KR101943434B1 - Marker composition and kit for diagnosising keloid tissue - Google Patents

Abstract

The biomarker composition according to an embodiment of the present invention includes an agent for detecting any one protein selected from the group consisting of Ezrin (EZRIN), phosphorylated Ephrin (phospho-EZRIN), and a combination thereof, Predict or diagnose the occurrence. The composition can be used as a data for predicting or diagnosing the development of keloid tissue, which can improve the convenience and accuracy of diagnosis and can help prevent or early treatment of the skin tissue of a patient who may develop keloid tissue .

Description

TECHNICAL FIELD [0001] The present invention relates to a biomarker composition for keloid tissue diagnosis and a kit comprising the biomarker composition for keloid tissue diagnosis,

The present invention relates to a biomarker composition useful for predicting or diagnosing occurrence of keloid tissue, a kit containing the same, and the like.

Ezrin is a member of the ERM (ezrin, radixin, moesin) family and is a protein involved in cell mobility, attachment, survival and cell death, which links the plasma membrane and the cytoskeleton.

Recent studies have shown that ezrin is involved in the morphology, growth, and motility of pancreatic malignancy through the Erk1 / 2 pathway, and pancreatic duct cell carcinoma (pancreatic ductal adenocarcinoma, PDAC) plays an early role in development and is known to be involved in the progression in the advancement stage.

Scar formation is a normal physiological response to injury in adults, and these scar tissue are generally caused by overgrowth of dense fibrous tissue during the inflammatory process. The wound healing and scar formation process can be classified into three stages: inflammation, proliferation, and remodeling and maturation. Hyperplastic scar tissue can be classified as keloid (keloid) and hypertrophic scar (hypertrophic scar).

While keloid tissue extends beyond the margins of the original wound, hypertrophic tissue differs from the border of the wound, and the cause of keloid tissue formation, It is not known. The keloid tissue can be removed surgically, and keloid tissue is often reformed after surgery.

On the other hand, changes in the extracellular matrix, such as formation and dissociation imbalance, may be the formation of hyperstimulated scar tissue, but do not fully understand the causal relationship and are presumed to be due to genetic and environmental factors.

1. Meng, Y., et al., Ezrin promotes invasion and metastasis of pancreatic cancer cells. J Transl Med, 2010. 8 (1): p. 61-74. 2. Makitie, T., et al., Ezrin as a prognostic indicator and its relationship to tumor characteristics in uveal malignant melanoma. Investigative ophthalmology & visual science, 2001. 42 (11): p. 2442-2449.

It is an object of the present invention to provide a biomarker composition useful for predicting or diagnosing occurrence of hypertrophic or calloid tissue and a diagnostic kit comprising the biomarker composition.

In order to achieve the above object, a biomarker composition for predicting or diagnosing the development of keloid tissue according to an embodiment of the present invention comprises a group consisting of EZRIN, phosphorylated EZRIN, An agent for detecting any one selected protein; A nucleotide that encodes ezrin, a nucleotide that encodes a protein that activates ezrin, and a preparation that detects any one of nucleotides selected from the group consisting of combinations thereof.

The agent for detecting the protein may be one which detects ezrin protein, phosphorylated phospho-EZRIN protein or both contained in epithelial tissues of the skin.

The biomarker composition comprises an agent that detects any protein selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN (phospho-EZRIN), and combinations thereof to provide information necessary for predicting the development of keloid tissue Or to provide information necessary for diagnosis.

The agent for detecting the protein may be one which detects the ezrin (EZRIN) protein contained in the skin epithelium.

According to another embodiment of the present invention, a kits for predicting or diagnosing occurrence of keloid tissue include a biomarker composition for predicting or diagnosing occurrence of keloid tissue.

The biomarker composition for predicting or diagnosing the development of keloid tissue is one that detects any protein selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN (phospho-EZRIN), and combinations thereof; Or an agent for detecting any one of nucleotides selected from the group consisting of a nucleotide encoding ezrin, a nucleotide encoding a protein activating ezrin, and a combination thereof.

A method of providing information necessary for the differential diagnosis of hypertrophic or keloid tissue according to another embodiment of the present invention is provided by a method selected from the group consisting of Ezrin (EZRIN), phosphorylated Ephrin (phospho-EZRIN) An agent for detecting any one of the proteins; Or a nucleotide encoding ezrin, to detect whether ezinin or phosphorylated azine is overexpressed, and to provide information necessary for differential diagnosis of hypertrophic or keloid tissue.

A method for screening a substance for preventing or treating keloid tissue formation according to another embodiment of the present invention is a method for screening a substance selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN and phospho-EZRIN A first step of preparing a screening cell that overexpresses one protein or a gene expressing the protein; The screening cell or the gene is treated with a substance that inhibits the overexpression of any one protein selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN (phospho-EZRIN), and combinations thereof to prepare a treatment group A second step; And a third step of comparing the degree of protein expression of the treatment group with the degree of protein expression by a cell or gene including an untreated control group not treated with the second step.

Hereinafter, the present invention will be described in more detail.

In a normal healing process, the skin is filled with fibrous proteins known as collagen and shows a rugged appearance characteristic of new wounds. And, collagen collapses and the wound is flattened and contracted. Unlike skin, which normally begins to heal wounds, when skin abnormality occurs, such as keloid tissue or hypertrophic scarring, the skin becomes overfilled with collagen, which appears to expand and expand the wound tissue.

Keloid is a deformed soft tissue that occurs during wound healing and is an abnormally formed scar tissue. It often occurs in deep skin wounds, and it often occurs in skin tissues such as chest and shoulders where high tension is applied. It usually occurs within a few years after wound creation, and has the characteristic that it extends beyond the original wound boundary.

Hypertrophic scars (hypertrophic scars) are similar to keloids in that they are one of the deformed soft tissues that occur during wound healing, but they are characterized by the fact that they do not extend beyond the boundaries of the original wound.

Hypertrophic scars and keloids also have a feature that the arrangement of newly formed collagen fibers is different from normal wound healing. In normal wound healing, newly formed collagen fibers in the net's dermis are arranged parallel to the skin surface, whereas in hypertrophic sclerosis and keloids the thickened and vitrified collagenous fibers gradually become thinner and spread over time, And is arranged parallel to the surface.

Little is known about the cause of keloid formation or why it hardly responds to treatment. Given the most frequent occurrence in African and Asian populations and the emergence of family history, it is assumed that genetic factors are involved. Surgical treatment, such as conventional surgery or cryosurgery, can remove keloid tissue, but recurrence is significant.

Histologically, there is some difference in optical microscope between hypertrophy and keloid. However, both keloid and hypertrophic scars activate collagen production and accumulation with increasing fibroblast density. It is difficult. Therefore, most of them will be diagnosed on the basis of clinical differences.

The inventors of the present invention conducted studies for the purpose of predicting the occurrence of "hypertrophic scars and / or keloids", which are not normal tissues, and providing information necessary for diagnosis of keloids as well as their clinical differences, (EZRIN) and phospho-EZRIN (phospho-EZRIN) in the keloid tissue and the hypertrophic scar tissue, respectively, ) Protein can be used to predict or diagnose the occurrence of keloid tissue, thus completing the present invention.

The biomarker composition for predicting or diagnosing the development of keloid tissue according to an embodiment of the present invention may be any protein selected from the group consisting of Ezrin (EZRIN), phosphorylated Ephrin (phospho-EZRIN) An agent to detect; Or a nucleotide encoding ezrin (EZRIN), a nucleotide encoding a protein that activates ezrin, and a combination of any of these nucleotides.

EZRIN is a member of the ERM family (ezrin, radixin, moesin), which links the plasma membrane and the cytoskeleton and is closely related to cell migration, attachment, survival and cell death It is known as protein. Phosphorylated ezrin (phospho-EZRIN) is the active state of ezrin.

The agent for detecting the protein may be one which is applied to a method for confirming the presence or absence of the protein in the biological sample and preferably includes an antibody that specifically binds to the protein. That is, the agent for detecting the protein may include, for example, Ezrin (EZRIN), phosphorylated EzRIN, or an antibody that specifically binds all of them.

The antibody refers to a specific protein molecule directed to an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein, and includes a polyclonal antibody, a monoclonal antibody, and a recombinant antibody . Since EZRIN and phosphorylated phospho-EZRIN proteins have been identified as described above, it is easy to produce antibodies by using techniques well known in the art. Polyclonal antibodies can be produced by methods well known in the art for obtaining sera containing antibodies by injection of the protein antigens into the animal and collection from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.

As an assay method for detecting the protein, an enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunohistochemistry Assay, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Assay, Immunoassay Assay, Immunoassay Assay, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Assay, Sorter, FACS, and protein chips.

The agent for detecting the nucleotide includes a nucleotide which encodes an azurin protein (e.g., mRNA), a nucleotide that encodes a protein that activates the azurin protein, and a nucleotide that quantitatively detects any one of nucleotides selected from the group consisting of these nucleosides And can be measured using, for example, primers or probes that bind to these nucleotides.

RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA; RNase protection assay, Northern blotting and DNA chip may be used.

The biological sample refers to a sample having a different level of the protein or the gene contained in the biological sample by keloid and / or hypertrophic scar formation, and specifically includes blood, skin tissue, etc., no.

Specifically, a sample containing skin epithelial tissue may be used as the biological sample. In order to predict or diagnose the possibility of keloid or hypertrophic scarring after surgery in the course of surgery involving cutaneous tissue resection, In order to utilize the composition, a biological sample containing a skin epithelial tissue that can be collected at the time of surgical excision can be applied as the above biological sample.

The agent for detecting the protein may be one which detects the content of the ezrin phosphorylated ezrin protein contained in the epithelium of the skin to provide information used for prediction or diagnosis of keloid tissue development.

In the skin epithelium with keloids, the level of expression of ezrin and phosphorylated ezrin is relatively high compared with that of normal tissue or hypertrophic tissue, especially a statistically significant higher value than that of normal tissues.

Therefore, by confirming the degree of expression of azurin and / or phosphorylated azurin in the skin tissue, it is possible to predict whether or not the wound tissue will progress to keloid. If the scar tissue has already developed, it can be used for diagnosis of keloid, .

The inventors of the present invention have also confirmed that the degree of expression of Ezinin protein affects the formation of keloid tissue and the expression of Ezinin protein in keloid and hypertrophic tissue is observed compared to normal tissue, , Which provides a method of providing information used in the diagnosis of keloids and hypertrophic scars by detecting Ezinin protein. In addition, the inventors of the present invention have utilized skin tissues obtained from women in the experiment, considering that the expression of Ezrin protein is affected by the level of estradiol, To reduce bias.

The keloid tissue development prediction or diagnosis kit according to another embodiment of the present invention is a kit that helps predict or diagnose keloid tissue development through analysis of a biological sample of a patient suspected of keloid tissue development. And a biomarker composition for predicting or diagnosing the occurrence of tissue. Specific details of the biomarker composition for predicting or diagnosing the occurrence of the keloid tissue are the same as those described above, and thus the description thereof will be omitted.

When these kits are used to predict or diagnose keloid tissue development, it is possible to predict the occurrence of abnormal scar tissue such as keloid or hypertrophic scars and to improve the diagnostic accuracy of keloid tissue, which is difficult to distinguish from hypertrophic scars, Treatment for or prevention of keloid tissue treatment can be advanced early.

The kit may be, but is not limited to, an immunoassay kit.

A method of providing information necessary for the differential diagnosis of hypertrophic or keloid tissue according to another embodiment of the present invention is provided by a method selected from the group consisting of Ezrin (EZRIN), phosphorylated Ephrin (phospho-EZRIN) An agent for detecting any one of the proteins; Or detecting a nucleotide encoding ezrin.

The method can provide information necessary for predicting or discriminating the occurrence of non-large thoracic or keloid tissues whether azurin, p-azurin or both are overexpressed in normal tissues in the tissue to be diagnosed.

A method for screening a substance for preventing or treating keloid tissue formation according to another embodiment of the present invention is a method for screening a substance selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN, A first step of preparing a screening cell for overexpressing a protein of the present invention; A second step of treating the screening cell with a substance that inhibits overexpression of any one protein selected from the group consisting of EZRIN, phosphorylated phospho-EZRIN, ; And a third step of comparing the degree of protein expression of the treatment group with the degree of protein expression by a cell or gene including an untreated control group not treated with the second step.

As the screening cell, a cell into which a gene inducing the expression or overexpression of the azin protein can be applied. Specifically, the cell can be applied to the NCBI GenBank Reference Sequence (NP_001104547) or the mRNA expressing the azin protein (NCBI GenBank Reference Sequence NM_001111077).

The screening method is a method for screening candidates for effective substances for the prevention or treatment of keloid tissue by detecting a substance that inhibits genes such as the protein (NCBI GenBank Reference Sequence: NP_001104547) or the mRNA expressing the protein (NCBI GenBank Reference Sequence NM_001111077) Can be screened effectively (see Table 1).

Ezine protein sequence NP_001104547 Homo sapiens (human), REFERENCE 1 (residues 1 to 586)
1 mpkpinvrvt tmdaelefai qpnttgkqlf dqvvktiglr evwyfglhyv dnkgfptwlk
61 ldkkvsaqev rkenplqfkf rakfypedva eeliqditqk lfflqvkegi lsdeiycppe
121 tavllgsyav qakfgdynke vhksgylsse rlipqrvmdq hkltrdqwed riqvwhaehr
181 gmlkdnamle ylkiaqdlem yginyfeikn kkgtdlwlgv dalglniyek ddkltpkigf
241 pwseirnisf ndkkfvikpi dkkapdfvfy aprlrinkri lqlcmgnhel ymrrrkpdti
301 evqqmkaqar eekhqkqler qqletekkrr etverekeqm mrekeelmlr lqdyeektkk
361 aerelseqiq ralqleeerk raqeeaerle adrmaalrak eelerqavdq iksqeqlaae
421 laeytakial leearrrked eveewqhrak eaqddlvktk eelhlvmtap ppppppvyep
481 vsyhvqeslq degaeptgys aelssegird drneekrite aeknervqrq lltlsselsq
541 ardenkrthn diihnenmrq grdkyktlrq irqgntkqri defeal Ezrine mRNA sequence NM_001111077 Homo sapiens (human), 1 (bases 1 to 3138)
1 ggcgtggtcc cgggacccgc cccgccgggg cttttgggag cgcgggcagc gagcgcactc
61 ggcggacgca agggcggcgg ggagcacacg gagcactgca ggcgccgggt tgggacagcg
121 tcttcgctgc tgctggatag tcgtgttttc ggggatcgag gatactcacc agaaaccgaa
181 aatgccgaaa ccaatcaatg tccgagttac caccatggat gcagagctgg agtttgcaat
241 ccagccaaat acaactggaa aacagctttt tgatcaggtg gtaaagacta tcggcctccg
301 ggaagtgtgg tactttggcc tccactatgt ggataataaa ggatttccta cctggctgaa
361 gctggataag aaggtgtctg cccaggaggt caggaaggag aatcccctcc agttcaagtt
421 ccgggccaag ttctaccctg aagatgtggc tgaggagctc atccaggaca tcacccagaa
481 acttttcttc ctccaagtga aggaaggaat ccttagcgat gagatctact gcccccctga
541 gactgccgtg ctcttggggt cctacgctgt gcaggccaag tttggggact acaacaaaga
601 agtgcacaag tctgggtacc tcagctctga gcggctgatc cctcaaagag tgatggacca
661 gcacaaactt accagggacc agtgggagga ccggatccag gtgtggcatg cggaacaccg
721 tgggatgctc aaagataatg ctatgttgga atacctgaag attgctcagg acctggaaat
781 gtatggaatc aactatttcg agataaaaaa caagaaagga acagaccttt ggcttggagt
841 tgatgccctt ggactgaata tttatgagaa agatgataag ttaaccccaa agattggctt
901 tccttggagt gaaatcagga acatctcttt caatgacaaa aagtttgtca ttaaacccat
961 cgacaagaag gcacctgact ttgtgtttta tgccccacgt ctgagaatca acaagcggat
1021 cctgcagctc tgcatgggca accatgagtt gtatatgcgc cgcaggaagc ctgacaccat
1081 cgaggtgcag cagatgaagg cccaggcccg ggaggagaag catcagaagc agctggagcg
1141 gcaacagctg gaaacagaga agaaaaggag agaaaccgtg gagagagaga aagagcagat
1201 gatgcgcgag aaggaggagt tgatgctgcg gctgcaggac tatgaggaga agacaaagaa
1261 ggcagagaga gagctctcgg agcagattca gagggccctg cagctggagg aggagaggaa
1321 gcgggcacag gaggaggccg agcgcctaga ggctgaccgt atggctgcac tgcgggctaa
1381 ggaggagctg gagagacagg cggtggatca gataaagagc caggagcagc tggctgcgga
1441 gcttgcagaa tacactgcca agattgccct cctggaagag gcgcggaggc gcaaggagga
1501 tgaagttgaa gagtggcagc acagggccaa agaagcccag gatgacctgg tgaagaccaa
1561 ggaggagctg cacctggtga tgacagcacc cccgccccca ccaccccccg tgtacgagcc
1621 ggtgagctac catgtccagg agagcttgca ggatgagggc gcagagccca cgggctacag
1681 cgcggagctg tctagtgagg gcatccggga tgaccgcaat gaggagaagc gcatcactga
1741 ggcagagaag aacgagcgtg tgcagcggca gctgctgacg ctgagcagcg agctgtccca
1801 ggcccgagat gagaataaga ggacccacaa tgacatcatc cacaacgaga acatgaggca
1861 aggccgggac aagtacaaga cgctgcggca gatccggcag ggcaacacca agcagcgcat
1921 cgacgagttc gaggccctgt aacagccagg ccaggaccaa gggcagaggg gtgctcatag
1981 cgggcgctgc cagccccgcc acgcttgtgt ctttagtgct ccaagtctag gaactccctc
2041 agatcccagt tcctttagaa agcagttacc caacagaaac attctgggct gggaaccagg
2101 gaggcgccct ggtttgtttt ccccagttgt aatagtgcca agcaggcctg attctcgcga
2161 ttattctcga atcacctcct gtgttgtgct gggagcagga ctgattgaat tacggaaaat
2221 gcctgtaaag tctgagtaag aaacttcatg ctggcctgtg tgatacaaga gtcagcatca
2281 ttaaaggaaa cgtggcagga cttccatctg tgccatactt gttctgtatt cgaaatgagc
2341 tcaaattgat tttttaattt ctatgaagga tccatctttg tatatttaca tgcttagagg
2401 ggtgaaaatt attttggaaa ttgagtctga agcactctcg cacacacagt gattccctcc
2461 tcccgtcact ccacgcagct ggcagagagc acagtgatca ccagcgtgag tggtggagga
2521 ggacacttgg attttttttt ttgttttttt tttttttgct taacagtttt agaatacatt
2581 gtacttatac accttattaa tgatcagcta tatactattt atatacaagt gataatacag
2641 atttgtaaca ttagttttaa aaagggaaag ttttgttctg tatattttgt taccttttac
2701 agaataaaag aattacatat gaaaaaccct ctaaaccatg gcacttgatg tgatgtggca
2761 ggagggcagt ggtggagctg gacctgcctg ctgcagtcac gtgtaaacag gattattatt
2821 agtgttttat gcatgtaatg gactatgcac acttttaatt ttgtcagatt cacacatgcc
2881 actatgagct ttcagactcc agctgtgaag agactctgtt tgcttgtgtt tgtttgtttg
2941 cagtctctct ctgccatggc cttggcaggc tgctggaagg cagcttgtgg aggccgttgg
3001 ttccgcccac tcattccttc tcgtgcactg ctttctcctt cacagctaag atgccatgtg
3061 caggtggatt ccatgccgca gacatgaaat aaaagctttg caaaggcacg aagcaaaaaa
3121 aaaaaaaaaa aaaaaaaa

The biomarker composition of the present invention and the kit containing the same can be used as predictive or diagnostic data for the development of keloid tissue, thereby improving the accuracy of early diagnosis and diagnosis, Prevention or early treatment may be possible. In addition, the screening method of the present invention can be utilized in the search for an effective substance for preventing or treating keloid tissue formation based on the control of target protein expression for the detection of keloid tissue.

FIG. 1 shows the results of histological examination of wound healing effect of POD 14 days after post-treatment day (POD) and post-treatment day (POD14) in a mouse experiment according to the embodiment of the present invention.
FIG. 2 is a graph showing the results of POD (post operation day), 10 days (POD10) and 14 days after post-treatment (POD14) Immunocytochemical analysis of the expression level of lean and p-azurin was performed.
Figure 3 is a photograph (x 100) showing H & E staining results of normal tissue (A), hypertrophic tissue (B), and keloid tissue (C) in an embodiment of the present invention.
Figure 4 is a photograph (x 100) showing the results of immunocytochemical analysis of p-azurin and ezrin protein in an embodiment of the present invention.
FIG. 5 shows the results of quantifying the results of FIG. 4 by H-scoring (* p <0.05, ** p <0.01).
6 is a graph showing the results of Western blotting of the expression levels of p-azurin and ezrin protein in the examples of the present invention (T567p-ezrin: 69 kDa and Ezrin: 81 kDa) p < 0.005)

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

1. Experimental Method

<Wound healing effect in mice>

The following animal experiments were conducted under the approval of the Institutional Animal Care and Use Committee (IACUC) of Soonchunhyang University.

Six weeks old female mice (BALB / c, 6 weeks old; Oriental bio) were anesthetized with zoletil (0.2 ml / kg) containing rumpun (9.8 ml / kg) A skin defect of 2.0 x 2.0 cm size was made using surgical scissors on the back of the patient. The epidermis, dermis and subcutaneous tissue were removed and the fascia (muscle fascia) was exposed. To prevent wound contracture, the wound border was sutured with a 6-0 suture (Ethicon, Somerville, NJ) and the wound border was fixed to the basal fascia. A mouse ( n = 6) treated in the same manner as the negative control group was also prepared. All experimental animals were individually raised after surgery.

<Preparation of human tissue sample>

The organization was collected from plastic surgeons and obstetrics and gynecology departments of tertiary hospitals in the Republic of Korea and was subsequently used in experiments after written consent to the organizational provider in accordance with a research protocol approved by the Institutional Review Board for Ethical (IRB).

All the tissues were collected from women, and the collected tissues were classified into three groups.

1) Normal group (normal, control, group 1, n = 8)

2) Hypertrophic scar (hypertrophic scar, group 2, n = 7)

3) keloid group (group 3, n = 7)

The wounded tissues were clinically diagnosed as hypertrophic scars and keloids, respectively, and were collected by surgery. These samples were frozen and stored immediately after collection at minus 80 ° C, and some tissues were fixed with 4% paraformaldehyde.

<Paraffin Section and Immunocellular Chemistry immunohistochemistry ) Analysis>

The collected tissues were fixed with formalin, paraffin-blocked and paraffin sections were prepared. Histological analysis was performed by performing hematoxylin and eosin staining (H & E) using tissue sections.

Color development was treated with 3,3'-diaminobenzidine and contrast dyeing with H & E staining. Immunohistochemical staining was performed using p-EZRIN and an immunocytochemical analysis kit for EZRIN (EXPOSE Mouse and Rabbit Specific HRP / DAB Detection immunohistochemistry kit, ab80436, Abcam Inc., Cambridge, MA, USA for p-EZRIN, 1: 200, Abcam, Inc , EZRIN 1: 200; Abcam, Inc., CA, USA). Mayer &apos; s hematoxylin was applied to the control stain and negative control was treated except for the primary antibody. Semi-parametric analysis was expressed as H-score, calculated by adding% of total area in randomly selected five fields, and confirmed by two independent observers.

< Protein filling  Extraction and Sampling>

The cell lysates were separated using a buffer at 4 ° C. Cells were detached from the rubber policeman in RIPA buffer with protease inhibitors, and the cell suspension was transferred to a 1.5 ml tube. The tubes were vortexed every 5 minutes on ice for 30 minutes and then centrifuged at 4 ° C for 20 minutes to obtain 13,000 g of protein. The total protein was obtained from the cellular debris (pellet) thus separated, and the total protein thus separated was transferred to a new tube and used for the following analysis. Protein concentration was measured using the dye-binding protein assay according to the Lowry method.

< Western blotting (Western blotting)

Cell lysates were obtained using SDS sample buffer, separated on a 10% polyacrylamide gel, and transferred to nitrocellulose membranes. Antibodies against ezrin (pZ-EZRIN) and p-EZRIN (b-actin) were purchased from abcam and sigma (A1978), respectively. HRP (horseradish peroxidase) was applied to the secondary antibody, and immunoreactive proteins were visualized on the X-ray film by applying an enhanced chemiluminescence detection system.

<Immune Cell Chemistry immunohistochemistry ) Analysis>

Mice were sacrificed at 7, 10 and 14 days after the start of the experiment, and the wound area was sampled to include the basal muscles. Each sampled tissue was used for immunocytochemical analysis. The specimens for tissue staining were immobilized with formalin and immersed in paraffin to prepare paraffin blocks, which were then sliced horizontally to a thickness of 4 μm and then immunostained with p-ezrin and ezrin antibodies Cell chemistry analysis was performed.

<Statistical Analysis>

Quantification of Western blot images was performed using the Image J program and quantified by the H-scoring method. Statistical analysis was performed using SPSS 19.00 (SPSS Inc. Chicago, IL, USA). Data were analyzed using Fisher's exact test and Mann-Whitney U test. p <0.05 was treated as statistically significant differences.

2. Experimental results

<Wound healing effect in mouse and Ezrin  Effect of Protein>

FIG. 1 shows the results of histological examination of wound healing effect of POD 14 days after post-treatment day (POD) and post-treatment day in mouse experiment, and FIG. 2 shows results of post- The expression levels of ezrin and p-ezrin in wound tissues of POD (post operation day), 10 days after post-treatment (POD10), and 14 days after post-treatment (POD14) This is a result.

Referring to FIGS. 1 and 2, p-ezrin protein and Ezrin protein were expressed in the totally re-formed skin tissue, and hair follicle and epidermal layer were increased in expression. This is a result of Ezlin's involvement in the recovery of granulation tissue. In other words, it is shown that Ezrin protein is involved in hair follicle formation, epidermal growth and connective tissue proliferation during wound healing process of skin tissue. Ezrin protein and p-ezrin protein in hair follicle and epidermal layer Both showed increasing results. In addition, it is thought that ezinin protein is involved in the late process of epidermal cell reforming, especially since it is observed in epidermal cells after 14 days from the treatment day.

<Clinical data of human samples>

7 keloid females, 7 hypertrophic females, and 8 normal females participated in this study and their clinical characteristics are shown in Table 2 below.

Normal group
(Normal, n = 8) Hypertrophic scar group
(Hypertrophic scar, N = 7) Keloid group
(Keloid, N = 7) P-value * Age (years) 35.2 ± 0.77 37.5 ± 2.12 31.7 ± 2.21 NS Key (cm) 157.7 ± 2.37 158.4 ± 1.45 160.6 ± 1.90 NS Weight (kg) 59.5 ± 2.16 59.3 ± 2.07 59.7 ± 3.73 NS BMI (Body Mass Index) 23.9 ± 0.86 23.6 ± 0.68 23.2 ± 1.66 NS

* Values mean mean ± SE, NS means no statistical significance.

The mean age was 35.25 ± 2.18 for the keloid group and 37.57 ± 5.62 for the hypertrophic group, similar to 35.25 ± 2.18 for the normal group. There were no statistically significant differences between the groups in mean age, height, body mass index and body mass index (BMI). All groups were composed of women, and most of the tissues were collected from cesarean delivery. Tissue was collected mainly from the abdominal region, although there were some differences in the location where tissues were collected between each group.

&Lt; Histological analysis results of human skin tissue samples >

Histological characteristics of the collected tissues were observed by H & E staining and the results are shown in FIG. In general, skin tissue consists of three layers: the epidermis, the middle dermis, and the deepest subcutis or hypodermis.

Referring to FIG. 3, which shows microscopic observation results, a lot of fibrous tissue and collagen accumulation was observed in wound tissues (hypertrophic and keloid tissues). On the other hand, keloid tissue showed denser tissue compared to hypertrophic tissue, and keloid structure was dense and thicker compared to surrounding normal skin tissue (see arrows in Fig. 3).

In particular, keloid tissue was observed to have the most irregular dense connective tissue in the dermis, and normal dermal tissue morphology was not observed in keloid tissue.

< Hypertrophic scar  From keloid Ezrin  Analysis of protein expression level>

Immunocytochemistry was performed using ezrin and p-ezrin antibodies in normal, hypertrophic and keloid groups, and the results are shown in FIGS. 4 and 5. FIG. Western blotting was also performed and the results are shown in Fig.

4 and 5, ezrin and p-ezrin protein expression is mainly located in the cytoplasm, and p-ezrin and p- The H-scores of the ezrin proteins were 4.98 ± 1.12 and 1.70 ± 0.42, respectively. (P-ezrin) and 7.32 ± 1.18 (ezrin) in the hypertrophic scar tissue and 6.65 ± 3.12 (p-ezrin) and 8.08 ± 4.57 (ezrin) in the keloid tissue, respectively.

The degree of protein expression was highest in keloid tissue, followed by hypertrophic tissue. Compared with normal tissues, the expression level of Ezrin protein was statistically significant in hypertrophic and keloid tissues, p = 0.003 in hypertrophic tissue and p = 0.037 in keloid tissue. Phosphorylated ezrin also showed similar results.

Referring to the Western blotting results in FIG. 6, the expression levels of phosphorylated Ezrin and Ezrin proteins in keloid tissues were significantly different from those in normal tissues.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (4)

An agent that detects any one protein selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN (phospho-EZRIN), and combinations thereof; Or a nucleotide encoding an ezrin. &Lt; RTI ID = 0.0 &gt; &lt; / RTI &gt; A kit for the diagnosis of hypertrophic scars, comprising a biomarker composition for diagnosis of hypertrophic scar tissue according to claim 1. An agent that detects any one protein selected from the group consisting of Ezrin (EZRIN), phosphorylated EzRIN (phospho-EZRIN), and combinations thereof; Or a nucleotide encoding ezrin, thereby providing information necessary for differential diagnosis of hypertrophic tissue. The biomarker composition for diagnosis of hypertrophic scar tissue according to claim 1, wherein the agent for detecting the protein or the agent for detecting a nucleotide is used for diagnosing keloid tissue at the same time.

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