WO2022138350A1 - Method for promoting regeneration of dermis by applying high-frequency electric stimulus - Google Patents

Method for promoting regeneration of dermis by applying high-frequency electric stimulus Download PDF

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WO2022138350A1
WO2022138350A1 PCT/JP2021/046152 JP2021046152W WO2022138350A1 WO 2022138350 A1 WO2022138350 A1 WO 2022138350A1 JP 2021046152 W JP2021046152 W JP 2021046152W WO 2022138350 A1 WO2022138350 A1 WO 2022138350A1
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high frequency
skin
stem cells
applying
electrodes
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PCT/JP2021/046152
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French (fr)
Japanese (ja)
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みなみ 山田
俊介 入山
勤 相馬
謙太朗 山▲崎▼
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株式会社 資生堂
ヤーマン株式会社
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Priority to CN202180078685.XA priority Critical patent/CN116669645A/en
Priority to JP2022572211A priority patent/JPWO2022138350A1/ja
Publication of WO2022138350A1 publication Critical patent/WO2022138350A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B18/00—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body
    • A61B18/04—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by heating
    • A61B18/12—Surgical instruments, devices or methods for transferring non-mechanical forms of energy to or from the body by heating by passing a current through the tissue to be heated, e.g. high-frequency current
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/18—Applying electric currents by contact electrodes
    • A61N1/32—Applying electric currents by contact electrodes alternating or intermittent currents

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  • the present invention relates to a beauty method using high frequency and a method for promoting dermis regeneration.
  • a high frequency beauty method is known that can improve wrinkles and sagging of the skin by passing a high frequency (RF) current to the skin such as the face and limbs.
  • RF high frequency
  • a high-frequency current is applied to the skin, the temperature of the dermis layer and subcutaneous tissue rises. It is expected that the heating action promotes the flow of blood and promotes the regeneration of collagen in the dermis layer by causing heat damage to the collagen in the dermis layer.
  • various beauty treatment devices using such high frequencies have been proposed (for example, Patent Document 1).
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2018-489
  • the conventional high-frequency cosmetology method promotes blood circulation and collagen regeneration based on the heating action of the dermis layer and subcutaneous tissue by high frequency, and is a temporary treatment. With increasing awareness of health in recent years, more fundamental improvement of function at the constitutional level is desired.
  • One of the objects of the present invention is to provide a cosmetic method based on the improvement of the stability of dermal stem cells obtained by applying high frequency to the skin.
  • a cosmetic method comprising applying high frequency to the skin to enhance the stability of dermal stem cells. Further, according to one aspect of the present invention, there is provided a cosmetic method comprising applying a high frequency to the skin to enhance the gene expression of ITGA6 in dermal stem cells. Further, according to one aspect of the invention, there is provided a cosmetic method comprising applying high frequency to the skin to enhance the production of V-type collagen and / or fibrillin-1 in dermal fibroblasts. Further, according to another aspect of the present invention, there is provided a method for stabilizing dermal stem cells, which comprises the step of applying high frequency to the skin.
  • Yet another aspect of the invention provides a method of enhancing ITGA6 gene expression in dermal stem cells, comprising the step of providing high frequency to the skin.
  • a method for enhancing the production of V-type collagen and / or fibrillin 1 in dermal fibroblasts which comprises a step of providing a high frequency to the skin.
  • a method of operating a high frequency beauty treatment apparatus having a pair of electrodes and a control circuit for applying a high frequency voltage to the pair of electrodes, according to the control circuit.
  • the operation of the high-frequency beauty treatment apparatus which comprises applying a high-frequency voltage to the pair of electrodes to enhance the stability of the dermal stem cells, and the application of the high-frequency voltage is performed in a state where the pair of electrodes are in contact with the skin.
  • the method is provided, According to still another aspect of the present invention, there is a method of operating a high frequency beauty processing apparatus having a pair of electrodes and a control circuit for applying a high frequency voltage to the pair of electrodes, wherein the pair of electrodes is used.
  • the application of the high frequency voltage comprises applying to the electrodes of the high frequency voltage which enhances the gene expression of ITGA6 in the dermal stem cells and / or the production of V-type collagen and / or fibrillin 1 in the dermal fibroblasts.
  • a method of operating a high frequency beauty treatment apparatus which is performed with a pair of electrodes in contact with the skin.
  • applying high frequency and “applying high frequency” mean applying a high frequency voltage to an object by applying a high frequency voltage between a pair of electrodes arranged in contact with the object at intervals. It means that a high frequency current is passed.
  • the word "about” used to indicate a numerical value means that it includes a numerical value of 5% before and after the numerical value modified by this word.
  • the present invention states that the application of high frequency (RF) to the skin greatly contributes to the improvement of the stability of dermal stem cells, for example, the improvement of the stability of dermal stem cells by enhancing the gene expression of ITGA6 in the dermal stem cells.
  • a cosmetic method including applying a high frequency to the skin to enhance the stability of dermal stem cells.
  • Increased ITGA6 gene expression improves dermal stem cell stability (eg, dermal stem cells are anchored near blood vessels), with concomitantly enhanced production of type V collagen and / or fibrillin-1 in dermal fibroblasts. .. Stabilization of dermal stem cells also affects skin tension, and application of RF currents to the skin contributes to skin tension.
  • the frequency of the high frequency applied to the skin is in the range of 0.5 MHz to 4 MHz, for example, 0.5 MHZ, 1 MHz, 2 MHZ, 3 MHz or 4 MHz, or the range defined by any of the above frequencies (eg, for example. Any frequency included in the range of 1 to 4 MHz) may be used. In some embodiments of the invention, the frequency is about 1 MHz.
  • the dermal stem cells that exist around the blood vessels work as a control tower that enhances the self-renewal power of the skin.
  • Integrin ⁇ 6 (ITGA6) plays a role in anchoring dermal stem cells to blood vessels via the basement membrane of blood vessels. Increased expression of ITGA6 stabilizes stem cells with increased adhesiveness around blood vessels, and as a result, promotes ECM production.
  • the application of high frequency to the skin may be combined with the application of drugs to the skin and various extracts.
  • applicable drugs and various extracts include inositol, retinol derivative, xylitol, hyaluronic acid, glycerin, yeast extract, cherry leaf extract, keihi extract, pine extract, bulgarian rose water, okra extract, ibukijakou extract, waremokou extract, and glycyrrhizin.
  • Dipotassium acid Dipotassium acid, Ginkgo biloba extract, Tencha extract, Yokuinin extract, Kanzo extract, Otaneninjin extract, Clara extract, Star fruit leaf extract, Carrot extract, Juyaku extract, Neem leaf extract, Saffron extract, Kina extract, Confree extract, Thyme extract, Fuyu strawberry , Yomena, Mube, Ryukyu Strawberry, Indian Yomena, Matebashii, Ogon Extract, Rosemary Extract, Anzu Nucleus Granules, Gentiana Extract, Hakka Powder, Taisou Extract, Hop Extract, Kiwi Extract, Roman Camille Extract, Apple Extract, Sanzashi Extract, Examples include Ukon extract, Yamamomo, Futomomo, and Konara. These can be used alone or in combination.
  • a high frequency device can be used to apply high frequency to the skin.
  • a form of the high frequency device will be described with reference to FIG.
  • the high frequency device 1 has an electrode unit 10 and a control circuit 20.
  • the electrode unit 10 has a first electrode 10a and a second electrode 10b connected to the control circuit 20.
  • a contact surface that comes into contact with the user's skin is provided on the electrode portion 10, and the first electrode 10a and the second electrode 10b are arranged at intervals from each other on the contact surface, whereby the first electrode 10a and the second electrode 10b are provided. They can be easily brought into contact with the skin at intervals.
  • the first electrode 10a and the second electrode 10b may be connected to the control circuit 20 via a wiring cable instead of the contact surface, whereby the first electrode 10a and the second electrode 10b can be freely arranged. can do.
  • the control control circuit 20 can have a power supply unit, a voltage control unit, and a resistance measurement unit.
  • the power supply unit applies a high frequency voltage (voltage indicating a high frequency) between the first electrode 10a and the second electrode 10b.
  • the power supply unit has an oscillation circuit that generates a high frequency voltage, a booster circuit that boosts the oscillated voltage, and the like.
  • a high frequency voltage of about 0.5 MHz to 4 MHz is applied to the first electrode 10a and the second electrode 10b. It is configured to apply between.
  • the voltage control unit controls the high frequency voltage applied by the power supply unit by controlling the power supply unit. Further, the voltage control unit uses the measurement result by the resistance measurement unit for voltage control.
  • the resistance measuring unit measures the resistance value (electrical resistance value) between the first electrode 10a and the second electrode 10b when the first electrode 10a and the second electrode 10b are in contact with the user's skin.
  • This resistance value changes depending on the condition of the user's skin and the type of external skin preparation such as lotion applied to the skin. For example, it is known that when a high-frequency current is passed through the skin of a human body, the skin generates heat and the impedance inside the skin decreases as the temperature of the skin rises. Therefore, as the temperature of the skin rises, the current flowing between the first electrode 10a and the second electrode 10b increases, and the measured resistance value decreases.
  • the resistance measuring unit measures the resistance value at a predetermined time interval (for example, every 0.5 seconds), and supplies the measured resistance value to the voltage control unit each time.
  • the voltage control unit controls the high-frequency voltage according to the resistance value measured by the resistance measurement unit, for example, so that the larger the measured resistance value, the higher the high-frequency voltage (increasing the amplitude of the high-frequency voltage).
  • the lower limit of the high frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 50 Vpp or more, more preferably 70 Vpp or more in the peak voltage.
  • the upper limit of the high frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 100 V or less, more preferably 90 Vpp or less in the peak voltage.
  • the applied high frequency voltage can be, for example, 88 Vpp.
  • the application of the high frequency voltage may be continuous application or pulse application.
  • application (ON) and non-application (OFF) of high frequency voltage are periodically repeated.
  • the cycle of repeating the pulse application may be 0.1 seconds to 2 seconds, for example, 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.5 seconds, 0.8 seconds, 1 second or 1 second. It can be .2 seconds.
  • the duty ratio which is the ON time ratio of the high frequency voltage in pulse application, is preferably in the range of 10% to 99%, more preferably in the range of 20% to 80%.
  • the duty ratio can be, for example, 40%.
  • the application conditions of high frequency such as high frequency voltage and duty ratio may be set by the user.
  • the high frequency device may have an input device operated by the user.
  • any device such as a switch can be used.
  • the control circuit 20 accepts the input operation through the input device, and according to the received input operation, the high frequency voltage, continuous application or pulse application, and pulse application are performed.
  • Control parameters such as cycle and duty ratio.
  • each parameter may be set independently, or a plurality of combinations of high frequency voltage, period and duty ratio in pulse application are preset in the control circuit 20. The user may be able to select a preset combination.
  • the high frequency device By using the high frequency device as described above, the high frequency can be easily applied to the user's skin.
  • the high frequency device can be operated as follows. First, the first electrode 10a and the second electrode 10b are placed in contact with the user's skin at intervals from each other. Next, the control circuit 20 applies a high-frequency voltage between the first electrode 10a and the second electrode 10b arranged in contact with the user's skin so as to enhance the stability of the dermal stem cells.
  • Radio Frequency Electrical Stimulation (RF) loading on fresh human skin and cells used radio frequency equipment and electrodes for cultured cells.
  • the adipose-derived stem cells were subjected to current loading for 0 minutes, 1 minute or 3 minutes from a high-frequency device as a set of three culture dishes placed on a plastic tray. Even in fresh human skin, a high-frequency device and electrodes for cultured cells were used, and a current load was applied from the high-frequency device for 0 minutes or 3 minutes.
  • Paraffin blocks, sectioned skin models, and fresh human skin were dehydrated and fixed with cold acetone according to the AMeX method, then replaced with acetone, methyl benzoate, and xylene in this order, and embedded in paraffin. Sections with a thickness of 3 ⁇ m were prepared and sections for tissue staining were prepared.
  • Paraffin sections prepared with various immunostaining thicknesses of 3 ⁇ m were deparaffinized with xylene and then hydrated with EtOH.
  • Antibodies to type V collagen (Acris, AM10159PU-N, V13F6, mouse monoclonal antibody), antibodies to cytokerald 14 (K-14) (Fitzgerald, 20R-CP002, guniea pig polyclonal antibody), antibodies to fibrillin 1 (Abcam, 11C1) .3, mouse monoclonal antibody), antibody against ⁇ 6 integrin (Santa Cruz, GOH-3, sc-19622, rat monoclonal antibody), antibody against CD31 (BD Pharming, 555444, sheep polyclonal antibody), antibody against CD34 (BD Pharming, Fluorescent immunostaining was performed using an antibody (Millipore, MAB2029, mouse monoclonal antibody) against melanoma-bound chondroitin sulfate proteoglycan (MCSP) (55039, mouse mono
  • Fat-derived stem cells (P / N 51-0070) were purchased and obtained from Invitrogen. The cells were cultured in MesenPRO RS TM Medium (cat. No.12746012, Thermo Fisher Scientific). One million cryopreserved cells in the second passage were seeded in T-75 flasks and precultured in MesenPRO RS TM Medium. After 3 days of culturing, the cells were detached and 100,000 cells were seeded in a 35 mm culture dish for current loading. The medium used was MesenPRO RS TM Medium, and the 35 mm culture dish for current loading was disinfected with 70% ethanol and rinsed with DPBS (-) twice immediately before use.
  • RNA extract was performed at 37 ° C. for 1 hour using 400 ⁇ l of CTS (trademark) CELLstart (trademark) Substrate (cat. No. A1014201, Thermo Fisher Scientific) per culture dish. Two days after sowing in a culture dish for current loading, high frequency current (RF) was applied when the density was reached again. The current-loaded cells were returned to the CO 2 incubator and further cultured overnight. Subsequently, 24 hours after the current loading, the cells were rinsed twice with D-PBS (-) and then the cells were lysed in an RNA extract (RNeasy mini kit, cat. No. 74104, QIAGEN).
  • the obtained cytolytic solution was homogenized with QIA shredder (cat. No. 79654, QIAGEN), and then total RNA was extracted and purified according to the protocol recommended by the manufacturer. Subsequently, the RNA concentration was measured with a NanoDrop 1000 ultra-trace spectrometer (Thermo Fisher Scientific), and the obtained total RNA was used as a template using SuperScript TM III Reverse Transcriptase (cat. No. 18080044, Thermo Fisher Scientific). cDNA synthesis was performed. Quantitative PCR is real-time PCR using Light Cycler 2.0 Instrument (Roche Diagnostics) and LightCycler® FastStart DNA MasterPlus SYBR Green I (cat. No. 03 515 885 001, Roche Diagnostics). Was done by. The primers of the gene used are shown below.
  • ITGA6 forward TTT GAA GAT GGG CCT TAT GAA (SEQ ID NO: 1)
  • ITGA6 reverse CCC TGA GTC CAA
  • AGA AAA ACC SEQ ID NO: 2
  • GAPDH forward GAG TCA ACG GAT TTG GTC GT (SEQ ID NO: 3)
  • GAPDH reverse TGG GAT TTC CAT TGA TGA CA (SEQ ID NO: 4)
  • Experiment 1-1 In Experiment 1, in the configuration shown in FIG. 1, a high-frequency device is provided in which the first electrode and the second electrode are connected to the control circuit by lead wires, respectively, so that the first electrode and the second electrode can be arranged at arbitrary positions.
  • the high frequency was applied to the adipose-derived stem cells as follows. First, adipose-derived stem cells were seeded in a culture vessel (Petri dish) in which the first electrode was placed at the bottom. Next, the first electrode and the second electrode were placed in contact with the culture medium of the adipose-derived stem cells in the culture vessel and at intervals from each other.
  • a high frequency having a frequency of 1 MHz and a peak voltage of 88 Vpp was applied to the culture solution via the first electrode and the second electrode for 1 minute.
  • the high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%.
  • the adipose-derived stem cells were cultured for 24 hours in an incubator. After culturing, adipose-derived stem cells were collected, RNA was extracted, and the gene expression of ITGA6 was confirmed by qPCR.
  • Experiment 1-1 and Experiment 1-2 are shown in Fig. 2-2.
  • the results of Experiments 1-3 and 1-4 are shown in FIG. 2-2.
  • control an application time of 0 minutes (hereinafter also referred to as "control").
  • the gene expression levels in Experiment 1-1 and Experiment 1-2 are shown as relative values when the gene expression level in the control is 1.
  • the expression level of ITGA6 is increased by applying high frequency.
  • the expression level is significantly increased (according to Dunnett's test, a significant difference was observed at the significance level of 5%). From the above, it was shown that the application of high frequency enhances the production of adhesion factors by dermal stem cells, and as a result, enhances the binding of dermal stem cells to blood vessels and contributes to the stabilization of dermal stem cells.
  • Experiment 2 Stabilization of dermal stem cells by applying high frequency to surplus skin
  • changes in ITGA6-positive cells were confirmed by using surgical surplus skin as a subject and applying a high frequency with a frequency of 1 MHz and an inter-peak voltage of 88 Vpp in the same manner as in Experiment 1.
  • the high frequency was applied for 3 minutes.
  • the high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%.
  • FIGS. 3-1 and 3-2 The images obtained in Experiment 2 on the third day of culture after RF application are shown in FIGS. 3-1 and 3-2.
  • the cultured tissue was visualized under the same conditions as above except that high frequency was not applied. It is also shown as "Control)".
  • the right column is an enlarged image of the square part in the left column. From FIG. 3-1 it was observed that the number of ITGA6-positive cells in the papillary layer increased compared to the control due to the RF load shown in the lower row. From FIG. 3-2, it was observed that the stem cell markers CD34 and MCSP were expressed in ITGA6-positive cells and their expression was increased by the RF loading shown in the lower row for each of CD34 and MCSP. Therefore, it can be said that stem cells are stabilized by applying RF.
  • Experiment 3 Enhancement of ECM production by applying high frequency to surplus skin after surgery
  • a surgical surplus skin was used as a subject, and a high frequency with a frequency of 1 MHz and a peak voltage of 88 Vpp was applied as in Experiment 2, and changes in dermis matrix production were confirmed.
  • the high frequency was applied for 3 minutes.
  • the high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%.
  • FIGS. 4-1 and 4-2 The images obtained in Experiment 3 on the third day of culture after RF application are shown in FIGS. 4-1 and 4-2.
  • the cultured tissue was visualized under the same conditions as above except that high frequency was not applied. It is also shown as "Control)". From FIGS. 4-1 and 4-2, it was observed that the expression of V-type collagen and fibrillin 1 by fibroblasts was increased by the RF load shown in the lower row as compared with the control. It is considered that the application of RF promotes the production of matrix and contributes to the tension of the skin.

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Abstract

The present invention provides an aesthetic method based on improvement of the stability of dermis stem cells by applying a high frequency to the skin. This aesthetic method comprises: applying a high frequency to the skin to increase the stability of dermis stem cells; and/or enhancing the expression of a gene related to the stability.

Description

高周波電気刺激の適用による真皮再生促進方法Method of promoting dermis regeneration by applying high-frequency electrical stimulation
　本発明は、高周波を利用した美容方法および真皮再生促進方法に関する。 The present invention relates to a beauty method using high frequency and a method for promoting dermis regeneration.
　顔や手足などの皮膚に対して高周波（ＲＦ）電流を流すことにより、皮膚のしわやたるみなどの改善を図ることが可能な高周波美容方法が知られている。皮膚に高周波電流を流すと、真皮層や皮下組織の温度が上昇する。その加熱作用によって、血液の流れを促進させること、および、真皮層のコラーゲンに熱ダメージを与えることで真皮層のコラーゲンの再生を促進させること等の効果が期待されている。近年は、そのような高周波を利用した様々な美容処理装置が提案されている（例えば、特許文献１）。 A high frequency beauty method is known that can improve wrinkles and sagging of the skin by passing a high frequency (RF) current to the skin such as the face and limbs. When a high-frequency current is applied to the skin, the temperature of the dermis layer and subcutaneous tissue rises. It is expected that the heating action promotes the flow of blood and promotes the regeneration of collagen in the dermis layer by causing heat damage to the collagen in the dermis layer. In recent years, various beauty treatment devices using such high frequencies have been proposed (for example, Patent Document 1).
　特許文献１：特開２０１８－４８９号公報 Patent Document 1: Japanese Unexamined Patent Publication No. 2018-489
　しかし、従来の高周波美容方法は、高周波による真皮層および皮下組織の加熱作用に基づく血行およびコラーゲン再生を促進するものであり、いわば一時的な処置であった。近年の健康に対する意識が高まる中、より根本的な、体質レベルでの機能の改善が望まれている。 However, the conventional high-frequency cosmetology method promotes blood circulation and collagen regeneration based on the heating action of the dermis layer and subcutaneous tissue by high frequency, and is a temporary treatment. With increasing awareness of health in recent years, more fundamental improvement of function at the constitutional level is desired.
　本発明は、皮膚に高周波を適用することによって得られる真皮幹細胞の安定性の改善に基づく美容方法を提供することを目的の一つとする。 One of the objects of the present invention is to provide a cosmetic method based on the improvement of the stability of dermal stem cells obtained by applying high frequency to the skin.
　本発明の一つの側面によれば、皮膚に高周波を適用して真皮幹細胞の安定性を高めることを含む美容方法が提供される。
　また、本発明の一つの側面によれば、皮膚に高周波を適用して真皮幹細胞におけるITGA6の遺伝子発現を増強させることを含む美容方法が提供される。
　さらに、本発明の一つの側面によれば、皮膚に高周波を適用して真皮線維芽細胞におけるV型コラーゲンおよび／またはフィブリリン1の産生を増強させることを含む美容方法が提供される。
　さらに、本発明の別の側面によれば、皮膚に高周波を適用する工程を含む、真皮幹細胞の安定化方法が提供され、
　本発明のさらに別の側面によれば、皮膚に高周波を提供する工程を含む、真皮幹細胞におけるITGA6の遺伝子発現の増強方法が提供され、
　本発明のさらに別の側面によれば、皮膚に高周波を提供する工程を含む、真皮線維芽細胞におけるV型コラーゲンおよび／またはフィブリリン1の産生の増強方法が提供される。
　また、本発明のさらに別の側面によれば、一対の電極と、前記一対の電極に高周波電圧を印加する制御回路と、を有する高周波美容処理装置の作動方法であって、前記制御回路により、前記一対の電極に、真皮幹細胞の安定性を高める高周波電圧を印加することを含み、前記高周波電圧の印加は、前記一対の電極を皮膚に接触させた状態で行なわれる、高周波美容処理装置の作動方法が提供され、
　本発明のさらに別の側面によれば、一対の電極と、前記一対の電極に高周波電圧を印加する制御回路と、を有する高周波美容処理装置の作動方法であって、前記制御回路により、前記一対の電極に、真皮幹細胞におけるITGA6の遺伝子発現、ならびに／または真皮線維芽細胞におけるV型コラーゲンおよび／もしくはフィブリリン1の産生を増強させる高周波電圧を印加することを含み、前記高周波電圧の印加は、前記一対の電極を皮膚に接触させた状態で行なわれる、高周波美容処理装置の作動方法が提供される。 According to one aspect of the invention, there is provided a cosmetic method comprising applying high frequency to the skin to enhance the stability of dermal stem cells.
Further, according to one aspect of the present invention, there is provided a cosmetic method comprising applying a high frequency to the skin to enhance the gene expression of ITGA6 in dermal stem cells.
Further, according to one aspect of the invention, there is provided a cosmetic method comprising applying high frequency to the skin to enhance the production of V-type collagen and / or fibrillin-1 in dermal fibroblasts.
Further, according to another aspect of the present invention, there is provided a method for stabilizing dermal stem cells, which comprises the step of applying high frequency to the skin.
Yet another aspect of the invention provides a method of enhancing ITGA6 gene expression in dermal stem cells, comprising the step of providing high frequency to the skin.
According to yet another aspect of the present invention, there is provided a method for enhancing the production of V-type collagen and / or fibrillin 1 in dermal fibroblasts, which comprises a step of providing a high frequency to the skin.
Further, according to still another aspect of the present invention, there is a method of operating a high frequency beauty treatment apparatus having a pair of electrodes and a control circuit for applying a high frequency voltage to the pair of electrodes, according to the control circuit. The operation of the high-frequency beauty treatment apparatus, which comprises applying a high-frequency voltage to the pair of electrodes to enhance the stability of the dermal stem cells, and the application of the high-frequency voltage is performed in a state where the pair of electrodes are in contact with the skin. The method is provided,
According to still another aspect of the present invention, there is a method of operating a high frequency beauty processing apparatus having a pair of electrodes and a control circuit for applying a high frequency voltage to the pair of electrodes, wherein the pair of electrodes is used. The application of the high frequency voltage comprises applying to the electrodes of the high frequency voltage which enhances the gene expression of ITGA6 in the dermal stem cells and / or the production of V-type collagen and / or fibrillin 1 in the dermal fibroblasts. Provided is a method of operating a high frequency beauty treatment apparatus, which is performed with a pair of electrodes in contact with the skin.
　（定義）
　本明細書において、「高周波の適用」および「高周波を適用する」とは、互いに間隔をあけて対象物に接触して配置された一対の電極間に高周波電圧を印加することによって、対象物に高周波電流を流すことを意味する。 (Definition)
As used herein, "applying high frequency" and "applying high frequency" mean applying a high frequency voltage to an object by applying a high frequency voltage between a pair of electrodes arranged in contact with the object at intervals. It means that a high frequency current is passed.
　また、数値を示す際に用いる「約」の語は、この語が修飾する数値の前後５％の数値を含むことを表す。 Also, the word "about" used to indicate a numerical value means that it includes a numerical value of 5% before and after the numerical value modified by this word.
　本発明によれば、真皮幹細胞の安定性の改善による美容効果をもたらすことができる。 According to the present invention, it is possible to bring about a cosmetic effect by improving the stability of dermal stem cells.
本発明に使用可能な高周波装置の一形態を示すブロック図である。It is a block diagram which shows one form of the high frequency apparatus which can be used in this invention. 実験１－１および実験１－２によるITGA6遺伝子発現量の変化を示すグラフである。It is a graph which shows the change of the ITGA6 gene expression level by Experiment 1-1 and Experiment 1-2. 実験１－３および実験１－４によるITGA6遺伝子発現量の変化を示すグラフである。It is a graph which shows the change of the ITGA6 gene expression level by Experiment 1-3 and Experiment 1-4. 実験２による、培養組織におけるITGA6の発現を可視化した画像である。上段の画像は対照（Control）の細胞、下段の画像はＲＦを適用した細胞を示している。It is an image which visualized the expression of ITGA6 in the culture tissue by Experiment 2. The upper image shows the control cells, and the lower image shows the cells to which RF has been applied. 実験２による、培養組織におけるITGA6、真皮幹細胞マーカーとしてCD34およびMSCPの発現を可視化した画像である。上段の画像は対照（Control）の細胞、下段の画像はＲＦを適用した細胞を示している。It is an image which visualized the expression of ITGA6, CD34 and MSCP as dermal stem cell markers in the cultured tissue by Experiment 2. The upper image shows the control cells, and the lower image shows the cells to which RF has been applied. 実験３による、ECM産生（V型コラーゲン）を可視化した画像である。上段の画像は対照（Control）の細胞、下段の画像はＲＦを適用した細胞を示している。It is an image which visualized ECM production (V type collagen) by Experiment 3. The upper image shows the control cells, and the lower image shows the cells to which RF has been applied. 実験３による、ECM産生（フィブリリン1）を可視化した画像である。上段の画像は対照（Control）の細胞、下段の画像はＲＦを適用した細胞を示している。It is an image which visualized ECM production (fibrillin 1) by Experiment 3. The upper image shows the control cells, and the lower image shows the cells to which RF has been applied.
　本発明は、皮膚への高周波（ＲＦ）の適用が、真皮幹細胞の安定性の改善、例えば、真皮幹細胞におけるITGA6の遺伝子発現の増強による真皮幹細胞の安定性の改善、に大きく寄与するという、本発明者らの新たな知見に基づくものであり、本発明の一態様として、皮膚に高周波を適用して真皮幹細胞の安定性を高めることを含む美容方法が提供される。ITGA6遺伝子発現の増強により真皮幹細胞の安定性が改善し（例えば、真皮幹細胞が血管付近に繋ぎとめられる）、それに伴って真皮線維芽細胞におけるV型コラーゲンおよび／またはフィブリリン1の産生も増強される。真皮幹細胞の安定化は皮膚の張りにも影響し、皮膚へのＲＦ電流の適用は、皮膚に張りを与えることに寄与する。 The present invention states that the application of high frequency (RF) to the skin greatly contributes to the improvement of the stability of dermal stem cells, for example, the improvement of the stability of dermal stem cells by enhancing the gene expression of ITGA6 in the dermal stem cells. Based on the new findings of the inventors, as one aspect of the present invention, there is provided a cosmetic method including applying a high frequency to the skin to enhance the stability of dermal stem cells. Increased ITGA6 gene expression improves dermal stem cell stability (eg, dermal stem cells are anchored near blood vessels), with concomitantly enhanced production of type V collagen and / or fibrillin-1 in dermal fibroblasts. .. Stabilization of dermal stem cells also affects skin tension, and application of RF currents to the skin contributes to skin tension.
　皮膚に適用する高周波の周波数は、０．５ＭＨｚ～４ＭＨｚの範囲、例えば、０．５ＭＨＺ、１ＭＨｚ、２ＭＨＺ、３ＭＨｚまたは４ＭＨｚのいずれかの周波数、または上記の任意の周波数により規定される範囲（例えば、１～４ＭＨｚの範囲）に含まれる任意の周波数が使用されてもよい。本発明の一部の形態においては、周波数は約１ＭＨｚである。 The frequency of the high frequency applied to the skin is in the range of 0.5 MHz to 4 MHz, for example, 0.5 MHZ, 1 MHz, 2 MHZ, 3 MHz or 4 MHz, or the range defined by any of the above frequencies (eg, for example. Any frequency included in the range of 1 to 4 MHz) may be used. In some embodiments of the invention, the frequency is about 1 MHz.
　血管の周囲に存在する真皮幹細胞は、肌の自己再生力を高める司令塔として働く。インテグリンα6（ITGA6）は、血管基底膜を介して真皮幹細胞を血管に繋ぎとめる役割を果たす。ITGA6の発現増加により接着性が高まった幹細胞が血管周囲に安定化し、結果としてECM産生が促される。 The dermal stem cells that exist around the blood vessels work as a control tower that enhances the self-renewal power of the skin. Integrin α6 (ITGA6) plays a role in anchoring dermal stem cells to blood vessels via the basement membrane of blood vessels. Increased expression of ITGA6 stabilizes stem cells with increased adhesiveness around blood vessels, and as a result, promotes ECM production.
　皮膚への高周波の適用は皮膚への薬剤や各種抽出液等の適用を組み合わせてもよい。例えば、適用できる薬剤や各種抽出液として、イノシトール、レチノール誘導体、キシリトール、ヒアルロン酸、グリセリン、酵母エキス、サクラリーフエキス、ケイヒエキス、マツエキス、ブルガリアローズウォーター、オクラエキス、イブキジャコウエキス、ワレモコウエキス、グリチルリチン酸ジカリウム、イチョウ葉エキス、テンチャエキス、ヨクイニンエキス、カンゾウエキス、オタネニンジンエキス、クララエキス、スターフルーツ葉エキス、ニンジンエキス、ジュウヤクエキス、ニームリーフエキス、サフランエキス、キナエキス、コンフリーエキス、タイムエキス、フユイチゴ、ヨメナ、ムベ、リュウキュウイチゴ、インドヨメナ、マテバシイ、オウゴンエキス、ローズマリーエキス、アンズ核粒、ゲンチアナエキス、ハッカ末、タイソウエキス、ホップエキス、キウイエキス、ローマカミツレエキス、リンゴエキス、サンザシエキス、ウコンエキス、ヤマモモ、フトモモ、コナラなどが例示される。これらは単独で又は複数組み合わせて使用することができる。 The application of high frequency to the skin may be combined with the application of drugs to the skin and various extracts. For example, applicable drugs and various extracts include inositol, retinol derivative, xylitol, hyaluronic acid, glycerin, yeast extract, cherry leaf extract, keihi extract, pine extract, bulgarian rose water, okra extract, ibukijakou extract, waremokou extract, and glycyrrhizin. Dipotassium acid, Ginkgo biloba extract, Tencha extract, Yokuinin extract, Kanzo extract, Otaneninjin extract, Clara extract, Star fruit leaf extract, Carrot extract, Juyaku extract, Neem leaf extract, Saffron extract, Kina extract, Confree extract, Thyme extract, Fuyu strawberry , Yomena, Mube, Ryukyu Strawberry, Indian Yomena, Matebashii, Ogon Extract, Rosemary Extract, Anzu Nucleus Granules, Gentiana Extract, Hakka Powder, Taisou Extract, Hop Extract, Kiwi Extract, Roman Camille Extract, Apple Extract, Sanzashi Extract, Examples include Ukon extract, Yamamomo, Futomomo, and Konara. These can be used alone or in combination.
　皮膚への高周波の適用には、高周波装置を用いることができる。高周波装置の一形態について、図１を参照して説明する。 A high frequency device can be used to apply high frequency to the skin. A form of the high frequency device will be described with reference to FIG.
　高周波装置１は、電極部１０と制御回路２０とを有する。電極部１０は、制御回路２０に接続された第１電極１０ａおよび第２電極１０ｂを有する。ユーザの皮膚に接触する接触面を電極部１０に設け、この接触面に、第１電極１０ａおよび第２電極１０ｂを互いに間隔をあけて配置することにより、第１電極１０ａおよび第２電極１０ｂを容易に皮膚に互いに間隔をあけて接触させることができる。第１電極１０ａおよび第２電極１０ｂを、接触面にではなく、配線ケーブルを介して制御回路２０と接続してもよく、こうすることによって、第１電極１０ａおよび第２電極１０ｂを自由に配置することができる。 The high frequency device 1 has an electrode unit 10 and a control circuit 20. The electrode unit 10 has a first electrode 10a and a second electrode 10b connected to the control circuit 20. A contact surface that comes into contact with the user's skin is provided on the electrode portion 10, and the first electrode 10a and the second electrode 10b are arranged at intervals from each other on the contact surface, whereby the first electrode 10a and the second electrode 10b are provided. They can be easily brought into contact with the skin at intervals. The first electrode 10a and the second electrode 10b may be connected to the control circuit 20 via a wiring cable instead of the contact surface, whereby the first electrode 10a and the second electrode 10b can be freely arranged. can do.
　制制御回路２０は、電源部、電圧制御部および抵抗測定部を有することができる。電源部は、第１電極１０ａと第２電極１０ｂとの間に高周波電圧（高周波を示す電圧）を印加する。電源部は、高周波の電圧を生成する発振回路、および発振された電圧を昇圧する昇圧回路等を有し、例えば、０．５ＭＨｚ～４ＭＨｚ程度の高周波電圧を第１電極１０ａと第２電極１０ｂとの間に印加するように構成される。ユーザの皮膚に第１電極１０ａおよび第２電極１０ｂを接触させた状態で第１電極１０ａと第２電極１０ｂとの間に高周波の電圧を印加することで、皮膚に高周波の電流を流すことができる。 The control control circuit 20 can have a power supply unit, a voltage control unit, and a resistance measurement unit. The power supply unit applies a high frequency voltage (voltage indicating a high frequency) between the first electrode 10a and the second electrode 10b. The power supply unit has an oscillation circuit that generates a high frequency voltage, a booster circuit that boosts the oscillated voltage, and the like. For example, a high frequency voltage of about 0.5 MHz to 4 MHz is applied to the first electrode 10a and the second electrode 10b. It is configured to apply between. By applying a high-frequency voltage between the first electrode 10a and the second electrode 10b with the first electrode 10a and the second electrode 10b in contact with the user's skin, a high-frequency current can be passed through the skin. can.
　電圧制御部は、電源部を制御することで電源部が印加する高周波電圧を制御する。また、電圧制御部は、電圧の制御に抵抗測定部による測定結果を用いる。抵抗測定部は、第１電極１０ａと第２電極１０ｂとがユーザの皮膚に接触した状態での第１電極１０ａと第２電極１０ｂとの間の抵抗値（電気抵抗値）を測定する。 The voltage control unit controls the high frequency voltage applied by the power supply unit by controlling the power supply unit. Further, the voltage control unit uses the measurement result by the resistance measurement unit for voltage control. The resistance measuring unit measures the resistance value (electrical resistance value) between the first electrode 10a and the second electrode 10b when the first electrode 10a and the second electrode 10b are in contact with the user's skin.
　この抵抗値は、ユーザの皮膚の状態、および皮膚に塗布される化粧水といった皮膚外用剤の種類等によって変化する。例えば、人体の皮膚に高周波電流を流した場合、皮膚が発熱し、皮膚の温度上昇に伴って皮膚内部のインピーダンスが減少することが知られている。従って、皮膚の温度上昇に伴って、第１電極１０ａおよび第２電極１０ｂの間に流れる電流が増加し、測定される抵抗値が小さくなる。抵抗測定部は、抵抗値の測定を決められた時間間隔（例えば０．５秒毎など）で行い、その度に測定した抵抗値を電圧制御部に供給する。 This resistance value changes depending on the condition of the user's skin and the type of external skin preparation such as lotion applied to the skin. For example, it is known that when a high-frequency current is passed through the skin of a human body, the skin generates heat and the impedance inside the skin decreases as the temperature of the skin rises. Therefore, as the temperature of the skin rises, the current flowing between the first electrode 10a and the second electrode 10b increases, and the measured resistance value decreases. The resistance measuring unit measures the resistance value at a predetermined time interval (for example, every 0.5 seconds), and supplies the measured resistance value to the voltage control unit each time.
　電圧制御部は、抵抗測定部で測定された抵抗値に応じて、例えば、測定された抵抗値が大きいほど高周波電圧を高くする（高周波電圧の振幅を大きくする）ように高周波電圧を制御する。 The voltage control unit controls the high-frequency voltage according to the resistance value measured by the resistance measurement unit, for example, so that the larger the measured resistance value, the higher the high-frequency voltage (increasing the amplitude of the high-frequency voltage).
　第１電極１０ａと第２電極１０ｂとの間に印加される高周波電圧の下限値は、ピーク間電圧で５０Ｖｐｐ以上であることが好ましく、より好ましくは７０Ｖｐｐ以上である。また、第１電極１０ａと第２電極１０ｂとの間に印加される高周波電圧の上限値は、ピーク間電圧で１００Ｖ以下であることが好ましく、より好ましくは９０Ｖｐｐ以下である。印加される高周波電圧は、例えば８８Ｖｐｐとすることができる。 The lower limit of the high frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 50 Vpp or more, more preferably 70 Vpp or more in the peak voltage. The upper limit of the high frequency voltage applied between the first electrode 10a and the second electrode 10b is preferably 100 V or less, more preferably 90 Vpp or less in the peak voltage. The applied high frequency voltage can be, for example, 88 Vpp.
　高周波電圧の印加は、連続印加であってもよいしパルス印加であってもよい。パルス印加の場合、高周波電圧の印加（ＯＮ）と不印加（ＯＦＦ）とが周期的に繰り返される。パルス印加の繰り返しの周期は、０．１秒～２秒であってよく、例えば０．１秒、０．２秒、０．３秒、０．５秒、０．８秒、１秒または１．２秒 とすることができる。パルス印加における高周波電圧のＯＮ時間比であるデューティー比は、１０％～９９ ％の範囲であることが好ましく、より好ましくは２０％～８０％の範囲である。デューティー比は、例えば４０％とすることができる。 The application of the high frequency voltage may be continuous application or pulse application. In the case of pulse application, application (ON) and non-application (OFF) of high frequency voltage are periodically repeated. The cycle of repeating the pulse application may be 0.1 seconds to 2 seconds, for example, 0.1 seconds, 0.2 seconds, 0.3 seconds, 0.5 seconds, 0.8 seconds, 1 second or 1 second. It can be .2 seconds. The duty ratio, which is the ON time ratio of the high frequency voltage in pulse application, is preferably in the range of 10% to 99%, more preferably in the range of 20% to 80%. The duty ratio can be, for example, 40%.
　高周波電圧およびデューティー比といった高周波の適用条件はユーザが設定可能であってもよい。高周波の適用条件をユーザが設定できるようにするため、高周波装置は、ユーザにより操作される入力デバイスを有することができる。入力デバイスとしては、例えばスイッチなど任意のデバイスを用いることができる。高周波の適用条件をユーザが設定可能である場合、制御回路２０は、入力デバイスを通じた入力操作を受け付け、その受け付けた入力操作に従って、高周波電圧、連続印加かパルス印加か、並びにパルス印加の場合の周期およびデューティー比などのパラメータを制御する。高周波の適用条件の設定に際し、各パラメータは、それぞれ独立して設定可能であってもよいし、パルス印加における高周波電圧、周期およびデューティー比について複数の組み合わせが制御回路２０にプリセットされており、そのプリセットされた組み合わせをユーザが選択するようになっていてもよい。 The application conditions of high frequency such as high frequency voltage and duty ratio may be set by the user. In order to allow the user to set the application conditions of the high frequency, the high frequency device may have an input device operated by the user. As the input device, any device such as a switch can be used. When the application condition of the high frequency can be set by the user, the control circuit 20 accepts the input operation through the input device, and according to the received input operation, the high frequency voltage, continuous application or pulse application, and pulse application are performed. Control parameters such as cycle and duty ratio. When setting the high frequency application conditions, each parameter may be set independently, or a plurality of combinations of high frequency voltage, period and duty ratio in pulse application are preset in the control circuit 20. The user may be able to select a preset combination.
　以上説明したような高周波装置を用いることで、ユーザの皮膚に容易に高周波を適用することができる。高周波装置は、次のように作動させることができる。まず、第１電極１０ａと第２電極１０ｂとを、互いに間隔をあけてユーザの皮膚に接触させて配置する。次に、制御回路２０により、真皮幹細胞の安定性を高めるように、ユーザの皮膚に接触させて配置した第１電極１０ａと第２電極１０ｂとの間に高周波電圧を印加する。 By using the high frequency device as described above, the high frequency can be easily applied to the user's skin. The high frequency device can be operated as follows. First, the first electrode 10a and the second electrode 10b are placed in contact with the user's skin at intervals from each other. Next, the control circuit 20 applies a high-frequency voltage between the first electrode 10a and the second electrode 10b arranged in contact with the user's skin so as to enhance the stability of the dermal stem cells.
　［高周波の適用による真皮幹細胞の安定性改善の確認］
　以下、発明者らが行った高周波の適用による真皮幹細胞の安定性改善の確認実験について説明する。 [Confirmation of improvement in dermal stem cell stability by applying high frequency]
Hereinafter, the experiments performed by the inventors to confirm the improvement of the stability of dermal stem cells by applying high frequency will be described.
　［材料および方法］
新鮮ヒト皮膚の培養
　インフォームドコンセントを行った被験者の眼瞼部の新鮮皮膚サンプルをクリニックより譲渡を受けた。William’s E培地（Thermo Fisher Science, Waltham, MA)あるいは、10% FBSを含むDMEM培地とEGFを抜去したHumedia-KG2 (KURABO, KK-2150S)の等量混合培養液で培養した。毎日培地交換を行い、３日目に皮膚片を回収した。 [material and method]
Culture of fresh human skin Fresh skin samples of the eyelids of subjects who gave informed consent were transferred from the clinic. The cells were cultured in William's E medium (Thermo Fisher Science, Waltham, MA) or an equal amount mixed culture medium of DMEM medium containing 10% FBS and Humedia-KG2 (KURABO, KK-2150S) from which EGF was removed. Medium was exchanged daily and skin pieces were collected on the third day.
高周波電気刺激（RF）の負荷方法
　新鮮ヒト皮膚および細胞への高周波電気刺激（ＲＦ）の負荷は、高周波装置と培養細胞用の電極を使用した。脂肪由来幹細胞は、プラスチック製のトレイの上に培養皿を３皿ずつ載せたものを１つのセットとして、高周波装置より、０分間、１分間または３分間の電流負荷を行った。新鮮ヒト皮膚においても、高周波装置と培養細胞用の電極を使用し、高周波装置より、０分間または３分間の電流負荷を行った。 Radio Frequency Electrical Stimulation (RF) Loading Methods Radio frequency electrical stimulation (RF) loading on fresh human skin and cells used radio frequency equipment and electrodes for cultured cells. The adipose-derived stem cells were subjected to current loading for 0 minutes, 1 minute or 3 minutes from a high-frequency device as a set of three culture dishes placed on a plastic tray. Even in fresh human skin, a high-frequency device and electrodes for cultured cells were used, and a current load was applied from the high-frequency device for 0 minutes or 3 minutes.
パラフィンブロック、切片作成
　回収した皮膚モデル、新鮮ヒト皮膚をAMeX法に従って、冷アセトンを用いて脱水固定後、アセトン、安息香酸メチル、キシレンの順で置換し、パラフィンに包埋した。３μｍの厚さで切片を作成し、組織染色用の切片を作成した。 Paraffin blocks, sectioned skin models, and fresh human skin were dehydrated and fixed with cold acetone according to the AMeX method, then replaced with acetone, methyl benzoate, and xylene in this order, and embedded in paraffin. Sections with a thickness of 3 μm were prepared and sections for tissue staining were prepared.
各種免疫染色
　３μｍの厚さで作成したパラフィン切片を、キシレンで脱パラフィン後、EtOHを使って水和化した。V型コラーゲンに対する抗体（Acris, AM10159PU-N, V13F6, mouse monoclonal antibody）及びサイトケラチン14（K-14）に対する抗体（Fitzgerald, 20R-CP002, guniea pig polyclonal antibody）、フィブリリン1に対する抗体（Abcam, 11C1.3, mouse monoclonal antibody）、α6インテグリンに対する抗体（Santa Cruz, GOH-3, sc-19622, rat monoclonal antibody）、CD31に対する抗体（BD Pharming, 555444, sheep polyclonal antibody）、CD34に対する抗体（BD Pharming, 55039, mouse monoclonal antibody）、メラノーマ結合コンドロイチン硫酸プロテオグリカン（MCSP）に対する抗体（Millipore, MAB2029, mouse monoclonal antibody）を用いて蛍光免疫染色を行った。 Paraffin sections prepared with various immunostaining thicknesses of 3 μm were deparaffinized with xylene and then hydrated with EtOH. Antibodies to type V collagen (Acris, AM10159PU-N, V13F6, mouse monoclonal antibody), antibodies to cytokerald 14 (K-14) (Fitzgerald, 20R-CP002, guniea pig polyclonal antibody), antibodies to fibrillin 1 (Abcam, 11C1) .3, mouse monoclonal antibody), antibody against α6 integrin (Santa Cruz, GOH-3, sc-19622, rat monoclonal antibody), antibody against CD31 (BD Pharming, 555444, sheep polyclonal antibody), antibody against CD34 (BD Pharming, Fluorescent immunostaining was performed using an antibody (Millipore, MAB2029, mouse monoclonal antibody) against melanoma-bound chondroitin sulfate proteoglycan (MCSP) (55039, mouse monoclonal antibody).
脂肪由来幹細胞のITGA6の遺伝子発現解析
　Invitrogenより脂肪由来幹細胞（P/N 51-0070）を購入取得した。MesenPRO RS(商標) Medium（cat. no.12746012，Thermo Fisher Scientific）にて培養した。継代2代目で凍結保存された同細胞100万個をT-75フラスコに播種し、MesenPRO RS(商標) Mediumで前培養を行った。培養３日後、細胞を剥離して電流負荷用の35 mm培養皿に10万個を播種した。なお、培地は同じくMesenPRO RS(商標) Mediumを使用、電流負荷用35 mm培養皿は使用する直前に70%エタノール消毒とDPBS(-)によるリンスを２回行った。また、メーカー推奨プロトコールに従い、培養皿1枚当たり400μl のCTS(商標) CELLstart(商標) Substrate （cat. no. A1014201，Thermo Fisher Scientific）を用いて37℃で1時間のコーティングを行った。電流負荷用の培養皿に播種した２日後、再度集密に達した時点で高周波電流（ＲＦ）を負荷した。電流を負荷した細胞はＣＯ２インキュベーターに戻してさらに一晩培養を行った。続いて、電流負荷の24時間後、D-PBS(-)でリンスを2回行ってからＲＮＡ抽出液（RNeasy mini kit，cat. no. 74104，QIAGEN）に細胞を溶解させた。得られた細胞溶解液をQIAshredder（cat. no. 79654，QIAGEN）でホモジナイズした後、メーカー推奨のプロトコールに従いtotal RNAを抽出・精製した。続いて、NanoDrop 1000超微量分光計（Thermo Fisher Scientific）でRNA濃度を測定し、得られたtotal RNAを鋳型にSuperScript(商標) III Reverse Transcriptase（cat. no. 18080044，Thermo Fisher Scientific）を用いてcDNA合成を行った。定量PCRはライトサイクラー 2.0 インスツルメント（ロシュ・ダイアグノスティックス）とLightCycler(登録商標) FastStart DNA MasterPlus SYBR Green I（cat. no. 03 515 885 001，ロシュ・ダイアグノスティック）を用いたリアルタイムＰＣＲにより行った。使用した遺伝子のプライマーを以下に示す。 Gene expression analysis of ITGA6 in adipose-derived stem cells Fat-derived stem cells (P / N 51-0070) were purchased and obtained from Invitrogen. The cells were cultured in MesenPRO RS ™ Medium (cat. No.12746012, Thermo Fisher Scientific). One million cryopreserved cells in the second passage were seeded in T-75 flasks and precultured in MesenPRO RS ™ Medium. After 3 days of culturing, the cells were detached and 100,000 cells were seeded in a 35 mm culture dish for current loading. The medium used was MesenPRO RS ™ Medium, and the 35 mm culture dish for current loading was disinfected with 70% ethanol and rinsed with DPBS (-) twice immediately before use. In addition, according to the protocol recommended by the manufacturer, coating was performed at 37 ° C. for 1 hour using 400 μl of CTS (trademark) CELLstart (trademark) Substrate (cat. No. A1014201, Thermo Fisher Scientific) per culture dish. Two days after sowing in a culture dish for current loading, high frequency current (RF) was applied when the density was reached again. The current-loaded cells were returned to the CO 2 incubator and further cultured overnight. Subsequently, 24 hours after the current loading, the cells were rinsed twice with D-PBS (-) and then the cells were lysed in an RNA extract (RNeasy mini kit, cat. No. 74104, QIAGEN). The obtained cytolytic solution was homogenized with QIA shredder (cat. No. 79654, QIAGEN), and then total RNA was extracted and purified according to the protocol recommended by the manufacturer. Subsequently, the RNA concentration was measured with a NanoDrop 1000 ultra-trace spectrometer (Thermo Fisher Scientific), and the obtained total RNA was used as a template using SuperScript ™ III Reverse Transcriptase (cat. No. 18080044, Thermo Fisher Scientific). cDNA synthesis was performed. Quantitative PCR is real-time PCR using Light Cycler 2.0 Instrument (Roche Diagnostics) and LightCycler® FastStart DNA MasterPlus SYBR Green I (cat. No. 03 515 885 001, Roche Diagnostics). Was done by. The primers of the gene used are shown below.
　プライマー名　　 配列
　ITGA6 forward：　TTT GAA GAT GGG CCT TAT GAA（配列番号１）
　ITGA6 reverse：　CCC TGA GTC CAA AGA AAA ACC（配列番号２）
　GAPDH forward：　GAG TCA ACG GAT TTG GTC GT （配列番号３）
　GAPDH reverse：　TGG GAT TTC CAT TGA TGA CA （配列番号４） Primer name sequence ITGA6 forward: TTT GAA GAT GGG CCT TAT GAA (SEQ ID NO: 1)
ITGA6 reverse: CCC TGA GTC CAA AGA AAA ACC (SEQ ID NO: 2)
GAPDH forward: GAG TCA ACG GAT TTG GTC GT (SEQ ID NO: 3)
GAPDH reverse: TGG GAT TTC CAT TGA TGA CA (SEQ ID NO: 4)
　［実験１：高周波の適用による幹細胞におけるITGA6の発現］
　真皮幹細胞の安定化は、若い肌を生み続ける力を高めると考えられる。そこで、真皮幹細胞の安定化に関わる因子として、細胞と細胞外マトリックスとの接着に関わるインテグリンα６の遺伝子発現量の変化を確認した。 [Experiment 1: Expression of ITGA6 in stem cells by applying high frequency]
Stabilization of dermal stem cells is thought to enhance the ability to continue to produce young skin. Therefore, as a factor involved in the stabilization of dermal stem cells, changes in the gene expression level of integrin α6 involved in the adhesion between cells and extracellular matrix were confirmed.
　［実験１－１］
　実験１では、図１に示した構成において、第１電極および第２電極をそれぞれリード線によって制御回路に接続し、第１電極および第２電極を任意の位置に配置できるようにした高周波装置を用い、以下のようにして、脂肪由来幹細胞に対して高周波を適用した。まず、第１電極を底部に配置した培養容器（シャーレ）に脂肪由来幹細胞を播種した。次いで、第１電極および第２電極を、培養容器内の脂肪由来幹細胞の培養液と接し、かつ、互いに間隔をあけて配置した。その後、第１電極および第２電極を介して培養液に、周波数１ＭＨｚ、ピーク間電圧８８Ｖｐｐの高周波を１分間適用した。高周波の適用は、繰り返し周期が１秒、デューティー比が４０％のパルス印加とした。その後、培養器にて脂肪由来幹細胞を２４時間培養した。培養後の脂肪由来幹細胞を回収してＲＮＡを抽出し、ｑＰＣＲによりITGA6の遺伝子発現を確認した。 [Experiment 1-1]
In Experiment 1, in the configuration shown in FIG. 1, a high-frequency device is provided in which the first electrode and the second electrode are connected to the control circuit by lead wires, respectively, so that the first electrode and the second electrode can be arranged at arbitrary positions. The high frequency was applied to the adipose-derived stem cells as follows. First, adipose-derived stem cells were seeded in a culture vessel (Petri dish) in which the first electrode was placed at the bottom. Next, the first electrode and the second electrode were placed in contact with the culture medium of the adipose-derived stem cells in the culture vessel and at intervals from each other. Then, a high frequency having a frequency of 1 MHz and a peak voltage of 88 Vpp was applied to the culture solution via the first electrode and the second electrode for 1 minute. The high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%. Then, the adipose-derived stem cells were cultured for 24 hours in an incubator. After culturing, adipose-derived stem cells were collected, RNA was extracted, and the gene expression of ITGA6 was confirmed by qPCR.
　［実験１－２］
　高周波の適用時間を３分間とした以外は実験１－１と同じ条件で遺伝子発現を確認した。 [Experiment 1-2]
Gene expression was confirmed under the same conditions as in Experiment 1-1 except that the application time of high frequency was set to 3 minutes.
　［実験１－３］
　高周波のピーク間電圧を６０Ｖｐｐとした以外は実験１－１と同じ条件で遺伝子発現を確認した。 [Experiment 1-3]
Gene expression was confirmed under the same conditions as in Experiment 1-1 except that the high frequency peak voltage was set to 60 Vpp.
　［実験１－４］
　高周波のピーク間電圧を６０Ｖｐｐとした以外は実験１－２と同じ条件で遺伝子発現を確認した。 [Experiment 1-4]
Gene expression was confirmed under the same conditions as in Experiment 1-2 except that the high frequency peak voltage was set to 60 Vpp.
　実験１－１および実験１－２の結果を図２－２に示す。また、実験１－３および実験１－４の結果を図２－２に示す。なお、高周波の適用による遺伝子発現の変化の効果を確認するため、高周波を適用しなかった以外は実験１－１と同じ条件で遺伝子発現を確認し、図２－１および図２－２には、その結果も適用時間０分（以下「コントロール」とも言う）として併せて示した。実験１－１および実験１－２の遺伝子発現量は、コントロールでの遺伝子発現量を１としたときの相対値で示した。 The results of Experiment 1-1 and Experiment 1-2 are shown in Fig. 2-2. The results of Experiments 1-3 and 1-4 are shown in FIG. 2-2. In order to confirm the effect of the change in gene expression due to the application of high frequency, gene expression was confirmed under the same conditions as in Experiment 1-1 except that high frequency was not applied. The results are also shown with an application time of 0 minutes (hereinafter also referred to as "control"). The gene expression levels in Experiment 1-1 and Experiment 1-2 are shown as relative values when the gene expression level in the control is 1.
　図２―１および図２－２より、次のことがいえる。ITGA6については、高周波を適用することによって、発現量が増加している。特に、図２－１より、発現量が有意に増加していることがわかる（ダネットの検定によると、有意水準５％で有意差が認められた）。以上より、高周波の適用は、真皮幹細胞による接着因子の産生を増強させ、結果的に、真皮幹細胞の血管への結合性を高め、真皮幹細胞の安定化に寄与することが示された。 The following can be said from Fig. 2-1 and Fig. 2-2. The expression level of ITGA6 is increased by applying high frequency. In particular, it can be seen from FIG. 2-1 that the expression level is significantly increased (according to Dunnett's test, a significant difference was observed at the significance level of 5%). From the above, it was shown that the application of high frequency enhances the production of adhesion factors by dermal stem cells, and as a result, enhances the binding of dermal stem cells to blood vessels and contributes to the stabilization of dermal stem cells.
　［実験２：手術余剰皮膚への高周波の適用による真皮幹細胞の安定化］
　実験２では、対象として手術余剰皮膚を用い、実験１と同様に、周波数１ＭＨｚ、ピーク間電圧８８Ｖｐｐの高周波を適用してITGA6陽性細胞の変化を確認した。高周波は、３分間適用した。高周波の適用は、繰り返し周期が１秒、デューティー比が４０％のパルス印加とした。 [Experiment 2: Stabilization of dermal stem cells by applying high frequency to surplus skin]
In Experiment 2, changes in ITGA6-positive cells were confirmed by using surgical surplus skin as a subject and applying a high frequency with a frequency of 1 MHz and an inter-peak voltage of 88 Vpp in the same manner as in Experiment 1. The high frequency was applied for 3 minutes. The high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%.
　実験２によって得られた、ＲＦ適用後、培養３日目の画像を図３－１、３－２に示す。なお、高周波の適用による効果を確認するため、高周波を適用しなかった以外は上記と同じ条件で培養組織を可視化し、図３－１および３－２には、その結果も上段に「コントロール（Control）」として併せて示した。右列は左列の四角部分を拡大した画像である。図３－１より、下段に示すＲＦ負荷により、乳頭層のITGA6陽性細胞がコントロールに比べて増えている様子が観察された。図３－２より、CD34およびMCSPのそれぞれについて、下段に示すＲＦ負荷により、ITGA6陽性細胞で幹細胞マーカーCD34やMCSPの発現が認められ、かつそれらの発現が上昇している様子が観察された。よって、ＲＦ適用により幹細胞が安定化すると言える。 The images obtained in Experiment 2 on the third day of culture after RF application are shown in FIGS. 3-1 and 3-2. In order to confirm the effect of applying high frequency, the cultured tissue was visualized under the same conditions as above except that high frequency was not applied. It is also shown as "Control)". The right column is an enlarged image of the square part in the left column. From FIG. 3-1 it was observed that the number of ITGA6-positive cells in the papillary layer increased compared to the control due to the RF load shown in the lower row. From FIG. 3-2, it was observed that the stem cell markers CD34 and MCSP were expressed in ITGA6-positive cells and their expression was increased by the RF loading shown in the lower row for each of CD34 and MCSP. Therefore, it can be said that stem cells are stabilized by applying RF.
　［実験３：手術余剰皮膚への高周波の適用によるECM産生の増強］
　実験３では、対象として手術余剰皮膚を用い、実験２と同様に、周波数１ＭＨｚ、ピーク間電圧８８Ｖｐｐの高周波を適用して真皮のマトリックス産生の変化を確認した。高周波は、３分間適用した。高周波の適用は、繰り返し周期が１秒、デューティー比が４０％のパルス印加とした。 [Experiment 3: Enhancement of ECM production by applying high frequency to surplus skin after surgery]
In Experiment 3, a surgical surplus skin was used as a subject, and a high frequency with a frequency of 1 MHz and a peak voltage of 88 Vpp was applied as in Experiment 2, and changes in dermis matrix production were confirmed. The high frequency was applied for 3 minutes. The high frequency was applied by applying a pulse with a repetition period of 1 second and a duty ratio of 40%.
　実験３によって得られた、ＲＦ適用後、培養３日目の画像を図４－１および４－２に示す。なお、高周波の適用による効果を確認するため、高周波を適用しなかった以外は上記と同じ条件で培養組織を可視化し、図４－１および４－２には、その結果も上段に「コントロール（Control）」として併せて示した。図４－１および４－２より、下段に示すＲＦ負荷により、線維芽細胞によるV型コラーゲンおよびフィブリリン1の発現がコントロールに比べて増えている様子が観察された。ＲＦの適用によりマトリックスの産生が促進され、肌の張りに寄与すると考えられる。 The images obtained in Experiment 3 on the third day of culture after RF application are shown in FIGS. 4-1 and 4-2. In order to confirm the effect of applying high frequency, the cultured tissue was visualized under the same conditions as above except that high frequency was not applied. It is also shown as "Control)". From FIGS. 4-1 and 4-2, it was observed that the expression of V-type collagen and fibrillin 1 by fibroblasts was increased by the RF load shown in the lower row as compared with the control. It is considered that the application of RF promotes the production of matrix and contributes to the tension of the skin.
　［実験１、２、３のまとめ］
　上述したとおり、実験１～３のそれぞれの結果より、高周波の適用が真皮幹細胞におけるITGA6の遺伝子発現の増強とそれによる真皮幹細胞の安定性の改善を導き、それに伴って線維芽細胞によるV型コラーゲンやフィブリリン1の産生が増強することが明らかになった。
  [Summary of Experiments 1, 2 and 3]
As described above, from the results of Experiments 1 to 3, the application of high frequency leads to the enhancement of ITGA6 gene expression in dermal stem cells and the resulting improvement in the stability of dermal stem cells, and accordingly, V-type collagen by fibroblasts. And fibrillin 1 production was found to be enhanced.
　１　　高周波装置
　１０　　電極部
　１０ａ　　第１電極
　１０ｂ　　第２電極
　２０　　制御回路
  1 High frequency device 10 Electrode part 10a 1st electrode 10b 2nd electrode 20 Control circuit

Claims (17)

  1. 　皮膚に高周波を適用して真皮幹細胞の安定性を高めることを含む、美容方法。 A cosmetological method that involves applying high frequency waves to the skin to increase the stability of dermal stem cells.
  2. 　皮膚に高周波を適用して真皮幹細胞におけるITGA6の遺伝子発現を増強させることを含む、美容方法。 A cosmetological method that involves applying high frequency to the skin to enhance ITGA6 gene expression in dermal stem cells.
  3. 　皮膚に高周波を適用して真皮線維芽細胞におけるV型コラーゲンおよび／またはフィブリリン1の産生を増強させることを含む、美容方法。 A cosmetological method comprising applying high frequency to the skin to enhance the production of V-type collagen and / or fibrillin-1 in dermal fibroblasts.
  4. 　真皮幹細胞の安定性を高める、請求項２または３に記載の美容方法。 The cosmetological method according to claim 2 or 3, which enhances the stability of dermal stem cells.
  5. 　高周波の周波数が約１ＭＨｚである、請求項１～４のいずれか一項記載の美容方法。 The beauty method according to any one of claims 1 to 4, wherein the high frequency frequency is about 1 MHz.
  6. 　高周波のピーク間電圧が５０～１００Ｖｐｐである、請求項１～５のいずれか一項に記載の美容方法。 The beauty method according to any one of claims 1 to 5, wherein the high frequency peak voltage is 50 to 100 Vpp.
  7. 　高周波の適用が、デューティー比１０％～９９％のパルス印加である、請求項１～６のいずれか一項に記載の美容方法。 The beauty method according to any one of claims 1 to 6, wherein the application of high frequency is pulse application with a duty ratio of 10% to 99%.
  8. 　前記パルス印加の周期は０．１秒～２秒である、請求項７に記載の美容方法。 The beauty method according to claim 7, wherein the pulse application cycle is 0.1 to 2 seconds.
  9. 　前記パルス印加の周期は１秒である、請求項８に記載の美容方法。 The beauty method according to claim 8, wherein the pulse application cycle is 1 second.
  10. 　皮膚の張りを向上させる、請求項１～９のいずれか一項に記載の美容方法。 The beauty method according to any one of claims 1 to 9, which improves the tension of the skin.
  11. 　皮膚に高周波を適用する工程を含む、真皮幹細胞の安定化方法。 A method for stabilizing dermal stem cells, including the step of applying high frequency to the skin.
  12. 　皮膚に高周波を適用する工程を含む、真皮幹細胞におけるITGA6の遺伝子発現の増強方法。 A method for enhancing ITGA6 gene expression in dermal stem cells, including the step of applying high frequency to the skin.
  13. 　皮膚に高周波を適用する工程を含む、真皮線維芽細胞におけるV型コラーゲンおよび／またはフィブリリン1の産生の増強方法。 A method for enhancing the production of V-type collagen and / or fibrillin 1 in dermal fibroblasts, which comprises the step of applying high frequency to the skin.
  14. 　一対の電極と、前記一対の電極に高周波電圧を印加する制御回路と、を有する高周波美容処理装置の作動方法であって、
    　前記制御回路により、前記一対の電極に、真皮幹細胞の安定性を高める高周波電圧を印加すること、
    　を含み、
    　前記高周波電圧の印加は、前記一対の電極を皮膚に接触させた状態で行なわれる、
    　高周波美容処理装置の作動方法。     A method of operating a high-frequency beauty treatment apparatus having a pair of electrodes and a control circuit for applying a high-frequency voltage to the pair of electrodes.
    By the control circuit, a high frequency voltage that enhances the stability of dermal stem cells is applied to the pair of electrodes.
    Including
    The application of the high frequency voltage is performed in a state where the pair of electrodes are in contact with the skin.
    How to operate the high frequency beauty treatment device.
  15. 　一対の電極と、前記一対の電極に高周波電圧を印加する制御回路と、を有する高周波美容処理装置の作動方法であって、
    　前記制御回路により、前記一対の電極に、真皮幹細胞におけるITGA6の遺伝子発現、ならびに／または真皮線維芽細胞におけるV型コラーゲンおよび／もしくはフィブリリン1の産生を増強させる高周波電圧を印加すること、
    　を含み、
    　前記高周波電圧の印加は、前記一対の電極を皮膚に接触させた状態で行なわれる、
    　高周波美容処理装置の作動方法。     A method of operating a high-frequency beauty treatment apparatus having a pair of electrodes and a control circuit for applying a high-frequency voltage to the pair of electrodes.
    By the control circuit, a high frequency voltage that enhances the gene expression of ITGA6 in dermal stem cells and / or the production of V-type collagen and / or fibrillin-1 in dermal fibroblasts is applied to the pair of electrodes.
    Including
    The application of the high frequency voltage is performed in a state where the pair of electrodes are in contact with the skin.
    How to operate the high frequency beauty treatment device.
  16. 　高周波のピーク間電圧が５０～１００Ｖｐｐである、請求項１４または１５に記載の作動方法。 The operating method according to claim 14 or 15, wherein the high frequency peak voltage is 50 to 100 Vpp.
  17. 　高周波電圧の印加が、１秒周期、デューティー比１０～９９％のパルス印加である、請求項１４～１６のいずれか一項に記載の作動方法。
          The operation method according to any one of claims 14 to 16, wherein the application of a high frequency voltage is a pulse application having a cycle of 1 second and a duty ratio of 10 to 99%.
PCT/JP2021/046152 2020-12-23 2021-12-14 Method for promoting regeneration of dermis by applying high-frequency electric stimulus WO2022138350A1 (en)

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Citations (4)

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US20170304642A1 (en) * 2008-06-29 2017-10-26 Venus Concept Ltd. Esthetic apparatus useful for increasing skin rejuvenation and methods thereof
JP2017222676A (en) * 2017-07-05 2017-12-21 マクカイ メモリアル ホスピタル Use of pedf-derived polypeptides for preventing and/or ameliorating skin aging
JP2018000489A (en) * 2016-06-30 2018-01-11 株式会社メディカ ボーテ Cosmetic device
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JP2018504976A (en) * 2015-02-03 2018-02-22 ジョンジュ ナ Treatment device for blood vessels in the skin
JP2018000489A (en) * 2016-06-30 2018-01-11 株式会社メディカ ボーテ Cosmetic device
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