WO2022138350A1 - 高周波電気刺激の適用による真皮再生促進方法 - Google Patents

高周波電気刺激の適用による真皮再生促進方法 Download PDF

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Publication number
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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Prior art keywords
high frequency
skin
stem cells
applying
electrodes
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English (en)
French (fr)
Japanese (ja)
Inventor
みなみ 山田
俊介 入山
勤 相馬
謙太朗 山▲崎▼
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Shiseido Co Ltd
Ya Man Ltd
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Shiseido Co Ltd
Ya Man Ltd
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Priority to CN202180078685.XA priority Critical patent/CN116669645A/zh
Priority to JP2022572211A priority patent/JPWO2022138350A1/ja
Publication of WO2022138350A1 publication Critical patent/WO2022138350A1/ja
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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

Definitions

  • 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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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170304642A1 (en) * 2008-06-29 2017-10-26 Venus Concept Ltd. Esthetic apparatus useful for increasing skin rejuvenation and methods thereof
JP2017222676A (ja) * 2017-07-05 2017-12-21 マクカイ メモリアル ホスピタル 皮膚老化を予防及び／又は改善するためのｐｅｄｆ−由来ポリペプチドの使用
JP2018000489A (ja) * 2016-06-30 2018-01-11 株式会社メディカ ボーテ 美容機器
JP2018504976A (ja) * 2015-02-03 2018-02-22 ジョンジュ ナ 皮膚内の血管の治療装置

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170304642A1 (en) * 2008-06-29 2017-10-26 Venus Concept Ltd. Esthetic apparatus useful for increasing skin rejuvenation and methods thereof
JP2018504976A (ja) * 2015-02-03 2018-02-22 ジョンジュ ナ 皮膚内の血管の治療装置
JP2018000489A (ja) * 2016-06-30 2018-01-11 株式会社メディカ ボーテ 美容機器
JP2017222676A (ja) * 2017-07-05 2017-12-21 マクカイ メモリアル ホスピタル 皮膚老化を予防及び／又は改善するためのｐｅｄｆ−由来ポリペプチドの使用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SEO KYU YOUNG, YOON MOON SOO, KIM DONG HYUN, LEE HEE JUNG: "Skin rejuvenation by microneedle fractional radiofrequency treatment in Asian skin; Clinical and histological analysis", LASERS IN SURGERY AND MEDICINE., WILEY- LISS, NEW YORK., US, vol. 44, no. 8, 1 October 2012 (2012-10-01), US , pages 631 - 636, XP055945052, ISSN: 0196-8092, DOI: 10.1002/lsm.22071 *

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