CN103857802A - System for screening skin-whitening material - Google Patents
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Abstract
Disclosed are a system for screening a skin-whitening material comprising one or more layers which include one or more cells of fibroblasts, keratinocytes, and melanocytes, and a method for screening a skin-whitening material using the system. In addition, the invention presents a composition for skin whitening which comprises a material screened by the method for screening a skin- whitening material as an active ingredient.
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
Up to now, in the time developing new skin whitening material, use following method,, be after melanophore (melanocyte) is directly processed by candidate substances to melanochrome founder cell, be confirmed whether that check melanin generates, or to melanophore irradiation ultraviolet radiation, confirm whether candidate substances has shielded the melanochrome now producing and generated signal.But, in fact in skin, melanophore is not Individual existence, but is subject to directly or indirectly other periphery cell or the physics of microhabitat or chemical affect and generate melanochrome, if only there is melanophore, cannot reproduce the state of above-mentioned actual skin tissue.
In addition, the generation essential factor of freckle, spot, these cutaneous pigmentation diseases of black mole or mechanism increase different from cause melanochrome generation due to ultraviolet ray, although there is the trend that this symptom can worsen due to uviolizing, even but not existing ultraviolet ray to wait the stimulation producing, melanic generation and dispersion also can continue to increase.Thus, in the case of the skin whitening material of developing based on existing ultraviolet irradiation method, in actual clinical, mostly cannot show the effect of the cutaneous pigmentation disease of suppressing people.In addition, if the skin at the pigmentation position in order to study cutaneous pigmentation disease to detected person carries out biopsy and uses, the energy of expense and cost is very large, and be difficult to obtain people's skin histology, in addition, different from uviolizing sample, the animal specimen that obtains cutaneous pigmentation disease is also very difficult.
Therefore, exist not only and construct, particularly exist the structure of the people's of cutaneous pigmentation disease skin histology be similar to very much and can screen simply the system of skin whitening material and the demand of screening method with people's skin histology.
Patent documentation 1: No. 10-0840144 (registration on June 16th, 2008) specification sheets of Korean registered patent.
Summary of the invention
One aspect of the present invention is, the skin whitening process for screening substances that can efficiently and simply screen clinically the skin whitening screening substances system of useful skin whitening material and utilize this system is provided.
Another aspect of the present invention is, provides and contains the material that screened by described skin whitening process for screening substances as effective constituent and the skin whitening composition of excellent whitening effect is shown.
In addition, another aspect of the present invention is, provides to contain to the melanochrome of skin to generate skin whitening composition or the external preparation for skin medicament composition of relevant gene self as effective constituent.
A scheme of the present invention is, the skin whitening screening substances system of the layer that comprises more than one cells of containing in inoblast (fibroblast), keratinocyte (keratinocyte) and melanophore (melanocyte) is provided, wherein said layer more than or equal one deck.
Another scheme of the present invention is, a kind of skin whitening process for screening substances is provided, it also comprises more than one the degree of expression or the step of phosphorylation degree in WIF1, WNT1, WNT5a and the NFATC2 confirming in more than one cells in inoblast, keratinocyte and melanophore after candidate substances is processed.
Another scheme of the present invention is, provide and contain the material that screened by the described skin whitening process for screening substances skin whitening composition as effective constituent, or contain skin whitening composition and the external preparation for skin medicament composition of WIF1 self as effective constituent.
The effect of invention
Skin whitening screening substances system involved in the present invention is owing to being designed to approach actual skin histology, even therefore under in vitro (in vitro) state, also can be with less time and expense and simply screening there is clinically the material of whitening effect.Especially, after melanophore being processed from by candidate substances, screen the existing method of whitening material by irradiation ultraviolet radiation different, skin whitening screening substances system involved in the present invention has and the condition that illustrates that the degree of gene expression of actual skin tissue of pigmentation disease and the phosphorylation degree of information transmitter substance are similar, therefore, in the case of utilizing system involved in the present invention, can filter out simply skin whitening material, particularly effectively improve the material of pigmentation disease.From being difficult to obtain people and the animal specimen this point for the research of pigmentation disease, system involved in the present invention can become for developing new skin whitening material, specifically developing system or the instrument that pigmentation disease is improved material.
Accompanying drawing explanation
Fig. 1 is the figure that the difference of the expression degree of the WIF1 gene between normal skin (N) and cutaneous pigmentation position (L) to detected person compares.
Fig. 2 is the figure that the difference of the expression degree of WNT1 between normal skin (N) and cutaneous pigmentation position (L) to detected person and WNT5a compares.
Fig. 3 is the figure that the difference of the expression degree to the WIF1 gene in melanophore (MC), keratinocyte (KC) and inoblast (FB) compares.
Fig. 4 is illustrated in and contains in the fibroblastic whitening screening system that suppresses WIF1 expression, the situation that the expression of tyrosine oxidase (tyrosinase) increases.
Fig. 5 is illustrated in and contains in the fibroblastic whitening screening system that suppresses WIF1 expression, the mobile situation about increasing of melanosome.
Fig. 6 is illustrated in and contains in the fibroblastic whitening screening system that suppresses WIF1 expression, the situation of NFATC2 dephosphorylation.
Fig. 7 is illustrated in the whitening screening system that contains the keratinocyte that suppresses WIF1 expression, the experimental result of the situation that the expression of tyrosine oxidase increases.
Fig. 8 is illustrated in the whitening screening system that contains the keratinocyte that suppresses WIF1 expression, the mobile situation about increasing of melanosome.
Fig. 9 is illustrated in the whitening screening system that contains the keratinocyte that suppresses WIF1 expression, the situation of NFATC2 dephosphorylation.
After Figure 10 represents that the people WIF1 to manufacturing by gene recombination technology processes in melanophore, melanocytic WIF1 expresses situation about increasing.
Figure 11 is illustrated in the melanophore that has brought out as shown in figure 10 WIF1 expression, the situation that the expression of tyrosine oxidase reduces.
Figure 12 is illustrated in the people WIF1 manufacturing by gene recombination technology after containing and processing in melanocytic whitening screening system, the mobile situation about reducing of melanosome.
Figure 13 is illustrated in the melanophore that has brought out as shown in figure 10 WIF1 expression, and the situation of phosphorylation occurs NFATC2.
Embodiment
In this manual, " skin " refer to and cover the tissue of animal body surface, not only refer to the tissue of the body surfaces such as coverage rate portion or health, is the concept of the broad sense including scalp and hair also.
The melanophore (melanocyte) of skin is to manufacture the cell of melanochrome (melanin) that determines skin color, is positioned on the basilar membrane working as the corium of skin and the boundary line of epidermis.From fetology angle, melanophore is that differentiation as described below forms,, originate from from neural crest cell (neural-crest cells), via melanophore stem cell (Melanocyte stem cell), as the melanoblast (melanoblast) of precursor cell and move to skin, and be finally divided into that can to generate melanic pigment cell be melanophore.
As mentioned above not Individual existence in skin of the melanophore after differentiation is fibroblastic impact of existing of the combination of keratinocyte and the corium that is arranged in below and generate melanochrome but be subject to the keratinocyte of the side of being located thereon.The melanochrome of being manufactured by melanophore, via cell dendron (dendrite), peritropous keratinocyte moves, and is dispersed in widely skin surface, waits adverse factor impact thereby protection skin is not subject to ultraviolet ray, presents skin color.Normal skin, in the time there is this stimulation of ultraviolet ray, generates more melanochrome, and increases melanic dispersion compared with normal circumstances, and then, if this stimulation disappears, melanic generation and dispersion recover lower level again.
As noted above, the whitening material of developing by existing method, the skin whitening material of wherein particularly developing based on ultraviolet irradiation method, owing to not reflecting actual skin condition, be melanophore in skin, be subject to other cells or around microhabitat physical chemical factor combined influence and generate this actual skin condition of melanochrome, so improving this one side of pigmentation disease of skin, mostly show poor clinically effect.Because cutaneous pigmentation disease has multiple generation reason and because special mechanism makes skin melanism, so, in order to develop the whitening material that can effectively improve cutaneous pigmentation disease, need to make and the similar experimental model of actual skin, and utilize this model development whitening material.
Describe the present invention in detail below.
A scheme of the present invention is, the skin whitening screening substances system of the layer that comprises more than one cells of containing in inoblast (fibroblast), keratinocyte (keratinocyte) and melanophore (melanocyte) is provided, described layer more than or equal one deck.Another scheme of the present invention is, a kind of skin whitening screening substances system that contains fibroblastic inoblast layer, the keratinocyte layer that contains keratinocyte and contain melanocytic melanophore layer that comprises is provided.Now, preferably for similar and inoblast is stacked in to orlop layer by layer, stacked melanophore layer above it, stacked keratinocyte layer above melanophore layer with actual skin condition.The skin whitening screening substances system that another scheme of the present invention is related, comprise contain fibroblastic inoblast layer and contain keratinocyte and melanophore in the layer of more than one cells, can in same layer, contain keratinocyte and melanophore.Now, inoblast can be stacked in to lower floor layer by layer, above it the stacked layer that contains melanophore and keratinocyte.Described skin whitening screening substances system is the system that is laminated with the 3 D stereo structure of skin cells layer, not only has melanophore, also has the cell of periphery, thus, can correctly reflect actual skin condition.The related skin whitening screening substances system of another scheme of the present invention also can be removed inoblast layer, and including containing the known layer melanophore and the keratinocyte with vital role that melanochrome is generated.
The inoblast layer of the related skin whitening screening substances system of a scheme of the present invention, is the layer for reproducing skin corium, is included in and in collagen protein, contains fibroblastic layer.Specifically, described inoblast layer can be in collagen liquid, to put into inoblast, condenses and the layer that manufactures.In another scheme of the present invention, described collagen protein can account for 0.001 % by weight～30 % by weight in the overall weight of skin whitening screening substances system, preferably 0.01 % by weight～20 % by weight, more preferably 0.1 % by weight～10 % by weight.In the situation that described scope contains collagen protein, be suitable for showing the desirable effect of the present invention, from expense/effect than this on the one hand, it is more suitable in described scope, to use.
In the related skin whitening screening substances system of a scheme of the present invention, the cell of more than one in inoblast, keratinocyte and melanophore comprises the cell in more than one the animal of selecting from the birds such as people, mouse, guinea pig, chicken and pig.
The skin whitening screening substances system that a scheme of the present invention is related, also can not only select to there is the material of skin whitening effect, specifically have and improve the material that causes the effect of skin melanism due to ultraviolet ray, can also effectively screen especially the cutaneous pigmentation diseases such as freckle, spot, black mole are had to the material that improves effect.
Present inventors find, occurring in the skin of pigmentation disease, regulating relevant extracellular signal transmission factor to embryonic development is WIF1(Wnt inhibitory factor 1, ACCESSION NP_009122, VERSION NP_009122.2 GI:111125011) gene expression reduce, specifically, the expression of WIF1 gene, in skin cells, not in the melanophore as melanochrome founder cell, reduce, but reduce in keratinocyte and inoblast, thus melanocytic melanochrome is generated and has impact.In addition, in the skin reducing in the expression of WIF1 gene, when generating the expression increase of relevant tyrosine oxidase to melanochrome, melanochrome increases to the diffusion of keratinocyte, therefore, has confirmed to have substantial connection between WIF1 and cutaneous pigmentation disease.Now, GSK-3 β (Glycogen synthasekinase-3 β) and phosphorylation and the MITF(microphthalmia-associated transcription factor of beta-catenin (β-catenin) are interfered in the expression that confirms WIF1 gene) expression, cutaneous pigmentation is had to impact.
Therefore, the skin whitening screening substances system that a scheme of the present invention provides the WIF1 that is suppressed to more than one cells in fibrocyte, keratinocyte and melanophore to express, specifically, provide the skin whitening screening substances system that suppresses keratinocyte or fibroblastic WIF1 expression.In another scheme of the present invention, described skin whitening screening substances system contains the cell that utilizes siRNA to express to suppress WIF1.In described screening system reflection keratinocyte or inoblast, WIF1 expresses the skin condition of the pigmentation disease of reducing, and can roughly reproduce similarly the skin condition that has pigment hemachromatosis with practical situation.Thus, in the situation that utilizing described screening system, the material with skin whitening function can be effectively selected, the material with the function of improving cutaneous pigmentation disease can be selected especially.
In addition, present inventors confirm occurring in the skin of 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) gene expression increase, as the NFATC2(nuclear factor of activated T cells of information transmitter substance, cytoplasmic, calcineurin-dependent 2) dephosphorylation, therefore, a scheme of the present invention is provided as fibrocyte, the skin whitening screening substances system of the cell that in keratinocyte and melanophore, more than one cell is the cell that increased of the expression of WNT1 and WNT5a or NFATC2 dephosphorylation.Described screening system reproduces and occurs the skin condition of pigmentation disease owing to can approaching very much practically, so can effectively select the material with skin whitening function, can select especially the material with the function of improving cutaneous pigmentation disease.
Before this, by the so-called candidate substances such as crude substance, its extract or chemosynthesis material are only processed melanophore, whether there is whitening effect thereby evaluate.In above-mentioned existing methodical situation, the candidate substances being diluted in cell culture fluid directly must be processed cell, in addition, candidate substances being made under the state of this actual formulation using of frost or emulsion, cannot process melanophore.Thus, in existing method, whether to there is actual whitening effect clinically in the formulation of implementing could confirm to contain candidate's whitening material after clinical trial.On the other hand, skin whitening screening substances system involved in the present invention certainly can be as existing method, candidate substances is diluted in nutrient solution and uses and evaluate it and have or not whitening effect, also can use the formulation such as frost or emulsion that contains candidate substances to evaluate it and have or not whitening effect.Now, for to formulation, evaluation has or not the skin whitening screening substances system of whitening effect, can be by contain more than one multiple layer in inoblast, melanophore and keratinocyte carry out stacked after, be exposed in air and create artificial skin.As mentioned above, skin whitening screening system involved in the present invention, owing to having and the similar physiology of actual skin, chemical feature, use the actual formulation using also can evaluate its whitening effect, therefore, can, in shorter time, efficiently and simply select the material in actual clinical with whitening effect with less expense.
A scheme of the present invention provides a kind of skin whitening process for screening substances, it has been having carried out candidate substances in the described skin whitening screening substances system of processing, and comprises more than one the degree of expression or the step of phosphorylation degree in WIF1, WNT1, WNT5a and the NFATC2 confirming in more than one cells in inoblast, keratinocyte and melanophore.As previously described, WIF1, WNT1, WNT5a and NFATC2 are relevant with the melanochrome expression of skin and the degree of diffusion.Specifically, the expression of expression minimizing, WNT1 or the WNT5a of WIF1 increases or the dephosphorylation degree of NFATC2 increases, and means with melanochrome and generates the expression increase that relevant enzyme is tyrosine oxidase, and melanochrome increases to the diffusion of keratinocyte.Thus, by confirm candidate substances in WIF1, WNT1, WNT5a and NFATC2 more than one the degree of expression or the degree of phosphorylation produce which kind of impact, can evaluate described candidate substances and whether there is skin whitening effect.
The related skin whitening process for screening substances of a scheme of the present invention, also can be after confirming the step of the degree that WIF1 expresses, and also contains the step that the material of the degree increase that WIF1 is expressed is judged as to skin whitening material.The skin whitening process for screening substances that another scheme of the present invention is related, also can be after confirming the step of the degree that WNT1 or WNT5a express, also contain the step that the material of the degree minimizing that WNT1 or WNT5a are expressed is judged as to skin whitening material.The skin whitening process for screening substances that another scheme of the present invention is related, also can, after the step of phosphorylation degree of confirming NFATC2, also contain the step that the material that the phosphorylation degree of NFATC2 is increased is judged as to skin whitening material.Especially, because the expression that contains WIF1 reduces, or the expression of WNT1 or WNT5a increases, or NFATC2 has carried out the inoblast of dephosphorylation, the skin whitening screening substances system of more than one cells in melanophore and keratinocyte, reproduce the actual skin condition of skin melanism, so by candidate substances being made to the WIF1 of the system after described adjustment, the phosphorylation degree of the expression degree of WNT1 or WNT5a gene or information transmitter substance NFATC2 changes and evaluates in which way, can confirm whether candidate substances is the material with whitening effect, can confirm especially whether candidate substances is the material that can improve cutaneous pigmentation disease.
A scheme of the present invention is provided as effective constituent and the skin whitening composition that contains the material filtering out by described skin whitening process for screening substances.As previously described, due to the material that the degree of the dephosphorylation of the expression minimizing of the expression increase of the WIF1 in more than one cells that make in inoblast, keratinocyte and melanophore or WNT1 or WNT5a or NFATC2 can be increased, be judged as the material that there is skin whitening effect, specifically there is cutaneous pigmentation disease and improve effect, so the composition that contains this material as effective constituent, can obtain skin whitening effect, specifically cutaneous pigmentation disease is improved effect.
Another scheme of the present invention, be skin-whitening composition or the external preparation for skin medicament composition of WIF1 self by the gene involved in the present invention described in containing is provided, thereby the tyrosine oxidase that suppresses WIF1 is expressed, check melanin is to the diffusion of keratinocyte, thereby can obtain skin whitening and cutaneous pigmentation disease is improved effect.
The related skin-whitening composition of another scheme of the present invention comprises make-up composition, medicament composition and food compositions.
For the formulation example of the related composition of a scheme of the present invention, be described below, but also can apply other various formulations, these examples do not limit the present invention, only for being specifically described.
［ formulation example 1 ］ nutrition astringent
The composition of recording according to following table 1, uses usual method to manufacture nutrition astringent.
[table 1]
［ formulation example 2 ］ nutritional emulsions
The composition of recording according to following table 2, uses usual method to manufacture nutritional emulsions.
[table 2]
［ formulation example 3 ］ nourishing cream
The composition of recording according to following table 3, uses usual method to manufacture nourishing cream.
[table 3]
［ formulation example 4 ］ facial mask
The composition of recording according to following table 4, uses usual method to manufacture facial mask.
[table 4]
［ formulation example 5 ］ ointment
The composition of recording according to following table 5, uses usual method to manufacture ointment.
[table 5]
, enumerate experimental example, embodiment and comparative example on one side below, on one side structure of the present invention and effect are described more specifically.But following experimental example, embodiment and comparative example only provide in order to help to understand illustration this purpose of the present invention, do not limit thus category of the present invention and scope.
The expression of the WIF1 gene at ［ experimental example 1 ］ cutaneous pigmentation position changes
Normal skin tissue (N) to detected person 13 people and the skin histology (L) at pigmentation position carry out biopsy (biopsy) and extract RNA in tissue, utilize RNA in this tissue, change by the expression of the gene of (real-time) PCR method investigation in real time.Its result as shown in Figure 1.
As can see from Figure 1, in the skin histology at pigmentation position, the expression of WIF1 minimizing compared with normal skin tissue.By this situation, can confirm WIF1 relevant to cutaneous pigmentation.
［ experimental example 2 ］ WNT1 at cutaneous pigmentation position and the expression of WNT5a change
Normal skin tissue (N) to detected person 13 people and the skin histology (L) at pigmentation position carry out biopsy and extract RNA in tissue, utilize RNA in this tissue, by the variation of (real-time) PCR method investigation WNT1 and WNT5a genetic expression in real time.Its result as shown in Figure 2.
As can see from Figure 2, in the skin histology at pigmentation position, the expression of WNT1 and WNT5a has increase compared with normal skin tissue.By this situation, can confirm WNT1 relevant to cutaneous pigmentation with WNT5a.
WIF1 genetic expression in ［ experimental example 3 ］ skin cells
Among the Medium 254 obtaining from Cascade Biologics company, put into and contain Buddhist ripple ester (PMA, phorbol 12-myristate 13-acetate) the human melanocytes growth additive (HMGs of 10ng/ml, human melanocyte growth supplement), set it as substratum and use, the 5%CO by the human skin melanophore separating from neonatal epidermis at 37 ℃
2in thermostat container, cultivate.In addition, by the Medium EpiLife(#M-EPI-500-CA obtaining from Cascade Biologics company) put into people's keratinocyte growth additive (HKGS, human keratinocyte growth supplement), set it as substratum and use, the 5%CO by the human keratinocytes separating from neonatal epidermis at 37 ℃
2in thermostat container, cultivate.Putting into DMEM(Dulbecco ' the s Modified Eagles medium of 10% foetal calf serum) substratum, put into the human skin fibroblast (fibroblast) separating from neonatal epidermis, at the 5%CO of 37 ℃
2under condition, cultivate.Isolation of RNA in each skin cells from cultivation described above, investigates the genetic expression of WIF1 by PCR in real time, its result as shown in Figure 3.
As can see from Figure 3, in inoblast (FB) and keratinocyte (KC), WIF1 has expression, but in melanophore (MC), there is no the expression of WIF1.By this situation, known in skin cells, WIF1 does not express in melanophore, but expresses in inoblast and keratinocyte.
The manufacture of ［ embodiment 1～3 and comparative example 1～2 ］ system
At the collagen protein 1(collagen of the important composition composition as corium I, 1mg/ml) put into 2.5 × 10 in solution
5the inoblast of/ml, injects them after 6 well culture plates with the amount of every hole 2ml, solidifies above, thereby make three-dimensional artificial dermis in the incubator of 37 ℃ with 1 hour.What on this artificial dermis, placement mixed with 1:1 amounts to 2.5 × 10
5individual melanophore and keratinocyte layer, thus three dimension system manufactured.Now, using possessing, siRNA carried out processing and suppressed fibroblastic system that WIF1 expresses as embodiment 1, will use the system as a comparison case 1 of normal fibroblast of human body skin.
In addition, siRNA is processed using the method identical with embodiment 1 essence utilizing and suppress keratinocyte that WIF1 expresses and melanophore and the three dimension system that is made into as embodiment 2, by the system of utilizing normal keratinocyte and melanophore to make as a comparison case 2.
And, the WIF1 making at end user's body recombination processes in substratum with 50ng/ml and makes in the melanophore of expression increase of WIF1, sneak into after keratinocyte, be placed on the artificial dermis made from the method identical with embodiment 1 essence and the system of making, as embodiment 3.
［ experimental example 4 ］ suppresses fibroblastic system of WIF1 expression and evaluates to containing
Whether the embodiment 1 for the system as involved in the present invention has correctly reflected actual skin condition, and as follows and comparative example 1 contrasts and evaluates.
The system that contains the fibroblastic embodiment 1 that suppresses WIF1 expression, by the detected by Western blot via protein electrophorese, confirm with actual cutaneous pigmentation position in the same manner, the enzyme in melanochrome generates with vital role is the expression of tyrosine oxidase, remarkable increase (with reference to Fig. 4) compared with comparative example 1.
In addition, fluorescent-labeled antibody Human Keratinocytes at the Keratin 14 (keratin 14) that utilizes identification only to express in keratinocyte dyes, utilize the tyrosinase-related protein matter 1(tyrosinase-related protein 1 expressing in identification melanophore, TRP 1) the antibody of being combined with the fluorescence of another kind of color melanophore is dyeed, utilize FACS(Fluorescence-activated cell sorting) confirm to have the quantity of keratinocyte of 2 kinds of fluorescence simultaneously, its result, embodiment 1 is as shown in cutaneous pigmentation position, melanochrome increases (with reference to Fig. 5) to the diffusion of keratinocyte compared with comparative example 1.In addition, now, by utilizing the protein immunoblot method of protein electrophorese, observe the NFATC2(nuclear factor of activated T cells as information transmitter substance in the system of embodiment 1, cytoplasmic, calcineurin-dependent 2) phosphorylation form (p-NFATC2) reduce (with reference to Fig. 6).
Based on above-mentioned situation, can confirm similar to system involved in the present invention stacked to inoblast, melanophore and keratinocyte and that dimensionally manufacture and actual skin condition, particularly can reproduce cutaneous pigmentation position.
［ experimental example 5 ］ evaluated the system that contains the keratinocyte that suppresses WIF1 expression
Whether the embodiment 2 for the system as involved in the present invention has correctly reflected actual skin condition, and as follows and comparative example 2 contrasts and evaluates.
To the system of the embodiment 2 that contains the keratinocyte that suppresses WIF1 expression, the result of evaluating with the method identical in fact with experimental example 4, confirm with actual cutaneous pigmentation position in the same manner, the enzyme in melanochrome generates with vital role is the expression of tyrosine oxidase, remarkable increase (with reference to Fig. 7) compared with comparative example 2.
In addition, the result of evaluating with the method identical in fact with experimental example 4 also confirms in embodiment 2, and as cutaneous pigmentation position, melanochrome diffusion increases (with reference to Fig. 8).And now, the form p-NFATC2 confirming after the phosphorylation of NFATC2 reduces, and confirms NFATC2 dephosphorylation (with reference to Fig. 9) has occurred.
According to foregoing, can confirm to utilize melanophore, keratinocyte and the system involved in the present invention dimensionally manufactured is also similar with actual skin condition, particularly can reproduce cutaneous pigmentation position.
［ experimental example 6 ］ processed and the melanocytic system of the WIF1 expression increase of cell self evaluated containing the WIF1 that utilizes human body recombination and make in direct substratum
For containing the system that the WIF1 of end user's body recombination making is processed in cell culture medium to the melanocytic embodiment 3 after 50ng/ml, utilize the protein immunoblot method based on protein electrophorese, the expression that confirms WIF1 increases (with reference to Figure 10).Now, by the method identical in fact with experimental example 4, the expression that confirms tyrosine oxidase in the system of embodiment 3 reduces (with reference to Figure 11).In addition, the result of comparing with comparative example 1, can confirm melanic diffusion in embodiment 3 and reduce (with reference to Figure 12), and the phosphorylation that also confirms NFATC2 increases (with reference to Figure 13).
According to foregoing, known have Close relation because WIF1 and NFATC2 and melanochrome generate, so candidate substances is processed by system involved in the present invention, then the phosphorylation degree that the increase degree of WIF1 being expressed and its next information transmitter substance are NFATC2 is evaluated, thereby can screen skin whitening material.
As mentioned above, skin whitening screening substances system involved in the present invention can be reproduced actual skin, therefore, even under in vitro state, also can effectively screen the material that shows excellent clinically whitening effect.In addition, because this system can correctly reflect the skin histology of human body reality, so also can be applied in the mechanism of the so-called albinism such as vitiligo or white hair and the research of improvement method.
Claims (17)
1. a skin whitening screening substances system, is characterized in that, comprises the layer that contains more than one cells in inoblast, keratinocyte and melanophore, described layer more than or equal one deck.
2. skin whitening screening substances system according to claim 1, is characterized in that, this skin whitening screening substances system comprises:
Contain described fibroblastic inoblast layer; And
Contain more than one the layer in described keratinocyte and melanophore.
3. skin whitening screening substances system according to claim 2, is characterized in that, described inoblast layer is in collagen protein, to contain fibroblastic layer.
4. skin whitening screening substances system according to claim 3, is characterized in that, described collagen protein accounts for 0.001 % by weight～30 % by weight of the overall weight of described skin whitening screening substances system.
5. skin whitening screening substances system according to claim 1, it is characterized in that, the cell of more than one in described inoblast, keratinocyte and melanophore is WIF1(Wnt inhibitory factor 1) the repressed cell of expression.
6. skin whitening screening substances system according to claim 1, is characterized in that, the cell of more than one in described inoblast, keratinocyte and melanophore is the cells after the expression of WNT1 or WNT5a increases.
7. skin whitening screening substances system according to claim 1, it is characterized in that, the cell of more than one in described inoblast, keratinocyte and melanophore is to make NFATC2(nuclear factor of activated T cells, cytoplasmic 2) cell after dephosphorylation.
8. skin whitening screening substances system according to claim 1, wherein, the cell of more than one in described inoblast, keratinocyte and melanophore is more than one cells of animal that come from the birds such as people, mouse, guinea pig, chicken and pig.
9. skin whitening screening substances system according to claim 1, is characterized in that, skin whitening material contains the skin whitening material of the pigmentation disease that is improved skin.
10. a skin whitening process for screening substances, it is characterized in that, comprise: in candidate substances is carried out to claim 1～9 after treatment in the related skin whitening screening substances system of any one, in WIF1, WNT1, WNT5a and NFATC2 in more than one the cell in described inoblast, keratinocyte and melanophore more than one the degree of expression or the step that phosphorylation degree is confirmed.
11. skin whitening process for screening substances according to claim 10, is characterized in that, after confirming the step of expression degree or phosphorylation degree, also contain the step that the material that the expression degree of WIF1 is increased is judged as to skin whitening material.
12. skin whitening process for screening substances according to claim 10, it is characterized in that, after confirming the step of expression degree or phosphorylation degree, also contain the step that the material that the expression degree of WNT1 or WNT5a is reduced is judged as to skin whitening material.
13. skin whitening process for screening substances according to claim 10, is characterized in that, after confirming the step of expression degree or phosphorylation degree, also contain the step that the material that the phosphorylation degree of NFATC2 is increased is judged as to skin whitening material.
14. 1 kinds of skin whitening compositions, is characterized in that, contain material that the screening method recorded by claim 10 filters out as effective constituent.
15. 1 kinds of skin whitening compositions, is characterized in that, contain WIF1 as effective constituent.
16. 1 kinds of external preparation for skin medicament compositions, is characterized in that, contain WIF1 as effective constituent.
17. according to the composition described in claim 15 or 16, it is characterized in that, described composition is make-up composition.
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| Application Number | Priority Date | Filing Date | Title |
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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 |
| 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 |
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| KR100840144B1 (en) | 2007-05-17 | 2008-06-23 | 경상대학교산학협력단 | Screening methods for candidate materials having whitening activity |
| CA2658074C (en) * | 2008-03-17 | 2017-11-07 | L'oreal | Functional pigmented skin equivalent |
| 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 |
| 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 |
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| DUBERTRET L. ET.AL: "Reconstruction of the human skin equivalent in vitro: a new tool for skin biology", <SKIN PHAMACOL> * |
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| JP2015501152A (en) | 2015-01-15 |
| KR102031161B1 (en) | 2019-10-14 |
| KR20130043069A (en) | 2013-04-29 |
| JP6069334B2 (en) | 2017-02-01 |
| CN103857802B (en) | 2017-03-08 |
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