KR101320262B1 - Method for screening anti-gray hair ingredient using stable cell lines and accelerated vitiligo mouse, and composition containing anti-gray hair ingredient detected thereby - Google Patents

Abstract

The present invention is a transformed cell line (stable cell line) constructed to monitor the expression of microphtalmia-associated factor (MITF), tyrosinase, and tyrosinase-related protein 2 (TRP2), which play a key role in melanin synthesis and melanocyte activity. ) And a method of screening for anti ^- femory substances consisting of vitiligo-modeled mice (C57bl / 6- Mitf ^{mi - vit} ), which promotes hair follicle development, and a composition for preventing anti-hairiness, containing the anti ^- hair follicles. More specifically, the transformed cell line of the present invention is a cell line obtained by stably transfecting plasmids capable of monitoring the expression level of MITF, tyrosinase and TRP2 genes into the chromosome of Melan-a cells, a kind of melanocytes. By using these cell lines, changes in MITF, tyrosinase and TRP2 can be simultaneously detected at the level of gene, protein, and total melanin levels. In addition, it is possible to evaluate the anti-white hair efficacy at the individual level by applying the anti-hair hair candidates derived from the cell lines to the back of the leukemia-promoted mouse with white hair development and visually assessing and measuring the change of melanin in the hair. . Therefore, the screening method of the present invention combines in vitro efficacy evaluation with transformed cell lines and in vivo efficacy evaluation with vitiligo mice, thereby increasing the accuracy of screening while reducing the time and cost of screening. Has the advantage of being reduced. In addition, the use of PDE (phosphodiesterase) inhibitors, including IBMX or caffeine, screened by the screening method of the present invention can be obtained excellent anti-hair effect.

Description

Method for screening anti-gray hair ingredient using stable cell lines and accelerated vitiligo mouse, and composition containing anti-gray hair ingredient detected thereby}

The present invention relates to a method for screening an anti-white hair substance using a transformed cell line and a vitiligo-promoted mouse, and a composition for preventing white hair containing the anti-white hair substance. More specifically, a transformed cell line capable of monitoring the expression changes of MITF, tyrosinase, and TRP2, which play the most important role in melanin synthesis and survival of melanocytes, at the cellular level, and the efficacy of candidate substances in vivo to inhibit white hair development were measured. It relates to a method for screening an anti-white hair substance composed of a vitiligo model mouse capable of. In addition, it relates to a composition for preventing white hair containing as an active ingredient PDE (phosphodiesterase) inhibitors, including IBMX or caffeine, screened by the screening method of the present invention.

MITF is a transcription factor that regulates the melanogenesis process and plays a key role in melanin synthesis and melanocyte survival. MITF has been reported so far five isoforms of A, B, C, D, H and M, but it is MITF-M that drives melanin synthesis in melanocytes. Therefore, this study was centered on MITF-M, and unless otherwise specified, the MITF of this patent refers to MITF-M.

When activated, MITF promotes melanin synthesis by activating the transcription of tyrosinase, TRP1 (Tyrosinase-related protein 1), and TRP2. In addition, MITF plays an important role in increasing the growth and survival of melanocytes by increasing the expression of Bcl-2 protein, which plays a role in preventing apoptosis (Cell 109:707-718,2002).

WS2A (Waardenburg syndrome type 2A) and TS (Tietz syndrome), which are representative pigmentation disorders, are genetic diseases caused by mutations in the MITF gene. Rev 84:1155-1228,2004). In the above examples, it can be seen that MITF is an essential element for pigment formation and activation of melanocytes.

Melanin is the final product produced by L-tyrosine through a complex multi-step transformation process in the melanosome of melanocytes. Tyrosinase is an important enzyme in the regulation of melanin synthesis, and plays a role in initiating melanin synthesis by converting L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). This process is a rate-limiting step of melanin synthesis and is an essential step in melanin synthesis in vitro or in vivo.

TRP1 and TRP2 were thought to be proteins that control the rate of melanin synthesis by helping tyrosinase until early 2004. However, during a study on the process of white hair development, it was found that unlike skin, melanocytes in hair follicles gradually die off with age, and it was announced that the decisive difference between skin and white hair follicle melanocytes is the presence or absence of TRP2 (L'Oreal Hair Biology Team, Intercontinental Hair Research Society (IMHRS)). According to this study, it can be seen that the decline of TRP2 in melanocytes proceeds to the death of hair follicle melanocytes, resulting in white hair. Therefore, it can be seen that TRP2 plays an important role in the survival of melanocytes along with the synthesis of melanin in the hair follicle.

White hair is a phenomenon that occurs because melanocytes cannot deliver melanin to the hair due to an abnormality in the melanocytes in the hair follicle. Various factors have been suggested as the cause of white hair, but the causes of white hair studied so far are largely first, reduction of hair follicle melanocytes due to the gradual death of melanocyte stem cells in the hair follicle; Second, death of hair follicle melanocytes due to a decrease in melanocyte killing inhibitory protein (Bcl-2); Third, it can be summarized into three categories: reduction in mobility and activity of the outer root sheat (ORS) melanocytes.

As can be seen from the results of the above studies, white hair is produced not only due to the decrease in the ability of the hair follicle melanocytes to synthesize melanin, but also because the survival, differentiation, and migration of the follicular melanocytes according to the hair cycle are not performed normally due to certain factors. Therefore, in order to be an effective anti-white hair substance, it must be able to continuously induce melanin synthesis without killing it by increasing the overall activity of melanocytes as well as the effect of enhancing melanin synthesis. In addition, for the development of effective white hair prevention raw materials, changes in MITF, which is an overall regulator of melanocyte activity, tyrosinase, which is the main enzyme of melanin synthesis, and TRP2, which plays an important role in the survival of melanocytes, can be observed quickly and easily. A screening system capable of verifying the efficacy of in vivo is essential.

Existing patents related to white hair prevention are melanin synthesis accelerator and skin external preparation (JP, Publication No. 2007-008888), health supplement composition for improving gray hair, and health supplement containing the same (KR, Publication No. 2005-0109345), and melanin production accelerator. And a composition for promoting melanin production (JP, Publication No. 2004-345959), a gray hair healing aid made from herbal medicinal materials (KR, Publication No. 2002-0095855), a hair cosmetic composition for inhibiting gray hair production containing dandelion extract (KR, Publication No. 2002-0068589), most of them focus only on substances that increase melanin synthesis of melanocytes. In addition, the screening method of the anti-white hair substance was limited to the method of measuring the activity level of tyrosinase, which mainly increases melanin synthesis, quantifying the amount of melanin produced, or determining the amount of melanin with the naked eye, so there was no attempt to comprehensively evaluate the activity of melanocytes. (JP, Publication No. 2002-212039 et al.).

In the patent published last year (Japanese Patent Application, Publication No. 2006-028143), MITF was selected as an index for screening for anti-white hair substances and the change in the expression of the MITF gene was measured by real-time PCR, but only melano One change in messenger RNA (mRNA) within the site has a limitation in that it is an in vitro screening method to find an anti-white hair substance. In another patent (Korean patent application, Publication No. 2006-0134302), a mouse model that caused white hair and a human body application experiment were conducted for the evaluation of white hair prevention substances, but the effect of suppressing white hair is evaluated by summing the scores according to the naked eye evaluation. There is a problem in that it cannot have objectivity.

Cell 109:707-718,2002 Physiol Rev 84:1155-1228,2004

In order to solve the above problem, the present inventors have transformed the expression of MITF, which is a major regulator of melanocyte physiological activity, tyrosinase, which is a major enzyme of melanin synthesis, and TRP2, which plays an important role in the survival of melanocytes. A screening method for anti-white hair substances was constructed consisting of vitiligo mice with promoted white hair development that can evaluate the anti-white hair efficacy of cell lines and candidate substances in vivo.

Accordingly, an object of the present invention is to combine the in vitro efficacy evaluation using transformed cell lines and the in vivo efficacy evaluation using vitiligo mice, thereby increasing the accuracy of screening for anti-white hair substances while increasing the accuracy of screening. It provides a screening method that can save time and money. In addition, it is to provide a composition for preventing white hair containing the material for preventing white hair screened through the screening method of the present invention.

In order to achieve the above object, the present invention is a microphthalmia-associated factor (MITF) promoter; Gaussian luciferase gene (GLuc); And it provides an expression vector pMITF-GLuc comprising at least one selected from the group consisting of neomycin resistance gene (NeoR) and ampicillin resistance gene (AmpR). The expression vector pMITF-GLuc is characterized by having the structure of FIG. 4.

The present invention is a tyrosinase (Tyrosinase) promoter; Gaussian luciferase gene (GLuc); And it provides an expression vector pTyrosinase-GLuc comprising at least one selected from the group consisting of neomycin resistance gene (NeoR) and ampicillin resistance gene (AmpR). The expression vector pTyrosinase-GLuc is characterized by having the structure of FIG. 5.

The present invention is a tyrosinase-related protein 2 (tyrosinase-related protein 2, TRP2) promoter; Gaussian luciferase gene (GLuc); And it provides an expression vector pTRP2-GLuc comprising at least one selected from the group consisting of neomycin resistance gene (NeoR) and ampicillin resistance gene (AmpR). The expression vector pTRP2-GLuc is characterized by having the structure of Figure 6.

In addition, the present invention Melan-a melanocyte cell line transformed with the expression vector pMITF-GLuc MITF-GLuc (accession number: KCLRF-BP-00162); Melan-a melanocyte cell line Tyrosinase-GLuc transformed with the expression vector pTyrosinase-GLuc (Accession No.: KCLRF-BP-00163); And it provides a Melan-a melanocyte cell line TRP2-GLuc (accession number: KCLRF-BP-00161) transformed with the expression vector pTRP2-GLuc.

The present invention comprises the steps of treating the transformed cell line with an anti-white hair candidate and culturing it in a medium; Measuring the amount of GLuc produced in the medium; Applying the anti-white hair candidate material to the back of a mouse with vitiligo in which the development of white hair is promoted by pulling the hair on the back; And measuring the amount of melanin from newly grown hairs on the back of the vitiligo mouse.

In the method for screening an anti-white hair substance of the present invention, the step of measuring the amount of GLuc produced includes transferring only the medium to the measurement plate without destroying the cells, and reacting the GLuc in the medium with the substrate to determine the amount of light emitted. It is characterized in that it is measured with a meter.

In the screening method for anti-white hair substances of the present invention, the screening method includes measuring the amount of GLuc produced in the medium, and then quantifying the amount of MITF, tyrosinase, and TRP2 protein from the cultured transformed cell line. ; And quantifying the total amount of melanin from the cultured transformed cell line.

In addition, the present invention provides a composition for preventing white hair, comprising as an active ingredient a PDE (phosphodiesterase) inhibitor screened through the screening method. In the composition for preventing white hair of the present invention, the PDE (phosphodiesterase) inhibitor is IBMX (3-isobutyl-1-methyl-xanthine) or caffeine.

According to the method for screening the anti-white hair substance of the present invention, it is possible to simultaneously grasp how the MITF, tyrosinase, and TRP2 that regulate melanocyte activity through the transformed cell line are changed at the level of the amount of genes, proteins, and total melanin, and the occurrence of white hair is The in vivo efficacy of the anti-white hair substance can be evaluated through the accelerated vitiligo mouse. That is, the method of the present invention combines the in vitro efficacy evaluation using the transformed cell lines and the in vivo efficacy evaluation using the vitiligo mouse, thereby increasing the accuracy of screening for anti-white hair substances and the time required for screening. It has the effect of reducing the cost and cost.

In addition, when the PDE (phosphodiesterase) inhibitor including caffeine or IBMX screened by the screening method of the present invention is used as an anti-white hair, excellent anti-white hair effect can be obtained.

1 is a diagram showing a mechanism for raising the intracellular cAMP level by inhibiting PDE, which degrades cAMP by IBMX in the cell.
Figure 2 is a diagram showing the relationship between the intracellular cAMP regulates MITF activity.
3 is a diagram showing the plasmid structure of pGLuc-Basic.
4 is a diagram showing the MCS of pGLuc-Basic.
5 is a diagram showing the plasmid structure of pCMV-GLuc.
6 is a diagram showing the plasmid structure of pMITF-GLuc.
7 is a diagram showing the plasmid structure of pTyrosinase-GLuc.
8 is a diagram showing the plasmid structure of pTRP2-GLuc.
Figure 9 shows the GLuc analysis results (Figure 9a) and total melanin amount (Figure 9b) after treatment with α-MSH 10nM and IBMX 100uM on stable cell lines transformed with the expression vector plasmids of the present invention. This is the graph shown.
10 is a graph showing the results of measuring the degree of MITF activation in transformed cell lines after treatment with various PDE inhibitors.
11 shows that when MITF siRNA was treated in transformed cell lines, not only MITF but also tyrosinase and TRP2 were inhibited at the gene level (GLuc activity, FIG. 11A), protein level (Western blot result, FIG. 11B), melanin synthesis level ( It is a photograph and a graph shown in FIG. 11c).
FIG. 12 is a photograph of a mouse after treatment with an anti-white hair substance in a mouse model of vitiligo in which the development of white hair is promoted (FIG. 12A) and a graph showing the quantification result of melanin in hair (FIG. 12B ).

Hereinafter, the present invention will be described in detail.

The method for screening an anti-white hair substance according to the present invention includes a transformed cell line for evaluating the in vitro efficacy of a candidate anti-white hair substance and a vitiligo-promoted vitiligo model mouse for evaluating in vivo efficacy. It consists of parts.

More specifically, Gluc, a plasmid capable of monitoring changes in MITF expression by binding a promoter of MITF in front of the gene of Gaussia Luciferase (GLuc), which can serve as a reporter of specific gene expression. Plasmids capable of monitoring changes in tyrosinase expression by binding a promoter of tyrosinase in front of the gene, and plasmids capable of monitoring changes in expression of TRP2 by binding a promoter of TRP2 in front of the Gluc gene were constructed. Transformed cell lines (hereinafter referred to as MITF-GLuc, Tyrosinase-GLuc, and TRP2-GLuc) that were stably introduced into the chromosome of Melan-a cells to monitor changes in the expression of MITF, tyrosinase, and TRP2 were established. The cell lines were deposited with the Korea Cell Line Research Foundation on June 8, 2007, and their accession number (in order of KCLRF-BP-00162, KCLRF-BP-00163, KCLRF-BP-00161).

In established cell lines, the degree of activity of each gene is proportional to the amount of GLuc generated, and the activity of each gene is measured by measuring the amount of light emitted when a certain amount of GLuc substrate is treated with a luminometer. You can compare the degrees.

GLuc is the same as conventional luciferases in that when it reacts with a substrate, it emits light of a specific wavelength, except that when it is produced, it is discharged out of the cell and stably exists in the culture medium. Therefore, using the transformed cell lines of the present invention, it is possible to measure the expression change of MITF, tyrosinase, and TRP2 by extracting a small amount of medium during cell culture and reacting with the substrate. This method is completely different from the existing luciferase experiments (Bandi, Renilla's luciferase), which must destroy cells, and does not affect the growth of cells, so their expression can be expressed in real time at a desired time. Can be monitored. In addition, since RNA and protein can be extracted using the remaining cells, changes in MITF, tyrosinase, and TRP2 can be monitored at the level of protein and total melanin.

Vitiligo mice (C57bl/6- Mitf ^{mi - vit} ) are mice in which a spontaneous mutation in the MITF gene occurs as age increases, resulting in white hair. This mouse model is characterized by the occurrence of white hair in the neck area, and when the age increases, white hair is generated in the tail area. From about 12 weeks of age, melanin cannot be delivered to the hair, so white hair develops in the neck and progresses toward the tail.It takes about 30 weeks for the white hair to completely develop on the back (neck to tail). Since 30 weeks is too long for screening for anti-white hair materials, the present inventors promoted the generation of white hair by depilation of the back. Hair pulling in mice promotes new hair growth by rapidly rotating the hair growth cycle of the hair follicle, thereby promoting the mutation of MITF in the hair follicle melanocytes, resulting in the development of white hair (Journal of Investigative Dermatology 87:299). -304,1986). In the present invention, focusing on the above characteristics, a mouse model of vitiligo with accelerated white hair development was established, and the melanin content in the hair grown after application of the substance was measured to quantitatively evaluate the anti-white hair effect of the substance.

On the other hand, the inventors of the present invention first discovered that PDE (phosphodiesterase) inhibitors, including IBMX or caffeine, have an anti-white hair effect by the screening method of the present invention. In particular, IBMX is a non-specific PDE (Phosphodiesterase) inhibitor, and serves to raise the intracellular cAMP level by inhibiting PDE, which plays a role of degrading cAMP in the cell, as illustrated in FIG. 1. The present inventors hypothesized that when the intracellular cAMP level is increased by PDE (phosphodiesterase) inhibitors including IBMX, the activity of MITF will also increase as shown in FIG. 2 cAMP-MITF up-regulation, The anti-white hair effect was confirmed by the screening method of the present invention.

In FIG. 2, indicated in pink is the cAMP⇒PKA & p70⇒⇒⇒MAPK pathway, indicating the activation of MITF through phosphorylation, and the blue color indicates the cAMP⇒⇒⇒⇒CREB pathway, indicating the activation of MITF transcription. That is, "IBMX⇒cAMP level increase⇒MITF protein amount and activity increase".

Hereinafter, the present invention will be described in more detail with reference to the following test examples, but the present invention is not limited to these examples.

[Test Example 1] Construction of a plasmid for observation of changes in the expression level of MITF, tyrosinase, and TRP-2

Each plasmid was prepared by combining the promoter of each gene extracted by PCR from genomic DNA into a multi-cloning site (MCS) of pGLuc-basic (New England Biolab, NEB). This work was commissioned by Neurogenex Co. Ltd, and detailed information of the constructed plasmids is as follows.

1) pGluc-Basic: As a plasmid expressing GLuc that does not have a specific promoter, it was used as a mother vector for the construction of other plasmids. Purchased from NEB. The plasmid structure is as shown in FIG. 3, and the MCS of pGLuc-Basic is indicated by a blue arrow in FIG. 4.

2) pCMV-GLuc: A GLuc expression plasmid having a CMV promoter, which is a universal promoter, which is always expressed, and was used as a positive control for the experiment. Purchased from NEB. The plasmid structure is shown in FIG. 5.

3) pMITF-GLuc: The PCR product of the MITF promoter (GenBank seq. ID: D82874) was prepared by ligation using EcoRI and BamHI sites in the MCS of pGLuc-Basic. PCR is EcoRI/MITF-forward primer 5'-GGC GAA TTC CTG CAG TCG GAA GTG GCA GTT A-3' (SEQ ID NO: 1), BamHI/MITF-reverse primer 5'-GGC GGA TCC AGA CTA TCC CTC CCT A 502bp product was obtained using CTA CTT TC-3' (SEQ ID NO: 2). The plasmid structure is shown in FIG. 6.

4) pTyrosinase-GLuc: The PCR product of the tyrosinase promoter (GenBank seq. ID: M27160) was prepared by ligation using EcoRI and BamHI sites in the MCS of pGLuc-Basic. PCR is EcoRI/Tyrosinase-forward primer 5'-GGC GAA TTC CTC TCA TTT GCA AGG TCA AAT CA-3' (SEQ ID NO: 3), BamHI/Tyrosinase-reverse primer for 5'-GGC GGA TCC TTC CTC TAG TCC TCA CAA GGT C-3' (SEQ ID NO: 4) was used to obtain a product of 398 bp. The plasmid structure is shown in FIG. 7.

5) pTRP2-GLuc: The PCR product of the TRP2 promoter (GenBank seq. ID: L38953) was prepared by ligation using EcoRI and BamHI sites in the MCS of pGLuc-Basic. PCR is EcoRI/TRP2- forward primer with 5'-GGC GAA TTC GAG CTC ACT GCA TCT ACT GAC T-3' (SEQ ID NO: 5), BamHI/TRP2- reverse primer with 5'-GGC GGA TCC GAG CTC TCT CTC TCT CTT ACT TT-3' (SEQ ID NO: 6) was used to obtain a product of 701bp. The plasmid structure is shown in FIG. 8.

[Test Example 2] Constructing a transformed cell line using Melan-a cells

In general, the process of constructing a transformed cell line includes determining the type and concentration of a selection drug to be used; Plasmid introduction into the cell (transfection) and primarily colony selection; And it is divided into the step of selecting the final colony. In the present invention, a transformed cell line was constructed using Melan-a cells, a kind of melanocytes, and neomycin G418 (Gibco) as an optional drug.

[2-1] Culture of Melan-a Cells and Determination of Optimal Concentration of Selective Drugs

_{Melan-a cells are 37 ℃, 10% CO 2} in RPMI 1640 medium containing 10% fetal bovine serum (FBS), 100 unit/ml penicillin-streptomycin (Gibco), 0.1uM TPA (Sigma). It was cultured under conditions. In general, the concentration of an optional drug for producing a transformed cell line is determined as a concentration at which about 80 to 90% of cells die after 2 weeks of drug treatment. Melan-a cells were treated with G418 and observed. At a concentration of 400 ug/ml, about 90% of the cells died in about 2 weeks. Based on this, the concentration of G418 was determined to be 400ug/ml.

[2-2] Plasmid transfection and primary colony selection by G418

Plasmid introduction was carried out using Lipofectamine 2000 (Lipo2000, Invitrogen) and referring to the experimental method provided by the manufacturer. Briefly, Melan-a cells are dispensed into 6 wells at ^{a concentration of 2×10 5} cells/well and incubated overnight in an incubator. A DNA-Lipo complex was formed by mixing 1 ug of plasmid and 2 ul of Lipo2000 in Opti-MEM (Gibco), and then the plasmid was introduced into the cells by spraying them on the cells. However, in order to increase the efficiency of transfection, a method of completely removing the cell medium and spraying the Opti-DNA-Lipo mixture was used, and after 4 to 6 hours, it was washed twice with PBS and replaced with the original culture medium. Incubated overnight at. About 24 hours after the plasmid was introduced, the medium was treated with G418 at a concentration of 400 ug/ml, and the medium was changed once every 3 days to induce the death of cells to which the plasmid was not introduced. After a period of about 2 weeks, it was observed that the surviving cells grew/divided to form colonies, and each colony was transferred to a 48-well dish using trypsin and cultured. In the subsequent cultivation process, a medium containing 400 ug/ml of G418 is always used.

[2-3] Final colony selection and treatment of anti-white hair candidate substances

When the primary selected colonies grow and occupy about 60% of the dish area, the GLuc value is measured in the medium and the colonies that do not show the GLuc value are discarded, and the remaining colonies are separated into strong/medium/weak according to the intensity of the GLuc value. Then continue culturing.

The separated colonies were treated with α-MSH (α-Melanocyte Stimulating Hormone, Sigma) and IBMX (3-isobutyl-1-methyl-xanthine, Sigma), which are known to increase melanin synthesis. Only colonies showing similar characteristics to the original Melan-a are finally selected, and unsuitable colonies are discarded.

Colonies selected through the above process were continuously scaled up to 13 cm T-flask and cultured, and the GLuc value was measured at each step to confirm whether the cell characteristics changed. After the verification of the cells is completed, the concentration of G418 is lowered to 100 ug/ml and cultured, and used in the experiment on treatment of anti-white hair candidates. Even at a concentration of 100 ug/ml, cell characteristics or homogeneity are not impaired. Several PDE (phosphodiesterase) inhibitors as candidates for white hair prevention under the hypothesis that an increase in intracellular cAMP level by an IBMX or phosphodiesterase (PDE) inhibitor including caffeine will increase the activity of MITF. Was used. PDE inhibitors include non-selective PDE inhibitors, IBMX; Caffeine; Theophylline; And pentoxifylline, a specific PDE inhibitor, which is a PDE1 specific inhibitor, 8-methoxymethyl-3-isobutyl-1-methylxanthine (hereinafter, 8-Meth-OH); EHNA,HCl, which are PDE2 specific inhibitors; Quazinone, a PDE3 specific inhibitor; Ro-20-1724, a PDE4 specific inhibitor; And Zaprinast, a PDE5 specific inhibitor. In this test example, IBMX and caffeine were purchased from Sigma and used at a concentration of 100 μM.

[Test Example 3] GLuc (Gaussian Luciferase) analysis

In order to quantify the amount of GLuc after treatment with the anti-white hair candidate substance, change in the amount of GLuc can be measured by moving only a small amount of medium while the cells are growing and reacting with the substrate to the measurement plate. Specifically, after picking a small amount of medium from the cell culture dish and transferring it to the measurement plate, add 1X GLuc asssy working solution (NEB) to this medium at a ratio of 4:1 and use a luminometer to generate the amount of light at 470 nM. Was measured.

The transformed cell lines (stable cell lines) were treated with α-MSH 10nM and IBMX 100uM, and GLuc analysis was performed at 24 and 48 hours. Figure 9 shows that the GLuc analysis results for the expression of MITF, tyrosinase, and TRP2 in the transformed cell line constructed in the present invention (Figure 9a) are proportional to the amount of melanin synthesis of the transformed cell lines 48 hours after treatment (Figure 9b). .

In the first graph of FIG. 9A, the reason that the activity of 48 hours treatment is lower than that of α-MSH treatment for 24 hours is because α-MSH (α-Melanocyte Stimulating Hormone) is a protein and is gradually degraded/denatured in the medium. . On the other hand, because IBMX is a synthesized chemical, its efficacy lasts up to 48 hours.

10 is a result of measuring the degree of MITF activation in a transformed cell line after treatment with various PDE inhibitors described in Test Example 2. FIG. As shown in FIG. 10, PDE inhibitors also increased the activity of MITF by increasing the intracellular cAMP level.

[Test Example 4] Gene expression inhibition experiment using MITF siRNA

In order to investigate the relationship between MITF, tyrosinase, and TRP2 by inhibiting the expression of the MITF gene, siRNA (small interferencing RNA) was synthesized, and each nucleotide sequence is as follows (Bioneer).

Sense: CCA GCA GCU UCA AAA ACA AGC AGC CAG (SEQ ID NO: 7)

Antisense: GGC UGC UUG UUU UUG AAG CUC CUG GAU (SEQ ID NO: 8)

siRNA was introduced into the cells using HiPerFect Transfection Reagent (Qiagen), and was carried out according to the experimental method provided by the manufacturer.

The constructed transformed cell line is dispensed into a 6-well plate so that the volume per well is 2.3 ml and the number of cells is 1×10 ⁵ . The next day, 37.5 ng of siRNA was diluted in 100 ul of serum-free culture medium to reach a final concentration of 5 nM, and then 3 ul of hiPerFect reagent was added thereto and reacted at room temperature for about 5-10 minutes to form a siRNA transfection complex. After dropping the resulting transfection complex onto the cells, gently shake the plate and incubate in an incubator.

After 3 days of siRNA introduction, the cells are washed twice with PBS and the cells are destroyed with Lysis buffer. In order to measure the degree of inhibition of the expression of the MITF gene, the activity of GLuc was measured, and the melanin pellet was dissolved with 1N NaOH, and the absorbance was measured at 404 nm to measure the total amount of melanin, and the supernatant was used the Lowry method. After quantification, Western blot was performed. MITF, tyrosinase (α-PEP7), and TRP2 (α-PEP8) antibodies were all used by calbiochem, and MITF was 1:500 and tyrosinase and TRP2 were diluted 1:1000.

FIG. 11 shows that when MITF siRNA was treated in transformed cell lines, not only MITF, but also tyrosinase and TRP2 were inhibited at the gene level (GLuc activity), protein level (Western blot result), and melanin synthesis level. 11A and 11B show that the expression of tyrosinase and TRP2 is inhibited at both the gene and protein levels by MITF siRNA treatment. Figure 11c shows that melanin synthesis is inhibited by MITF siRNA. The above results confirmed once again that MITF is important as a major regulator of melanocyte activity.

[Test Example 5] Evaluation of the efficacy of preventing white hair in vivo using vitiligo mice with promoted white hair development

Vitiligo mice (C57bl/6- Mitf ^{mi - vit} ) were purchased from The Jackson Lab in the United States and used. A method of screening for an anti-white hair substance using a mouse with promoted white hair development is as follows. The dorsal hairs of 12-week-old mice are removed by depilation. However, the width of the area where the hair is removed is the same for each individual. From the day after the hair is pulled, apply white hair prevention candidates twice a day to the hairless area. After about 3 weeks, if the difference in the anti-white hair effect between the candidate substances is visually identified, newly grown hairs are collected and the amount of melanin in the hair is measured. The amount of melanin in the hair is measured using a proteolytic enzyme, Esperase (Novazyme). A reaction buffer was prepared by dissolving esperase to a concentration of 1NPU/ml in a buffer (50mM Tris-HCl, 5mM DTT, pH 9.3). 5mg of mouse hair was added to 1ml of the reaction buffer and allowed to react for 13 hours while shaking at a speed of 1,000rpm at 37℃, and then the hair and the reaction solution were separated by instantaneous centrifugation. The reaction solution thus obtained is placed in a 96 well plate, and the absorbance is measured at a wavelength of 405 nM to measure the amount of melanin in the reaction solution.

12 is a photograph and graph showing the results of measuring the efficacy of the anti-white hair substance through the naked eye observation and quantification of melanin in the hair when the white hair prevention material is treated in a mouse model of vitiligo in which the development of white hair is promoted. 12A is a photograph of a hair growth when a candidate anti-white hair substance is applied for 3 weeks, and FIG. 12B is a graph measuring the amount of melanin in the grown hair. As the vehicle of the experiment, a mixture of EtOH:1,3-BG:DW = 3:2:5 (volume ratio), and IBMX, which showed the best activity enhancing efficacy in screening through a transformed cell line, was used as a candidate for preventing white hair. C57bl/6 mice with black hair were used as the positive control (C57CTL) of the experiment.

Hereinafter, examples of the formulation of the composition for preventing white hair of the present invention will be described, but this is not intended to limit the invention, but is merely intended to be described in detail.

[Formulation Example 1] Hair Tonic

ingredient Weight ratio (%) ethanol 50 menthol 0.02 glycerin 3 Salicylic acid 0.05 IBMX 0.1 to 5 Flavoring and coloring Appropriate amount Purified water Balance (to 100)

[Formulation Example 2] Hair lotion

ingredient Weight ratio (%) Cetostearyl alcohol 2.0 EDTA 2Na 0.2 Hydroxyethylcellulose 0.5 Mineral oil 5.0 IBMX 0.1 to 5 antiseptic Appropriate amount Flavoring and coloring Appropriate amount Purified water Balance (to 100)

[Formulation Example 3] Hair nourishing lotion

ingredient Weight ratio (%) Beeswax 4.0 Polysorbate 60 1.5 Sorbitansquioleate 0.7 Mineral oil 10.0 glycerin 3.0 Butylene glycol 3.0 Propylene glycol 3.0 Carboxyvinyl polymer 0.1 Triethanolamine 0.2 IBMX 0.1 to 5 antiseptic Appropriate amount Flavoring and coloring Appropriate amount Purified water Balance (to 100)

[Formulation Example 4] Hair Shampoo

ingredient weight% Purified water Balance (To 100) IBMX 0.1 to 5 Sodium lauryl sulfite 36.0 Cocamidopropylbetaine 8.0 Palmitidine malate 2.0 Glycol stearate 1.5 Polyquaternium 10 0.5 Cyctic Acid 0.1 glycerin 2.0 Preservatives, colors and flavors Appropriate amount

[Formulation Example 5] Hair conditioner and treatment

ingredient weight% Purified water Balance (To 100) IBMX 0.1 to 5 Propylene glycol 2.0 Cetyltrimethylammonium chloride 1.0 Cetyl alcohol 3.0 Stearyl alcohol 3.0 Mineral oil 0.5 Cyctic Acid 0.2 Polydimethylsiloxane 1.0 Preservatives, colors and flavors Appropriate amount

Korea Cell Line Research Foundation KCLRFBP00161 20070608 Korea Cell Line Research Foundation KCLRFBP00162 20070608 Korea Cell Line Research Foundation KCLRFBP00163 20070608

Attach electronic file of sequence list

Claims (4)

Melan-a melanocyte cell line Tyrosinase-GLuc transformed using the expression vector pTyrosinase-GLuc (KCLRF-BP-00163).
Treating the transformed cell line of claim 1 with an anti-hairpin candidate and culturing in a medium;
Measuring the amount of GLuc present in the medium;
Applying the candidate for preventing hair loss on the back of the vitiligo mouse in which hair growth is promoted by plucking the back hair; And
And measuring the amount of melanin from the newly grown hair on the back of the vitiligo mouse.
3. The method of claim 2,
The step of measuring the production amount of GLuc, anti-hair follicle material, characterized in that by measuring the amount of light emitted by reacting GLuc in the medium with a substrate without moving the medium only to the measurement plate without destroying cells Screening method.
3. The method of claim 2,
The screening method comprises the steps of quantifying the amount of MITF, tyrosinase, and TRP2 protein from the cultured transgenic cell line after measuring the amount of GLuc present in the medium; And
The method for screening anti-white hair material, characterized in that it further comprises the step of quantifying the total amount of melanin from the cultured transformed cell line.

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