JP2009161509A - Hair loss inhibitor and hair depigmentation inhibitor associated with type xvii collagen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide hair loss inhibitors and hair depigmentation inhibitors. <P>SOLUTION: The hair loss and depigmentation inhibitors comprise (i) a type XVII collagen having an amino-acid sequence represented by sequence number: 1, (ii) a type XVII collagen variant having at least 80% amino acid sequence identical to the amino acid sequence of sequence number: 1, and having activity to inhibit hair loss, (iii) a type XVII collagen encoded by a nucleotide sequence represented by sequence number: 2, or (iv) a type XVII collagen variant encoded by a nucleic acid which hybridizes with a nucleotide sequence represented by sequence number: 2 or complementary strands thereof under a stringent condition and having activity to inhibit hair loss. This can inhibit hair loss or depigmentation of hair. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a hair loss inhibitor for XVII collagen, a hair depigmentation inhibitor, a composition for hair loss inhibition, a composition for hair depigmentation inhibition, and a method for inhibiting hair loss or hair depigmentation. The present invention relates to an ectopic differentiation inhibitor for pigment stem cells, an agent for inhibiting pigment stem cell disappearance, an agent for inhibiting abnormal differentiation of hair follicle stem cells, and an agent for inhibiting disappearance of hair follicle stem cells.

  In addition to protecting individuals from the outside world, the skin plays an important role in identifying and defending individuals by the color and pattern of hair and skin. The skin consists of three layers of the epidermis, dermis, and subcutaneous adipose tissue from the outside, and the hair follicle extends continuously from the epidermis toward the subcutaneous adipose tissue. The constant part of the hair follicle is maintained throughout the hair cycle, whereas the transient part periodically repeats its growth and retraction in synchronization with the hair cycle. In addition, pigment cells produce melanin pigments when differentiated in the skin, and pass them to surrounding keratinocytes to supply pigments to the skin and hair. And play a role such as defense.

  In addition, gray hair is the most prominent aging phenomenon experienced by all of us. It was known as a phenomenon that the number of pigment cells producing pigment decreased in hair follicles that grew white hair, but the mechanism was hardly known. In 1990, Cotsarelis et al. Proposed that hair follicle stem cells exist in the bulge region because many hair follicle stem cells are found in the bulge region (Non-patent Document 1). The present inventor has found a pigment stem cell so far (Non-patent Document 2) and has clarified that maintenance of the pigment stem cell is essential as a mechanism of gray hair (Non-patent Document 3).

  On the other hand, hair is derived from the skin and is greatly affected by skin changes and defects. Furthermore, it is known that the hair loss phenomenon tends to increase with aging not only in men but also in women even when hair loss due to skin diseases is not included. In particular, in male pattern baldness, the hair follicles in the frontal region and the parietal region regress and lead to hair loss. It is known that such regression of hair follicles is due to the effect of testosterone (male hormone). A hair growth agent that targets male hormones has been developed (Patent Document 1). In addition, a hair restorer using a hydrolyzate of collagen protein that promotes hair elongation in the shaved skin has also been developed (Patent Document 2). In addition, as a factor of hair follicle regression, morphological changes in the connective tissue of the skin including the hair follicle also contribute to the hair loss phenomenon. In addition, for the purpose of research on BP, which is a type of autoimmune disease in the skin where blisters (fissures) are formed under the skin, XVII type collagen gene deficient (COL17KO) mice, which are BP autoantigens, have been developed. In this COL17KO mouse, it has been reported that mice with a low proportion of white hair and a large proportion of white hair are observed along with the formation of blisters as a BP symptom and disorder of the epidermis (Non-patent Document 4).

Cotsarelis G. et al., Cell 61: 1329-1337.1990 Nishimura EK et al. Nature. 416 (6883): 854-60, 2002 Nishimura EK et al. Science. 307 (5710): 720-724. 2005 Nishie W et al. Nat Med 13: 378-383, 2007 JP 2004-248632 A Japanese Patent No. 3247774

  Various methods such as vasodilators and plant extracts have been taken so far for the purpose of suppressing hair loss and depigmentation of hair, but none of them have solved the above problems. Development of hair loss inhibitors and hair depigmentation inhibitors is desired in the field of pharmaceutical and bio-related product development.

  In addition, it is not clear what mechanism causes depigmentation (whitening) and hair loss of hair with aging. Non-patent document 4 briefly mentions that COL17KO mice have a small body hair and a large proportion of white hair. However, non-patent document 4 causes whitening or hair loss over time. There is no description as to whether or not it is a research report on hair loss and gray hair. In the first place, it is not a research report on hair loss and depigmentation of hair. Non-Patent Document 4 does not interpret the mechanism of hair whitening and hair loss.

  However, elucidation of these mechanisms is desired in the field of drug development and bio-related product development for the purpose of hair loss treatment, skin and hair regeneration aging control, gray hair treatment, or skin homeostasis maintenance.

  The present invention has been made in view of the above circumstances, and an object thereof is to provide a hair loss inhibitor or a hair depigmentation inhibitor.

  Another object of the present invention is to elucidate the above mechanism and provide a technique useful for hair loss treatment, gray hair treatment, skin and hair regeneration aging control, skin homeostasis maintenance and the like.

  In the process leading to the present invention, the present inventor has found that pigment stem cells and hair follicle stem cells disappear prior to whitening and hair loss seen with aging in XVII type collagen gene deficient (COL17KO) mice. . Therefore, when human XVII collagen was specifically expressed in a region containing hair follicle stem cells in COL17KO mice, abnormal differentiation of pigment stem cells and hair follicle stem cells was suppressed, and no abnormality was observed in morphology or distribution, and hair loss And it was confirmed that no whitening was observed. From the above, it became clear that hair loss and whitening were suppressed by XVII collagen, and the present invention was completed.

  To repeat the explanation, focusing on the mechanism of hair loss and whitening, in the process leading to the present invention, the inventor first analyzed in detail the expression of type XVII collagen in the skin, A region that was specifically expressed was found. Furthermore, when COL17KO mice were observed focusing on the pigment stem cells, the pigment stem cells were ectopically differentiated into mature pigment cells in the bulge region prior to whitening and hair loss, followed by the disappearance of the pigment stem cells. Admitted. Furthermore, when the hair follicle stem cells were examined, the hair follicle stem cells constitute bulge regions as undifferentiated keratinocytes, and the undifferentiated keratinocytes that constitute the bulge regions undergo abnormal mature differentiation. It was also observed that the hair follicle stem cells disappear. From the above, it has been found that type XVII collagen is essential for the maintenance of pigment stem cells and hair follicle stem cells, and white hair and hair loss occur due to insufficient maintenance. From the above results, it has been clarified that the defect of XVII type collagen is whitened and the whitening is caused by the disappearance of pigment stem cells, and abnormal differentiation of pigment stem cells and hair follicle stem cells is suppressed by XVII type collagen. Thus, it was proved that the pigment stem cells and the hair follicle stem cells are maintained by suppressing the disappearance thereof, thereby completing the present invention.

  That is, according to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, And an XVII type collagen variant having an activity of suppressing hair loss, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof. There is provided a hair loss inhibitor comprising a XVII type collagen variant encoded by a nucleic acid that hybridizes under mild conditions and having an activity of suppressing hair loss. In the present invention, as shown in the examples described later, hair loss is suppressed by XVII type collagen.

  Furthermore, according to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, And an XVII collagen variant having an activity of suppressing depigmentation of hair, (iii) XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complement Provided is a hair depigmentation inhibitor comprising an XVII type collagen variant encoded by a nucleic acid that hybridizes to a strand under stringent conditions and having an activity of inhibiting hair depigmentation. In the present invention, depigmentation of hair is suppressed by XVII type collagen, as shown in Examples described later.

  In addition, according to the present invention, a promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an amino acid sequence shown in SEQ ID NO: 1 downstream of the promoter sequence Or a nucleotide sequence encoding a type XVII collagen variant having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and having an activity of inhibiting hair loss, or (ii) The nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or the nucleotide sequence encoding the XVII type collagen variant that hybridizes under stringent conditions and has the activity of suppressing hair loss A composition for inhibiting hair loss comprising a vector having It is. In the present invention, as shown in the examples described later, hair loss is suppressed by XVII type collagen.

  In addition, according to the present invention, a promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an amino acid sequence shown in SEQ ID NO: 1 downstream of the promoter sequence A nucleotide sequence encoding a type XVII collagen, or an amino acid sequence of SEQ ID NO: 1 having at least 80% amino acid sequence identity and having an activity of suppressing depigmentation of hair, Or (ii) XVII type that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has the activity of suppressing depigmentation of hair Hair comprising a vector having a nucleotide sequence encoding a collagen variant Composition for the dye-suppression is provided. In the present invention, depigmentation of hair is suppressed by XVII type collagen, as shown in Examples described later.

  Moreover, according to the present invention, hair loss or hair comprising administering the above-mentioned hair loss inhibitor, hair depigmentation inhibitor, a composition for hair loss inhibition or a composition for hair depigmentation inhibition A method for inhibiting the depigmentation of is provided. In the present invention, hair loss or depigmentation of hair is suppressed by the type XVII collagen, as shown in Examples described later.

  According to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, And an XVII type collagen variant having an activity of suppressing ectopic differentiation of pigment stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2, or A chromosomal stem cell ectopic differentiation inhibitor comprising a variant XVII collagen encoded by a nucleic acid that hybridizes under stringent conditions to the complementary strand and having an activity of inhibiting ectopic differentiation of pigment stem cells. Provided. In the present invention, ectopic differentiation of pigment stem cells is suppressed by XVII type collagen or a mutant thereof, as shown in Examples described later.

  Furthermore, according to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, And an XVII type collagen variant having an activity of suppressing the disappearance of pigment stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complementary strand There is provided a pigment stem cell disappearance inhibitor comprising a variant XVII collagen which is encoded by a nucleic acid that hybridizes under stringent conditions and has an activity of inhibiting the disappearance of pigment stem cells. In the present invention, disappearance of pigment stem cells is suppressed by XVII type collagen or a mutant thereof, as shown in Examples described later.

  According to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, And an XVII type collagen variant having an activity of suppressing abnormal differentiation of hair follicle stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or the same Provided is an agent for suppressing abnormal differentiation of hair follicle stem cells, which comprises a variant XVII collagen encoded by a nucleic acid that hybridizes to a complementary strand under stringent conditions and has an activity of suppressing abnormal differentiation of hair follicle stem cells. . In the present invention, abnormal differentiation of hair follicle stem cells is suppressed by XVII type collagen or a mutant thereof, as shown in Examples described later.

  According to the present invention, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, And an XVII collagen variant having an activity of suppressing the disappearance of hair follicle stem cells, (iii) XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complement Provided is a hair follicle stem cell disappearance inhibitor comprising a variant XVII collagen encoded by a nucleic acid that hybridizes to a strand under stringent conditions and having an activity of inhibiting the disappearance of hair follicle stem cells. In the present invention, disappearance of hair follicle stem cells is suppressed by XVII-type collagen or a mutant thereof, as shown in Examples described later.

  In addition, the hair loss inhibitor for the above-mentioned type XVII collagen, hair depigmentation inhibitor, pigment stem cell ectopic differentiation inhibitor, pigment stem cell disappearance inhibitor, hair follicle stem cell abnormal differentiation inhibitor, hair follicle stem cell The disappearance inhibitor is one embodiment of the present invention, and the present invention may be any combination of the above components. In addition, the composition for inhibiting hair loss, the composition for inhibiting hair depigmentation, the method for inhibiting hair removal or hair depigmentation, etc. of the present invention have the same configuration and have the same effects. Play.

  According to the present invention, as shown in the examples below, human XVII collagen or a mutant thereof can be used to remove hair, depigment hair, ectopic differentiation of pigment stem cells, disappearance of pigment stem cells, hair follicle stem cells Abnormal differentiation or loss of hair follicle stem cells is suppressed.

(Background of the invention)
In Non-Patent Document 4 described above, it is reported that COL17KO mice, which are BP disease model mice, have mice with a small proportion of white hair and a large proportion of white hair, but it is unclear whether there is any change over time. Non-Patent Document 4 is not a research report on depilation and depigmentation of hair in the first place. Therefore, no specific description is given regarding the state of the body hair of the COL17KO mouse, such as what state it was at the age of the day. That is, COL17KO mice had a small amount of white hair from the beginning of birth, or a large proportion of white hair, or a sufficient amount of black hair from the beginning of birth, but the amount of white hair gradually decreased. It has not been explained whether or not there was an increase. Here, in general, “hair removal” means a phenomenon in which the hair had a sufficient amount at the beginning but gradually decreased, and “white hair” originally had black hair. This means a phenomenon in which white hair gradually increases. Therefore, it can be said that Non-Patent Document 4 does not interpret the mechanism of hair depigmentation and hair removal.

  Therefore, the present inventor first observes the hair of the mouse over time, observes in detail whether the hair is abnormal from the time of birth, and when the hair abnormality begins. It became clear that hair growth began, followed by hair loss and progressed progressively. Furthermore, in order to clarify the mechanism of hair depigmentation and hair loss with aging, the expression of type XVII collagen in the skin was analyzed in detail. The expression was specifically observed in the hair follicle bulge region (a group of cells corresponding to hair follicle stem cells). Furthermore, COL17KO mice in which hair loss and whitening occur with aging were examined with a focus on pigment stem cells. Then, prior to whitening and hair loss, the pigment stem cells ectopically differentiate into mature pigment cells in the bulge region, and then disappear, further, surrounding hair follicle stem cells that are important for pigment stem cell maintenance In addition to the fact that hair follicle stem cells constitute bulge regions as undifferentiated keratinocytes, the undifferentiated keratinocytes that constitute the bulge regions usually suppress the epidermis, sebaceous glands, etc. It was confirmed that the hair follicle stem cells disappeared by showing abnormal maturation differentiation with respect to the differentiation direction. In addition, when human XVII-type collagen was expressed using a promoter (keratin 14 promoter) that is specifically expressed in a region containing hair follicle stem cells in COL17KO mice, abnormal differentiation of pigment stem cells and hair follicle stem cells was suppressed. -No abnormality was observed in the distribution, and it was confirmed that no hair loss or whitening was observed. The above findings strongly suggest that the present invention can be applied to the development of pharmaceuticals and bio-related products targeting skin XVII collagen for the purpose of hair loss treatment and gray hair treatment. These results could not be inferred from Non-Patent Document 4 where research reports on BP disease mechanisms and treatment methods have been made, and were completely unexpected findings.

  From the above results, it was clarified that pigment stem cells and hair follicle stem cells disappeared due to deficiency of human XVII collagen, resulting in hair loss and whitening. Furthermore, it has been clarified that the loss of hair follicle stem cells and pigment stem cells can be suppressed by XVII collagen, and hair loss and whitening can be suppressed. Moreover, it became clear that pigment stem cells and hair follicle stem cells can be maintained by suppressing abnormal differentiation and disappearance of hair follicle stem cells and pigment stem cells by XVII collagen, thereby completing the present invention.

Hereinafter, embodiments of the present invention will be described in detail.
(Overview)
In this embodiment, (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and hair loss XVII type collagen variant having inhibitory activity, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) conditions stringent to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand A hair loss inhibitor comprising a variant XVII type collagen encoded by a nucleic acid that hybridizes with and having an activity of suppressing hair loss. As shown in the Examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing hair loss, the hair loss inhibitor itself containing it also suppresses hair loss.

  Furthermore, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and A variant of type XVII collagen having an activity of suppressing depigmentation of hair, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complementary strand It is a hair depigmentation inhibitor comprising an XVII type collagen variant that is encoded by a nucleic acid that hybridizes under stringent conditions and has an activity of inhibiting hair depigmentation. As shown in the examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing depigmentation of hair, the depigmentation inhibitor of the hair containing it itself is also depigmented of hair. Control.

  Further, in the present embodiment, a promoter sequence that controls to express XVII type collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an amino acid sequence shown in SEQ ID NO: 1 downstream of the promoter sequence Or Xii type collagen having nucleotide sequence encoding at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing hair loss, or (ii) sequence The nucleotide sequence shown in No. 2 or the nucleotide sequence of SEQ ID No. 2 or a nucleotide sequence encoding an XVII type collagen variant that hybridizes under stringent conditions and has an activity of suppressing hair loss A composition for inhibiting hair loss comprising a vector havingAs shown in the Examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing hair loss, the composition for expressing the XVII collagen or the XVII collagen variant in a region containing hair follicle stem cells. The object itself also suppresses hair loss.

  Further, in the present embodiment, a promoter sequence that controls to express XVII type collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an amino acid sequence shown in SEQ ID NO: 1 downstream of the promoter sequence A nucleotide sequence encoding a type XVII collagen, or an XVII type collagen variant having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing depigmentation of hair, or (Ii) XVII collagen that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has the activity of suppressing depigmentation of hair Hair removal comprising a vector having a nucleotide sequence encoding a variant It is a composition for fluorination suppressed. As shown in the Examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing hair loss, the composition for expressing the XVII collagen or the XVII collagen variant in a region containing hair follicle stem cells. The object itself also suppresses depigmentation of hair.

  In addition, the present embodiment includes the administration of the above-mentioned hair loss inhibitor, hair depigmentation inhibitor, a composition for hair loss inhibition or a composition for hair depigmentation, This is a method for suppressing depigmentation. As shown in the examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing hair loss and a function of suppressing depigmentation of hair, there is also a method comprising administering it. Suppresses hair loss or depigmentation of hair.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and XVII type collagen variant having activity to suppress ectopic differentiation of pigment stem cells, (iii) XVII type collagen encoded by nucleotide sequence shown in SEQ ID NO: 2, or (iv) nucleotide sequence of SEQ ID NO: 2 or the same A chromosomal stem cell ectopic differentiation inhibitor containing a XVII collagen variant that encodes a nucleic acid that hybridizes to a complementary strand under stringent conditions and has an activity of suppressing ectopic differentiation of pigment stem cells . As shown in the examples described later, since the type XVII collagen or a variant thereof has a function of suppressing ectopic differentiation of pigment stem cells, the ectopic differentiation inhibitor containing the same is also an ectopic region of pigment stem cells. Inhibits sexual differentiation.

  Furthermore, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and An XVII type collagen variant having an activity to suppress differentiation into a mature pigment cell in the bulge region of a pigment stem cell, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) SEQ ID NO: A XVII type collagen variant that encodes a nucleic acid that hybridizes to a nucleotide sequence of 2 or a complementary strand thereof under stringent conditions and has an activity of suppressing differentiation into a mature chromosomal cell in a bulge region of a chromogenic stem cell It is an agent for inhibiting differentiation into mature pigment cells in the bulge region of pigment stem cells. As shown in Examples below, type XVII collagen or a mutant thereof has a function of promoting stem cell maintenance by suppressing differentiation into a pigmented stem cell in the bulge region and subsequent cell loss. Therefore, the ectopic differentiation inhibitor itself containing it also suppresses ectopic differentiation of pigment stem cells. As a result, ectopic differentiation of pigment stem cells into mature pigment cells in the bulge region is suppressed, and disappearance of pigment stem cells is suppressed.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and An XVII type collagen variant having an activity of suppressing the disappearance of pigment stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complementary strand It is an agent for suppressing the disappearance of pigment stem cells, which contains an XVII type collagen variant that is encoded by a nucleic acid that hybridizes under stringent conditions and has the activity of suppressing the disappearance of pigment stem cells. As shown in the Examples described later, since the type XVII collagen or a variant thereof has a function of suppressing the disappearance of pigment stem cells, the cell death inhibitor itself containing the same suppresses the disappearance of pigment stem cells.

  Furthermore, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and (X) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iii) an XVII type collagen variant having an activity of suppressing disappearance subsequent to differentiation into mature pigment cells in the bulge region of pigment stem cells, or ( iv) A nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and suppresses disappearance that occurs subsequent to differentiation into mature pigment cells in the bulge region of pigment stem cells Involves differentiation into mature pigment cells in the bulge region of pigment stem cells, including active type XVII collagen variants An inhibitor of loss that occur have. As shown in the examples described later, the type XVII collagen or a variant thereof has a function of promoting stem cell maintenance by suppressing disappearance following differentiation into mature pigment cells in the bulge region of pigment stem cells. In addition, the cell death inhibitor itself containing it also suppresses the disappearance of pigment stem cells. As a result, the disappearance that occurs following the differentiation of pigment stem cells into mature pigment cells in the bulge region is suppressed, and the maintenance of pigment stem cells is promoted.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and A variant of type XVII collagen having an activity of suppressing abnormal differentiation of hair follicle stem cells, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleotide sequence of SEQ ID NO: 2 or its complement A hair follicle stem cell abnormal differentiation inhibitor comprising a variant XVII collagen encoded by a nucleic acid that hybridizes to a chain under stringent conditions and having an activity of suppressing abnormal differentiation of hair follicle stem cells. As shown in the examples described later, since the type XVII collagen or a variant thereof has a function of inhibiting and / or promoting abnormal differentiation of hair follicle stem cells, the abnormal differentiation inhibitor containing the same itself is also a hair follicle stem cell. Suppresses abnormal differentiation.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and A variant XVII collagen having an activity of suppressing differentiation of hair follicle stem cells into mature epidermal follicular sebaceous gland cells, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) XVII encoded by a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has an activity of suppressing differentiation of hair follicle stem cells into mature epidermal follicular sebaceous cells It is an agent for inhibiting differentiation of hair follicle stem cells into mature epidermal hair sebaceous gland cells, including a type I collagen variant. As shown in the examples described later, the hair follicle stem cells constitute a bulge region, and the type XVII collagen or a variant thereof shows that the hair follicle stem cells mature and differentiate in an abnormal direction from undifferentiated keratinocytes. It has a function of suppressing. Therefore, the abnormal differentiation inhibitor itself containing XVII type collagen having such a function also suppresses abnormal differentiation of hair follicle stem cells. As a result, abnormal differentiation of hair follicle stem cells into mature cells in the bulge region is suppressed, and loss of hair follicle stem cells is suppressed.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and An XVII type collagen variant having an activity of suppressing the disappearance of hair follicle stem cells, (iii) an XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleotide sequence of SEQ ID NO: 2 or a complementary chain thereof A hair follicle stem cell disappearance inhibitor comprising a variant XVII collagen encoded by a nucleic acid that hybridizes under stringent conditions and having an activity of inhibiting the disappearance of hair follicle stem cells. As shown in the examples described later, since the type XVII collagen or a variant thereof has a function of suppressing and / or promoting the disappearance of hair follicle stem cells, the disappearance inhibitor itself containing the hair follicle stem cells also disappears. Suppress.

  Moreover, this embodiment has (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1, and A variant XVII collagen having an activity of suppressing loss of hair follicle stem cells due to abnormal maturation differentiation from undifferentiated keratinocytes constituting the bulge region of the stem cells, (iii) encoded by the nucleotide sequence shown in SEQ ID NO: 2 From an undifferentiated keratinocyte that encodes a type XVII collagen, or (iv) a nucleic acid that hybridizes to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand under stringent conditions and constitutes the bulge region of a stem cell Bulge region of hair follicle stem cells, comprising a type XVII collagen mutant having an activity to suppress hair follicle stem cell disappearance due to abnormal maturation differentiation An inhibitor of hair follicle stem cell loss due to abnormal maturation differentiation from undifferentiated keratinocytes that constitute. As shown in the examples described later, the type XVII collagen or a variant thereof has a function of suppressing disappearance due to mature differentiation from undifferentiated keratinocytes constituting the bulge region of hair follicle stem cells to the epidermis, sebaceous glands, etc. Therefore, the disappearance inhibitor itself containing it also suppresses the disappearance of hair follicle stem cells. As a result, the disappearance of the hair follicle stem cells in the bulge region due to mature differentiation from the undifferentiated keratinocytes constituting the bulge region is suppressed, and the maintenance of the hair follicle stem cells is promoted.

(Type XVII collagen)
As used herein, the term “type XVII collagen” includes native sequence type XVII collagen. The type XVII collagen described herein may be prepared by recombinant or synthetic methods or isolated from various sources.

  “Native sequence type XVII collagen” is a polypeptide having the amino acid sequence of naturally occurring type XVII collagen. Such native sequence type XVII collagen can be produced by recombinant or synthetic means and can be isolated from various sources.

(XVII type collagen variant polypeptide)
“XVII type collagen variant” means a type XVII collagen having 80% or more amino acid sequence identity with the full-length native sequence type XVII collagen disclosed herein. Such a XVII type collagen variant includes XVII in which one or more (for example, 2, 3,...) Amino acid residues are added, substituted, or deleted at the N- or C-terminus of the full-length natural amino acid sequence. Type collagen is included. Generally, a type XVII collagen variant has a full-length natural amino acid sequence disclosed herein, 80% or more amino acid sequence identity with the full-length natural sequence XVII collagen sequence disclosed herein, preferably 90% or more amino acid sequence identity. More preferably 95% or more amino acid sequence identity. In addition, SEQ ID NO: 1 shown below shows the amino acid sequence of the full-length natural sequence type XVII collagen.

  As defined herein, “percent (%) amino acid sequence identity” may align sequences and introduce gaps so that maximum percent sequence identity is obtained, and conservative substitutions are considered as part of sequence identity. Defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues of the type XVII collagen sequence, not considered part. Selection of alignments for purposes of determining percent amino acid sequence identity is performed using methods well known to those skilled in the art, such as publicly available computers such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. This is possible by using software. One skilled in the art can determine appropriate parameters to measure alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

(XVII collagen variant polynucleotide)
A “variant polynucleotide” is a nucleic acid molecule that encodes a polypeptide that has the activity inherent to the polypeptide, and is relative to the nucleotide sequence that encodes the full-length native sequence type XVII collagen polypeptide sequence disclosed herein. It has 80% or more sequence identity. Usually, a type XVII collagen variant polypeptide nucleotide has a nucleic acid sequence identity of 80% or more, preferably 90%, to the nucleic acid sequence encoding the full-length native sequence type XVII collagen polypeptide sequence disclosed herein. It has identity, more preferably 95% nucleic acid sequence identity. Variants do not contain the native nucleotide sequence. Note that SEQ ID NO: 2 shown below contains a full-length sequence encoding human XVII collagen.

  “Percent (%) nucleic acid sequence identity” relative to the type XVII collagen-encoding nucleic acid sequence identified herein may align the sequences and introduce gaps to obtain maximum percent sequence identity. Defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides of a type I collagen polypeptide-encoding nucleic acid sequence. Selection of alignments for purposes of determining percent nucleic acid sequence identity is well known to those skilled in the art, eg publicly available such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software This is possible by using computer software. One skilled in the art can determine appropriate parameters to measure alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

  In other embodiments, the type XVII collagen variant polynucleotide encodes a type XVII collagen variant polypeptide, preferably encoding the polypeptide set forth in SEQ ID NO: 1 under stringent hybridization and wash conditions. A nucleic acid molecule that hybridizes to a nucleotide sequence. The XVII collagen variant polypeptide may be one encoded by a XVII collagen variant polynucleotide.

(hair)
The term “hair” as used herein refers to a skin appendage composed of a hair shaft and hair root mainly composed of keratin, and includes, for example, head hair and body hair.

(Hair loss)
The term “hair removal” as used herein means that a part or all of hair falls off due to, for example, aging or illness. Various factors can be considered as the cause. In addition to the rise of male hormones and morphological changes in the connective tissue of the skin including hair follicles, loss of hair follicle stem cells can also contribute.

(Suppression of hair loss)
The suppression of hair loss here is different from promoting the elongation of shortened hair such as hair that has been removed, such as suppressing hair loss caused by factors such as morphological changes in the connective tissue of the skin, etc. Say.

(Depigmentation of hair)
The depigmentation of hair here means that the hair that originally has melanin pigments approaches the white color of hair as the supply of melanin pigments from pigment cells decreases. This includes, for example, the change of hair to white hair and whitening of body hair.

(Skin structure)
The skin is divided into three layers, the epidermis, the dermis, and the subcutaneous tissue, starting from the surface, covering the entire surface of the body, bordering the outside world, and protecting internal organs such as internal organs from external stimuli and impacts. is doing. The epidermis is composed of a stratum corneum, a granule layer, a spiny layer, and a basal layer. The basal layer is in the lowermost layer of the epidermis, and is in contact with the dermis in a waveform. The basal cells constituting the basal layer have a single-layer structure in which cubic or columnar cells are arranged in a line, and several pigment cells are scattered. Melanin pigment is produced from pigment cells by stimulation such as exposure to ultraviolet rays. In the basal layer, nutrients are replenished from the capillaries of the dermal papilla, and cell division is always carried out, and cell formation and growth are repeated.

(Stem cell)
A stem cell refers to an undifferentiated cell that exhibits high self-sustaining ability (self-replicating ability) and can supply differentiated progeny cells. Examples include pigment stem cells that supply pigment cells as progeny cells, and hair follicle stem cells that have the ability to form bulge regions as undifferentiated keratinocytes and differentiate into skin appendages such as epidermis, sweat glands, and sebaceous glands. .

(niche)
A niche is an ecological place for stem cells. Usually, the localized site of tissue stem cells is called a niche. In a niche environment, stem cells are maintained in an undifferentiated state. For example, the niche of pigment stem cells and hair follicle stem cells exists in the bulge region. Usually, some of the progeny cells grown from the pigment stem cells in the bulge region move from the bulge region to the hair matrix, where they differentiate and mature into pigment cells to supply the melanin pigment to the hair.

(Abnormal differentiation of pigment stem cells)
Abnormal differentiation of pigment stem cells means that chromosomal stem cells have melanin granules and acquire a dendritic morphology in a bulge region (niche) that is originally maintained in an undifferentiated state in wild-type mice. It means to mature and differentiate. Normally, this ectopic differentiated cell disappears without being maintained. For example, it means that pigment stem cells lose undifferentiation in the bulge region and differentiate into mature pigment cells.

(Abnormal differentiation of hair follicle stem cells)
Abnormal differentiation of hair follicle stem cells means that hair follicle stem cells differentiate in an abnormal differentiation direction without self-maintaining. For example, the hair follicle stem cells cannot maintain stem cell properties in the bulge region, and are differentiated from normal controls such as hair follicles, epidermis and sebaceous glands.

(Dosage form)
Here, the hair loss inhibitor or hair depigmentation inhibitor of the present embodiment is considered to have the effect of oral administration or transdermal administration. As its dosage form, for example, powders, pills, tablets, Capsules, creams, liquids for internal use, injections may be used, and addition to hair tonics, hair lotions, hair creams, aerosols, ointments, shampoos, rinses, etc. is also conceivable.

(vector)
The vector can be any vector that can be conveniently subjected to recombinant DNA procedures, and the choice of vector often depends on the host cell into which the vector is to be introduced. Thus, the vector may be a self-replicating vector, ie a vector that exists as extrachromosomal material whose replication is independent of chromosomal replication. Such vectors include, for example, a plasmid, phage, cosmid, mini, chromosome, or virus. Alternatively, the vector may be one that, when introduced into a host cell, is integrated into the host cell genome and replicated with one or more chromosomes integrated therein. Examples of suitable vectors are bacterial expression vectors and yeast expression vectors. Examples of the viral vector include, but are not limited to, vectors derived from viruses such as adenovirus, retrovirus, papoba virus such as SV40, vaccinia virus, fowlpox virus, and pseudorabies virus. With the expression vector constructed in the present invention, type XVII collagen can be expressed at any place.

(promoter)
The term “promoter” as used herein refers to a sequence that controls the expression of a polypeptide present downstream of the promoter sequence. In this case, a promoter that can be expressed particularly in a region containing hair follicle stem cells is desirable. Examples include the K14 promoter and the K5 promoter. In the examples described later, the keratin 14 promoter is used as a promoter that controls specific expression in the epidermal basal layer including hair follicle stem cells.

(Method of administration)
The administration method referred to here is a method of administering the above-mentioned agent or composition. Here, the hair loss inhibitor or hair depigmentation inhibitor of this embodiment exhibits its effect by oral administration or transdermal administration. There are possibilities, for example, powders, pills, tablets, capsules, creams, liquids for internal use, hair tonics, hair lotions, hair creams, aerosols, ointments, shampoos, rinses, etc. . In addition, as an injection, a method of administration by subcutaneous injection or the like by a doctor or nurse is also conceivable.

(Function and effect)
The operational effects of this embodiment will be described below.
The present embodiment is based on the knowledge obtained as a result of experiments conducted for the purpose of elucidating the mechanisms of whitening and hair loss associated with aging and providing techniques useful for hair loss treatment and hair removal treatment. That is, as shown in the examples described later, when the pigment stem cells and hair follicle stem cells were examined in detail, it was clear that the pigment stem cells and hair follicle stem cells disappeared prior to whitening and hair loss associated with aging. Furthermore, when human XVII collagen originally expressed in hair follicle stem cells is deficient, it becomes clear that pigment stem cells maintained by hair follicle stem cells disappear, and hair follicle stem cells begin to disappear. It was. As a result, it became clear that white hair and hair loss proceed simultaneously because stem cells cannot supply differentiated cells.

  The hair loss inhibitor of this embodiment has (i) XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. And an XVII type collagen variant having an activity of suppressing hair loss, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a string in the nucleotide sequence of SEQ ID NO: 2 or its complementary strand And a XVII type collagen variant that encodes a nucleic acid that hybridizes under a gentle condition and has an activity of suppressing hair loss. Since the XVII type collagen or the XVII type collagen variant has an action of suppressing hair loss as shown in Examples described later, the hair loss inhibitor itself of the present embodiment including the same also has the effect of suppressing hair loss.

  Furthermore, the hair loss inhibitor of this embodiment comprises (i) XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. And a variant of type XVII collagen having the activity of suppressing hair loss due to disappearance of hair follicle stem cells, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) SEQ ID NO: 2 By loss of hair follicle stem cells, including a XVII type collagen variant that is encoded by a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of or a complementary strand thereof and has an activity of suppressing hair loss due to loss of hair follicle stem cells It may be an inhibitor of hair loss. Also in this case, as shown in the examples described later, since the XVII collagen or the XVII collagen mutant has a function of suppressing the disappearance of hair follicle stem cells, the hair loss inhibitor itself containing the hair follicle stem cells is also a hair follicle stem cell. There is an effect of suppressing disappearance, and as a result, there is an effect of suppressing hair loss.

  Further, the depigmentation inhibitor of the present embodiment includes (i) XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1. An XVII collagen variant having the ability to inhibit hair depigmentation, (iii) the XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) the SEQ ID NO: 2 It includes a type XVII collagen variant that is encoded by a nucleic acid that hybridizes to a nucleotide sequence or its complementary strand under stringent conditions and has an activity of suppressing depigmentation of hair. The hair depigmentation inhibitor of the present embodiment, as shown in the examples described later, has the effect that the XVII type collagen or the XVII type collagen variant suppresses the depigmentation of the hair, and therefore the hair containing it. The depigmentation inhibitor itself has the effect of promoting depigmentation of the hair.

  Furthermore, the hair depigmentation inhibitor of this embodiment comprises (i) a type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) 80% or more amino acids relative to the amino acid sequence of SEQ ID NO: 1. An XVII type collagen variant having sequence identity and having an activity of suppressing depigmentation of hair due to disappearance of pigment stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or ( iv) A type XVII collagen variant encoded by a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and having an activity of suppressing depigmentation of hair due to disappearance of pigment stem cells It may be a hair depigmentation inhibitor due to disappearance of pigment stem cells. Also in this case, as shown in the examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing the disappearance of the pigment stem cells, the hair depigmentation inhibitor itself containing the pigment is also a pigment. The effect of suppressing the disappearance of stem cells is exhibited, and as a result, the effect of suppressing depigmentation of hair is exhibited.

  In addition, the composition for inhibiting hair loss according to the present embodiment includes a promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and (i) a sequence downstream of the promoter sequence. Encodes an XVII collagen having the amino acid sequence shown in No. 1 or an XVII collagen variant having an amino acid sequence identity of at least 80% with the amino acid sequence of SEQ ID No. 1 and having an activity of suppressing hair loss Nucleotide sequence, or (ii) the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2, or the complementary strand thereof, hybridizes under stringent conditions and has an activity of suppressing hair loss Vectors having nucleotide sequences encoding variants are included. The composition for inhibiting hair loss according to the present embodiment, as shown in the examples described later, has the effect that the XVII collagen or the XVII collagen variant has the effect of inhibiting hair loss, so that the XVII type having such a function. A composition that expresses collagen or a type XVII collagen variant in a region-specific manner containing hair follicle stem cells also has an effect of suppressing hair loss.

  In addition, the composition for hair loss suppression according to this embodiment includes a promoter sequence that controls expression of type XVII collagen or a variant thereof in the epidermal basal layer, and (i) SEQ ID NO: 1 downstream of the promoter sequence. A nucleotide sequence encoding an XVII type collagen having the amino acid sequence shown in the following, or an XVII type collagen variant having an amino acid sequence identity of at least 80% with the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing hair loss, Or (ii) a type XVII collagen variant that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has an activity of suppressing hair loss A vector having the encoding nucleotide sequence is included. The composition for inhibiting hair loss according to the present embodiment, as shown in the examples described later, has the effect that the XVII collagen or the XVII collagen variant has the effect of inhibiting hair loss. Therefore, the XVII type having such a function. The composition itself that expresses collagen or XVII collagen variant in the epidermal basal layer also has the effect of suppressing hair loss.

  In addition, the composition for suppressing hair loss according to the present embodiment comprises a keratin 14 promoter sequence that controls to express XVII type collagen or a variant thereof specifically in epidermal basal cells, and (i) downstream of the promoter sequence. Coding the XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, or the XVII type collagen variant having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing hair loss Or (ii) the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, hybridizes under stringent conditions and has the activity of suppressing hair loss Including a vector having a nucleotide sequence encoding a collagen variant It may be. Also in this case, the composition for inhibiting hair loss according to the present embodiment has a function that the XVII type collagen or the XVII type collagen mutant inhibits hair loss, as shown in the examples described later. The composition itself capable of expressing the functional XVII collagen or XVII collagen variant specifically in epidermal basal cells also has the effect of suppressing hair loss.

  In addition, the composition for inhibiting depigmentation of hair according to the present embodiment includes a promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and downstream of the promoter sequence. (I) XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, or at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of suppressing depigmentation of hair A nucleotide sequence encoding a type XVII collagen variant, or (ii) a nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof under a stringent condition, and Nucleotide encoding XVII type collagen mutant having activity to suppress depigmentation Including a vector having a row. Since the composition for inhibiting depigmentation of hair according to this embodiment has an action of inhibiting depigmentation of hair, as shown in the Examples below, the XVII type collagen or the XVII type collagen variant has the effect of inhibiting hair depigmentation. The composition itself that expresses the XVII type collagen or XVII type collagen variant having such a function in a region-specific manner containing hair follicle stem cells also has an effect of suppressing depigmentation of hair.

  In addition, the composition for inhibiting depigmentation of hair according to this embodiment comprises a promoter sequence that controls expression of type XVII collagen or a variant thereof in the epidermal basal layer, and (i) downstream of the promoter sequence. XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, or XVII collagen having an amino acid sequence identity of at least 80% with the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing depigmentation of hair Nucleotide sequence encoding a variant, or (ii) the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, hybridizes under stringent conditions, and depigmentation of hair Has a nucleotide sequence encoding a type XVII collagen variant that has the activity of inhibiting Comprising the vector. Since the composition for inhibiting depigmentation of hair according to this embodiment has an action of inhibiting depigmentation of hair, as shown in the Examples below, the XVII type collagen or the XVII type collagen variant has the effect of inhibiting hair depigmentation. The composition itself that expresses the XVII type collagen or XVII type collagen variant having such a function in the epidermis basal layer also has an effect of suppressing depigmentation of hair.

  In addition, the composition for suppressing depigmentation of hair according to the present embodiment includes a keratin 14 promoter sequence that controls expression of XVII collagen or a variant thereof specifically to epidermal basal cells, and downstream of the promoter sequence. (I) XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, or having at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, and having an activity of suppressing depigmentation of hair A nucleotide sequence encoding a type XVII collagen variant, or (ii) a nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence of SEQ ID NO: 2 or a complementary strand thereof under a stringent condition, and hair Nucleo Encoding XVII Type Collagen Variant Having Activity to Suppress Depigmentation of Protein It may include a vector having a de sequence. Also in this case, the composition for inhibiting depigmentation of hair according to the present embodiment is that the XVII collagen or XVII collagen variant inhibits depigmentation of the hair, as shown in the examples described later. Since it has a function, the composition itself that can express the XVII type collagen or the XVII type collagen variant having such a function in an epidermal basal cell-specific manner also has an effect of suppressing depigmentation of hair.

  Further, the present embodiment is for hair removal or hair comprising administering the above-mentioned hair loss inhibitor, hair depigmentation inhibitor, a composition for hair loss inhibition or a composition for hair depigmentation inhibition. A method of inhibiting depigmentation of As shown in the examples described later, since the XVII collagen or the XVII collagen variant has a function of suppressing hair loss and a function of suppressing depigmentation of hair, there is also a method comprising administering it. It has an effect of suppressing hair loss or depigmentation of hair.

  Furthermore, the ectopic differentiation inhibitor for pigment stem cells of the present embodiment includes (i) a type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) the amino acid sequence of SEQ ID NO: 1 and at least 80% amino acid XVII type collagen variant having sequence identity and having the activity of suppressing ectopic differentiation of pigment stem cells, (iii) XVII type collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) sequence A XVII type collagen variant that encodes a nucleic acid that hybridizes to the nucleotide sequence of No. 2 or a complementary strand thereof under stringent conditions and has an activity of suppressing ectopic differentiation of pigment stem cells. The ectopic differentiation inhibitor for pigment stem cells of the present embodiment, as shown in Examples described later, has an effect that XVII collagen suppresses ectopic differentiation of pigment stem cells. The differentiation inhibitor itself also suppresses ectopic differentiation of pigment stem cells, and as a result, has the effect of suppressing disappearance of pigment stem cells.

  Furthermore, the ectopic differentiation inhibitor for pigment stem cells of the present embodiment includes (i) a type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) the amino acid sequence of SEQ ID NO: 1 and at least 80% amino acid An XVII collagen variant having sequence identity and having an activity of suppressing differentiation into mature pigment cells in the bulge region of pigment stem cells, (iii) XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2 Or (iv) encoded by a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has the activity of suppressing differentiation into mature pigment cells in the bulge region of pigment stem cells It may be an agent for inhibiting differentiation into mature pigment cells in the bulge region of pigment stem cells, which contains a type XVII collagen variantAlso in this case, the ectopic differentiation inhibitor of the pigment stem cell of the present embodiment is such that the XVII type collagen or the XVII type collagen variant is transformed into a mature pigment cell in the bulge region of the pigment stem cell, as shown in Examples below. Therefore, the ectopic differentiation inhibitor itself containing it has the effect of suppressing ectopic differentiation of the pigment stem cells, and as a result, the effect of suppressing the disappearance of the pigment stem cells. Play.

  Further, the pigment stem cell death inhibitor of this embodiment is (i) XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, or (ii) the amino acid sequence of SEQ ID NO: 1 and at least 80% amino acid sequence An XVII type collagen variant having the identity and having the activity of suppressing the disappearance of pigment stem cells, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) of SEQ ID NO: 2. It includes a type XVII collagen variant that encodes a nucleic acid that hybridizes to a nucleotide sequence or its complementary strand under stringent conditions and has an activity of suppressing the disappearance of pigment stem cells. The pigment stem cell death inhibitor of the present embodiment, as shown in the examples described later, has the effect that the XVII collagen or the XVII collagen variant suppresses the disappearance of the pigment stem cells, and therefore the disappearance suppression including the same. The agent itself also suppresses the disappearance of the pigment stem cells, and as a result, has the effect of promoting the maintenance of the pigment stem cells.

  Furthermore, the cell death inhibitor of pigment stem cells of the present embodiment includes (i) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) at least 80% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1. And a type XVII collagen variant having the ability to suppress disappearance subsequent to differentiation into mature pigment cells in the bulge region of pigment stem cells, (iii) encoded by the nucleotide sequence shown in SEQ ID NO: 2 Type XVII collagen, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and subsequent to differentiation into mature pigment cells in the bulge region of pigment stem cells In the bulge region of a pigment stem cell, which contains an XVII type collagen mutant having an activity to suppress disappearance caused by It may be an inhibitor of loss that occurs subsequent to differentiation into mature pigment cells. Also in this case, the pigment stem cell death inhibitor of the present embodiment is differentiated into mature pigment cells in the bulge region of the pigment stem cells, as shown in the Examples below. Therefore, the cell death inhibitor containing it itself has the effect of suppressing the cell death of the pigment stem cell, and as a result, the effect of suppressing the disappearance of the pigment stem cell. .

  In addition, the agent for suppressing abnormal differentiation of hair follicle stem cells of the present embodiment includes (i) XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) the amino acid sequence of SEQ ID NO: 1 and at least 80% amino acid sequence An XVII type collagen variant having the identity and having an activity of suppressing abnormal differentiation of hair follicle stem cells, (iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) SEQ ID NO: A XVII type collagen variant that encodes a nucleic acid that hybridizes to a nucleotide sequence of 2 or a complementary strand thereof under stringent conditions and has an activity of suppressing abnormal differentiation of hair follicle stem cells. Since the agent for inhibiting abnormal differentiation of hair follicle stem cells according to this embodiment has an effect that collagen type XVII suppresses abnormal differentiation of hair follicle stem cells and promotes maintenance of hair follicle stem cells, as shown in Examples described later. In addition, the abnormal differentiation inhibitor itself containing the same suppresses abnormal differentiation of hair follicle stem cells, and as a result, has the effect of suppressing disappearance of hair follicle stem cells.

  In addition, the agent for suppressing abnormal differentiation of hair follicle stem cells of the present embodiment includes (i) XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, (ii) the amino acid sequence of SEQ ID NO: 1 and at least 80% amino acid sequence An XVII type collagen variant having identity and having an activity of suppressing differentiation into mature cells in the bulge region of hair follicle stem cells, (iii) type XVII encoded by the nucleotide sequence shown in SEQ ID NO: 2 A nucleic acid that hybridizes under stringent conditions to collagen or (iv) the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and inhibits differentiation into mature cells in the bulge region of hair follicle stem cells It may be an agent for inhibiting differentiation into mature cells in the bulge region of hair follicle stem cells, which contains a type XVII collagen variant having the activity ofAs shown in the examples described later, hair follicle stem cells constitute a bulge region, and XVII collagen or XVII collagen mutants have hair follicle stem cells matured from undifferentiated keratinocytes in an abnormal direction. It has a function of suppressing. Therefore, also in this case, the abnormal differentiation inhibitor itself of this embodiment containing XVII type collagen or XVII type collagen mutant having such a function similarly suppresses abnormal differentiation of hair follicle stem cells, It has the effect of suppressing the disappearance of hair follicle stem cells.

  In addition, the hair follicle stem cell disappearance inhibitor of the present embodiment is (i) a type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) at least 80% amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1. An XVII collagen variant having the ability to inhibit the loss of hair follicle stem cells, (iii) the XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) of SEQ ID NO: 2. It includes a XVII type collagen variant that encodes a nucleic acid that hybridizes to a nucleotide sequence or its complementary strand under stringent conditions and has an activity of suppressing the disappearance of hair follicle stem cells. The hair follicle stem cell disappearance inhibitor of the present embodiment, as shown in the examples described later, has the effect that type XVII collagen suppresses the disappearance of hair follicle stem cells and promotes the maintenance of hair follicle stem cells. The disappearance-suppressing agent itself contains the effect of suppressing the disappearance of the hair follicle stem cells, and as a result, promoting the maintenance of the hair follicle stem cells.

  In addition, the hair follicle stem cell disappearance inhibitor of the present embodiment is (i) a type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, and (ii) at least 80% amino acid sequence identical to the amino acid sequence of SEQ ID NO: 1. An XVII type collagen mutant that has the ability to suppress disappearance due to abnormal maturation differentiation from undifferentiated keratinocytes constituting the bulge region of hair follicle stem cells to the epidermis, sebaceous glands, etc., (iii) Encoded by the type XVII collagen encoded by the nucleotide sequence shown in No. 2; or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID No. 2 or its complementary strand; and XVII type collagen having activity to suppress disappearance due to abnormal mature differentiation from undifferentiated keratinocytes constituting bulge region to epidermis and sebaceous glands Including variants, it may be lost inhibitor by abnormal mature differentiated from undifferentiated keratinocytes constituting the bulge area of hair follicle stem cells into epidermal and sebaceous glands and the like. In this case, as shown in the Examples described later, the hair follicle stem cells constitute a bulge region, and the XVII type collagen or the XVII type collagen mutant has the hair follicle stem cells in an abnormal direction from the undifferentiated keratinocytes. It has a function of suppressing disappearance due to mature differentiation. Therefore, the disappearance inhibitor of this embodiment containing the XVII type collagen or the XVII type collagen variant having such a function also similarly suppresses the disappearance of hair follicle stem cells, thereby promoting the maintenance of hair follicle stem cells. The effect of doing.

  As mentioned above, although embodiment of this invention was described with reference to the amino acid sequence shown to sequence number 1, and the nucleotide sequence shown to sequence number 2, these are the illustrations of this invention, and various structures other than the above are employ | adopted. You can also.

  For example, the hair loss inhibitor or hair depigmentation inhibitor of this embodiment does not need to consist only of type XVII collagen or a variant thereof, and is known to have a hair loss inhibitory effect or hair depigmentation effect. Or various components known to have beneficial activity on the skin, especially the scalp, and body hair, particularly hair. In that case, it is expected that a further excellent hair removal inhibitory effect or hair depigmentation effect can be obtained by the additive effect or synergistic effect with these various components.

  For example, UV blocking agents such as sunscreens; vitamins (A, C, or E) and derivatives thereof (retinyl palmitate, tocopheryl acetate, or tocopheryl palmitate); ceramides; proteins and protein hydrolysates Ureas and allantoins; sugars and sugar derivatives such as reducing or oxidized sugars; plant-derived extracts (from Iridaceae or soybeans) or microorganism-derived extracts; Hydroxy acids, in particular hydroxycarboxylic acids or ketocarboxylic acids (fruit acids, salicylic acid) and their esters (5-n-octanoylsalicylic acid); diazoxides, spirooxazones or phospholipids (lecithin, in particular defatted lecithin); nicotinic acid derivatives (nicotine) Acid esters, in particular tocopheryl nicotinate, benzyl nicotinate and C1-C22 alkyl nicotinates such as methyl nicotinate or hexyl nicotinate; pyrimidine derivatives (2,4-diamino-6-piperidinopyrimidine 3-oxide, pyrimidine 3-oxide derivatives, 2,4-diaminopyrimidine 3-N-oxide); antiandrogens (steroidal or non-steroidal 5α-reductase inhibitors, cyprosterone acetate, azelaic acid and its salts and derivatives, flutamide, prostagland Gins such as PGF-2α or PGE2 in salt or ester forms, and analogs thereof such as latanoprost; fungicides, antifungals, or antidandruff (selenium derivatives, ketoconazole, octopirox, triclocarban, triclosa , Pyrithione zinc, itraconazole, asiatic acid, hinokitiol, mipyrosin, tetracycline, especially erythromycin, clinicin hydrochloride, benzoyl peroxide or benzyl peroxide, minocycline); calcium channel antagonists and potassium channel agonists; hormones; steroidal anti-inflammatory agents (gluco Corticoids, corticosteroids), and non-steroidal anti-inflammatory agents (glycyrrhetinic acid and α-bisabolol, benzydamine); retinoid RXR receptor agonists and retinoid antagonists; free radical scavengers and antioxidants (butylhydroxyanisole or butylhydroxytoluene); Antiseborrheic agent; Antiparasitic agent; Antiviral agent; Anti-Eczema agent; Caroteno Lactones and their corresponding salts; essential fatty acids (linolenic acid, eicosatetraenoic acid, linolenic acid, and eicosatrienoic acid) or their esters and amides; essential oils; phenols and polyphenols such as Flavonoids; etc. can be suitably used together with type XVII collagen or a variant thereof.

  In addition, in the method for suppressing hair loss or depigmentation of hair according to this embodiment, the above-mentioned various agents or compositions are administered, and these administration subjects are mammals regardless of gender and age. Although not particularly limited, it is desirable to administer prophylactically or therapeutically, particularly when there is a concern about the progress of hair loss or depigmentation of hair. In addition, when there is a tendency for hair removal or depigmentation of hair to progress in relatives, there is a possibility that the gene of XVII collagen or a mutant thereof is genetically defective or the expression of the gene is poor. Therefore, it is particularly preferable as an administration target. Furthermore, for these administration subjects, as a result of examining the gene using various well-known techniques in advance, the gene of XVII type collagen or its mutant is actually missing or the expression of the gene It is more preferable to target male humans who have been confirmed to be poor.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further, this invention is not limited to these.

Example 1 Analysis of COL17KO mice Test method (1) Experimental animal The Dct-lacZ transgenic mouse used in this study was provided by Dr. Ian Jackson, MRC, UK, and the CAG-CAT-EGFP mouse was provided by Prof. Junichi Miyazaki, Osaka University, Dct ^{tml ( Cre) Bee} mouse is provided by Dr. Freedrich Beerman, K14-hCOL17 transgenic mouse is provided by Prof. Kim Yancey of Wisconsin Medical University, and COL17KO mouse is provided by Prof. Hiroshi Shimizu, Graduate School of Medicine, Hokkaido University.

(2) Immunohistochemistry The back skin of mice was fixed with 4% paraformaldehyde or frozen and embedded in OCT compound (Sakura Finetechnical), then cut into 8 μm sections, and blocked with 3% skim milk at room temperature for 30 minutes. . Thereafter, the primary antibody was appropriately diluted, reacted at 4 ° C. overnight, washed with PBS, and then reacted with the secondary antibody.

  Primary antibodies used for immunostaining were anti-human XVII collagen antibody (N18, Santa Cruz), anti-mouse XVII collagen antibody (provided by Professor Toshihiro Tanaka, Shiga Medical University), anti-β-galactosidase antibody (Cappel) Anti-keratin 15 antibody (Covance), anti-CD34 antibody (eBioscience), anti-α6 integrin antibody (BD Pharmingen), and anti-S100a6 antibody (LAB VISION). As the secondary antibody, AlexaFlor 488, 568, 594 (Molecular Probes) was used.

(3) H. E. Staining The back skin of the mouse was fixed with Buan and embedded in paraffin, and then a 4 μm section was prepared and stained with hematoxylin and eosin according to a conventional method.

(4) Separation of pigment cells The back skin of CAG-CAT-EGFP; Dct ^{tml (Cre) Bee} mice in which pigment cells including 6-day-old pigment stem cells are GFP-positive is collected, and 1000 U / ml dispase ( After reacting overnight at 4 ° C. in (SANKO JUNYAKU) / PBS, the dermis was removed under a stereomicroscope. Thereafter, 0.25% trypsin was added, reacted at 37 ° C. for 10 minutes, neutralized with fetal calf serum, and then GFP-positive pigment cells were separated using a flow cytometry (FACS) method.

(5) Flow cytometry (FACS) analysis For separation of pigment cells, a GFP positive cell fraction was collected by the above-described method. For keratinocytes, a skin suspension prepared by enzyme treatment was reacted with the CD34 and α6 integrin antibodies described above. Bay Bioscience JSAN or Becton Dickinson FACS caliber was used for FACS analysis.

(6) Analysis of mouse XVII type collagen mRNA expression by RT-PCR RNA was extracted from mouse back skin and GFP positive pigment cells using TRIzol (GIBCO). 3 μg of RNA was reverse transcribed using THERMOSCRIPT RT-PCR System (GIBCO) to synthesize cDNA. Mouse XVII collagen mRNA expression was analyzed using the following primers. Moreover, the expression of GAPDH mRNA was measured as an internal standard.
-Primer sequence for mouse XVII collagen forward primer: 5'-actcgcctttttttacaacca (SEQ ID NO: 4)
reverse primer: 5′-gagcaggacccccattgtatt (SEQ ID NO: 5)
・ GAPDH
forward primer: 5′-accacagtccatgcccatcac (SEQ ID NO: 6)
reverse primer: 5′-tccaccacctgtgtgctgta (SEQ ID NO: 7)

(Results and discussion)
Although many COL17KO mice die within 2 weeks after birth, the surviving individuals were normal in appearance until around 4 weeks after birth, and were covered with black hair (FIG. 1a). After that, however, whitening gradually began to occur. Around 3 months after birth, whitening and hair loss became noticeable, and the body hair became longer than usual. Around 6 months after birth, the whole body became almost completely grayed, and finally around 10 months, hair loss occurred throughout the body (FIG. 1b).

  We have previously shown that poor maintenance of pigment stem cells can cause whitening. Therefore, in order to investigate whether the cause of whitening of COL17KO mice is due to insufficient maintenance of pigment stem cells, a Dct-LacZ transgene capable of marking pigment stem cells was introduced into COL17KO mice, and the distribution of pigment stem cells in COL17KO mice and The morphology was observed. As a result, it was revealed that type XVII collagen is strongly expressed in the bulge region and the basal cells of the epidermis and the pigment stem cells are present in a state surrounded by hair follicle stem cells (FIG. 1c). The distribution and morphology of pigment stem cells in COL17KO mice did not show any abnormality immediately after birth, but the number of pigment stem cells gradually decreased from about 3 months after birth, and at the same time the sarcoma had some melanin granules. Larger differentiated forms of mature pigment cells with multiple protrusions were seen (FIGS. 1d, e). The pigment stem cells disappeared completely around 5 months when the whole body became white. From the above results, it was concluded that the cause of whitening in COL17KO mice was due to insufficient maintenance of pigment stem cells.

Generally, it is known that type XVII collagen is expressed in epithelial cells forming hemidesmosomes. In order to examine the presence or absence of expression of type XVII collagen in pigment cells of the skin including pigment stem cells, in mice expressing GFP in the pigment cells (CAG-CAT-EGFP; Dct ^{tml (Cre) Bee} mouse), RNA was collected and the expression of mouse XVII collagen mRNA was analyzed using RT-PCR, and it was confirmed that there was no expression of XVII collagen mRNA in pigment cells including pigment stem cells (FIG. 1f). The above results suggest that there is no expression of type XVII collagen in pigment stem cells, and that the cause of the loss of pigment stem cells seen in COL17KO mice may be due to abnormal XVII collagen expression in cells surrounding the pigment stem cells It was done.

  In order to examine the presence or absence of abnormal hair follicle stem cells, COL17KO mice and control back skin were first examined at each age. H. E. According to the findings of the stained tissue image, thickening of the epidermis and an increase in sebaceous glands were observed from about 3 months after birth (FIG. 2a). Furthermore, around 6 months, many hair follicles were cysted or atrophyed (figure 2b). Finally, around 10 months, skin appendages such as hair follicles almost disappeared (FIG. 2c). These findings suggested that type XVII collagen is required for the maintenance of hair follicles. In addition, these H.P. E. The findings of the stained histological images are H. of mice in which c-Myc was activated by basal cells reported as a mouse depleted of stem cells in the epidermis and mice in which Rac1 was knocked out in the skin. E. It was found to be very similar to the findings of the stained tissue image. Therefore, considering the possibility that hair follicle stem cells are also depleted in COL17KO mice, staining with anti-keratin 15 antibody, anti-CD34 antibody, anti-α6 integrin antibody, anti-S100a6 antibody as hair follicle stem cell markers, and FACS Analysis was performed to confirm depletion of hair follicle stem cells (FIGS. 2d-f).

  From the above results, it was clearly demonstrated for the first time in this example that the defect of type XVII collagen was whitened and caused by the disappearance of pigment stem cells. That is, it has been clarified that type XVII collagen in hair follicle stem cells is essential not only for maintaining hair follicle stem cells but also for maintaining pigment stem cells.

  Although it is partially repeated, it has been reported in Non-Patent Document 4 that mice with a small body hair and a high proportion of white hair are observed in COL17KO mice.・ This is not a research report on depigmentation of hair. Therefore, no specific description is given regarding the state of the body hair of the COL17KO mouse, such as what state it was at the age of the day. That is, COL17KO mice had a small amount of white hair from the beginning of birth, or a large proportion of white hair, or a sufficient amount of black hair from the beginning of birth, but the amount of white hair gradually decreased. It has not been explained whether or not there was an increase. One reason is that many COL17KO mice died within 2 weeks of birth, making it difficult to observe adequately.

  In contrast, in this example, many COL17KO mice die within 2 weeks of life, but the surviving individuals are normal in appearance until about 4 weeks of age and are covered with black hair. Was revealed by sufficient follow-up (FIG. 1a). After that, further follow-up observation gradually showed whitening, and at around the age of 3 months, whitening and hair loss became noticeable and the body hair became longer than usual. It was made clear that Finally, a long-term follow-up of about one year revealed that the whole body was almost completely whitened around 6 months of age, and finally hair loss occurred throughout the whole body around 10 months. Collagen XVII deficiency Successfully demonstrated for the first time that is the cause of whitening and hair loss in mammals. Until now, long-term follow-up of the body hair state of such COL17KO mice has not been performed, and for the first time in this example, the defect of collagen XVII is the cause of whitening and hair loss in mammals. It became clear.

Example 2 Analysis of COL17KO, K14-hCOL17 transgenic mice (1) Experimental animals COL17KO was provided by Professor Hiroshi Shimizu, Graduate School of Medicine, Hokkaido University.

(Production of COL17KO, K14-hCOL17 transgenic mice)
From the mouse 129Sv / Ev genomic library (Stratagne), a 14.7 kb mouse XVII collagen gene was cloned, from which a 11.5 kb restriction enzyme NheI-NotI fragment was subcloned. A targeting vector (Stratagne) was prepared by incorporating the PGK / Neo cassette from exon 2 to intron 3 in this fragment, and the recombinant vector was introduced into embryonic hepatocytes of 129Sv / Ev mice for precise targeting. The obtained embryonic cells were injected into the blastcycle of C57BL / 6J mice to produce chimeric mice. This chimeric mouse and C57BL / 6J mouse were further mated, and F1 heterozygous animals were crossed over 4 generations or more to produce heterozygous mice (COL17 ^{m − / −} ).

Transgenic mice (COL17 ^{m + / +, h +} mice) heterologous to C57BL / 6J that specifically express human XVII collagen only in the subepithelial layer by incorporating XVII collagen cDNA downstream of the K14 promoter Mice (COL17 ^{m +/−} mice) were bred. Mice (COL17 ^{m +/−, h +} mice) having heterogeneous human XVII collagen transgenes obtained as a result are mated to produce COL17KO, K14-hCOL17 transgenic mice (COL17 ^{m − / −, h +} mice). did.

(2) Immunohistochemistry The back skin of mice was immunostained in the same manner as in Example 1. Primary antibodies used for immunostaining are anti-mouse type XVII collagen antibody (provided by Professor Toshihiro Tanaka, Shiga Medical University) and anti-β-galactosidase antibody (Cappel). For staining of Dct, a mouse having Dct-lacZ transgene was stained with an anti-β-galactosidase antibody.

(Results and discussion)
In order to confirm that abnormalities of hair follicle stem cells cause abnormalities of pigment stem cells in COL17KO mice, COL17KO mice (COL17 ^{m − / −, h +} mice) into which a transgene expressing human XVII collagen was expressed by the keratin 14 promoter were used. Examined. In this mouse, it was confirmed that human XVII collagen was expressed in keratinocytes including bulge, but neither human nor mouse XVII collagen was expressed in other cells including pigment cells (Fig. 3a and b (some data not shown)). In this mouse, hair loss and whitening were not observed even after 5 months of age (Figs. 3a and b), and no abnormalities were observed in the morphology and distribution of Dct-positive pigment stem cells (Figs. 3c and d). ). Based on the above results, loss of XVII collagen caused hair loss and whitening, and that hair loss and whitening were caused by loss of hair follicle stem cells and / or pigment stem cells, and expression of type XVII collagen in hair follicle stem cells It was shown that the loss of hair follicle stem cells and pigment stem cells can be prevented by the treatment. That is, it has been clarified that the type XVII collagen in hair follicle stem cells is essential not only in maintaining hair follicle stem cells but also in maintaining pigment stem cells and suppressing hair loss and depigmentation of hair.

  That is, according to this example, by expressing XVII collagen in a hair follicle stem cell of a mammal genetically deficient in XVII collagen, a sufficient amount of XVII collagen is supplied to the hair follicle stem cell. It became clear for the first time in mice that hair loss and whitening were not observed even after 5 months of age, and that there was no abnormality in the morphology and distribution of pigment stem cells. Until now, long-term follow-up of the hair state and the morphology / distribution of pigment stem cells of COL17KO mice into which a transgene expressing human XVII collagen is introduced with such a keratin 14 promoter has not been performed. According to the examples, it was clarified that, when a sufficient amount of type XVII collagen was supplied to hair follicle stem cells for the first time, hair loss and whitening could be suppressed in order to prevent the loss of hair follicle stem cells and pigment stem cells. .

  In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are within the scope of the present invention.

It is a figure which shows the whitening and hair loss seen in COL17KO mouse | mouth, and XVII type | mold collagen expression in a control mouse | mouth. (a) Photograph of COL17KO mouse. From the top, mice at 2 weeks, 6 weeks, 2 months, 3 months, 6 months and 10 months are shown. Progressive hair loss and whitening were observed. (b) Photo of 10 month control. (c) Left: XVII collagen is strongly expressed in the bulge region and epidermal basal cells. Right: Pigment stem cells are surrounded by hair follicle stem cells. (d) LacZ-stained images of controls at 6 days, 3 months and 5 months after birth. (e) LacZ stained image of knockout mouse (KO mouse). In KO mice, LacZ-positive dye stem cells are gradually reduced as compared to control mice, and differentiated images become conspicuous. After 5 months, almost all positive cells disappear. (f) RT-PCR. Expression of type XVII collagen is not observed in GFP positive pigment cells. It is a figure which shows the loss | disappearance of the hair follicle stem cell which is a niche of a pigment stem cell. (A) From about 3 months after birth, thickening of the epidermis and an increase in sebaceous glands were observed. (B) From around 6 months of age, images of hair follicles forming cysts were also observed. (C) At 10 months of age, skin appendages such as hair follicles disappear. (D) α6 integrin and CD34 double positive hair follicle stem cells were significantly reduced in COL17KO mice. (E) Staining with hair follicle stem cell marker of control tissue. (F) Staining of COL17KO mouse tissue with hair follicle stem cell marker. In COL17KO mice, keratin 15, CD34, α6 integrin and S100a6 positive cells were not observed. It is a figure which shows the hair state in COL17 ^{m − / −, h +} mouse and COL17KO mouse, the expression of human type XVII collagen, and the morphology and distribution of stem cells. (A) In COL17KO mice, expression of type XVII collagen was not observed, and progressive whitening and hair loss were observed. (B) In COL17 ^{m − / −, h +} mice, expression of human XVII collagen was observed in the basal layer of the epidermis and hair follicle, and hair loss and whitening were not observed. showed that. (C) In COL17KO mice, pigment stem cells were observed on the 6th day of birth, but disappearance of the pigment stem cells was observed at 5 months of age. (D) In COL17 ^{m − / −, h +} mice, no disappearance of stem cells was observed at 6 days of birth or at 5 months of age.

Claims (18)

(I) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1,
(Ii) an XVII type collagen variant having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing hair loss;
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and hair loss XVII type collagen mutant having activity of inhibiting
A hair loss inhibitor.   The hair loss inhibitor according to claim 1, wherein the hair loss is hair loss due to disappearance of hair follicle stem cells. (I) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1,
(Ii) a type XVII collagen variant having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing depigmentation of hair;
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and hair A type XVII collagen mutant having an activity of suppressing depigmentation of
A hair depigmentation inhibitor comprising:   The hair depigmentation inhibitor according to claim 3, wherein the depigmentation of the hair is depigmentation due to disappearance of pigment stem cells. A promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, or a sequence downstream of the promoter sequence A nucleotide sequence encoding a variant XVII collagen having at least 80% amino acid sequence identity with the amino acid sequence of No. 1 and having an activity of suppressing hair loss, or (ii) a nucleotide sequence shown in SEQ ID NO: 2 Or a nucleotide sequence that encodes a type XVII collagen variant that hybridizes to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand under stringent conditions and has an activity of suppressing hair loss,
A composition for inhibiting hair loss, comprising a vector having   The composition according to claim 5, wherein the promoter is a promoter that controls to be expressed in the basal layer of the epidermis.   The composition according to claim 6, wherein the promoter is a keratin 14 promoter having the nucleotide sequence shown in SEQ ID NO: 3. A promoter sequence that controls expression of type XVII collagen or a variant thereof in a region containing hair follicle stem cells, and (i) an XVII type collagen having the amino acid sequence shown in SEQ ID NO: 1, or a sequence downstream of the promoter sequence A nucleotide sequence encoding a type XVII collagen variant having at least 80% amino acid sequence identity with the amino acid sequence of No. 1 and having an activity of inhibiting depigmentation of hair, or
(Ii) XVII collagen that hybridizes under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 2, or the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and has the activity of suppressing depigmentation of hair A nucleotide sequence encoding a variant,
A vector having
A composition for suppressing depigmentation of hair.   The composition according to claim 8, wherein the promoter is a promoter that controls to be expressed in the basal layer of the epidermis.   The composition according to claim 9, wherein the promoter is a keratin 14 promoter having the nucleotide sequence shown in SEQ ID NO: 3. (I) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1,
(Ii) an XVII type collagen variant having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing ectopic differentiation of pigment stem cells;
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and a dye An agent for inhibiting ectopic differentiation of pigment stem cells, comprising an XVII collagen mutant having an activity of suppressing ectopic differentiation of stem cells.   The ectopic differentiation inhibitor according to claim 11, wherein the ectopic differentiation of the pigment stem cells is differentiation into mature pigment cells in a bulge region. (I) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1,
(Ii) a type XVII collagen variant having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing the disappearance of pigment stem cells;
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and a dye An XVII type collagen mutant having an activity of suppressing the disappearance of stem cells,
An agent for suppressing the disappearance of pigment stem cells.   The disappearance inhibitor according to claim 13, wherein the disappearance of the pigment stem cell is a loss that occurs following differentiation into a mature pigment cell in a bulge region. (I) type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1,
(Ii) an XVII type collagen variant having an amino acid sequence identity of 80% or more with respect to the amino acid sequence of SEQ ID NO: 1 and having an activity of suppressing abnormal differentiation of hair follicle stem cells;
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and hair An XVII type collagen mutant having an activity of suppressing abnormal differentiation of a stem cell,
An agent for suppressing abnormal differentiation of hair follicle stem cells, comprising: The hair follicle stem cells constitute bulge regions as undifferentiated keratinocytes,
The abnormal differentiation inhibitor according to claim 15, wherein the abnormal differentiation of the hair follicle stem cell is abnormal mature differentiation from an undifferentiated keratinocyte constituting a bulge region to an epidermis or sebaceous gland. (I) Type XVII collagen having the amino acid sequence shown in SEQ ID NO: 1, or (ii) 80% or more amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1, and loss of hair follicle stem cells A type XVII collagen variant having inhibitory activity,
(Iii) type XVII collagen encoded by the nucleotide sequence shown in SEQ ID NO: 2, or (iv) a nucleic acid that hybridizes under stringent conditions to the nucleotide sequence of SEQ ID NO: 2 or its complementary strand, and hair An XVII type collagen mutant having an activity of suppressing disappearance of the stem cell,
An agent for suppressing the disappearance of hair follicle stem cells. The hair follicle stem cells constitute bulge regions as undifferentiated keratinocytes,
The disappearance inhibitor according to claim 17, wherein the disappearance of the hair follicle stem cells is disappearance by mature differentiation from undifferentiated keratinocytes constituting the bulge region to epidermis, sebaceous glands and the like.