KR100672853B1 - Method for Screening Responsiveness to Drugs Effective in Treatment or Prevention of Vitiligo and Method for Prognosis of Vitiligo - Google Patents

Abstract

The present invention is directed to a drug effective for the treatment or prevention of vitiligo by using a transcription initiator 4A1 (eIF4A1) gene, a ribosomal protein L13 (L13) gene, and an RNA polymerase mediator (MRT) gene for transcription. The present invention relates to a method and kit for screening for, a method and kit for screening an effective ingredient effective for the treatment or prevention of vitiligo, and a method for prognostic determination and prognostic determination of vitiligo.

Corticosteroids, skin disease, vitiligo, ATRA, vitamin C, trichloroacetic acid, calcipotriol, eIF4A1, reactivity, prognosis

Description

Method for Screening Responsiveness to Drugs Effective in Treatment or Prevention of Vitiligo and Method for Prognosis of Vitiligo}             

1 is a gel photograph showing fractional expression patterns using a kit comprising annealing regulatory primers (ACPs) of ATRA-treated and excipient-treated vitiligo epidermal samples. The difference in specific mRNA expression between normal epidermis (N) and excipient treated epidermis (V) was partially or completely recovered by ATRA treatment (A). The bands (arrows) and their expression levels differed between patients (as in clone 3), but 10 bands from six patients showed expression restored to normal levels by ATRA treatment.

Figure 2 is a DNA sequence and primer sequence of the translational initiator 4A1 (eIF4A1) of eukaryotic cells. One of the mRNA bands showing the amount of expression recovered by ATRA application was cloned. The sequence (underlined) between SEQ ID NOs: 841 to 1261 exhibited at least 99% homology with eIF4A1, which is approximately 1.4 kb in size. Primer sets were designed for RT-PCR, one of which is capitalized and the other in bold.

3A is a gel photograph showing the relationship between semi-quantitative RT-PCR assay results and clinical responsiveness. Four known genes, ie the translational initiator 4A1 (eIF4A1) of the eukaryotic cell, ribosomal protein L13 (L13), the mediator of RNA polymerase for transcription (MRT) and the ribosomal phosphoprotein PO (PO) gene RT-PCR was performed using 5 patients' samples using primers for GAPDH as an internal standard. The expression level of eIF4A1 mRNA in excipient-treated depigmented epidermis (V) was lower than that of normal pigmented epidermis (N). ATRA treatment (A) resulted in elevated levels of eIF4A1 expression to normal levels in three patients (patients # 1, # 2, # 3) with favorable clinical responsiveness to ATRA. On the other hand, there was no difference in the expression level of eIF4A1 in the other two patients (patients # 4 and # 5) with no difference in clinical responsiveness between ATRA and excipient treatment. L13, MRT and PO mRNAs showed similar correlations in certain patients.

3B is a graph showing the relationship between semi-quantitative RT-PCR assay results and clinical responsiveness. Relative amounts of mRNA were obtained by calculating the ratio of each mRNA amount to the amount of GAPDH. The ratio of normalized densitometer values between depigmented and normal pigmented epidermis was analyzed in excipient-treated and ATRA-treated samples. From this ratio, the association with clinical responsiveness was investigated and found to correlate with eIF4A1 mRNA in all five patients.

4 is a graph showing real-time PCR results of eIF4A1.

5 is a gel photograph showing the results of Western blotting analysis for eIF4A1 protein expression. N is normally pigmented and excipient treated epidermal cells, V is excipient treated depigmented epidermal cells, and A is ATRA treated depigmented epidermal cells. Recombinant eIF4A1 protein was used as a positive control. β-actin was used as internal control.

6 is a result of Western blotting analysis showing the expression pattern of eIF4A1 protein in the normal and lesion.

The present invention relates to a method and kit for screening responsiveness to a drug effective for the treatment or prevention of vitiligo, a method and kit for screening an active ingredient effective for the treatment or prevention of vitiligo, and a method for prognostic determination and prognostic determination of vitiligo will be.

Retinoids affect cell differentiation and proliferation. Retinoids are metabolic or synthetic derivatives of vitamin A, and the activity of vitamin A is achieved by three main compounds, retinol, retinyl aldehyde and retinoic acid (RA). When combined with local corticosteroids and all-trans-retinoic acid (tretinoin: ATRA) to suppress corticosteroid-induced skin atrophy (1-3), pain is a common type of alum. Very rapid repigmentation is seen in the particular patient receiving it. It is not clear why ATRA exerts better effects in certain patients and why RA's effects indicate inconsistencies.

Although the effects of RA on keratinocytes and fibroblasts have been studied, few studies have been conducted on the effects of RA on normal human melanocytes (4). The role of RA in the constitutive melanin production of normal human melanocytes is contradictory: RA does not affect melanogenesis (5, 6), inhibits melanogenesis (7, 8) or Increase pigmentation (9). Results showing increased pigmentation were obtained in micropigs or mice, but similar results were confirmed in humans (9). Variation in responsiveness to RA treatment requires large amounts of sampling from cultures of different sources to define the action of RA on melanocytes (7). In addition, the published effects of RA on melanocyte numbers tend to contradict. RA inhibits the proliferation of normal human melanocytes growing in serum-deficient medium (5), but RA increases the number of endogenous mouse melanocytes and ultraviolet B-activated mouse melanocytes (9).

The effect of RA on melanogenesis may be direct (5, 7) or by cell-cell interactions between melanocytes, keratinocytes and fibroblasts (6, 8). The effect of RA on keratinocytes is relatively consistent. ATRA and / or 9-cis causes proliferation of normal human keratinocytes cultured in submerged culture systems (10). Although the exact molecular mechanism by which retinoids control keratinocyte proliferation is not known, heparin-binding EGF-like growth factor (HB-EGF), a member of the EGF family of growth factors, at least partially RA-induced keratinocyte proliferation And mediate epidermal fructose formation (11).

The inventors of the present invention sought to find a biological substance that can serve as a marker for responsiveness to the drug of vitiligo. As a result, the present inventors have completed the present invention by confirming that three genes are differentially expressed according to reactivity. .

Accordingly, it is an object of the present invention to provide a method for screening responsiveness to drugs effective for the treatment or prevention of vitiligo.

Another object of the present invention is to provide a method for screening responsiveness to all-trans-retinoic acid (ATRA) for use in the treatment of vitiligo.

Still another object of the present invention is to provide a kit for screening reactivity for a drug effective for treating or preventing vitiligo.

Another object of the present invention is to provide a biological label for reactivity with a drug effective for the treatment or prevention of vitiligo.

Another object of the present invention is to provide a method for screening an effective ingredient effective for the treatment or prevention of vitiligo.

Another object of the present invention is to provide a kit for screening an effective ingredient effective for the treatment or prevention of vitiligo.                         

It is another object of the present invention to provide a prognosis method of vitiligo.

Another object of the present invention to provide a kit for determining the prognosis of vitiligo.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention provides a method for screening responsiveness to a drug effective for the treatment or prevention of vitiligo, comprising: (a) the skin treated by the drug Isolating total RNA from epidermal cells isolated from diseased patients; (b) synthesizing cDNA from the isolated total RNA; (c) a primer set that hybridizes to (i) the translation initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene and (iii) an RNA polymerase for transcription PCR using at least one primer set selected from the group consisting of a primer set hybridized to a mediator (MRT) gene of the above and the synthesized cDNA as a template; (d) electrophoresis of the PCR product to obtain a band of the PCR product; And (e) determining that the electrophoretic band pattern is responsive to the drug when the electrophoretic band pattern exhibits substantially the same pattern as the positive control obtained from normal epidermal cells.

The inventors have found that eIF4A1, L13 and MRT correlate closely with human responsiveness to drugs effective for the treatment or prevention of vitiligo.

Of these proteins, the biological function of eIF4A1 is well known, but L13 and MRT are unknown proteins, except that L13 is a 60S ribosomal subunit protein that is involved in the extension of mRNA translation (12). eIF4A is one of the transcription factors involved in the binding of mRNA to the complex at 43S development (13, 14). mRNA translation is a major regulatory point in gene expression. Binding to the 5'-end cap structure of cytoplasmic mRNA of all eukaryotic cells is a major regulatory step in mRNA translation. eIF4A is one subunit of the heterotrimer translation initiator, eIF4F (15-17), which is one of the proteins that binds the cap (13). eIF4A has ATP-dependent RNA helicase activity, which releases secondary structures of mRNAs to promote translation (18, 19). eIF4A is present in abundance than other cap-binding proteins and is present as part of the eIF4F complex and as free eIF4A. Unlike other components of the eIF4F complex, eIF4A activity is not regulated by phosphorylation in mammalian cells (20), mainly by regulating the amount of mRNA (21, 22). eIF4A is encoded by two genes eIF4A1 and IF4A2. eIF4A1 mRNA is synthesized and most efficiently transduced in growing cells, whereas eIF4A2 mRNA synthesis and translation is favorably associated with growth-stopped state (23).

EIF4A1, L13 and MRT with these biological functions are closely correlated with the human responsiveness to drugs effective for the treatment or prevention of vitiligo.

The present invention applies to vitiligo.

The reactivity tested by the present invention is a drug used for the treatment or prevention of skin diseases requiring administration of corticosteroids, preferably all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid and calcipotriol (calcipotriol). Most preferably, it is ATRA.

In the methods of the invention, the step of isolating total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning. A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol. Biol. Rep. , 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology , John Willey & Sons (1987); And Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)). For example, Trizol can be used to easily isolate total RNA in cells.

The second step of the invention is the step of synthesizing cDNA from the isolated total RNA. Since the total RNA of the present invention is isolated from human epidermal cells, cDNA can be easily synthesized by using oligo-dT primers and reverse transcriptases using poly-A tails at the ends of mRNAs. ( PNAS USA , 85: 8998 (1988); Libert F, et al., Science , 244: 569 (1989); and Sambrook, J. et al., Molecular Cloning.A Laboratory Manual , 3rd ed. Cold Spring Harbor Press (2001).

The primer set used in the PCR step of the present invention is a primer set hybridized to the eIF4A1 gene, a primer set hybridized to the L13 gene, a primer set hybridized to the MRT gene, or a combination of these primer sets. Most preferably, it is a primer set that hybridizes to the eIF4A1 gene that is most correlated with reactivity.

As used herein, the term “primer” refers to a single-strand that can serve as an initiation point for template-directed DNA synthesis under suitable conditions (ie, four different nucleoside triphosphates and polymerases) at a suitable temperature. Oligonucleotides. Suitable lengths of primers are typically 15-30 nucleotides, although varying depending on various factors, such as temperature and the use of the primer. Short primer molecules generally require lower temperatures to form a hybrid complex that is sufficiently stable with the template.

The sequence of the primer does not need to have a sequence that is completely complementary to some sequences of the template, and it is sufficient that the primer has sufficient complementarity within a range capable of hybridizing with the template to perform a primer-specific function. Therefore, the primer set in the present invention does not need to have a sequence that is perfectly complementary to the eIF4A1 gene, L13 gene, or MRT gene, which is a template, and has sufficient complementarity within a range capable of hybridizing to these gene sequences to act as a primer. Is enough. For example, a primer set that hybridizes to the eIF4A1 gene is a primer set that hybridizes to the SEQ ID NO: 1 sequence, and a primer set that hybridizes to the L13 gene is a primer set that hybridizes to the SEQ ID NO: 2 sequence, and that hybridizes to the MRT gene. A primer set is a primer set that hybridizes to SEQ ID NO: 3 sequence. As used herein, the term "primer set" refers to a primer pair consisting of a forward primer and a reverse primer.

The design of such primers can be readily carried out by those skilled in the art with reference to the sequence of the template, for example, using a program for primer design (eg, PRIMER 3 program).

Hybridization (annealing) in the PCR process is generally carried out under stringent conditions that allow for specific binding between the primer and the target sequence. These stringent conditions are suitable buffer concentrations and temperatures to allow primers to hybridize to complementary sequences specific (M. Kanehisa, Nucleic Acids Res. , 12: 203 (1984)). Stringent conditions are sequence-dependent and vary depending on the environment. Longer sequences hybridize specifically at higher temperatures. In general, stringent conditions are selected at temperatures about 5 ° C. below the melting point (T _m ) for the particular sequence.

PCR procedures in the present invention can be carried out according to conventional PCR methods, the details of which are disclosed in US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. For example, it is carried out according to a constant temperature program in a suitable buffer comprising Taq polymerase, dNTPs, cDNA template, primer set and MgCl ₂ .

The amplification product generated by the PCR process is subjected to the electrophoresis process, the strength of the formed band is visually observed or quantified by a densitometer. When the band pattern thus observed shows substantially the same pattern as the positive control obtained in normal epidermal cells, it is determined that the band pattern is responsive to the drug. The positive control targets the epidermis separated from the human body without skin disease, and the electrophoretic band pattern is obtained by performing steps (a)-(d) on the normal sample. Substantially the same pattern of bands means that when compared with the naked eye, the intensity of the band of the sample and the band of the normal epidermis are judged to be substantially the same, or when the values quantified by densitometry are compared. It represents the case. When quantified by a densitometer, it is preferable to compare the relative ratios with respect to an internal standard, and the internal standard is the GAPDH (glyceraldehyder 3-phosphate dehydrogenase) gene.

According to a more specific aspect of the present invention, the present invention provides a method for screening responsiveness to all-trans-retinoic acid (ATRA) for use in the treatment of alum skin disease comprising the following steps: (a) ATRA Separating total RNA from epidermal cells isolated from alum skin disease patients treated by; (b) synthesizing cDNA from the isolated total RNA; (c) PCR using a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of the eukaryotic cell and the synthesized cDNA as a template; And (d) electrophoresing the PCR product to obtain a band of PCR product; (e) determining that the electrophoretic band pattern is responsive to the drug when the electrophoretic band pattern exhibits substantially the same pattern as the positive control obtained from normal epidermal cells.

According to another aspect of the present invention, the present invention provides a primer set that hybridizes to (i) a transcription initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene, and ( Iii) screening for responsiveness to drugs effective for the treatment or prevention of vitiligo comprising at least one primer set selected from the group consisting of a primer set hybridized to an RNA polymerase mediator (MRT) gene for transcription. It provides a kit for.

The kit of the present invention is a kit for carrying out the method of the present invention described above. Therefore, overlapping content in the methods and kits of the present invention is omitted in order to avoid excessive complexity of the present specification.

Kits of the present invention may further comprise reagents for DNA amplification and may include, for example, buffers, DNA polymerases, DNA polymerase cofactors and dNTPs. Optimum amounts of such reagents can be readily determined by one skilled in the art.

According to another aspect of the invention, the invention provides a method for screening responsiveness to all-trans-retinoic acid (ATRA) for use in the treatment of vitiligo comprising: (a) a vitiligo patient treated by ATRA Obtaining a cell homogenate comprising total protein from epidermal cells isolated from the; (b) reacting the homogenate with an anti-eIF4A1 antibody; (c) detecting an antigen-antibody immune response signal between the protein in the homogenate and the anti-eIF4A1 antibody; And (d) determining that the signal of step (c) is responsive to ATRA when the signal of step (c) exhibits substantially greater intensity compared to negative control obtained from epidermal cells isolated from vitiligo patients not treated by ATRA.

According to another aspect of the invention, the invention provides a kit for screening for reactivity against all-trans-retinoic acid (ATRA) comprising an anti-eIF4A1 antibody.

The method of the present invention can be carried out at the protein level as well as at the mRNA level, as described above. When the method of the invention is carried out at the protein level, basically an antigen-antibody immune reaction is used.

In the method of the present invention, the step of obtaining the cell homogenate containing the total protein from the epidermal cells can be carried out in various ways according to conventional methods known in the art. For example, epidermal cells may be impregnated with liquid nitrogen and then pulverized to obtain a cell homogenate by lysing the powder obtained in a buffer containing a protease.

The method of the present invention basically comprises immunoassay methods (eg, radioimmunoassay methods, radioimmunoprecipitation methods, enzyme-linked immunosorbent methods (ELISA), dot blot analysis, Western blot, using antigen-antibody responses). , Quantitatively or qualitatively according to inhibition or competition assays and Sandswitch assays ( Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-). linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984).

For example, when following a radioimmunoassay, the generation of an immune complex formed by binding to an antigen eIF4A1 protein in a sample using an anti-eIF4A1 antibody labeled with a radioisotope (eg P ³² , S ³⁵ ). By measuring, the eIF4A1 protein in the sample can be detected qualitatively or quantitatively (preferably quantitatively).

In addition, in accordance with the ELISA method, (i) adsorbing an anti-eIF4A1 antibody to a well plate; (Ii) adding a sample to the well plate to react and wash; (Iii) adding and reacting the anti-eIF4A1 antibody to which the chromophore or the fluorescent material is bound; And (iii) determining whether the anti-eIF4A1 antibody to which the chromophore or the fluorescent material is bound is bound to the eIF4A1 protein in the sample.

The color development enzymes include, but are not limited to, alkaline phosphatase, β-galactosidase, horse radish peroxidase (HRP), and the like. In addition, when alkaline phosphatase is used, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF are used as substrates. Substrate and the like, when using a horse radish peroxidase, chloronaphthol, aminoethylcarbazole, diaminobenzidine, luminol, ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) as a substrate o-Phenylenediamine (OPD) is used.

On the other hand, when the present invention is carried out according to the Western blotting method, the present invention comprises the steps of: (i) denaturing proteins obtained from epidermal cells (eg, denaturing with SDS and 2-mercaptoethanol); (Ii) SDS-PAGE the denatured protein; (Iii) transferring the denatured protein on the SDS-PAGE completed gel to the NC membrane; (Iii) reacting the protein on the NC membrane with an anti-eIF4A1 antibody that is a primary antibody; (Iii) reacting the secondary antibody with the primary antibody to which the enzyme catalyzing the color reaction is bound; (Iii) inducing a color reaction by adding a substrate of an enzyme bound to the secondary antibody; And (iii) measuring the degree of color development developed by the color reaction.

According to the method of the invention, it is determined that the signal of step (c) is responsive to ATRA when the signal of step (c) exhibits substantially greater intensity compared to the negative control obtained from epidermal cells isolated from vitiligo patients not treated by ATRA. Done. These signal intensities can be compared qualitatively (visually) or quantitatively (eg, using a densitometer or spectrophotometer).

Kits for screening reactivity to ATRA of the present invention may include, in addition to the anti-eIF4A1 antibody, other components according to immunoassay methods. For example, when the kit of the present invention is an ELISA kit and HRP is used as a chromophore, chromium naphthol, aminoethylcarbazole, diaminobenzidine, luminol, ABTS, or OPD may be included.

According to still another aspect of the present invention, the present invention provides a transfection initiator 4A1 (eIF4A1) of a eukaryotic cell or a nucleic acid molecule encoding the same; (Ii) ribosomal protein L13 (L13) or a nucleic acid molecule encoding it; And (iii) a biological label for responsiveness to a drug effective for the treatment or prevention of vitiligo comprising a biological material selected from the group consisting of an RNA polymerase mediator (MRT) or a nucleic acid molecule encoding the same for transcription. To provide.

The biological label of the present invention relates to a novel use of nucleic acid molecules encoding the above eIF4A1, L13 and MRT. The overlapping information in the biological labeling of the present invention and the method of the present invention is omitted in order to avoid excessive complexity of the present specification.

As used herein, the term "nucleic acid molecule" has the meaning of comprehensively including DNA (gDNA and cDNA) and RNA molecules, the nucleotides that are the basic structural units in the nucleic acid molecule is modified as well as natural nucleotides, sugar or base sites Analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews , 90: 543-584 (1990)).

According to another aspect of the present invention, the present invention provides a method for screening an active ingredient effective for the treatment or prevention of vitiligo, comprising the steps of: (a) subjecting a sample compound or biological molecule to be tested Treating the epidermal cells isolated from the patient with the disease; (b) mediators of (i) translational initiator 4A1 (eIF4A1) gene, (ii) ribosomal protein L13 (L13) gene, and (iii) RNA polymerase for transcription in epithelial cells treated (MRT) ) The amount of expression of at least one gene selected from the group consisting of genes; And (c) when the irradiated expression amount exhibits substantially the same expression amount as compared with the positive control obtained from normal epidermal cells, determining the effective ingredient as effective for the treatment or prevention of vitiligo.

The present invention relates to novel uses of the biological labels of the invention described above, eIF4A1, L13 and MRT, or nucleic acid molecules encoding these proteins. eIF4A1, L13 and MRT can be used to screen effective ingredients effective for the treatment or prophylaxis of vitiligo because there is a distinct difference in expression levels for specific drug candidates.

The overlapping contents in the screening method of the present invention and the reactive screening method of the present invention described above are omitted in order to avoid excessive complexity of the present specification.

Examining the expression level of eIF4A1, L13 and MRT can be carried out largely at the nucleotide level and the protein level.

If performed at the nucleotide level, expression analysis can be performed by the RT-PCR method described above.

If performed at the protein level, it is carried out using antibodies against eIF4A1, L13 and MRT proteins. Such antibodies (polyclonal antibodies and monoclonal antibodies) are prepared by methods known in the art using eIF4A1, L13 and MRT proteins as antigens (Harlow, E. and Lane, D., Antibodies: A Laboratory Manual , Cold Spring Harbor Press, New York, 1988; Zola, H., Monoclonal Antibodies: A Manual of Techniques , CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY , Wiley / Greene, NY, 1991 ). Preferably, the antibody used in the present invention is a monoclonal antibody. Expression analysis, ie, analysis of intracellular protein amount, can be performed quantitatively or qualitatively by conventional immunoassay methods (see Enzyme Immunoassay , ET Maggio, ed., CRC Press, Boca Raton, Florida, 1980; and Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology , Vol. 1, Walker, JM ed., Humana Press, NJ, 1984).

For example, when following a radioimmunoassay, the antibody of the invention labeled with a radioisotope (e.g., P ³² , S ³⁵ ) can be used to bind to an antigen in a sample, e.g., eIF4A1, L13 or MRT protein. The production of the formed immune complexes can be measured to detect the protein in the sample qualitatively or quantitatively. In addition, in accordance with the ELISA method, (i) adsorbing an antibody having an ability to bind eIF4A1, L13 or MRT protein to a well plate; (Ii) adding a sample to the well plate to react and wash; (Iii) adding and reacting the antibody of the present invention; (Iii) washing the well plate and reacting by adding a secondary antibody bound to a chromophore or a fluorescent substance; And (iii) determining whether or not the secondary antibody is bound. The color development enzymes include, but are not limited to, alkaline phosphatase, β-galactosidase, horse radish peroxidase, and the like. In addition, when using alkaline phosphatase, bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), ECF substrate, etc. are used as a substrate, and when using a horse radish peroxidase, Chloronaphthol, aminoethylcarbazole, diaminobenzidine, luminol, ABTS (2,2'-Azine-di [3-ethylbenzthiazoline sulfonate]) and the like are used as the substrate.

Finally, when the expression amount analyzed by the above procedure shows substantially the same expression level compared to the positive control obtained from normal epidermal cells, it is determined as an active ingredient.

According to another aspect of the present invention, the present invention provides a primer set that hybridizes to (i) a transcription initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene, and ( Iii) providing a kit for screening an active ingredient effective for the treatment or prevention of vitiligo comprising at least one primer set selected from the group consisting of a primer set hybridized to an RNA polymerase (MRT) gene for transcription do.

According to another aspect of the present invention, the present invention provides an antibody having a binding capacity to (i) a translation initiator 4A1 (eIF4A1) of eukaryotic cells, (ii) an antibody having a binding capacity to ribosomal protein L13 (L13) and (iii) Provided is a kit for screening an active ingredient effective for the treatment or prevention of vitiligo comprising at least one antibody selected from the group consisting of an antibody having the ability to bind to an RNA polymerase (MRT) of RNA polymerase for transcription.

Kit of the present invention is for carrying out the above-described screening method of the active ingredient, the common matters are omitted in order to avoid excessive complexity of the present invention.

According to another aspect of the present invention, the present invention provides a method of prognosis of vitiligo comprising the following steps: (a) epidermal cells or lesions with vitiligo symptoms in a normal skin region of a patient with vitiligo symptoms Separating total RNA from epidermal cells of the site; (b) synthesizing cDNA from the isolated total RNA; (c) PCR using a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of the eukaryotic cell and the synthesized cDNA as a template; (d) electrophoresis of the PCR product to obtain a band of the PCR product; And (e) in the band of step (d) (iii) the PCR band obtained from epidermal cells in the normal skin area of the patient with vitiligo shows reduced band strength compared to the positive control obtained from normal epidermal cells. It is determined that vitiligo is highly likely to develop in the normal skin area, or (ii) when the PCR band obtained from epidermal cells of the lesion site shows increased intensity compared to the previous, vitiligo is determined to be improved. Steps.

According to another aspect of the present invention, the present invention provides a method of prognosis of vitiligo comprising the following steps: (a) epidermal cells or lesions with vitiligo symptoms in a normal skin region of a patient with vitiligo symptoms Obtaining a cell homogenate containing total protein from epidermal cells of the site; (b) reacting the homogenate with an anti-eIF4A1 antibody; (c) detecting an antigen-antibody immune response signal between the protein in the homogenate and the anti-eIF4A1 antibody; And (d) in the signal of step (c), (iii) the signal obtained from epidermal cells in the normal skin area of the patient with vitiligo shows reduced signal strength compared to the positive control obtained from normal epidermal cells. Determining that vitiligo is highly likely to develop in the normal skin area, or (ii) determining that vitiligo is improved when the signal obtained from epidermal cells at the lesion site shows increased intensity as compared to the previous .

According to another aspect of the present invention, the present invention provides a kit for prognostic determination of vitiligo comprising a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of eukaryotic cells.

According to another aspect of the present invention, the present invention provides a kit for prognostic determination of vitiligo comprising an anti-eIF4A1 antibody.

As used herein, the term "prognosis" refers to the future state of vitiligo.

According to the prognostic determination method of the present invention carried out at the RNA level, the finally obtained PCR band is analyzed. (Iii) Positive PCR bands obtained from epidermal cells in the normal skin area of patients with vitiligo symptoms are obtained from normal epidermal cells. It is determined that vitiligo is highly likely to develop in the normal skin area when the band intensity is reduced compared to the control group (i.e., the control group with a large expression level of the eIF4A1 gene). That is, a vitiligo patient exhibiting such band intensity patterns may be determined to have a high probability of spreading vitiligo to their normal skin area.

Also, among the finally obtained PCR bands, (ii) when the PCR bands obtained from epidermal cells of the lesion site in which vitiligo develop, show increased intensity compared to the PCR bands obtained through the same process before obtaining this PCR band, It can be determined that vitiligo improves, and that vitiligo worsens if it exhibits reduced band strength.

According to the prognostic determination method of the present invention performed at the protein level, among the finally obtained antigen-antibody immune response signals, (i) the signal obtained from epidermal cells in the normal skin area of patients with vitiligo symptoms is obtained from normal epidermal cells. When the signal intensity is reduced compared to the control group, it can be determined that vitiligo is more likely to develop in the normal skin area.

In addition, (ii) when the signal obtained from the epidermal cells of the lesion site shows an increased intensity compared with the signal obtained through the same process before obtaining the present signal, it can be determined that vitiligo is improved, and the reduced intensity is shown. In this case, it can be determined that vitiligo is worsening.

According to the method of the present invention, the prognosis of vitiligo can be determined relatively accurately.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example

Experiment method

RNA Isolation from Suction-blister Epidermis of Alum Patients

Six patients (two men and four women) diagnosed with the general type of alum were involved in this experiment. These patients were between 21 and 52 years old (mean age, 38 years old). The patients were not treated for delivery. Placebo-controlled, pair-comparison, left-right studies with ATRA and excipients were conducted for 3-6 months. Following written consent, epidermal samples were obtained from the patient by inhalation. Normal pigmented epidermis of the same patient was obtained as a control. Inhalation blisters were made by applying a negative pressure of 200-250 mmHg against normal and depigmented epidermis with or without ATRA treatment. Loops of the inhalation blisters of all patients were used for RNA isolation.

GeneFishing ^TM  Reverse transcription-polymerization reaction

Total RNAs were isolated from the inhalation-blister epidermis by gentle homogenization using Trizol (GibcoBRL, 15596-026, NY). Reverse transcription was carried out using the GeneFishing ^™ DEG kit (Seegene, DEK 3101 & 3104, South Korea) as follows: 3 μg of isolated RNAs containing RNase-deficient distilled water and 10 μM dT-ACP1 (final volume 9.5). [Mu] l) and then the tubes were placed in DNA Thermal Cycler 9600 (Perkin Elmer). In order to identify bands that are differentially expressed between samples, the same amount of RNA was compared. The mixture was incubated at 80 ° C. for 3 minutes, then cooled on ice and briefly spun. 20 μl of a reaction solution consisting of 5 × RT buffer, 2 mM dNTP, RNase inhibitor and M-MLV reverse transcriptase (Promega, M170B, WI) was added to the mixture. The tube was then incubated at 42 ° C. for 90 minutes, heated at 94 ° C. for 2 minutes, then cooled on ice and briefly spun. The first cDNA strand synthesized was diluted with 80 μl of RNase-deficient distilled water.

cDNA samples were stored at −20 ° C. until use. 10 × buffer without MgCl ₂ , 25 mM MgCl ₂ , 5 μM Aviary ACPs, 10 μM dT-ACP2, 2 mM dNTP, 2.5 U Taq DNA Polymerase Gincraft (Biotherm, GC-002-250, France) and 50 ng PCR amplification was performed in DNA Thermal Cycler 9600 (Perkin Elmer) using the same GeneFishing ^™ DEG kit (Seegene, DEK 3101 & 3104, South Korea) in 50 μl of the reaction solution consisting of the first cDNA strand. Each kit includes 20 different Aviary Annealing Control Primers (ACPs). The thermal cycler was preheated at 94 ° C. before mounting the tube. The program of PCR amplification is as follows: 5 cycles at 94 ° C., 3 cycles at 50 ° C. and 1 minute at 72 ° C., 40 cycles at 94 ° C., 40 seconds, 40 seconds at 65 ° C. and 40 seconds at 72 ° C. , 5 minutes at 72 ° C. as final elongation reaction. DNA fragments formed by PCR were separated by electrophoresis on 2% agarose gel. The formed bands were stained with ethidium bromide and then photographed with a film of polaroid under ultraviolet light and analyzed with a densitometer (Pharmacia).

Cloning and Sequencing

Fractionally expressed bands were extracted from the gels as agar using the GENECLEAN II kit, GLASSMILK gel extraction kit (Q-BIOgene, # 1001-400, UK). Each DNA fragment was cloned using the TOPO TA cloning kit (Invitrogen, K4500-01, CA) as follows: pCR PCR product with 3 adenine protruding from the end  2.1-TOPO  The vector was inserted and the recombinant vector was transformed into competent E. coli . Amplified DNA was extracted using AccuPrep Plasmid Extraction Kit (Bioneer, # K-3030-1, South Korea). DNA sequencing was performed by Macrogen Company (South Korea).

Primer sequence

The DNA sequences of each gene were compared and confirmed by comparison with the sequences known in GenBank (NIH, MD). Primer sequences for each gene were designed using the PRIMER 3 program (MIT, MA). The synthesis of the primers was carried out at Bioneer Company (South Korea).

Semi-quantitative RT-PCT analysis

CDNA was synthesized against RNAs extracted from five of six alum patients using the original cDNA strand synthesis kit (AMV) for RT-PCR (Boehringer Mannheim, 1483 188, Germany). Sense and antisense primers were designed based on subcloned cDNA sequences (see FIG. 1). Primers for human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as internal standards. The GAPDH sense primers are 5'-ACCACAGTCCATGCCATCAC-3 'and the antisense primers are 5'-TCCACCACCCTGTTGCTGTA-3', which produces 452-bp of product.

PCR amplification was performed using DNA Thermal Cycler 9600 (Perkin Elmer) in 40 μl reaction solution consisting of 10 × reaction buffer, 2.5 mM MgCl ₂ , 250 μM dNTPs, 100 ng PCR primer, 0.5 U Taq DNA polymerase and 2050 ng DNA. Was carried out. PCR amplification for three primer sets (3-1, 64-1 and 77-4) was performed 25 cycles of 1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C. The program for 14-1 is 30 cycles of 1 minute at 94 ° C, 1 minute at 57 ° C and 1.5 minutes at 72 ° C. For GAPDH, 25 cycles of 1 minute at 94, 1 minute at 59 ° C, and 1 minute at 72 ° C. DNA fragments prepared by PCR were isolated by electrophoresis on 2% agarose gel. The bands were photographed using a polaroid film under ultraviolet light after ethidium bromide staining. The optical density of each band was analyzed with a densitometer (Pharmacia). The relative amount of mRNA was confirmed by calculating the ratio of the amount of each mRNA to the amount of GAPDH.

Real-Time PCR

Total RNAs isolated from seven alum patients were used. CDNA was synthesized from RNA using First Strand cDNA Synthesis Kit (Boehringer Mannheim). TaqMan  TaqMan using Universal PCR Master Mix Kit (Perkin Elmer)  PCR was performed. TaqMan with oligonucleotide PCR primers and FAM as 5 'reporter and TAMRA as 3' reporter  Probes were designed using the Primer-Express program (Perkin Elmer) and synthesized at MuenBio Inc. (Baltimore, MD, USA). The sequence is as follows: eIF41 sense primer, 5'-CTGGCCAGAGGCATTGATGT-3 ', eIF41 antisense primer, 5'-CCACACCTTTACGGCCAAAC-3' and eIF41 TaqMan  Probe, 5'-TTTCTTTAGTCATCAACTATGACCTTCCCACCAA-3 '.

Because of the difference in the total amount of RNA added to each reaction, the endogenous control was used as the active standard to normalize the quantification of mRNA targets. Β-actin mRNA was assessed using human hu β-actin (20X) kit (Perkin Elmer). Primer and TaqMan  Fluorescence probes were added to 0.9 and 0.25 μM, respectively. The total PCR volume is 20 μl, which contains 1 μl of cDNA, which is equivalent to 50 ng of total RNA. Three reaction tubes were set for each sample and transcript. The tube was placed in an ABI Prism 7900HT System, which was programmed with 40 continuous cycles consisting of 2 minutes at 50 ° C., 1 / 10th cycle at 95 ° C., followed by 15 seconds at 95 ° C., and 40/1 chloro at 60 ° C. will be. Data was analyzed using the SDS 2.1 program (Perkin Elmer). The comparative Ct method uses an arithmetic formula to obtain the same result for relative quantification. Normalized to the intrinsic standard and the amount of target relative to the adjuster is given by 2 ^−ΔΔCT .

The results obtained from the three reactions were analyzed using the Wilcoxon's Signed Ranks Test to investigate the difference between ATRA-treated and excipient-treated samples. P -values less than 0.05 are considered statistically significant.

Preparation of Anti-rheIF4A1 Antibodies

For cloning of recombinant human eIF4A1 (rheIF4FA1), human keratinocytes were isolated (Boyce ST., Et al., J. Invest Dermatol 81: 33s-40s (1983)). Total RNA was extracted from human keratinocytes initially cultured by TRIZOL (Invitrogen) method. CDNA was prepared using oligo-dT primers and MMLV RT-PCR kit (Promega). EIF4A1 cDNA was amplified by PCR using 5'-ATGTCTGCGAGCCAGGATTCC-3 'as forward primer and 5'-TCAGATGAGGTCAGCAACATTG-3' as reverse primer. The temperature cycle used in the PCR is as follows: 10 minutes at 95 ° C., then 30 seconds at 95 ° C., 30 seconds at 58 ° C. and 35 cycles of 1.5 minutes at 72 ° C., then 10 minutes at 72 ° C. PCR products were cloned into the PCR2.1-TOPO vector (Invitrogen). Cloned genes were sequenced and compared to known sequence data for human eIF4A1 (GenBank accession no. BC009585). Polyclonal anti-eIF4A1 antibody was prepared using the cloned rheIF4F1 gene (Labfrontier Co., Korea).

Western blotting analysis for eIF4A1

Epidermal samples were frozen by impregnation with liquid nitrogen. The frozen tissue is ground to powder and ice-cold homogenization buffer [50 mM Tris-base (pH 7.4), 150 mM NaCl, 10 mM EDTA, 0.1% Tween-20 and protease inhibitor (0.1 mM phenylmethylsulfonylfluoride , 5 μg / ml aprotinin and 5 μg / ml leupetin). Homogenates were centrifuged at 12,000 x g for 30 minutes and the supernatant was collected. Protein concentration of the supernatant was determined using the DC Protein Assay Kit (Bio-Rad Laboratories, Inc. USA). Equal amounts of protein (20 μg) were separated by 7% SDS-PAGE and transferred to nitrocellulose membrane. The membrane was placed in a nonfat dry milk blocking solution in Tris-buffered saline (TTBS) containing 10 mM Tris (pH 7.6), 150 mM NaCl and 0.1% Tween-20 for 1 hour at room temperature. The rabbit polyclonal anti-eIF4A1 antibody diluted 1: 4000 was reacted with the membrane overnight at 4 ° C. in blocking solution. After the reaction, the membrane was reacted with anti-rabbit horseradish peroxidase-conjugated antibody (Pharmingen, USA), washed with TTBS and diluted 1: 2000, for 1 hour at room temperature in a blocking solution. The membrane was washed for 30 minutes in TTBS, then treated with enhanced chemiluminescent solution (ECL kit; Amersham Life Science, UK) for 1 minute and then exposed to X-ray film (Hyperfilm, Amersham). To monitor the amount of protein loaded in each lane, the membranes were treated with antibodies against β-actin. The membrane was stripped with stripping buffer (0.1 M glycine, pH 2.5), and then the membrane was reacted with mouse monoclonal anti-actin antibody (Sigma, USA) diluted 1: 5000 at room temperature for 1 hour in blocking solution. The membrane was then reacted with a secondary antibody in the same manner as above. Immediately after exposure to the film, protein bands were analyzed by densitometry.

Comparison of eIF4A1 Protein Expression in Lesions and Normal Patients with Vitiligo

Irrespective of the response to ATRA treatment, we analyzed the difference in the expression level of eIF4A1 protein in the lesion and normal of vitiligo itself. The normal and lesion epidermis of 22 patients were used as samples, and the analysis method is almost the same as the Western blotting method. Β-actin was used as an internal control.

Experiment result

Fractionally Expressed cDNA Amplification Products

The expression levels of specific mRNAs for normal pigmented epidermis, excipient treated depigmented epidermis and ATRA treated depigmented epidermis from the same patient were compared. The expression levels of approximately 10 mRNAs expressed were different in normal pigmented and depigmented epidermis. The expression level was lower in the depigmented epidermis than in the normal pigmented epidermis, and the extent of this difference was different between patients (FIG. 1). Reduced mRNA expression in the excipient-treated epidermis compared to normal pigmented epidermis was partially or fully recovered after the ATRA treatment (arrow) and others were reduced (arrow head) (FIG. 1).

Detection of genes from cloning and sequencing

Bands of mRNAs showing clear results were amplified using the TOPO TA Cloning Kit (Invitrogen, K4500-01, CA). The sequences of the four genes correspond to parts of known genes. They are consistent with the translational initiator 4A1 (eIF4A1) (Figure 2), ribosomal protein L13 (L13), RNA polymerase mediator for transcription (MRT) and ribosomal phosphoprotein PO (PO) gene sequences in eukaryotic cells. (Table 1).

Genes and primers showing elevated expression levels by ATRA treatment in alum Clone PCR primers Amplification size (bp) mRNA size (kb) Similarity 3-1 ³⁵⁰ 5'-AAGGTGGTCATGGCACTAGG-3 '5'-CTGGCCGTGTGTTTGATATG-3' 228 1.4 eIF4A1 AT ¹ 5'-GTCTGCGAGCCAGGATTCC-3 '5'-CAATGTTGCTGACCTCATCT-3' 1217 - eIF4A1 14-1 ⁹⁷ 5'-AAGCAGCTTGAGGCATCATT-3 '5'-TCCCTGACCATCTGAGAACC-3' 280 1.2 MRT 64-1 ²²⁷ 5'-GTTCGGTACCACACGAAGGT-3 '5'-CAAACTCATCCTCTTCCCCA-3' 196 1.1 L13 77-4 ⁷⁰¹ 5'-AATGGCAGCATCTACAACCC-3 '5'-CTTGCTGAAAAGGTCAAGGC-3' 218 1.2 PO

Correlation with RT-PCR Results and Clinical Responsiveness

The expression level of eIF4A1 mRNA in the depigmented epidermis was lower than that of normal pigmented epidermis. In experiments with two sets of primers (FIG. 2) the results were similar, each amplifying the full length sequence (about 1.3 kb). The expression level was partially recovered in 3 of 5 patients (Patients # 1, # 2, # 3) who showed favorable clinical responsiveness to ATRA. On the other hand, there was no difference in eIF4A1 mRNA expression by ATRA in two patients (patients # 4, # 5), which showed no difference between clinical responsiveness to ATRA and responsiveness in excipient treatment (FIGS. 3A and 3B). L13, MRT, and PO mRNAs showed similar correlations in 1, 1, and 2 patients, respectively (FIGS. 3A and 3B).

Real-time PCR Results and Western Blot Analysis of eIF4A1

Real time PCR was performed on 7 vitiligo patients. In Figure 4, patients 1, 2, 4, and 5 are patients with good clinical responsiveness to ATRA, and patients 6, 7, and 8 are patients without clinical responsiveness to ATRA. Results from real-time PCR of eIF4A1 in normal pigmented and excipient-treated depigmented epidermis were not always consistent with those obtained from semi-quantitative RT-PCR. The difference between ATRA-treated and excipient-treated epidermis was significantly greater than the results obtained from semi-quantitative RT-PCR. The amount of eIF4A1 mRNA in four patients (patients 1, 2, 4 and 5) showing good responsiveness to ATRA was higher in the ATRA-treated epidermis than in the excipient-treated epidermis, while the other 3 patients (patient numbers 6, 7 and 8) low or similar results were obtained.

Western blots were performed for patients with good clinical responsiveness to ATRA (Patient No. 9) and non-responsive patients (Patient No. 10). In both patients, the excipient treated normal pigmented epidermis showed a higher amount of eIF4A1 protein than the excipient treated depigmented epidermis (see FIG. 5). Patients with good clinical responsiveness to ATRA (Patient No. 9) showed high amounts of 47 kDa eIF4A1 protein in ATRA-treated epidermis, while patients with no response to ATRA (Patient No. 10) had similar expression of eIF4A1 protein. The amount is shown.

Comparison of eIF4A1 Protein Expression in Lesions and Normal Patients with Vitiligo

As can be seen in Figure 6, the eIF4A1 protein was expressed more in the normal epidermis than the epidermis in which vitiligo developed. Therefore, it can be seen that the measurement of the expression level of eIF4A1 protein can be utilized for prognostic determination of vitiligo.

The present invention provides methods and kits for screening responsiveness to drugs effective for the treatment or prevention of vitiligo. The present invention also provides a biological label for responsiveness to drugs effective for the treatment or prevention of vitiligo. On the other hand, the present invention provides a method and kit for screening an effective ingredient effective for the treatment or prevention of vitiligo. The present invention also provides a prognostic determination method of vitiligo. According to the method of the present invention, since the reactivity to vitiligo can be analyzed before administration, tailor-made drug administration can be performed, and thus, the most effective drug treatment can be suggested. In addition, according to the method of the present invention, it is possible to quickly screen candidates of drugs effective for vitiligo. According to the prognostic determination method of the present invention, the future state of vitiligo can be predicted relatively accurately.

Reference

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Claims (22)

A method of screening for responsiveness to a drug effective for the treatment or prevention of vitiligo comprising the following steps: (a) isolating total RNA from epidermal cells isolated from the vitiligo patient treated with the drug; (b) synthesizing cDNA from the isolated total RNA; (c) a primer set that hybridizes to (i) the translation initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene and (iii) an RNA polymerase for transcription PCR using at least one primer set selected from the group consisting of a primer set hybridized to a mediator (MRT) gene of the above and the synthesized cDNA as a template; (d) electrophoresis of the PCR product to obtain a band of the PCR product; And (e) determining that the drug is responsive to the drug when the electrophoretic band pattern exhibits substantially the same electrophoretic band pattern as compared to the positive control obtained from normal epidermal cells. The method of claim 1, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid and calcipotriol. The method of claim 1, wherein the primer set is a primer set that hybridizes to an eIF4A1 gene. The method of claim 1, wherein step (e) is performed by visually comparing the intensity of the electrophoretic band or by quantifying with a densitometer. A method for screening responsiveness to all-trans-retinoic acid (ATRA) for use in the treatment of vitiligo comprising the following steps: (a) separating total RNA from epidermal cells isolated from vitiligo patients treated with ATRA; (b) synthesizing cDNA from the isolated total RNA; (c) PCR using a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of the eukaryotic cell and the synthesized cDNA as a template; And (d) electrophoresis of the PCR product to obtain a band of the PCR product; (e) determining that the drug is responsive to the drug when the electrophoretic band pattern exhibits substantially the same electrophoretic band pattern as compared to the positive control obtained from normal epidermal cells. (Iii) a primer set that hybridizes to the translational initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene, and (iii) an mediator of RNA polymerase for transcription ( A kit for screening responsiveness to a drug effective for the treatment or prevention of vitiligo comprising at least one primer set selected from the group consisting of primer sets hybridized to the MRT) gene. 7. The kit of claim 6, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid and calcipotriol. The kit of claim 6, wherein the primer set is a primer set that hybridizes to the eIF4A1 gene. delete delete delete delete A kit for screening reactivity for all-trans-retinoic acid (ATRA) comprising an anti-eIF4A1 antibody. A method for screening an active ingredient effective for the treatment or prevention of vitiligo comprising the following steps: (a) treating a sample compound or biological molecule of interest to an epidermal cell isolated from a patient having said disease; (b) mediators of (i) translational initiator 4A1 (eIF4A1) gene, (ii) ribosomal protein L13 (L13) gene, and (iii) RNA polymerase for transcription in epithelial cells treated (MRT) ) The amount of expression of at least one gene selected from the group consisting of genes; And (c) when the irradiated expression amount shows substantially the same expression amount as compared with the positive control obtained from normal epidermal cells, determining the effective ingredient for the treatment or prevention of vitiligo. 15. The method of claim 14, wherein the drug is selected from the group consisting of all-trans-retinoic acid (ATRA), vitamin C, trichloroacetic acid and calcipotriol. 15. The method of claim 14, wherein step (c) is performed by visually comparing the intensity of the electrophoretic band or by quantification with a densitometer. (Iii) a primer set that hybridizes to the translational initiator 4A1 (eIF4A1) gene of eukaryotic cells, (ii) a primer set that hybridizes to ribosomal protein L13 (L13) gene, and (iii) an mediator of RNA polymerase for transcription ( MRT) Kit for screening an active ingredient effective for the treatment or prevention of vitiligo comprising at least one primer set selected from the group consisting of a primer set hybridized to the gene. (I) an antibody capable of binding to translational initiator 4A1 (eIF4A1) of eukaryotic cells, (ii) an antibody capable of binding to ribosomal protein L13 (L13), and (iii) a mediator of RNA polymerase for transcription (MRT) A kit for screening an active ingredient effective for the treatment or prevention of vitiligo comprising at least one antibody selected from the group consisting of antibodies having an ability to bind to. Prognosis method of vitiligo in mammals other than humans, comprising the following steps: (a) separating total RNA from epidermal cells in the normal skin area of the patient with vitiligo or epithelial cells in the lesion site with vitiligo; (b) synthesizing cDNA from the isolated total RNA; (c) PCR using a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of the eukaryotic cell and the synthesized cDNA as a template; (d) electrophoresis of the PCR product to obtain a band of the PCR product; And (e) in the band of step (d) (iii) the PCR bands obtained from epidermal cells in the normal skin area of the patient with vitiligo show reduced band strength compared to the positive control obtained from normal epidermal cells Determining that vitiligo is highly likely to develop in the normal skin area, or (ii) when the PCR band obtained from epidermal cells of the lesion site shows increased intensity as compared to the previous, vitiligo is improved. step. Prognosis method of vitiligo in mammals other than humans, comprising the following steps: (a) obtaining a cell homogenate comprising total protein from epidermal cells in the normal skin area of the patient with vitiligo or epithelial cells in the lesion site with vitiligo; (b) reacting the homogenate with an anti-eIF4A1 antibody; (c) detecting an antigen-antibody immune response signal between the protein in the homogenate and the anti-eIF4A1 antibody; And (d) when the signal obtained from epidermal cells in the normal skin area of the patient with vitiligo symptoms shows a reduced signal intensity compared to the positive control obtained from normal epidermal cells. Determining that vitiligo is highly likely to develop in normal skin areas, or (ii) determining that vitiligo is improved if the signal obtained from epidermal cells at the lesion site shows increased intensity as compared to the previous. A kit for prognostic determination of vitiligo comprising a primer set hybridized to the translational initiator 4A1 (eIF4A1) gene of eukaryotic cells. Prognostic kit for vitiligo comprising anti-eIF4A1 antibody.

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