JP2013177368A - Human amelogenin partial peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel peptide having an activity similar to that of the human amelogenin.SOLUTION: A partial peptide includes up to the C-terminal 29 amino acids of the complete-length human amelogenin, rh174, and includes C-terminal 7 amino acids thereof, has a cell growth promoting effect and a cell differentiation promoting effect similar to those of amelogenin, and can be used in various clinical applications requiring amelogenin effects.

Description

  The present invention provides a partial peptide of amelogenin protein which is a kind of tooth enamel protein.

  Amelogenin is a protein present in tooth enamel. At the time of tooth development, enamel has about 30% protein, about 90% of which is amelogenin. Other enamel proteins include ameloblastin and enamelin. Amelogenin is an important enamel protein secreted during the formation of teeth in the living body, and has a function of inducing crystals of hydroxyapatite, which is the main component of enamel. In addition, amelogenin is not limited to enamel blasts, but also Marasse epithelial cells (Non-patent Document 1), odontoblasts (Non-patent Document 2), osteoblasts, bone cells, chondrocytes, bone marrow-derived cells, osteoclasts It is known that it is also expressed in cells (Non-patent Document 3). In amelogenin knockout mice, remarkable root resorption is observed, suggesting that amelogenin acts as a regulator of root resorption (Non-patent Document 4). In addition, human amelogenin is a cell proliferation of cementoblasts (Non-patent document 5), periodontal ligament cells (Non-patent document 5) and bone marrow-derived mesenchymal stem cells (Non-patent document 6), and calcification of cementoblasts (non-patent document 5). It is known to have a physiologically active function that promotes Patent Document 7).

  Based on the above physiological activity, enamel protein containing amelogenin has been clinically applied as a regenerative therapeutic agent in recent years, and can be used for periodontal tissue therapeutic agents, wound healing promoters, root filling materials, jaw bone regeneration, etc. It has been proposed (Patent Documents 1 to 5). In addition, there are those already marketed as pharmaceuticals such as Emdogain (trademark) which is porcine enamel protein as a periodontal tissue regeneration material and Xelma (trademark) as an intractable wound healing promoter (Non-patent Documents 8 and 9). ).

  Both enamel protein and amelogenin used for pharmaceutical use are biological materials or full-length amelogenin proteins. Biological materials are expensive, their quality is not stable, and there is a risk of contamination with unknown pathogens. In addition, full-length proteins have a large molecular weight, so there are restrictions on preparations and poor absorption into the body. The amino acid sequence of the full length protein of amelogenin is known, but there is no information about the active site.

Special table 2003-535905 gazette JP-T-2002-504520 Japanese Patent No. 4726300 JP 2004-143165 A JP 2010-189399 A

Fong CD, Hammarstrom L. Expression of amelin and amelogenin in epithelial root sheath remnants of fully formed rat molars. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2000 Aug; 90 (2): 218-23. Papagerakis P, MacDougall M, Hotton D, Bailleul-Forestier I, Oboeuf M, Berdal A. Expression of amelogenin in odontoblasts. Bone. 2003 Mar; 32 (3): 228-40. Haze A, Taylor AL, Blumenfeld A, Rosenfeld E, Leiser Y, Dafni L, Shay B, Gruenbaum-Cohen Y, Fermon E, Haegewald S, Bernimoulin JP, Deutsch D. Amelogenin expression in long bone and cartilage cells and in bone marrow progenitor cells. Anat Rec (Hoboken). 2007 May; 290 (5): 455-60. Hatakeyama J, Sreenath T, Hatakeyama Y, Thyagarajan T, Shum L, Gibson CW, Wright JT, Kulkarni AB.The receptor activator of nuclear factor-kappa B ligand-mediated osteoclastogenic pathway is elevated in amelogenin-null mice.J Biol Chem. 2003 Sep 12; 278 (37): 35743-8. Epub 2003 Jul 8. Huang YC, Tanimoto K, Tanne Y, Kamiya T, Kunimatsu R, Michida M, Yoshioka M, Yoshimi Y, Kato Y, Tanne K. Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow. Cell Tissue Res. 2010 Nov; 342 (2): 205-12. Epub 2010 Oct 22. Kunimatsu R, Tanimoto K, Tanne Y, Kamiya T, Ohkuma S, Huang YC, Yoshimi Y, Miyauchi M, Takata T, Tanne K. Amelogenin enhances the proliferation of cementoblast lineage cells. J Periodontol. 2011 Nov; 82 (11): 1632-8. Doi: 10.1902 / jop.2011.110031. Epub 2011 Apr 12. 7) Tanimoto K, Kunimatsu R, Tanne Y, Huang YC, Michida M, Yoshimi Y, Miyauchi M, Takata T, Tanne K. Differential Effects of Amelogenin on Mineralization of Cementoblasts and Periodontal Ligament Cells. J Periodontol. 2011 Sep 26. Epub ahead of print] MDOGAIN (registered trademark) gel package insert Clinical Interventions in Aging 2008: 3 (2) 263-272

  An object of the present invention is to provide a novel peptide having activity similar to that of human amelogenin.

  The present invention provides an amelogenin peptide comprising a peptide fragment of 7 to 29 amino acids from the C-terminus of full-length human amelogenin rh174. The cDNA and amino acid sequence of full-length human amelogenin rh174 is published by GenBank at GI6715562.

  The sequence of a peptide fragment of 29 amino acids from the C-terminal of the full-length human amelogenin peptide rh174 is SEQ ID NO: 11: PMQPLPPMLPDLTLEAWPSTDKTKREEVD (C29). The peptide of the present invention is the C29 peptide or a partial peptide thereof, comprising SEQ ID NO: 1 TKREEVD (C7).

Preferred peptides of the present invention include those having any of the following sequences:
Sequence number 1: TKREEVD (C7)
Sequence number 2: KTKREEVD (C8)
Sequence number 3: DKTKREEVD (C9)
Sequence number 4: TDKTKREEVD (C10)
Sequence number 5: STDKTKREEVD (C11)
Sequence number 6: PSTDKTKREEVD (C12)
Sequence number 7: WPSTDKTKREEVD (C13)
Sequence number 8: AWPSTDKTKREEVD (C14)
Sequence number 9: EAWPSTDKTKREEVD (C15)
Sequence number 10: LEAWPSTDKTKREEVD (C16)
In the following description and drawings, when referring to each peptide individually, the terms in parentheses are used and referred to as “C7 peptide” or simply “C7”. In addition to the above, for example, peptides having up to 19 amino acids are exemplified.

  The amelogenin peptide of the present invention exhibits an activity similar to that of amelogenin, but is composed of amino acids having a low molecular weight and high hydrophilicity. Therefore, the amelogenin peptide of the present invention can be used for the same clinical use as amelogenin, and has an excellent effect of high safety and high tissue permeability compared to amelogenin preparations and biological material-derived enamel protein preparations. Have.

  The present invention also provides a pharmaceutical composition containing the amelogenin peptide. The pharmaceutical composition of the present invention includes, for example, bone, cartilage formation promoter, bone, cartilage regeneration promoter, periodontal tissue formation promoter, periodontal tissue regeneration promoter, wound healing promoter, graft uptake promoter, etc. Can be used as

It is a figure which shows the effect | action of the peptide of this application with respect to the proliferation of a human origin cementoblast. C7 to C11 correspond to the peptides of SEQ ID NOs: 1 to 5, respectively, and C16 corresponds to the peptide of SEQ ID NO: 10. The vertical axis represents absorbance, and the horizontal axis represents the amount of peptide added (μg / ml). * p <0.05, ** p <0.01 (vs. control) The effect | action of the peptide of this application with respect to the gene expression of the alkaline phosphatase (ALP) of a human-derived cementoblast is shown. When the human-derived cement blast cells were cultured in the presence of the peptide of the present application (0.1 μg / ml), the expression level of the gene was measured and compared with the result of the additive-free control. * p <0.05, ** p <0.01 (vs. control) The effect | action of the peptide of this application with respect to the alkaline phosphatase (ALP) protein expression of a human-derived cementoblast is shown. The amount of intracellular ALP when human-derived cementoblasts were cultured in the presence of the peptide of the present application (0.1 μg / ml) was measured. * p <0.05, ** p <0.01 (vs. control) The effect of the present peptide on the expression of osteoprotegerin (OPG) in human-derived cementoblasts is shown. When the human-derived cement blasts were cultured in the presence of the peptide of the present application (0.1 μg / ml), the expression level of the OPG gene was measured and compared with the result of the additive-free control. * p <0.05, ** p <0.01 (vs. control) It is a figure which shows the effect | action of this application peptide with respect to NF-kappa B activation receptor (RANKL) gene expression in a human origin cementoblast. The expression level of the RANKL gene when human-derived cementoblasts were cultured in the presence of the peptide of the present application (0.1 μg / ml) was measured and compared with the results of the additive-free control. * p <0.05, ** p <0.01 (vs. control) The effect | action of this application peptide with respect to the mineralization of a human-derived cement blast cell is shown. Dark red (shown in dark gray in FIG. 5) by Ariza Red indicates that cell calcification was induced. As a result of culturing human-derived cementoblasts in the presence of the peptide of the present application (0.1 μg / ml), it was shown that the peptide of the present application enhances calcification. It is a figure which shows the effect | action of this application peptide with respect to the bone formation related protein expression of a human-derived cementoblast. Cement blasts derived from humans are cultured in the presence of each peptide of the present application (0.1 μg / ml), and type I collagen (col1), osteocalcin (OCN), bone sialo, which are proteins detected at the early stage of cell calcification The expression level of the protein (BSP) was measured by Western blot and compared with the result of the additive-free control. The effect | action of this application peptide with respect to the cell growth ability in a human origin periodontal ligament cell is shown. The amount of DNA synthesis when human periodontal ligament cells were cultured in the presence of each peptide of the present application (0.1 μg / ml) was measured. * p <0.05, ** p <0.01 (vs. control) It is a figure which shows the effect | action of this application peptide with respect to the calcium concentration of a human cementoblast and a human periodontal ligament cell. Human cementoblasts and human periodontal ligament cells were cultured in the presence of each peptide of the present application (0.1 μg / ml), and the intracellular calcium concentration was measured. * p <0.05, ** p <0.01 (vs. control) It is a figure which shows the effect | action of this application peptide with respect to the amount of DNA synthesis of a human osteosarcoma origin osteoblast-like cell. The SaOS2 cell line was cultured in the presence of each peptide of the present application (0.1 μg / ml), and the amount of DNA synthesis was measured. ** p <0.01 (vs. control)

  The peptide of the present application is a partial peptide of up to 29 amino acids at the C-terminus, including 7 amino acids at the C-terminus of full-length human amelogenin (rh174). The sequence of human amelogenin full-length protein (rh174) is known (GeneBank GI6715562).

  The peptide of the present invention can be produced by ordinary peptide synthesis. Examples of such methods include Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; Peptide Synthesis, Maruzen Co., 1975; Maruzen Co., Ltd., 1985; continued development of pharmaceuticals, Volume 14, Peptide Synthesis, Hirokawa Shoten, 1991).

  The peptide of the present invention can also be produced by producing a polynucleotide (DNA or RNA) corresponding to the amino acid sequence of the peptide of the present invention and using genetic engineering techniques using the polynucleotide.

  The peptides of the present invention include salts. Examples of the salt include salts formed by acid addition, such as inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.) or organic carboxylic acids (acetic acid, propionic acid, maleic acid, succinic acid, apples) Acid, citric acid, tartaric acid, salicylic acid, trifluoroacetic acid, etc.), glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid and other acidic sugars, hyaluronic acid, chondroitin sulfates, heparan sulfate, heparin, 6-position desulfated heparin, 2 Examples thereof include acidic desulfated heparin, N-desulfated heparin, acidic polysaccharides such as alginic acid, and salts such as organic sulfonic acids (methanesulfonic acid, p-toluenesulfonic acid, etc.). Of these salts, medically acceptable salts are preferred.

  The salt of the peptide of the present invention may also include a salt formed with a basic substance, for example, an inorganic base such as an alkali metal salt (sodium salt, lithium salt, potassium salt, etc.), alkaline earth metal salt, ammonium salt, etc. And salts with organic bases such as diethanolamine salt and cyclohexylamine salt are medically acceptable salts.

  In addition, peptides obtained by modifying the peptide of the present invention are also encompassed by the peptide of the present invention. Examples of such a peptide include a peptide modified with an α-amino group or an α-carboxyl group, a peptide modified with a side chain functional group, and the like. Examples of the modification include modification with a modifying group and a protecting group generally used in the field of peptide chemistry, and substitution with D-amino acid which is an optical isomer.

  The peptide of the present invention has cell growth promoting action and cell differentiation promoting action similar to amelogenin, and can be used for various clinical uses expected for amelogenin.

  Here, “expected clinical use for amelogenin” includes treatment of periodontal diseases, fractures, bone defects, osteoporosis, joint diseases, orthopedic diseases (such as severe osteochondritis, osteoarthritis), Examples include treatment of teeth and missing teeth, periodontal treatment, orthopedic treatment (cultured cell transplantation, bone perforation, osteochondral column transplantation, etc.), bone transplantation by tumor extraction, and improved biocompatibility of bone filling Is done. Therefore, the amelogenin peptide of the present invention can be used as an active ingredient of a pharmaceutical used for such clinical use. The amelogenin peptide of the present invention has a calcification effect on cells having a hard tissue forming ability. In the present invention, “cells having the ability to form hard tissue” are exemplified by cement blasts, osteoblasts, odontoblasts and the like. That is, the peptide of the present invention is excellent in the action of promoting differentiation into hard tissues such as bone, enamel, dentin, and cementum, and is a bone or cartilage formation promoter or regeneration promoter, tooth formation promoter or regeneration. It is particularly preferably used as an accelerator. Pharmaceutical compositions used for such applications are also included in the present invention.

  The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier together with one or more of the peptides of the present invention. The concentration of the peptide of the present invention in the pharmaceutical composition is not limited, and may be appropriately determined according to the application site and the expected effect. The dosage is not particularly limited and should be individually set according to the purpose of administration, the type of disease, the patient's symptoms, sex, age, body weight, administration site, administration method, and the like.

  The pharmaceutical composition of the present invention can be appropriately selected from administration methods such as injection (subcutaneous, intradermal, intravenous, intraperitoneal, etc.), instillation, instillation, implantation, transdermal, oral, inhalation, etc. according to the purpose. can do. Depending on the method of administration, injections (solutions, suspensions, emulsions, solid preparations for use, etc.), tablets, capsules, granules, powders, solutions, lipolytic agents, ointments, gels, An external powder, spray, inhalation powder, eye drop, eye ointment, suppository, pessary and the like can be appropriately selected and formulated. In particular, it is preferably formulated as a dental preparation, such as a root filler, a periodontal tissue treatment agent, and an alveolar bone defect filling material.

  The pharmaceutical agent of the present invention does not adversely affect the peptide of the present invention and does not adversely affect the effects of the present invention, such as other pharmaceutically active ingredients such as hyaluronic acid, stabilizers, emulsifiers, osmotic pressure, etc. Use ingredients normally used in medicine, such as regulators, pH regulators, buffers, isotonic agents, preservatives, soothing agents, colorants, excipients, binders, lubricants, disintegrants, etc. Can do.

  Further, by fixing the peptide of the present invention on the surface of an appropriate insoluble carrier or mixing with an appropriate material, cell proliferation is promoted, cell differentiation is promoted, and bone tissue / cartilage tissue or periodontal tissue is formed. The drug may be used for the purpose of promoting or promoting regeneration, improving biocompatibility, and the like. As such an insoluble carrier or material, various shapes including beads, films, plates, monofilaments, non-woven fabrics, sponges, woven fabrics, knitted fabrics, short fibers, tubes, hollow fibers and the like can be used. Further, it can be used as a medical composite material for implants, bone cements, bone fillers, root canal fillers, fracture plates, artificial joints, and the like.

  When the peptide of the present invention is immobilized on the surface of such a material, the immobilization method is not particularly limited, and is generally used as a method for preparing an immobilized enzyme such as a physical adsorption method, an ionic bond method, a covalent bond method, or a comprehensive method. A method (an immobilized enzyme, 1975, published by Kodansha, pages 9 to 75) can be used.

The invention is illustrated in more detail in the following examples.
[material]
(1) Human full-length amelogenin rh174: GI6715562 cDNA expressed in E. coli is used to purify the produced protein. In the following examples and drawings, human full-length amelogenin rh174 may be referred to as “rh174”.
(2) Peptides of SEQ ID NOs: 1 to 5 and 10 (C7 to C11 and C16): synthesized by conventional methods.
(3) Cement blasts derived from human: A cell line established by the Hiroshima University Graduate School of Oral and Maxillofacial Pathology was used.
(4) Human periodontal ligament cells: A cell line established by the Hiroshima University Graduate School of Oral and Maxillofacial Pathology was used.
(5) Human myeloma-derived osteoblast-like cell line SaOS-2: purchased from DS Pharma Biomedical.

Effect on the proliferation of human-derived cementoblasts Measurement of cell proliferation ability (DNA synthesis amount) Comparison of the effect of the peptide of the present application (C7-C16 peptide) and human full-length amelogenin (rh174) on the amount of human cementoblast DNA synthesis investigated. Human cementoblasts were seeded in 96-well plates (FALCON) at a density of 1 × 10 ³ cells / well, and cultured using α-MEM containing 10% FBS under conditions of 37 ° C. and 5% CO ₂ . Four days after the start of the culture, each peptide of the present application or full-length amelogenin rh174 was added to the medium at a concentration of 0.001-10 μg / ml.

  24 hours after the addition, cell proliferation activity was quantified using Cell Proliferation ELISA BrdU Kit (Roche Diagnost-ics, Basel, Switzerland). That is, after the cell layer was washed twice with PBS, 5-bromo-2'-deoxyuridine (BrdU) (Roche Diagnotics) was added to PBS and incubated at 37 ° C for 2 hours. After removing excess BrdU, an anti-BrdU antibody was added, and the absorbance of the resulting color product was measured using a microplate reader (BIO-RAD) at a wavelength of 450 nm.

  The results are shown in FIG. As is apparent from FIG. 1, the peptide of the present invention has a cementoblast proliferating ability equivalent to that of full-length human amelogenin.

Effects on human differentiation of cell-derived blasts Quantification of bone differentiation markers PCR analysis Human cementoblasts were seeded on 6-well plates (FALCON) at a density of 1 × 10 ⁶ cells / well and contained α-containing 10% FBS. Culturing was performed using MEM under conditions of 37 ° C. and 5% CO ₂ . After reaching confluence, C7-C16 peptide and rh174 at a concentration of 0.1 μg / ml were added to the culture.

After 24 hours, total RNA was extracted from the cell layer using Trizol ^{(registered trademark)} Reagent (Invitrogen). RNA was quantified by measuring absorbance at a wavelength of 260 nm using a spectrophotometer (Gene Spec, Hitachi, Tokyo). Next, cDNA was synthesized from 1 μg of total RNA using ReverTraAce ^{(registered trademark)} (Toyobo, Osaka) and oligo (dt) 20 primer (Toyobo), and osteogenesis-related factors alkaline phosphatase (ALP) and osteosynthesis were synthesized. The expression level of each gene was quantitatively analyzed by the Light Cycler system (Roche Diagnostics) using protegerin (OPG) primers. The results were expressed as a ratio to the gene expression level in the control (no addition). The results are shown in FIG. 2A and FIG.

Measurement of ALP amount Human cementoblasts were seeded on 6-well plates (FALCON) at a density of 1 × 10 ⁶ cells / well, and α-MEM containing 10% FBS was used at 37 ° C and 5% CO _2. In culture. After reaching confluence, C7-C16 peptide and rh174 at a concentration of 0.1 μg / ml were added.

  24 hours after the addition, the cell layer was washed twice with PBS, 0.2% Triton-X-100 was added, and the mixture was allowed to stand at room temperature for 5 minutes. Thereafter, the cells were pulverized with Sonic Vibra Cell (Sonic & Materials Inc). The amount of ALP in the obtained extract was quantified using pNPP Phoshatase Assay Kits (Bioassay Systems, Hayward, CA, USA). 50 μl of Reconstituted Reagent was added to 50 μl of the extract, and incubated at 37 ° C. for 30 minutes. Using a microplate reader (BIO-RAD), the absorbance of the obtained color product at a wavelength of 405 nm was measured. The result is shown in FIG. 2B.

  Increased expression of alkaline phosphatase and osteoprotegerin in cementoblasts means that differentiation into bone is promoted. The C7-C16 peptide of the present application has an action similar to that of human full-length amelogenin and promotes cell bone differentiation.

Effects on NF-kappa B activating receptor (RANKL) expression in human-derived cementoblasts
Human cementoblasts were seeded on a 6-well plate (FALCON) at a density of 1 × 10 ⁶ cells / well, and cultured using 10% FBS-containing α-MEM under conditions of 37 ° C. and 5% CO ₂ . After reaching confluence, it was changed to α-MEM containing 10% FBS, and C7-C16 peptide and rh174 at a concentration of 0.1 μg / ml were added. After 24 hours, total RNA was extracted from the cell layer using Trizol ^{(registered trademark)} Reagent (Invitrogen). RANKL gene expression level was measured by quantitative PCR analysis (above). The results were expressed as a ratio to the gene expression level in the control (no addition).

  The results are shown in FIG. Increased RANKL expression means enhanced bone resorption. It turns out that the peptide of this application suppresses the expression of RANKL like human amelogenin, and suppresses bone resorption.

Effects of human-derived cementoblasts on calcification deposits
Alizarin red staining
HCEM was seeded on a 6-well plate (FALCON) at a density of 1 × 10 ⁶ cells / well, and C7-C16 peptide or rh174 was added from the second day of the culture, and the medium was changed every two days. The culture was carried out in the same manner without adding anything to the control. On the 15th day from the start of culture, the cell layer was washed with 10 mM Tris-HCl (pH 7.5) containing 0.9% NaCl, fixed with 95% ethanol, and treated with 1% Alizarin red staining solution. . The stained cell layer was photographed with a digital camera.

  The results are shown in FIG. The portion indicated by dark red (dark gray in the figure) means that calcified substances are deposited in the cells. The peptides of the present invention induced cement blast mineralization. In particular, the tendency was remarkable in peptides of C9 or higher.

Effects of human-derived cementoblasts on the expression of bone formation-related proteins Investigate the effects of peptides on the expression of type I collagen (col1), osteocalcin (OCN), and bone sialoprotein (BSP), proteins detected in the early stages of calcification It was.
Human cementoblasts were seeded on a ^6- well plate at a density of 1 × 10 ⁶ cells / well and cultured in αMEM containing 10% FBS. After confluence, the FBS concentration was 0%, and C7-C16 peptide or rh174 was added at a concentration of 0.1 μg / ml, and the culture was continued. 24 hours after the addition of the peptide, the cells were dissolved in Triton buffer containing 0.1% protease inhibitor and the protein solution was recovered.

A sample solution for SDS electrophoresis was added to 10 μg of the obtained protein solution, and it was solubilized by heating at 100 ° C. for 3 minutes to obtain a sample for immunochemical detection. After separation by electrophoresis on 12.5% SDS-polyacrylamide gel e-PAGEL ^{(registered trademark)} , the product was transferred onto a PVDF membrane using iBlot gel transfer system (Invitrogen). The membrane was immersed in Block Ace (Snow Brand Milk Products, Sapporo), treated at room temperature for 30 minutes, and then col1 (sc-8784, Santa Cruz Biotechnology INC., Santa Cruz, CA, USA), OCN (sc-18319) as the primary antibody. , Santa cCuz Biotechnology Inc.), BSP (LSL-LB-4335, Cosmo bio co. LTD, Tokyo) and using a GetGet Signal ^{(registered trademark)} Solution 1 (Toyobo ⁾ so that the final antibody dilution is 1: 1000. , Osaka) and incubated overnight at 4 ° C. The membrane was washed with wash buffer {0.5% Tween-20-containing phosphate buffer solution (PBS)}, and col1 and OCN as secondary antibodies were Donkey anti-Goat IRDye ^{(registered trademark)} 800CW (LI-COR Bioscience, Lincoln , NE, USA), the BSP using Goat anti-Rabbit IRDye ^{(TM) 680 (LI-COR Bioscience)} , a final dilution of primary antibodies to each IgG antibody 1: 5000 to become as CanGet Signal ⁽ ( ^{Registered Trademark)} solution2 (Toyobo) and shaken at room temperature for 1 hour. Thereafter, the membrane was thoroughly washed with a wash buffer, and observed with a near infrared fluorescence detection apparatus ODYSSEY (LI-COR Bioscience). The total protein amount was calculated from the βactin amount. The band of each protein is shown in FIG.

  As shown in FIG. 6, there was no difference in the expression level of β-actin between each peptide added group and the non-added control group. The expression levels of type I collagen (col1) and bone sialoprotein (BSP) were increased in the C9, C10, C11, C16, and rh174 added groups compared to the non-added control. An increase in the expression level of osteocalcin (OCN) was observed in all peptide-added groups compared to the non-added control.

Effect on periodontal ligament cell increase Measurement of cell proliferation ability (DNA synthesis amount) The effects of the addition of the present peptide and human amelogenin (rh174) on the DNA synthesis of human periodontal ligament cells were compared.
Human periodontal ligament cells were seeded in a 96-well plate (FALCON) at a density of 1 × 10 ³ cells / well and cultured in DMEM containing 10% FBS at 37 ° C. and 5% CO 2. Four days after the start of the culture, C7 to C16 peptide and rh174 were added at a concentration of 0.1 μg / ml. The culture was carried out in the same manner without adding anything to the control.

  24 hours after the addition, cell proliferation activity was quantified using Cell Proliferation ELISA BrdU Kit (Roche Diagnost-ics, Basel, Switzerland). That is, after the cell layer was washed twice with PBS, 5-bromo-2'-deoxyuridine (BrdU) (Roche Diagnotics) was added to PBS and incubated at 37 ° C for 2 hours. After removing excess BrdU, an anti-BrdU antibody was added, and the absorbance of the resulting color product was measured using a microplate reader (BIO-RAD) at a wavelength of 450 nm.

The results are shown in FIG. The peptide of the present invention promoted proliferation of human periodontal ligament cells.

Effect on calcium concentration of human cementoblasts and human periodontal ligament cells Human cementoblasts and human periodontal ligament cells are seeded on a 6-well plate (FALCON) at a density of 1 × 10 ⁶ cells / well, and the culture starts 2 days. From the eyes, C11, C16 peptide and rh174 were added, and the medium was changed every two days. The culture was carried out in the same manner without adding anything to the control. On the 14th day, the cell layer was washed with 10 mM Tris-HCl buffer (pH 7.0), 10% formic acid was added, and the mixture was extracted at room temperature for 30 minutes to obtain a sample for calcium measurement.
Calcium concentration is measured by measuring the absorbance of the colored product at a wavelength of 570 nm using a microplate reader (BIO-RAD) using Calcium C-Test Wako (Wako Pure Chemical Industries), which is a colorimetric method based on the OCPC method. did. The calcium concentration was calculated using a standard curve prepared by diluting the kit standard solution. The results are shown in FIG.

  In human cementoblasts, the addition of C11, C16 peptide and rh174 significantly increased the calcium concentration. On the other hand, no significant change was observed in periodontal ligament cells by any addition. Thus, C11, C16 peptides and rh174 were shown to increase cementoblast calcification without affecting calcification in periodontal ligament cells.

Effect on proliferation of human osteosarcoma-derived osteoblast-like cells The effect of peptide addition on the amount of DNA synthesis of human osteosarcoma-derived osteoblast-like cells was examined. SaOS2 cell line is seeded in 96-well plate (FALCON) at a density of 1 × 10 ³ cells / well, and after reaching confluence, the serum concentration is 0% and C9, C11, C16 peptides at a concentration of 0.1 μg / ml And rh174 were added. 24 hours after the addition, the amount of DNA synthesis was evaluated using Cell proliferation ELISA BrdU Kit (Roche Diagnostics, Basel, Switzerland). 5-bromo-2-deoxyuridine (BrdU, Roche Diagnotics) was added to α-MEM and incubated at 37 ° C. for 2 hours to be incorporated into DNA during HCEM synthesis. After removing excess BrdU, an anti-BrdU antibody was added, and the absorbance of the resulting color product was measured using a microplate reader VERSAmax (Molecular Devices) at a wavelength of 450 nm. The results are shown in FIG.

  In the peptide C9, C11, C16 and rh174 added groups, significant enhancement of the proliferation ability was recognized as compared with the non-added control. * <0.01 (vs. control)

Claims (9)

A human amelogenin peptide which is the whole or a partial peptide of SEQ ID NO: 11 and comprises the sequence of SEQ ID NO: 1. The amelogenin peptide according to claim 1, wherein the number of amino acids is up to 19. The amelogenin peptide according to claim 1, which is the peptide according to any one of SEQ ID NOs: 1 to 10. The pharmaceutical composition which contains the peptide in any one of Claims 1-3 as an active ingredient. The pharmaceutical composition according to claim 4, which is a cell differentiation promoting agent. The pharmaceutical composition according to claim 4, which is a cell growth promoter. The pharmaceutical composition according to claim 5, which promotes calcification of cells having a hard tissue forming ability. The pharmaceutical composition according to claim 4, which is a bone or cartilage formation promoter or regeneration promoter. The pharmaceutical composition according to claim 4, which is a periodontal tissue formation promoter or regeneration promoter.