung low-differentiation squamous carcinoma and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113493769A (en) * | 2021-06-15 | 2021-10-12 | 武汉大学 | Human tongue squamous carcinoma cell line and application thereof |
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