JP2000229889A - Screening of anti-white hair agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To screen anti-white hair agents by utilizing a migration phenomenon of melanocyte to a hair papilla by bringing a specific melanocyte to a culture supernatant liquid of hair papilla and observing the degree of migration of melanocyte to the cultured supernatant liquid. SOLUTION: (A) Melanocyte is cultured in the presence of a test article. The cultured melanocyte is brought into contact with (B) a culture supernatant liquid of a hair papilla cell. Whether the component A is migrated to the component B is observed or the degree of the migration is observed to screen anti- white hair agents. For example, the component B is obtained by vertically cutting a scalp piece excised from a normal Japanese head orthopedically right under corium, extracting hair follicle, collecting a hair papilla from the isolated human hair papilla, leaving the hair papilla to stand in a culture laboratory dish, subculturing only a cell outgrowing from the implanted hair papilla in a culture flask and collecting a cultured supernatant liquid in subculture of three to seven times.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for screening an anti-white hair agent.

[0002]

2. Description of the Related Art The black color of hair is provided by melanin, a black pigment, and melanin is biosynthesized in melanocytes by a group of enzymes of the melanin biosynthesis system such as tyrosinase. Cutaneous melanocytes are distributed at a relatively low density in the skin, whereas hair follicle melanocytes are distributed at a relatively high density in the dermal papilla of the hair follicle. It is considered that the hair papilla causes a migration phenomenon of hair follicle melanocytes to the hair papilla as a cause of this. As a method for screening an anti-grey-haired agent, a method has been proposed in which tyrosinase is involved in the biosynthetic pathway of melanin, a black pigment, and a substance that promotes the synthesis of tyrosinase is searched for (Japanese Patent Application Laid-open No. No. 9-263540).

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to provide a completely new anti-whitening agent screening method by utilizing the migration phenomenon of melanocytes to the dermal papilla.

[0004]

Means for Solving the Problems The present inventors have conducted studies to elucidate the melanocyte migration phenomenon. As a result, when hair papilla cells are cultured in vitro, a protein-like migration factor that induces melanocyte migration is studied. Is produced, whereby it is possible to artificially cause the migration of melanocytes, and furthermore, it has been found that a substance which enhances the melanocyte migration by incubation with melanocytes has an activity as an anti-white hair agent. The present invention has been completed.

Accordingly, the present invention provides a method for screening an anti-grey-hair agent, comprising culturing melanocytes in the presence of a test substance, contacting the cultured melanocytes with a culture supernatant of hair papilla cells, and It is intended to provide a method characterized by observing whether or not it moves toward the culture supernatant or the degree of the movement.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Preparation, culture and culture of dermal papilla cells
Preparation of Supernatant A scalp section was plastically surgically excised from a normal Japanese head (mainly from male occipital region). DMEM after resection
Store on ice while immersed in a medium or the like, and treat as much as possible on the same day. The following operations are performed in a clean bench under a stereoscopic microscope. The obtained scalp section is cut up and down just below the dermis using a sharp scalpel. Since the hair follicles are buried in the fat layer, they are pulled out one by one using sharp tweezers. Pulled hair follicles are placed in DMEM until just before treatment.

From the isolated human hair follicle, the hair papilla is collected as sharply as possible using sharp forceps and the tip of a syringe needle. The collected dermal papilla was added to a normal culture dish at a rate of about 3 to 10 (DMEM) + 10% (FB
Set aside during S). In this state, the hair was allowed to stand for 2 weeks without vibration in the presence of 5% CO ₂ , and Out hair was placed out of the implanted hair papilla.
Only the grown cells are subcultured into a 25 cm ² culture flask, and the culture supernatant is collected between 3 and 7 passages.

When collecting the culture supernatant, the cells in the confluent state are rinsed with DMEM containing no FBS for at least 1 hour, and then fresh DMEM (containing no FBS) is added thereto. ~ 18 hours)
After standing, the medium (serum-free DMEM) is collected and centrifuged at 10,000 rpm for 15 minutes with a cooling centrifuge, and the supernatant is collected and used. The culture supernatant can be used immediately after preparation, or can be used after being frozen and stored at, for example, -135 ° C and thawed as necessary.

Preparation of hair follicle melanocytes In the same manner as in the preparation of the dermal papilla, the hair follicles were isolated and 0.5% collagenase (type V, Sigma) was added to a petri dish.
a) Prepare 0.5 ml of (prepared in the primary culture medium), and transfer the hair follicle to it. Incubate at 37 ° C for 30 minutes.
Peel off the connective follicle root sheath using tweezers under a stereoscopic microscope. The isolated hair follicles were placed in PBS (-) and the final concentration was 0.
Add trypsin to 1% and incubate at 37 ° C for 10 minutes.

After centrifugation to remove trypsin, 2 ml of a medium is added and dispersed by pipetting. MGM4 (Clonetic) + 2% in collagen-coated incubator
Start the culture in FCS medium. The cells are mostly keratinocytes initially thought to be derived from the outer root sheath, but some of them begin to differentiate and detach in about 2 weeks. In that gap, melanocytes appear and occupy the majority of the petri dish within a few weeks.

Thereafter, the cells are transferred to a 25 cm ² culture flask, and thereafter, 2% FCS + MGM4 and HFM (Morinaga Chemical Laboratory) are mixed in equal amounts, and the culture is continued in a medium containing 1% FCS. . In this system, hair follicle-derived melanocytes can be obtained that grow exponentially in the range of about 1 × 10 ⁶ to 4 × 10 ⁶ about one month after passage. The average cell doubling time is about 7 days. Skin melanocytes are Clo
A normal skin melanocyte called “NHEM2486” purchased from Netich and cultured in the attached MGM3 medium was used.

Culture of melanocytes in the presence of test substances Skin melanocytes in logarithmic growth phase (NHEM248
6) were plated at a concentration of about ⁵ × 10 5 / 25cm ² flask, and cultured with MGM3 medium. 0.1% of test substance having anti-grey-hair effect (in this case, salmon extract)
(Equivalent to 2 ppm of the dried residue), and the cells were cultured for one week. Cells to which no test substance was added were similarly prepared and used as a control.

Screening operation In order to examine the migratory effect of the test substance on melanocytes, the culture medium of dermal papilla cells prepared as described above is brought into contact with the melanocytes treated with the test substance. In order to prevent the passive (diffusion) movement of melanocytes, the above contact is made by the porous membrane 3 as shown in FIG.
It is preferable to go through The pore size of the porous membrane is such that hair follicle melanocytes can be notified based on chemotaxis, and preferably about 8 to 12 μm.

Specifically, in FIG. 1, a well of a plate 1 having a well 2 is filled with a dermal papilla cell culture supernatant, a porous membrane 3 is covered thereon, and the well 2 is placed thereon.
A plate 5 having a through hole 4 is placed at a position compatible with the above, and a culture solution containing melanocytes is put into the through hole and allowed to stand. The standing is performed at about 35 to 38 ° C. for about 14 to 20 hours. Thereafter, the porous membrane is detached from the plate, and the melanocytes adhering to the membrane surface in contact with the dermal papilla cell culture supernatant may be observed with a microscope. In the above experiment, melanocytes cultured in the absence of the test substance may be used as a control, and the melanocytes attached to the membrane surface may be observed under a microscope in the same manner as above, and the two may be compared.

[0015]

Next, the present invention will be described more specifically with reference to examples. Embodiment 1 FIG . Confirmation of migratory activity of melanocytes (1) Preparation of human skin melanocytes Human skin melanocytes were obtained from Clonetech.
Cells designated NHEM2486 were purchased and used.

(2) Preparation of Human Hair Papillae Culture Supernatant Human hair papilla was collected from a hair follicle isolated from the scalp which was plastically surgically removed from a normal Japanese scalp. Human hair follicles were prepared as follows. A scalp section was used which was resected plastically from a normal Japanese head (mainly from a male occipital region). After the excision, the cells were stored on ice while immersed in a DMEM medium or the like, and processed on the same day. The following operations were performed in a clean bench under a stereoscopic microscope. The obtained scalp section was cut up and down under the dermis using a sharp scalpel, and the hair follicles were buried in the fat layer. Put it inside.

From the isolated human hair follicles, the papilla was collected as sharply as possible using sharp tweezers and the tip of a syringe needle. The collected hair papilla was allowed to stand in DMEM + 10% FBS at a ratio of about 3 to 10 in a normal culture dish. In this state, the sample was allowed to stand for 2 weeks without vibration in the presence of 5% CO ₂ (if any form of vibration was applied during the standing, the implantation rate of the dermal papilla decreased), and the sample was implanted. Only the cells grown outgrown from the dermal papilla were subcultured into a 25 cm ² culture flask, and the culture supernatant was collected between passages 3 and 7.

When collecting the culture supernatant, the cells in the confluent state are rinsed with DMEM containing no FBS for at least 1 hour, and then fresh DMEM (containing no FBS) is added thereto. ~ 18 hours)
After standing, the medium (serum-free DMEM) was collected and centrifuged at 10,000 rpm for 15 minutes with a cooling centrifuge, and the supernatant was fractionated and used. The culture supernatant was aliquoted and stored at −135 ° C., except when performing an experiment for measuring the difference in migration activity depending on the storage state and immediately measuring the migration activity.

(3) Confirmation of Melanocyte Migratory Property 28 μL of the culture supernatant of human dermal papilla cells collected using serum-free DMEM was placed in the lower chamber of a 48-well chemotaxis chamber. The lower layer chamber was covered with a 12 μm-hole polycarbonate filter with the glossy surface facing up, and after covering with plastic packing, the upper chamber was placed. It was confirmed that there were no bubbles between the liquid in the lower layer chamber and the filter. Later, the upper and lower chambers were screwed tightly.

Human skin melanocytes were detached from the incubator by trypsin treatment, treated with a corresponding trypsin inhibitor, centrifuged, and placed in serum-free DMEM at 350,000.
The cells were suspended at 0 cells / mL. The suspended cells were left at 37 ° C. until immediately before use. Add 43 μL of cell suspension to each chamber well (upper chamber). Spread Kim towel moistened with distilled water on an appropriately sized tupperware, put the whole chamber in it, occupy the lid of the tupper completely, treat the whole with ethanol for disinfection, and incubate at 37 ° C in 5% CO ₂ incubator. Overnight (average 16-18 hours).

The next day, the upper chamber was removed, and the filter was immersed in 70% ethanol for 7 minutes to fix the cells.
Then, the cells were immersed in distilled water for 7 minutes, and cells that had not migrated were removed with a scraper attached to the chemotaxis chamber (cells with a glossy surface). After the filter was dried at room temperature for about 1 hour, it was immersed in a 5% GIMZA solution for 20 minutes, washed with distilled water, and then dried again.

The whole filter was sealed in a slide glass, and the number of migrated cells was counted under an inverted microscope. Since the nucleus of the cells migrated by the liquid in the lower chamber has a blue-violet color, the number was defined as the number of migrated cells in each well. The N for each test was at least 3 wells, and the average value was calculated as the migration activity of the test. The surface of the filter was photographed as needed. As a control, the same experiment as above was performed using a fresh medium instead of the human dermal papilla cell culture supernatant.

The results are shown in FIG. 1 and FIG. In these photographs, the round points are the pores of the filter and the points without the oval indicate the Giemsa-stained nuclei of melanocytes. As shown in FIG. 1, when a human dermal papilla culture supernatant is used, the melanocytes present in the upper layer are transferred to the dermal papilla cell culture supernatant through the porous membrane, and the filter is in contact with the culture supernatant. While melanocytes adhered to the surface, as shown in FIG. 2, when a fresh medium was used, the melanocytes did not move, and as a result, a substance that attracted melanocytes was found in the culture supernatant of hair papilla cells. It was revealed that it was being produced.

Embodiment 2 FIG. Screening of anti-white hair agent (1) Preparation of salmon extract 500 g of salmon skin was soaked in ethanol at room temperature for one week, and ethanol in the extract was distilled off to obtain 9.7 g of salmon extract. .

(2) Treatment of melanocytes with salamander extract Human skin melanocytes (NHEM2486; from Caucasian skin; Clonet) were placed in two 25 cm ² flasks under the same conditions.
semi-confluent (obtained from ich company)
⁵ cells / flask), and 0.1% of the salmon extract (equivalent to 2 ppm of the dry residue) was added to one flask, and the other flask was added without the salmon extract. Cultured for one week.

Next, the migration ability of the human skin melanocytes was measured by the same method as described in Example 1. As a result, assuming that the migration ability (the number of cells passing through the porous membrane) of the skin melanocytes that were not treated with the salamander extract was 100, the migration ability of the skin melanocytes treated with the salamander extract (the porous membrane (The number of cells passed) was about 230, confirming that the salamander extract enhances the melanocyte migration ability. FIG. 6 shows the results.

(3) Effect of salamander extract on proliferation of human skin melanocytes Human skin melanocytes (NHEM2486; from Caucasian skin; obtained from Clonetech) were cultured in MGM3 medium at 1 × 10 ⁴ cells in a 96-well culture plate. Per well / well for 2 days, and MBM2 (Cloneti).
2% FBS, 5 μg / ml insulin, 0.5
μg / ml hydrocortisone, 50 μg / ml gentamicin, 50 μg / ml amphotericin-B)
For 2 days, and various amounts (0 to 10 ppm as dry residue concentration) of a salamander extract were added, followed by further culturing for 2 days. Assuming that the number of melanocytes in the case where the sansho extract was not added (addition amount 0 ppm) was 100, the relative number of melanocytes cultured by adding the sansho extract was as shown in FIG. The melanocyte proliferation-promoting effect of the product was observed.

(4) Effect of Salmon Extract on Tyrosinase Activity of Human Hair Follicle Melanocytes Human hair follicle melanocytes were prepared from human scalp as follows. After isolating the hair follicles in the same manner as in the preparation of the dermal papilla, 0.5% collagenase (type V,
Sigma) (prepared in the primary culture medium) was prepared, and the hair follicle was transferred thereto. Incubated at 37 ° C for 30 minutes. The connective woven root sheath was peeled off using tweezers under a stereoscopic microscope. The isolated hair follicle was placed in PBS (−), and trypsin was added to a final concentration of 0.1%.
Incubated at 10 ° C for 10 minutes. After centrifugation, trypsin was removed, and 2 ml of the medium was added and dispersed by pipetting.
MGM4 (Clone) in a collagen-coated incubator
cultivation was started in a 2% FCS medium.

The cells are mainly composed of keratinocytes which are considered to be originally derived from the outer root sheath, but a part of the cells began to differentiate and detach in about two weeks. In that gap, melanocytes appeared and occupied most of the petri dish within a few weeks. Thereafter, the cells were transferred to a 25 cm ² culture flask, and thereafter, equal amounts of 2% FCS + MGM4 and HFM (Morinaga Seikagaku Kenkyusho) were mixed, and culturing was continued in a medium containing 1% FCS. After culturing in this system for about one month after subculture, hair follicle-derived melanocytes that grow logarithmically in the range of about 1 × 10 ⁶ to 4 × 10 ⁶ could be obtained. The average cell doubling time is about 7 days.

NHEM2486 in a 96-well culture plate
The medium was seeded with 6000 cells / well of human hair follicle melanocytes, cultured for 2 days, and then cultured in MBM2 medium (Clone).
tich), 2% FBS, 5 μg / ml insulin,
Deplate in 0.5 μg / ml hydrocortisone, 50 μg / ml gentamicin, 50 μg / ml amphotericin-B) for 2 days and add the desired quantification (0 ppm (no addition) to 20 ppm) of sanshon extract. And two more
After culturing for days, tyrosinase activity was measured.

The measurement of tyrosinase was performed as follows. The remaining cells from which the culture solution was removed were washed twice with PBS (−), and the cells were added to 45 μl of 1% Triton X-10.
After adding 0 / PBS (-1) and shaking for 30 seconds, 5 μl of 10 mM DL-DPA / PBS (−)-T was added.
After adding riton, incubating at 37 ° C. for 1 hour, the absorbance at 475 nm was measured. FIG. 5 shows the relative activity of tyrosinase due to the addition of the salmon extract, where the tyrosinase activity when the salmon extract was not added (0 ppm) was 100. As is clear from this table, the salmon extract enhanced the activity of tyrosinase, an enzyme of the melanin biosynthesis system.

(5) Gray Hair Prevention Effect of Salmon Extract As a test subject, forty four men and women of 40 to 60 years old who have white hair for each sample, twice a day (morning and night) for four consecutive months, as shown in Table 1. And the lotion without sansho extract (control) was used separately on the left and right scalp by the half-head method. investigated. While applying this lotion every day, the ratio of preventing the generation of gray hair was determined based on the amount of hair 1
The number of white hairs per 000 hairs was counted.

[0033]

[Table 1]

Production Method A salmon extract, a polyvinylpyrrolidone / vinyl acetate copolymer, a preservative, and a fragrance were added to ethyl alcohol and uniformly dissolved. The aqueous phase (purified water, silicone derivative, glycerin) previously dissolved therein was added thereto and dissolved.

[0035]

[Table 2] The results of this test are shown in the following table.

[0036]

[Table 3] As described above, the melanocyte migration ability enhancing effect of the test substance is well correlated with the anti-grey-hair property, and it is clear that screening of the anti-white hair agent is possible by measuring the melanocyte migration ability-enhancing action of the test substance. It became.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of an apparatus used in the method of the present invention.

FIG. 2 is a micrograph showing that melanocytes show migration to the culture supernatant of dermal papilla cells, and a drawing substitute photograph showing the form of an organism.

FIG. 3 is a micrograph showing that melanocytes do not migrate to a fresh medium, and is a drawing substitute photograph showing the form of an organism.

FIG. 4 is a graph showing the effect of salmon extract on skin melanocyte proliferation.

FIG. 5 is a graph showing the effect of sanshon extract on tyrosinase production of hair follicle melanocytes.

FIG. 6 is a graph showing the effect of the extract of salmon extract for enhancing melanocyte migration.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Akihiro Ishino 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture Inside Shiseido Daiichi Research Center Co., Ltd. (72) Inventor Yoshiki Moriya 2-12-, Fukuura, Kanazawa-ku, Yokohama-shi, Kanagawa 1 Shiseido Second Research Center Co., Ltd. (72) Inventor Yuki Yamase 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture In-house Shiseido First Research Center (72) Inventor Yoshiharu Tsuji Nippa-cho, Kohoku-ku, Yokohama, Kanagawa 1050 Shiseido Daiichi Research Center Co., Ltd. (72) Inventor Norio Kojima 7-5-5 Ginza, Chuo-ku, Tokyo Shiseido Co., Ltd. (72) Inventor Eiji Ifuku Niiwa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture 1050 Shiseido Daiichi Research Center Co., Ltd. (72) Inventor Naomi Tanaka 105 Niwamachi, Kohoku-ku, Yokohama, Kanagawa 0 F-term in Shiseido Daiichi Research Center (reference) 2G045 AA40 BA14 BB04 BB20 CB01 CB16 FA16 GC01 GC30

Claims (2)

[Claims] In a method for screening an anti-grey-hair agent, melanocytes are cultured in the presence of a test substance, the cultured melanocytes are brought into contact with a culture supernatant of hair papilla cells, and the melanocytes are cultured in the culture supernatant. A method of observing whether or not to move toward, or the degree of movement. 2. The cultured melanocytes and the culture supernatant of dermal papilla cells are brought into contact with each other via a porous membrane, and whether the cultured melanocytes migrate to the culture supernatant through pores of the membrane. 3. The method of claim 2, wherein the degree of migration is monitored.