JP2011225519A - Polypeptide and protein comprising the polypeptide, and periodontal tissue-regenerating agent containing them as active ingredients - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new polypeptide exhibiting the cell differentiation activities of a human periodontal membrane cell in the degree nearly equal to that of a polypeptide represented by SEQ ID NO:1(H-2), and to provide a periodontal tissue-regenerating agent containing the new polypeptide and the protein comprising the polypeptide as active ingredients, and capable of effectively recovering the periodontal tissue such as the human periodontal membrane.SOLUTION: The polypeptide includes the amino acid sequence represented by SEQ ID NO:35(H-2') or SEQ ID NO:36. The polypeptide is preferably synthesized from a C terminus-side peptide of a human enamel sheath protein formed from an N terminus-side of a human sheathlin by degradation by a protease.

Description

The present invention relates to a polypeptide that leads to an enamel sheath protein having cell differentiation activity of human periodontal ligament cells, ie, periodontal ligament regeneration ability.
The present invention also relates to a periodontal tissue regenerating agent that can effectively recover periodontal tissues such as periodontal ligament by using such a polypeptide or a protein comprising the polypeptide as an active ingredient.

In this specification, an amino acid sequence or a base sequence is represented by a SEQ ID number.
Further, in the present specification, the term “protein” means a proteinaceous segment, which consists of about 150 or more consecutive amino acids and contains 70% or more of the amino acids encoded by the gene. The term “polypeptide” means a proteinaceous segment consisting of up to about 3 to 150 consecutive amino acids.

As shown in the schematic diagram of teeth and periodontal tissue (Fig. 1), regeneration of periodontal tissue involves the regeneration of cementum in which collagen fiber bundles are initially embedded (Cementum Regeneration) to trace the root growth. , Sometimes referred to as “CR”), followed by periodontal ligament regeneration and alveolar bone regeneration.
Since it became clear that enamel protein, which has an effect on the formation of enamel, was also involved in the induction and regeneration of periodontal tissue, the preparation of the enamel protein (commercially, porcine enamel matrix A derived product (Enamel Matrix Derivatives) as a main component and marketed as a registered trademark “EMDOGAIN ^™ ” has been clinically used for periodontal regeneration in periodontal disease treatment.

However, since these enamel protein preparations are multi-component systems (mainly composed of three types of proteins: 25 kDa amelogenin, 89 kDa enamelin, and 65 kDa sheathrin), they are related to the mechanism of their physiological activity and to them. In many cases, the molecular period has not been fully elucidated and periodontal ligament regeneration does not always reach the expected level during actual treatment. In addition, since extracts from porcine juvenile enamel are currently used, they are heated before preparation of the drug, but there is also a risk of infection with type E viruses or unidentified viruses.
In subsequent studies, of the above three proteins, only sheathrin (not amelogenin or enamelin) was able to regenerate cementum and periodontal ligaments in animal experiments using dogs with buccal cleft bone defects. In addition, it was clarified from the fact that human periodontal ligament cell differentiation activity was promoted in a culture system experiment using human periodontal ligament cells. In order to directly use the enamel protein for periodontal disease treatment, there is much room for improvement.

As soon as the sheathrin is secreted from the enamel blast, the 17 kDa enamel sheath protein (hereinafter sometimes referred to as “ESP”) derived from the N-terminus, the 25 kDa acidic protein derived from the center of the molecule , Which is broken down into three segments of a 19 kDa calcium binding protein derived from the C-terminus.
So far, of these three segments, 17 kDa ESP has been involved in the construction of enamel trabecular sheath (sheath) during tooth development, and once by protease from the N-terminal side of human or porcine sheathlin. It has been clarified that it is a product produced by cleavage of. That is, the 65 kDa sheathrin is a parent protein of 17 kDa ESP and may contain a portion other than the portion directly involved in periodontal tissue regeneration compared with 17 kDa ESP. Or 447 in the case of humans) is not necessarily required for periodontal tissue regeneration.

The present inventors previously described "170 amino acid sequences represented by SEQ ID NO: 10" out of 421 amino acid sequences constituting porcine sheathlin and 447 amino acid sequences constituting human sheathlin. Among them, it was found that “196 amino acid sequence represented by SEQ ID NO: 11” includes a site with a stronger periodontal tissue regeneration action (see Patent Document 1),
Subsequently, by synthesizing a short polypeptide of about 3 to 66 based on these two or more amino acid sequences composed of 170 or more, which part of the amino acid sequence is actually a human periodontal ligament (Human PerioDontal Ligament, hereinafter sometimes referred to as “HPDL”) It has been investigated whether it promotes the cell differentiation activity of cells most (see Patent Document 2).

  In the above Patent Document 2, as a result of examination of various short polypeptides represented by SEQ ID NOs: 1 to 34 by the present inventors, SEQ ID NO: 1 and SEQ ID NO: It is described that the polypeptide having the amino acid sequence represented by 2 enhances the alkaline phosphatase (Alkaline Phosphatase, hereinafter sometimes referred to as “ALP”) activity of HPDL cells. In cell culture systems, it is a standard marker for cell differentiation of osteoblast cells.)

JP 2004-143165 A Japanese translation of PCT publication 2008-53474

J Dent Res 2003; 82: 982-986 J Dent Res 2002; 81: 103-108

As described above, in clinical practice, as an active ingredient of a periodontal tissue regenerating agent, it can be applied quickly and with excellent periodontal tissue regeneration, and directly involved in periodontal tissue regeneration. It is always desirable to develop a protein that does not contain extra proteins other than the protein to be used.
Specifically, for example, there is a strong demand for the development of a periodontal tissue regenerating agent that minimizes the side effects caused by extra proteins not involved in periodontal tissue regeneration and the risk of viral infection.

In view of such a demand, the present invention aims to provide a novel polypeptide capable of exhibiting cell differentiation activity of human periodontal ligament cells as much as the polypeptide represented by SEQ ID NO: 1 described above. . The present invention also provides a periodontal tissue regenerating agent capable of effectively recovering periodontal tissues such as periodontal ligament by using such a novel polypeptide or a protein comprising the polypeptide as an active ingredient. Provision is also an issue.
In the present invention, the periodontium means cementum, periodontal ligament, gingiva, alveolar bone and the like (see FIG. 1 described above). Therefore, the periodontal tissue regenerating agent of the present invention has the purpose of regenerating those tissues, but is particularly excellent mainly in the regeneration action of cementum and periodontal ligament, and other periodontal tissues are secondary. Have a regenerative action.

In order to solve the above-mentioned problems, the present inventors firstly made it impossible to prepare a sample of human tooth enamel (thickness of about 2.0 to 2.5 mm). Focusing on the fact that dentin odontoblasts, which are the main components of, express enamel protein in the same way as enamel blasts, odontoblasts were prepared from human premolars extracted by orthodontic treatment as samples.
Next, by cloning the sheathrin mRNA from the odontoblast and determining the base sequence based on the database, it was found that the amino acid called Gly at position 154 of SEQ ID NO: 11 was missing. As a result of repeated examination,
Periodontal ligament cells having a polypeptide containing the missing portion (polypeptide in which the 154th Gly of SEQ ID NO: 11 is missing) is the same as the polypeptide represented by the aforementioned SEQ ID NO: 1. As a result, the present invention was achieved.

The present invention has been made based on such knowledge, and the gist thereof is as follows.
(1) A polypeptide containing the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36 without the 154th Gly.
(2) The polypeptide according to (1) above, which is synthesized from a peptide of the C-terminal side of human enamel sheath protein produced by protease degradation from the N-terminal side of human sheathlin.
(3) A protein comprising the polypeptide according to (1) or (2).
(4) A periodontal tissue regenerating agent comprising the polypeptide according to (1) or (2) or the protein according to (3) as an active ingredient.

The polypeptide represented by SEQ ID NO: 35 (SEQ ID NO: 36) of the present invention is equivalent to the polypeptide represented by SEQ ID NO: 1 (SEQ ID NO: 11) described in Patent Documents 1 and 2. The periodontal ligament regeneration ability can be expressed, and since it has one amino acid less than these known polypeptides, it has an excellent effect that it is easy to synthesize artificially (chemically).
Since the periodontal tissue regenerating agent of the present invention contains such a polypeptide or a protein comprising the polypeptide as an active ingredient, it can exhibit an excellent periodontal tissue regenerating action quickly by simply applying a small amount. In addition, there is no risk of side effects caused by extra proteins not involved in periodontal tissue regeneration, risk of viral infection caused by protein extracts derived from pigs, and the like.

It is a schematic diagram explaining the name of a tooth | gear and periodontal tissue. Amino acid sequence of porcine and human enamel sheath protein (ESP). It is the graph which showed the difference in the ALP induction activity of the HPDL cell by the presence or absence of addition of 1α-25 dihydroxy-vitamin D ₃ (VD). (A) shows the difference in ALP activity in HPDL cells depending on the presence or absence of VD addition of TGF-β1, and (B) shows the synthetic ESP peptides H-1, H-2, H-3, H-4. , ALP activity in HPDL cells when H-5 VD was added, (C) shows ALP activity in HPDL cells when VD of these synthetic ESP peptides was not added. It is the graph which showed the ALP induction activity of the HPDL cell by TGF- (beta) 1, BMP-2, and a synthetic ESP peptide. It is a graph which shows the influence at the time of adding TGF- (beta) 1 receptor inhibitor (SB) in the ALP induction activity of TGF- (beta) 1, synthetic ESP peptide H-2. (A) shows the ALP-inducing activity of TGF-β1, and (B) shows the ALP-inducing activity of H-2. It is the result of investigating the calcification activity of H-2 peptide and TGF-β1, (A) is a photograph stained with alizarin red S, and (B) shows the measured amount of calcium (ng / cm ² ). (A) shows the expression of bone sialoprotein gene, (B) shows the expression of osteocalcin gene, and (C) shows the result of semiquantitative PCR using a light cycler, respectively.

The polypeptide of the present invention contains the amino acid sequence shown by SEQ ID NO: 35 or SEQ ID NO: 36.
FIG. 2 shows the amino acid sequences of porcine and human enamel sheath protein (ESP) (the shaded area represents the site obtained as a result of alternative splicing). In FIG. 2, “p” is a porcine ESP sequence (SEQ ID NO: 10), “h” is a human ESP sequence (SEQ ID NO: 11) obtained by the database, and “h ′” is an odontoblast It is a human ESP sequence (SEQ ID NO: 36) determined from the cDNA sequence obtained from the sample.
SEQ ID NO: 35 refers to the 13 amino acid sequences (SDKPPKPELPVDF) corresponding to the 144th to 156th stages in “h ′ (SEQ ID NO: 36)” shown in FIG.

As apparent from FIG. 2, SEQ ID NO: 36 is composed of 195 amino acids, and thus has one amino acid less than SEQ ID NO: 11 composed of 196 amino acids described in Patent Documents 1 and 2. And SEQ ID NO: 35 consisting of 13 amino acids has one amino acid less than SEQ ID NO: 1 consisting of 14 amino acids corresponding to the 144th to 157th stages in SEQ ID NO: 11.
That is, the polypeptide represented by SEQ ID NO: 35 (SEQ ID NO: 36) of the present invention is more than the polypeptide represented by SEQ ID NO: 1 (SEQ ID NO: 11) described in Patent Documents 1 and 2. However, since each one amino acid is less, it has an excellent effect that it is easy to synthesize artificially (chemically).

As shown in FIG. 2 (in the figure, 165th to 190th stages of “h”, 164th to 189th stages of “h ′”), the amino acid sequence of human ESP is near (rear) near the C-terminal side. , 26 residues corresponding to exons 8 and 9 have an extra peptide. Exons corresponding to these extra peptides are not found in other mammals, including pigs, but are derived from two duplicates of exon 7 on human DNA. In addition, there are people in AMBN (ameloblastin) exon 7 has a GAG codon corresponding to Gly ^154. That is, as a polypeptide that expresses human ESP, a polypeptide lacking the 154th Gly (SEQ ID NO: 35 or SEQ ID NO: 36) and a sequence containing the Gly (SEQ ID NO: 1) Or there is a mutation with SEQ ID NO: 11).

  In the present invention, the above-mentioned polypeptide can be chemically synthesized based on a conventional method (for example, prepared as a recombinant protein using a plasmid having DNA encoding the polypeptide), You may synthesize | combine from the C terminal peptide of the human enamel sheath protein produced | generated by protease from the N terminal side.

The present invention also provides a protein comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36.
A protein basically consists of a single amino acid sequence, but such a single amino acid sequence exists with several additional amino acid residues, for example, about 1 to about 100 additional residues. Sometimes.
This additional amino acid residue (sequentially encoded sequence) is one that is naturally associated with the polypeptide, or something like a heterogeneous amino acid residue / peptide sequence. Such proteins can have several additional amino acid residues, or can include hundreds or more additional amino acid residues.

The periodontal tissue regenerating agent of the present invention contains the above-described polypeptide or a protein comprising the polypeptide as an active ingredient.
At this time, the polypeptide or a protein comprising the polypeptide (hereinafter sometimes referred to as “enamel sheath protein according to the present invention” for convenience) may be associated with the aforementioned amelogenin or enamelin, A complex formed by assembling these may exhibit an excellent periodontal tissue regeneration action.

The enamel sheath protein according to the present invention is sufficiently effective for regenerating periodontal tissue by containing 0.001 to 3 mg, preferably about 0.01 to 1 mg, as an active ingredient in 100 mg of the periodontal tissue regenerating agent of the present invention. Can be demonstrated.
If the amount of the active ingredient is less than the above, the periodontal tissue regeneration action may not be sufficiently obtained. If the amount is more than the above, the effect is saturated, but the cost becomes high and economically disadvantageous. Become.

When formulating, the enamel sheath protein according to the present invention may be sterilized as necessary to inactivate the protease contained therein and prevent infection of the individual virus or the like. preferable. If the sterilization treatment is performed at a temperature higher than necessary for a long time, the enamel sheath protein as an active ingredient may be denatured and deactivated. Therefore, generally at about 60 to 100 ° C. for about 10 to 60 minutes. Sterilization is recommended.
The enamel sheath protein according to the present invention is an active ingredient of the periodontal tissue regenerating agent of the present invention, and of course has an action related to enamel formation. After treatment of dental diseases such as periodontal disease, it can be used as a therapeutic agent for the purpose of regenerating the removed periodontal tissue, depending on the gingival retraction by brushing or the denture used at the time of treatment It can be used as a therapeutic agent aiming at regenerating periodontal tissue in response to tooth root exposure and the like, and can also be used as a biomaterial constituting an implant surface.

The enamel sheath protein according to the present invention may be used as it is or dissolved in pure water or the like. However, the enamel sheath protein may be used in the mouth without any problem, and has a high viscosity PGA (propylene glycol alginic acid). Salt) or hyaluronic acid, etc., it is held at the site to be applied and fixed easily. In addition to the enamel sheath protein, which is an active ingredient, in order to make it easy to use for the above-mentioned applications, anti-inflammatory agents, bulking agents, fragrances, cell growth growth factor (TGF), bone morphogenetic factor (BMP), etc. A growth factor or the like may be added to obtain a periodontal tissue regenerating agent.
Moreover, it can be expected that the periodontal tissue regeneration action can be obtained more effectively by combining the treatment using the periodontal tissue regeneration agent of the present invention and the treatment such as GTR method.

[Materials and methods]
The peptides synthesized based on the amino acid sequence of the C-terminal side of human enamel sheath protein (ESP) were examined for the enhancement and mineralization ability of alkaline phosphatase (ALP) during the culture of HPDL cells.

[Determination of human ESP sequence]
Recombinant enamel sheath protein or physiologically active peptide exhibiting regenerative (CR) activity of cementum can be prepared based on human sheathlin (ameloblastine) cDNA prepared from human odontoblasts (see Non-patent Document 1).
Human odontoblasts were obtained from healthy premolars extracted for orthodontic treatment by the method described in Non-Patent Document 2. The freshly extracted tooth was divided by “bone only” in the long axis direction, the pulp was removed with tweezers, and the odontoblasts remaining on the surface of the pre-ivory were removed. Odontoblasts express amelogenin, enamelin, sheathlin, enamelin (MMP-20), KLK4 (which is a protease), etc., so total RNA can be expressed using the Stratagene Total RNA Miniprep Kit and its protocol (Stratagene, La Jolla, CA, USA).

  cDNA was synthesized using Ready-To-Go You-Prime First-Strand Beads (Amersham-Pharmacia Biotech, Piscataway, NJ, USA). PCR primers for human enamel sheath protein were designed based on the nucleotide sequences in the GenBank database. The two primer pairs were 5′-TGAAGGACCTGATACTGATCC (SEQ ID NO: 37) and 5′-TGATTTGCTCCAAAAGGCACG (SEQ ID NO: 38), and the amplification product was 718 bp.

[Synthetic peptide]
Several polypeptides were synthesized based on the sequences of human and porcine ESP C-terminal peptides. Their sequences are shown in Table 1.
In Table 1, P-1 peptide was used as a control because it had the highest ALP-inducing activity among porcine synthetic ESP peptides in previous studies.

[Cell culture of HPDL cells and ST2 cells]
Normal periodontal ligament cells,
・ 10% calf serum (FBS, Asahi Technoglass, Chiba, Japan),
Either with or without 1α-25 dihydroxy-vitamin D ₃ (Calbiochem, La Jolla, CA, USA),
1% antibacterial agent (100 U / mL penicillin G and 100 μg / mL streptomycin sulfate; Gibco BRL, Grand Island, NY, USA),
The mixture was cultured at 37 ° C. in an α-minimal essential medium (α-MEM) containing 5% under humidified conditions containing 5% carbon dioxide.
ST2 cells (Riken Cell Bank, Tsukuba, Japan), which are osteoblast-like cells derived from mouse bone marrow cells, were also cultured under the same conditions as the periodontal ligament cells.

[Analysis of Alkaline Phosphatase Activity]
In the cell culture system using HPDL cells and ST2 cells cultured under the above conditions, the ALP-inducing activities of one porcine synthetic peptide and 11 human synthetic peptides shown in Table 1 were analyzed as described below. .
As positive controls, recombinant osteogenesis factors “bone induction factor-2 (hereinafter referred to as“ BMP-2 ”: TECHNE Co., MN, USA) 1 μg / mL” and “transforming growth factor-β1” And “TGF-β1”: R & D Systems, Inc., MN, USA) 0.5 or 1 ng / mL ”.

For HPDL cells, first, using a 96-well plate, about 5 × 10 ⁵ cells were seeded in one well and incubated for 24 hours. Thereafter, the medium contains 10 nM of 1α-25 dihydroxy-vitamin D ₃ (hereinafter sometimes abbreviated as “VD”) and contains either a synthetic peptide dissolved in pure water or a positive control (growth factor). The medium was replaced with α-MEM medium.
After 96 hours of incubation, the cells were washed once with phosphate buffered saline, 100 mM 2-amino-2methel-1,3 containing 5 mM MgCl ₂ and 10 mM p-nitrophenyl phosphate as a substrate. The ALP activity was measured by incubation at 37 ° C. for 10 minutes in -propanediol-HCl buffer (pH 10.0). At this time, 0.2 M NaOH was added to stop the reaction, and then the absorbance at 405 nm was measured using a plate reader. The concentration of the synthetic peptide when added to these HPDL cell culture systems is 25 or 50 μg / mL, and the effect of the TGF-β1 receptor inhibitor (SB431542, 10 μM) on the ALP-inducing activity of the synthetic peptide is also examined. In order to be added to these HPDL cell culture systems.

FIG. 3 shows the difference in alkaline phosphatase (ALP) inducing activity of human periodontal ligament (HPDL) cells with and without 1α-25 dihydroxy-vitamin D ₃ (VD).
FIG. 3 (A) shows ALP activity of transforming growth factor-β1 (TGF-β1) in HPDL cells with and without VD. As shown in FIG. 3 (A), TGF-β1 increased the ALP activity of HPDL cells in a dose-dependent manner when VD was added.
FIG. 3 (B) shows synthetic ESP peptides H-1, H-2, H-3, H-4, H-5 (as shown in Table 1, SEQ ID NO: 3, NO: 1, NO: 2, NO: 4, NO: 5) shows ALP activity in HPDL cells when VD is added.
FIG. 3 (C) shows ALP activity in HPDL cells of synthetic ESP peptides (H-1 to H-5) when VD is not added.

  As shown in FIG. 3, it was found that the level of ALP activity in HPDL cells was enhanced when VD was added in any of the cell culture systems added with synthetic ESP peptide and TGF-β1.

FIG. 4 shows the ALP-inducing activity of HPDL cells by synthetic ESP peptide and positive controls TGF-β1 and BMP-2.
As shown in FIG. 4, synthetic peptides H-2 (SEQ ID NO: 1) and H-2 ′ (SEQ ID NO: 35) had the highest activity compared to other synthetic peptides. The data in FIG. 4 represents the average (means ± SEM) of five culture results. * Represents a significant difference (p <0.01) with respect to H-2. All were culture systems to which 10 nM VD was added, synthetic peptides were examined at a concentration of 50 μg / mL, and TGF-β1 was examined at a concentration of 1 ng / mL.
In addition, despite the cell culture system to which VD was added, no enhancement was observed in the level of BLP-2 ALP activity.

FIGS. 3 and 4 show that multiple synthetic peptides induced ALP activity in HPDL cells, but higher concentrations were required than TGF-β1, and ALP activity was none of that of TGF-β1. It was n’t expensive.
Further, as shown in FIG. 4, the H-2 peptide had the highest ALP-inducing activity of HPDL cells among human synthetic peptides and P-1 peptides. All peptides smaller than the H-2 peptide were less active except for the H-2 ′ peptide.

When a TGF-β1 receptor inhibitor (SB431542 (hereinafter sometimes abbreviated as “SB”) in the ALP-inducing activity of TGF-β1 and synthetic peptide H-2 (SEQ ID NO: 1) was added to FIG. HPDL cells were cultured by adding 10 nM VD, SB was added at a concentration of 10 μM, and TGF-β1 was examined at concentrations ranging from 0 to 5 ng / mL.
As shown in FIG. 5 (A), TGF-β1 inhibitor SB431542 significantly inhibited the ALP-inducing activity of TGF-β1, but as shown in FIG. 5 (B), the synthetic peptide H- The ALP-inducing activity of HPDL cells increased in response to 2 was not inhibited by the addition of SB and was not affected. This suggests that the cell differentiation activity of the synthetic peptide is induced via a receptor different from the TGF-β1 receptor.

  Although the results are not shown, the ALP activity of ST2 cells was enhanced by BMP-2, not by TGF-β1, and not by any synthetic peptide.

[Calcification activity of TGF-β1 and synthetic peptide H-2]
Recombinant TGF-β1 and synthetic peptide H-2 (SEQ ID NO: 1) were analyzed for calcification activity of HPDL cells as described below. Since synthetic peptide H-2 had higher ALP-inducing activity than any synthetic peptide, it was decided to examine the mineralization activity of the peptide.
1 × 10 ⁵ HPDL cells were seeded in a 6-well plate and cultured for 24 hours. Thereafter, the medium was divided into 50 μM ascorbic acid, 10 mM β-glycerophosphate, 10 nM 1α-25 dihydroxy-vitamin D ₃ (differentiation medium), and samples (TGF-β1: 1 ng / mL, H-2: 25 μg / mL), respectively. The growth medium was changed.
The cells were cultured for 28 days while changing the medium every 72 hours, the medium was discarded, and the calcification activity was examined with alizarin red S staining and calcium content.

[Analysis method]
For alizarin red S staining, cells cultured for 28 days were fixed with 100% methanol and stained with alizarin red S for 10 minutes. The staining solution for alizarin red S is prepared by dissolving 1% alizarin red S (sodium alizarin sulfonate: Sigma) in pure water and adjusting the pH to 6.4 with 0.1 N aqueous ammonia.
In order to measure the amount of calcium, the cell portion was dissolved in 0.5N hydrochloric acid. The solution was reacted with Calcium C-test kit (Wako Pure Chemical Industries Ltd, Osaka, Japan), and the absorbance at 570 nm was measured using a plate reader. The DNA base sequence of cDNA corresponding to human enamel sheath protein was determined by ALF DNA zSequencer (Pharmacia, LKB ALF, Sweden).

FIG. 6 shows the results of calcification activity of H-2 peptide and TGF-β1. “Cont” in FIG. 6 represents a control to which no sample (TGF-β1 or H-2) was added.
FIG. 6 (A) is a photograph stained with alizarin red S, and FIG. 6 (B) is a measured calcium amount (ng / cm ² ).

  As shown in FIGS. 6 (A) and (B), the H-2 peptide was found to induce calcification more than the control (which also calcifies), but the induction level is a positive control. It was lower than that of TGF-β1.

[Semi-quantitative PCR using a light cycler]
Regarding the expression of osteopontin, osteocalcin, and bone sialoprotein, which are calcified tissue markers related to the differentiation state of HPDL cells, from culture of HPDL cells in culture medium inducing calcification for 4 days, 14 days, 28 days The total mRNA obtained was examined.

Total RNA was extracted from cultured HPDL cells using RNAzol ^™ B (Tel-Test Inc., Friendswood, TX, USA). cDNA was synthesized from 3 μg of total RNA of HPDL cells cultured for 4 to 21 days using the oligo-dT primer and You-primed First-Strand Beads Kit (Amersham-Pharmacia Biotech, Piscataway, NJ, USA) according to the protocol. PCR primers were synthesized based on human mRNA sequences. The primer pairs are as follows:
Osteopontin: 5′-TGACCTCTGTGAAAACAGCGT-3 ′ (SEQ ID NO: 45) and 5′-TGTACATTGTGAAGCTG TGAA-3 ′ (SEQ ID NO: 46) (301 bp),
Osteocalcin: 5′-TTGTGTCCAAGCAGGAGGGCA-3 ′ (SEQ ID NO: 47) and 5′-ACATCCATAGGGCTGG GAGGT-3 ′ (SEQ ID NO: 48) (304 bp),
Bone sialoprotein: 5′-GCAGAAGTGGATGAAAACGA-3 ′ (SEQ ID NO: 49) and 5′-TGGTGGTAGTATTCTGACCA-3 ′ (SEQ ID NO: 50) (448 bp).
The primer set was amplified using glyceraldehyde-3phosphate dehydrogenase (Clontech, Palo Alto, CA, USA) mRNA as a control.
cDNA was prepared routinely using a DNA master SYBR Green I kit and a light cycler (Roche Molecular Biochemicals, Mannheim, Germany). The relative amount of each mRNA was determined using half of the PCR product and these were corrected by relative comparison of GAPDH mRNA.

FIG. 7 shows the results of semi-quantitative PCR measurement using a light cycler.
FIG. 7A shows the expression of bone sialoprotein gene in HPDL cells, FIG. 7B shows the expression of osteocalcin gene, and FIG. 7C shows the expression of osteopontin gene in comparison with the value of “4C”.
“4C” is a control cultured for 4 days,
“4T” is cultured with TGF-β1 for 4 days,
“4H” is cultured in H-2 for 4 days.
“14C” is a control cultured for 14 days,
“14T” is cultured for 14 days in TGF-β1,
“14H” is cultured in H-2 for 14 days.
“28C” is a control cultured for 28 days,
“28T” is cultured for 28 days in TGF-β1,
“28H” represents 28-day culture with H-2.
TGF-β1 was examined at a concentration of 1 ng / mL, and H-2 peptide was examined at a concentration of 25 μg / mL.

As shown in FIGS. 7A to 7C, all of the bone sialoprotein gene, osteocalcin gene, and osteopontin gene are expressed and cultured in both the control and the case where TGF-β1 or H-2 is added. (Expression level) increased while
Their expression when H-2 peptide was added was very close to that of TGF-β1, and compared to the control value, the expression of these bone sialoprotein gene, osteocalcin gene, and osteopontin gene was promoted. Indicated.

[Statistical analysis]
All values in this example were averaged (the means ± SEM). Statistical processing was determined using Student's unpaired t-test, and p <0.05 was considered significant.

The periodontal tissue regenerating agent comprising the polypeptide or protein of the present invention as an active ingredient has an excellent periodontal tissue regenerating action.
Therefore, after treating dental diseases such as periodontal disease, the therapeutic agent intended to regenerate the removed periodontal tissue, gingival retraction by brushing, and tooth root exposure by the denture used at the time of treatment It can also be used as a therapeutic agent aiming at regenerating periodontal tissue in response to the above, a biomaterial constituting an implant surface, and the like.
Moreover, it can be expected that the periodontal tissue regeneration action can be obtained more effectively by combining the treatment using the periodontal tissue regeneration agent of the present invention and the treatment such as GTR method.

Claims (4)

  A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 35 or SEQ ID NO: 36.   The polypeptide according to claim 1, wherein the polypeptide is synthesized from a C-terminal peptide of human enamel sheath protein produced by protease degradation from the N-terminal side of human sheathlin.   A protein comprising the polypeptide according to claim 1 or 2.   A periodontal tissue regenerating agent comprising the polypeptide according to claim 1 or 2 or the protein according to claim 3 as an active ingredient.