阅读说明：本技术 瘢痕疙瘩或者增生性瘢痕预防或者治疗用组合物 (Composition for preventing or treating keloid or hypertrophic scar ) 是由 全世华 郑淏允 吴娜拉 金伦希 韩职铉 文弦阿 于 2019-12-04 设计创作，主要内容包括：本发明涉及一种增生性瘢痕的预防及治疗用药学组合物。发明人明示了硫氧还蛋白5、PRRC1、钙囊素、半乳糖凝集素1、丝氨酰胺A、真核起始因子-5A、膜联蛋白A2及脂肪酸结合蛋白-5的表达抑制,可成为增生性瘢痕的改善及治疗的新的目标。在本发明中制造硫氧还蛋白5、PRRC1、钙囊素、半乳糖凝集素1、丝氨酰胺A、真核起始因子-5A、膜联蛋白A2及脂肪酸结合蛋白-5-特异性siRNA,确认了增生性瘢痕的治疗可能性。因此,所述蛋白质或者编码此的基因的抑制在增生性瘢痕诱导细胞消灭并减少胶原蛋白的表达,从而可非常有用的使用在疤痕治疗。(The invention relates to a pharmaceutical composition for preventing and treating hypertrophic scars. The inventor clearly shows that the inhibition of the expression of thioredoxin 5, PRRC1, calcerocystin, galectin 1, serine amide A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5 can become a new target for improving and treating hypertrophic scars. Thioredoxin 5, PRRC1, calcein, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5-specific siRNA were produced in the present invention, and the possibility of treating hypertrophic scars was confirmed. Therefore, inhibition of the protein or the gene encoding the same induces cell destruction and reduces collagen expression in hypertrophic scars, and thus can be very useful for scar treatment.)

1. A pharmaceutical composition for preventing or treating keloids or hypertrophic scars, which comprises, as an active ingredient, a substance that inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide A, eukaryotic initiation factor-5A, annexin A2 and fatty acid-binding protein-5, or inhibits the expression of genes encoding the one or more proteins.

2. The pharmaceutical composition for the prevention or treatment of keloids or hypertrophic scars according to claim 1, wherein the active ingredient is short hairpin RNA, small interfering RNA, microribonucleic acid, antisense oligonucleotide, ribozyme, criprpr-cas 9, deoxyribostam, peptide nucleic acid, peptide, antibody, aptamer, natural extract or chemical substance.

3. The pharmaceutical composition for the prevention or treatment of keloids or hypertrophic scars according to claim 2, wherein the effective ingredient is a short hairpin RNA, a small interfering RNA, a micro ribonucleic acid or an antisense oligonucleotide that inhibits the expression of the gene.

4. A screening method for a prophylactic or therapeutic substance for keloid or hypertrophic scars, wherein the screening method comprises the steps of:

(a) a step of subjecting the tissue derived from the patient with keloid or hypertrophic scar to a test substance treatment; and

(b) analyzing the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, sersamine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5 or the expression level of a gene encoding the protein from the test substance-treated tissue or cell,

judging the test substance as a prophylactic or therapeutic substance for keloid or hypertrophic scar, when the activity of the protein in normal tissues or the expression state of the gene encoding the same is different.

5. A diagnostic composition for keloids or hypertrophic scars, comprising a substance for measuring the expression level of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, sersamine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid-binding protein-5, or one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, sersamine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid-binding protein-5.

6. A method of keloid or hypertrophic scar diagnosis, wherein said diagnosis comprises the steps of:

measuring the expression level of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, sersamine a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5, or one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, sersamine a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5, in a sample obtained from an individual having or expected keloid or hypertrophic scar; and

comparing the measurement result with the expression level of the gene or protein in a sample from a normal individual.

7. A method for treating an individual having a keloid disease or a hypertrophic scar, comprising administering a medical composition comprising a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calpain, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calpain, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5, as an active ingredient of the individual having a keloid or hypertrophic scar.

Technical Field

The present invention relates to a composition for preventing or treating keloid or hypertrophic scar.

Background

When deep dermis is damaged due to surgery or trauma, collagen in the dermis, which maintains skin tension, is excessively proliferated, thereby penetrating thin skin after wound healing, leaving a trace of skin recovery, which is called "common scar". This is the result of healing of the wound, or when there is an impairment in the functioning of the proper regulation and inhibition of the wound healing process, an abnormally dense growth of fibrous tissue, which can generate Hypertrophic scars (hypertrophical scars) or keloids (keloids). Hypertrophic scars do not go beyond the wound and gradually disappear over time, but keloids have a difference that over time grow wider than the damaged area, invading normal skin. For the treatment of hypertrophic scars or keloids, attempts have been made to include surgical treatment, radiation treatment, steroid treatment, occlusive dressing by silica gel, laser treatment, but these have only limited effects and no established method has been established.

The cause of the generation of hypertrophic scars or keloids is known as excessive activation of cell migration in the wound healing phase, or excessive formation of tissue cells and capillaries by abnormal proliferation, or excessive accumulation of collagen due to failure to normally decompose. Registered patent No. 10-1505294 discloses a starfish hatching extract useful for hydrolyzing collagen to reduce scar formation, and registered patent No. 10-1697396 discloses a method for treating hypertrophic scars using a compound targeting Connective Tissue Growth Factor (CTGF) associated with fibrosis.

There is little information on the formation of collagen and biomarkers involved in cell proliferation, which are accumulated more than necessary in the process of healing scars. By selecting such a biomarker, it is possible to accurately diagnose an abnormal scar such as a hypertrophic scar and screen a substance targeting the biomarker, thereby inhibiting and treating the formation of a hypertrophic scar or a keloid.

Disclosure of Invention

Problems to be solved by the invention

The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of keloids (keloids) or Hypertrophic scars (Hypertrophic scars).

It is a further object of the present invention to provide a method for the prevention or treatment of keloids (keloids) or Hypertrophic scars (Hypertrophic scars).

It is another object of the present invention to provide a method for screening a prophylactic or therapeutic substance for keloids (keloids) or Hypertrophic scars (Hypertrophic scars).

It is another object of the present invention to provide a method for diagnosing keloids (keloids) or Hypertrophic scars (Hypertrophic scars).

Means for solving the problems

For the above purpose, the inventors compared a plurality of protein expression differences exhibited in hypertrophic scar tissue and normal tissue, thereby assuming that a protein marker exhibiting an abnormal expression state is a cause of hypertrophic scars, and selected a protein exhibiting a different expression state from that of normal tissue.

Thus, the present invention provides a diagnostic composition for keloids or hypertrophic scars, which comprises a substance for measuring the expression level of one or more genes selected from the group consisting of thioredoxin 5(TXNDC5), PRRC1, calcein (S100a11), Galectin 1(Galectin 1), serine amide a (filamin a), eukaryotic initiation factor-5A (eIF-5A), Annexin a2(Annexin a2) and fatty acid binding protein-5 (FABP5), or one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, Galectin 1, serine amide a, eukaryotic initiation factor-5A, Annexin a2 and fatty acid binding protein-5.

Also, the present invention provides a method for diagnosing keloid or hypertrophic scars, comprising the steps of: measuring the expression level of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, sersamine a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5, or one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, sersamine a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5 in the tissue of keloid or hypertrophic scar; and comparing the measurement result with the expression amount of the gene and protein from a normal tissue.

The measurement of the expression level of the gene or protein can be carried out by various methods known in the art. For example, RT-PCR (Sambrook et al, Molecular cloning. A Laboratory Manual,3rd ed. Cold Spring Harbor Press (2001)), northern blotting (Peter B. Kaufma et al, Molecular and Cellular Methods in Biology and Medicine, 102) 108, CRC Press), hybridization reactions using cDNA microarrays (Sambrook et al, Molecular cloning. A Laboratory Manual,3rd ed. Cold Spring Harbor Press (2001)), immunoblotting, or in vivo (in situ) hybridization reactions (Sambrook et al, Molecular cloning. A Laboratory Manual,3rd ed. Cold Spring Harbor Press (2001)).

Furthermore, the inventors identified the expression of the selected protein in keloid or hypertrophic scars and normal tissues as immunoblot and immunohistochemical staining. Reduced expression of type I collagen, alpha-SMA and PCNA proteins was confirmed when the individual genes encoding the proteins were reduced (knock-down) in keloid or hypertrophic scar tissue. These results support that inhibition of the protein or reduction of the gene encoding it is very useful for keloid or hypertrophic scar treatment.

Thus, the present invention provides a pharmaceutical composition for preventing or treating keloids (keloids) or Hypertrophic scars (Hypertrophic scars), comprising, as an active ingredient, a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5.

Also, the present invention provides a method for treating an individual having a keloid disease or a hypertrophic scar, comprising administering a medical composition comprising (a) a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5, as an active ingredient of the individual having a hypertrophic scar or a keloid wound.

In the present invention, the substance inhibiting the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5 may include shRNA (short hairpin RNA ), siRNA (small interference RNA), miRNA (microRNA ), Crispr-cas9, Crispr-cpf1, ribozyme (ribozyme), deoxyribozyme (DNAzyme), PNA (peptide nucleic acid), and antisense oligonucleotide, and the substance inhibiting the activity of a protein expressed from the gene may include an antibody, an aptamer, a natural extract, or a chemical substance, and any substance that inhibits the expression of the gene and the activity of the protein may be included without limitation.

The term "shRNA (short hairpin RNA)" used in the present specification is RNA having a single line of 45 to 70 nucleotides in length, and when oligonucleotide DNA to which a 3 to 10-base linker (linker) is ligated is synthesized between the sense (sense) and complementary nonsense of a target gene siRNA base sequence and then cloned into a plasmid vector, or shRNA is inserted into and expressed in retrovirus lentivirus (lentivirus) and adenovirus (adenovirus), shRNA having a circular hairpin structure is prepared, and the effect of RNAi is expressed by intracellular Dicer conversion into siRNA.

The term "siRNA" in the present invention means a nucleic acid molecule that interferes with RNA or mediates gene silencing (see WO00/44895, WO01/36646, WO99/32619, WO01/29058, WO99/07409 and WO 00/44914). siRNA can inhibit the expression of a target gene, and thus is provided as an effective gene reduction method or gene therapy method. siRNAs were first discovered in plants, worms, Drosophila and parasites, and siRNAs were recently developed/utilized for mammalian cell research (Degot S, et al 2002; Degot S, et al 2004; Ballout L, et al 2005).

The siRNA molecule of the present invention may have a double-stranded structure in which a sense line (a (correlating) sequence corresponding to the mRNA sequence of each gene) and an antisense line (a sequence complementary to the mRNA sequence of each gene) are located on opposite sides of each other. Also, the siRNA molecule of the present invention may have a single-stranded structure having self-complementary (self-complementary) meaning and an antisense strand. The siRNA is not limited to complete pairing of double-stranded RNA portions formed only by pairing between RNAs, and may include portions in which no pairing is formed by mispairing (corresponding bases are not complementary), bulging (no base corresponding to one strand), and the like. The total length is 10 to 100 bases, preferably 15 to 8 bases, more preferably 20 to 70 bases, most preferably 20-30 bases.

The term "mirna (microrna)" as used in the present invention is a single line RNA molecule of 21 to 25 nucleotides, which is a regulatory substance that controls gene expression of eukaryotes by inhibition in a step of destruction or detoxification of target mRNA. These mirnas are formed by a two-step process. The primary miRNA transcript (primary miRNA) is produced into a stem-loop structure of about 70 to 90 bases in the nucleus by the RNase type III enzyme of Drosha, i.e., a pregna, and then moved to the cytoplasm, and cleaved by Dicer enzyme to produce a mature miRNA of 21 to 25 bases. The miRNA thus formed complementarily binds to mRNA to function as a post-transcriptional gene suppressor (post-transcriptional gene suppressor), and induces translational inhibition and mRNA instability. mirnas are involved in a variety of physiological phenomena and diseases.

The term "antisense oligonucleotide" as used herein means a DNA or RNA containing a complementary nucleic acid sequence in a sequence of a specific mRNA, or a complementary sequence in an mRNA to which these derivatives bind, and which functions to prevent translation of the mRNA as a protein. The antisense sequence of the present invention means a DNA or RNA sequence that is complementary to each of the genes and can bind to the mRNA of each of the genes, and may interfere with the necessary activities for translation, cytoplasmic translocation, maturation, or all other overall biological functions of the mRNA of the genes. The length of the antisense nucleic acid may be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

According to a specific example of the present invention, the substance may be siRNA. More specifically, the siRNA for decreasing thioredoxin-5 can be composed of sense5'-GGCCCUAACUAGAGUUCUAtt-3' (SEQ ID NO.1) and antisense 5'-UAGAACUCUAGUUAGGGCCtt-3' (SEQ ID NO. 2); the two siRNAs for reducing PRRC1 consist of sense 5'-CAAGAAGACCCUAGAAUUAtt-3' (SEQ ID NO.3), antisense 5'-UAAUUCUAGGGUCUUCUUGtt-3' (SEQ ID NO.4), sense5' -UAUCAAAUCUGGUGAAtt-3' (SEQ ID NO.5) and antisense siRNA UUCACCCAGAAUUUGUAtt-3 ' (SEQ ID NO. 6); and the siRNA for reducing the calcein is formed by the base sequences of sense GAACUAGCUGCCACAAtt-3' (SEQ ID NO.7) and antisense 5'-UUGUGAAGGCAGCUAGUUCtt-3' (SEQ ID NO. 8).

The term "treatment" in this specification means (i) prevention of keloid or hypertrophic scars; (ii) inhibiting or ameliorating the formation of keloids or hypertrophic scars; and (iii) alleviation of the associated disease or disorder in terms of inhibiting or ameliorating the formation of keloids or hypertrophic scars. The term "therapeutically effective amount" in the present specification means an amount sufficient to achieve the pharmacological effect.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating keloid (keloid) disease or hypertrophic scar, comprising (a) a therapeutically effective amount of a substance that inhibits, as an active ingredient, the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2 and fatty acid binding protein-5; and (b) pharmaceutically acceptable carriers (carriers).

Pharmaceutically acceptable carriers included in the compositions of the invention are included as commonly used in formulations including: lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. The pharmaceutical composition of the present invention may include, in addition to the ingredients, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like.

The pharmaceutical composition of the present invention is preferably administered parenterally, for example, by intravenous administration, intraperitoneal administration, intratumoral administration, intramuscular administration, subcutaneous administration, hepatic portal vein administration, hepatic artery administration or local transdermal administration.

The appropriate dosage of the pharmaceutical composition of the present invention can be varied depending on factors such as the preparation method, the administration method, the age, body weight, sex, degree of disease symptoms, diet, administration time, administration route, excretion rate and response sensitivity of the patient, and the effective dosage for the target treatment can be easily determined and prescribed by a generally skilled physician.

The pharmaceutical composition of the present invention is formulated using pharmaceutically acceptable carriers and/or excipients, according to a method conveniently carried out by those skilled in the art, to be manufactured in a unit volume form or to be contained in a multi-volume container. In this case, the dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium or an extract, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention can be used for a skin external preparation for keloid or hypertrophic scar improvement or treatment. In this case, the formulation thereof is not particularly limited depending on the body part. Specifically, for example, it may be a cosmetic composition having a dosage form of a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a mask, a gel, or a skin-adhesive type cosmetic, and may be a transdermal administration dosage form such as an emulsion, an ointment, a gel, a cream, a patch, or a spray. In addition, in the external preparation composition having each formulation, other components than the pharmaceutical composition of the present invention can be easily selected and combined by those skilled in the art according to the formulation of other external preparations for skin or the purpose of use, and in this case, when used together with other raw materials, an enhancing effect can be produced.

Further, the present invention may provide a screening method for a prophylactic or therapeutic substance for keloid (keloid) disease or hypertrophic scar, comprising the following steps. As (a) a step of performing a test substance treatment on keloid or hypertrophic scar cells; and (b) analyzing the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine amide a, eukaryotic initiation factor-5A, annexin a2, and fatty acid binding protein-5 or the expression level of a gene encoding said protein from the tissue or cell treated with said test substance, and determining said test substance as a prophylactic or therapeutic substance for keloid or hypertrophic scar when the activity of said protein or the expression level of the gene encoding said protein is different from that of a normal tissue.

According to the method of the present invention, the test substance to be analyzed is first contacted with the keloid or hypertrophic scar tissue. The term "test substance" used in reference to the screening method of the present invention means an unknown substance used in screening in order to examine the expression amount of the gene or the amount of the protein or whether the activity is affected. The test substance includes, but is not limited to, chemical substances, nucleotides, antisense RNA, shRNA, miRNA, siRNA (small interference RNA), and natural extracts.

Then, the tissue or cell to be treated with the test substance is analyzed for the expression level of the gene and the protein in the cell. When the expression of the gene or the activity or expression of the protein is decreased in the cell as a result of the measurement, the test substance can be judged as a prophylactic or therapeutic substance for keloid or hypertrophic scar.

Effects of the invention

The present invention clarifies the protein which can become the cause of the keloid or the hypertrophic scar and participates in the abnormal collagen formation of the wound part, and the hypertrophic scar can be accurately diagnosed by using the corresponding protein or the gene for coding the protein. Further, a substance which inhibits the expression and activity of the protein or gene is provided, whereby improvement or treatment of keloid-type or hypertrophic scars can be effectively utilized.

Drawings

Figure 1 is a graph showing immunohistochemical staining of a postcomplement protein to hypertrophic scar tissue in humans.

FIG. 2 shows the results of confirming the protein inhibitory efficiency of siRNA targeting three postcomplement proteins by immunoblotting treatment.

Fig. 3 is a graph showing the results of confirming the expression state of collagen by immunoblotting after hypertrophic scar tissue in which the complement protein was inhibited.

Detailed Description

The present invention will be described in more detail below with reference to examples. These examples are provided only to illustrate the present invention more specifically, and those skilled in the art will appreciate that the scope of the present invention is not limited to these examples in light of the emphasis of the present invention.

Examples

Preparation of human dermal fibroblasts, normal tissue and hypertrophic scar tissue

Skin tissues used for the experiment were obtained from 3 patients, and IHC was performed using normal and scar tissues of male and female, and the effect of transformation by siRNA was observed using primary fibroblast cells (primary fibroblast cells) derived from each tissue.

After washing the tissue with 70% ethanol (ethanol), the tissue for IHC was isolated by building (trimming) to remove fat and cutting (chopping). After further trimming of the remaining tissue, it was minced and put into a solution mixed with collagenase (collagenase), trypsin (trypsin) and EDTA, and the cells were detached at 37 ℃ and 100 rpm. The isolated cells were cultured in F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and gentamicin (gentamycin).

Comparison of expression of post-complement proteins by Immunohistochemical (IHC) experiments on hypertrophic scar tissue

Tissues obtained from Biopsy (Biopsy) were placed in an O.C.T compound (Cell Poth, KMA-0100-00A) and frozen in dry ice. The frozen tissue was fixed in 1:1 buffer by Acetone (Acetone) and methanol (methanol). After releasing the buffer, 0.5% Triton X-100 was treated at room temperature for 10 minutes to allow permeation, and then the peptidase activity was inhibited by using ultravisible hydrogen peroxide (Thermo kit/Ultravision LP detection system). After 10 minutes of reaction in a normal temperature suprablock (Ultravision block) buffer, the corresponding primary antibody was treated and treated overnight at 4 ℃ (overnight). Thioredoxin 5(Abcam, Abcam 155684), PRRC1(Abcam/Ab12544), calpain (Abcam/Ab97329), galectin 1(Abcam, Abcam 108389), serine amide A (Millipore/MAB1680), eIF5-A (Abcam/Ab32014), annexin A2(Cell singnaling/8235), and fatty acid binding protein-5 (Abcam/Ab37267) were used as the primary antibody, respectively. After treating a Primary anti-enhancer (Primary anti-enhancer) at room temperature for 10 minutes, an enzyme-labeled secondary antibody (HRP polymer) was reacted at room temperature for 15 minutes in a light-blocked state. After removal of HRP polymer, all tissues from which PRRC1 was removed were treated by AEC (Spring, ASS-125), PRRC1 by DAB (Thermo, TA-125-HDX) for 1 min. After staining the tissue by Mayer's hematoxylin at room temperature and dehydrating, mounting (blunting) was performed by Canada balsam mixed Xylene (Canada balsam mixed Xylene), and then observed by a microscope.

IHC results showed that thioredoxin 5, PRRC1, calcerin, galectin 1, serine amide A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5 showed increased expression in hypertrophic scar tissue compared to normal tissue. Differences in expression levels are shown depending on the positions of dermis (dermis) and epidermis (epidermis) and layers, and the results of differences in expression of postcomplement proteins are shown in table 1 below and fig. 1.

[ TABLE 1 ]

Table of results of comparison of human fibroblast normality/scar to protein expression and position

Candidate	Differential results of expression
		Thioredoxin 5	Scar epidermis, increased dermal expression
PRRC1	Scar epidermis, increased dermal expression
		Calcium bursin	Scar epidermis, increased dermal expression
Galectin 1	Increased scar dermal expression
		Serine amide A	Scar epidermis, increased dermal expression
Eukaryotic initiation factor-5A	Scar epidermis, increased dermal expression
		Annexin A2	Scar epidermis, increased dermal expression
Fatty acid binding protein-5	Increased scar epidermal expression

Form-to-form conversion effect change of post-supplementation protein specific siRNA-expression change of collagen type I, a-SMA and PCNA of fibroblast of hypertrophic scar tissue

Among the proteins selected by the ICH results, the expression of collagen, fibroblast proliferation-related proteins, and the like, which are overexpressed in scars, were analyzed after the expression of each gene was reduced (knock-down) by siRNA for three kinds (thioredoxin 5, PRRC1, calcerin).

Specifically, siRNA using thioredoxin-5 reduction can be represented by sense5'-GGCCCUAACUAGAGUUCUAtt-3' (SEQ ID NO.1) and antisense 5'-UAGAACUCUAGUUAGGGCCtt-3' (SEQ ID NO. 2); the two siRNAs for reducing PRRC1 consist of sense 5'-CAAGAAGACCCUAGAAUUAtt-3' (SEQ ID NO.3), antisense 5'-UAAUUCUAGGGUCUUCUUGtt-3' (SEQ ID NO.4), sense5' -UAUCAAAUCUGGUGAAtt-3' (SEQ ID NO.5) and antisense siRNA UUCACCCAGAAUUUGUAtt-3 ' (SEQ ID NO. 6); and the siRNA for reducing the calcein consists of sense GAACUAGCUGCCACAAtt-3' (SEQ ID NO.7) and antisense 5'-UUGUGAAGGCAGCUAGUUCtt-3' (SEQ ID NO. 8).

Specifically, fibroblasts isolated from hypertrophic scar tissue were cultured in F12 medium supplemented with 10% FBS, and then transfected (transfection) with the siRNA that reduces each protein. 48 hours after transfection of siRNA, the lysate (lysate) was extracted and immunoblotting (western blot) was performed to observe changes in expression of collagen and the like. As a result, it was confirmed that the target protein transfected by siRNA was decreased (FIG. 2), and the effect of decrease in the target protein on the function and growth of fibroblasts was confirmed by changes in the expression of collagen-1, α -SMA and PCNA protein. As a result of the observation, it was confirmed that the expression of collagen-1, α -SMA and PCNA proteins was suppressed when the expression of all the three genes was inhibited (FIG. 3).

In conclusion, inhibition of collagen synthesis and fibroblast proliferation, which are overproduced in hypertrophic scars, are effectively inhibited and cell destruction is induced by inhibition of the respective proteins, and thus, it was confirmed that the postsupplementation protein may be a target for improvement or treatment of hypertrophic scars.

While certain aspects of the present invention have been described in detail, those skilled in the art will appreciate that these specific techniques are merely preferred embodiments and are not intended to limit the scope of the invention. The substantial scope of the present invention can be defined by the claims and equivalents thereof.

Sequence listing

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