JP2009000092A - Human photoaged skin model - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a human photo aged skin model useful for evaluating cosmetics or the like in terms of anti-photoageing or improving effects, and to provide an animal model and a method for producing them. <P>SOLUTION: The method for making the human photoaged skin model is the one where UV rays, having wavelengths B of 40 to 100 mJ/cm<SP>2</SP>, are continuously irradiated on the transplanted skin of an immunodeficient non-human animal transplanted with the human skin for 6 weeks or longer. The human photoaged skin model and the animal model are produced by the method. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a human photoaging model skin provided with human photoaging skin, a model animal, and a method for producing them.

  Human skin ages and wrinkles and sags with age. In particular, in skin exposed to sunlight, such as the face, neck, and shoulders, wrinkles, sagging, freckles, and freckles become more prominent than other areas that do not receive light due to the effects of chronic ultraviolet rays. At the same time, the elasticity of the skin, darkening of the skin, an increase in yellowing, and a decrease in the amount of horny water are also induced by the influence of ultraviolet rays. The phenomenon of peculiar skin aging that occurs in these UV-exposed areas is called photoaging.

  Therefore, in order to elucidate the mechanism of such skin photoaging, and to evaluate the adverse effects of photoaging, i.e., cosmetics and pharmaceuticals useful for the prevention and improvement of wrinkles, blemishes, freckles, etc. The existence of a model skin for photoaging that reflects more faithfully is useful.

  The most commonly used method for creating photo-aged model skin and animal models is the system that is exposed to chronic UV rays. To date, UV rays are irradiated daily on the back skin of hairless mice and the skin of the rat sole for several weeks. There are known methods (Non-Patent Document 1 and Non-Patent Document 2) and methods for irradiating a three-dimensional skin model with a single ultraviolet ray in vitro (Non-Patent Document 3).

  In addition, as a model using human skin, a method of performing single UV irradiation on nude mice transplanted with human skin is known (Non-Patent Document 4), but a model system that continuously irradiates for several weeks every day. is not. Therefore, depending on this method, the skin to be used is derived from humans, but due to ultraviolet irradiation in a short period of time, it is not possible to cause a sufficient photoaging state in the transplanted human skin. It is insufficient.

Thus, no human model skin or model animal having human photoaged skin that faithfully reflects human photoaged skin has been obtained.
Bissett DL, Hannon DP, Orr TV, Photochem Photobiol. 1987; 46 (3): 367-78, Imayama S, Nakamura K, Takeuchi M, Hori Y, Takema Y, Sakaino Y, Imokawa G, J Dermatol Sci. 1994 7 (1): 32-8 Nelson D, Gay RJ, Photochem Photobiol. 1993; 57 (5): 830-7 Del Bino S, Vioux C, Rossio-Pasquier P, Jomard A, Demarchez M, Asselineau D, Bernerd F, Br J Dermatol. 2004; 150 (4): 658-67

  The present invention relates to providing a human photoaging model skin, a human photoaging model animal useful for evaluating the effects of preventing and improving aging of cosmetics, quasi drugs and the like, and a method for producing the same.

  The present inventors examined skin aging in human skin transplanted animals, and provided human photoaged skin very close to photoaging by continuously irradiating the transplanted skin with a specific amount of B-wavelength ultraviolet radiation for 6 weeks or longer. We found that model animals can be created.

That is, the present invention provides a human photoaging model skin characterized by continuously irradiating the transplanted skin of an immunodeficient non-human animal transplanted with human skin with 40 to 100 mJ / cm ² of B wavelength ultraviolet radiation for 6 weeks or more. It relates to the creation method.

  The present invention also relates to a human photoaging model skin prepared by the above method.

  The present invention also relates to a human photoaging skin model animal prepared by the above method.

  According to the method of the present invention, human photoaging model skin and model animals that stably exhibit the skin aging state can be created in a short period of time, and by using this, elucidation of human skin aging mechanism and prevention / improvement of skin aging The substance can be evaluated more accurately.

In the method for producing human photoaging model skin of the present invention, 40 to 100 mJ / cm ² of B wavelength ultraviolet rays are continuously irradiated for 6 weeks or more to the transplanted skin of an immunodeficient animal transplanted with human skin.

  In the present invention, human photoaging model skin refers to an aging state that is very close to natural aging (for example, wrinkles, sagging, blotches, freckles, reduced skin elasticity, skin blackening, increased yellowing, horny water content, It means the skin in a human photoaged skin model animal provided with human photoaged skin exhibiting a decrease, etc.).

As the animal in the method of the present invention, an immunodeficient non-human animal is used. An immunodeficient mouse such as a SCID mouse (for example, BALB cA-nu / scid, B-17 / Icr-Scid, etc.), an athymic nude mouse, F344 Jc1- Examples include immunodeficient rats such as rnu, and immunodeficient mice are preferred, and SCID mice are more preferred from the standpoint that they can withstand long-term irradiation with ultraviolet rays.
These animals are preferably raised under the condition of 1 animal / cage in an SPF environment. Such animals are available from Nippon Clare Co., Ltd. and Taconic (NY).

  As human skin to be transplanted into such immunodeficient non-human animals, it is preferable to use non-lighted skin, for example, a newborn foreskin or an abdominal skin if derived from an adult. The skin may be circumcision, skin supplied by cosmetic surgery, or cadaver skin supplied by a skin center.

  The human skin is collected aseptically in a thickness of 2 to 5 mm, and in a DMEM containing an appropriate culture medium such as L-glutamine and antibiotic / antimycotic (Invitrogen, CA) until it is transplanted to an animal. It is preferably stored at 2 to 4 ° C. and stored in a non-overcrowded state.

  Skin transplantation to animals may be performed according to a known method (Demarchez M, Hartmann DJ, Herbage D, Ville G, Dev Biol. 1987; 121 (1): 119-29.), For example, Can be performed.

After anesthetizing the animal with isofluorane / oxygen, Nembutal, etc., a skin defect of 2 × 2 cm to 3 × 3 cm is created on the back. At this time, it is preferable to shave. Thereafter, a human skin graft of the same size is transplanted into the skin defect on the back of the animal. Next, 10-20 needles are sutured with nylon thread. After suturing, it is preferable to add a local anesthetic such as “sensorcaine” (bupivacaine hydrochloride) to the boundary between the transplanted skin and the mouse skin to treat sensory defatting.
Mice are maintained in a 37 ° C. incubator from anesthesia to waking up.

  The irradiation with ultraviolet rays is preferably started after healing of the transplanted skin is completed, that is, after about 10 weeks have passed since the transplantation.

  The ultraviolet rays to be irradiated are B-wavelength ultraviolet rays in the range of 290 to 320 nm, and those having a peak in the vicinity of 302 nm are preferably used.

  In terms of operability, the irradiation can be performed at a distance from the ultraviolet lamp to the animal (transplanted skin) of about 5 to 80 cm, preferably about 30 to 50 cm.

The irradiation dose may be a 40~100mJ / cm ^2, for example ^{40~60mJ / cm 2, 60~100mJ / cm} 2, include 80~100mJ / cm ^2.
The said irradiation dose can be suitably determined on the basis of the minimum erythema amount (MED) of the transplanted skin, and may be determined from the irradiation dose corresponding to 0.8 to 1.6 MED, for example. The minimum amount of erythema is determined by irradiating the transplanted skin with various amounts of ultraviolet rays and visually determining erythema after 24 hours.
Moreover, although irradiation is continuously irradiated for 6 weeks or more, for example, 6 to 12 weeks are mentioned, Preferably it is 6 to 10 weeks, More preferably, it is 6 to 8 weeks.

Moreover, until the irradiation start first weeks to week 3, increased weekly by 10 mJ / cm ^2, the third week later to maintain 60~100mJ / cm ^2, eliminate the effect of acute UV This is preferable. Nachi Suwa, irradiation dose of the first week after the beginning of the ultraviolet irradiation, 40 mJ / cm ^2, the second week is 50 mJ / cm ^2, the third week was set to 60 mJ / cm ^2, irradiation start third week thereafter 60-100 mJ / cm ² is preferable.

  In addition, it is good to perform such ultraviolet irradiation 5-6 times a week with the irradiation frequency of the said dose of ultraviolet rays once a day.

  Human transplanted skin that has been irradiated with ultraviolet rays under the conditions described above develops an aging state immediately after the end of irradiation.

  The aging state thus developed is very close to photo-aged skin such as wrinkle formation as shown in FIG. 1, and this state lasts for 8 weeks. In the skin, collagen I, collagen III, collagen IV, collagen VI, elastin (oxytalan fiber), matrix metalloproteinase (MMP) 1, MMP13, keratin 6, keratin 12, keratin 16, filaggrin, loricrin It is accompanied by changes in protein expression that occur in the skin due to photoaging, such as increased or decreased expression of proteins such as involucrin.

  The human photoaging model skin and model animal of the present invention thus obtained are useful for elucidating the human skin aging mechanism, evaluating skin aging prevention / improving substances, regenerative medicine, and the like.

1. Skin transplantation method
Skin was transplanted into 4-6 week old immunodeficient mice (female). Mice were kept under aseptic conditions throughout the duration of the experiment. Immediately before transplanting the skin, the back hair was removed using an electric shaver and anesthetized with isofluorane / oxygen (3% / 0.8 liter). During the transplantation operation, anesthesia was maintained in an isofluorane / oxygen (2% / 0.7 liter) environment. The skin on the back of the mouse was removed so that a skin defect (wound bed) with a diameter of about 2.0-3.0 cm was formed, and the skin derived from the abdomen was sutured and transplanted. The supplied skin was maintained in DMEM containing L-glutamine and antibiotic / antimycotic (Invitrogen, CA) until transplantation (2-4 ° C.). After the suture, sensorcaine was added to the boundary between the transplanted skin and the mouse skin to treat sensory defatting. Mice were maintained in a 37 ° C. incubator from anesthesia until waking up.

2. B wavelength ultraviolet (UVB) irradiation
UVB irradiation began at least 10 weeks after the transplant was completed. A UVB lamp with a peak near the wavelength of 302 nm was used, and the distance from the lamp to the transplanted skin was about 30 cm.

Since the minimum erythema dose of grafted skin (MED) was found to be equivalent to 40 mJ / cm ^2, irradiation of chronic UVB was to begin at 40 mJ / cm ^2. Irradiation was carried out for 5 days a week for 6 weeks. The dose was increased by 10 mJ / cm ² by 3 weeks and maintained at 60 mJ / cm ² from 3 to 6 weeks. During the irradiation, the mouse was allowed to move freely in a 100 cm ² transparent container.

3. Evaluation of aging skin condition (1) After the start of irradiation, the surface of the transplanted skin is observed with the naked eye using the reflected transmitted light by Charm View (registered trademark; Moritex, Japan), and digital images are taken every two weeks. I took a picture.

(2) A replica of the transplanted skin was prepared using a replica agent, and the obtained replica was subjected to roughness analysis by the following method.

<Replica roughness analysis>
Replicas were made before the start of the study and at the 3rd and 6th weeks from the start. When replicas were made, anesthesia was maintained in an isofluorane / oxygen (2% / 0.7 liter) environment.
The replica of the transplanted skin obtained by SILFLO (Flexico developments) is analyzed using a replica agent (GC exafine) and applied with a plate or the like so as to be flattened as much as possible. It was.
The three-dimensional shape of the replica was measured with PRIMOS Compact (GF Messtechnik). After measurement, the swell was removed with a filter.

  In the line roughness analysis, an analytical line which is a straight line having a length of 7-9 mm of the obtained replica was drawn, and a line roughness parameter value of this portion was obtained. At that time, I tried not to take in the shape of the ridge except for the wrinkle. In the surface analysis, a rectangular range of about 4-5 mm × 7-9 mm was specified for the central portion of the previous replica, and surface roughness analysis was performed within that range. In this case, the range was arbitrarily selected so as not to capture the shape such as the wrinkles as much as possible.

As parameters, Ra and Sa (arithmetic mean roughness) and Rz and Sz (ten-point mean roughness) were used for comparison. Ra and Rz represent one-dimensional parameters, and Sa and Sz represent two-dimensional parameters.
Ra, Sa: Arithmetic mean roughness
Rz, Sz: Ten-point average roughness (the sum of the highest peak height from the highest peak to the fifth highest and the highest valley depth from the deepest valley to the fifth highest)

(3) Gene expression analysis The transplanted skin was collected 24 hours after the last UVB irradiation. Total RNA was extracted with Trizol reagent (Invitrogen, CA) and purified with RNeasy mini kit (Qiagen, CA). The purity of the RNA sample (28s / 18s) was confirmed using an Alien bioanalyzer. The target cRNA labeled with biotin was performed according to the protocol of Affymetrix. Briefly, double-stranded cDNA was prepared using T7 (-dT) 24 primer 5'-GGCCAGTGAATTGTAATACGACTCACTAT AGGGAGGCGG-3 '(SEQ ID NO: 1) and Superscript II reverse transcriptase. CRNA from each sample was hybridized to Human Genome U133 plus 2.0 chip.

4). Results (1) Appearance FIG. 1 shows a comparison with the skin appearance control (UVB non-irradiated group) at the time when the transplanted skin was irradiated with UVB for 6 weeks. As is clear from FIG. 1, the skin surface shape did not change significantly in the control, whereas the shape change on the wrinkle-like line was observed and increased when UVB irradiation was performed.

(2) Replica analysis

  FIG. 2 shows a comparison with the control of the three-dimensional roughness analysis result of the skin replica after UVB irradiation 3 and 6 weeks irradiation when using abdominal skin. As is clear from FIG. 2, the control shows almost no change in the roughness parameters Ra and Sa (arithmetic mean roughness, FIGS. 2A and C), Rz and Sz (10-point mean roughness, FIGS. 2B and D). That is, no change was observed in the roughness of the skin surface, but in the group irradiated with UVB, an increase in Ra, Rz, Sa and Sz values was observed when UVB was irradiated for 6 weeks. That is, it shows that the wrinkle-like appearance unevenness change shown in FIG. 1 appeared.

(3) Gene expression profile After confirming the degree of RNA purification, a gene expression profile in an abdominal skin graft irradiated with UVB was prepared using a control group not irradiated with UVB as a control. The amount of change in mRNA transcript concentration between the control group and the group irradiated with UVB was shown as a log-log plot. In the abdominal skin graft, genes whose expression was increased by UVB irradiation were keratin 6, keratin 16, keratin 12, MMP13, MMP1, involucrin and filaggrin. The genes whose expression was suppressed were collagen I, collagen III, collagen IV and collagen VI.

The figure which showed the external appearance change of the skin transplanted with the SCID mouse | mouth in a control (A, C, and E) and a UVB irradiation group (B, D, and F). A and B: 0 weeks after the start of UVB irradiation, C and D: 3 weeks after the start of irradiation, E and F: 6 weeks after the start of irradiation (Bar = 3 mm). Arithmetic average roughness Ra and Sa (A, C) and ten-point average roughness obtained from the three-dimensional roughness analysis of replicas at 0, 3 and 6 weeks after the start of UVB irradiation when using abdominal skin The graph which showed Rz and Sz (B, D). Square marks indicate controls, and circle marks indicate UVB irradiation groups. Error Bar = ± SD (n = 4-6), * p <0.05 ** p <0.01 (compared to week 0).

Claims (7)

A method for preparing human photoaging model skin, comprising continuously irradiating B-100-ultraviolet rays of 40 to 100 mJ / cm ² for 6 weeks or more to the transplanted skin of an immunodeficient non-human animal transplanted with human skin.   The method according to claim 1, wherein the immunodeficient non-human animal is a SCID mouse.   The method according to claim 2, wherein the SCID mouse is BALB cA-nu / scid or B-17 / Icr-Scid.   The method according to any one of claims 1 to 3, wherein the ultraviolet ray is irradiated continuously for 6 to 8 weeks. The dose of B-wavelength ultraviolet, until the irradiation start first weeks to week 3, increased weekly by 10 mJ / cm ^2, the third week after 1 to claim to maintain 60~100mJ / cm ² 5. The method according to any one of 4 above.   The human photoaging model skin created by the method of any one of Claims 1-5.   A human photoaging skin model animal produced by the method according to any one of claims 1 to 5.