JP2006220609A - Evaluation method for skin aging with fibulin-5 as indicator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a skin examination method for evaluating skin aging state. <P>SOLUTION: The skin examination method for evaluating senescent state of a subject is characterized, by determining that the aging of skin has been progressing, when the amount of presence of fibulin-5 in skin to be examined has fallen off significantly, while comparing with the development in contrast skin. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention provides a test method for evaluating the skin aging state using fibulin-5 as an index.

  Skin aging is divided into two types. That is, natural aging and photoaging. Natural aging is a basic biological process common to all living organisms, characterized by the deterioration of skin function and structure with age (such as epidermal atrophy and flattening of the epidermis-dermis junction). Lavker, RMJ Invest. Dermatol. 73: 59-66, 1979). Histologically, skin in its natural aging state has an extracellular matrix that is atrophic with reduced levels of elastin (Braverman, IM et al., J. Invest. Dermatol. 78: 434-443, 1982). . On the other hand, it is well known that photoaging is a result of constant exposure to sunlight. Sunlit skin (eg, skin on the face and neck) ages clearly faster than torso skin that is relatively protected from sunlight, and various clinical features of sunlit skin (eg, wrinkles). (Gilchrest, BAJ Am. Acad. Dermatol. 21: 610-613, 1989); Griffiths, CE, Br. J. Dermatol, rough skin, rough skin, spots, pigment changes, telangiectasia, neoplasia, etc.) 127: 37-42, 1992) and histological features (eg cell atypia, loss of polarity, flattening of the epidermis-dermis junction, collagen loss, skin fibrosis, elastic fibrotic material to the dermis (Abnormal deposition of) (Kligman, AM et al., J. Am. Acad. Dermatol. 15: 836-859, 1986; Lavker, RMJ Invest. Dermatol. 73: 59-66, 1979). Damage to skin collagen and elastin (extracellular matrix) is evidence of long-term exposure to sunlight ultraviolet light and is believed to be the cause of wrinkles found in sun-treated skin (Kligman, AM et al. 1986 (supra)).

  The fibrin gene family includes five different genes, from which nine or more protein products are produced through alternative splicing (Timpl, R. et al., Nat. Rev. Mol. Cell Biol. 4: 479-489, 2003). Fibulin is a widely expressed secreted protein found in blood and in the basement membrane and stroma of most tissues. Fibrin is self-associating in such places (Balbona, K. et al., J. Biol. Chem. 267: 20120-20125, 1992; Pan, TC et al, J. Biol. Chem. 123: 1269- 1277, 1993) and / or interacting with a variety of extracellular matrix elements (eg fibronectin, laminin, nidogen, aggrecan, versican, endostatin, elastin) (Aspberg, A. et al., J. Biol. Chem). 274: 20444-20449, 1999; Nakamura, T. et al., Nature 415: 171-175, 2002; Timpl, R. et al., 2003 (supra); Yanagisawa, H. et al., Nature 415: 168-171, 2002). Fibulin is thought to be involved in the construction and stabilization of extracellular matrix structures and has been previously implicated in the regulation of organogenesis, angiogenesis, fibrogenesis, and tumor formation (Roark, Ef et al., J. Histochem. Cytochem. 43: 401-411, 1995; Tran, H. et al., J. Biochem. 270: 19458-19464, 1995; Zhang, HY et al, Dev. Dyn. 205 : 348-364, 1996).

  The latest members of the fibrin family are fibulin-5 [EVEC (Kowal, RC et al., Circ. Res. 84: 1166-76, 1999) or DANCE (Nakamura, T. et al., J. Biol. Chem. 274: 22476-22483, 1999)]. Fibulin-5 is a glycoprotein consisting of 448 amino acids and has interesting structural features. Fibulin-5 is a proline-rich insert in the RGD motif that binds to integrin, six epidermal growth factor-like repeat regions that bind to calcium, and the first epidermal growth factor-like repeat region that binds to calcium. And a globular C-terminal domain (Kowal, RC et al., 1999 (supra); Nakamura, T. et al., 1999 (supra)). Functionally, fibulin-5 binds to αvβ3 integrin, αvβ5 integrin, α9β1 integrin (Nakamura, T. et al., 2002 (supra)) and adheres to endothelial cells through the RGD motif (Nakamura, T. et al ., 1999 (supra)). Inactivation of the fibulin-5 gene in mice causes severe elastinopathies in the skin, lungs, and vasculature (Nakamura, T. et al., 2002 (supra); Yanagisawa, H. et al., 2002 (supra)). ).

Nakamura, T. et al., Nature 415: 171-175, 2002 Yanagisawa, H. et al., Nature 415: 168-171, 2002

  The purpose of the research underlying the present invention was to examine the change of elastic fibers in the skin aging process. Fibulin-5 is a secreted extracellular matrix protein that serves as a scaffold for elastic fibers, but it is clear how fibulin-5 is distributed in human skin and how it changes with age. is not. In order to investigate how fibulin-5 is involved in skin aging, the present inventor analyzed the age-related changes in the presence of fibulin-5 in human skin with other elastic fiber elements (elastin, fibrillin-1 It was compared with the age change of (Fibrillin-1) and Fibulin-2). In the dermal reticular layer of the skin taken from the upper arm protected from sunlight of a 17-year-old subject, not only fibulin-5 but also other elastic fiber elements were present, whereas in the dermal papillary layer fibrin- No. 5 showed a candlestick-like structure perpendicular to the epidermis, and a non-stained area was present directly under the epidermis. This is similar to the presence state of elastin, but is different from the presence state of fibrillin-1. Fibulin-5 in the dermal reticulate layer decreased with age in skin protected from sunlight on the thigh, abdomen, and upper arm, and disappeared in some cases. In sunlit skin, fibulin-5 was significantly reduced in the dermis of the cheek skin, even in 20-year-old subjects. Irradiation with 2MED UVB significantly reduces fibulin-5 levels, moderately lower fibulin-2 levels and slightly lower elastin levels compared to non-irradiated control skin did. Interestingly, fibulin-5, like other elastic fiber elements, had increased deposits in sunlight elastic fibrosis. These results suggest that fibulin-5 is an excellent marker of skin aging and that early reduction of fibulin-5 may be related to age changes in other elastic fiber elements. Yes.

As a result, the present application provides the following inventions.
(1) A skin test method for evaluating the aging state of a subject's skin, wherein the expression level of fibulin-5 in the skin to be examined is significantly lower than the expression in the control skin A skin inspection method, characterized in that it is determined that the aging state of the skin is progressing.
(2) The skin examination method according to (1), wherein the aging of the skin is caused by natural aging and / or exposure to ultraviolet B waves (UVB).
(3) The skin examination method according to (1) or (2), wherein the measurement of the expression level of fibulin-5 is carried out by measuring the amount of fibulin-5 in the skin to be examined.
(4) The skin test method according to (3), wherein the amount of fibulin-5 is measured by an immunoassay using an antibody specific for fibulin-5.
(5) The skin test method according to (4), wherein the antibody is a polyclonal antibody.
(6) The skin test method according to (1) or (2), wherein the expression level of fibulin-5 is determined by measuring the amount of mRNA encoding fibulin-5 extracted from the skin.
(7) The skin test method according to (6), wherein the mRNA is measured by a polymerase chain reaction method.
(8) The skin test method according to (7), wherein the polymerase chain reaction method is a real-time polymerase chain reaction method.

  According to the present invention, it is possible to provide a skin inspection method for evaluating the skin aging state.

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention provides a method for evaluating skin aging status. This method measures the expression level of fibulin-5 in the skin to be examined, compares the expression level with the expression level in the control skin, and the expression level of fibulin-5 in the skin to be examined is in the control skin. If the expression level is significantly increased compared to the expression level, it is judged that the aging state of the skin is progressing. If the progress of skin aging can be detected at an early stage, the advancement of treatment can advantageously prevent the progress of skin aging. As an evaluation criterion for the aging state of the skin, for example, fibulin-5 expression in skin cells is 10% or more, or 20% or more, or 30% or more, or 50% or more, or 70 compared to those in control skin cells. % Or 100% or more, it may be determined that “the aging state of the skin is progressing”.

  The skin to be examined may be the dermis of any part of the face, neck, arms, limbs, etc. that is concerned about the progress of aging. The control skin may be, for example, the epidermis of a part of the same individual that is difficult to be exposed to, for example, ultraviolet rays and relatively difficult to age, such as the dermis of the abdomen and buttocks.

  The expression level of fibulin-5 in the skin can be determined, for example, by directly measuring the amount of fibulin-5 in the skin. Preferably, this measurement utilizes an antibody specific for fibulin-5, and is well known in the art, such as immunohistochemical staining using fluorescent materials, dyes, enzymes, etc., Western blotting, immunoassay, such as Various methods such as ELISA and RIA can be used. The antibody specific for fibulin-5 used in the above measurement may be a monoclonal antibody or a polyclonal antibody. Polyclonal antibodies are particularly preferred. Methods for producing monoclonal and polyclonal antibodies are well known to those skilled in the art. For example, a description of antibodies specific for fibulin-5 is Charbonneau, NL et al.,. Biol. Chem., 2740-2749, 2003; Reinhardt, DP , J. Mol. Biol. 258: 104-116, 1996.

  It can also be determined by extracting RNA from the skin and measuring the amount of mRNA encoding fibulin-5. The base sequence of the gene encoding fibulin-5 is known and is described, for example, in Nakamura, T. et al., 1999 (supra). Extraction of mRNA and measurement of the amount thereof are also well known in the art. For example, RNA is quantified by quantitative polymerase chain reaction (PCR), for example, real-time polymerase chain reaction (rPCR). Alternatively, fibulin-5 expression can be determined through in situ hybridization methods.

  Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this.

Materials and Methods Subjects All studies were approved by St. Marianna University School of Medicine and Shiseido's Ethics Committee. Written informed consent was submitted by all subjects at St. Marianna University Hospital. One male and four females provided skin samples on the thigh, two males and two females provided upper arm skin samples, and one male and two females provided cheek skin ( Age range is 5 to 76 years old). Samples were prepared from excess skin cut during plastic surgery. Written informed consent was submitted by two healthy male volunteers to conduct a one-time UVB study. A punch biopsy sample (3 mm) was taken from the skin that was not exposed to sunlight, and from the skin of the buttocks 2 days after irradiation with 2 MED UVB.

Antibodies Characterization of monoclonal antibodies against elastin, fibrillin-1, and fibrin-2 have been previously characterized (Charbonneau, NL, 2003 (supra); Reinhardt, DP et al., 1996 (supra)). Using a pCEP-Pu vector as described in Markova, DY et al., Am. J. Hum. Genet. 72: 98-1004, 2003; Pan, TC et al, 1993 (supra). Rabbit antiserum was obtained from recombinant fibulin-2 and fibulin-5 expressed in 293-EBNA cells.

Immunohistochemistry
According to the AMeX method (Sato, Y. et al, Am. J. Pathol. 125: 431-435, 1986), skin fragments and biopsy samples were fixed with cold acetone and embedded in paraffin. Paraffin was removed from 3 μm thick sections using xylene, rehydrated and pretreated with 3% hydrogen peroxide for 15 minutes to stop endogenous peroxidase activity. Standard 10% rabbit serum (Histfine, Tokyo, Japan) was used to block nonspecific binding and samples were incubated at room temperature for 30 minutes. Add primary monoclonal antibody and incubate overnight at 4 ° C, then incubate sections with EnVision (DAKO, Carpinteria, CA), then stain with DAKO's liquid DAB + based chromogen Then, cell nuclei were stained with hematoxylin.

Experimental result
Fig. 1 shows the presence of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the skin dermis protected from sunlight in young human dermis protected from sunlight . . Presence of elastin (a and e), fibulin-5 (b and f), fibrillin-1 (c and g), fibulin-2 (d and h) in the upper arm skin protected from sunlight in a 17-year-old woman The condition was examined by immunohistochemical means using specific antibodies as described in “Materials and Methods”.
In the dermal reticular layer of the skin taken from the upper arm protected from sunlight of a 17-year-old woman, there are not only fibulin-5 but also other elastic fiber elements (eg elastin, fibulin-2, fibrillin-1). (FIGS. 1a to 1d). Fibulin-2, unlike fibrillin-1, was not detected in either children's skin or adult skin (data not shown). In the dermal papilla layer, fibulin-5 was a branched candlestick-like structure perpendicular to the epidermis, similar to the structure of other elastic fiber elements (FIGS. 1e-1h). However, when looking at the area directly under the epidermis, fibulin-5 and elastin were not present (FIGS. 1e and 1f), but fibrillin-1 was observed as a fiber structure at that position (FIG. 1g). This fibrillin-1 may have been inserted into the epidermal basement membrane. The fiber structure of fibulin-2 in the dermal papilla layer (FIG. 1h) was not as clear as that of elastin, fibrillin-1, and fibulin-5 (FIGS. 1e-1g), but fibrin-2 in the dermal reticulate layer The fiber structure (FIG. 1d) was clearer than the fiber structures of elastin, fibrillin-1 and fibrin-5 (FIGS. 1a to 1c).

FIG. 2 shows the changes in the expression of elastin, fibulin-5, fibrillin-1 and fibulin-2 in the dermis of the skin protected from sunlight. Shows age change. Elastin (a, e, i, m, q), fibrillin-5 (b, f, j, n, r), fibrillin-1 (c, g, k, o) in the thigh dermis protected from sunlight , S) and fibulin-2 (d, h, l, p, t) were examined by immunohistochemical means using specific antibodies described in “Materials and Methods”. The age change of elastic fiber elements was examined in the dermis of skin collected from subjects aged 5 to 75 years.
In the dermis taken from the thighs protected from sunlight of subjects aged 5, 13, 16 years, fibulin-5 shows a candelabrum-like structure in the dermal papilla layer and other elasticity in the dermis reticular layer. It was associated with elastic fibers composed of fiber elements (eg, elastin, fibulin-2, fibrillin-1) (FIGS. 2a to 21). However, the staining intensity of fibulin-5 (Fig. 2n) in the dermal reticular layer of skin collected from a 34-year-old subject is lower than that of other elastic fiber elements (Fig. 2m, Fig. 2o, Fig. 2p). It was. Fibulin-5 (FIG. 2r) was almost absent from the dermal reticular layer of skin taken from a 75 year old woman. Similarly, fibulin-5 combined with elastic fibers is present in the dermal reticular layer of the upper arm skin protected from sunlight in a 36 year old subject, in the dermal reticular layer taken from 11 year old, 17 year old and 24 year old subjects. (Data not shown). In addition, fibulin-5 bound to elastic fibers was significantly less than fibrillin-1 and fibulin-2 in the dermal reticular layer of skin taken from the abdomen of 34 and 75 year old subjects (data not shown). In contrast, in the dermal papilla layer, fibulin-5 maintained the staining intensity, but the number of stained fibers appeared to decrease with age (FIGS. 2n and 2r). Elastin also had a much larger age-dependent decrease in the dermal papillary layer than in the dermal reticular layer (Figure 2q).

FIG. 3 shows the age change of the expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the skin exposed to sunlight. Elastin (a, e, i), fibulin-5 (b, f, j), fibrillin-1 (c, g, k), fibulin-2 (d, h, l) in the dermis of sun-exposed cheek skin ) Was examined by immunohistochemical means using specific antibodies described in “Materials and Methods”. The age change of elastic fiber elements was examined in the dermis of the skin from subjects aged 20 to 76 years.
In sunlit skin, fibulin-5 was almost lost in the dermis of the cheek skin, even in 20 and 45 year old subjects (FIGS. 3b and 3f). This change occurred much earlier than the sun-protected skin (Figure 2). The elastic fiber structure in the dermis of the 45-year-old skin (Figs. 3e, 3g, 3h) is much wider and looser than the elastic fiber structure in the dermis of the 20-year-old skin (Figs. 3a, 3c, 3d). Seemed to be. This is similar to the elastic fibers observed in the dermis of a 76-year-old subject (Figs. 3i-3l), suggesting that there may be a progression of solar elastic fibrosis in the 45-year-old skin. ing. Interestingly, an increase in fibulin-5 deposition in solar elastic fibrosis was observed, as was the case with other elastic fiber elements (FIG. 3j). However, in skin exposed to sunlight, fibulin-5 decreased with aging and did not result in sunlight elastic fibrosis (FIGS. 3b and 3f).

Reduction of fibulin-5 in the dermis after UVB irradiation FIG. 4 shows the decrease in fibulin-5 in the dermis after UVB irradiation. The presence of elastin (a, b, g, h), fibrillin-5 (c, d, i, j), and fibrillin-1 (e, f, k, l) in the dermis of the buttock skin is expressed as “materials and By the immunohistochemical means using the specific antibody described in “Method”, the untreated sites (af) and UVB-irradiated sites (gl) were compared.
The effect of UVB irradiation on the distribution of fibulin-5 in the skin of the buttock taken from two volunteer men was reduced because dermal fibulin-5 decreased more quickly in sun-skinned skin than in sun-protected skin. Examined. Once irradiated with 2 MED UVB, the level of fibulin-5 (Figs. 4i and 4j) is significantly reduced in the dermis compared to the unirradiated skin (Figs. 4a to 4f), The level of fibulin-2 (FIGS. 4k and 4l) was moderately decreased and the level of elastin (FIGS. 4g and 4h) was slightly decreased.

DISCUSSION Through this study, the present inventor has found that the content of fibulin-5 in the dermal reticulate layer decreases with aging, and the rate of the decrease is other elastic fiber elements (e.g. elastin, fibulin-2, fibrillin-1). It was revealed that it was bigger than. The decrease in fibulin-5 was increased by UVB irradiation and occurred much earlier in sun-skinned skin than in sun-protected skin. Thus, UVB appears to be one of the major factors that cause elastic fiber damage during aging. Moreover, the early loss of fibulin-5 may be related to changes in elastic fibers during aging, particularly changes in elastic fibers due to photoaging.

  It has been reported that fibrin-5-deficient mice have prominent elastinopathies due to disordered elastic fibers, resulting in loose skin, abnormal blood vessels, and emphysema (Nakamura, t. Et al). ., 2002 (supra); Yanagisawa, H. et al., 2002 (supra)). Since fibulin-5 has an N-terminal domain that binds to integrin, it is thought to contribute to the organization of elastic fibers by playing a role in stably attaching cells to elastic fibers (Nakamura, t. Et al., 2002 (supra). Through this study, the inventor has found that fibulin-5 decreases in the dermal reticulated layer and eventually disappears during the aging process. When fibulin-5 is lost, the interaction between the dermal cells and the elastic fibers is weakened, so that the stability of the elastic fibers is reduced, and the elastic fibers may be atrophied while aging.

  Fibulin-5 deposition decreased faster in sun-skinned skin than in sun-protected skin. Furthermore, degradation of fibulin-5 deposited in the dermis was induced by UVB irradiation. Metalloproteinase mRNA, protein, and activity that degrade the matrix are known to be induced in living human skin within a few hours after UVB irradiation, and may degrade skin collagen and elastin. (Fisher, GJ etal., Nature 379: 335-339, 1996). Fibulin-2 has been reported to be degraded by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leukocyte elastase, and mast cell chymase (Sasaki, T. et al. al., Eur. J. Biochem. 240: 427-474, 1996). A small amount of fibrin-2 degradation products were also detected in photoelastic fibrosis, probably reflecting increased proteinase activity in light-damaged skin (Hunzelmann, N. et al. , Br. J. Dermatol., 145: 217-222, 2001). Although the sensitivity of fibulin-5 to proteinases has not yet been reported, proteinases induced by exposing the skin to UV may be involved in the degradation of fibulin-5 in the dermis.

  Characteristic of skin damaged by light is the change associated with the accumulation of elastic fiber component substances in the dermis (Lavker, R.M. J. Invest. Dermatol. 73: 59-66, 1979). Previous immunohistochemical studies have reported increased deposition of elastin, versican, hyaluronic acid, fibrillin, and fibulin-2 in areas with sunlight elastic fibrosis (Bernstein, EF et al., 72: 662-669, 1995; Bernstein, EF et al., Br. J. Dermatol. 135: 255-62, 1996; Hunzelmann, N. et al., 2001 (supra). Found in this study that fibrin-5 deposition increased with age, but also increased in sunlight elastic fibrosis, with increased expression of elastic fiber components and damage to light-damaged skin. The mechanism of abnormal deposition in the dermis is not yet known, as fibulin-5 increase was seen at a later stage of deposition of elastic fiber components and after an extreme decrease in the dermis, thus controlling fibulin-5 gene expression, Or Control of fibulin-5 protein synthesis or fibulin-5 deposition may be different from the control of other elastic fiber components, revealing the role of fibulin-5 in the development of solar elastic fibrosis Therefore, further research is needed.

  In summary, the inventor has shown for the first time that fibrin-5 deposition in the dermis decreases with age, albeit increased in solar elastic fibrosis. Furthermore, sudden UV exposure promotes the disappearance of fibulin-5 in the dermis. Therefore, fibulin-5 is considered to be an excellent marker of skin aging, particularly photoaging.

The immunohistochemical dyeing | staining figure which shows the presence state of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of the skin protected from sunlight of the young person. The immunohistochemical dyeing | staining figure which shows the age change of the expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the dermis of the skin protected from sunlight. The immunohistochemical dyeing | staining figure which shows the age change of the expression of elastin, fibulin-5, fibrillin-1, and fibulin-2 in the skin exposed to sunlight. The immunohistochemical dyeing | staining figure which shows the reduction | decrease of fibulin-5 in the dermis after UVB irradiation.

Claims (8)

  A skin examination method for evaluating the aging state of a subject's skin, wherein the expression level of fibulin-5 in the skin to be examined is significantly lower than the expression in the control skin. A skin test method, characterized in that it is determined that an aging state is progressing.   The skin examination method according to claim 1, wherein the skin aging is caused by natural aging and / or exposure to ultraviolet B wave (UVB).   The skin test method according to claim 1 or 2, wherein the expression level of fibulin-5 is measured by measuring the amount of fibulin-5 in the skin to be examined.   4. The skin test method according to claim 3, wherein the amount of fibulin-5 is measured by an immunoassay using an antibody specific for fibulin-5.   The skin test method according to claim 4, wherein the antibody is a polyclonal antibody.   The skin examination method according to claim 1 or 2, wherein the expression level of fibulin-5 is determined by measuring the amount of mRNA encoding fibulin-5 extracted from the skin.   The skin test method according to claim 6, wherein the mRNA is measured by a polymerase chain reaction method.   The skin test method according to claim 7, wherein the polymerase chain reaction method is a real-time polymerase chain reaction method.