CN101469320A - Method for constructing oral squamous cell carcinoma animal model - Google Patents
Method for constructing oral squamous cell carcinoma animal model Download PDFInfo
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Abstract
The invention relates to a method for constructing animal models, in particular to a method for constructing oral mucosa carcinoma animal models. The method adopts the technical proposal that the method for constructing the oral mucosa carcinoma animal models comprises the following steps: firstly, culturing a tumor tissue and tumor cells; and identifying biological characteristics of in vitro growth of the cells. The method for constructing the oral mucosa carcinoma animal models has the advantages that: firstly, Rca-B cell lines and Rca-T cell lines constructed by the method all come from single cloned tumor cells induced by SD pure mouse chemical carcinogens, and have the same genetic background and stable biological characteristics; and secondly, the characteristics of the cell lines such as hypodermal tumor formation in a nude mouse and high experimental liver transfer rate provide two practical animal models for study on prevention and treatment of oral mucosa epicytoma.
Description
Technical field
The present invention relates to a kind of method that makes up animal model, specifically about a kind of method that makes up oral squamous cell carcinoma animal model.
Background technology
Oral mucosa epithelium squama cancer is the modal class malignant tumour of oromaxillo-facial region.5 years survival rates of patient are paced up and down about in the of 50% after based on the complex therapy of operation.The cell model that set up that a kind of biology performance is stable, can form transplanted tumor in the body comes the generation and the biological characteristics of anthropomorphic dummy's oral mucosa squama cancer, is the essential condition of carrying out oral squamous cell carcinomas basis, diagnosis and treatment preclinical study.The method of setting up at present mouth neoplasm clone and tumor animal model thereof both at home and abroad mainly contains following 4 kinds: 1. spontaneous animal tumor model and clone, spontaneous generation or a class tumour and the relevant clone thereof set up by additional carcinogen induction culturing in the laboratory animal population; The epithelium malignant tumour VX-2 of rabbit uses this method to set up; 2. bringing out property of chemical carcinogens animal tumor model and clone thereof adopt the animal malignant tumour that various chemical carcinogenses bring out and use the clone that such tumour is set up; Regrettably up to the present, still do not have foundation and the application that stabilate is learned the oral epithelium cancer of characteristic both at home and abroad.3. human tumour cell line and immune deficiency animal-transplanted tumor model thereof.Clone is set up in people's malignant tumor tissue subculture in vitro separately cultivation, tumor tissues or clone are transplanted in immune deficiency animal tissuess such as nude mice or SCID mouse, set up transplanted tumor model in the body; This is the most frequently used technology of cancer cell of oral cavity model and method, existing both at home and abroad polyclonal cellular system's foundation and application.4. transgenic animal tumor model and tumor cell line, adopt genetically manipulated technology (transgenic technology and gene knockout technology) to change the hereditary property (mainly being the amplification of oncogene and the silence of cancer suppressor gene) of animal, produce spontaneous tumor, and then set up animal tumor model and tumor cell line.Prior art has following shortcoming: spontaneous animal tumor model incidence is extremely low, and repeatability is also very poor.The heterogenous animal tumor model shortcoming that the human tumour cell line sets up be can not fine simulation tumour the spontaneous generation process, and xenogenesis inoculation back oncobiology characteristic changes bigger.Chemical carcinogens inductive animal mouth neoplasm and clone can be simulated tumorigenic natural process, but external only seldom several clones, its biological characteristics is carried out systematic study, the biological characteristics instability of clone is difficult to routine and uses as the clone model.
Summary of the invention
The objective of the invention is to: a kind of method that makes up oral squamous cell carcinoma animal model is provided.
The objective of the invention is to realize by following technological method: the applied chemistry carcinogen brings out the canceration of SD rat oral mucosa, and set up SD rat epithelial cancer cell line on this basis, set up the stable monoclonal cell strain of biological characteristics through repeated screening, and its biological characteristics is identified comprehensively.(Rca-T Rca-B) as SD rat oral mucosa epithelial cancer cell line, is that model is compared with in the past cancer cell of oral cavity to oral mucosa epithelium malignant tumour, and generating process of its source tumour can reflect natural process and the mechanism that the mucosal epithelium cancer forms better.This clone biological characteristics is stable, and formed mouse transplanted tumor tumor formation rate is high, and good reproducibility.The successful foundation of this cell model will be to generation, the developer molecule Mechanism Study of oral cavity mucosal epithelium cancer, and screening diagnosis and treatment molecular target and explore new methods of treatment ideal model is provided.
Technical scheme of the present invention is as follows:
A kind of method that makes up oral squamous cell carcinoma animal model may further comprise the steps: (1) tumor tissues and cell cultures; (2) the cell in-vitro growth biological characteristics is identified; Wherein said step (1) comprises that the 4NQO drinking technique brings out rat, can go down to posterity to cultivate the external removal of the tumour cell inoblast of mouse, and screening mono-clonal tumour cell obtains the cell that mono-clonal is originated at last respectively.The cells in vitro continuous passage in mono-clonal source surpassed for 120 generations.4NQO concentration is 0.02 grams per liter.The cells in vitro continuous passage in mono-clonal source obtains rat root of the tongue mucosal epithelium squamous cell carcinoma system and rat cheek mucosal epithelium squamous cell carcinoma system above 120 generations.Described step (2) comprises following biological characteristics identification experiment: (1) uses upgrowth situation and the form that differs with differential interference inverted biologic microscope observation of cell; (2) cytokeratin and vimentin detect; (3) cell Electronic Speculum Ultrastructural observation; (4) cell growth curve and population doubling time; (5) dull and stereotyped colony/cloning efficiency; (6) cell cycle distribution; (7) chromosome karyotype analysis.
The disclosed a kind of method that makes up oral squamous cell carcinoma animal model of the present invention, its advantage shows: Rca-B that (1) the present invention sets up and Rca-T clone are all from the purebred rat chemical carcinogens of SD inductive mono-clonal tumour cell, genetic background is identical, and biological characteristics is stable; (2) Rca-B and Rca-T clone provide 2 very practical animal models in characteristics such as the subcutaneous one-tenth knurl of nude mice and experimental lung metastasis rate are high for the epitheliomatous study on prevention of oral mucosa; (3) the 4NQO drinking technique SD rat cheek mucosal epithelium cancer and the lingual mucous membrane epithelial cancer of bringing out has and the similar pathogenic course of human oral mucosal epithelium cancer, for the morbidity molecular mechanism of research oral squamous cell carcinoma provides rare, optimal cell and animal model; (4) the single cell clone culture technique is used in this invention from the miscellaneous tumour cell, successfully sets up tumour cell monoclonal cell system; Each tumour cell all derives from a common progenitor cell in the clone of being set up, and its cytogenetics background is identical, the 21st, 65 generation cell chromosome be 84; Biological character difference is few, and biological characteristics is relatively stable; (5) with visible a large amount of tonofibril in the visible endochylema of electron microscopic observation, iuntercellular has abundant desmosome, hemidesmosome, meets epithelial characteristics under the ultrastructure; (6) the application cell keratin stain has determined that the source of cell is an epithelial cell, and is tesselated epithelium.
Description of drawings
Fig. 1: Rca-T cell in-vitro growth situation, Rca-T (A) phase microscope (* 100) see that cell is polygonal property or fusiformis adherent growth.
Fig. 2: Rca-B cell in-vitro growth situation, Rca-B (B) cell all is polygon, similar round, the nuclear location projection, inlays and arranges adherent growth (differential interference microscope * 100).
Fig. 3: the keratin stain result of the 65th generation Rca-T cell, cytoplasm are the brown yellow granule positive staining, and albuminous cell all is strong positive and expresses (SABC * 100).
Fig. 4: the vimentin coloration result of the 65th generation Rca-T cell, cytoplasm are the brown yellow granule positive staining, and albuminous cell all is strong positive and expresses (SABC * 100).
Fig. 5: the keratin stain result of the 65th generation Rca-B cell, cytoplasm are the brown yellow granule positive staining, and albuminous cell all is strong positive and expresses (SABC * 100).
Fig. 6: the vimentin coloration result of the 65th generation Rca-B cell, cytoplasm are the brown yellow granule positive staining, and albuminous cell all is strong positive and expresses (SABC * 100).
Fig. 7: Rca-T cell ultrastructure observations, scanning electron microscope shows: cell surface all has abundant microvillus structure, (SEM * 5K).
Fig. 8: Rca-T cell ultrastructure observations, the projection Electronic Speculum shows: endochylema contains abundant tonofibril bundle and a large amount of plastosome and rough surfaced endoplasmic reticulum and typical desmosome structures, (TEM * 3K).
Fig. 9: Rca-T cell ultrastructure observations, the projection Electronic Speculum shows: endochylema contains abundant tonofibril bundle and a large amount of plastosome and rough surfaced endoplasmic reticulum and typical desmosome structures, (TEM * 3K).
Figure 10: Rca-B cell ultrastructure observations, scanning electron microscope shows: cell surface all has abundant microvillus structure, (SEM * 6K).
Figure 11: Rca-B cell ultrastructure observations, the projection Electronic Speculum shows: endochylema contains abundant tonofibril bundle and a large amount of plastosome and rough surfaced endoplasmic reticulum and typical desmosome structures, (TEM * 6K).
Figure 12: Rca-B cell ultrastructure observations, the projection Electronic Speculum shows: endochylema contains abundant tonofibril bundle and a large amount of plastosome and rough surfaced endoplasmic reticulum and typical desmosome structures, (TEM * 6K).
The 21st generation of Figure 13: cell growth curve: Rca-T, 65 generation cell growth curve figure.
Figure 14: cell growth curve: Rca-B cell, the 21st generation, 65 generation cell growth curve figure.
Figure 15: the cell plate clone forms the result: the clone that the Rca-T cell cultures formed in 14 days.
Figure 16: the cell plate clone forms the result: the clone that the Rca-B cell cultures formed in 14 days.
Figure 17: nude mice subcutaneous vaccination cell becomes the pathomorphism of tumor tissue: cell forms cancer nests, and is not of uniform size, and heteromorphism is (Rca-B, HE * 100) obviously.
Figure 18: cell experiment lung metastasis pathological examination: the agglomerating distribution of lung metastatic tumour cell, visible normal lung bubble structure (Rca-B, HE * 100).
Embodiment
Below in conjunction with embodiment the present invention is further described.
Experimental example one tumor tissues and cell cultures
1, material: cytokeratin (cytokeratin, CK) monoclonal antibody (clone AE1/AE3) and vimentin (vimentin, Vim) monoclonal antibody is available from DAKO company, working concentration is the 1:200 diluent.(4-nitroquinoline-1-oxide is 4NQO) available from Sigma company for 4-nitroquinoline-1-oxide compound; Cycle TEST
TMPLUS DNA test kit is available from U.S. company BD.Female sd inbred rats and Nu/nu nude mice are all available from Chinese Academy of Sciences's Shanghai Experimental Animal Center.
2, method
1) add in the drinking-water 4-nitroquinoline-1-oxide compound (4-nitroquinoline-1-oxide, 4NQO), making its final concentration is 0.02 grams per liter; Raise 80 SD rats.Observation raised for 36 weeks, had 61 survivals, and had tumour to form.Cut oral mucosa epithelial tumor tissue under the aseptic condition, be defined as oral mucosa malignant tumour person, capable respectively tumor tissues vitro culture of former generation through quick frozen section.Cultivation results only No. 23 mouse (pathological diagnosis is an oral cavity root of the tongue mucosal epithelium malignant tumour) and No. 41 mouse (pathological diagnosis is an oral cavity Jia portion mucosal epithelium malignant tumour) has cell amplification and can cultivate by subculture in vitro separately.
2) with the tumour cell of above-mentioned No. 23 and No. 41 mouse cultivating of can going down to posterity, external repeatedly with mechanical curettage method and trypsin digestion removal inoblast; Limiting dilution assay screening mono-clonal tumour cell.Limiting dilution assay screening mono-clonal tumour cell: during cell cultures to 2 month, collecting cell to 5-10/ml, is inoculated in 96 orifice plates with every hole 200 μ l with nutrient solution diluting cells density, and mark contains the hole of individual cells after 24 hours.Began to observe, seek cell clone on the 4th day, the monoclonal cell hole is continued to cultivate; After 2 weeks each clone cell being moved to 12 orifice plates cultivates; After being paved with, cell moves to 6 orifice plates again; Move to 25cm at last
2Continuous passage is cultivated in the culturing bottle.Culture condition is the RPMI RPMI-1640 that contains 10% foetal calf serum, 37 ℃, 5%CO
2Cultivate in the incubator.Obtain the cell in mono-clonal source at last respectively, external so far continuous passage all surpassed for 120 generations; With its difference called after Rca-T clone (being SD rat root of the tongue mucosal epithelium squamous cell carcinoma system) and Rca-B clone (being SD rat cheek mucosal epithelium squamous cell carcinoma system).
Experimental example two cell in-vitro growth biological characteristics qualification results
1, material, key instrument: cytokeratin (cytokeratin, CK) monoclonal antibody (clone AE1/AE3) and vimentin (vimentin, Vim) monoclonal antibody is available from DAKO company, working concentration is the 1:200 diluent.Biological inverted microscope (TE300, Japanese NICKON company); Transmission electron microscope (transmission electon microscope, TEM, CM-120 type transmission electron microscope, Dutch PHILIP company); Scanning electron microscope (scanning electron microscope, SEM; The QUANTA-200 environmental scanning electronic microscope, Dutch PHILIP company).Flow cytometer (FACSCalibur, U.S. company BD).
2, method
1) use upgrowth situation and the form that differs with differential interference inverted biologic microscope observation of cell: the Rca-T cell mostly is polygon or fusiformis epithelioid cell, adherent growth (see figure 1); The Rca-B cell is polygon or similar round epithelioid cell, inlays arrangement, adherent growth, nucleus position projection (see figure 2).
2) cytokeratin (CK) and vimentin (Vim) detect: Rca-T cell (seeing Fig. 3,4) and Rca-B cell (Fig. 5,6) interior CK, Vim dyeing all are the stronger positive, and positive rate is more than 96%.
3) transmission electron microscope and scanning electron microscope Ultrastructural observation:
Cell is conventional the cultivation when growing to 80-90% and converging in culturing bottle, 4 ℃ of precooling PBS washings, 2% glutaraldehyde of 4 ℃ of precoolings on ice before fixing 2h, send that Electron Microscopy Room is fixing behind rinsing, osmic acid, rinsing, dehydration, oxyethane are replaced, soak into, embedding, section, make the transmission electron microscope sample after the dyeing, transmission electron microscope observing, take pictures.When cell grows into individual layer on the culture dish cover glass, the PBS washing, 2% glutaraldehyde of 4 ℃ of precoolings is fixing 2h on ice, send Electron Microscopy Room to make the scanning electron microscope sample after fixing behind rinsing, the osmic acid, rinsing, gradient alcohol dehydration, drying, conduction (ion sputtering), scanning electron microscopic observation, takes pictures.
Cell Electronic Speculum Ultrastructural observation result: the cellular form under Rca-T cell (seeing Fig. 7,8,9) and Rca-B cell (seeing Figure 10,11, the 12) Electronic Speculum.(Fig. 7,10) cell is polygon or fusiformis under the scanning electron microscope, has a large amount of endochylema projections to be connected with iuntercellular, and there is abundant microvillus on the surface.(Fig. 8,9,11,12) see that cell has abundant microvillus under the transmission electron microscope, and rough surfaced endoplasmic reticulum is abundant in the endochylema, and kernel is obvious, and iuntercellular has the tonofibril of typical Desmosomes and Hemidesmosomes, bunchy.
4) cell growth curve and population doubling time: get the 21st generation, the 65th generation cell, be diluted to single cell suspension, every hole 3000/200 μ l are inoculated in 96 orifice plates, and establish blank, totally 8 blocks of plates.From second day beginning, the same time of every day got a plate and adds tetrazolium salts (MTT) solution 20 μ l, added dimethyl sulfoxide (DMSO) 200 μ l behind the cultivation 5h, after vibration 20min dissolves fully to crystallization, measured absorbance (A) on the 490nm wavelength enzyme-linked immunosorbent assay instrument.Draw cell growth curve and calculate population doubling time.
Cell growth curve and population doubling time: Rca-T cell (seeing Figure 13) and the 21st generation of Rca-B (seeing Figure 14), 65 generation cell growth curve.The 21st generation of Rca-T cell, 65 generation cell population doubling time be 23.13h and 22.94h; The 21st generation of Rca-B cell and 65 doubling times in generation then are respectively 26.34h and 25.44h.Statistical result showed, the 21st generation of every kind of cell strain and 65 generation cell the population doubling time no difference of science of statistics.
5) the dull and stereotyped colony forming efficiency of cell: get the cell of the 65th generation logarithmic phase growth, make single cell suspension, be inoculated in 6 25cm by 300/bottle
2In the plastic culture bottle, stop cultivating when the visible colony of naked eyes grows up to after 2 weeks, 1:3 acetate methanol (fresh preparation) is fixing, Giemsa dyeing, clone's number of 〉=50 cells of calculating.Cloning efficiency=clone's number/inoculating cell number * 100%.
Dull and stereotyped colony/cloning efficiency: the plate clone rate of formation in the 65th generation of Rca-T cell (seeing Figure 15) is 67.3% (seeing Figure 14); The Rca-B cell is 56.3% (seeing Figure 16).
6) the flow cytometer cell cycle is detected: collect cultivate 48h the 65th generation cell, PBS washing 1 time adds the physiological saline of 4 ℃ of precoolings of 2ml, makes single cell suspension, adds the dehydrated alcohol of 6ml-20 ℃ of precooling, ice bath 30min puts 4 ℃ of refrigerator overnight.Obtained cell suspension, CycleTEST
TMThe dyeing of PLUS DNA test kit, the cells were tested by flow cytometry cell cycle.Adopt Cell Quest software to obtain data, Mod FIT software analysis.
Cell cycle distribution: the flow cytometer detected result shows that the Rca-T cell cycle distribution is that G1 phase cell accounts for 70.15%, G2/M phase cell 9.21%, S phase cell 20.64%.The Rca-B cell G1 phase is 66.72%, G2/M phase 13.15%, S phase 20.13%.The chromosomal ploidy of Rca-T and Rca-B all has only a tetraploid main peak.
7) chromosome analysis: the chromosome specimen of preparation 21,65 and 101 generation metaphase in cell division.Get index division stage cell and add colchicine, final concentration is 0.033 μ g/ml, the incubator overnight incubation, the phase cell is divided in piping and druming gently, move into the 10ml centrifuge tube, through 0.075mol/L KCl hypotonic 2 times, each 30min, after the fresh stationary liquid of the acetate methanol of 1:3 pre-fixes 3 times, add the featheriness of 1ml stationary liquid and beat evenly, the precooling slide glass drips sheet, dry air, fresh Giemsa liquid dyeing 20min, flushing, airing, mounting.The oil mirror selects finely disseminated karyomit(e) to take pictures down.The karyomit(e) of 200 cells of counting is analyzed its mode and is carried out the karyomit(e) grouping.
Chromosome karyotype analysis: the 21st generation of Rca-T cell, 65 generation cell chromosome numbers (normal 2N=42) between 21～105, there is heteroploid cell to exist, tetraploid cell occupies the majority, and modal number is 84.The 21st generation of Rca-B, 65 generation the cell chromosome count results all be the tetraploid caryogram, modal number is for also being 84.
Experimental example three nude mice subcutaneous vaccinations become knurl and experimental lung metastasis result
1) subcutaneous vaccination becomes the knurl experiment to show: Rca-T and Rca-B clone, and respectively in nude mice subcutaneous vaccination 3 * 10
6Cell was put to death animal after 20 days, and 16 subcutaneous injection points of each clone all become knurl, and tumor formation rate is 100%.The subcutaneous one-tenth tumor tissue of all tumor tissue pathology's sections observation, be typical squamous cell cancer structure, the cell atypia is big, and multinuclear tumor giant cell and pathologic mitosis are seen more, as seen subcutaneous one-tenth tumor tissue has cancer nests to form, and can see keratin pearl (seeing Figure 17).
2) experimental lung metastasis experimental result: Rca-T and Rca-B cell line cell, capable respectively nude mice tail intravenous inoculation 3 * 10
6Behind the individual cell, inoculate 21 days and dissect lung tissue, HE dyeing confirms that 10 nude mices of every group lung all occurs and shift (seeing Figure 18), and the experimental lung metastasis rate is all 100%.
Claims (5)
1, a kind of method that makes up oral squamous cell carcinoma animal model may further comprise the steps:
(1) tumor tissues and cell cultures;
(2) the cell in-vitro growth biological characteristics is identified;
Wherein said step (1) comprises that the 4NQO drinking technique brings out rat, can go down to posterity to cultivate the external removal of the tumour cell inoblast of mouse, and screening mono-clonal tumour cell obtains the cell that mono-clonal is originated at last respectively.
2, method according to claim 1 is characterized in that: the cells in vitro continuous passage in mono-clonal source surpassed for 120 generations.
3, method according to claim 1 is characterized in that: 4NQO concentration is 0.02 grams per liter.
4, method according to claim 2 is characterized in that: the cells in vitro continuous passage in mono-clonal source obtains rat root of the tongue mucosal epithelium squamous cell carcinoma system and rat cheek mucosal epithelium squamous cell carcinoma system above 120 generations.
5, method according to claim 1 is characterized in that: described step (2) comprises following biological characteristics identification experiment:
(1) uses upgrowth situation and the form that differs with differential interference inverted biologic microscope observation of cell;
(2) cytokeratin and vimentin detect;
(3) cell Electronic Speculum Ultrastructural observation;
(4) cell growth curve and population doubling time;
(5) dull and stereotyped colony/cloning efficiency;
(6) cell cycle distribution;
(7) chromosome karyotype analysis.
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| CN103239479A (en) * | 2013-03-19 | 2013-08-14 | 邱启裕 | Animal model establishment method of human primary skin squamous epithelial cell cancer stem cell |
| CN106399252A (en) * | 2016-08-31 | 2017-02-15 | 上海交通大学医学院附属第九人民医院 | Oral squamous carcinoma cell strain as well as preparation method and applications thereof |
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| CN113201495A (en) * | 2021-05-07 | 2021-08-03 | 上海交通大学医学院附属第九人民医院 | Murine oral squamous cell carcinoma cell line, preparation method and application thereof |
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| CN103239479A (en) * | 2013-03-19 | 2013-08-14 | 邱启裕 | Animal model establishment method of human primary skin squamous epithelial cell cancer stem cell |
| CN103239479B (en) * | 2013-03-19 | 2014-07-02 | 邱启裕 | Animal model establishment method of human primary skin squamous epithelial cell cancer stem cell |
| CN106399252A (en) * | 2016-08-31 | 2017-02-15 | 上海交通大学医学院附属第九人民医院 | Oral squamous carcinoma cell strain as well as preparation method and applications thereof |
| CN111802326A (en) * | 2020-07-09 | 2020-10-23 | 中山大学附属口腔医院 | Construction method of Kras mutation related oral mucosa malignant animal model |
| CN111802326B (en) * | 2020-07-09 | 2022-06-17 | 中山大学附属口腔医院 | Construction method of Kras mutation related oral mucosa malignant change animal model |
| CN112251410A (en) * | 2020-10-23 | 2021-01-22 | 中国医学科学院肿瘤医院 | Mouse-derived gastric cancer cell line NCCG1, and establishment method and application thereof |
| CN113201495A (en) * | 2021-05-07 | 2021-08-03 | 上海交通大学医学院附属第九人民医院 | Murine oral squamous cell carcinoma cell line, preparation method and application thereof |
| CN114686434A (en) * | 2022-03-14 | 2022-07-01 | 北京航空航天大学 | Model for evaluating tumor cell invasion in vitro and application thereof |
| CN114686434B (en) * | 2022-03-14 | 2024-05-28 | 北京航空航天大学 | Model for in vitro evaluation of tumor cell invasion and application thereof |
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Application publication date: 20090701 |