JP4617183B2 - Screening method for chemical peeling agent - Google Patents

Description

  The present invention relates to a method for detecting an epidermal cell and dermal fibroblast proliferation action, and further relates to a method for screening a chemical peeling agent capable of improving skin wrinkles, spots, kumi, acne and the like.

  Chemical peeling is to apply a drug to the skin and peel the surface by its action. Known chemical peeling agents (chemical peeling agents) include, for example, glycolic acid, salicylic acid, trichloracetic acid, and lactic acid. Then, skin peeling such as wrinkles, stains, rashes and acne is performed by promoting the regeneration of the skin through subsequent wound treatment and accompanying inflammatory reaction using chemical peeling.

  Although chemical peeling is known for its action, there is still little knowledge about the mechanism. Non-Patent Document 1 discloses that cytokine IL-1α released from keratinocytes promotes collagen synthesis in dermal fibroblasts in chemical peeling using α-hydroxyacetic acid (glycolic acid). Patent Document 1 discloses that α-hydroxyacetic acid grows dermal fibroblasts, but does not disclose the relationship with chemical peeling and the mechanism thereof.

  Meanwhile, it has recently been found that mammalian skin cell membranes have equivalents to receptors for various neuronal information transmitters found in nerve cell membranes. However, although the mechanism of information transmission in the nervous system has been elucidated considerably, the function of these receptors in skin cells has hardly been studied.

For example, in 1997, Caterina et al. Cloned a VR1 receptor, which is a neuronal receptor (hereinafter referred to as VR1) (non-patent document 2). VR1 is also called a capsaicin receptor, and is a cation channel TRPV1 having six transmembrane domains structurally belonging to the TRP (Transient Receptor Potential) channel family. VR1 is activated by capsaicin, which is a component of red pepper fruit, and heat and protons generated as a result of tissue damage, and causes cations, mainly calcium ions, to be transported. The ions flow toward the lower concentration gradient, causing depolarization first and release of neurotransmitters from nerve endings. This VR1 is also present in epidermal cells, and it has been clarified that the calcium ion concentration in the epidermal cells varies due to VR1 stimulation (Patent Document 2). However, no knowledge has been obtained that VR1 of epidermal cells is associated with proliferation of epidermal cells and dermal fibroblasts.
JP-A-5-112422 JP 2002-372530 A Exp Dermatol, 12 (Supply 2), 57-63, 2003.Okano Y, Funasaka Y. et al. Nature 389, 816-824, 1997. Caterina MJ, Schumacher MA, TominaGA M, Rosen TA, Levine JD, Juius D.

  As mentioned above, little is known about the chemical peeling mechanism. The present inventors considered that if the mechanism of epidermal cell and dermal fibroblast proliferation can be clarified, the epidermal cell and dermal fibroblast proliferation action of the test substance can be easily and rapidly detected. .

  Therefore, the present invention clarifies the mechanism of epidermal cells and dermal fibroblast proliferation by chemical peeling agents, and can easily and rapidly detect the epidermal cells and dermal fibroblast proliferation action of the test substance. It is an object of the present invention to provide a novel method for detecting epidermal cells and dermal fibroblast proliferation and a method for screening chemical peeling agents.

As a result of intensive studies in view of the above problems, the inventors succeeded in elucidating the mechanism of epidermal cell and dermal fibroblast proliferation by glycolic acid (hereinafter referred to as GA), which is a typical chemical peeling agent. The mechanism is “GA stimulates VR1 of epidermal cells → Ca ²⁺ inflows into epidermal cells → ATP is released from epidermis → epidermal cells proliferate / IL-1α is released from epidermis → dermal fibroblasts proliferate” Is. It has not been reported so far that the activation of VR1 by stimulation of acid or the like promotes the proliferation of epidermal cells and dermal fibroblasts, and the relationship between VR1 and the proliferation of epidermal cells and dermal fibroblasts It was particularly important to elucidate the above mechanism.

  And by elucidating this mechanism, the activity of VR1 in epidermal cells was evaluated, and it was found that a candidate for a chemical peeling agent could be selected, and the present invention was completed. That is, the present invention is as follows.

The screening method of claim 1, wherein the chemical peeling agent of the present invention, in contact with the epidermal cells harvested or cultured with the test substance, and evaluating the variation of the concentration of calcium ions in VR1 activity cells of epidermal cells as an indicator a substance that gives VR1 activity epidermal cells, characterized in that it comprises a step of selecting as a candidate of the chemical peeling agent.

According to the screening method of claim 1, wherein the chemical peeling agent of the present invention, to evaluate the VR1 activity, by selecting a substance to be a candidate of the chemical peeling agent, conventionally referred to will be selected from a large number of compounds Compared with this method, a chemical peeling agent having an effective action can be found quickly and accurately. In addition, a substance having a strong chemical peeling action can be selected from a substance that provides high VR1 activity, and a substance having a low chemical peeling action can be selected from a substance that provides low VR1 activity. Therefore, by evaluating VR1 activity, screening of chemical peeling agents according to the purpose such as strengthening chemical peeling action or weakening chemical peeling action to suppress skin irritation, tingling sensation and pain by chemical peeling agent. it can.

  Hereinafter, the present invention will be described in detail.

The present invention is based on the mechanism of proliferation of epidermal cells and dermal fibroblasts by GA, which has been elucidated by the inventors. The mechanism is “GA stimulates VR1 of epidermal cells → Ca ²⁺ inflows into epidermal cells → ATP is released from epidermis → epidermal cells proliferate / IL-1α is released from epidermis → dermal fibroblasts proliferate” In a series of steps, VR1 is activated by the stimulation of GA, whereby proliferation of epidermal cells and dermal fibroblasts is promoted.

  The method for detecting the epidermal cell and dermal fibroblast proliferation action of the present invention utilizes the above-described VR1-mediated proliferation mechanism of epidermal cells and dermal fibroblasts, and the epidermis of the test substance brought into contact with the epidermal cells. The cell and dermal fibroblast proliferation action is correlated with the VR1 activity of epidermis cells to detect the epidermal cell and dermal fibroblast proliferation action of the test substance. Alternatively, when VR1 activity of epidermal cells is recognized by contact with the test substance, it is determined that the test substance has a proliferating action on epidermal cells and dermal fibroblasts.

  Here, the epidermal cells are cells that form the epidermis part of the skin. The epidermal cells may be cultured epidermal cells. Keratinocytes, which are one of the epidermal cells, are suitable in the present invention because the cultured cells are easily available. The origin of epidermal cells is not limited, and a wide range of animals such as humans and mice can be applied. Also, dermal fibroblasts are not limited to specific ones.

  Although the method for evaluating VR1 activity in the present invention is not limited to a specific method, for example, the activity of VR1 can be evaluated using fluctuations in intracellular calcium ion concentration as an index. As a method for detecting the fluctuation of the calcium ion concentration, a conventionally known method can be used. For example, a reagent (calcium indicator) that is colored according to the calcium ion concentration is contacted with epidermal cells. Then, by evaluating the change in the color, a variation in the calcium ion concentration can be detected. As calcium indicators, Fura-2AM, Indo-1, Fluo-3, Rhod-2, etc. whose fluorescence intensity is changed by binding with calcium are known. Moreover, calcium sensitive photoproteins, such as aequorin, can also be used as a calcium indicator.

  As described above, when the calcium ion concentration in the epidermal cell is increased by combining the calcium ion concentration in the epidermal cell and the VR1 activity by the method of detecting the fluctuation of the calcium ion concentration, it is determined that the VR1 activity is present. can do. It can also be determined that the greater the increase in the calcium ion concentration in the epidermal cells, the higher the VR1 activity, and the lower the increase in the calcium ion concentration in the epidermal cells, the lower the VR1 activity.

  Then, by correlating the epidermal cell and dermal fibroblast proliferation action of the test substance brought into contact with the epidermal cells with the VR1 activity of the epidermis cell, the epidermal cell and dermal fibroblast proliferation action of the test substance can be easily and quickly performed. Can be detected. It should be noted that the higher the VR1 activity in the epidermal cells, the larger the epidermal cell and dermal fibroblast proliferation action of the test substance, and the lower the VR1 activity in the epidermal cell, the proliferative action of the test substance epidermal cells and dermal fibroblasts. Becomes smaller.

  Next, the screening method for the chemical peeling agent of the present invention will be described. The screening method for the chemical peeling agent of the present invention uses the above-described method for detecting the epidermal cell and dermal fibroblast proliferation action, and includes a step of selecting a substance that is a candidate for the chemical peeling agent.

  First, a test substance is brought into contact with epidermal cells. Next, VR1 activity of epidermal cells is evaluated. For evaluation of VR1 activity, variation in intracellular calcium ion concentration can be used as an index. As described above, the higher the VR1 activity in the epidermal cells, the greater the epidermal cell and dermal fibroblast proliferation action of the test substance, and the lower the VR1 activity in the epidermal cells, the lower the epidermal cell of the test substance. And the dermal fibroblast proliferation action is reduced. Then, based on the VR1 activity of epidermal cells, a substance that is a candidate for a chemical peeling agent is selected.

  Here, when the activity of VR1 is high, the substance has a strong chemical peeling action, and when the activity of VR1 is low, the substance has a weak chemical peeling action. Therefore, by evaluating the activity of VR1, a chemical peeling agent according to the purpose such as strengthening the chemical peeling action or weakening the chemical peeling action to suppress skin irritation, tingling sensation, and pain due to the chemical peeling agent. Can be screened.

  Furthermore, the method for activating epidermal cells and dermal fibroblasts of the present invention uses the chemical peeling agent selected by the screening method for chemical peeling agents of the present invention, and activates epidermal cells and dermal fibroblasts. be able to. And the activation of the epidermis cell and dermal fibroblast can be adjusted by selecting the chemical peeling agent suitable for the objective.

  The method for activating epidermal cells and dermal fibroblasts of the present invention provides VR1 activity to epidermal cells. By giving VR1 activity to the epidermal cells, activation of the epidermal cells and dermal fibroblasts can be achieved reliably, contributing to the proliferation of the epidermal cells and dermal fibroblasts. The means for providing VR1 activity to epidermal cells is not limited to a specific means.

  The chemical peeling agent of the present invention utilizes the growth mechanism of epidermal cells and dermal fibroblasts via VR1, and contains a component that imparts VR1 activity to epidermal cells. By containing a component that gives VR1 activity to epidermal cells, it is possible to reliably achieve activation of epidermal cells and dermal fibroblasts. Then, by applying the chemical peeling agent of the present invention to the skin, epidermal cells and dermal fibroblasts proliferate, and it is possible to improve skin wrinkles, stains, wrinkles, acne and the like.

  Furthermore, the present invention provides an apparatus (not shown) for detecting epidermal cell and dermal fibroblast proliferation action. The detection apparatus includes detection means for detecting VR1 activity in epidermal cells. Any detection means may be used as long as it can detect the VR1 activity, and the detection means is not limited to a specific one. However, since the VR1 activity and the calcium ion concentration are related, the detection means is the epidermis. What detects the fluctuation | variation of the calcium ion concentration in a cell is preferable.

  More specifically, for example, the detection means can be configured by an apparatus capable of measuring the fluorescence intensity ratio. In this case, a variation in the calcium ion concentration can be easily detected by contacting a known calcium indicator and epidermal cells and measuring the change in the fluorescence intensity ratio.

  Furthermore, the detection apparatus for epidermal cell and dermal fibroblast proliferation action according to the present invention is a determination means for determining that an epidermal cell has VR1 activity when the fluctuation of the calcium ion concentration detected by the detection means exceeds a predetermined value. It is equipped with. The determination means receives a detection signal from the detection means, and determines the presence or absence of VR1 activity of the epidermis cells based on the magnitude of fluctuation of the detection signal, and can be configured by a computer program. In addition, you may further provide display means, such as a display, which displays the determination result by this determination means.

  Thus, according to the detection apparatus for the epidermal cell and dermal fibroblast proliferation action of the present invention, the epidermal cell and dermal fibroblast proliferation action can be easily and quickly detected by detecting the VR1 activity in the epidermis cell. can do. Moreover, it can be easily determined that the epidermal cell has VR1 activity by determining that the epidermal cell has VR1 activity when the fluctuation of the calcium ion concentration detected by the detection means exceeds a predetermined value. .

  Hereinafter, specific examples will be described, but the present invention is not limited to the following examples.

(Experiment 1) GA application experiment to mouse skin Objective: To examine changes in epidermal growth activity and skin tissue morphology by GA application.

  Method: The back of a normal hairless mouse (HR-1) was divided into right and left with a midline, 50 μl of 40% (w / v) GA solution was applied to only one side for 10 minutes, and then washed with warm water. Four types of pH of 1.3, 2.0, 2.4, and 2.8 were used for GA. This application was repeated every 3 to 4 days for a total of 3 times. The skin was collected 48 hours after the last application. In addition, as an artificial skin barrier function destruction model, another mouse was subjected to tape stripping (repeating the cellophane tape and peeling work four times), and the skin was collected after 2 hours. As a control for each mouse, the uncoated skin was collected. These skin tissues were embedded in a paraffin block according to a conventional method, and sliced sections were prepared. This section was stained with HE (hematoxylin-eosin) to compare the morphology of the tissues.

  Results: FIG. 1 is an HE-stained image 48 hours after GA three times application. In each HE-stained image, the slightly darker colored upper portion is the epidermis and the lower portion is the dermis. Experiment 1 revealed the difference between tape stripping causing inflammation and chemical peeling by GA. In both cases, epidermal thickening was observed and the proliferation activity of epidermal cells was increased, but the action of GA was weaker. The effect of epidermal thickening was stronger as the pH of GA was lower. No inflammation was caused by GA (pH 2.8 to 2.0). Tape stripping destroys the skin barrier, but GA has little change in the amount of stratum corneum moisture evaporation, and hardly destroys the skin barrier. From the above results, it was confirmed that GA has the action of promoting mild epidermal cell proliferation without causing inflammation or skin barrier destruction.

(Experiment 2) GA application experiment to test skin Objective: To analyze changes in cell proliferation activity after GA application to test skin and its mechanism.

  Method: The test skin is a three-dimensional skin model consisting of a human skin epidermis keratinocyte (an epidermal keratinocyte) and a collagen gel embedded with dermal fibroblasts. Has a thickness closer to that of human skin than mouse, and it is easy to measure information-transmitting substances while culturing. A stratum corneum is also formed on the epidermis, and it is possible to apply and remove the drug.

  Therefore, Toyobo test skin (LSE-High) was cultured according to the attached protocol. The attached silicon ring is mounted on the epidermis, and 200 μl of 40% GA solution (pH 1.3, 2.0, 2.4, 2.8) is put in the center of the ring and after a certain time (1.5 minutes, 3 Min, 5 min), and GA was removed by suction. Further, the surface was washed three times with a culture solution to completely remove water by suction, and the culture was continued. The culture medium was collected 10 minutes and 1 hour after the GA treatment, and replaced with a new culture medium.

  Subsequently, the culture solution and test skin were collected after 24 or 48 hours. Two hours before harvesting, BrdU (5-bromo-2'-deoxy-uridine) was added to the culture (final concentration 50 μM) to label the DNA of replicating cells. The collected culture medium was used for measurement of ATP and cytokine. For ATP, ATP Bioluminescence Assay Kit CLS II manufactured by Roche Diagnostics, ATP Bioluminescent Assay Kit manufactured by Sigma, ATP Determination Kit manufactured by Molecular Probes, etc. were used for quantification according to the luminescence method according to the protocol of each company. . IL-1α and FGF-7 were quantified by ELISA using a Quantikine Immunoassay Kit manufactured by R & D Systems. The center of the test skin was collected with a biopsy punch having a diameter of 8 mm and embedded in a paraffin block according to a conventional method to prepare a sliced slice. This section was immunostained with an anti-BrdU antibody, and the number of cell nuclei labeled with BrdU was counted as an index of cell proliferation activity.

  Capsazepine was added to the culture solution in an experiment to examine the influence of the VR1 antagonist, and capsaicin was added to the culture solution in an experiment to examine the influence of the VR1 agonist, respectively, so that the GA treatment was performed. In order to examine the influence of only the addition of each drug, an experiment was similarly performed on a drug that was added to the culture solution and was not subjected to GA treatment.

  In this experiment, a method for measuring the number of BrdU positive epidermal cells and dermal fibroblasts was used as a method for evaluating cell proliferation. BrdU is incorporated into replicating DNA as an analog of thymidine and is used to identify proliferating cells. When the tissue is collected 1-2 hours after adding BrdU and stained with an antibody against BrdU, the nucleus of the proliferating cell is stained. The more nuclei, the higher the proliferation activity.

result:
(1) Cell proliferation activity (number of BrdU positive cells) by GA (pH 2.8)
FIG. 2 shows an anti-BrdU stained image 24 hours after application of GA (pH 2.8), and FIG. 3 shows the number of BrdU positive cells 24 hours after application of GA (pH 2.8). FIG. 4 shows the effect of VR1 antagonists on epidermal proliferation activity.

  As shown in FIG. 2, the number of cells that incorporated BrdU and stained by GA application increased. In FIG. 2, in each anti-BrdU stained image, the upper band-shaped portion corresponds to the epidermis and the lower portion corresponds to the dermis, and the stained cells are dark dots as indicated by arrows. Is appearing. Moreover, as shown in FIG. 3, the number of BrdU positive cells in the epidermis increased by GA application. In particular, it significantly increased (p <0.05) after treatment with GA for 1.5 minutes. In addition, the number of BrdU positive cells was also increased by GA in dermal fibroblasts. From these results, it was confirmed that the growth activity of epidermal cells and dermal fibroblasts was enhanced by GA. Furthermore, as shown in FIG. 4, the increase in the number of BrdU positive cells was suppressed by capsazepine, a VR1 antagonist.

(2) Quantification of free ATP in medium FIG. 5 shows the release of ATP into the medium by application of GA, and FIG. 6 shows the influence of VR1 antagonists and agonists on the release of ATP. As shown in FIG. 5, it was found that ATP was released into the medium by application of GA. ATP was released most by 10 minutes after GA treatment, and the amount of ATP reached a high level at pH 1.3. Further, as shown in FIG. 6, it was found that ATP release by GA was inhibited by capsazepine as a VR1 antagonist and capsaicin as a VR1 agonist.

(3) Quantification of free IL-1α and FGF-7 in the medium FIG. 7 shows IL-1α release and FGF-7 release into the medium by GA application. As shown in FIG. 7, it was confirmed that IL-1α and FGF-7 were released into the medium by application of GA. Release of IL-1α and FGF-7 increased not after the addition of GA but after 24 hours.

  It has been reported that when ATP is given to keratinocytes, cytokine production such as IL-1α is promoted via P2 receptor (Japanese Patent Application No. 2004-074638), and IL-1α and FGF-7 are involved in the epidermis-dermis interaction. Have been reported to promote each other's growth (Szabowski A et al., Cell 103, 745-755, 2000). Furthermore, it has been reported that low concentrations of ATP have a cell proliferating action in keratinocytes (Aina V. H. Grcig et al., J. Invest. Dermatol. 120, 1007-15, 2003). Therefore, it is considered that after the release of ATP, IL-1α is released from the epidermis and FGF-7 is released from the dermis, thereby promoting the proliferation of epidermal cells and dermal fibroblasts.

(4) Summary From the above results (1), (2), (3) and known literature, it was confirmed that GA promotes the proliferation of epidermal cells and dermal fibroblasts via VR1. In addition, ATP is involved in the action, and it is found that IL-1α is released from the epidermis and FGF-7 is released from the dermis by the release of ATP, thereby promoting the proliferation of epidermal cells and dermal fibroblasts. It was.

  In addition, as shown in FIG. 6, it is thought that it is because of desensitization that ATP release by GA is inhibited by capsaicin which is a VR1 agonist. Desensitization is also found in nerves that have been exposed to capsaicin for a long time, and muscle paints have been developed using this.

(Experiment 3) Intracellular calcium influx of epidermal keratinocytes by GA Objective: To examine whether VR1 as a calcium channel opens at the cellular level using calcium influx as an index.

Method: Performed according to the method of Inoue et al. (Inoue K., et.al., BBRC 291, 124-129, 2002). Here is an outline. Normal human epidermal keratinocytes were purchased from Kurabo Industries and cultured according to the attached protocol. When 8-well-chamber slide is seeded with keratinocytes and becomes 80-90% confluent, it is equilibrated with a culture solution containing 10 μM of calcium indicator (Fura-2AM; manufactured by Molecular Probes), and buffered BSS (Balanced salt solution) Washed with NaCl 150 mM, KCl 5 mM, CaCl ₂ 1.8 mM, NaH ₂ PO ₄ 1.2 mM, MgCl ₂ 1.2 mM, D-glucose 10 mM, HEPES 25 mM, adjusted to pH 7.4 with NaOH), and inverted fluorescence microscope Fluorescence images (wavelength 340 nm, 380 nm) were recorded over time while perfusing underneath, and the fluorescence intensity ratio was calculated. As the acid solution such as GA, an acid solution obtained by dissolving 100 to 500 mM of acid in BSS composition excluding HEPES and adjusting the pH with NaOH was used. Stimulation with an acid was performed by flowing an acid solution for 5 to 10 seconds, and the same cells were stimulated twice. In experiments to examine the effects of VR1 antagonists, after perfusion with BSS containing 10 μM capsazepine, stimulation was similarly performed with an acid solution containing 10 μM capsazepine.

  Results: FIG. 8 is a graph showing changes in intracellular calcium ions (fluorescence intensity ratio) over time. It was found that calcium ion influx occurred immediately after GA was directly applied to keratinocytes, and the influx was inhibited by capsazepine, a VR1 antagonist. From this result, it was confirmed that the receptor channel for GA on keratinocytes is VR1.

(Experiment 4) Detection of ATP release from human skin Objective: To confirm that ATP is released immediately after GA is applied to human skin, ex vivo.

  Method: The surplus skin from the plastic surgery of the face was used. The skin was cut into 1 × 1.5 cm squares after excision and divided into a control (only the washing operation without GA application) group and a GA application group. In the GA application group, a 40% GA solution (pH 2.8) was applied on the epidermal stratum corneum for 3 minutes. Thereafter, the GA solution was wiped off, washed with PBS, floated with the stratum corneum on a petri dish containing BSS, and ATP in BSS was measured after 10 and 25 minutes. The measurement method was the same as that described in Experiment 2.

  Results: FIG. 9 is a graph showing ATP release from human skin by GA application. Even in human skin, ATP was released within 10 minutes immediately after GA application.

From the results of Experiments 1 to 4, “GA stimulates VR1 of epidermal cells → Ca ²⁺ inflows into epidermal cells → ATP is released from epidermis → epidermal cells proliferate / IL-1α is released from epidermis → dermal fibroblasts” It was confirmed that epidermal cells and dermal fibroblasts proliferate by the mechanism of “cell proliferation”.

It is a HE dyeing | staining image 48 hours after the application | coating of GA3 times in experiment 1. FIG. NT indicates control and T indicates treatment with GA. It is an anti-BrdU dyeing | staining image 24 hours after GA (pH 2.8) application | coating in Experiment 2. FIG. 6 is a graph showing the number of BrdU positive cells 24 hours after application of GA (pH 2.8) in Experiment 2. 2 is a graph showing the influence of a VR1 antagonist on epidermal proliferation activity in Experiment 2. GA treatment time 3 minutes, pH 2.8. 6 is a graph showing ATP release into the medium by GA application in Experiment 2. FIG. The figure on the left shows GA at pH 2.8, and the figure on the right shows a treatment time of 3 minutes. 6 is a graph showing the influence of VR1 antagonists and agonists on ATP release in Experiment 2. GA treatment time 3 minutes, pH 2.4. It is a graph which shows IL-1 (alpha) and FGF-7 release | release in the culture medium by GA application | coating in the experiment 2. FIG. It is a graph which shows the change of inflow of the calcium ion in the keratinocyte by GA (pH 2.8) addition in Experiment 3. The horizontal axis represents time and the vertical axis represents the fluorescence intensity ratio. The larger the fluorescence intensity ratio, the greater the inflow of calcium into the keratinocytes. GA was allowed to act for the time indicated by the arrow. 6 is a graph showing ATP release from human skin by GA application in Experiment 4.

Claims (1)

A test substance is brought into contact with the collected or cultured epidermal cells, and the change in calcium ion concentration in the VR1 active cells of the epidermal cells is evaluated as an index. A substance that imparts VR1 activity to the epidermal cells is selected as a chemical peeling agent candidate. screening how the chemical peeling agent characterized by comprising the step of selecting as a.