CN105255833A - Oral mucosa epithelium model and in-vitro construction method thereof - Google Patents
Oral mucosa epithelium model and in-vitro construction method thereof Download PDFInfo
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Abstract
The invention provides an oral mucosa epithelium model and an in-vitro construction method thereof and belongs to the technical field of tissue engineering biological materials. The in-vitro oral mucosa epithelium model is formed through stratification of one or two of oral mucosa squamous cancer cell lines TR146, Tca8113, SCC-4, SCC-9, SCC-25,HN-6 and CAL-27 with an air-liquid interface culture method adopting different culture systems at different stages after in-vitro amplification according to cytobiology and engineering principles.
Description
Technical field
The invention belongs to organizational engineering technical field of biological materials, be specifically related to a kind of can the security of carrying out the products such as dental care products, micromolecular compound, activated protein, medicine equipment, chemical, disposable sanitary articles of external alternative Oral mucosa keratinocyte and both effectiveness detection and the security of other materials contacted with Oral mucosa keratinocyte and the Oral mucosa keratinocyte model of Efficiency assessment and construction process thereof.
Background technology
In the present invention, Oral mucosa keratinocyte model is application cell biology and engineering principles, a small amount of seed cell is built after amplification in vitro and forms, its structure and Normal oral mucosa epithelial structure height similar, structure is from top to bottom followed successively by granular layer, spinous layer and stratum basale, equal anoderm, there is complete epithelial structure, except can be used for substituting or repair pathology, defective tissue, outside reconstruction physiological function, reconstruction in vitro oral mucosa also can be used as vitro detection model, to dental care products, micromolecular compound, activated protein, medicine equipment, chemical, security and both effectiveness detection and other material safety contacted with oral mucosa and the Efficiency assessment of the products such as disposable sanitary articles.
At present, Oral mucosa keratinocyte is in the security of dental care products and both effectiveness detection, and drug osmotic ability detects, and tobacco component carciongenic potency detects, and each side such as oral cavity pathogen pathogenesis are more and more paid close attention to.But at present in research, Oral mucosa keratinocyte is originated mainly animal oral cavity mucous membrane, corrective surgery excision oral cavity mucous membrane tissue.Both there is certain application drawback, the oral cavity mucous membrane tissue of animal-origin can not accurately react human experimentation, and result statistics shows to only have the results of animal of 50% to meet somatic data result; In addition, the ethics problem that a large amount of animal is used for every test is under suspicion, and has forbidden the experimentation on animals of makeup, chemical etc. in the world.Mainly there is difficulty of drawing materials, limited source in the oral cavity mucous membrane tissue in human body source, can not meet oral cavity related products security in-vitro evaluation quantity demand.
Early stage in the research of organizational project oral mucosa, investigator utilizes Buccal mucosa cell vitro culture to form epithelium diaphragm, is directly used in mucous membrane tissue defect repair.People's gingival epitheliums such as Lauer, utilize 3T3 cell for trophoderm, cultivate in vitro and the complete epithelium diaphragm comprising 3 ~ 6 confluent monolayer cells within 3 ~ 4 weeks, can be formed, detected result finds that the epithelial differentiation cultivated is good, similar with healthy tissues, but the application of xenogenesis trophocyte, may cause potential danger to human body in clinical repair.After have investigator in non-trophoblast situation, utilize tissue block method to cultivate Oral mucosa keratinocyte diaphragm be applied to clinical, be implanted into after in patient body, success ratio reaches 65%.But usually there is fibroblastic pollution when tissue block method cultivates as previously mentioned, affect epithelial growth.
The people end user oral cavity mucous membrane tissue vitro culture such as MitchellKlausner build oral mucosa single-layer model, the barrier function of this model is analyzed, have detected the cytoactive of this model and the release conditions of cytokine under NaF and tetra-sodium (PPI) stimulate, and use this model to carry out ET-50 analysis to commercial serial dental care products; FrancisKoschier etc. use the alcohol of the oral mucosa model research of external structure different concns to the hormesis of oral mucosa, and oral mucosa model is to the receptivity of alcohol, and alcohol stimulates rear model on the impact of the receptivity of caffeine.But these models equal end user source oral cavity mucous membrane tissue, has source difficulty equally, cannot realize external a large amount of production, the shortcoming of meeting the need of market.
For overcoming the defect in above seed cell source, there is investigator to adopt oral mucosa squamous cancer cell strain TR146 as seed cell, after amplification in vitro, being built into oral mucosa model.The strain of oral mucosa squamous cancer cell is Buccal mucosa cell comparatively ripe at present, and clone is stablized controlled, can solve the problems such as seed cell source difficulty, cell stability.The people such as such as HanneM rckNielsen utilize TR146 cells in vitro to build oral mucosa model and study, but this model has a series of defect: one, the structure time is long, the a collection of model of external structure need reach one month, the long structure time can uncontrollable factor in increase process, affects the stability of model; Two, the single nutritional needs that can not adapt to cell different steps of culture system, causes model claddingization poor, and stratum basale is without hemidesmosome structure, and structure heterogeneity is complete, and barrier function weakens, the serious application reducing model; Three, be mainly used in drug osmotic Absorption Study, application direction is single.
Summary of the invention
in view of this, in order to solve the problems of the prior art, the invention providesa kind of Oral mucosa keratinocyte model and vitro construction method thereof, application cell biology and engineering principles, use in the strain of oral mucosa squamous cancer cell TR146, Tca8113, SCC-4, SCC-9, SCC-25, HN-6, CAL-27 one or both, after amplification in vitro, adopt gas surface culture method, use different culture system in different steps, make its cladding, organizer cavitas oris externa mucosal epithelium model.
Content of the present invention is as follows:
A kind of Oral mucosa keratinocyte model, described model cell has the syndeton of cell-ECM, cell-basement membrane, and have expressed Oral mucosa keratinocyte associated protein.
Further, Oral Mucosal Cells used is one or both in the strain of oral mucosa squamous cancer cell TR146, Tca8113, SCC-4, SCC-9, SCC-25, HN-6, CAL-27.
A vitro construction method for Oral mucosa keratinocyte model, comprises following methods step:
The amplification cultivation of step one, oral mucosa squamous cancer cell
Get any one or two kinds of oral mucosa squamous cancer cell strains, after recovery amplification, use P10 ~ P20 for cell, make single cell suspension with trysinization, adjustment cell density 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, is inoculated in culturing bottle, adds nutrient solution I cultivation of 10% foetal calf serum, rocks culturing bottle gently, after making cell dispersal evenly, is placed in 37 DEG C, 5%CO
2cultivate under condition;
Described nutrient solution I is DMEM, and glucose content contained by it is 1.0 ~ 4.5g/L, and glutamine content is 1.0 ~ 10.0mM;
Inoculation culture under step 2, Buccal mucosa cell liquid
When using a kind of oral mucosa squamous cancer cell strain to build, cell in vegetative period of taking the logarithm, makes single cell suspension with nutrient solution II, and adjustment cell density is 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, inoculates, and is placed in 37 DEG C, 5%CO
2cultivate 1 ~ 5 hour under condition; After proceed to liquid under cultivate, incubation time is 1 ~ 10 day, and every day changes liquid;
When using two kinds of oral mucosa squamous cancer cell strains to build, cell in vegetative period of taking the logarithm respectively, makes single cell suspension with nutrient solution II, mixes in the ratio of 3:1 ~ 1:3, adjustment cell density 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, inoculates, and is placed in 37 DEG C, 5%CO
2cultivate 1 ~ 5 hour under condition; After proceed to liquid under cultivate, incubation time is 1 ~ 10 day, and every day changes liquid;
Described nutrient solution II, for in nutrient solution I, adding human epidermal growth factor's concentration is 0.1 ~ 100.0ng/mL, insulin concentration is 0.1 ~ 100.0ng/mL, hydrocortisone concentration is 0.1 ~ 10.0 μ g/mL, Triphosaden concentration is 1.0 ~ 100.0 μ g/mL, transferrin concentrations is 1.0 ~ 100.0ng/mL, Concentration of Progesterone is 1.0 ~ 100.0nM, butyl p-hydroxybenzoate concentration is 0.1 ~ 10.0 μ g/mL, butanediamine concentration is 1.0 ~ 100.0 μMs, ox pituitary gland extractive substance concentration is 1.0 ~ 100.0 μ g/mL, Toxins,exo-, cholera concentration is 1.0 ~ 100.0 μ g/L, amphotericin B concentration is 1.0 ~ 50.0mg/L, penicillin concn is 1.0 ~ 100.0IU/mL, Streptomycin sulphate concentration is 1.0 ~ 100.0 μ g/mL,
The proliferation and differentiation in step 3, Oral mucosa keratinocyte model gas-liquid face is cultivated
Discarded by the nutrient solution of above-mentioned little indoor, be replaced by nutrient solution III, carry out the cultivation of gas-liquid face, incubation time is 1 ~ 10 day, and every day changes liquid; Be replaced by nutrient solution IV again, proceed gas-liquid face and cultivate, incubation time is 1 ~ 10 day, and every day changes liquid; Finally be replaced by nutrient solution V, proceed gas-liquid face and cultivate, incubation time is 1 ~ 10 day, and every day changes liquid, and cultivate and terminate, the external structure of Oral mucosa keratinocyte model terminates;
Described nutrient solution III, in nutrient solution II, adds CaCl
2concentration is 20.0 ~ 100.0 μ g/mL, and vitamin C concentration is 10.0 ~ 50.0 μ g/mL;
Described nutrient solution IV, in nutrient solution II, adds CaCl
2concentration is 50.0 ~ 150.0 μ g/mL, and vitamin C concentration is 50.0 ~ 100.0 μ g/mL;
Described nutrient solution V, in nutrient solution II, adds CaCl
2concentration is 150.0 ~ 300.0 μ g/mL, and vitamin C concentration is 100.0 ~ 200.0 μ g/mL.
Further, this models applying is in the security of the products such as dental care products, micromolecular compound, activated protein, medicine equipment, chemical, disposable sanitary articles and both effectiveness detection, drug osmotic ability detects, tobacco component carciongenic potency detects, oral cavity pathogen pathogenesis.
The invention has the beneficial effects as follows:
Adopt the Oral mucosa keratinocyte model that method provided by the invention builds, have the following advantages: one, oral mucosa squamous cancer cell system stablizes controlled, the problems such as seed cell source difficulty, cell stability can be solved, external structure, repeatability is good, can realize industrialization preparation, two, the use of single cell, solves the problem of heterogenous cell, inoblast pollution, three, gas-liquid face subsection filter method, ensure that the Different Nutrition demand of cell in different steps, shortens the structure time, reduces production cost, four, the interpolation of the factor in substratum, ensure that the normal cladding of model, thus define the cell-ECM (desmosome) similar with human oral cavity mucosal epithelium height, cell-basement membrane (hemidesmosome) syndeton, and normal expression Oral mucosa keratinocyte associated protein, the impact of this self structure and extracellular microenvironment can the barrier function of lift scheme further, and the final external Oral mucosa keratinocyte model built has highly similar structure and function to human oral mucosal epithelium, five, use the Oral mucosa keratinocyte model built, human oral mucosal epithelium, mouse Oral mucosa keratinocyte tissue detects partial chemical product in " global chemical homogeneous classification and labeling system ", result shows, the Oral mucosa keratinocyte model using method provided by the invention to prepare accurately can judge whether this chemical has pungency, its result is consistent with the result of determination of end user oral cavity mucosal epithelium, and use the judgement of mouse Oral mucosa keratinocyte tissue to occur false positive and false negative result, therefore, Oral mucosa keratinocyte model can the reflection detected result of objective reality, can be good at alternative animal model, become the core tool of vitro test.In sum, use Oral mucosa keratinocyte model prepared by method provided by the invention, the demand of existing market to vitro detection can be met, expand market application direction greatly, its application mainly comprises: one, dental care products is as pungency, the toxicity detection of the formulas such as toothpaste, collutory, mouth spray and dental material, and the both effectiveness detection of functional component in formula.Two, the security of the products such as micromolecular compound, activated protein, medicine equipment, chemical, disposable sanitary articles and both effectiveness detection.Three, the detection of the osmotic absorption ability of drug osmotic receptivity, infiltration accelerating agent.Four, the detection of tobacco component pungency, carinogenicity, research smoking is on the impact of human oral mucous membrane disease.Five, oral cavity pathogen pathogenesis etc.
Accompanying drawing explanation
Fig. 1 carries out Histological section HE stained photographs after Oral mucosa keratinocyte model construction terminates.
Fig. 2 terminates to carry out transmission electron microscope (TEM) to its stratum basale and scan the hemidesmosome structure photo obtained afterwards for Oral mucosa keratinocyte model construction.
Embodiment
Below by concrete embodiment, the invention will be further described, not as limitations on claims.
Embodiment 1:
The present embodiment highlights the vitro construction method step that oral mucosa squamous cancer cell strain TR146 is Oral Mucosal Cells:
The amplification cultivation of step one, oral mucosa squamous cancer cell TR146:
Get oral mucosa squamous cancer cell strain TR146, after recovery amplification, use P10 for cell, make single cell suspension with trysinization, adjustment cell density 5.0 × 10
5individual/mL, is inoculated in culturing bottle, adds nutrient solution I cultivation of 10% foetal calf serum, rocks culturing bottle gently, after making cell dispersal evenly, is placed in 37 DEG C, 5%CO
2cultivate under condition;
Described nutrient solution I is DMEM, and glucose content contained by it is 4.5g/L, and glutamine content is 2.0mM;
Inoculation culture under step 2, Buccal mucosa cell liquid
To take the logarithm Oral Mucosal Cells in vegetative period, make single cell suspension with nutrient solution II, adjustment cell density is 5.0 × 10
5individual/mL, inoculates, and inoculum size is 0.1mL, is placed in 37 DEG C, 5%CO
2cultivate 1 hour under condition; After proceed to liquid under cultivate, incubation time is 2 days, and every day changes liquid;
Described nutrient solution II, for adding human epidermal growth factor (0.1ng/mL) in nutrient solution I, Regular Insulin (0.1ng/ml), hydrocortisone (0.1 μ g/mL), Triphosaden (10.0 μ g/mL), Transferrins,iron complexes (10.0ng/mL), progesterone (1.0nM), butyl p-hydroxybenzoate (0.2 μ g/mL), butanediamine (10.0 μMs), ox pituitary gland extractive substance (10.0 μ g/mL), Toxins,exo-, cholera (10.0 μ g/L), amphotericin B (10.0mg/L), penicillin (10.0IU/mL), Streptomycin sulphate (10.0 μ g/mL);
The proliferation and differentiation in step 3, Oral mucosa keratinocyte model gas-liquid face is cultivated
Discarded by the nutrient solution of above-mentioned little indoor, be replaced by nutrient solution III, carry out the cultivation of gas-liquid face, incubation time is 9 days, and every day changes liquid; Be replaced by nutrient solution IV again, proceed gas-liquid face and cultivate, incubation time is 9 days, and every day changes liquid; Finally be replaced by nutrient solution V, proceed gas-liquid face and cultivate, incubation time is 9 days, and every day changes liquid, and cultivate and terminate, the external structure of Oral mucosa keratinocyte model terminates;
Described nutrient solution III, for adding CaCl in nutrient solution II
2(30.0 μ g/mL), vitamins C (10.0 μ g/mL);
Described nutrient solution IV, for adding CaCl in nutrient solution II
2(100.0 μ g/mL), vitamins C (50.0 μ g/mL);
Described nutrient solution V, for adding CaCl in nutrient solution II
2(200.0 μ g/mL), vitamins C (100.0 μ g/mL).
Specifically, the strain of oral mucosa squamous cancer cell is adopted in above-mentioned steps, compare traditional seed cell source, clone is stablized controlled, can solve the problems such as seed cell source difficulty, cell stability, external structure, repeatability is good, can realize industrialization preparation, and ripe single clone uses, solve from oral cavity mucous membrane tissue be separated cause heterogenous cell, inoblast pollute problem, become the core tool of vitro test and be widely used.
The nutrient solution II adopted in above-mentioned steps, on the basis of traditional nutrient solution, with the addition of the various factor, and wherein human epidermal growth factor (EGF) can move by stimulate cell growth, suppresses the appearance of aging gene, delays epithelial cell aging; Regular Insulin can irritation cell to the absorption of uridine and glucose to synthesize RNA, protein and lipid, Regular Insulin can also be combined and multiple pathways metabolism in regulating cell by the insulin receptor on cytolemma, increase the synthesis of lipid acid and glucose, cell growth plays an important role; Transferrins,iron complexes in conjunction with iron ion, can reduce its toxicity and is utilized by cell; Hydrocortisone may have short cell attachment and proliferation function concurrently, can break up by inducing cell during cell density height.Cultivate for tradition the single substratum (such as using DMEM substratum merely) used to be merely able to provide amino acid, VITAMIN, inorganic salt, organic compound, micro-defect, the interpolation of these factors, ensure that the nutritional needs of cell, facilitate the growth of cell.
Adopt the method for gas-liquid face subsection filter in above-mentioned steps, use different culture system in different steps, make its cladding, organizer cavitas oris externa mucosal epithelium model (Fig. 1).In the method, gas-liquid face is cultivated and is divided into three phases, and the nutrient solution of use is respectively nutrient solution III, IV, V, is, in nutrient solution II, with the addition of CaCl with the difference of nutrient solution II
2and vitamins C, and CaCl
2and ascorbic concentration progressively increases.CaCl
2different Effects can be produced to the propagation of Buccal mucosa cell, differentiation under different concns; Vitamins C can stimulate generation and the lamellar body content maturation of sphingophospholipid, as increased the synthesis of glycosylation ceramide, promoting epithelial cell differentiation, and then improving epithelial barrier function.In nutrient solution III stage, CaCl
2and ascorbic concentration is lower, the Cell tracking of now epithelial cell formation is less, cell is in rapid amplification state, and the factors such as human epidermal growth factor, Regular Insulin, hydrocortisone also promote the propagation of cell, this condition ensure that the factor concentration needed for cell proliferation, and makes epithelial cell start cladding.In nutrient solution IV stage, CaCl
2and ascorbic concentration raises, under this condition, CaCl
2and there is multiple change in vitamins C induction epithelial cell, comprises and form cladding, desmosome, hemidesmosome and stick together connection, and the Oral mucosa keratinocyte associated protein such as normal expression Keratin sulfate, silk polymeric protein, loricrin, Ca
2+the essential condition of cell differentiation procedure, the especially formation of cladding and desmosome, hemidesmosome and gathering; Now the factor such as human epidermal growth factor, Regular Insulin, hydrocortisone also ensure that the normal proliferative of cell, and simultaneously when cell density increases, hydrocortisone has also played the effect of Cell differentiation inducing activity.In nutrient solution V stage, CaCl
2and ascorbic concentration raises further, epithelial cell differentiation degree strengthens further, and desmosome, hemidesmosome are connected a large amount of formation with sticking together, and epithelium associated protein great expression, ensure that the complete cladding of model.
In sum, compared with the construction process of traditional single substratum, single training method: first, the factor contamination that the method different steps is added is all different, ensure that the Different Nutrition demand of cell different steps in multiple stratification processes, model incubation time is greatly shortened.Second, the interpolation of gas-liquid face subsection filter and the factor, also ensure that the normal cladding of model, thus define the cell-ECM (desmosome) similar with human oral cavity mucosal epithelium height, cell-basement membrane (hemidesmosome) syndeton: stratum basale first can be made to form the hemidesmosome structure (Fig. 2) similar with Normal oral mucosa physiological structure height.Hemidesmosome is the privileged site of cell and junction, extracellular, and model-based bottom is attached on polycarbonate leaching film by hemidesmosome, and this similar, in the mode of connection of epithelium and reticular tissue, makes model be combined with filter membrane closely; Secondly, the normal cladding of model can also form cell and the good desmosome structure of iuntercellular, and desmosome is the important feature maintaining Cell tracking, ensures intercellular tight junction; Desmosomes and Hemidesmosomes makes tissue have stronger anti-tensile, anti-pressure ability, and structure is more homogeneous complete, finally makes model barrier function strengthen; 3rd, normal claddingization can ensure the normal expression of Oral mucosa keratinocyte associated protein, such as Keratin sulfate, silk polymeric protein, loricrin etc., the structure stable state of normal expression to Oral mucosa keratinocyte of these albumen has vital role, and this extracellular microenvironment can the barrier function of lift scheme further.The external Oral mucosa keratinocyte model of final structure has highly similar structure and function to human oral mucosal epithelium, compensate for the defect that traditional use oral mucosa squamous cancer cell prepares the method for model, the shortening structure time, reduce costs while, ensure that the security of model and both effectiveness.
Embodiment 2:
The present embodiment highlights the vitro construction method step that oral mucosa squamous cancer cell strain SCC-25 is Oral Mucosal Cells:
The amplification cultivation of step one, oral mucosa squamous cancer cell SCC-25:
Get oral mucosa squamous cancer cell strain SCC-25, after recovery amplification, use P10 for cell, make single cell suspension with trysinization, adjustment cell density 5.0 × 10
5individual/mL, is inoculated in culturing bottle, adds nutrient solution I cultivation of 10% foetal calf serum, rocks culturing bottle gently, after making cell dispersal evenly, is placed in 37 DEG C, 5%CO
2incubator is cultivated;
Described nutrient solution I is DMEM, and glucose content contained by it is 4.5g/L, and glutamine content is 2.0mM;
Inoculation culture under step 2, Buccal mucosa cell liquid
To take the logarithm cell in vegetative period, make single cell suspension with nutrient solution II, adjustment cell density is 5.0 × 10
5individual/mL, inoculates, and inoculum size is 0.1mL, is placed in 37 DEG C, 5%CO
2cultivate 1 hour under condition; After proceed to liquid under cultivate, incubation time is 2 days, and every day changes liquid;
Described nutrient solution II, for on the basis of nutrient solution I, add human epidermal growth factor (50.0ng/mL), Regular Insulin (50.0ng/ml), hydrocortisone (8.0 μ g/mL), Triphosaden (100.0 μ g/mL), Transferrins,iron complexes (100.0ng/mL), progesterone (75.0nM), butyl p-hydroxybenzoate (8.0 μ g/mL), butanediamine (75.0 μMs), ox pituitary gland extractive substance (75.0 μ g/mL), Toxins,exo-, cholera (100.0 μ g/L), amphotericin B (100.0mg/L), penicillin (100.0IU/mL), Streptomycin sulphate (100.0 μ g/mL),
The proliferation and differentiation in step 3, Oral mucosa keratinocyte model gas-liquid face is cultivated
Discarded by the nutrient solution of above-mentioned little indoor, be replaced by nutrient solution III, carry out the cultivation of gas-liquid face, incubation time is 3 days, and every day changes liquid; Be replaced by nutrient solution IV again, proceed gas-liquid face and cultivate, incubation time is 3 days, and every day changes liquid; Finally be replaced by nutrient solution V, proceed gas-liquid face and cultivate, incubation time is 3 days, and every day changes liquid, and cultivate and terminate, the external structure of Oral mucosa keratinocyte model terminates;
Described nutrient solution III, for adding CaCl in nutrient solution II
2(30.0 μ g/mL), vitamins C (10.0 μ g/mL);
Described nutrient solution IV, for adding CaCl in nutrient solution II
2(100.0 μ g/mL), vitamins C (50.0 μ g/mL);
Described nutrient solution V, for adding CaCl in nutrient solution II
2(200.0 μ g/mL), vitamins C (100.0 μ g/mL).
Embodiment 3:
The present embodiment highlights the vitro construction method step that the strain of oral mucosa squamous cancer cell HN-6, CAL-27 are Oral Mucosal Cells:
The amplification cultivation of step one, oral mucosa squamous cancer cell HN-6, CAL-27:
Get the strain of oral mucosa squamous cancer cell HN-6, CAL-27, respectively after recovery amplification, use P10 for cell, make single cell suspension with trysinization, adjustment cell density 5.0 × 10
5individual/mL, is inoculated in culturing bottle respectively, adds nutrient solution I cultivation of 10% foetal calf serum, rocks culturing bottle gently, after making cell dispersal evenly, is placed in 37 DEG C, 5%CO
2incubator is cultivated;
Described nutrient solution I is DMEM, and glucose content contained by it is 4.5g/L, and glutamine content is 2.0mM;
Inoculation culture under step 2, Buccal mucosa cell liquid
To take the logarithm respectively HN-6, CAL-27 cell in vegetative period, make single cell suspension with nutrient solution II, adjustment cell density is 5.0 × 10
5individual/mL, mix in the ratio of 1:1, inoculate, inoculum size is 0.1mL, is placed in 37 DEG C, 5%CO
2cultivate 1 hour under condition; After proceed to liquid under cultivate, incubation time is 2 days, and every day changes liquid;
Described nutrient solution II, for on the basis of nutrient solution I, add human epidermal growth factor (50.0ng/mL), Regular Insulin (50.0ng/ml), hydrocortisone (8.0 μ g/mL), Triphosaden (100.0 μ g/mL), Transferrins,iron complexes (100.0ng/mL), progesterone (75.0nM), butyl p-hydroxybenzoate (8.0 μ g/mL), butanediamine (75.0 μMs), ox pituitary gland extractive substance (75.0 μ g/mL), Toxins,exo-, cholera (100.0 μ g/L), amphotericin B (100.0mg/L), penicillin (100.0IU/mL), Streptomycin sulphate (100.0 μ g/mL),
The proliferation and differentiation in step 3, Oral mucosa keratinocyte model gas-liquid face is cultivated
Discarded by the nutrient solution of above-mentioned little indoor, be replaced by nutrient solution III, carry out the cultivation of gas-liquid face, incubation time is 2 days, and every day changes liquid; Be replaced by nutrient solution IV again, proceed gas-liquid face and cultivate, incubation time is 2 days, and every day changes liquid; Finally be replaced by nutrient solution V, proceed gas-liquid face and cultivate, incubation time is 2 days, and every day changes liquid, and cultivate and terminate, the external structure of Oral mucosa keratinocyte model terminates;
Described nutrient solution III, for adding CaCl in nutrient solution II
2(30.0 μ g/mL), vitamins C (10.0 μ g/mL);
Described nutrient solution IV, for adding CaCl in nutrient solution II
2(100.0 μ g/mL), vitamins C (50.0 μ g/mL);
Described nutrient solution V, for adding CaCl in nutrient solution II
2(200.0 μ g/mL), vitamins C (100.0 μ g/mL).
Claims (4)
1. an Oral mucosa keratinocyte model, is characterized in that: described model cell has the syndeton of cell-ECM, cell-basement membrane, and have expressed Oral mucosa keratinocyte associated protein.
2. Oral mucosa keratinocyte model according to claim 1, is characterized in that: Oral Mucosal Cells used is one or both in the strain of oral mucosa squamous cancer cell TR146, Tca8113, SCC-4, SCC-9, SCC-25, HN-6, CAL-27.
3. a vitro construction method for Oral mucosa keratinocyte model, is characterized in that: comprise following methods step:
The amplification cultivation of step one, oral mucosa squamous cancer cell
Get any one or two kinds of oral mucosa squamous cancer cell strains, after recovery amplification, use P10 ~ P20 for cell, make single cell suspension with trysinization, adjustment cell density 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, is inoculated in culturing bottle, adds nutrient solution I cultivation of 10% foetal calf serum, rocks culturing bottle gently, after making cell dispersal evenly, is placed in 37 DEG C, 5%CO
2cultivate under condition;
Described nutrient solution I is DMEM, and glucose content contained by it is 1.0 ~ 4.5g/L, and glutamine content is 1.0 ~ 10.0mM;
Inoculation culture under step 2, Buccal mucosa cell liquid
When using a kind of oral mucosa squamous cancer cell strain to build, cell in vegetative period of taking the logarithm, makes single cell suspension with nutrient solution II, and adjustment cell density is 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, inoculates, and is placed in 37 DEG C, 5%CO
2cultivate 1 ~ 5 hour under condition; After proceed to liquid under cultivate, incubation time is 1 ~ 10 day, and every day changes liquid;
When using two kinds of oral mucosa squamous cancer cell strains to build, cell in vegetative period of taking the logarithm respectively, makes single cell suspension with nutrient solution II, mixes in the ratio of 3:1 ~ 1:3, adjustment cell density 5.0 × 10
5individual/mL ~ 5.0 × 10
6individual/mL, inoculates, and is placed in 37 DEG C, 5%CO
2cultivate 1 ~ 5 hour under condition; After proceed to liquid under cultivate, incubation time is 1 ~ 10 day, and every day changes liquid;
Described nutrient solution II, for in nutrient solution I, adding human epidermal growth factor's concentration is 0.1 ~ 100.0ng/mL, insulin concentration is 0.1 ~ 100.0ng/mL, hydrocortisone concentration is 0.1 ~ 10.0 μ g/mL, Triphosaden concentration is 1.0 ~ 100.0 μ g/mL, transferrin concentrations is 1.0 ~ 100.0ng/mL, Concentration of Progesterone is 1.0 ~ 100.0nM, butyl p-hydroxybenzoate concentration is 0.1 ~ 10.0 μ g/mL, butanediamine concentration is 1.0 ~ 100.0 μMs, ox pituitary gland extractive substance concentration is 1.0 ~ 100.0 μ g/mL, Toxins,exo-, cholera concentration is 1.0 ~ 100.0 μ g/L, amphotericin B concentration is 1.0 ~ 50.0mg/L, penicillin concn is 1.0 ~ 100.0IU/mL, Streptomycin sulphate concentration is 1.0 ~ 100.0 μ g/mL,
The proliferation and differentiation in step 3, Oral mucosa keratinocyte model gas-liquid face is cultivated
Discarded by the nutrient solution of above-mentioned little indoor, be replaced by nutrient solution III, carry out the cultivation of gas-liquid face, incubation time is 1 ~ 10 day, and every day changes liquid; Be replaced by nutrient solution IV again, proceed gas-liquid face and cultivate, incubation time is 1 ~ 10 day, and every day changes liquid; Finally be replaced by nutrient solution V, proceed gas-liquid face and cultivate, incubation time is 1 ~ 10 day, and every day changes liquid, and cultivate and terminate, the external structure of Oral mucosa keratinocyte model terminates;
Described nutrient solution III, in nutrient solution II, adds CaCl
2concentration is 20.0 ~ 100.0 μ g/mL, and vitamin C concentration is 10.0 ~ 50.0 μ g/mL;
Described nutrient solution IV, in nutrient solution II, adds CaCl
2concentration is 50.0 ~ 150.0 μ g/mL, and vitamin C concentration is 50.0 ~ 100.0 μ g/mL;
Described nutrient solution V, in nutrient solution II, adds CaCl
2concentration is 150.0 ~ 300.0 μ g/mL, and vitamin C concentration is 100.0 ~ 200.0 μ g/mL.
4. the Oral mucosa keratinocyte model prepared of method according to claim 3, it is characterized in that: this models applying is in the security of the products such as dental care products, micromolecular compound, activated protein, medicine equipment, chemical, disposable sanitary articles and both effectiveness detection, drug osmotic ability detects, tobacco component carciongenic potency detects, oral cavity pathogen pathogenesis.
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| CN107164309A (en) * | 2017-06-18 | 2017-09-15 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-containing medium external structure |
| CN107312743A (en) * | 2017-06-18 | 2017-11-03 | 广东博溪生物科技有限公司 | A kind of gingival epithelium model and its vitro construction method |
| CN107326003A (en) * | 2017-06-18 | 2017-11-07 | 广东博溪生物科技有限公司 | The 3D models and its construction method of a kind of utilization serum-free medium external structure |
| CN107326003B (en) * | 2017-06-18 | 2020-09-25 | 广东博溪生物科技有限公司 | 3D model constructed in vitro by using serum-free culture solution and construction method thereof |
| CN108342322A (en) * | 2018-02-24 | 2018-07-31 | 宁夏医科大学总医院 | The method for establishing primary people's endometrial epithelial cell liquid phase culture model |
| CN108546673A (en) * | 2018-04-24 | 2018-09-18 | 济南磐升生物技术有限公司 | A kind of serum-free Buccal mucosa cell culture solution and application |
| CN108546673B (en) * | 2018-04-24 | 2021-10-22 | 济南磐升生物技术有限公司 | Serum-free oral mucosa epithelial cell culture solution and application |
| CN109880792A (en) * | 2019-03-14 | 2019-06-14 | 广州薇美姿实业有限公司 | A kind of human oral cavity mucous membrane model and preparation method thereof |
| CN112048477A (en) * | 2020-08-20 | 2020-12-08 | 山东银丰生命科学研究院 | Method for establishing EBV virus infection artificial respiratory epithelium model |
| CN112048477B (en) * | 2020-08-20 | 2023-06-30 | 山东银丰生命科学研究院 | Method for establishing EBV virus infection artificial respiratory tract epithelial model |
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Application publication date: 20160120 |