CN114480398A

CN114480398A - siRNA and application thereof in preparing medicine for improving and/or treating leucoderma

Info

Publication number: CN114480398A
Application number: CN202210257753.5A
Authority: CN
Inventors: 邹璞玉; 肖嵘; 曾茁桐; 邓前程; 史雅倩
Original assignee: Second Xiangya Hospital of Central South University
Current assignee: Second Xiangya Hospital of Central South University
Priority date: 2022-03-16
Filing date: 2022-03-16
Publication date: 2022-05-13

Abstract

The invention relates to the technical field of biological medicines, in particular to siRNA and application thereof in preparing a medicine for treating leucoderma. The nucleotide sequence of the siRNA is shown as SEQ ID NO.1, or the nucleotide sequence of the siRNA is shown as SEQ ID NO:1 or a nucleotide sequence which is substituted, deleted or added with one or more nucleotides and has the same or similar functions, or a nucleotide sequence which is complementary with the sequence shown in SEQ ID NO. 1. The siRNA can specifically inhibit OCLN in leucoderma CD8⁺Expression on T cells and significantly reduced CD8⁺T cell adhesion to melanocytes and contributes to the reduction of CD8⁺The killing effect of the T cells on melanocytes provides a new target for the research of leucoderma medicament treatment.

Description

siRNA and application thereof in preparation of medicine for improving and/or treating leucoderma

Technical Field

The invention relates to the technical field of biological medicines, in particular to siRNA and application thereof in preparing a medicine for improving and/or treating leucoderma.

Background

Vitiligo is an acquired depigmentation disease and is also the most common cause of depigmentation. Worldwide prevalence is about 1%, and the prevalence of Chinese population is 0.56%. It can appear at any age without sex difference, and the incidence rate of people with dark skin color is higher than that of people with light skin color.

Autoimmune diseases are closely related to the onset of vitiligo, and thus vitiligo, a pigmentary disease, is widely considered to be an autoimmune skin disease. And cytotoxic CD8⁺The killing of melanocytes by T cells is an essential condition in the occurrence and development of vitiligo.

Occn-encoded occludin is the first transmembrane protein found in the tight junction structure. It is an approximately 65kDa protein, comprising four transmembrane domains and two extracellular loops. The tight junction structure is a complex of multiple proteins consisting of transmembrane and cytoplasmic scaffold proteins, the expression and localization of which determine the integrity of barriers such as the blood-brain barrier and the intestinal epithelial barrier. OCLN is an important component of the tight junction complex, and is mainly expressed in epithelial cells, endothelial cells, and keratinocytes. OCLN is in a self-ligand form, and is combined with OCLN of adjacent cells to help epithelial cells, endothelial cells and the like to close the intercellular space, so that the stability of the blood brain barrier and the intestinal physical barrier is improved.

Alexander et al showed that after stimulation of normal T cells with PMA and A23187, activated T cells, whether CD4⁺T cells or CD8⁺Expression of both T cells and OCLN is elevated, and thus OCLN may also have a major impact on activated T lymphocyte function, in addition to playing an important role in the formation and regulation of tight junction cellular bypass permeability barriers. RNAi technology refers to a technique in which, in an organism cell, an mRNA having a homologous sequence is recognized by double-stranded RNA, i.e., small interfering RNA, either exogenous or endogenous, and specifically cleaved to block its translation, thereby finally suppressing the expression of a gene that transcribes the mRNA. At present, the technology is applied to the treatment of various diseases and tumors, and has great advantages. However, siRNA research aiming at vitiligo is less, and no report is found yet for inhibiting the expression of OCLN as a target for vitiligo treatment.

Disclosure of Invention

In view of the above, the present invention provides siRNA and its application in preparing a medicament for improving and/or treating vitiligo.

In order to achieve the above object, the present invention provides the following technical solutions:

the siRNA molecule has a nucleotide sequence of any one of (a) to (c):

(a) as shown in SEQ ID NO. 1;

(b) in SEQ ID NO:1, or a nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in the formula 1 and has the same or similar functions;

(c) a nucleotide sequence complementary to the sequence shown in SEQ ID NO. 1.

In the present invention, the nucleotide sequence of the antisense strand of the siRNA molecule is any one of d in (a) to (b).

In the present invention, the complementarity is complete complementarity or partial complementarity.

Wherein the partial complementarity is that at least 19 consecutive bases are complementary to the sequence shown in SEQ ID NO. 1.

The invention also provides application of the siRNA in preparing a medicament for improving and/or treating leucoderma or in improving and/or treating leucoderma.

The improvement and/or treatment of vitiligo comprises at least one of (1) to (3):

(1) inhibition of OCLN Gene at CD8⁺Expression on T cells;

(2) reduction of CD8⁺T cell adhesion to melanocytes;

(3) reduction of CD8⁺Killing of melanocytes by T cells.

The invention constructs OCLN over-expression recombinant plasmid, which is transfected into CD8 by electrotransformation⁺T cells, then co-cultured with skin explants. As a result, it was found that CD8⁺Increased OCLN expression in T cells, CD8⁺Adhesion ratio after co-culture of T cells with melanocytes and CD8 in skin explants⁺T cell infiltration was significantly higher than that of the normal control group (normal CD of untreated healthy people)8⁺T cells). And the leucoderma patients CD8 after OCLN siRNA transfection⁺OCLN expression was significantly reduced in T cells compared to the patient group, CD8⁺The adhesion ratio of the T cell and melanocyte co-culture is obviously reduced; CD8 in skin explants after transfection of OCLN siRNA compared to normal groups⁺T cell infiltration was significantly reduced.

The above results indicate that OCLN small interfering RNA shown in SEQ ID NO.1 inhibits CD8⁺Binding of T cell surface OCLN to melanocyte surface OCLN such that administration of said small interfering OCLN RNA inhibits CD8⁺T cells adhere to melanocytes.

The core sequence of the recombinant plasmid of the OCLN gene is shown in SEQ ID NO. 1.

In the application provided by the invention, the medicament is in a dosage form of a dressing, a patch, an injection or an oral preparation.

The invention also provides a medicine for improving and/or treating leucoderma, which comprises the siRNA and pharmaceutically acceptable auxiliary materials.

In addition, the invention also provides a method for improving and/or treating leucoderma, which comprises the following steps: administering to the subject an siRNA, or a medicament comprising an siRNA, as described herein.

The nucleotide sequence of the siRNA provided by the invention is shown as SEQ ID NO.1, or the nucleotide sequence is shown as SEQ ID NO:1 or a nucleotide sequence which is substituted, deleted or added with one or more nucleotides and has the same or similar functions, or a nucleotide sequence which is complementary with the sequence shown in SEQ ID NO. 1. Research shows that the siRNA can specifically inhibit OCLN in leucoderma CD8⁺Expression on T cells and significantly reduced CD8⁺T cell adhesion to melanocytes and contributes to the reduction of CD8⁺The killing effect of the T cells on melanocytes provides a new target for the research of leucoderma medicament treatment.

Drawings

FIG. 1 shows CD8 of a normal control group and a transfected OCLN overexpression plasmid provided by the present invention⁺Protein expression level of OCLN in T cells (n-3, P)<0.05)；

FIG. 2 shows a patient suffering from vitiligo according to the present inventionPeripheral blood CD8⁺T cell and OCLN siRNA transfected leucoderma patient peripheral blood CD8⁺mRNA expression level of OCLN in T cells (n-3, P)<0.05)；

FIG. 3 shows melanocytes and differently treated CD8 according to the present invention⁺Adhesion of T cell co-cultures; melanocytes were labeled with MELAN-A, red for MELAN-A positive staining, green for CD8 positive staining, and blue for DAPI staining;

FIG. 4 shows CD8 processed differently as provided by the present invention⁺After co-culture of T cells with skin explants, CD8⁺T cell infiltration into skin explants. Melanocytes were marked with MELAN-A, red for MELAN-A positive staining, green for CD8 positive staining, blue for DAPI staining, yellow for OCLN positive staining, and white at yellow and blue co-stained sites.

Detailed Description

The invention provides siRNA and application thereof in preparing a medicament for improving and/or treating leucoderma. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

In the invention, 9 cases of the peripheral blood of the vitiligo patients are used, all the patients meet the vitiligo diagnosis standard, and other autoimmune diseases, no other kidney diseases, no urinary tract infection and no other serious diseases in the near term are excluded. Matching age and gender. Age and sex matched normal controls had 15 peripheral blood samples and 9 skin samples at the exposed sites.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following is a set of regulatory CD8 provided by the present invention⁺Methods and materials for T cell adhesion to melanocytes are further described.

Example 1: the OCLN over-expression recombinant plasmid and the small interfering RNA preparation of the invention.

The OCLN over-expression recombinant plasmid is synthesized by the agency of Kingsler Biotechnology GmbH, and the sequence is shown as SEQ ID NO. 2.

The small interfering RNA molecule is synthesized by Guangzhou Ruibo biotechnology limited company, and the sequence is shown as SEQ ID NO. 1.

Example 2: detecting CD8⁺Recombinant plasmid for OCLN overexpression in T cells and transfection condition of OCLN siRNA

In this example, 3 cases of peripheral blood of a vitiligo patient and 3 cases of peripheral blood of a healthy control are used.

1. Peripheral blood mononuclear cell separation:

separating peripheral blood mononuclear cells by adopting a density gradient centrifugation method.

2、CD8⁺T cell isolation:

whirling in beautiful sky^TMCD8⁺T cell magnetic beads, CD8 was isolated⁺T cells.

3. OCLN overexpression plasmids and OCLN siRNA transfection

Using Lonza electrotransfer reagent, plasmid and small interfering RNA molecules were electrotransfected into CD8⁺T cells.

4. Cell transfection efficiency assay:

transfection of Normal CD8 with an OCLN overexpression plasmid⁺After T cells, we detected changes in OCLN protein expression in the cells by the western blot method. The results are shown in FIG. 1.

Transfection of vitiligo patient CD8 with OCLN siRNA⁺After T cells, we detected the mRNA expression level of OCLN in the cells by Real-time PCR. The results are shown in FIG. 2.

The results showed that the over-expression OCLN plasmid transfected CD8⁺After T cells, OCLN expression is significantly increased; transfection of Small interfering RNA molecules into CD8+ T cellsAfter cells, the expression of OCLN is obviously reduced, which shows that the small interfering RNA molecule can specifically inhibit the OCLN in leucoderma CD8⁺Expression on T cells.

Example 3: melanocytes and differently treated CD8⁺Adhesion of T cell cocultures

(1) When the melanocytes are passaged to the 2 nd to 4 th generations, the melanocytes are digested by 0.25% pancreatin, counted, seeded in a 12-well plate with a slide plate, and cultured for 1 day with 2 × 104 melanocytes per well to make the melanocytes completely adhere to the wall.

(2) After 24h, the M254 medium was aspirated, and normal CD8+ T cells, normal CD8+ T cells overexpressing OCLN (after 24h of treatment), patient CD8+ T cells, and patient CD8+ T cells transfected with OCLN siRNA (after 24h of treatment) were added to melanocyte-seeded 12-well plates at a ratio of 1X 105 CD8+ T cells per well to CD8+ T cells of 1: 5. The culture was continued for 24 h.

(3) After 24h, the well was aspirated, rinsed 3 times with PBS gently for 3min each time, and fixed with paraformaldehyde for 15 min. The paraformaldehyde solution was aspirated and gently rinsed 3 times with PBS for 3min each.

(2) 0.5% TritonX-100 was incubated for 20 min.

(3) The cells were rinsed 3 times with PBS gently for 3min each.

(4) Starting from the serum block, the procedure followed 2.3.9 multispectral immunohistochemistry. The number of melanocytes and the number of CD8+ T cells adhering to melanocytes were counted per high power of the mirror field to obtain a CD8+ T cell adhesion ratio, which is the number of CD8+ T cells/melanocyte in field, and the obtained adhesion ratio represents the number of CD8+ T cells adhering to each melanocyte. The results are shown in FIG. 3.

The results show that CD8 was treated differently⁺After the T cells and the melanocytes are co-cultured, the CD8 of the leucoderma patients is discovered⁺The adhesion ratio of the T cells and the melanocytes after coculture is obviously higher than that of a normal control group; OCLN over-expressed CD8⁺The adhesion ratio of T cells co-cultured with melanocytes was also significantly higher than that of normal control group. The leucoderma patient CD8 transfected with OCLN siRNA of the invention⁺Adhesion ratio of T cells co-cultured with melanocytes compared with patient groupThe drop is significant. The siRNA provided by the invention can obviously reduce CD8⁺T cells adhere to melanocytes.

Example 4: co-culture of differently treated CD8+ T cells with skin explants

(1) Preparing a skin explant: trimming subcutaneous fascia and fat of normal human skin, and cutting skin into 1-2mm²The small and large fragments are suspended on DMEM medium and cultured for 24 hours.

(2) Normal CD8⁺T cells overexpress OCLN recombinant plasmid, Normal CD8⁺T cells were transfected with OCLN siRNA and cultured for 24 h.

(3) After 24h, the DMEM medium in which the skin explants were cultured was removed and normal CD8 was removed⁺T-cell, OCLN-overexpressing CD8⁺T cells, OCLN siRNA transfected CD8⁺T cells were added to 12-well plates containing skin explants at 1 x 10 per well⁵An individual CD8⁺T cells were cultured for 24 h.

(4) After 24h, fixing the skin explants with formaldehyde to prepare paraffin sections.

(5) Multispectral immunohistochemistry was used to detect the expression of Melan-A, CD8, OCLN in skin explants. The results are shown in FIG. 4.

The results show that the present invention treats CD8 differently⁺Co-culture of T cells with skin explants revealed OCLN-overexpressing CD8⁺Co-culture of T cells with skin explants in which CD8 was present⁺T cell infiltration was significantly higher than normal controls; and OCLN siRNA transfected CD8⁺Co-culture of T cells with skin explants in which CD8 was present⁺T cell infiltration was significantly lower than normal controls. Indicating that OCLN siRNA-transfected CD8⁺The killing of melanocytes by T cells is reduced.

From the above results, it can be seen that the OCLN small interfering RNA molecule of the invention can inhibit CD8⁺Expression of OCLN in T cells, thereby reducing CD8⁺T cell adhesion to melanocytes, CD8⁺The T cells kill the melanocytes and reduce the melanocytes, has good treatment effect on the vitiligo, and has good clinical application prospect in preventing, improving and treating the vitiligo.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Sequence listing

<110> Xiangya II Hospital of Zhongnan university

<120> siRNA and application thereof in preparation of drugs for improving and/or treating leucoderma

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gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420

attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900

gtttaaactt aagcttggta ccgagctcgg atccgccacc atgtcatcca ggcctcttga 960

aagtccacct ccttacaggc ctgatgaatt caaaccgaat cattatgcac caagcaatga 1020

catatatggt ggagagatgc atgttcgacc aatgctctct cagccagcct actcttttta 1080

cccagaagat gaaattcttc acttctacaa atggacctct cctccaggag tgattcggat 1140

cctgtctatg ctcattattg tgatgtgcat tgccatcttt gcctgtgtgg cctccacgct 1200

tgcctgggac agaggctatg gaacttccct tttaggaggt agtgtaggct acccttatgg 1260

aggaagtggc tttggtagct acggaagtgg ctatggctat ggctatggtt atggctatgg 1320

ctacggaggc tatacagacc caagagcagc aaagggcttc atgttggcca tggctgcctt 1380

ttgtttcatt gccgcgttgg tgatctttgt taccagtgtt ataagatctg aaatgtccag 1440

aacaagaaga tactacttaa gtgtgataat agtgagtgct atcctgggca tcatggtgtt 1500

tattgccaca attgtctata taatgggagt gaacccaact gctcagtctt ctggatctct 1560

atatggttca caaatatatg ccctctgcaa ccaattttat acacctgcag ctactggact 1620

ctacgtggat cagtatttgt atcactactg tgttgtggat ccccaggagg ccattgccat 1680

tgtactgggg ttcatgatta ttgtggcttt tgctttaata attttctttg ctgtgaaaac 1740

tcgaagaaag atggacaggt atgacaagtc caatattttg tgggacaagg aacacattta 1800

tgatgagcag ccccccaatg tcgaggagtg ggttaaaaat gtgtctgcag gcacacagga 1860

cgtgccttca cccccatctg actatgtgga aagagttgac agtcccatgg catactcttc 1920

caatggcaaa gtgaatgaca agcggtttta tccagagtct tcctataaat ccacgccggt 1980

tcctgaagtg gttcaggagc ttccattaac ttcgcctgtg gatgacttca ggcagcctcg 2040

ttacagcagc ggtggtaact ttgagacacc ttcaaaaaga gcacctgcaa agggaagagc 2100

aggaaggtca aagagaacag agcaagatca ctatgagaca gactacacaa ctggcggcga 2160

gtcctgtgat gagctggagg aggactggat cagggaatat ccacctatca cttcagatca 2220

acaaagacaa ctgtacaaga ggaattttga cactggccta caggaataca agagcttaca 2280

atcagaactt gatgagatca ataaagaact ctcccgtttg gataaagaat tggatgacta 2340

tagagaagaa agtgaagagt acatggctgc tgctgatgaa tacaatagac tgaagcaagt 2400

gaagggatct gcagattaca aaagtaagaa gaatcattgc aagcagttaa agagcaaatt 2460

gtcacacatc aagaagatgg ttggagacta tgatagacag aaaacagatt acaaggatga 2520

cgacgataag tgataaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 2580

tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 2640

ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 2700

gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg 2760

ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta 2820

tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 2880

gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct 2940

cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 3000

atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag 3060

tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 3120

tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga 3180

tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa 3240

atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc 3300

tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 3360

aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 3420

accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 3480

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tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 3600

ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt 3660

cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 3720

ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 3780

tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 3840

ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 3900

gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 3960

caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 4020

atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 4080

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gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 4200

gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa 4260

aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 4320

gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 4380

ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 4440

cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca 4500

acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 4560

tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 4620

tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4680

caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4740

tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 4800

ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 4860

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4920

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4980

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 5040

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 5100

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 5160

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5220

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5280

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5340

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 5400

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5460

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5520

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5580

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5640

gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5700

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5760

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5820

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5880

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5940

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 6000

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 6060

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 6120

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 6180

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 6240

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 6300

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 6360

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 6420

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 6480

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6540

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6600

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6660

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6720

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6780

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6840

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6900

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc 6959

Claims

An siRNA molecule having a nucleotide sequence of any one of (a) to (c):

(a) as shown in SEQ ID NO. 1;

(b) in SEQ ID NO:1, or a nucleotide sequence which is obtained by substituting, deleting or adding one or more nucleotides in the nucleotide sequence shown in the formula 1 and has the same or similar functions;

(c) a nucleotide sequence complementary to the sequence shown in SEQ ID NO. 1.
2. An siRNA molecule according to claim 1, wherein said complementarity is full or partial complementarity.
3. An siRNA molecule according to claim 2 characterized in that said partial complementarity is that at least 19 consecutive bases are complementary to the sequence shown in SEQ ID NO 1.
4. Use of the siRNA molecule of any one of claims 1-3 in the manufacture of a medicament for ameliorating and/or treating vitiligo, or in the amelioration and/or treatment of vitiligo.
5. The use according to claim 4, wherein the amelioration and/or treatment of vitiligo comprises at least one of (1) to (3):

(1) inhibition of OCLN Gene at CD8⁺Expression on T cells;

(2) reduction of CD8⁺T cell adhesion to melanocytes;

(3) reduction of CD8⁺Killing of melanocytes by T cells.
6. The use according to claim 4, wherein the medicament is in the form of a poultice, a patch, an injection or an oral formulation.
7. A medicament for improving and/or treating vitiligo, comprising the siRNA of any one of claims 1-3 and a pharmaceutically acceptable adjuvant.