JP6366415B2 - Cell activator comprising Orai3 gene expression inhibitor or ORAI3 protein function inhibitor - Google Patents

Description

  The present invention relates to a skin cell activator comprising an Orai3 gene expression inhibitor or an ORAI3 protein function inhibitor. In yet another aspect, the present invention also relates to a screening method for a skin cell activator using Orai3 gene expression as an index.

  Skin undergoes various aging phenomena under the influence of aging, exposure to sunlight (ultraviolet rays), eating habits, stress, and the like. Examples of the skin aging phenomenon include pigmentation such as spots, freckles, and liver spots, dullness, dryness, wrinkles and the like. Skin aging occurs separately or in conjunction with the skin epidermis, dermis, and subcutaneous tissue, but the epidermis present in the outermost layer and the dermatological aging involved in wrinkles and pigmentation are particularly problematic. It is said. In the epidermis, it is known that while the number of stratum corneum increases as an aging phenomenon, the barrier function of the skin decreases, and thereby, a dry state is exhibited (Non-patent Documents 1 and 2). As a result, skin troubles such as rough skin and dry skin occur, and the problem of beauty is caused by the loss of texture and flexibility. In the dermis, the shrinkage, flexibility, moisture retention, etc. that the skin originally maintains by reducing or degenerating connective tissue and elastic fibers due to internal factors such as aging and external factors such as ultraviolet rays and active oxygen The function of the skin declines, causing various skin problems such as wrinkles and sagging, tension and elasticity.

  Research has been made to delay the progression of the skin aging phenomenon and reverse it if possible, for example, searching for substances that remove antioxidants and substances that promote the production of elastic fibers ( Patent Documents 1 and 2). For the development of beauty products, research at such a macro level has been conducted, but the aging phenomenon at the cellular level has not yet been fully elucidated.

On the other hand, the Orai family is known as a kind of calcium channel. Unlike other calcium channels, the Orai family of calcium channels is a four-transmembrane membrane protein. Orai1 is identified as a protein that senses Ca ²⁺ depletion in the endoplasmic reticulum as a constituent molecule of the Ca ²⁺ release activated Ca ²⁺ (CRAC) channel that is activated when the Ca ²⁺ store in the endoplasmic reticulum is depleted. Identified by studies using RNAi technology following STIM1. Orai1 has been shown to be present in the cell membrane. Immediately after the discovery of Orai1, Orai2 and Orai3 were discovered as human homologous genes, and evolutionarily, Orai3 was revealed to be the newest homologous gene that emerged during differentiation into mammals. Therefore, although Orai3 is predicted to have a new biological function according to the environmental adaptation of mammals, its function still remains unknown (Non-patent Document 3).

Japanese Patent No. 5468906 Japanese Patent No. 4842105

The Journal of Clinical Investigation (1995) Vol. 95, 1995, 2281-2290 Journal of Geriatric Dermatology (1993) Vol.1, 111-120 Journal of Physiology (2012) Vol.590, Part2, 241-257 Gerontolgy (2013) No.59, 159-164 Journal of Investigative Dermatology (2003) Vol. 121, 1557-1558 Journal of Investigative Dermatology (2002) Vol.119, 1128-1136 Journal of Investigative Dermatology (2001) Vol. 116, No.1, 50-56

  An object of this invention is to develop the chemical | medical agent which can activate a skin cell while specifying the mechanism in connection with aging at the cell level in skin. Furthermore, it aims at providing the screening method of the substance which can specify the mechanism in connection with aging in a cell, and can thereby delay or reverse the progress of cell aging.

As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have obtained for the first time the finding that recovery from Ca ²⁺ responses in epidermal cells to external stimuli is delayed with aging. Based on this finding, the cause of slowing recovery from the Ca ²⁺ response was investigated. By knocking down Orai3, a type of Ca ²⁺ channel, the recovery from the Ca ²⁺ response was accelerated. I found out. Surprisingly, when Orai3 knockdown cells were subjected to scratch assay, a wound healing model, with the expectation that rapid recovery from the Ca ²⁺ response was involved in cell rejuvenation, the cell proliferation rate was surprisingly increased. The present inventors have discovered that the ability to heal wounds is increased and have arrived at the present invention.

The invention therefore relates to:
[1] A skin cell activator comprising an ORAI3 protein or Orai3 gene function inhibitor in skin.
[2] The ORAI3 protein or Orai3 gene function inhibitor is selected from the group consisting of an ORAI3-specific antibody and an RNAi-inducible nucleic acid, antisense nucleic acid, ribozyme, and their expression vectors that specifically inhibit the expression of Orai3 The skin cell activator according to item 1, which is one or more of the above.
[3] The skin cell activator according to item 1 or 2, wherein the skin cell activator is a skin barrier function enhancer or a wound healing promoter.
[4] A step of adding a candidate drug to cultured cells,
A method for screening a cell activator comprising a step of directly or indirectly measuring Orai3 gene expression in cultured cells after addition of a candidate drug, and a step of comparing with gene expression in cultured cells before addition of the candidate drug.
[5] The method for screening a cell activator according to Item 4, wherein the step of directly measuring the gene expression of Orai3 is a method of measuring the amount of Orai3 mRNA or the amount of ORAI3 protein.
[6] The step of indirectly measuring the gene expression of Orai3 includes the following:
Step of measuring an intracellular Ca ²⁺ concentration before and after the pressure stimulation applied to cultured cells, and the intracellular Ca ²⁺ concentration time from beyond the intermediate value between the peak value and the steady-state value before returning to the intermediate value (half The method according to item 4, comprising a step of associating a value range) or a value of intracellular Ca ²⁺ concentration after a lapse of a predetermined time with the gene expression of Orai3.
[7] The screening method for a skin cell activator according to any one of items 4 to 6, wherein the skin cell activator is a skin barrier function enhancer or a wound healing promoter.
[8] A method for producing a cultured skin sheet, comprising a step of adding an Orai3 gene expression inhibitor or an Orai3 protein function inhibitor to skin cultured cells or a three-dimensional skin culture sheet.
[9] The function inhibitor of the ORAI3 protein or Orai3 gene is selected from the group consisting of an ORAI3-specific antibody, and an RNAi-inducible nucleic acid, antisense nucleic acid, ribozyme, and their expression vectors that specifically inhibit the expression of Orai3 The manufacturing method of the cultured skin sheet of item 8 which is 1 or more types.
[10] A method for growing cultured cells, comprising a step of adding an Orai3 gene expression inhibitor or an Orai3 protein function inhibitor to cultured cells.
[11] The method for proliferating cultured cells according to Item 10, further comprising a step of stimulating the cultured cells.
[12] A method for activating skin cells, comprising administering an ORAI3 protein or Orai3 gene function inhibitor.
[13] The function inhibitor of the ORAI3 protein or Orai3 gene is selected from the group consisting of an ORAI3-specific antibody, and an RNAi-inducible nucleic acid, antisense nucleic acid, ribozyme, and their expression vectors that specifically inhibit the expression of Orai3 Item 13. The skin cell activation method according to Item 12, which is one or more types.
[14] A cosmetic method comprising administering an ORAI3 protein or Orai3 gene function inhibitor.
[15] The function inhibitor of the ORAI3 protein or Orai3 gene is selected from the group consisting of an ORAI3-specific antibody and an RNAi-inducible nucleic acid, antisense nucleic acid, ribozyme, and their expression vector that specifically inhibits the expression of Orai3 Item 15. The cosmetic method according to Item 14, wherein the cosmetic method is one or more types.
[16] A method for treating a wound, comprising administering an ORAI3 protein or an Orai3 gene function inhibitor.
[17] The function inhibitor of the ORAI3 protein or Orai3 gene is selected from the group consisting of an ORAI3-specific antibody and an RNAi-inducible nucleic acid, antisense nucleic acid, ribozyme, and their expression vector that specifically inhibits the expression of Orai3 Item 17. The wound treatment method according to Item 16, wherein the wound treatment method is one or more types.
[18] Use of an ORAI3 protein or Orai3 gene function inhibitor for the manufacture of a medicament or cosmetic for skin anti-aging.
[19] The ORAI3 protein or Orai3 gene function inhibitor is selected from the group consisting of ORAI3-specific antibodies and RNAi-inducible nucleic acids, antisense nucleic acids, ribozymes, and their expression vectors that specifically inhibit Orai3 expression. 19. Use according to item 18, wherein the use is one or more.

  By suppressing the expression of Orai3 gene and the function of Orai3 protein, cell activation effects including enhancement of skin barrier function or promotion of wound healing ability can be exhibited.

FIG. 1A shows a series of photographs showing changes in intracellular Ca ²⁺ concentration after a hydraulic load on cells (epidermal keratinocytes) obtained from a 24-year-old or 63-year-old healthy person. 1B and 1C are graphs showing changes in intracellular Ca ²⁺ concentration in cells obtained from a 24-year-old healthy person (FIG. 1B) and a 63-year-old healthy person (FIG. 1C). FIG. 1D is a graph showing the correlation between the time (half value width) from when the intracellular Ca ²⁺ concentration exceeds the intermediate value between the peak value and the steady value to the return to the intermediate value, and the age of the donor of the cell. It is. FIG. 2A is a graph showing the correlation between the age of a cell donor and the level of Orai3 mRNA. FIG. 2B shows the correlation between the time (half width) from when the intracellular Ca ²⁺ concentration exceeds the intermediate value between the peak value and the steady value to the time when the intracellular Ca ²⁺ concentration returns to the intermediate value, and the mRNA level of Orai3. It is a graph. FIG. 3 shows that Orai3 gene expression was suppressed by introduction of Orai3 siRNA into keratinocytes. FIG. 3A shows changes in the amount of mRNA, and FIG. 3B shows changes in the amount of protein. FIG. 4A is a graph showing the effect of rejuvenation on the Ca ²⁺ response induced by water pressure stimulation when the expression of Orai3 is suppressed in keratinocytes obtained from an elderly person (63 years old). FIG. 4B shows the time (half-value width) from when the intracellular Ca ²⁺ concentration calculated from the graph of FIG. 4A exceeds the intermediate value between the peak value and the steady value until it returns to the intermediate value. It is the graph shown by the average. FIG. 5A is a photograph showing the localization of Orai3 in a frozen section of healthy human skin. FIG. 5B shows a negative control. FIG. 6 is a graph showing the gene expression level of Orai3 in cultured keratinocytes in the growth phase and cultured keratinocytes induced to differentiate. FIG. 7A is a photograph showing the results of a scratch assay for cultured keratinocytes knocked down by Orai3. FIG. 7B is a graph showing the wound healing effect 7 hours after scratching.

  The present invention relates to a skin cell activator comprising an ORAI3 protein or Orai3 gene function inhibitor. In the present invention, the amino acid sequence of ORAI3 is known as SEQ ID NO: 1, for example. As for the nucleotide sequence of the Orai3 gene, SEQ ID NO: 2 is known as an open reading frame (ORF), SEQ ID NO: 3 is known as the sequence of mRNA to be transcribed, and the genome sequence is also known (ENSG00000175938). Both the amino acid and nucleotide sequences of ORAI3 are registered in GenBank as Accession number Nm — 152288. In the present invention, the ORAI3 or Orai3 gene includes a mutant of the ORAI3 or Orai3 gene, for example, a mutant based on mutation, polymorphism, alternative splicing, degeneracy and the like. Specifically, ORAI3 in the present invention may be a protein comprising the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence containing a deletion, substitution or addition of one or several amino acids in the amino acid sequence. Good. Alternatively, ORAI3 in the present invention is 90% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, and most preferably 99% or more identity with the amino acid sequence shown in SEQ ID NO: 1. And a variant having an in vivo function equivalent to that of ORAI3. The Orai3 gene in the present invention is 90% or more, preferably 95% or more, more preferably 97% of the nucleotide sequence encoded by the amino acid sequence of SEQ ID NO: 1, more specifically the nucleotide sequence shown by SEQ ID NO: 2. %, More preferably 98% or more, and most preferably 99% or more of the nucleotide sequence having the same in vivo function as that of ORAI3 after translation.

ORAI3 is a calcium channel, but surprisingly, knocking down ORAI3 speeds up the recovery of the stimulated epidermal keratinocytes from the Ca ²⁺ response and further improves the ability of skin wounds to heal It has been found by the present inventors. It has also been found by the inventors that the time taken to recover from stimulated keratinocytes from the Ca ²⁺ response increases with age. It is also known that the wound healing ability decreases with aging (Non-Patent Document 4). Therefore, it is considered that cell activation is possible by suppressing the gene expression of Orai3 by knockdown or the function of ORAI3, and further, rejuvenation of aged cells is enabled. Therefore, a substance capable of suppressing the function of the ORAI3 protein or the Orai3 gene can be used as a cell activator for cells, preferably skin cells, particularly epidermal cells. The cell activator of the present invention can be used as an anti-aging agent, aging retrograde agent, or rejuvenating agent for the skin. Such a substance may be used not only in vivo but also ex vivo or in vitro. Moreover, since the substance which suppresses the function of ORAI3 protein or Orai3 gene improves wound healing ability, it can be used as a wound healing promoter.

Moreover, regulatory Ca ²⁺ concentration gradient in the epidermis, has been shown to decrease with age (non-patent document 5), epidermal Ca ²⁺ concentration gradient, a differentiation formation of the epidermis of the stratum corneum (Non-patent document 6). Further, it is known that the number of stratum corneum increases with aging, but the skin barrier function is weakened and a dry state is exhibited (Non-patent Document 2). Combining these conventional techniques with the findings revealed by the present inventors, it is considered that the Ca ²⁺ concentration gradient in the epidermis that decreases with aging is caused by the action of the Orai3 gene involved in skin aging. It is done. Therefore, it is considered that a substance that suppresses the function of the ORAI3 protein or the Orai3 gene affects the intraepidermal Ca ²⁺ concentration gradient, thereby appropriately maintaining the stratum corneum formation. Thereby, the substance that suppresses the function of the ORAI3 protein or the Orai3 gene can be involved in the improvement of the skin barrier function, and can be used as a skin barrier function enhancer.

  In the present invention, cell activation including an ORAI3 protein or Orai3 gene function inhibitor refers to enhancing cell activity in skin, particularly epidermal cells, and may include anti-aging and rejuvenation effects. Examples of the activation effect include improvement of cell proliferation, stimulus responsiveness, temperature change and resistance to stress by chemical substances. Since the cell activator of the present invention can enhance cell proliferation, particularly in response to external stimuli such as wounds and burns, the cell activation includes improvement of wound or burn healing. In addition, by activating cells, skin barrier functions are enhanced, skin conditions such as skin texture and wrinkles are improved, and whitening and dullness are improved by promoting epidermal turnover (specifically, epidermal cells incorporating melanin pigments) Effects such as emission promotion).

  The Orai3 gene function inhibitor may be any substance as long as it can suppress the expression of Orai3 gene, and may be referred to as an Orai3 gene expression inhibitor. Examples of the Orai3 gene expression inhibitor include RNAi-inducible nucleic acids, antisense nucleic acids, ribozymes, and their expression vectors that specifically inhibit Orai3 gene expression. Suppression of the function of the Orai3 gene is expressed as the amount of mRNA or protein, and if it shows a statistically lower value compared to the control, it can be said that the function of the Orai3 gene was suppressed. When function suppression is shown, these expression levels are preferably 80% or less, more preferably 50% or less, and particularly preferably 20% or less with respect to the control.

The function inhibitor of the ORAI3 protein may be any substance as long as it is a substance capable of suppressing the function of the Ca2 ⁺ channel formed by the ORAI3 protein, preferably a plurality of ORAI3. Examples include functional neutralizing antibodies. Since the ORAI3 protein functions as a Ca ²⁺ channel, the suppression of the function of the ORAI3 protein can be measured by the behavior of Ca ²⁺ . In particular, ORAI3 works to sustain the Ca ²⁺ response by stimulation in skin cells, so the amount of Ca ²⁺ is measured over time, and the intracellular Ca ²⁺ concentration after Ca ²⁺ response is the peak value. The value of intracellular Ca ²⁺ concentration after elapse of a predetermined time, for example (1 minute, 2 minutes, or 3 minutes), from the time when the intermediate value with the steady value is exceeded to the time when the intermediate value is returned (half width). If such a value is statistically lower than that of the control, it can be said that the function of the ORAI3 protein is suppressed. When showing functional inhibition, these values are preferably 80% or less, more preferably 60% or less, particularly preferably 40% or less, relative to the control.

  The RNAi-inducible nucleic acid used in the present invention refers to a polynucleotide capable of inducing RNA interference by being introduced into a cell, and is usually 19 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 to 19 nucleotides. It may be RNA, DNA comprising 23 nucleotides, or a chimeric molecule of RNA and DNA, and any modification may be applied. RNAi interference may occur for mRNA or RNA immediately after transcription prior to processing, that is, RNA of a nucleotide sequence including exons, introns, 3 ′ untranslated regions, and 5 ′ untranslated regions. . The RNAi method that can be used in the present invention includes (1) directly introducing a short double-stranded RNA (siRNA) into a cell, or (2) incorporating short-hairpin type RNA (shRNA) into various expression vectors. Or (3) creating a vector for expressing siRNA by inserting a short double-stranded DNA corresponding to siRNA between the promoters into a vector having two promoters arranged in opposite directions, RNAi may be induced by a technique such as introduction into cells. The RNAi nucleic acid may include siRNA, shRNA, or miRNA that enables cleavage of ORAI3 RNA or suppression of its function, and these RNAi nucleic acids may be directly introduced using liposomes or the like, or these RNAi nucleic acids May be introduced using an expression vector that induces.

The siRNA for Orai3 used in the present invention can be synthesized using a well-known chemical synthesis technique based on the Orai3 sequence that is the target of siRNA. For example, chemically synthesized using an automatic DNA (/ RNA) synthesizer utilizing a DNA synthesis technique such as a solid phase phosphoramidite method, or a contract synthesis company related to siRNA (for example, Life Technologies) It is also possible to synthesize by consigning. According to the embodiment of the present invention, the siRNA of the present invention is induced from its precursor short-hairpin type double-stranded RNA (shRNA) through processing by an intracellular RNase, Dicer. Also good. In the present invention, the siRNA used is RNA corresponding to DNA comprising 19 to 30 nucleotides, preferably 19 to 25 nucleotides, more preferably 19 to 23 nucleotides derived from SEQ ID NO: 2 or its complementary sequence. . One or several such RNAs may be added to the 5 ′ or 3 ′ end, such as 2 additional sequences (tt or uu), thereby preventing degradation in the cell and increasing stability. Can do. For example, the following sequences can be used as Orai3 siRNAs, but are not intended to be limited to the following, and sequences complementary to the following sequences may be used:
5′-GCUACAAGCAGGAACUAGA-3 ′ (SEQ ID NO: 4)
5′-GCACCUCUUUGCACUCAUG-3 ′ (SEQ ID NO: 5)
5′-GAAGUUGUCCUGGUUGGUU-3 ′ (SEQ ID NO: 6)
5′-GUGGCUACCUCCCUUAGUC-3 ′ (SEQ ID NO: 7)
5′-CAUCCACAACCUCAACUCUtt-3 ′ (SEQ ID NO: 8)
5′-GCUACAAGCAGGAACUAGAtt-3 ′ (SEQ ID NO: 9)
5′-GGUUGGUUGGGUCAAGUUUtt-3 ′ (SEQ ID NO: 10)
5′-GCUGUGAGCAACAUCCACAtt-3 ′ (SEQ ID NO: 11)
5′-UUGAAGCUGUGAGCAACAU-3 ′ (SEQ ID NO: 12)
5′-GGGUCAAGUUUGUGCCCCAU-3 ′ (SEQ ID NO: 13)
5′-GCUACAAGCAGGAACUAGA-3 ′ (SEQ ID NO: 14)
5′-GCACCUCUUUGCACUCAUG-3 ′ (SEQ ID NO: 15)
5′-GAAGUUGUCCUGGUUGGUU-3 ′ (SEQ ID NO: 16)
5′-GUGGCUACCUCCCUUAGUC-3 ′ (SEQ ID NO: 17)
5′-UUGAAGCUGUGAGCAACAUtt-3 ′ (SEQ ID NO: 18)
5′-CACCAGUGGCUACCUCCCUUAtt-3 ′ (SEQ ID NO: 19)
5′-TCCUUAGCCCUUGAAAUACAAtt-3 ′ (SEQ ID NO: 20)
5′-CUGCCCUGGGCACCUUUCU-3 ′ (SEQ ID NO: 21)
5′-GCAACAUCCACAACCUCAA-3 ′ (SEQ ID NO: 22)
Among these, from the viewpoint of confirmation of Orai3 gene suppression, one or more selected from the group consisting of SEQ ID NOs: 4 to 10 is preferably used as the ORAI3 siRNA, and those of SEQ ID NOs: 4 and / or 9 are particularly preferable. .

  An antisense nucleic acid may be used as an active ingredient of the composition of the present invention. This antisense nucleic acid is either RNA or DNA containing a sequence of mRNA corresponding to the Orai3 gene containing the nucleotide sequence of SEQ ID NO: 2, or a sequence complementary to a partial sequence thereof. The partial sequence includes a sequence consisting of about 30 or more, 50 or more, 70 or more, 100 or more, 150 or more, 200 or more, or 250 or more and less than the full length, for example, 50 to 150 nucleotides in the sequence of the Orai3 gene or mRNA. be able to. This sequence may include substitution, addition, or deletion of one or a plurality of nucleotides as long as the function as an antisense nucleic acid is not hindered, or a nucleotide analog may be used. The nucleotides of antisense nucleic acids can include modified nucleotides having groups such as halogen (fluorine, chlorine, bromine or iodine), methyl, carboxymethyl or thio groups in addition to natural nucleotides. Antisense nucleic acids can be synthesized using well-known DNA / RNA synthesis techniques or DNA recombination techniques. When synthesizing by DNA recombination technology, it is necessary to amplify the target sequence by performing polymerase chain reaction (PCR) using a vector DNA containing the sequence of SEQ ID NO: 2 as a template and a primer sandwiching the sequence to be amplified. And can be cloned into a vector to produce antisense DNA. Alternatively, the thus obtained DNA having an amplified target sequence can be inserted into a vector, the vector is introduced into a eukaryotic or prokaryotic cell, and antisense RNA can be obtained using the transcription system. As the vector, the virus vectors and plasmid vectors described above can be used.

  Ribozymes can also be used as the function inhibitor of the Orai3 gene of the present invention. Ribozyme refers to RNA having an enzymatic activity that cleaves nucleic acids, and cleaves RNA at a desired position by placing sequences complementary to the sequence to be cleaved on both sides of the sequence having enzymatic activity. Can do. On the other hand, recent studies have revealed that oligo DNA having the base sequence of the enzyme active site also has nucleic acid cleavage activity, so in this specification, as long as it has sequence-specific nucleic acid cleavage activity. It is used as a concept including DNA. Specifically, the ribozyme can specifically cleave mRNA encoding the Orai3 gene or a sequence in the initial transcript. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the portion having the hammerhead structure (about 10 bases in total) are combined with the sequences before and after the desired cleavage site of mRNA. By using complementary sequences, it is possible to specifically cleave only the target mRNA. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. (Nucleic Acids Res., 29 (13): 2780-2788 (2001)).

  The function inhibitor of the Orai3 gene can be used in the form of a vector that can be expressed in cells. The expression vector used includes an Orai3 gene siRNA, an antisense nucleic acid, a polynucleotide encoding a ribozyme, and a promoter operably linked to the polynucleotide. As the promoter, any promoter can be used as long as it enables transcription of an expression target under control in the cell. For example, a polIII promoter (eg, tRNA promoter, U6 promoter, H1 promoter) Mammalian promoters (eg, CMV promoter, CAG promoter, SV40 promoter) and the like are used. The expression vector of the present invention further includes a selection marker gene (a gene that confers resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, phosphinothricin, a gene that complements an auxotrophic mutation, etc.). May be.

The backbone of the expression vector of the present invention is not particularly limited as long as it can transcribe a controlled expression target in mammalian cells such as humans, and examples thereof include plasmid vectors and virus vectors. Suitable vectors for administration to mammals include viral vectors such as retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, pox virus, poliovirus, Sindbis virus, Sendai virus and the like. Of these, virus vectors derived from retroviruses, adenoviruses, adeno-associated viruses, and vaccinia viruses are preferred.

  The ORAI3 protein function inhibitor may be any substance as long as the function of the ORAI3 protein can be suppressed. For example, it may be an antibody against the ORAI3 protein or a fragment thereof. The antibody of the present invention can be obtained, for example, by screening using the sequence of the extra-membrane region of ORAI3 as an epitope. In the present invention, the term antibody is used in the broadest sense, and includes a monoclonal antibody, a polyclonal antibody, an antibody variant, and an artificial antibody as long as a desired specific binding property is shown. The antibody in the present invention may be an antibody derived from any animal such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. In addition, the antibody in the present invention is any antibody such as a modified antibody in which a part of the amino acid sequence is substituted, a modified antibody in which various molecules are bound, an antibody fragment, a low molecular antibody, etc. Also good. Furthermore, the antibody which performed affinity maturation with respect to these antibodies is also mentioned. All of these antibodies can be produced using known methods.

  Polyclonal antibodies can be generated by immunizing mammals such as rabbits, chickens, mice, rats, sheep and the like according to a standard method using the epitope according to the present invention. Polyclonal antibodies against ORAI3 can be produced, for example, according to the description of Chapter 9 of Current Protocols in Immunology, Wiley and Sons Inc. In another embodiment, a polyclonal antibody that binds to ORAI3 of the present invention is generated by immunizing a mammal with ORAI3 and screening the generated antibody group using ORAI3 or an epitope peptide thereof. be able to.

  A monoclonal antibody among the antibodies of the present invention refers to an antibody of a single clone antibody population. Monoclonal antibodies include chimeric antibodies, humanized antibodies, human antibodies, bispecific antibodies, peptidomimetics of antibodies, modified antibodies, artificial antibodies and conjugated antibodies and fragments thereof. The monoclonal antibody against ORAI3 may be an antibody derived from any animal such as a mouse antibody, a human antibody, a rat antibody, a rabbit antibody, a goat antibody, or a camel antibody. In addition, the monoclonal antibody against ORAI3 in the present invention can be any antibody such as a modified antibody in which a part of the amino acid sequence is modified, an antibody modified product in which various molecules are bound, an antibody fragment, a low molecular antibody, etc. It may be an antibody. The monoclonal antibody of the present invention can be produced using any known method such as a hybridoma method, a phage display method, and a genetic engineering method.

  In the present invention, for the purpose of reducing the heterologous antigenicity to humans, for example, chimeric antibodies, humanized antibodies, bispecific antibodies, peptide mimetics of antibodies substituted with peptide mimetics, modified antibodies, artificial antibodies , And conjugate antibodies with these antibodies can also be used, and these antibodies can be produced using known methods.

  The antibody used in the present invention may be, for example, a conjugated antibody bound to various molecules such as non-peptidic polymers such as polyethylene glycol (PEG), radioactive substances, and toxins. Such a conjugated antibody can be obtained by chemically modifying the obtained antibody. Chemical modification methods have already been established in this field. The antibodies in the present invention also include these conjugated antibodies (DJKing., Applications and Engineering of Monoclonal antibodies., 1998 TJ International Ltd, Monoclonal Antibody-Based Therapy of Cancer., 1998 Marcel Dekker Inc; Chari et al , Cancer Res., 1992 Vol152: 127; Liu et al., Proc Natl Acad Sci USA., 1996 Vol 93: 8681).

In the present invention, apart from the whole antibody as described above, it may be an antibody fragment or a modified product thereof as long as it has an ORAI3 epitope binding property and exhibits Ca ²⁺ activity. For example, antibody fragments include Fab fragments, Fv fragments, F (ab ′) ₂ fragments, Fab ′ fragments, or single chain Fv (scFv) in which Hv and L chain Fvs are linked by an appropriate linker. It is done.

  As the production system for antibody production, either an in vitro or in vivo production system can be used. Examples of in vitro production systems include production systems that use eukaryotic cells such as animal cells, plant cells, or fungal cells, and production systems that use prokaryotic cells such as bacterial cells such as Escherichia coli and Bacillus subtilis. As animal cells to be used, mammalian cells such as CHO, COS, myeloma, BHK, HeLa, Vero, and commonly used cells, insect cells, plant cells and the like may be used. Examples of in vivo production systems include production systems using animals and production systems using plants. When animals are used, for example, there are production systems using mammals and insects. Examples of mammals that can be used include goats, pigs, sheep, mice, and cows (Vicki Glaser, SPECTRUM Biotechnology Applications, 1993). Moreover, as an insect, a silkworm can be used, for example. When using a plant, for example, tobacco can be used.

  The resulting antibody can be purified to homogeneity. For separation and purification of antibodies, separation and purification methods used for ordinary proteins may be used. For example, antibodies can be separated and purified by appropriately selecting and combining chromatography columns such as affinity chromatography, filters, ultrafiltration, salting out, dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, Cold Spring Harbor Laboratory, 1988), but is not limited thereto. Examples of columns used for affinity chromatography include a protein A column and a protein G column. Examples of the carrier resin to which protein A is bound include Hyper D (Pall Corporation), POROS (Life Technologies), Sepharose FF (GE Healthcare) and the like.

  The skin cell activator, skin barrier function enhancer or skin wound healing promoter of the present invention can be blended in pharmaceuticals, quasi drugs, or cosmetics. The cosmetics to be blended may be any products as long as they can be applied to the skin, and examples thereof include lotions, cosmetic liquids, emulsions, creams, packs and the like. On the other hand, when blended in a pharmaceutical product or quasi-drug, the skin cell activator, skin barrier function enhancer or skin wound healing promoter may be prepared in any dosage form. It is preferably prepared as a skin external preparation because it acts on cells present in the skin. Such a pharmaceutical composition may further contain known adjuvants such as excipients, pH adjusters, bases, and preservatives. When blended in a pharmaceutical composition or a cosmetic composition, the dose of the active ingredient to be blended can be appropriately determined through experiments.

  In yet another aspect, the present invention relates to a cosmetic method comprising administering an ORAI3 protein or Orai3 gene function inhibitor. In the skin, an ORAI3 protein or Orai3 gene function inhibitor can be administered transdermally by applying it directly to the skin.

The scratch assay performed in the present invention can examine the wound healing ability of skin cells (Non-patent Document 7). Specifically, the scratch assay is a method for measuring the rate at which scratches are formed on cultured skin cells with a needle or pipette, and the scratches are repaired. Although not intended to be bound by theory, when cells in a cultured state are scratched, cells existing at the boundary of the scratch cause Ca ²⁺ influx by the stimulation of the scratch and repair the scratch. It is thought that signal transduction occurs. In aging cells, it is considered that it takes time to repair scratches as a result of the failure of such signals to be transmitted well due to the action of ORAI3.

In another aspect of the present invention, the present invention relates to a cell activator screening method using Orai3 gene expression as an index. Since Orai3 gene expression is an index of skin aging, substances that reduce Orai3 gene expression in cultured skin cells, especially epidermal cultured cells, are selected as drugs that have a cell activation effect that can suppress or reverse skin aging can do. Therefore, such a cell activator screening method includes, for example, the following steps:
Adding a candidate drug to the cultured cells;
A step of directly or indirectly measuring Orai3 gene expression in cultured cells after addition of the candidate drug, and a step of comparing with gene expression in cultured cells before addition of the candidate drug. The step of culturing for a predetermined period may be included after the step of adding the candidate drug. The drug thus selected as a cell activator is also a skin barrier function enhancer or a wound healing promoter, and can also be referred to as a skin cell aging inhibitor or retrograde agent or rejuvenation agent. Gene expression in cultured cells before addition of a candidate drug may be measured by a step of measuring from a cultured cell sample obtained before the candidate drug addition step, or gene expression is measured in advance in a cultured cell system to be used. It may be left. Direct measurement of gene expression may be performed by measuring the transcribed mRNA by Northern blot or real-time PCR technique, or by translated protein by Western blot or fluorescent immunochemical technique. It may be done by measuring the amount. On the other hand, since the Orai3 gene functions to sustain the Ca ²⁺ response of cells generated by stimulation, the gene expression is indirectly measured by using an index representing the persistence of the Ca ²⁺ response. Is possible. As an index representing the persistence of the Ca ²⁺ response, the time (half-time) from when the intracellular Ca ²⁺ concentration after the Ca ²⁺ response exceeds the intermediate value between the peak value and the steady value to the intermediate value is returned. Value range), or the value of intracellular Ca ²⁺ concentration after a predetermined period of time can be used. Therefore, as a screening method using indirect measurement of gene expression,
Adding a candidate drug to the cultured cells;
In cell culture after a candidate drug addition, a step of measuring an intracellular Ca ²⁺ concentration before and after application of the stimulus, and the intermediate value after exceeding the intermediate value of the intracellular Ca ²⁺ concentration peak and steady-state value There is a method including a step of comparing the time until returning (half width) or the value of intracellular Ca ²⁺ concentration after elapse of a predetermined time with the value in cultured cells before addition of a candidate drug.
Candidate drugs used in these screenings may be substances such as any cosmetic material library or pharmaceutical compound library. The stimulus applied to the cell may be any stimulus as long as it can elicit a Ca ²⁺ response, and may be a mechanical stimulus, a pressure stimulus such as a hydraulic stimulus, a drug stimulus, or an electrical stimulus.

  The skin cell activator of the present invention can provide a skin cell rejuvenation effect. Therefore, it can be directly applied to the skin, and in in vitro and ex vivo experiments, cell proliferation can be enhanced by adding at the time of culturing skin cultured cells. In yet another aspect, the skin cell activator of the present invention can be used when culturing cells obtained from a skin sample obtained from adult skin to produce a cultured skin sheet for autologous transplantation. .

Example 1: Cell Culture Cells were cultured in Epilife-KG2 medium (Kurabo) using epidermis-derived keratinocytes prepared from the skin of healthy donors (14 women) of various ages. This was seeded in a 35 mm diameter dish with a glass bottom coated with collagen and used for a calcium imaging experiment.

Example 2: Hydraulic load and calcium imaging After culturing human epidermis-derived keratinocytes in a medium containing 5 μM fura-2AM (calcium indicator) at 37 ° C. for 45 minutes, the cells were washed with a buffer solution and set on an inverted fluorescence microscope. A buffer solution is placed in a glass capillary with a thin tip, connected to a microinjector (Femtojet, manufactured by Eppendorf), and the tip of the capillary (outer diameter of 30 microns) is obliquely above the single cell at an angle of about 45 degrees. The water pressure was applied to one cell by setting the water flow to be ejected and blowing 100 hectopascal pressure for 1 second. In the cells before and after application of water pressure, Fura2 fluorescence images (F340, F380) were taken with an ORCA-R2 CCD camera (Hamamatsu Photonics) every 2 seconds. A representative example of the fluorescence image is shown in FIG. 1A. Using image analysis software (Aqua Cosmos, manufactured by Hamamatsu Photonics), the Fura2 fluorescence intensity ratio (F340 / F380) of the cytoplasmic part of one cell subjected to water pressure was calculated. C: Changes in Ca ²⁺ concentration after water pressure loading in cells obtained from the age of 63 are shown in graphs (FIGS. 1B and C). From the graph of the Ca ²⁺ response induced by water pressure stimulation, the time from when the intracellular Ca ²⁺ concentration exceeds the intermediate value between the peak value and the steady value until it returns to the intermediate value (half-value width), and 2 minutes The value of intracellular Ca ²⁺ concentration after the lapse was calculated. FIG. 1D is a graph showing the correlation between the half-width and donor age (N = 11, P <0.05, Pearson's product moment correlation coefficient (R) = 0.854), and the half-width increases with aging. It was shown that. Thus, it was shown that the return after Ca ²⁺ inflow is delayed in the Ca ²⁺ response after stimulation with aging.

Example 3: RT-PCR
Total RNA was extracted from the cells using EZ1 RNA cell mini kit (manufactured by Qiagen), and cDNA was synthesized from the RNA using reverse transcriptase. mRNA was quantified by real-time quantitative PCR using a LightCycler 480 and a universal probe (Roche Diagnostics). The primers and universal probes listed in the table below were used.

The age of the donors of the cells used and the Orai3 mRNA levels were plotted on a graph (FIG. 2A). Furthermore, the half-width after stimulation with water pressure in each cell measured in Example 2 and the mRNA level of Orai3 were plotted in a graph (FIG. 2B). It was shown that Orai3 expression increases with age (N = 14, P <0.05). Furthermore, after the Ca ²⁺ response induced by water pressure stimulation, the time (half width) from when the intracellular Ca ²⁺ concentration exceeds the intermediate value between the peak value and the steady value to return to the intermediate value is the expression of the Orai3 gene. (N = 14, P <0.05).

Example 4: Confirmation of cell activation effect by knockdown of Oari3 gene by siRNA
For siRNA knockdown experiments, siRNA is mixed with transfection reagent (Lipofectamine RNAiMAX, manufactured by Life Technologies) in the growth phase (50-80% confluent) cells and added to the medium. The cells were cultured for 12 to 24 hours at a final concentration of 5 nM. The sequence of Orai3-siRNA used is shown in Table 1 below. The siRNAs of SEQ ID NOs: 4-7 are from Dhermacon, and the siRNAs of SEQ ID NOs: 8-10 are from Ambion (now Life Technologies). For siRNA control experiments, non-specific sequence siRNA (negative control siRNA, manufactured by Dhermacon (catalog number: D-001810-10-05) or Ambion (catalog number 4390843)) was used.

  Orai3 siRNA was introduced, RNA was extracted 24 hours later, and the amount of Orai3 mRNA was quantified by the RT-PCR method of Example 3. Orai3-siRNA reduced the amount of Orai3 mRNA to about 10% compared to negative control-siRNA (FIG. 3A).

Immunoprecipitation and Western Blot Two days after the introduction of siRNA, proteins were extracted from the cells by the following method, immunoprecipitated with Orai3 antibody, and then Western blotted. Two days after siRNA introduction, cells were extracted in an extraction solution (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.3 mM PMSF, 1 μg / ml pepstatin A, 10 μg / ml leupeptin). After crushing, the supernatant was collected and the protein was quantified, and 300 μg of total protein was used for one reaction. This was reacted with Orai3 antibody (SAB3500121, Sigma-Aldrich) and protein G beads, and the immunoprecipitated protein (protein bound to the beads) was separated by SDS-PAGE (10% gel) and transferred to a PVDF membrane. This was reacted with another Orai3 antibody (HPA015502, manufactured by Sigma-Aldrich), and the band was detected with an X-ray film by a luminescence method. This detected a 31 kd Orai3 band. Orai3-siRNA showed that the amount of Orai3 protein was reduced to about 50% compared to the negative control (FIG. 3B).

Water Load and Calcium Imaging In cells in which the above Orai3 siRNA or control siRNA was introduced into keratinocytes obtained from an elderly person (63 years old), the water pressure application and calcium imaging of Example 2 were performed, and Ca induced by water pressure stimulation. A graph of ²⁺ response was acquired (FIG. 4A). The time (half value width) from when the intracellular Ca ²⁺ concentration exceeded the intermediate value between the peak value and the steady value to the return to the intermediate value was shown as an average of 6 times (FIG. 4B). Gene knockdown Orai3, return of Ca ²⁺ response faster, similar to the graph of Ca ²⁺ responses in cells obtained from young subjects (Fig. 1B). In addition, FIG. 1D shows that the half-value width shown to correlate with aging also decreases. Thereby, the effect of cell rejuvenation was observed by suppressing the function of Orai3.

Example 5: Localization of Orai3 in skin and confirmation of increase in Orai3 by differentiation induction
Immunohistochemical staining Frozen sections of healthy human skin were prepared, fixed with 100% methanol, reacted with Orai3 antibody (ACC-065, manufactured by Alomone Labs), further reacted with Alexa594-secondary antibody, and then fluorescence microscope And I took a picture with a CCD camera (Fig. 5). FIG. 5A is a negative control using Orai3 antibody and FIG. 5B is a negative control with no added Orai3 antibody. It has been shown that Orai3 is localized in the epidermis and its expression increases especially as differentiation progresses.

Differentiation induction of cultured cells When the density of cultured keratinocytes became 100% confluent, differentiation was induced by setting the calcium concentration in the medium to 1.8 mM, and the cells were cultured for 40 to 48 hours. Next, the cells were seeded in a 6-well plate, and the cells were collected in the cell growth phase or differentiation phase (2 days after differentiation induction), and the expression level of Orai3 was examined in the same manner as in Example 3 (FIG. 6). It was shown that Orai3 expression increases with epidermal cell differentiation.

Example 6: Scratch assay Cultured keratinocytes were seeded in 6-well plates and transfected with Orai3 siRNA or control siRNA according to Example 4 to knockdown the Orai3 gene. The knocked-down cells were further cultured, and when they became 100% confluent, the center of each well was pulled linearly with a pipette tip (P1000), and the cells were peeled off to create one scratch (scratch). The culture was continued with the medium changed, and a phase contrast image of the cells in the scratch portion was taken every few hours. The photographs immediately after scratching and after 7 hours are shown in FIG. 7A. The area in which the cells migrated and proliferated to the scratched portion from this image to repair the wound was quantified by ImageJ image analysis software, and is shown in FIG. 7B. By suppressing the function of Orai3, cell proliferation was promoted in the scratch assay. This indicates that the inhibition of the function of Orai3 has a wound healing effect or a cell activation effect.

Claims (6)

A skin cell activator comprising an ORAI3 protein or Orai3 gene function inhibitor in the skin , wherein the ORAI3 protein or Orai3 gene function inhibitor specifically binds to the ORAI3 protein, and Orai3 A skin cell activator that is at least one selected from the group consisting of RNAi-inducible nucleic acids, antisense nucleic acids, ribozymes, and their expression vectors that specifically inhibit expression . The skin cell activator according to claim 1 , wherein the skin cell activator is a skin barrier function enhancer or a wound healing promoter. Adding a candidate drug to cultured skin cells;
A method for screening a skin cell activator, comprising directly or indirectly measuring Orai3 gene expression in cultured skin cells after addition of a candidate drug, and comparing with gene expression in cultured skin cells before addition of the candidate drug . The screening method for a skin cell activator according to claim 3 , wherein the step of directly measuring the gene expression of Orai3 is a method of measuring the amount of Orai3 mRNA or the amount of ORAI3 protein. The step of indirectly measuring the gene expression of Orai3 includes the following:
A step of measuring intracellular Ca ²⁺ concentration before and after application of stimulation to cultured skin cells, and a time from when the intracellular Ca ²⁺ concentration exceeds an intermediate value between a peak value and a steady value to return to the intermediate value, or The method for screening a skin cell activator according to claim 3 , comprising a step of associating the value of intracellular Ca ²⁺ concentration after a predetermined time with the expression of Orai3 gene. The screening method for a skin cell activator according to any one of claims 3 to 5 , wherein the skin cell activator is a skin barrier function enhancer or a wound healing promoter.