CN107267441A - A kind of method for setting up the three-dimensional cutaneous model for anti-inflammatory antiallergic efficacy assessments - Google Patents
A kind of method for setting up the three-dimensional cutaneous model for anti-inflammatory antiallergic efficacy assessments Download PDFInfo
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Abstract
A kind of method for building up of three-dimensional cutaneous model of the present invention, comprises the following steps：1) extracellular matrix macromolecule hydrogel solution is prepared；2) fibroblast is mixed with extracellular matrix macromolecule hydrogel solution, is put into culture cell and cultivates；3) keratinocyte is inoculated with, by gas-liquid interface culture, obtains including the three-dimensional cutaneous model of skin corium, epidermis and cuticula.The inventive method can effectively set up three-dimensional cutaneous model, and preparation method is easy, can be used for the screening of skin anti-inflammatory antiallergic medicine, application prospect is excellent.
Description
Technical field
The present invention relates to the method for building up of skin model, in particular it relates to for setting up the new of anti-inflammatory antiallergic efficacy assessments
The method of type three-dimensional cutaneous model.
Background technology
Skin stops first of outpost of the tax office of external substance interference as the vital tissue organ for covering and protecting body surface, holds
It is vulnerable to the infringement of the factors such as wound, burn, inflammation, ulcer, tumor post-operation and congenital disorders, while can be by some special anti-
It should be on the defensive.The incidence of disease of the anaphylaxis dermatosis such as whole world dermatitis, eczema is more than 10%.Resist at present in progress skin anti-inflammatory
The screening of sensitizing drug or cosmetic material is very inconvenient, and cost is also very high, and mice caused by dimethylbenzene xylene ear swelling, egg white cause rat foot
Plantar swelling, swollen hyperplasia of rat granuloma chronic inflammation model, Murine local lymph node priming experiments are conventional animal experimental models.
However, promulgate ban on zoopery in cosmetic product with the cosmetics regulation in Europe, the chemistry of anti-inflammatory antiallergic is allowed
The screening and exploitation of product and cosmetic material are more difficult to realize.Simultaneously as the complexity of sensitization of skin and inflammation mechanism, same
It is difficult to reflect that whole sensitization causes scorching process comprehensively in vitro test system, accordingly, it would be desirable to develop more experiment in vitro for chemicals
Prediction is provided with skin anti-inflammatory antiallergic effect of personal care product and is assessed.
The content of the invention
In order to solve foregoing problems, the technical scheme is that a kind of construction method there is provided three-dimensional cutaneous model and
It is applied.
The method for building up of three-dimensional cutaneous model of the present invention, comprises the following steps：
1) extracellular matrix macromolecule hydrogel solution is prepared；
2) fibroblast is mixed with extracellular matrix macromolecule hydrogel solution, is put into culture cell and cultivates；
3) keratinocyte is inoculated with, by air-liquid interface culture, is obtained comprising skin corium, epidermis and cuticula
Three-dimensional cutaneous model.
Step 1) in, the extracellular matrix macromolecule hydrogel solution is hyaluronic acid, polyethyleneglycol diacrylate
With the mixed solution of type i collagen, wherein, the weight ratio of hyaluronic acid, polyethyleneglycol diacrylate and type i collagen is 4:1:1,
Preferably, their concentration is respectively 6.67mg/ml, 1.67mg/ml, 1.67mg/ml.
Step 2) in, while fibroblast is mixed with extracellular matrix macromolecule hydrogel solution, add 1.25
×105~2 × 105Individual mast cell, mixing；
Preferably, the mast cell is prepared as follows：Cicatricial tissue is taken, subcutaneous fat is removed, is cut into small pieces,
Digestive juice digestion 4h is added, then is sheared to milky suspension, filtrate is collected by filtration；The centrifugal filtrate under the conditions of 4 DEG C, abandons supernatant
Liquid, is resuspended, then centrifuges, and abandons supernatant, and suspension cell is inoculated with the DMEM nutrient solutions containing 10%FBS and cultivated, hypertrophy is produced
Cell；The digestive juice is the mixed solution of NTx enzyme and hyaluronidase, and wherein the concentration of NTx enzyme is 0.3mg/
Ml, the concentration of hyaluronidase is 100U/ml.
Step 2) in, add 5 × 10 per 1ml hydrogel solutions5~8 × 105Individual fibroblast, is put into culture cell,
It is placed in culture plate and cultivates, hydrogel is formed after culture 30min, the time of culture is 5.
Step 3) in, the amount of the keratinocyte of inoculation is：Step 2) in using 12 orifice plate culture when, per hole add 2
×105~4 × 105Keratinocyte.
Step 3) in, the method for air-liquid interface culture is：
It is inoculated with after keratinocyte, is cultivated 5 in the case where DMEM culture mediums flood culture cell, then in DMEM
Culture medium is cultivated 14 in the case of being in culture cell bottom；
Or after inoculation keratinocyte, first cultivate 1, add in the case where DMEM culture mediums flood culture cell
Immunocyte continues to cultivate 4, then in the case where DMEM culture mediums are in culture cell bottom to cultivating around cell
Culture 14 days, that is, prepare；Wherein, the amount of the keratinocyte of inoculation is：Step 2) in using 12 orifice plate culture when, often
Hole adds 2 × 105~5 × 105Immunocyte, the immunocyte is monocyte, BMDC, T lymphocytes or huge
Phagocyte.
The fibroblast is prepared as follows：Dermis of skin layer tissue is taken, is cut into small pieces, using containing 10%
FBS DMEM medium cultures, change a nutrient solution in every 2 days therebetween, after cultivating 6 days, reclaim cell, pass through flow cytometer
To cell screening fibroblast, you can；
The keratinocyte is prepared as follows：Take epiderm skin layer tissue, be cut into small pieces, using containing
10%FBS DMEM medium cultures, change a nutrient solution in every 2 days therebetween, after cultivating 6 days, reclaim cell, thin by streaming
Born of the same parents' instrument is to cell screening keratinocyte, you can；
The monocyte is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes list
Individual nucleus layer, using RPMI-1640 culture mediums, after adherent 2h, removes cell that is not adherent or not pasting jail, what is scraped is adherent
Cell is monocyte；
The granulocyte is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes grain thin
Born of the same parents' layer, washing, using RPMI-1640 medium cultures, is produced；
The BMDC is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes
Mononuclearcell layer, using RPMI-1640 culture mediums, after adherent 2h, removes cell that is not adherent or not pasting jail, collects adherent
Cell, immunomagnetic beads CD14+ monocytes, with containing 10%FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4
RPMI-1640 nutrient solutions be placed in 37 DEG C, 5%CO25-6d is cultivated in incubator, BMDC is obtained；
The T lymphocytes are prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes list
Individual nucleus layer, using RPMI-1640 culture mediums, after adherent 2h, collects not adherent cell, adds the RPMI- containing 10%FBS
1640 culture medium, produces T lymphocytes；
The macrophage is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes list
Individual nucleus layer, using RPMI-1640 medium culture 1-2d, adds final concentration of 125ng/mL phorbol exters (PMA)
RPMI-1640 culture mediums carry out cell differentiation, cultivate 2-3d, produce macrophage.
Present invention also offers the three-dimensional cutaneous model that preceding method is set up.
Present invention also offers purposes of the foregoing three-dimensional cutaneous model in screening skin anti-inflammatory antiallergic medicine.
Present invention also offers a kind of method for screening treatment skin anti-inflammatory antiallergic medicine, comprise the following steps：
A, the three-dimensional cutaneous model set up as the method previously described；
B, candidate is applied to three-dimensional cutaneous model described in a steps；
C, with the potential anti-inflammatory antiallergic medicine of three-dimensional cutaneous model evaluation.
Set up to provide based on sensitization of skin inflammation experiment integration testing system and integrate inspection policies, research and development contain immunocyte
Skin model for anti-inflammatory antiallergic efficacy detection.Set up a standardization using the skin model having been built up, it is safe efficient
External skin-protection health products three-dimensional cutaneous appraisement system, as one of the method for substituting conventional animal experiment, to single chemicals
With Product Safety and the in-vitro evaluation method of both effectiveness detection, verify that it tests stability, repeatability and as replacement
The feasibility of method, provides important references for three-dimensional cutaneous research, is chemicals and the skin antiallergic anti-inflammatory of personal care product
Effect provides prediction and assessed, while providing the foundation with reference significance in terms of zoopery method is substituted for China.
Three-dimensional cutaneous (3D-Skin) is a kind of more perfect tissue engineering skin, is by dermal fibroblast, table
The artificial skin that skin keratinocyte is mixed with extracellular matrix substitute.Tissue engineering skin is removed in Clinical practice
In addition, because its stability is high, the development easy to operate for also promoting it in non-clinical study work.Ground in skin biology
Study carefully field, skin senescence, light protection, test etc. field instead of animal testing, the three-dimensional cutaneous of external structure is respectively provided with very high
Application value.Three-dimensional cutaneous model can meet different intercellular interactions, can be between in vitro and in vivo
One important bridge is provided.
The inventive method can effectively set up three-dimensional cutaneous model, and preparation method is easy, can be used for skin anti-inflammatory
The screening of antiallergic medicine, application prospect is excellent.
According to the above of the present invention, according to the ordinary technical knowledge and customary means of relevant art, not
Depart under the premise of above-mentioned basic fundamental thought of the invention, the modification of other diversified forms can also be made, replace or change.
Brief description of the drawings
Figure 1A, C undergrowth fibroblast；The well-grown fibroblast of B, D
Fig. 2A, C undergrowth keratinocyte；The well-grown keratinocyte of B, D
The independent HyStemTM of Fig. 3 A (the hyaluronic acid HA of sulfydryl modification)；B independent ExtralinkTM (polyethylene glycol dipropyls
Olefin(e) acid ester, PEGDA)；The independent type i collagens of C；D hyaluronic acid-collagen hydrogels of the present invention
Fig. 4 three-dimensional cutaneous model and HE dyeing
Fig. 5 three-dimensional cutaneous modeling figures
The structure schematic diagram of the full cortex skin models of Fig. 6
Fig. 7 derived from peripheral blood immunocyte cultures
Fig. 8 contains the three-dimensional cutaneous mould of primary peripheral blood source immunocyte (monocyte, lymphocyte, granulocyte etc.)
Type culture
Fig. 9 contains mast cell three-dimensional cutaneous model such as
Figure 10 three-dimensional cutaneous containing mast cell model
Figure 11 inflammatory factors Testing index (solid phase chip)
Figure 12 HE dyeing, scanning electron microscopic observation
Cell survival rate after Figure 13 is handled through tested material
Embodiment
The preparation of the three-dimensional cutaneous model of the present invention of embodiment 1
Skin holostrome is included into immunocyte (lymphocyte, BMDC, granulocyte, monocyte, macrophage in vitro
Cell etc.) all types cell and biologic bracket material is compound constructs with skin similar to regular skin structure form
Method.
The construction method of 1 three-dimensional cutaneous model of the present invention
The source of 1.1 various cells and preparation
1) acquisition of fibroblast and keratinocyte：
The skin histology of normal person is taken, tissue is carried out disinfection, corium layer tissue and epidermis layer tissue fritter is isolated
(3mm×3mm).Tissue fritter is put in sterile petri dish, addition culture medium (the DMEM culture mediums containing 10%FBS).Group
Knit fritter adherent at the veneer of culture dish, a part of cell can be attached to culture dish superficial growth, change within every 2 days once train therebetween
Nutrient solution, and by its growing state of micro- sem observation, after the cultures of 6 days, cell is reclaimed, by flow cytometer to cell
Screened, obtain fibroblast and keratinocyte.Preceding method kind, the condition of cell culture is 37 DEG C, 5%CO2
Cultivated in incubator
The growth figure of acquisition fibroblast and keratinocyte is as shown in Figure 1B, D, Fig. 2 B, D.
2) acquisition of primary peripheral haemocyte：
In aseptic collection venous blood 6ml injection anticoagulant heparin pipes, gently shake up, add at room temperature isometric PBS or
Hank, s liquid, gently piping and druming are mixed.6ml lymphocytes separating solutions are placed in centrifuge tube, then by the blood sample after dilution in separation
Ullage, 18-20 DEG C, 2000rpm centrifuges 30min, divides four layers after centrifugation from ttom of pipe to liquid level, is followed successively by red blood cell and grain
Cellular layer, layering liquid layer, mononuclearcell layer, plasma layer.
Wherein mononuclearcell cultural method is：The flaxen diluting plasma in upper strata is removed, gently suctioned out milky
Mononuclearcell layer, is put into new centrifuge tube, adds PBS and washes twice, plus RPMI-1640 culture mediums (containing 10%FBS) training
Support mononuclearcell.
Wherein monocyte cultural method is：Above-mentioned mononuclearcell is moved into culture in blake bottle, and (RPMI-1640 is cultivated
Base), after adherent 2h, not adherent cell is sucked, the cell for not pasting jail is washed down, attached cell is scraped for monocyte, plus RPMI-
1640 culture mediums (containing 10%FBS) culture monocyte.
Wherein granulocyte cultural method is：The flaxen diluting plasma in upper strata is removed successively, milky single core is thin
Born of the same parents' layer is removed, and separating layer is removed, and gentle aspiration goes out granulocyte, is put into new centrifuge tube, is added PBS and is washed twice, plus RPMI-
1640 culture mediums (containing 10%FBS) culture granulocyte.
Wherein BMDC cultural method is：Above-mentioned mononuclearcell is moved into blake bottle (RPMI-1640 culture mediums)
After culture, adherent 2h, not adherent cell is sucked, adherent monocyte is collected, immunomagnetic beads CD14+ monokaryons are thin
Born of the same parents, be placed in the RPMI-1640 nutrient solutions containing 10%FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4 37 DEG C, 5%
CO2Cultivated in incubator, cell can be divided into BMDC after 5-6d.
Wherein T separation of lymphocytes method is：Above-mentioned mononuclearcell is moved into blake bottle and cultivated, after adherent 2h, is collected
Not adherent cell, adds the RPMI-1640 nutrient solutions containing 10%FBS, produces T lymphocytes.
The cultural method of wherein macrophage is：Above-mentioned monocyte, which is moved into, cultivates 1-2d in blake bottle, add final concentration
RPMI-1640 culture mediums for 125ng/mL phorbol exters (PMA) carry out cell differentiation, cultivate 2-3d, that is, are divided into macrophage thin
Born of the same parents.
Derived from peripheral blood immunocyte culture is as shown in Figure 7.
3) acquisition of primary mast cell
Perform the operation people's cicatricial tissue for cutting, remove subcutaneous fat, with the clear skin graft of Tyrode liquid and be cut into 1mm2Size
Fritter, adds digestive juice (0.3mg/ml NTx enzyme and 100U/ml hyaluronidase) digestion 4h, then shear to milky white
Color suspension, is collected by filtration filtrate.The centrifugal filtrate under the conditions of 4 DEG C, abandons supernatant, and suspended precipitation again with cold Tyrode liquid,
Centrifuge again, abandon supernatant, suspension cell is inoculated with into the DMEM nutrient solutions containing 10%FBS, mast cell is produced.
1.2 modeling method
1) extracellular matrix macromolecule hydrogel solution is prepared：
Hyaluronic acid-collagen hydrogels：Shape solid HyStemTM (sulfydryls are freezed in HyStem kits (Sigma)
The hyaluronic acid HA of modification) and ExtralinkTM (polyethyleneglycol diacrylate, PEGDA) respectively with after deionized water dissolving
10mg/mL storing liquids are obtained, type i collagen (Sigma) is formulated as 10mg/ml, and pH value is 7.2 storage liquid.HA, PEGDA and I type
Collagen (4:1:1) after being well mixed, hydrogel solution is produced.
Hydrogel solution solidifies, you can obtain hydrogel three-dimensional support, its schematic diagram is as shown in Figure 3 D.
2) three-dimensional cutaneous model is built
A, the first：Full cortex skin model
Build schematic diagram as shown in Figure 6：
Fibroblast is inoculated with hydrogel solution, 5 × 10 are added per 1ml hydrogel solutions5~8 × 105It is individual thin
Born of the same parents, are gently put into culture cell (transwell) and move into 12 well culture plates, be placed in incubator and cultivated, after 30 minutes
Hydrogel solution solidify to form hydrogel, as containing fibroblastic support, and addition DMEM culture mediums flood culture cell,
Culture builds corium, after cultivating 5, and being inoculated with keratinocyte in its rack surface (adds 2 × 10 per hole5~4 × 105), lead to
Cross air-liquid interface culture formation epidermis：Specifically first culture 5 days in the case where DMEM culture mediums flood culture cell, then
Cultivate 14, that is, prepare in the case where being in culture cell bottom in DMEM culture mediums.
Skin model is carried out after FFPE processing, HE dyeing is carried out, morphology contrast is carried out with normal human skin, sentences
Breaking, it cultivates situation.Three-dimensional cutaneous model and HE dyeing are as shown in Figure 4；Three-dimensional cutaneous modeling figure is as shown in Figure 5.
B, second：The three-dimensional cutaneous model of the immunocyte containing derived from peripheral blood
Build schematic diagram as shown in Figure 8：
Fibroblast is inoculated with hydrogel solution, 5 × 10 are added per 1ml hydrogel solutions5~8 × 105It is individual thin
Born of the same parents, are gently put into culture cell (transwell) and move into 12 well culture plates, be placed in incubator and cultivated, after 30 minutes
Hydrogel solution solidify to form hydrogel, as containing fibroblastic support, and addition DMEM culture mediums flood culture cell,
Culture builds corium, after cultivating 5, and being inoculated with keratinocyte in its rack surface (adds 2 × 10 per hole5~4 × 105It is individual thin
Born of the same parents), epidermis is formed by air-liquid interface culture：Specifically first cultivate 1 in the case where DMEM culture mediums flood culture cell
Day, immunocyte (monocyte, BMDC, T lymphocytes or macrophage) is added to cultivating around cell, often
Hole adds 2 × 105~5 × 105Individual cell continues to cultivate 4, then in the case where DMEM culture mediums are in culture cell bottom
Culture 14 days, that is, prepare.
C, the third：Three-dimensional cutaneous model containing mast cell
Build schematic diagram as shown in Figure 9：
Fibroblast, mast cell are inoculated with hydrogel solution, 5 × 10 are added per 1ml hydrogel solutions5~8
×105Individual fibroblast and 1.25 × 105~2 × 105Individual mast cell, is gently put into culture cell (transwell) and moves
Enter in 12 well culture plates, be placed in incubator and cultivated, hydrogel solution solidify to form hydrogel after 30 minutes, as containing into
The support of fibrocyte, addition DMEM culture mediums flood culture cell, and culture builds corium, after cultivating 5, in its rack surface
It is inoculated with keratinocyte and (2 × 10 is added per hole5~4 × 105), epidermis is formed by air-liquid interface culture：Specifically first exist
DMEM culture mediums are cultivated 5 in the case of flooding culture cell, and the situation of culture cell bottom is then in DMEM culture mediums
Lower culture 14 days, that is, prepare.
2 model inspections
1) HE dyeing and the constructed skin texture of organization engineering skin observation constructed by detection method and various immune
The formational situation of cell；
A) the three-dimensional cutaneous mould containing primary peripheral blood source immunocyte (monocyte, lymphocyte, granulocyte etc.)
Type
Culture 24-72h separates skin model with immune small cell, and is done skin model surface washing with physiological saline
It is net and be ready for HE dyeing；Immunocyte after separation is used for the expression for detecting cell surface marker；Culture supernatant is used for
Inflammatory factor is detected.
HE is dyed：Take the sample routine paraffin wax of neutral formalin fixation to embed, serial section done on paraffin slicing machine, mount in
On the slide handled through APES, section is placed in baking in 60 DEG C of constant temperature roasters, sealing, om observation after HE dyeing.
The parameter of Flow cytometry derived from peripheral blood immunocyte (monocyte, lymphocyte, granulocyte etc.)：From
Cell is taken out in Tissue Culture Plate, is moved into respectively in corresponding EP pipes per hole, is collected using centrifuge (250g, 5min, 4 DEG C)
Cell, is cleaned once with FACS buffer solution (PBS+0.1% BSA), and cell is resuspended.Add containing monoclonal antibody dilution
Upper machine testing after supernatant, plus the dilution mixing of PBS liquid is removed in liquid, room temperature lucifuge reaction 20m in, centrifugation, using FACS-Calibur
(U.S. company BD product), argon laser, power is 15mw, excitation wavelength 488nm.
Testing result：
Neutrophil leucocyte：Gr-1, cD11b
Mature T cells (CD3+), T helper-inducers cell (CD3+/CD4+),
Monocyte CD 14+
Granulocyte CD 15+
BMDC：CD11c+, MHCII+
Macrophage：CD 14+, CD11b+
B) the three-dimensional cutaneous model containing mast cell
HE is dyed：Take the sample routine paraffin wax of neutral formalin fixation to embed, serial section done on paraffin slicing machine, mount in
On the slide handled through APES, section is placed in baking in 60 DEG C of constant temperature roasters, sealing, om observation after HE dyeing.
Mast cell (toluidine blue) is dyed：Routinely dewaxing is to water for paraffin section, slightly water after 30min in toluidine blue dye liquor
Wash, break up in glacial acetic acid liquid, distillation washing after-blow is done, and dimethylbenzene is transparent, light Microscopic observation after neutral gum sealing.
Mast cell three-dimensional cutaneous model such as Figure 10.
2) with known stimulant detection model, the change of detection biomarker expression.
Stimulant control group：Lipopolysaccharides (LPS), 2,4- nonadienes
Protection control group：Minoxidil
Negative control thing：Distilled water
By (second of the skin model of preincubate：The three-dimensional cutaneous model of the immunocyte containing derived from peripheral blood) it is placed in inoculation
It is placed in 12 porocyte culture plates；Sample tested material is applied to skin model surface, it is rear in incubator to be incubated 24h, every group
Sample carries out 3 groups of parallel tests；Skin model surface tested material is rinsed well with physiological saline after 24h and MTT is ready for
Test, scanning electron microscopic observation, HE dyeing, the change of FCM analysis index of correlation；Supernatant uses solid phase chip Proteome
Profiler Human Cytokine Array Kit (BD) detect the expression of relevant inflammatory factors.Inflammatory factor Testing index
As shown in figure 11.
As a result as shown in Figure 12~13：
By monitoring Cell viability, metamorphosis, Cytokine profile, cell surface marker after exposure tested material
Thing expression and relative intensity of fluorescence, judge chemicals or skin anti-inflammatory antiallergic effect of personal care product.
2, the 4- nonadienals or 2ug/ml LPS that 500uM is added on three-dimensional cutaneous model are cultivated 6 hours, then removed
Nutrient solution, adds nutrient solution 48h again, and section is fabricated to afterwards and carries out observation shooting.Contrast is cut without processing control group in vertical profile
Observed on piece more due to skin cavity caused by allergy and inflammation；Observe that skin surface is formed in cross-sectional section
More scales of skin that peel off shape skin damaged.The entirety for having damaged skin for the three-dimensional cutaneous that LPS and 2,4- nonadienal induce, to being damaged three
The minoxidil that skin model adds 0.1mM is tieed up, upside is cultivated 6 hours, skin cavity is had with surface scales of skin that peel off skin damaged
The reparation of effect.
Using preceding method to the first foregoing full cortex skin model and the third contain the three-dimensional cutaneous of mast cell
Model is detected that LPS and 2,4- nonadienal can equally induce the damage of three-dimensional cutaneous, and impaired three-dimensional cutaneous model is added
Plus 0.1mM minoxidil, upside is cultivated 6 hours, skin cavity has also obtained effective reparation with surface scales of skin that peel off skin damaged.
Experimental result illustrates that three-dimensional cutaneous model of the present invention is successfully established, the screening available for skin anti-inflammatory antiallergic medicine.
Should be it is demonstrated experimentally that the present invention establishes three-dimensional cutaneous model, available for screening skin anti-inflammatory antiallergic medicine, before
Scape is excellent.
Claims (10)
1. a kind of method for building up of three-dimensional cutaneous model, it is characterised in that：Comprise the following steps：
1) extracellular matrix macromolecule hydrogel solution is prepared；
2) fibroblast is mixed with extracellular matrix macromolecule hydrogel solution, is put into culture cell and cultivates；
3) keratinocyte is inoculated with, by air-liquid interface culture, obtains including the three-dimensional of skin corium, epidermis and cuticula
Skin model.
2. according to the method described in claim 1, it is characterised in that：Step 1) in, the extracellular matrix macromolecule hydrogel
Solution is the mixed solution of hyaluronic acid, polyethyleneglycol diacrylate and type i collagen, wherein, hyaluronic acid, polyethylene glycol two
The weight ratio of acrylate and type i collagen is 4:1:1, it is preferable that their concentration be respectively 6.67mg/ml, 1.67mg/ml,
1.67mg/ml。
3. according to the method described in claim 1, it is characterised in that：Step 2) in, fibroblast and extracellular matrix is high
While molecule hydrogel solution is mixed, 1.25 × 10 are added5~2 × 105Individual mast cell, mixing；
Preferably, the mast cell is prepared as follows：Cicatricial tissue is taken, subcutaneous fat is removed, is cut into small pieces, is added
Digestive juice digests 4h, then shears to milky suspension, and filtrate is collected by filtration；The centrifugal filtrate under the conditions of 4 DEG C, abandons supernatant, weight
It is outstanding, then centrifuge, supernatant is abandoned, suspension cell is inoculated with the DMEM nutrient solutions containing 10%FBS and cultivated, mast cell is produced；
The digestive juice is the mixed solution of NTx enzyme and hyaluronidase, and wherein the concentration of NTx enzyme is 0.3mg/ml, thoroughly
The concentration of the sour enzyme of bright matter is 100U/ml.
4. the method according to claim 1 or 3, it is characterised in that：Step 2) in, add 5 × 10 per 1ml hydrogel solutions5
~8 × 105Individual fibroblast, is put into culture cell, is placed in culture plate and cultivates, and forms hydrogel after culture 30min, trains
The foster time is 5.
5. according to the method described in claim 1, it is characterised in that：Step 3) in, the amount of the keratinocyte of inoculation is：Step
During 12 orifice plate culture of rapid 2) middle use, 2 × 10 are added per hole5~4 × 105Keratinocyte.
6. according to the method described in claim 1, it is characterised in that：Step 3) in, the method for air-liquid interface culture is：
It is inoculated with after keratinocyte, cultivates 5, then cultivated in DMEM in the case where DMEM culture mediums flood culture cell
Base is cultivated 14 in the case of being in culture cell bottom；
Or after inoculation keratinocyte, first cultivate 1, add immune in the case where DMEM culture mediums flood culture cell
Cell continues to cultivate 4, then cultivated in the case where DMEM culture mediums are in culture cell bottom to cultivating around cell
14, that is, prepare；Wherein, the amount of the keratinocyte of inoculation is：Step 2) in using 12 orifice plate culture when, add per hole
Enter 2 × 105~5 × 105Immunocyte, the immunocyte is that monocyte, BMDC, T lymphocytes or macrophage are thin
Born of the same parents.
7. the method according to claim 1 or 6, it is characterised in that：
The fibroblast is prepared as follows：Dermis of skin layer tissue is taken, is cut into small pieces, using containing 10%FBS
DMEM medium cultures, therebetween every 2 days change a nutrient solution, culture 6 days after, reclaim cell, pass through flow cytometer pair
Cell screening fibroblast, you can；
The keratinocyte is prepared as follows：Epiderm skin layer tissue is taken, is cut into small pieces, using containing 10%
FBS DMEM medium cultures, change a nutrient solution in every 2 days therebetween, after cultivating 6 days, reclaim cell, pass through flow cytometer
To cell screening keratinocyte, you can；
The monocyte is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes single core
Cellular layer, using RPMI-1640 culture mediums, after adherent 2h, removes cell that is not adherent or not pasting jail, the attached cell scraped
For monocyte；
The granulocyte is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes granulocyte layer,
Washing, using RPMI-1640 medium cultures, is produced；
The BMDC is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes single
Nucleus layer, using RPMI-1640 culture mediums, after adherent 2h, removes cell that is not adherent or not pasting jail, collects adherent thin
Born of the same parents, immunomagnetic beads CD14+ monocytes, with containing 10%FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4's
RPMI-1640 nutrient solutions are placed in 37 DEG C, 5%CO25-6d is cultivated in incubator, BMDC is obtained；
The T lymphocytes are prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes single core
Cellular layer, using RPMI-1640 culture mediums, after adherent 2h, collects not adherent cell, adds the RPMI-1640 containing 10%FBS
Nutrient solution, produces T lymphocytes；
The macrophage is prepared as follows：Blood is taken, is handled using lymphocytes separating solution, centrifuges, takes single core
Cellular layer, using RPMI-1640 medium culture 1-2d, adds the RPMI- of final concentration of 125ng/mL phorbol exters (PMA)
1640 culture mediums carry out cell differentiation, cultivate 2-3d, produce macrophage.
8. the three-dimensional cutaneous model that claim 1~7 any one methods described is set up.
9. purposes of the three-dimensional cutaneous model described in claim 8 in screening skin anti-inflammatory antiallergic medicine.
10. a kind of method for screening treatment skin anti-inflammatory antiallergic medicine, it is characterised in that：Comprise the following steps：
A, the three-dimensional cutaneous model set up according to the method described in claim 1~7 any one；
B, candidate is applied to three-dimensional cutaneous model described in a steps；
C, with the potential anti-inflammatory antiallergic medicine of three-dimensional cutaneous model evaluation.
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