JP2007020559A - Hair quality rating method with runx1(runt-related transcription factor 1) as indicator - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a hair quality rating method at biochemical/molecular biological levels. <P>SOLUTION: The hair quality rating method comprises assaying mRNA derived from RUNX1 gene in the hair by real-time polymerase chain reaction technique, wherein the mRNA uses as indicator for the hair. This method can also used as an immunoassay of the RUNX1 levels in the hair using an antibody specific to RUNX1. These methods can be conducted using assaying kits respectively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a hair quality evaluation method using RUNX1 (runt-related transcription factor 1) as an index.

  Problems related to hair include those related to the health condition of hair roots and hair follicles such as thin hairs and hair loss, and those related to physical properties of hair such as hard, soft, non-abrasive / stubborn, split ends, prickly hairs, and pasasuku. In addition, even if it is a problem related to the physical properties of hair, it is caused by direct damage to hair, such as damage caused by external factors, such as permanent or bleach treatment, exposure to ultraviolet light, exposure to air pollutants, combing friction, etc. And the case where an internal element in hair shaft formation is involved. The hair care in the latter case requires not only improving the quality of the hair but also taking care to improve the health of the hair root and the hair follicle itself. Therefore, even if it is said that the state of the hair is not good, the care method varies depending on the cause.

  Conventional hair quality evaluation is based on subjective methods that rely on the tactile sensation of salespersons such as hairdressers and shop fronts, or various physical property measuring instruments such as moisture meters, hardness meters, and Young's modulus measuring devices. Use objective methods such as physical measurement. Any of these methods can be used to provide appropriate advice. However, these evaluation methods cannot elucidate the root cause of the quality of hair and derive appropriate hair care advice that is more suitable for the hair.

  The hair itself is made up of a cuticle covering the surface, fur inside it and occupying most of the hair, and medulla in the center. It has been clarified that cutil contributes greatly to the firmness and firmness of hair (Akira Sogabe et al., J. Soc. Cosmet. Chem. Japan, Vol. 36, No. 3 (2002)). pp. 207-216 “Study on physical properties of hair 1—Measurement of short and long diameters of hair and evaluation method of bending stress”). Cuticles are composed of various components such as fiber proteins such as acidic hair keratins Ha1 and Ha2, basic keratins Hb1 and Hb2, and modified enzymes such as transglutaminase, peptidylarginine diminase, and granule component tricohiarin S100. Is known. However, the relationship between each of these components and the hair quality, for example, the hair lump and the hair, has not been fully elucidated. If the mechanism that influences the hair quality is fully elucidated at the biochemical / molecular biology level, it may be possible to provide a product that has a more remarkable effect than existing hair quality improving agents.

  JP 2002-97116 discloses cell activators containing taurine as an active ingredient, and International Publication WO 2002/034253 describes cell activators containing N-methyl taurine as an active ingredient and hair scalp / strain improving agents. Has been. The effects of these agents are described as hair cell control, hair growth period extension, hair cell proliferation activation, hair patch / strain improvement by activation of cell proliferation. The improvement effect is still evaluated by a physical property test (torsion torque) of hair. Therefore, there is a need for a novel hair patch / strain improving agent based on activity evaluation at the biochemical / molecular biology level.

JP-A-8-20523 JP2001-302543 JP2002-97116 WO 2002/034253 J. Invest. Dermatol, 122: 147-158, 2004

  An object of the present invention is to provide a hair quality evaluation method at the biochemical / molecular biology level.

  In view of the above problems, the present inventor has previously investigated the correlation between hardness, which is a measure of hair strain / beam, specifically Young's modulus, and the expression of various hair-related genes. The hair keratin gene, particularly the KAP5 gene Specifically, it is found that the higher the expression of the KAP5.1-5 gene, the higher the Young's modulus of the hair, that is, that the hair is applied to the hair, and that the hair feels stiff. A patent application has been filed regarding a method of using expression of 1-5 genes as an index of hair quality (Japanese Patent Application No. 2004-348325).

  More recently, bioinformatics analysis of the promoter region of the KAP10 / KAP12 gene belonging to the KAP family has shown that several enhancer elements are present in that region. It has been found that the binding element of the transcription factor RUNX1 (also known as “AML1” (acute myeloid leukemia gene)) is included (Michael A. Rogers, et al., J. Invest. Dermatol. 122: 147-158, 2004). In view of this fact, the present inventor examined the relationship between RUNX1 and the KAP5 gene that affects hair strain and hair, and confirmed that the expression of the KAP5 gene is enhanced depending on the expression level of RUNX1. It was done.

  As a result, in the first aspect, the present invention provides a method for evaluating hair, which uses the expression of the RUNX1 gene in hair as an index of hair.

  In a preferred embodiment, the measurement is performed by measuring mRNA derived from the RUNX1 gene in hair. More preferably, the measurement of such genes is performed by the real time polymerase chain method.

  In another embodiment, the assessment is performed by measuring the amount of RUNX1 in the hair. In a more preferred embodiment, the measurement is performed by an immunological measurement method using an antibody specific for RUNX1, such as ELISA or RIA.

  In another aspect, the present invention provides a kit or apparatus for performing hair quality assessment. Such a kit or device is characterized in that the evaluation is performed by measuring the expression of the RUNX1 gene in the hair as an index of hair.

  Furthermore, since the Young's modulus of the hair increases significantly as the expression level of the KAP5 gene increases, a drug that enhances the expression of RUNX1, which can enhance the expression of the KAP5 gene, improves the hardness of the hair.・ It is naturally expected to improve this. Therefore, it is considered that RUNX1 or a gene encoding the same can be used as an index for a screening method for a drug that improves hair hardness and improves hair sticking / straining.

  According to the present invention, it is possible to provide a hair quality evaluation method at the gene level.

Hair quality evaluation method The present invention aims to evaluate the hardness of hair, generally a physical property called “beam” or “koshi” feeling. As described above, the present inventor measured the Young's modulus of hair in order to examine the correlation between the hardness of the hair and various genes extracted from the hair, and the measurement results and the expression levels measured by RT-PCR of the various genes Was analyzed statistically using Spearman's rank correlation coefficient (Zar, JHJAmer.Stat.Assoc.67: 578-580, 1970 "Significance testing of the Spearman rank correlation coefficient") It was found that the Young's modulus is statistically significantly increased when the expression level of the KAP5 gene is large (Japanese Patent Application No. 2004-348325), and this time, it was revealed that the expression of the KAP5 gene is enhanced by RUNX1. .

  The RUNX family is a transcription factor that has recently attracted attention in the fields of development, differentiation and canceration. The RUNX protein has a DNA binding domain called a RUNT domain and a regulatory region on the N-terminal side, and has a transcription control region and each matrix binding site on the C-terminal side. The DNA binding activity by the RUNT domain is increased by binding of the DNA non-binding transcriptional regulatory factor CBFβ to the RUNT domain. RUNX1 is the most frequent target of chromosomal translocation in human leukemia, and is known to be involved in hematopoietic cell-specific gene expression and to control the differentiation / proliferation of hematopoietic cells. The amino acid sequence of the RUNT domain in RUNX1 is considered to have 90% or more homology with other RUNX family proteins, RUNX2 and 3, and almost the same three-dimensional structure.

  The KAP5 gene (Homo sapiens genomic DNA, chromosome 11 clone: RP11-684B2, complete sequences. ACCESSION: AP000867) belonging to the human keratin-related protein (KAP) 5 family is a human EST whose base sequence is already known. Not. The relationship between the enhanced expression of the KAP5 gene and hair hardness was unknown. Although not particularly bound by any theory, for example, the putative protein encoded by the KAP5.2 gene is cysteine-rich (about 35.5%) and is rich in the low molecular amino acids glycine and serine (18.8 each). % And 24.2%) because it consists of 186 amino acids, it does not form secondary structures such as α-helix, can easily penetrate between keratin fibers, and has many hydrogen bonds by OH groups of many serine residues. May also contribute greatly to the mechanical strength of the hair. Similarly, other KAP5 genes are rich in cysteine, glycine, and serine, and the expression product has a structure similar to that of KAP5.2 and is considered to contribute greatly to the mechanical strength of hair.

  The hair according to the present invention mainly refers to the hardness of the hair, that is, a “beam” or “strain” feeling. The hardness of hair can be quantified by measuring its “bending hardness” or “bending stress” using, for example, a bending stress measuring device such as a torque sensing type bending stress measuring device KES-SH (Kato Tech). Preferably, in order to minimize the factors depending on the hair diameter and shape, the hair diameter is also measured and expressed in Young's modulus. The Young's modulus of hair can be determined, for example, as described in the following examples.

  Expression of the RUNX1 gene in hair may be determined by measuring the amount of the protein in the hair. Preferably, this measurement uses an antibody specific for RUNX1, and is a well-known method in the art, for example, an immunostaining method using a fluorescent substance, a dye, an enzyme, etc., a Western blot method, an immunoassay method such as an ELISA method, Various methods such as RIA can be used. For example, it can also be determined by extracting total RNA in hair and measuring the amount of mRNA encoding RUNX1. Extraction of mRNA and measurement of the amount thereof are also well known in the art. For example, RNA is quantified by quantitative polymerase chain reaction (PCR), for example, real-time polymerase chain reaction (RT-PCR). Selection of suitable primers for RT-PCR can be performed by methods well known to those skilled in the art.

  According to the hair quality evaluation method according to the present invention, for example, depending on whether the expression level of the RUNX1 gene measured as described above is higher or lower than a predetermined reference value, the hair is hard or soft at the gene level. It is possible to determine whether there is a feeling of “beam” or “strain”. The reference value may be, for example, an average value of the expression level of each gene of hair derived from a statistically significant number of people, for example, 5 or more, preferably 10 or more, more preferably 20 or more. The reference value can vary depending on the sex, age, race, and other factors of the individual being evaluated. For example, when examining the hair quality of a 20-year-old Japanese woman, the reference value is preferably an average value of a desired gene expression level of a group of Japanese women of the same generation (for example, 15 to 30 years old). Whether the expression level of each gene is higher or lower than the standard value is, for example, significantly higher if it is at least 10%, or 20%, or 30%, or 50%, or 100% higher than the standard value. It can be significantly lower if it is at least 10%, or 20%, or 30%, 50%, or 90% lower than the standard value.

  In addition to the gene (RUNX1 isoform B; NM_001001890; SEQ ID NO: 1) and its transcriptional variant (RUNX1 isoform A; NM_001754; SEQ ID NO: 2), the RUNX1 referred to in the present invention includes, for example, RUNX gene 1 In which one or several nucleotides are substituted, added, or deleted, and the enhanced expression thereof is correlated with the Young's modulus of hair, or RUNX gene, 60% or more, preferably 70% or more, more preferably 90% or more, still more preferably 95% or more, most preferably 99% or more of homologous genes whose increased expression correlates with the Young's modulus of hair, or the above genes under high stringency conditions And mutant genes whose enhanced expression correlates with Young's modulus of hair. The hybridization here can be performed according to a well-known method or a method according thereto, for example, the method described in J. Sambrook et al., Molecular Cloning 2nd, Cold Spring Harbor Lab. Press, 1989, and highly stringent hybridization. The conditions refer to conditions including, for example, a sodium concentration of about 10 to 40 mM, preferably about 20 mM, and a temperature of about 50 to 70 ° C., preferably about 60 to 65 ° C.

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

Method for measuring KAP5.2 gene expression level Method for preparing cDNA from extracted hair specimen About 1 cm of the hair root was extracted in liquid nitrogen and left in liquid nitrogen as it was. Five frozen hair roots were collected, put into a 1.5 ml tube containing 1 ml of RNA extract ISOGEN (Nippon Gene), and sufficiently stirred with a vortex mixer. After adding 200 μl of chloroform and sufficiently stirring again with a vortex mixer, centrifugation (15000 rm, 15 minutes) was performed with a small microcentrifuge, and about 500 μl of an aqueous phase containing RNA was recovered. 50 μl of 3M sodium acetate and 1 μl of etaprecipitate (Nippon Gene) were added to the collected solution and stirred thoroughly. Further, 1 ml of isopropanol was added and stirred, followed by centrifugation (15000 rm, 20 minutes) with a small microcentrifuge to precipitate total RNA. After discarding the supernatant, 75% ethanol was added, and the mixture was again centrifuged (15000 rm, 10 minutes) with a small microcentrifuge. The supernatant was discarded and the precipitate was air-dried and dissolved in 50 μl of nuclease free water. The contaminating genomic DNA was removed by DNase I treatment, and total RNA was recovered again by ethanol precipitation and dissolved in 20 μl of nuclease free water. Of these, 1.0 μl was used to measure the RNA concentration by NanoDrop (Technologies, Inc.). CDNA was synthesized from 100 ng of the obtained total RNA and used for quantification of the expression level of each gene by PCR.

Quantification of gene expression by real-time PCR method Synthetic cDNA, primers specific to the gene to be detected and quantified, and real-time PCR reagent (Roche) are mixed, and real-time PCR is performed at 350S using the LightCycler Quick System. -Amplified the fragment of the gene to be quantified. At this time, the same gene fragment of known concentration (corresponding to 10 ² to 10 ¹¹ copies of PCR product) is similarly amplified by real-time PCR to create a calibration curve, and the expression level of the gene to be detected and quantified contained in the sample is determined. Calculated as copy number per unit (1 μg) RNA.

The following combinations of primers were used as primers specific to the KAP5.2 gene and not amplifying genomic DNA.
Sense ACTGTAGCTGTGTCCTGA (SEQ ID NO: 5)
Antisense GATGAAGATGAAGGGTGGA (SEQ ID NO: 6)

Measurement of hair hardness (Young's modulus) Method of preparing sample for measurement For each hair, attach water-resistant paper tags to both ends (interval 3 cm) and enter the number. After lightly washing with a shampoo, rinsing with water and immersing in ion exchange water, it was air-dried in a constant temperature and humidity chamber (25 ° C., humidity 50%).

Measurement of hair diameter In a constant temperature / humidity chamber (25 ° C, humidity 50%), the hair diameter measuring device SK-2000 (Kato Tech / Shiseido) using laser light is used to separate the major and minor diameters of hair samples at intervals of 2 mm. Was measured over a length of 20 mm, and these average values were taken as the major and minor diameters of the hair sample.

Bending stress measurement The bending stress of the hair sample was measured in a constant temperature / humidity chamber (25 ° C., humidity 50%) using a torque sensing type bending stress measuring device KES-SH (Kato Tech). The bending speed was set to 0.5 cm ⁻¹ / sec., And the bending stress (M) of each hair sample at the curvature (ρ) ± 1.0 to 2.0 cm ⁻¹ was measured.

Calculation of Young's Modulus Measure the hair diameter and bending stress for each hair sample, and calculate the Young's modulus (E) for each hair sample using the following formula using the moment of inertia (I). Calculated.
E = M ・ ρ / I
At this time, assuming that the hair cross section approximates an ellipse and further bends on the short diameter side when the hair bends, the cross-sectional secondary moment (I) is expressed by the following equation.
I = π / 4 · a ³ · b (a is the short radius of the hair, b is the long radius of the hair)

  For comparison with gene expression, Young's modulus was calculated for each of the five hair samples from which RNA was extracted together, and the average value was used.

The relationship between the expression level of the KAP5.2 gene and the Young's modulus of the hair is shown in FIG. Spearman rank correlation coefficient r _s between KAP5.2 gene expression level and hair Young's modulus was 0.487 (minimum r _s = 0.447 (Spearman's test table with P <0.05) ).

  From the above experiments, in a human test by 20 panelists, a significant correlation was observed between hair hardness (Young's modulus) and human KAP5.2 gene expression.

Examination of the relationship between Young's modulus of hair and expression of KAP5.1 and 5.4 genes
The hardness (Young's modulus) of the hair of the 11 panelists was measured as described above, and Young's modulus of 11.4 or higher was “high Young's modulus hair”, in other words, “hairy / stiff” hair, 10.2. The following are grouped into hair with low Young's modulus, in other words, hair without “brush / feel”, and the expression levels of KAP5.1 and KAP5.4 genes for each group were determined by real-time PCR as described above. Examined.
The primers used are as follows:
Human KAP5.1
Sense tctcttccca agtcaactgc (SEQ ID NO: 3)
Antisense agagtgttgg acaggcaaag (SEQ ID NO: 4)
Human KAP5.4
Sense ttctccagct catcatccat (SEQ ID NO: 9)
Antisense ggtcagacct tgcatctcag (SEQ ID NO: 10)

  The result is shown in FIG. FIG. 2A shows the average value and standard deviation (p <0.05) of “high Young's modulus hair” (High) and “low Young's modulus hair” (Low). This figure shows that there is a significant difference in Young's modulus between the “high Young's modulus hair” group and the “low Young's modulus hair” group by the above grouping. FIG. 2 (b) shows the difference in expression level between the “high Young's modulus hair” (High) and the “low Young's modulus hair” (Low) for the KAP5.1 gene. It is clear that the expression of KAP5.1 gene is increased in high Young's modulus hair compared to low Young's modulus hair. FIG. 2 (c) shows the difference in the expression level between “high Young's modulus hair” (High) and “low Young's modulus hair” (Low) for the KAP5.4 gene. It is clear that the expression of the KAP5.4 gene is increased in the hair with a high Young's modulus compared to the hair with a low Young's modulus, and from the comparison with FIG. 2 (b), the KAP5. The difference in the expression levels of the 4 genes was also significant compared to that of the KAP5.1 gene. From the above results, it has been clarified that the expression of KAP5.1 and KAP5.4 genes, particularly KAP5.4 gene, is significantly increased in hair with a high Young's modulus, that is, hair with abundant feel. Therefore, it was found that the measurement of the expression of KAP5.1 and KAP5.4 genes is also significant as an index of hair quality evaluation.

  As described above, the KAP5.1 to 5.5 genes are a group of genes that belong to the human keratin-related protein KAP5 family and have relatively high homology to each other. The high degree of homology between the amino acid sequences of the putative proteins encoded by each gene is apparent from FIGS. 3 and 4 and the following table.

  In addition, as described above, each putative protein encoded by the KAP5.1-5 gene is all cysteine-rich and rich in low-molecular amino acids glycine and serine. Since it does not form the next structure, it can easily penetrate between keratin fibers, and many hydrogen bonds can be formed by the OH groups of many serine residues, which greatly contributes to the mechanical strength of hair. it is conceivable that. The following table shows a partial amino acid composition of the KAP5.1-5 gene estimation coding protein.

Therefore, based on the high degree of homology between genes in the KAP5 gene family and the structural similarity between the coding proteins, the above results obtained for KAP5.1, 5.2 and 5.4 suggest that KAP5.3 and 5. It can be inferred that the hair quality can also be evaluated by examining the expression of 5.

Identification of human KAP5 gene expression site by in situ hybridization method (ISH method) Preparation of KAP5.1-5 Riboprobes cDNA prepared from extracted hair, KAP5.1, KAP5.2, KAP5.3, KAP5.4, For each of KAP5.5, each KAP5 gene fragment was amplified using the primers shown in the following table, with the T7 polymerase promoter sequence added to the antisense side.

Each amplified gene fragment was purified with a commercially available kit (Wizard SV Gel and PCR clean-up System: Promega), and then each KAP5 gene-specific riboprobe was prepared with T7 polymerase and DIG RNA labeling kit (Roche). .

Identification of the expression site of each KAP gene Neutral formalin-fixed and paraffin-embedded sample of scalp tissue is sliced into 5 μm with a microtome to produce tissue sections (slide glass) containing hair follicles, and Ventana's automatic staining device (Ventana HX system discovery) and reagent ribomap kit for in situ hybridization (Ventana) were reacted with each KAP5 gene-specific riboprobe (200 ng per slide). Furthermore, the tissue section reacted with the probe was reacted with an alkaline phosphatase-labeled anti-DIG antibody (Roche) on an automatic staining apparatus. Remove the slide glass with the tissue section from the automatic staining device, wash with Tris-HCl buffer (pH 9.0), and react with BM-purple (Roche) for about 5 hours. The expression site of each KAP5 gene is blue-purple. It was visualized as a stained site. As a result, it was confirmed that all of the KAP5.1, KAP5.2, KAP5.3, KAP5.4, and KAP5.5 genes were specifically present in the cuticle site.

Identification of RUNX1 expression site in human growth hair follicle by immunostaining method After fixing scalp tissue with neutral formalin for 1 week, it was dehydrated with ethanol series, embedded in paraffin, and cut 6 μm thick. The above sections were deparaffinized and hydrophilically treated with xylene / ethanol series, and heated and boiled using a microwave oven in a 10 mM citrate-sodium hydroxide (pH 6.0) solution to activate the RUNX1 antigen. It was. Anti-RUNX1 antibody (Abcam) as a primary antibody, 3-amino-9-ethylcarbazole (AEC) that stains immunohistochemically by the streptavidin-biotin-peroxidase complex method and exhibits red deposition as a chromogenic substrate ) Was used. As a result, RUNX1 immunostaining was observed in the keratinized band of the hair shaft portion of the human growth hair follicle and the nucleus of the cell of the outer root sheath. In the cuticle layer, the immunostaining property of RUNX1 was confirmed on the hair bulb side from the site where the gene expression of KAP5 family, KAP5.1-5.5, which is related to the beam / koshi, was observed. These KAP5 genes This suggests the possibility of regulating the transcription of.

Measurement of RUNX1 transcriptional regulatory activity using KAP5.1 reporter assay Immortalized outer root sheath cells (IORS) (Japanese Patent Laid-Open No. 2000-89) are placed in a 24-well multi-well plate so that the number of cells becomes 100,000 to 160,000 per well. Seeded and cultured overnight at 37 ° C. and 5% CO ₂ . The medium used was keratinocyte-SFM medium (Gibco).
Day 1: Plasmid DNA introduction (transient). According to the manual of FuGENE6 Transfection Reagent (ROCHE), the cultured IORS cells were mixed with a reporter plasmid pGL3-KAP5.1 (firefly luciferase) (Japanese Patent Application No. 2003-416987) and a commercially available internal standard plasmid pRL-TK (renilla luciferase). A commercially available mouse RUNX1 gene (GenBank #: BC069929) (SEQ ID NO: 12) expression plasmid or negative control β-galactosidase gene expression plasmid (pCMVSport β-gal, Invitrogen) was added for transformation.
Day 2: Assay. The luciferase activity in each well was measured using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's manual. The luminometer used was AutoLumat LB953 (BERTHOLD).
The result is shown in FIG. When a negative control β-galactosidase gene expression plasmid was co-introduced, no change in KAP5.1 transcriptional activity was observed regardless of the amount introduced. On the other hand, when the mouse RUNX1 gene expression plasmid was co-introduced, an increase in KAP5.1 transcriptional activity was observed depending on the amount introduced, and the increase was about 2.5 times at 0.1 μg.

Effect of RUNX1 on endogenous KAP5.1 gene expression of immortalized outer root sheath cells (IORS) Immortalized outer root sheath cells (IORS) (Japanese Patent Laid-Open No. 2000-89) in 1 well in a 6-well multiwell plate The cells were seeded at 500,000 to 800,000 cells, and cultured overnight at 37 ° C. and 5% CO ₂ . As the medium, Keratinocyte-SFM medium (Gibco) was used.

Day 1: Plasmid DNA introduction (transient)
According to the manual of FuGENE6 Transfection Reagent (ROCHE), the cultured IORS cells were transformed into a commercially available mouse RUNX1 gene (GenBank #: BC069929) (SEQ ID NO: 12) expression plasmid or negative control β-galactosidase gene expression plasmid (pCMVSport β- gal, Invitrogen) was added for transformation.

Day 2: Quantification by real-time PCR 1 ml of RNA extract ISOGEN (Nippon Gene) was added to the cultured IORS cells, dissolved, and collected in a 1.5 ml microtube. Stir well with a vortex mixer, add 200 μl of chloroform, stir again thoroughly with a vortex mixer, and then centrifuge (15000 rm, 15 minutes) with a small microcentrifuge to recover approximately 500 μl of RNA containing water phase . 50 μl of 3M sodium acetate and 1 μl of etaprecipitate (Nippon Gene) were added to the collected solution and stirred thoroughly. Further, 1 ml of isopropanol was added and stirred, followed by centrifugation (15000 rm, 20 minutes) with a small microcentrifuge to precipitate total RNA. After discarding the supernatant, 75% ethanol was added, and the mixture was again centrifuged (15000 rm, 10 minutes) with a small microcentrifuge. The supernatant was discarded and the total RNA precipitate was air-dried and dissolved in 50 μl of nuclease free water. The contaminating genomic DNA was removed by DNase I treatment, and total RNA was recovered again by ethanol precipitation and dissolved in 20 μl of nuclease free water. Of these, 1.0 μl was used to measure the RNA concentration by NanoDrop (Technologies, Inc.). CDNA was synthesized from 3 μg of the obtained total RNA and used for quantification of the expression level of each gene by real-time PCR.

  The synthesized cDNA, primers specific to the gene to be detected and quantified, and real-time PCR reagent (Roche) are mixed, and real-time PCR is performed at 350S with the LightCycler Quick System to amplify the gene fragment to be detected and quantified. did. At this time, the change in the expression level of KAP5.1 and GAPDH when the RUNX1 expression plasmid was introduced was evaluated with reference to the gene expression level when the β-galactosidase expression plasmid having no transcriptional regulatory activity was introduced (100). .

The primers used are as follows:
Human KAP5.1
Sense tctcttccca agtcaactgc (SEQ ID NO: 3)
Antisense agagtgttgg acaggcaaag (SEQ ID NO: 4)
Human KAP5.4
Sense gagtcaacgg atttggtcgt (SEQ ID NO: 14)
Antisense tgggatttcc attgatgaca (SEQ ID NO: 15)

The result is shown in FIG. FIG. 7 (a) shows confirmation of KAP5.1 gene expression in IORS cells. When the reverse transcription reaction was performed, the DNA fragment of the KAP5.1 gene was amplified (RT +), whereas when the reverse transcription reaction was not performed, amplification was not observed (RT−).
FIG. 7 (b) shows that when the RUNX1 expression plasmid was introduced, an increase in (endogenous) KAP5.1 gene expression in IORS cells was observed depending on the amount introduced, and the increase was about 2.5 times at 1 μg. On the other hand, the expression amount of GADPH gene quantified as an internal standard did not change even when either RUNX1 or β-galactosidase was introduced.

Relationship between expression level of KAP5.2 gene and Young's modulus of hair. Relationship between expression level of KAP5.1 and KAP5.4 genes and Young's modulus of hair. Comparison of amino acid sequences of putative coding proteins of human KAP5.1-5.5 genes. Amino acid sequence homology of the putative coding protein of the human KAP 5.1-5.5 gene. Regulation of expression of KAP5 genes. The involvement of RUNX1 in KAP5.1 transcription regulation by co-introducing a RUNX1 expression plasmid into a KAP5.1 reporter assay system and overexpressing RUNX1 to evaluate the transcriptional activity of the KAP5.1 gene is shown. RUNX1 transcription is introduced into an immortalized outer root sheath cell by introducing an expression plasmid to overexpress RUNX1, and the expression of KAP5.1 gene and GADPH (internal standard control) is evaluated by a real-time PCR method. Involvement in regulation.

Claims (5)

  A method for evaluating hair, wherein the expression of RUNX1 in hair is used as an index of hair.   The method according to claim 1, wherein the measurement is performed by measuring mRNA derived from the RUNX1 gene in hair by a real-time polymerase chain method.   The method of claim 1, wherein the evaluation is performed by measuring the amount of RUNX1 in the hair.   The method according to claim 1, wherein the evaluation is performed by an immunoassay method using an antibody specific for RUNX1.   A kit for evaluating hair quality, wherein the evaluation is performed by measuring the expression of RUNX1 in hair as an index of hair quality.