CN103857802B - Skin-whitening screening substances system - Google Patents
Skin-whitening screening substances system Download PDFInfo
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- CN103857802B CN103857802B CN201280051510.0A CN201280051510A CN103857802B CN 103857802 B CN103857802 B CN 103857802B CN 201280051510 A CN201280051510 A CN 201280051510A CN 103857802 B CN103857802 B CN 103857802B
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- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
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- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
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Abstract
The invention discloses a kind of skin-whitening screening substances system including the layer containing one or more of fibroblast, keratinocyte and melanocyte cell, wherein said layer is than or equal to one layer, and the skin-whitening process for screening substances using described system.In addition, the invention discloses a kind of containing the material being filtered out by skin-whitening process for screening substances as effective ingredient skin-whitening compositionss.
Description
Technical field
The present invention relates to a kind of skin-whitening screening substances system and skin-whitening process for screening substances.
Background technology
So far, when developing new skin-whitening material, using following methods, i.e. by candidate substances to melanin
Cellulation is melanocyte（melanocyte）After directly being processed, it is confirmed whether to suppress melanin to generate, or to black
Plain cell irradiation ultraviolet, confirms whether candidate substances shield the melanin now producing and generate signal.But, actually exist
In skin, melanocyte is not individualism, but is directly or indirectly subject to other periphery cells or microhabitat
Physically or chemically affect and generate melanin, the shape of above-mentioned actual skin tissue if only melanocyte, cannot be reproduced
State.
In addition, the generation of these cutaneous pigmentation diseases of freckle, speckle, melanotic nevus will because or mechanism lead to due to ultraviolet
Melanin generates to be increased different although there is the trend that this symptom can deteriorate because ultraviolet irradiates, even if there is not purple
The stimulation of the generations such as outside line, melanic generation and dispersion also may proceed to increase.Thus, based on existing ultraviolet irradiation side
In the case of the skin-whitening material that method is developed, in actual clinical, the skin pigment that mostly cannot show suppression people sinks
The effect of disease.If additionally, in order to study cutaneous pigmentation disease and the skin at pigmentation position to detected person
Carry out biopsy and use, then the energy of expense and cost is very big, and hardly result in the skin histology of people, in addition, with
Ultraviolet irradiates sample difference, and the animal specimen obtaining cutaneous pigmentation disease is also highly difficult.
Accordingly, there exist the skin group not only constructing, being especially in the presence of the people of cutaneous pigmentation disease with the skin histology of people
The construction knitted is very approximate and can simply screen the system of skin-whitening material and the demand of screening technique.
Patent documentation 1：Ebrean Registered Patent the 10-0840144th（On June 16th, 2008 registers）Description.
Content of the invention
One aspect of the present invention is, provides and can efficiently and simply screen clinically useful skin-whitening material
Skin-whitening screening substances system and using this system skin-whitening process for screening substances.
Another aspect of the present invention is, provides containing the material being screened by described skin-whitening process for screening substances
As effective ingredient and illustrate the skin-whitening compositionss of excellent whitening effect.
In addition, another aspect of the present invention is, provides and generate related gene itself work containing to the melanin of skin
Skin-whitening compositionss for effective ingredient or external preparation for skin medicament composition.
One scheme of the present invention is, provides including containing fibroblast（fibroblast）, keratinocyte
（keratinocyte）And melanocyte（melanocyte）One or more of the layer of cell skin-whitening screening substances system
System, wherein said layer is than or equal to one layer.
Another scheme of the present invention is, provides a kind of skin-whitening process for screening substances, and it is entering to candidate substances
After row is processed, also include validating that in one or more of fibroblast, keratinocyte and melanocyte cell
The degree of the expression of one or more of WIF1, WNT1, WNT5a and NFATC2 or the step of phosphorylation degree.
Another scheme of the present invention is, provides containing the thing being screened by described skin-whitening process for screening substances
Matter is as the skin-whitening compositionss of effective ingredient, or combines as the skin-whitening of effective ingredient containing WIF1 itself
Thing and external preparation for skin medicament composition.
The effect of invention
Skin-whitening screening substances system involved in the present invention due to be designed as close to reality skin histology, be therefore
Make in vitro（in vitro）It is also possible to simply screen with less time and expense, clinically there is whitening effect under state
The material of fruit.Especially, screen whitening material by irradiation ultraviolet radiation with after melanocyte is processed with candidate substances
Existing method different, skin-whitening screening substances system involved in the present invention has and the actual skin illustrating pigmentation disease
The similar condition of the phosphorylation degree of the degree of gene expression of skin tissue and messenger substance, therefore, is utilizing the present invention
In the case of involved system, can simply filter out skin-whitening material, particularly be effectively improved pigmentation disease
Material.For hardly resulting in for the people of pigmentation disease research and animal specimen this point, system involved in the present invention
Can become for developing new skin-whitening material, specifically exploitation pigmentation disease improves system or the instrument of material.
Brief description
Fig. 1 is the normal skin to detected person（N）With cutaneous pigmentation position（L）Between WIF1 gene expression
The figure that the difference of degree is compared.
Fig. 2 is the normal skin to detected person（N）With cutaneous pigmentation position（L）Between WNT1 and WNT5a
The figure that the difference of expression degree is compared.
Fig. 3 is to melanocyte（MC）, keratinocyte（KC）And fibroblast（FB）In WIF1 gene table
Reach the figure that the difference of degree is compared.
Fig. 4 represents in the fibroblastic whitening screening system containing suppression WIF1 expression, tryrosinase
（tyrosinase）Expression increase situation.
Fig. 5 represents in the fibroblastic whitening screening system containing suppression WIF1 expression, the movement of melanosome
Increased situation.
Fig. 6 represents in the fibroblastic whitening screening system containing suppression WIF1 expression, NFATC2 dephosphorylation
Situation.
Fig. 7 is to represent in the whitening screening system of the keratinocyte containing suppression WIF1 expression, tryrosinase
The experimental result of the situation that expression increases.
Fig. 8 represents in the whitening screening system of the keratinocyte containing suppression WIF1 expression, the shifting of melanosome
Dynamic increased situation.
Fig. 9 represents in the whitening screening system of the keratinocyte containing suppression WIF1 expression, NFATC2 dephosphorylation
Situation about changing.
After Figure 10 represents the people WIF1 being manufactured by gene recombination technology is processed in melanocyte, melanocyte
WIF1 expression increase situation.
Figure 11 represents in the melanocyte inducing WIF1 expression as shown in Figure 10, the feelings of the expression minimizing of tryrosinase
Condition.
Figure 12 is represented in the people WIF1 that will be manufactured by gene recombination technology containing melanocytic whitening screening system
In processed after, the situation of the mobile minimizing of melanosome.
Figure 13 represents that, in the melanocyte inducing WIF1 expression as shown in Figure 10, NFATC2 occurs the feelings of phosphorylation
Condition.
Specific embodiment
In this manual, " skin " refers to cover the tissue of animal body surface, refers not only to cover the bodies such as face or body
The tissue of table, is the broadest concept also including scalp and hair.
The melanocyte of skin（melanocyte）It is to manufacture the melanin determining skin color（melanin）Cell,
It is located on the basement membrane working as the corium of skin and the boundary line of epidermis.Knowable to fetology angle, melanocyte is
Differentiation as described below, i.e. from neural crest cell（neural-crest cells）Origin, via melanocyte stem cell
（Melanocyte stem cell）, as precursor melanoblast（melanoblast）And move to skin, and
Being divided into eventually and can generating melanic pigment cell is melanocyte.
Melanocyte not individualism in skin after breaking up as mentioned above, and the angle being affected by and being positioned above
Matter forms fibroblastic impact present in the combination that cell is keratinocyte and underlying corium and gives birth to
Become melanin.The melanin being manufactured by melanocyte, via cells Dendritic（dendrite）And peritropous keratinocyte
Mobile, being widely dispersed in skin surface, thus protecting skin not affected by harmful factors such as ultraviolet, assuming skin face
Color.Normal skin, when there is this stimulation of ultraviolet, generates more melanin compared with normal condition, and increases melanin
Dispersion, then, if this stimulation disappear, melanic generate and dispersion again recover reduced levels.
Whitening material as noted above, being developed by existing method, is wherein based particularly on ultraviolet irradiation method
The skin-whitening material developed, due to not reflecting actual skin condition, that is, melanocyte is subject to other thin in skin
The combined influence of the physical chemical factor of the microhabitat of born of the same parents or surrounding and generate this actual skin condition of melanin, so
Improve this one side of pigmentation disease of skin, mostly show clinically poor effect.Due to cutaneous pigmentation disease
There are multiple producing cause and skin melanism is made due to special mechanism, so, skin can be effectively improved in order to develop
The whitening material of pigmentation disease, needs to make the experimental model similar with actual skin, and utilizes this model development whitening thing
Matter.
Below, describe the present invention in detail.
One scheme of the present invention is, provides including containing fibroblast（fibroblast）, keratinocyte
（keratinocyte）And melanocyte（melanocyte）One or more of the layer of cell skin-whitening screening substances system
System, described layer is than or equal to one layer.Another scheme of the present invention is, provides one kind to include containing fibroblastic one-tenth
Fibrocyte layer, the keratinocyte layer containing keratinocyte and the skin containing melanocytic melanocyte layer
Whitening screening substances system.Now, preferred to similar with actual skin condition and fibroblast cell layer is layered in orlop,
It is laminated melanocyte layer above it, be laminated keratinocyte layer above melanocyte layer.Another scheme of the present invention
Involved skin-whitening screening substances system, including containing fibroblastic fibroblast cell layer and containing cutin shape
Become the layer of one or more of cell and melanocyte cell, keratinocyte can be contained within the same layer and melanocyte is thin
Born of the same parents.At this point it is possible to fibroblast cell layer is layered in lower floor, it is laminated above it containing melanocyte and keratinocyte
Layer.Described skin-whitening screening substances system is the system of the 3 D stereo construction being laminated with skin cell layers, not only has black
Plain cell, also has the cell of periphery, thus, it is possible to correct reflect actual skin condition.Another scheme institute of the present invention
The skin-whitening screening substances system being related to can also remove fibroblast cell layer, and includes generating tool containing known to melanin
The melanocyte playing an important role and keratinocyte are in interior layer.
The fibroblast cell layer of the skin-whitening screening substances system involved by one scheme of the present invention, is for reproducing
The layer of skin corium, including in collagen protein contain fibroblastic layer.Specifically, described fibroblast cell layer can be
Fibroblast, the layer being condensed and being manufactured into is put in collagen liquid.In another scheme of the present invention, described
Collagen protein can account for 0.001 weight %～30 weight % in the overall weight of skin-whitening screening substances system, preferably
0.01 weight %～20 weight %, more preferably 0.1 weight %～10 weight %.The feelings containing collagen protein in described scope
Under condition, it is suitable to show the effect desired by the present invention, for expense/effect is than this one side, use in described scope
Relatively suitably.
In skin-whitening screening substances system involved by a scheme of the present invention, fibroblast, cutin are formed
The cell of one or more of cell and melanocyte include from the birds such as people, Mus, guinea pig, chicken and pig select one kind with
On animal in cell.
The skin-whitening screening substances system involved by one scheme of the present invention is it is also possible to not only select there is skin U.S.
The material of white effect, specifically there is improvement the material of the effect of skin melanism is led to due to ultraviolet, can also be effectively
Screening especially has the material of improvement to the cutaneous pigmentation disease such as freckle, speckle, melanotic nevus.
Present inventors find, in skin pigmentation disease, adjust related cell to embryonic development
External signal transmission factor is WIF1（Wnt inhibitory factor 1, ACCESSION NP_009122, VERSION
NP_009122.2 GI:111125011）The expression of gene reduces, and specifically, the expression of WIF1 gene, in Skin Cell
In, not reduce in the melanocyte as melanin cellulation, but in keratinocyte and fibroblast
Reduce, thus melanocytic melanin is generated and have an impact.In addition, in the skin that reduces of expression of WIF1 gene, and black
While pigment generates the expression increase of related tryrosinase, melanin increases to the diffusion of keratinocyte, therefore, really
Accept and there is substantial connection between WIF1 and cutaneous pigmentation disease.Now, GSK-3 β is interfered in the expression confirming WIF1 gene
（Glycogen synthasekinase-3β）And beta-catenin（β-catenin）Phosphorylation and MITF
（microphthalmia-associated transcription factor）Expression, cutaneous pigmentation is had an impact.
Therefore, a scheme of the present invention provides in suppression fibroblast, keratinocyte and melanocyte
Plant the skin-whitening screening substances system of the WIF1 expression of above cell, specifically, provide suppression keratinocyte or one-tenth
The skin-whitening screening substances system of fibrocellular WIF1 expression.In another scheme of the present invention, described skin-whitening
Screening substances system contains using siRNA to suppress the cell of WIF1 expression.Described screening system reflection keratinocyte or
The skin condition of the pigmentation disease that WIF1 expression reduces in fibroblast, can be substantially similarly existing again with practical situation
The skin condition of pigmentation disease.Thus, in the case of using described screening system, can effectively select beautiful with skin
The material of contour painting energy, especially can select the material with the function of improving cutaneous pigmentation disease.
In addition, present inventors confirm in skin pigmentation disease, proto-oncogene（proto-
oncogene）Precursor WNT1（Wingless-type MMTV integration site family, member1,
ACCESSION NP_005421, VERSION NP_005421.1 GI:4885655）And protein WNT5a（Wingless-
Type MMTV integration site family, member 5a, ACCESSION NP_001243034,
VERSION NP_001243034.1 GI:371502087）The expression of gene increases, as the NFATC2 of messenger substance
（nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 2）De-
Phosphorylation, therefore, scheme of the present invention provides in fibroblast, keratinocyte and melanocyte more than one
The skin-whitening screening substances of the cell that cell has been the cell that increased of expression or the NFATC2 dephosphorylation of WNT1 and WNT5a
System.Described screening system due to closely can practically reproducing the skin condition that pigmentation disease occurs it is possible to
Effectively select the material with skin-whitening function, especially can select the thing with the function of improving cutaneous pigmentation disease
Matter.
Before this, by by so-called candidate substances such as natural materials, its extract or chemical synthesis materials only to melanocyte
Cell is processed, thus evaluating whether there is whitening effect.Based in the case of above-mentioned existing method it is necessary to will be diluted in thin
Candidate substances in born of the same parents' culture fluid are directly processed to cell, in addition, candidate substances are being made frost or this reality of emulsion
It is impossible to process to melanocyte in the state of the dosage form using.Thus, in existing method, to implement clinical trial
Just can confirm that the actual whitening effect whether having in the dosage form containing candidate's whitening material clinically afterwards.On the other hand, this
Bright involved skin-whitening screening substances system is it is of course possible to, as existing method, candidate substances are diluted in culture fluid
Using and evaluate it and have or not whitening effect, it is possible to use the dosage form such as the frost containing candidate substances or emulsion is evaluated it and is had or not whitening
Effect.Now, for dosage form evaluation is had or not with the skin-whitening screening substances system of whitening effect, can be will to contain into fiber
After multiple layers of one or more of cell, melanocyte and keratinocyte are laminated, expose and manufacture in atmosphere
Become artificial skin.As described above, skin-whitening screening system involved in the present invention, similar with actual skin due to having
Physiology, chemical feature, its whitening effect can also be evaluated using actually used dosage form, therefore, it is possible to more
In short time, the material in actual clinical with whitening effect efficiently and is simply selected with less expense.
One scheme of the present invention provides a kind of skin-whitening process for screening substances, and it is being processed to candidate substances
Described skin-whitening screening substances system in, include validating that in fibroblast, keratinocyte and melanocyte
The degree of the expression of one or more of WIF1, WNT1, WNT5a and NFATC2 in more than one cells or phosphorylation degree
Step.As previously described, WIF1, WNT1, WNT5a and NFATC2 are expressed with the melanin of skin and the degree of diffusion is relevant.
Specifically, the expression of WIF1 reduces, the expression of WNT1 or WNT5a increases or the dephosphorylation degree of NFATC2 increases, meaning
Taste the expression that the enzyme relevant with melanin generation is tryrosinase to be increased, and melanin increases to the diffusion of keratinocyte.
Thus, by confirming degree or the phosphoric acid of the expression to one or more of WIF1, WNT1, WNT5a and NFATC2 for the candidate substances
Which kind of impact is the degree changed produce, and can evaluate whether described candidate substances have skin whitening effects.
The skin-whitening process for screening substances involved by one scheme of the present invention is it is also possible in the journey confirming WIF1 expression
After the step of degree, also contain by so that WIF1 expression degree increase the material step that is judged as skin-whitening material.This
Invention the skin-whitening process for screening substances involved by another scheme it is also possible to confirm WNT1 or WNT5a expression journey
After the step of degree, also contain so that the material that the degree of WNT1 or WNT5a expression reduces is judged as skin-whitening material
Step.The skin-whitening process for screening substances involved by another scheme of the present invention is it is also possible in the phosphoric acid confirming NFATC2
After the step of change degree, also contain so that the material that the phosphorylation degree of NFATC2 increases is judged as skin-whitening material
Step.Especially, because the expression containing WIF1 reduces or the expression of WNT1 or WNT5a increases or NFATC2 is carried out
The skin-whitening screening substances of one or more of the fibroblast of dephosphorylation, melanocyte and keratinocyte cell
System, has reproduced the actual skin condition of skin melanism, so the WIF1 of the system after candidate substances are made with described adjustment,
The phosphorylation degree of the expression degree of WNT1 or WNT5a gene or messenger substance NFATC2 changes in which way to be commented
Valency, can confirm that whether candidate substances are the material with whitening effect, especially can confirm that whether candidate substances are to change
The material of kind cutaneous pigmentation disease.
One scheme of the present invention is provided to contain as effective ingredient and is sieved by described skin-whitening process for screening substances
The skin-whitening compositionss of the material selected.As previously described, due to fibroblast, keratinocyte can will be made
And the expression of the WIF1 in one or more of melanocyte cell increase or WNT1 or WNT5a expression reduce or
The material that the degree of the dephosphorylation of NFATC2 increases, is judged as with skin whitening effects, specifically has skin pigment
The material of hemachromatosis improvement, so the compositionss containing this material as effective ingredient, is obtained in that skin-whitening is imitated
Really, specifically cutaneous pigmentation disease improvement.
Another scheme of the present invention, is U.S. of WIF1 itself containing described gene involved in the present invention by offer
Use compositionss or external preparation for skin medicament composition in vain, thus suppressing the tryrosinase of WIF1 to express, suppression melanin is to cutin shape
Become the diffusion of cell such that it is able to obtain skin-whitening and cutaneous pigmentation disease improvement.
The skin-whitening composition involved by another scheme of the present invention includes cosmetic composition, medicament composition and food
Product compositionss.
For the dosage form example of the compositionss involved by a scheme of the present invention, it is described below but it is also possible to answer
With other various dosage forms, these examples do not limit the present invention, are only used for being specifically described.
［ preparation example 1 ］ nutrition astringent
Composition according to described in table 1 below, manufactures nutrition astringent using usual method.
【Table 1】
［ preparation example 2 ］ nutritional emulsions
Composition according to described in table 2 below, manufactures nutritional emulsions using usual method.
【Table 2】
［ preparation example 3 ］ nourishing cream
Composition according to described in Table 3 below, manufactures nourishing cream using usual method.
【Table 3】
［ preparation example 4 ］ facial film
Composition according to described in table 4 below, manufactures facial film using usual method.
【Table 4】
［ preparation example 5 ］ ointment
Composition according to described in table 5 below, manufactures ointment using usual method.
【Table 5】
Below, experimental example, embodiment and comparative example are enumerated, while the structure to the present invention and effect are carried out more specifically
Ground explanation.But following experimental example, embodiment and comparative example carry only to assist in the illustration this purpose understanding the present invention
For, it is not so limited scope of the invention and scope.
The expression change of the WIF1 gene at ［ experimental example 1 ］ cutaneous pigmentation position
Normal skin tissue to detected person 13 people（N）Skin histology with pigmentation position（L）Carry out biopsy
（biopsy）And extract RNA in tissue, using RNA in this tissue, by real-time（real-time）PCR method investigates gene
Expression change.Its result is as shown in Figure 1.
It will be noted from fig. 1 that in the skin histology at pigmentation position, the expression of WIF1 and normal skin tissue's phase
Than minimizing.By this situation, can confirm that WIF1 is related to cutaneous pigmentation.
The expression change of WNT1 and WNT5a at ［ experimental example 2 ］ cutaneous pigmentation position
Normal skin tissue to detected person 13 people（N）Skin histology with pigmentation position（L）Carry out biopsy and
Extract RNA in tissue, using RNA in this tissue, by real-time（real-time）PCR method investigates WNT1 and WNT5a gene
The change of expression.Its result is as shown in Figure 2.
From fig. 2 it can be seen that in the skin histology at pigmentation position, the expression of WNT1 and WNT5a and normal skin
Skin tissue has compared increase.By this situation, can confirm that WNT1 and WNT5a is related to cutaneous pigmentation.
WIF1 gene expression in ［ experimental example 3 ］ Skin Cell
From the Medium 254 that Cascade Biologics company obtains, put into containing phorbol exters（PMA,
phorbol 12-myristate 13-acetate）The human melanocytes growth additive of 10ng/ml（HMGs, human
melanocyte growth supplement）, as culture medium use, will from neonatal epidermis detached fell
Skin melanocyte is in 37 DEG C of 5%CO2Cultivate in calorstat.In addition, will obtain from Cascade Biologics company
Medium EpiLife（#M-EPI-500-CA）In put into Human keratinocytes growth additive（HKGS, human
keratinocyte growth supplement）, as culture medium use, will from neonatal epidermis detached people
Skin keratin forms the 5%CO that cell is at 37 DEG C2Cultivate in calorstat.DMEM in the hyclone putting into 10%
（Dulbecco’s Modified Eagles medium）In culture medium, put into detached application on human skin from neonatal epidermis
Fibroblast（fibroblast）, in 37 DEG C of 5%CO2Under the conditions of cultivate.Each Skin Cell after as described above culture
The middle gene expression separating RNA, investigating WIF1 by real-time PCR, its result is as shown in Figure 3.
It can be seen in figure 3 that in fibroblast（FB）And keratinocyte（KC）Middle WIF1 has expression, but black
Plain cell（MC）In there is no the expression of WIF1.By this situation it is known that in Skin Cell, WIF1 is not in melanocyte
Expression, but express in fibroblast and keratinocyte.
The manufacture of ［ embodiment 1～3 and comparative example 1～2 ］ system
Collagen protein 1 in the important composition composition as corium（Collagen I, 1mg/ml）2.5 are put in solution ×
105The fibroblast of/ml is inject in 6 well culture plates them with the amount of every hole 2ml after, little with 1 in 37 DEG C of incubator
When more than solidify, thus making three-dimensional artificial corium.This artificial dermis is placed with 1:1 mixing amount to 2.5 × 105Individual black
Plain cell and keratinocyte layer, thus manufacture three dimension system.Now, will be provided with siRNA having been carried out process and suppress
Fibroblastic system of WIF1 expression, as embodiment 1, the system of the normal fibroblast using human body skin is made
For comparative example 1.
In addition, by using the cutin shape suppressing WIF1 expression to siRNA so that the method substantially identical with embodiment 1 is processed
The three dimension system becoming cell and melanocyte and being fabricated to, will be using normal keratinocyte and melanocyte used as embodiment 2
The system as a comparison case 2 that cell makes.
And, made so that 50ng/ml is processed in the medium in the WIF1 being made using human body recombination
In the melanocyte that the expression of WIF1 increases, after being mixed into keratinocyte, it is placed on the method substantially identical with embodiment 1
The system made on the artificial dermis making, as embodiment 3.
［ experimental example 4 ］ is evaluated to the fibroblastic system containing suppression WIF1 expression
Whether the embodiment 1 as system involved in the present invention is correctly reflected to the skin condition of reality, as follows
Shown contrast with comparative example 1 and evaluated.
The system of the fibroblastic embodiment 1 containing suppression WIF1 expression, by the albumen via protein electrophorese
Immunoblotting, confirms identically with actual cutaneous pigmentation position, has important function in melanin generates
Enzyme is the expression of tryrosinase, dramatically increases compared with comparative example 1（With reference to Fig. 4）.
In addition, in the Keratin 14 using identification expression only in keratinocyte（keratin 14）Fluorescent labeling
Antibody Human Keratinocytes are dyeed, using the tyrosinase-related protein matter 1 of expression in identification melanocyte
（tyrosinase-related protein 1, TRP 1）The antibody that is combined of the fluorescence with another kind of color to melanocyte
Dyeed, using FACS（Fluorescence-activated cell sorting）Confirm the angle with 2 kinds of fluorescence
Matter forms the quantity of cell, as a result, embodiment 1 is as shown in cutaneous pigmentation position, melanin is to keratinocyte
Diffusion increase compared with comparative example 1（With reference to Fig. 5）.In addition, now, by using the protein immunoblot side of protein electrophorese
Method is it was observed that as the NFATC2 of messenger substance in the system of embodiment 1（nuclear factor of activated
T cells, cytoplasmic, calcineurin-dependent 2）Phosphorylation form（p-NFATC2）Reduce（Reference
Fig. 6）.
Based on above-mentioned situation, can confirm that the stacking of fibroblast, melanocyte and keratinocyte is three-dimensional
The system involved in the present invention that is manufactured into similar with actual skin condition, particularly can reproduce cutaneous pigmentation portion
Position.
［ experimental example 5 ］ is evaluated to the system of the keratinocyte containing suppression WIF1 expression
Whether the embodiment 2 as system involved in the present invention is correctly reflected to the skin condition of reality, as follows
Shown contrast with comparative example 2 and evaluated.
System to the embodiment 2 of the keratinocyte containing suppression WIF1 expression, with substantially the same with experimental example 4
The result evaluated of method, confirm identically with actual cutaneous pigmentation position, have in melanin generates
The enzyme of important function is the expression of tryrosinase, dramatically increases compared with comparative example 2（With reference to Fig. 7）.
In addition, the result evaluated with the method substantially the same with experimental example 4, also confirm in example 2, such as
The such melanin diffusion of cutaneous pigmentation position increases（With reference to Fig. 8）.And, after now, confirming the phosphorylation of NFATC2
Form p-NFATC2 reduce, confirm NFATC2 and there occurs dephosphorylation（With reference to Fig. 9）.
According to the above, can confirm that the institute of the present invention dimensionally manufacturing using melanocyte, keratinocyte
The system being related to is also similar with actual skin condition, particularly can reproduce cutaneous pigmentation position.
［ experimental example 6 ］ makes cell to processing in direct culture medium containing the WIF1 making using human body recombination
The melanocytic system that the WIF1 expression of itself increases is evaluated
For containing black after being processed into 50ng/ml using the WIF1 that human body recombination makes in cell culture medium
The system of the embodiment 3 of plain cell, using the protein immunoblot method based on protein electrophorese, the expression confirming WIF1 increases
Plus（With reference to Figure 10）.Now, by the method substantially the same with experimental example 4, confirm tryrosinase in the system of embodiment 3
Expression reduce（With reference to Figure 11）.In addition, compared with comparative example 1 as a result, it is possible to confirm melanic in embodiment 3
Diffusion reduces（With reference to Figure 12）, further acknowledge that the phosphorylation of NFATC2 increases（With reference to Figure 13）.
According to the above it is known that there is Close relation because WIF1 and NFATC2 is generated with melanin, so passing through
System involved in the present invention is processed to candidate substances, then the increase degree to WIF1 expression and its next information transmission
Material is that the phosphorylation degree of NFATC2 is evaluated, such that it is able to screen skin-whitening material.
As described above, skin-whitening screening substances system involved in the present invention can reproduce the skin of reality, therefore, that is,
Make under ex vivo situation it is also possible to effectively screen the material showing clinically excellent whitening effect.Further, since this is
System can correctly reflect the actual skin histology of human body, so can also be applied to the so-called pigment such as vitiligo or poliosis lacking
In the research of the mechanism of weary disease and ameliorative way.
Claims (7)
1. a kind of skin-whitening screening substances system is it is characterised in that include：
Containing fibroblastic fibroblast cell layer；With
Containing keratinocyte and melanocytic layer,
Wherein, the cell of one or more of described fibroblast, keratinocyte and melanocyte is WNT1 or WNT5a
Expression increase cell and/or NFATC2（nuclear factor of activated T cells, cytoplasmic
2）The cell of dephosphorylation, and
Wherein, the cell of one or more of described fibroblast, keratinocyte and melanocyte is WIF1（Wnt
inhibitory factor 1）The repressed cell of expression.
2. skin-whitening screening substances system according to claim 1 it is characterised in that described fibroblast cell layer be
Fibroblastic layer is contained in collagen protein.
3. skin-whitening screening substances system according to claim 2 is it is characterised in that described collagen protein accounts for described skin
0.001 weight %～30 weight % of the overall weight of skin whitening screening substances system.
4. skin-whitening screening substances system according to claim 1, wherein, described fibroblast, cutin are formed carefully
The cell of one or more of born of the same parents and melanocyte comes from one or more of the birds and pig such as people, Mus, guinea pig, chicken
The cell of animal.
5. skin-whitening screening substances system according to claim 1 is it is characterised in that skin-whitening material contains improvement
The skin-whitening material of the pigmentation disease of skin.
6. a kind of skin-whitening process for screening substances is it is characterised in that include：Right after candidate substances are processed will
Ask in the skin-whitening screening substances system involved by any one in 1～5, to described fibroblast, keratinocyte
And the journey of the expression of one or more of WIF1, WNT1, WNT5a and NFATC2 in the cell of one or more of melanocyte
Degree or the step that confirmed of phosphorylation degree；
After confirming the step of expression degree or phosphorylation degree, also include the thing that the expression degree making WIF1 is increased
Matter, the material that the expression degree making WNT1 or WNT5a is reduced or the expression degree making WNT1 or WNT5a is reduced
The step that material is judged as skin-whitening material.
7. purposes in manufacturing skin-whitening compositionss for the material being filtered out by the screening technique described in claim 6.
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| KR20110106614 | 2011-10-18 | ||
| KR10-2011-0106614 | 2011-10-18 | ||
| KR1020120114965A KR102031161B1 (en) | 2011-10-18 | 2012-10-16 | Screening System of candidate material for skin-whitening |
| KR10-2012-0114965 | 2012-10-16 | ||
| PCT/KR2012/008555 WO2013058578A2 (en) | 2011-10-18 | 2012-10-18 | System for screening skin-whitening material |
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| KR102024802B1 (en) * | 2017-05-02 | 2019-09-24 | 태이생명과학 주식회사 | Method for Screening Substance for Improving Skin Condition |
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| CN101426934A (en) * | 2006-04-25 | 2009-05-06 | 株式会社资生堂 | Method of predicting skin spot formation and method of screening skin spot formation inhibitor with the use of spot site-increasing genes as indication |
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| CA2658074C (en) * | 2008-03-17 | 2017-11-07 | L'oreal | Functional pigmented skin equivalent |
| KR20100039077A (en) * | 2008-10-07 | 2010-04-15 | 숙명여자대학교산학협력단 | Screening system using ndrg2 as a novel regulator gene of pigmentation in developing agents for application of skin hyper-pigmentation treatment |
| JP2011092179A (en) | 2009-09-30 | 2011-05-12 | Toyobo Co Ltd | 3-dimensional cultured skin model containing melanocyte, and use of the same |
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| CN101426934A (en) * | 2006-04-25 | 2009-05-06 | 株式会社资生堂 | Method of predicting skin spot formation and method of screening skin spot formation inhibitor with the use of spot site-increasing genes as indication |
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| KR20130043069A (en) | 2013-04-29 |
| JP6069334B2 (en) | 2017-02-01 |
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