KR101637497B1 - Peptide for promoting bone formation and use thereof - Google Patents

Abstract

The present invention relates to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and a composition for promoting bone formation comprising the same as an active ingredient. The peptide according to the present invention promotes osteoadhesion and osteogenesis, Can be used for implant treatment and inhibition of inflammation caused by oral bacteria by increasing the proliferation of tissue, and can be applied to skin aging and wound healing by promoting growth and migration of fibroblasts.

Description

Bone-forming peptides and uses thereof

The present invention relates to bone forming peptides and uses thereof.

Bone tissue is a dynamic tissue that constantly undergoes bone formation (bone formation / osteoblast differentiation) and bone resorption (bone resorption / osteoclast differentiation). While osteoclasts absorb bone, osteoblasts play a role in synthesizing and filling bone matrix. Therefore, bone mass depends on the relative function of these cells. The amount of bone resorption and amount of bone formation is always balanced in normal adults.

In recent 10 ~ 20 years, as the life span of human beings has been extended and the quality of life has been improved, the domestic and foreign implant market has grown greatly and become popular. The implant is an artificial tooth or a third tooth, which is inserted into the jawbone that has increased its volume to be sufficiently wrapped by bone grafting, osteogenesis, and other operations such as tooth decoloration / erosion or jaw bone removal It is a dental treatment that restores the function of the natural teeth by implanting a biocompatible implant body. After the osteo-integration, which is a morphological, physiological and direct coupling between the jawbone with the normal function and the surface of the implanted implant body, is performed, the bone is subjected to a bone remodeling process of the peri-implant jaw. This osseointegration is the most important index that determines the success of the implant. If the osseointegration does not occur within a short period of time, the success rate of the implant is lowered due to the failure of the initial osseointegration. Therefore, in order to achieve successful implant placement, it is necessary to enhance osseointegration and bone formation capability.

The success rate of implants depends on the initial bone fusion between implant material and bone cells. Recently, a method of directly applying a physiologically active substance such as BMP (Bone Matrix Protein), which is a protein belonging to the TGF-β family, which promotes the proliferation and differentiation of osteoblast cells, directly to the implant surface or to the vicinity of the surgical site has been used . It is well known that BMP proteins play an important role in osteoblast differentiation and growth. Since the BMP family proteins have been reported to play important roles in osteogenesis, bone differentiation, and alveolar bone formation, many experiments have been conducted. In addition to the use of BMP proteins in animals as well as animals, osteoblasts are involved in bone formation and differentiation, Regeneration, and strengthening of the human body. However, BMP is not only expensive, but also rapidly dissolves and disappears rapidly due to enzymes present in the body, making it difficult to effectively apply the BMP. It is difficult to apply it because it can not remain effectively, the half-life is short, and the standardization is not performed.

Periodontal disease is often referred to as taste, depending on the progress of the disease gingivitis (gingivitis) and periodontitis (periodontitis) is classified as. The periodontal disease, which is relatively mild and has a rapid recovery, is called gingivitis. When the inflammation progresses to the gums and gums, it is called periodontitis. There is a V-shaped gap between the gums and the teeth. The periodontal disease is the periodontal disease that causes the oral bacteria to attack the underside of the gaps and damage the periodontal ligaments and nearby tissues. If the initial treatment is not properly performed, The tooth loss occurs.

According to the Korean Dental Association, more than 90% of Korean children have experienced dental caries. Dental caries is the most common disease among children and adults. In addition, more than 80% of adults have periodontitis.

Causes of tooth decay and periodontitis vary, but lactic acid produced by degradation of carbohydrates such as sugar or starch of food residue attached to teeth by the fermentation action of bacteria usually resides in the oral cavity, Occurs. As a result, the anaerobic microorganism cells multiply and the gum tissues are destroyed by the toxin components generated by the periodontal bacteria, resulting in the shaking and dropping of the teeth. Streptococcus mutans and Porphyromonas gingivalis are typical pathogenic microorganisms causing tooth decay and periodontitis.

Currently, a method using various antibiotics or a method of suppressing pathogenic bacteria by fluorine is used as a method for suppressing tooth decay and periodontitis. However, a method using antibiotics is a method in which microorganisms become resistant , Diarrhea, vomiting, and the like, as well as the problem that the growth of the microorganisms in the oral cavity is inhibited. Antimicrobial activity of antimicrobial substances derived from natural products, which have been known so far, has been reported to have a problem that antimicrobial activity is poor, thermal stability is poor, and antimicrobial activity is rapidly deteriorated.

Accordingly, the present inventors have completed the present invention by experimentally confirming that peptides composed of VKKYRPKYCGSC have excellent bone formation effects while studying peptides for developing a composition for bone formation.

It is an object of the present invention to provide a peptide for promoting bone formation.

It is also an object of the present invention to provide a polynucleotide encoding said peptide and a vector comprising said polynucleotide.

It is also an object of the present invention to provide a composition for promoting osteogenesis, a composition for preventing or treating periodontal disease, and a composition for antiinflammation comprising the peptide.

In order to achieve the above object, the present invention provides a peptide for promoting bone formation.

Also, an object of the present invention is to provide a polynucleotide encoding said peptide and a vector comprising said polynucleotide.

Also, the object of the present invention is to provide a composition for promoting osteogenesis, a composition for preventing or treating periodontal disease, and a composition for anti-inflammation comprising the peptide.

The osteogenic peptide according to the present invention promotes bone adhesion and osteogenesis, thereby increasing osteoblast differentiation and proliferation of dental tissues, and can be used for implant treatment and inhibition of inflammatory action by oral bacteria. The growth and migration of fibroblasts Thereby promoting skin aging and wound healing.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of measurement of BMP receptor binding ability of peptides of the present invention. FIG.
FIG. 2 is a graph showing the results of measuring the cell growth promotion rate of the peptide of the present invention. FIG.
FIG. 3 shows the results of ALP staining of the peptides of the present invention. FIG.
4 is a graph showing the results of measurement of ALP enzyme activity of the peptides of the present invention.
FIG. 5 is a graph showing the results of RT-PCR analysis of the expression of the osteoblast late differentiation-related gene of the peptide of the present invention.
FIG. 6 is a graph showing the results of measuring the degree of calcification of the osteoblast extracellular matrix of the peptide of the present invention. FIG.
FIG. 7 is a graph showing the expression of the osteoblast-related gene of the peptide of the present invention measured by RT-PCR.
FIG. 8 is a graph showing the results of measuring the inhibitory effect of the peptides of the present invention on inhibition of osteoblast differentiation by ALP method.
FIG. 9 is a graph showing the results of RT-PCR measurement of the inhibitory effect of the peptides of the present invention on inhibition of late-differentiation of osteoblast-induced osteoblast differentiation.
FIG. 10 is a graph showing the results of RT-PCR measurement of the inhibitory effect of the peptides of the present invention on the inflammatory cytokine and MMP1 increase.
Fig. 11 is a graph showing the recovery effect of osteoblast growth inhibition by LPS of the peptide of the present invention.
FIG. 12 is a graph showing the results of ALP staining of the peptide of the present invention, in which LPS inhibited the inhibition of osteoblast differentiation.
FIG. 13 is a graph showing the results of RT-PCR measurement of the inhibitory effect of the peptide of the present invention on LPS-inhibited osteoblast differentiation.
FIG. 14 is a graph showing the results of measurement of the inhibitory effect of LPS of the peptide of the present invention on inflammatory cytokine and MMP1 increase by RT-PCR. FIG.

Hereinafter, the present invention will be described in detail.

The present invention provides a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and a composition for promoting osteogenesis containing the peptide as an effective ingredient.

Natural amino acids include glycine, alanine, serine, aspartic acid, glutamic acid, valine, threonine, arginine, , Lysine, proline, leucine, phenylalanine, isoleucine, histidine, tyrosine, methionine, cystein, tryptophan tryptopane) asparagine, glutamine and the like. The peptide of the present invention is composed of VKKYRPKYCGSC (SEQ ID NO: 1), which can be synthesized by solid phase peptide synthesis (Merrifield, Biochemistry 3 (1964) 1385) commonly used in the art.

V, K, Y, R, P, C, G and S of the peptide are valine, lysine, thyrosine, arginine, proline, cystein, , Glycine and serine, the amino acid sequence of the peptide of the present invention is as follows.

Val-Lys-Lys-Tyr-Arg-Pro-Lys-Tyr-Cys-Gly-Ser-Cys

Amino acids commonly used are those in which the N-terminal is protected and the reactive side chain is protected. As the protecting group for protecting the N-terminus, a 9-fluorenylmethyloxycarbonyl group (Fmoc) may be used. As the protecting group for protecting the reactive side chain, triphenylmethyl, t-butylester, butyloxycarbonyl, pentamethyl 6-sulfonyl and the like can be used. Resins such as 2-chlorotrityl chloride (CTC), 4-benzyloxybenzyl alcohol (Wang) or trialkoxybenzhydrylamine (Rink Amide) can be used as the solid phase resin used for the reaction .

The peptides of the present invention are synthesized from the C-terminus to the N-terminus. When coupling the carboxyl group of the amino acid with HATU (N - [(dimethylamino) -1H-1,2,3-triazolo [4,5-b] pyridin-1-ylmethylene] -N-methylmethanaminium hexafluorophosphate N- (1-hydroxy-7-azabenzotriazole, HOAt) or HBTU (N - [(1H-benzotriazol-1-yl) (dimethylamino) methylene] -N-methylmethanaminium hexafluorophosphate N- And N, N'-diisopropylcarbodiimide (DIC) and 1-hydroxy-7-hydroxybenzotriazole (HOBt) -Azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) is added to the reaction mixture before activation. After the coupling, the protecting group at the N-terminus is removed with piperidine, and the coupling reaction of the following amino acids is carried out. The peptidyl resin synthesized in this way is separated from the solid resin by the cleavage solution containing Trifluroacetic acid (TFA) and the protecting group bound to the side chain is removed.

The present invention also provides a polynucleotide encoding a peptide consisting of the amino acid sequence represented by SEQ ID NO: 1.

The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides present in single-stranded or double-stranded form. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes not only a nucleotide sequence encoding the peptide but also a sequence complementary to the sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences. This means a sequence capable of hybridizing with a nucleotide sequence encoding a peptide of SEQ ID NO: 1 under stringent conditions known in the art.

The polynucleotide may also be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The polynucleotide encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is determined by aligning the nucleotide sequence with any other sequence as closely as possible, and analyzing the aligned sequence using algorithms commonly used in the art to obtain a sequence having at least 80% homology, At least 90% homology or at least 95% homology.

The present invention also provides a recombinant vector comprising the polynucleotide.

The term "vector" means means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retroviral vectors, and adeno-associated viral vectors. The vector that can be used as the recombinant vector may be a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series, and pUC19), phage (e.g.,? gt4? B,? -charon,?? z1 and M13) or viruses (e.g., CMV, SV40 and the like).

The polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 in the recombinant vector may be operatively linked to a promoter. The term "operatively linked" refers to the functional linkage between a nucleotide expression control regulatory sequence (e.g., a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence may thereby regulate transcription and / or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or as a vector for expression. The expression vector may be any conventional vector used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed by a variety of methods known in the art.

The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) , A ribosome binding site for initiation of translation, and a transcription / translation termination sequence. When the eukaryotic cell is used as a host, the origin of replication that functions in the eukaryotic cells contained in the vector is f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, CMV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

The peptide thus synthesized specifically binds to the BMP receptor and exhibits a strong binding ability to the BMP receptor. It promotes osteoadhesion and osteogenesis through growth and differentiation of osteoblasts and fibroblasts, protects against oral bacteria Lt; / RTI >

Accordingly, the present invention provides a composition for promoting osteogenesis, a composition for preventing or treating periodontal disease, and a composition for anti-inflammation comprising the peptide, polynucleotide or recombinant vector as an active ingredient.

The composition for preventing or treating periodontal disease includes a pharmaceutical composition or a quasi-drug composition. The quasi drug composition may be, for example, in the form of a toothpaste or an oral rinse, but is not limited thereto.

The periodontal disease includes, but is not limited to, gingivitis, periodontitis, periodontal pocket or periodontal abscess.

The periodontal disease is caused by oral bacteria, preferably, but not exclusively, by S. mutans or P. gingivalis .

The composition of the present invention may be prepared by incorporating at least one further pharmaceutically acceptable carrier in addition to the above peptide, polynucleotide or recombinant vector. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components. If necessary, an antioxidant, , And other conventional additives such as a bacteriostatic agent may be added. In addition, it can be formulated into injectable formulations such as aqueous solutions, suspensions, emulsions, injection solutions, pills, capsules, granules, powders or tablets by additionally adding diluents, dispersants, surfactants, binders and lubricants. Further, it can be suitably formulated according to each disease or ingredient, using methods disclosed in Remington's Pharmaceutical Science (recent edition) or Mack Publishing Company, Easton PA, as appropriate methods in the art.

The composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously or topically) according to the desired method, and the dose is appropriately determined depending on the body weight, age, sex, Time, method of administration, excretion rate, and severity of the disease. The term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The pharmaceutical composition of the present invention is useful as an effective ingredient or amount of a pharmaceutical composition that induces a biological or medical response in a tissue system, an animal or a human, as considered by the researcher, veterinarian, physician or other clinician, Or a therapeutically effective amount which is sufficient to induce relief. It will be apparent to those skilled in the art that the therapeutically effective dose and the frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For the desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 0.05 to 0.1 mg / kg / day, preferably 0.01 to 0.1 mg / kg / day, It can also be administered in divided doses.

The pharmaceutical composition of the present invention may be administered to a subject in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In addition, the present invention provides a bone graft material and a tissue engineering support wherein the surface of the peptide, polynucleotide or recombinant vector is coated with a composition for promoting bone formation.

 The bone graft materials and supports that can be used in the present invention include all kinds and forms of bone graft materials and polymer scaffolds used in the art. Preferably, the bone graft materials and the porous bone blocks , Bone hydroxyapatite powder and porous block, tricalcium phosphate powder and porous block, monocalcium phosphate powder and porous block, bone graft made of silicon dioxide (silica), bone-filling implant made of a mixture of silica and polymer, chitosan, polylactic acid And a porous support, titanium, and a three-dimensional porous support, but the present invention is not limited thereto. At this time, it is preferable that the surfaces of the bone graft material and the support are modified to facilitate attachment of active peptides.

It is preferable that the composition for promoting bone formation is chemically bonded to the surface of the bone graft material and the support to coat the surface of the bone graft material and the support surface with 0.01 to 1 μM per cm 2, 0.5 μM, and most preferably 0.1 μM per surface area of the bone graft material and the supporter, but the present invention is not limited thereto.

When the composition of the present invention is immobilized on the surfaces of the bone graft material, the support, the shielding film, or the implant as described above, the composition exhibits activity while being present at a desired concentration, so that the therapeutic effect can be enhanced.

In addition, the present invention provides an anti-aging cosmetic composition comprising the above peptide, polynucleotide or recombinant vector as an active ingredient. The cosmetic composition may be used for preventing skin aging and improving skin wrinkles, and may be in the form of a skin, a lotion, a cream, an essence, an ointment, a gel, a stick, a powder and a spray.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and the production examples.

Example 1. Preparation of sample

Solid phase peptide synthesis (Merrifield, Biochemistry 3 (1964) 1385) was performed to prepare a peptide composed of the amino acid sequence of SEQ ID NO: 1 of the present invention.

First, 700 mg of chlorotrityl chloride resin (CTL resin, Nova biochem Cat No. 01-64-0021) was charged into a reaction vessel, 10 ml of methylene chloride (MC) was added, and the mixture was stirred for 3 minutes. , And 10 ml of dimethylformamide (DMF) was added thereto. After stirring for 3 minutes, the solvent was removed again. Then, 10 ml of a dichloromethane solution (DCM) was added to the reaction vessel, and 200 mmole of Fmoc-Cys (Trt) -OH (Bachem, Swiss) and 400 mmole of diisopropylethylamine (DIEA) And reacted with stirring for 1 hour. After the reaction, methanol and DIEA (2: 1) were dissolved in DCM and reacted for 10 minutes. The reaction mixture was washed with excess DCM / DMF (1: 1) and the solution was removed. 10 ml of DMF was added thereto and stirred for 3 minutes. . 10 ml of a deprotection solution (20% Piperidine / DMF) was then added to the reaction vessel and stirred for 10 minutes at room temperature before the solution was removed. The same amount of the deprotection solution was added and the reaction was maintained for 10 minutes. Then, the solution was removed, and the Cys-CTL resin was prepared by washing twice with DMF, once with MC, and once with DMF, for 3 minutes each. 200 ml of Fmoc-Ser (tBu) -OH (Bachem, Swiss), 200 mmole of HoBt and 200 mmole of Bop were placed in a new reactor, and the mixture was stirred and dissolved. Then, 400 mmole DIEA -Diisopropylethylamine) was added in portions over 2 times and stirred for at least 5 minutes until all solids had dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel containing the deprotected resin and allowed to react for 1 hour at room temperature with stirring. After the reaction solution was removed, the reaction solution was stirred three times for 5 minutes with DMF solution. The solution was again removed, and a small amount of the reaction resin was taken and the degree of reaction was checked using a Kaiser test (Nihydrin test). Thereafter, the reaction solution was subjected to a deprotection reaction twice in the same manner as described above to prepare a Ser-Cys-CTL resin. The prepared resin was thoroughly washed with DMF and MC and once again subjected to a Kaiser test. Then, the following amino acid attachment was carried out in the same manner as above. Fmoc-Tyr (tBu), Fmoc-Lys (Boc), Fmoc-Pro, Fmoc-Arg (Pbf), and Fmoc-Tyr The Fmoc-Lys (Boc), Fmoc-Lys (Boc) and Fmoc-Val were sequentially reacted in this order, and the Fmoc-protecting group was reacted with the deprotecting solution twice for 10 minutes. After that, acetic anhydride, DIEA, and HoBt (Hydroxybenzotriazole) were added and acetylation was performed for 1 hour. Then, the prepared peptidyl resin was washed three times with DMF, MC and methanol, and then nitrogen gas was slowly flowed and dried. a _{(phosphorus pentoxide, P 2 O 5} ) under the reduced pressure to the vacuum dried after the fugitive solution [TFA (Trifluroacetic acid) 95% , distilled water 2.5%, thio anisole (Thioanisole) 2.5%] occasionally at room temperature into a 30 ml The reaction was maintained for 2 hours with shaking, the resin was filtered by filtering, and the resin was washed with a small amount of solution and then combined with the mother liquor. This was distilled using a reduced pressure to leave about half of the total volume, and 50 ml of cold ether was added to precipitate the precipitate. The precipitate was collected and washed with 2 more cold ether. Lys-Tyr-Arg-Pro-Lys-Tyr-Cys-Gly-Ser-Cys-OH peptide 1 was purified by preparative HPLC to obtain 0.88 g of purified NH ₂ -Val- %). The peptide was measured using a molecular weight analyzer, and it was confirmed that the peptide had a molecular weight of 1431.7 (theoretical value: 1431.75 Da).

Experimental Example 1. Confirmation of BMP receptor specific binding ability

BMP receptor binding assay was performed to measure the binding ability of the peptides prepared in Example 1 to BMP receptors. The results are shown in FIG.

As shown in Fig. 1, the peptides of the present invention were found to bind specifically to BMP receptors as compared to negative control. This indicates that the peptides of the present invention can bind to BMP receptors and activate the lower signaling system of BMP receptors.

EXPERIMENTAL EXAMPLE 2. Examination of Promoting Growth of Tissue Cells and Osteoblasts

The growth promoting effects of the peptides prepared in Example 1 on human periodontal ligament fibroblast (HPLF) and osteoblast (C2C12, murine osteoblast) were examined.

First, each cell was put into a well of a 96-well plate at 2 × 10 ³ cells / well (final media volume: 100 ml), and cultured in a 5% CO ₂ incubator at 37 ° C. for 24 hours. And replaced with serum-free medium. Each of the wells was treated with the above peptides by concentration, and then cultured in an incubator for 72 hours. After the incubation, 10 ml of MTT solution (5 mg / ml in DW) was added to each well, followed by further incubation for 4 hours. Then, the culture solution was removed, and 100 ml of DMSO solution was added to dissolve the generated Formazin . The absorbance of the solution was measured at 540 nm using a spectrophotometer. At this time, the cell viability (%) of each cell line was calculated by the following formula (1) based on the negative control group treated with distilled water instead of the sample, and the result is shown in FIG.

[Equation 1]

Cell survival rate (%) = [(OD _sample ) / OD _control ] x 100

The peptides of the present invention were found to increase the growth of fibroblasts, osteoblasts and embryonic fibroblasts in size ligament.

Experimental Example 3. Confirmation of osteoblast differentiation promoting ability

3-1. Promoting osteoblast differentiation promoting ability

In order to confirm the ability of the peptide prepared in Example 1 to promote osteoblast differentiation early, the staining and activity of ALP (Alkaline phosphatase), an early marker protein of osteogenic cells, were measured. First, the osteoblast cells (C2C12) in 24-well plate 2x10 ⁴ each frequency divider to 1 days culture, and culture medium (with 10% FBS) to replace, and at the same time by treating the peptide 37 ℃, 3 days under the conditions of 5% CO ² Lt; / RTI > For ALP staining, the cells were fixed with fixative solution (25 mM citrate buffer, 3% formaldehyde, 65% acetone) and 200 ml of staining reagent (Sigma 85C-1 kit) was added for 30 min at 37 ° C. The staining was observed with an optical microscope, and the results are shown in Fig.

To measure ALP enzyme activity, peptides were added and incubated for 3 days, and then lysed with a cell lysis buffer (10 mM Tris, pH 8.0, 1 mM MgCl 2, 0.5% Triton X-100). Then, phosphatase After incubation with 10 mM phosphatase substrate, the absorbance was measured at 405 nm. The results are shown in FIG.

As shown in FIG. 3 and FIG. 4, it was confirmed that the peptide significantly increased ALP staining and enzyme activity as compared with the negative control group.

3-2. Promoting osteoblast differentiation promoting ability

RT-PCR was performed to confirm the ability of the peptide prepared in Example 1 to promote late differentiation of osteoblast cells to compare mRNA production amounts of the extracellular matrix proteins of the late differentiation markers of osteogenic cells.

The cells were cultured under the same conditions as Experimental Example 3-1, and the cells were collected on the fifth day of culture and RNA was extracted using Trizol® reagent (Intron). For the synthesis of cDNA from RNA, 5 mg of RNA, 1 ml of random hexamer and DEPC-treated water were added and reacted at 65 ° C for 5 minutes. 5x first strand buffer, 0.1M DTT, 10mM dNTP and reverse transcriptase ml < / RTI > at 42 < 0 > C for 1 hour. For the termination of the reaction, cDNA was finally obtained by heating at 95 DEG C for 5 minutes. Then, PCR was carried out using 2 ml of cDNA as a template, adding 10 pmole of a primer specific to each gene, mixing 10 × taq buffer, 10 mM dNTP, and i-taq DNA polymerase. The PCR product was loaded on 1% agarose gel, electrophoresed for 20 minutes, and irradiated with a UV illuminator. The PCR product was amplified 30 times at 94 ° C. for 30 seconds, 60 ° C. for 30 seconds and 72 ° C. for 30 seconds. And the results are shown in Fig.

As shown in FIG. 5, the peptide significantly increased the expression of alkaline phosphatase, osteopontin and osteocalcin, which are post-osteoblast differentiation extracellular matrix markers, as compared with the negative control group Respectively. Was significantly increased when compared with the positive control group BMP2.

3-3. Confirm osteoblast calcification promoting ability

Alizarin red S staining was performed to confirm whether or not osteoblastic osteoblastization of the peptide prepared in Example 1 proceeded well, and the degree of calcification of osteoblast extracellular matrix was confirmed.

On the 7th day after incubation, the cells were fixed with 70% ethanol for 1 hour and stained with 2% Alizarin Red S solution (pH 4.1-4.3). After thoroughly washing with PBS, the sample was observed using a microscope, and a solution of 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) was dissolved to measure the absorbance at 562 nm , And the results are shown in Fig.

As shown in FIG. 5, it was confirmed that the bone morphogenesis by the peptide was improved.

Experimental Example 4. Confirmation of promoting ability to attach osteoblast

RT-PCR was carried out in the same manner as described in Experimental Example 3-2 to confirm whether the peptide prepared in Example 1 increased the expression of adhesion-related genes of osteoblast cells. The results are shown in FIG. 6 Respectively.

As shown in FIG. 6, the peptide is expressed in the expression of Procollagen Type 1, Collagen 1, Osteoadherin, Integrin? 3, Was significantly increased compared to the negative control group. This indicates that the peptide of the present invention promotes the adhesion of the osteoblast by promoting the expression of the adhesion-related gene of the osteoblast.

Experimental Example 5. Treatment of periodontal disease and anti-inflammatory effect

In order to confirm whether the peptide prepared in Example 1 has a periodical disease treatment effect and an anti-inflammatory effect, Lipopolysaccaride (LPS) present on the surface of s.mutans and p . Gingivalis , typical oral bacteria causing tooth decay and periodontal disease, Were used to create an inflammatory environment in the dendritic cells, and the peptides were treated.

5-1-1. S.Mutans Inhibitory effect of bacterial inhibition on early differentiation of osteoblast

Alkaline phosphatase (ALP) staining and activity were measured in the same manner as in Experimental Example 4-1 to confirm whether or not the osteoblast cells were restored to their initial differentiation. The results are shown in FIGS. 7 and 8.

It was confirmed that, said peptide is the increase again early osteoblast differentiation inhibition by s.mutans bacteria, in the group treated with the other hand to the differentiation of osteoblasts by reducing s.mutans bacteria as shown in Figures 7 and 8 .

5-1-2. S.Mutans Inhibitory effect of bacterial inhibition on late differentiation of osteoblast

The RT-PCR test was carried out in the same manner as in Experimental Example 4-2 to confirm whether or not the osteoblast was inhibited in late differentiation. The results are shown in FIG.

As shown in Fig. 9, the expression of osteogenic osteoblast-related genes was decreased by s.mutans while the inhibition of osteoblast differentiation-related genes was restored by S. mutans in the group treated with the peptides Respectively.

5-1-3. S.Mutans Inhibitory effect of bacteria on osteoblast cell inflammation

An increase in the inflammatory cytokines of osteoblasts occurs by S. mutans . It has been known that the increase in the size of the periodontal ligament affects to the formation of the bone tissue, and the whole periodontal tissue is largely affected. RT-PCR was performed in the same manner as in Experimental Example 3-2 to confirm the inhibition of osteoclast inflammation. The results are shown in FIG.

As shown in FIG. 10, inflammatory cytokines such as COX2, IL-1β, IL-8, TNF-α and MMP1, which are extracellular matrix proteases, were increased by S. mutans , , And it was confirmed that MMP1 was decreased when chronic periodontal disease progressed. This indicates that the peptide of the present invention can help prevent and treat periodontal disease by inhibiting the inflammatory reaction.

5-2-1. Recovery effect of osteoblast growth inhibition by LPS

In order to inhibit osteoblast growth, LPS was treated, and the peptide prepared in Example 1 was treated. After that, the MTT test was performed to confirm whether osteoclast inhibition was restored. The results are shown in FIG. 11 .

As shown in FIG. 11, it was confirmed that osteoclast death was increased by LPS, whereas LPS-induced apoptosis was suppressed in the group treated with peptide.

5-2-2. Inhibitory effect of LPS on inhibition of early differentiation of osteoblast

In order to confirm whether or not restoration of osteoblast differentiation was inhibited, ALP (Alkaline phosphatase) staining was performed in the same manner as in Experimental Example 4-1, and the results are shown in FIG.

As shown in FIG. 12, osteoblast differentiation was reduced by LPS, while inhibition of osteoblast differentiation by LPS was increased again in the group treated with the peptide.

5-2-3. LPS-induced restoration of osteoblast differentiation

RT-PCR was performed in the same manner as in Experimental Example 4-2 to confirm whether or not the osteoblast was inhibited in late differentiation. The results are shown in FIG.

As shown in FIG. 13, the expression of late-differentiation-related osteoblast-related genes was reduced by LPS, while the inhibition of osteoblast differentiation-related genes by LPS was restored in the group treated with the above peptides.

5-2-4. Inhibitory effect of LPS on osteoblast inflammation

RT-PCR was performed in the same manner as in Experimental Example 4-2 to confirm the inhibition of osteoclast inflammation, and the results are shown in FIG.

As shown in FIG. 14, inflammatory cytokines such as COX2, IL-1β, IL-8 and TNF-α were increased by LPS, but it was confirmed that inflammatory factors were significantly decreased by the peptides. This indicates that the peptide of the present invention can help prevent and treat periodontal disease by inhibiting the inflammatory reaction.

Hereinafter, formulation examples of the composition of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1. Pharmaceutical preparation

1-1. Manufacture of Powder

20 mg of the peptide of the present invention

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

10 mg of the peptide of the present invention

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

10 mg of the peptide of the present invention

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

10 mg of the peptide of the present invention

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Manufacture of liquid agent

10 mg of the peptide of the present invention

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation Example 2. Cosmetic preparation

2-1. Manufacture of softening longevity

10 mg of the peptide of the present invention

Glycerin 30 g

20 g of butylene glycol

Propylene glycol 20 g

10 g of carboxyvinyl polymer

100 g ethanol

Triethanolamine 10 g

Trace amount of perfume

Fine amount of purified water

2-2. Manufacture of nutrition cream

10 mg of the peptide of the present invention

100 g of wax

Polysorbate 60 15 g

50 g of sorbitan sesquioleate

100 g of liquid paraffin

Squalane 50 g

Caprylic / capric triglyceride 50 g

20 g of triethanolamine

Trace amount of perfume

Fine amount of purified water

<110> CAREGEN CO., LTD. <120> Peptide for promoting bone formation and use thereof <130> 1-3 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 12 <212> PRT <213> Bone matrix protein <400> 1 Val Lys Lys Tyr Arg Pro Lys Tyr Cys Gly Ser Cys   1 5 10

Claims (11)

1. A peptide for promoting bone formation, comprising the amino acid sequence represented by SEQ ID NO: 1. A polynucleotide encoding the peptide of claim 1. A recombinant vector comprising the polynucleotide of claim 2. A composition for promoting osteogenesis comprising the peptide of claim 1, a polynucleotide encoding the peptide, or a recombinant vector comprising the polynucleotide as an active ingredient. A bone graft material wherein the composition of claim 4 is coated on the surface. A tissue engineering support, wherein the composition of claim 4 is coated on the surface. A pharmaceutical composition for preventing or treating periodontal disease comprising the peptide of claim 1, a polynucleotide encoding the peptide, or a recombinant vector comprising the polynucleotide as an active ingredient. The pharmaceutical composition according to claim 7, wherein the periodontal disease is gingivitis, periodontitis, periodontal pocket, or periodontal abscess. The pharmaceutical composition according to claim 7, wherein the periodontal disease is caused by oral bacteria. A quasi-drug composition for preventing or improving periodontal disease, comprising the peptide of claim 1, a polynucleotide encoding the peptide, or a recombinant vector comprising the polynucleotide as an active ingredient. A pharmaceutical composition for antiinflammatory comprising the peptide of claim 1, a polynucleotide encoding the peptide, or a recombinant vector comprising the polynucleotide as an active ingredient.

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