JPH07265069A - Human pericemental cell adhesive factor - Google Patents

Abstract

PURPOSE:To obtain a human pericemental cell adhesive factor effective in treating periodontosis requiring the regeneration of a bone and the pericementum by local or systemic administration or improving the implant take ratio of an artificial dental root due to its ability to induce the adhesion of the pericementum. CONSTITUTION:The characteristic of this human pericemental cell adhesive factor comprises (1) manifestation of 60+ or -20kDa molecular weight measured by the gel chromatography, (2) having an affinity for a hydroxyapatite column and eluted with 0.5+ or -0.1M phosphoric acid (pH7.4) and (3) manifesting a single band at the position of 70+ or -5kDa apparent molecular weight by the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue. Furthermore, this method for producing the adhesive factor is provided and a medicinal composition contains the human pericemental cell adhesive factor as an active ingredient.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel purified human periodontal ligament cell adhesion factor, a method for purifying the same, and a pharmaceutical composition containing the purified human periodontal ligament cell adhesion factor as an active ingredient.

[0002]

2. Description of the Related Art The adhesion between the periodontal ligament and the cementum / dentin is important for the regeneration of the periodontal ligament in the treatment of periodontal disease and the regeneration of teeth by implanting artificial dental roots (Inoue Takashi et al. Dental Outlook, 1986, 67, 1027-1037). From such a point of view, experiments have been conducted on adhesion and implant engraftment of artificial material or hydroxyapatite, which is a constituent of cementum / dentin, and periodontal ligament cells.

However, fibronectin (Toru Takigami, Journal of Japan Periodontology Society, 1989, 31, 773-79) has been clarified as a factor inducing adhesion of periodontal ligament cells.
3), osteopontin (Somerman MJ et al., J. Periodo
ntol, 1989, 60, 73-77), fibroblast growth factor (bF)
GF) (Terranova VP et al., J. Periodontol, 1989, 60,293.
-301).

On the other hand, as a protein produced from periodontal ligament cells, transforming growth factor β
(TGF-β) -like substance (Mitsuyoshi Kubota, Kanagawa Dental Science, 1989,
24,157-168), alkaline phosphatase (Ohshima M
Et al., J. Nihon Univ. Sch. Dent., 1988, 30, 208-217;
Kawase T et al., Adv. Den. Res., 1988, 2, 234-239),
Osteonectin (Somerman MJ et al., Archs. Oral Bi
ol., 1990, 35, 241-247) is known.

The aforementioned fibronectin is produced by various cells existing in vascular endothelial cells and other connective tissues and is involved in adhesion of almost all cells (Toshihiko Nagai, Biochemistry, 1991, 63, 507-512). Osteopontin is present in bone tissue but is also confirmed to be present in other tissues and has a cell adhesion signal (RGD sequence). The action is said to be related to general cell adhesion (Shimokawa et al., The Bone, 19
89, 3, 79-88). In addition, fibroblast growth factor (bFG
F) is produced by fibroblasts, acts on bone and cartilage progenitor cells, and is involved in differentiation and proliferation (Suzuki, protein nucleic acid enzyme, 19
91, 36, 1259-1267).

[0006]

However, in normal periodontal tissues, adhesion between tooth dentin and periodontal ligament cells is important, and periodontal ligament cell adhesion factors that act locally are produced by periodontal ligament cells. It is presumed that the periodontal ligament cell adhesion factor produced by periodontal ligament cells has not been identified so far, and the isolation and purification thereof has not yet been performed.

The first object of the present invention is to provide a purified human periodontal ligament cell adhesion factor. Second of the present invention
An object of the present invention is to provide a method for purifying human periodontal ligament cell adhesion factor. A third object of the present invention is to provide a pharmaceutical composition containing purified human periodontal ligament cell adhesion factor as an active ingredient.

[0008]

Means for Solving the Problems The present inventors have accomplished the present invention as a result of intensive research for solving the above problems. That is, the gist of the present invention is (1) purified human periodontal ligament cell adhesion factor derived from human periodontal ligament cell culture supernatant, (2) method for purifying human periodontal ligament cell adhesion factor, and (3) purified human The present invention relates to a pharmaceutical composition containing a periodontal ligament cell adhesion factor as an active ingredient.

The purified human periodontal ligament cell adhesion factor derived from the human periodontal ligament cell culture supernatant of the present invention has the following physicochemical characteristics. (1) 60 ± 2 by gel chromatography
It has a molecular weight of 0KD, has an affinity for (2) hydroxyapatite column, and has 0.5 ± 0.1M phosphoric acid (pH
7.4), and (3) SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining show a single band at an apparent molecular weight of 70 ± 5 KD.

The purified human periodontal ligament cell adhesion factor of the present invention has the following biological characteristics. (1) The purified human periodontal ligament cell adhesion factor of the present invention is
Significantly enhances adhesion of human periodontal ligament cells to hydrophobic 96 well microplates. (2) At that time, a significant development of adhered human periodontal ligament cells is observed by observation with a phase contrast microscope. (3) The human periodontal ligament cell adhesion-inducing activity of the purified human periodontal ligament cell adhesion factor of the present invention is hardly affected by the addition of the GRGDS synthetic peptide containing the RGD sequence which is the receptor binding site of fibronectin. This indicates that the purified human periodontal ligament cell adhesion factor of the present invention does not induce cell adhesion by the cell adhesion signal (RGD sequence) represented by fibronectin. (4) The human periodontal ligament cell adhesion-inducing activity of the purified human periodontal ligament cell adhesion factor of the present invention is not inhibited by the addition of anti-fibronectin antibody, anti-vitronectin antibody or anti-fibronectin receptor antibody. This means that the purified human periodontal ligament cell adhesion factor of the present invention is not a fibronectin or vitronectin-like substance, and the adhesion of human periodontal ligament cells by the purified human periodontal ligament cell adhesion factor of the present invention is a fibronectin receptor or It shows that it is not mediated by the vitronectin receptor.

The purified human periodontal ligament cell adhesion factor of the present invention has (A) an apparent molecular weight of 70 ± 5 KD on SDS-polyacrylamide gel electrophoresis under reduction, and (B) an RGD sequence. Fibronectin, vitronectin, laminin, I rather than being involved in adhesion
Since it is not a ligand of the integrin family like CAM-1 and has an affinity for (C) hydroxyapatite, it has been conventionally known as a substance that induces adhesion of periodontal ligament cells. ), Osteopontin (44KD),
It is a physiologically active substance different from fibroblast growth factor (bFGF) (16-32KD).

The method for purifying the human periodontal ligament cell adhesion factor of the present invention comprises the steps of purifying the culture supernatant of human periodontal ligament cells by gel chromatography and hydroxyapatite column chromatography. The human periodontal ligament cells used here can be cultured in a medium in the presence of fetal bovine serum or in a serum-free medium (for example, MCDB107 medium), but in order to isolate human periodontal ligament cell adhesion factor, a human serum-free medium is used. It is preferable to culture. As the human periodontal ligament cells, periodontal ligament cells collected from a dental treatment patient can be used, but it is also possible to use established human periodontal ligament cells.

The culture supernatant of human periodontal ligament cells is concentrated in advance to a content of 40 KD or more, for example, by ultrafiltration in order to be subjected to a purification step by gel chromatography. In this case, it is preferable to use a filtration membrane, such as Amicon Diaflow Membrane YM-10, which does not leak the contents of 40 KD or more. For use in gel chromatography, a buffer is prepared by a commonly used method such as dialysis or ultrafiltration. During the concentration operation and the buffer preparation operation, various proteolytic enzyme inhibitors (eg, phenylmethylsulfonyl fluoride (PMSF), aprotinin, tranexamic acid, benzamide, etc.) can be added.

The concentrated culture supernatant is fractionated by gel chromatography using the above-prepared buffer to obtain a fraction having a molecular weight of 60 ± 20 KD. As a column for gel chromatogram, for example, PO-60K manufactured by JASCO Corporation can be used.

The fraction of 60 ± 20 KD is converted into a buffer of 0.01 M phosphoric acid (pH 7.4) by a method such as dialysis and ultrafiltration, and then purified by hydroxyapatite column chromatography. Be used for the process. That is, chromatography was carried out with a phosphoric acid concentration gradient of 0.01 M to 1.0 M to obtain 0.5 ± 0.1
The fraction eluted with M is obtained. As the hydroxyapatite column, for example, Bio-Gel HTP (trade name) can be used. This fraction is SD under reducing
S-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining gives a single band at 70 ± 5 KD.

Unlike the above-mentioned purification method, the order of the purification step by gel chromatography and the purification step by hydroxyapatite column chromatography may be exchanged. That is, the concentrated culture supernatant of human periodontal ligament cells containing substantially 40 KD or more of content was first fractionated by hydroxyapatite chromatography to obtain a fraction eluted with 0.5 M phosphoric acid, and then the gel. It is also possible to obtain a purified human periodontal ligament cell adhesion factor by a method of obtaining a 60 ± 20 KD fraction by chromatography.

The human periodontal ligament cell adhesion factor thus obtained is further purified by a known means such as ion exchange chromatography, a hydrophobic group such as 2 to 2 carbon atoms.
Further purification can be achieved by using hydrophobic chromatography using a silica gel-based carrier bearing 0 alkyl groups, phenyl groups, reverse phase chromatography, or the like, or a combination thereof.

The purified human periodontal ligament cell adhesion factor of the present invention is used for bone regeneration and periodontal regeneration in periodontal disease treatment,
It is effective for bone regeneration in the treatment of osteoporosis, promotion of implant implantation of artificial dental roots, or promotion of bone formation when local bone formation is required such as fracture. As for osteoporosis,
Examples include postmenopausal or senile osteoporosis, and secondary osteoporosis due to diabetes, renal dialysis, steroids and the like.

The growth of periodontal ligament cells is important for the regeneration of tooth roots, and the growth of periodontal ligament cells promotes bone formation. For the growth of such periodontal ligament cells, adhesion of periodontal ligament cells to teeth and bone tissue is essential, and the purified human periodontal ligament cell adhesion factor of the present invention is for the purpose of inducing adhesion of periodontal ligament cells. It is administered orally or parenterally to the periodontal region. Further, in the artificial tooth root implant, an artificial material in which the purified human periodontal ligament cell adhesion factor of the present invention is impregnated and carried on the surface or inside of the implant material can be implanted, and the purified human tooth root cell adhesion factor of the present invention can also be implanted. The human periodontal ligament cell adhesion factor can be orally or parenterally administered to the periphery of the implant.

Further, activation of osteoblasts in bone formation is brought about by adhesion of osteoblasts to hydroxyapatite, collagen, proteoglycan, etc. which constitute the bone matrix. Human periodontal ligament cells are similar to osteoblasts, and the purified human periodontal ligament cell adhesion factor of the present invention can be involved in osteoblast adhesion. Therefore, when local bone formation is required such as healing of a fracture, the purified human periodontal ligament cell adhesion factor of the present invention is orally or parenterally administered to the fracture site for the purpose of inducing osteoblast adhesion. Administer

The pharmaceutical composition of the present invention contains purified human periodontal ligament cell adhesion factor as an active ingredient, and suitable carriers and excipients known to be pharmaceutically acceptable. It is possible to contain one or more of the above bases.
The administration method can be local administration, or transmucosal administration, for example, nasal, rectal, orally, or parenterally, intramuscularly, subcutaneously, and intravenously. The pharmaceutical composition of the present invention is produced by a method known per se, for example, by conventional mixing, lysis, lyophilization, and about 0.1 to 100%, particularly 1 to 50% of purified human periodontal ligament cells. Contains an adhesion factor. The form of the preparation is not particularly limited and may be, for example, an injection, a tablet, a dragee, an ampoule, a vial, a spray, or a suppository. The purified human periodontal ligament cell adhesion factor of the present invention generally depends on the patient's weight, symptoms, administration method and the like, and is generally about 100 μg to about 5000 μ based on the judgment of a doctor.
g / kg body weight / day is administered.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. The abbreviations used in the examples have the following meanings. HPLF: human periodontal ligament cells, HPLF-CM: culture supernatant of human periodontal ligament cells,
BSA: bovine serum albumin, FCS: fetal bovine serum,
PBS (−): Phosphate b without Ca and Mg
uffer saline, manufactured by Nissui Pharmaceutical Co., Ltd.

Example 1 (Culture of human periodontal ligament cells) Human periodontal ligament was collected from orthodontic patients aged 10 to 12 years old from a healthy and completely erupted first premolar extracted for the purpose of treatment. That is, the removed first premolar was stored in a medium containing an antibiotic prepared in advance. When collecting the periodontal ligament, in order to avoid mixing of tissues other than the periodontal ligament as much as possible, avoid the cervical part containing the gingiva and the apex part containing other connective tissues, and use a scalpel to remove the central part of the root. Peeled off. The detached tissue piece was analyzed by Saito et al. And Kawase et al. (Kawase T et al., Adv. Dent. Res., 1
988,22,234-239), 24 well tissue culture
In each well of the plate (Falcon 3047), 5% fetal calf serum (FCS: Flow Laboratories), 50 μg / ml ascorbic acid (Wako Pure Chemical Industries, Ltd.) and antibiotics (10
Medium (Dulbecco's Modified Eagle Medium) (DME containing 0 units / ml penicillin and 100 μg / ml streptomycin, Gibco # 600-5 / 40)
M, Nissui Pharmaceutical Co., Ltd.).

Thereafter, the tissue piece was allowed to stand still at the center of the bottom of the well, and 15 mm round thermanox.
Cover slips (round thermanox coverslip) (Lux 54
14) was put on. This tissue piece is 37 ° C, humidity 95% or more,
The cells were cultured under the conditions of 5% carbon dioxide gas and 95% air, the medium was replaced every 3 to 4 days, cells were migrated from the tissue pieces, and the culture was continued for about 14 days until the migrated cells reached confluence. Confluent cells also adhere and grow on the bottom of the well and the surface of the coverslip, and both the well and the coverslip contain 0.25% trypsin (1/250, Difco Laboratories) to obtain subcultured cells. P
Cells treated with BS (-) and floating cells were collected by centrifugation, seeded again, and passaged repeatedly. In the present invention, HPLF which was passaged 5-8 times was used for the experiment.

(Collection of human periodontal ligament cell culture supernatant) The HPLF obtained by the above method was subcultured, and HPLF-CM was collected using the HPLF grown to confluence. That is, when the confluent HPLF medium was replaced, 50 μg / ml of ascorbic acid and antibiotics (100 units / ml penicillin and 100 μg / ml) were used.
ml streptomycin, Gibco # 600-5 / 4
The cell layer was washed twice with a serum-free medium MCDB107 (Far East Pharmaceutical Co., Ltd.) containing 0), and then cultured for 24 hours in the same medium. After that, the culture supernatant was collected, and 1,000 × g,
After centrifugation for 5 minutes to remove contaminating cells, the supernatant was frozen and stored at -20 ° C as HPLF-CM. After thawing, 10,000 × g, to remove insoluble components,
Centrifugation was performed at 4 ° C. for 10 minutes, and the supernatant was concentrated using an ultrafiltration membrane (Diaflow Membrane YM-10, Amicon Co., Ltd.).

(Separation of culture supernatant of human periodontal ligament cells by gel chromatography) HPLF-CM 0.1m concentrated 100 times by the ultrafiltration operation in the above method.
l was further passed through a filter having a pore size of 0.22 μm (Millex GV, manufactured by Nippon Millipore Industry Co., Ltd.) to give a sample for high performance liquid chromatography (HPLC). That is, gel chromatography (GPC) column PO-60K for HPLC (manufactured by JASCO Corporation)
PBS (-) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., Ltd.)
Was equilibrated with a flow rate of 0.25 ml for 1 minute and divided into test tubes of a fraction collector (Gilson Model 203, MS Equipment Co., Ltd.) every 1 minute for separation operation. That is, the amount of protein was monitored by absorption at 280 nm, and the whole was divided into 8 fractions. As a result, the molecular weight is 60 ± 2
1.2 ml was obtained as fraction III corresponding to 0KD (Fig. 1). In addition, HPLF-CM, gel chromatography I
~ Fractions up to VIII, 50% FCS, and Blank
100 μl of each (serum-free medium) was added to the hydrophobic 96w
In addition to the well plate, the number of human periodontal ligament cells adhered by the method described in Example 3 to be described later was determined by DN per well.
It was measured by quantifying the amount of A. As a result, as shown in FIG. 2, high cell adhesion-inducing activity was observed in Fraction III.

(Separation by Hydroxyapatite Chromatography) 5 lots of Fraction III having a molecular weight of 60 ± 20 KD obtained by gel chromatography in the above method were collected to make 6 ml, and pH 7.4 was obtained using an ultrafiltration membrane YM-10. , PBS adjusted to 0.01M phosphate concentration
Replace the buffer with (-), and then add a pore size of 0.22.
Through a μm filter (Millex GV, manufactured by Nippon Millipore Industrial Co., Ltd.), high performance liquid chromatography (H
PLC) sample. That is, the HPLC column is a hydroxyapatite column (Bio-GelHT).
P) using PBS (-) at pH 7.4 and a phosphoric acid concentration of 0.01 M to 1.0 M, and a continuous concentration gradient (0.01
(M-1.0M / 40 minutes). It is divided into test tubes of a fraction collector (Gilson Model 203, MS Equipment Co., Ltd.) every 1 minute, and a separation operation is performed to obtain a fraction (HTP-0.5M) of 0.5M corresponding to a phosphoric acid concentration of 0.5M.
25 ml was obtained.

Example 2 (SDS-polyacrylamide electrophoresis) The fraction (HTP-0.5M) obtained in Example 1 and the HPLF-CM before fractionation obtained in Example 1 were similarly subjected to a hydroxyapatite column. The 0.5 M phosphoric acid elution fractions obtained by fractionating with (Bio-Gel HTP) were each concentrated about 10 times using Centricon 10 manufactured by Amicon. 20 μl of this concentrated solution was added to Selected Method in Cellular
Immunology (Selected Method in Cellular Immunolo
gy) (BBMishell and SMShiigi, p416, 1980, Fr.
eeman & Co.) and treat the sample with a sample buffer containing 2-mercaptoethanol.
Load on 5% polyacrylamide gel, buffer 25 mM Tris, 190 mM glycine, 0.1% S
Using DS, electrophoresis was performed at a current of 25 mA for 3 hours. After the electrophoresis, the gel was stained with Coomassie Brilliant Blue Acetic Acid solution according to a conventional method. As a result, the fraction (HTP-0.5
Regarding M) (lane B), a single band was observed on 67KD of molecular weight marker (lane A) of the Pharmacia Electrophoresis calibration kit that was electrophoresed (FIG. 3). Considering the protein separation ability of SDS-polyacrylamide electrophoresis, the molecular weight of the protein shown above 67 KD was determined to be 70 ± 5 KD.

Example 3 (Human periodontal ligament cell adhesion-inducing activity) Cell adhesion experiments were carried out according to the method of Kawase et al. (See Tissue Culture, 1990, 16, 42-46). That is, 1 × 10 ⁶ cells of HPLF
The cells were planted in s / 100 mm dish, cultured for 3 days, confirmed by a phase contrast microscope that HPLF was in the growth stage, and used for the experiment. HPLF-CM, after gel chromatography fractionation, molecular weight 60 ± 20 KD Fraction III (GP
C-Fr.III), a fraction eluted with 0.5 M phosphoric acid by hydroxyapatite chromatography (HTP-
0.5M), and 100 μm of each fraction (HTP-Pass) that has no affinity for the hydroxyapatite column.
1 for each well of a hydrophobic 96-well plate
Coating was performed at 7 ° C. for 1 hour. After that, the added sample was removed, and BSA (Fraction V, Mikes: Seikagaku Corporation) inactivated by heat treatment at 80 ° C. for 15 minutes was added to each well and left in an incubator at 37 ° C. for 30 minutes. At the same time, the growth-stage HPLF was treated with PBS (−) containing 0.25% trypsin, and the released HPLF was 2.5 × 10 ⁴ ce / well.
ll inoculated with 50 μg / ml ascorbic acid and antibiotics (100 units / ml penicillin and 100 μl).
g / ml streptomycin, Gibco # 600-5
/ 40) in a serum-free medium MCDB107 (Far East Pharmaceutical Co., Ltd.) was further cultured for 20 hours.

The number of cells was analyzed by measuring the amount of DNA, and the spread state of the cells was observed with a phase contrast microscope. D per cell
Since the NA amount was constant, the number of adherent cells was calculated from the DNA amount. The DNA measurement method was carried out by applying the method of Puzaus JE and Goodman DBP (see Anal. Biochem., 1978, 86, 50-55). That is, in order to measure the number of adherent cells,
The cell layer of the hydrophobic 96-well plate was dried at 60 ° C. for 1 hour together with the standard DNA amount. In the meantime, 2M diaminobenzoic acid (DABA, Aldrich) was mixed with activated carbon (Merck) for removal of the fluorescence measurement inhibitor, and
Centrifugation was performed at 0 × g for 5 minutes at room temperature, and the supernatant was filtered through a filter having a pore size of 0.22 μm. 45 μl of the filtered 2M diaminobenzoic acid solution was added to each well in which the cells were dried, and the reaction was performed for 30 minutes in a dryer at 60 ° C. After that, 0.6N perchloric acid 200μ
1 was added to each well, and a microplate fluorescence spectrophotometer (MRF-100, JASCO Corporation) was used.
Excitation was performed at 405 nm, and fluorescence intensity measurement was performed at 500 nm. However, the standard sample is DNA derived from salmon sperm (Sigma
Company) was used. As a result, as shown in FIG. 4, HPLF-
CM, GPC-Fr.III, and HTP-0.5M showed high human periodontal ligament cell adhesion-inducing activity.

Example 4 (Effect of synthetic peptide on cell adhesion) The basic adhesion signal of cell adhesion was determined by Hautanen A et al. In RGD (Arg-
Gly-Asp) sequence is the most important (J. Biol. C
hem., 1989, 264, 1437-1442), synthetic peptide GR
The effect was searched using GDS (Gly-Arg-Gly-Asp-Ser: Peptide Research Institute). That is, in the cell adhesion experiment in Example 3, 50 μg / ml of ascorbic acid and antibiotics (100 units / ml penicillin and 1 μg / ml of which the final concentration of GRGDS was adjusted to 0.2 mM) were used.
00 μg / ml streptomycin, Gibco # 60
HPL pretreated with serum-free medium MCDB107 (Kyoto Pharmaceutical Co., Ltd.) containing 0 to 5/40) at 37 ° C. for 20 minutes
The cell adhesion inducing activity of each sample was measured using F.
That is, Blank (serum-free medium), HPLF-CM
(Human periodontal ligament cell culture supernatant) and HTP-0.5M
(100 μl of 0.5 M phosphoric acid elution fraction of hydroxyapatite column chromatography), and FN (fibronectin) (manufactured by Koken Co., Ltd., human plasma fibronectin) 1.2 μg / ml and VN (vitronectin) (Koken) 1.2 μg / ml of human plasma vitronectin manufactured by K.K. Co., Ltd. was added to each hydrophobic 96-well plate, and the number of adherent human periodontal ligament cells was quantified to determine the amount of DNA per well to determine the cell adhesion-inducing activity It was measured. On the other hand, G
To see the effect of RDGS peptide, the same procedure was performed using human periodontal ligament cells that had been previously treated with GRDGS 0.2 mM as described above (addition of GRDGS). The result is shown in Fig. 5.
Shown in. The results are shown in FIG.
The inhibition rate of the peptide is shown. As is clear from FIG.
The inhibition of human periodontal ligament cell adhesion by GRDGS peptide was found to be strong in fibronectin, vitronectin, and serum-free medium, but extremely weak in HTP-0.5M.

Example 5 (Effect of various antibodies on cell adhesion) Anti-fibronectin against human periodontal ligament cell adhesion-inducing activity of the fraction (HTP-0.5M) eluted with 0.5M phosphoric acid by hydroxyapatite chromatography Antibodies (Biomedical Technology, rabbit anti-human fibronectin), anti-vitronectin antibody (Transformation Research, rabbit anti-fibronectin receptor) and anti-fibronectin receptor antibody (Hail Chemical Pharmaceuticals, rabbit anti-human laminin) Considered the impact. That is, in the cell adhesion experiment in Example 3, HPLF2.5 × 10 ⁴ cells was used.
For l, each antibody was adjusted to a final concentration of 1 μg / ml, 50 μg / ml of ascorbic acid and antibiotics (100 units / ml penicillin and 100 μg / m).
l streptomycin, Gibco # 600-5 / 4
Incubation was further performed for 20 hours in serum-free medium MCDB107 (Kyokuto Pharmaceutical Co., Ltd.) containing 0). When the cell adhesion rate was measured by the amount of DNA in the same manner as in Example 3, it was 285 ± 25 ng / well for the control HTP-0.5M. On the other hand, with anti-fibronectin antibody, 236 ± 4
5 ng / well, 263 ± for anti-vitronectin antibody
37 ng / well, 242 ± 42 ng / well for anti-fibronectin receptor antibody. In contrast to the adhesion-inducing action (215 ± 23 ng / well) of fibronectin (1.2 μg / ml) that was performed at the same time, the anti-fibronectin antibody was 45 ± 5 ng / well, and the anti-fibronectin receptor antibody was 50 ± 6 ng / well, which significantly suppressed the adhesion. did.

Example 6 (Injection) Aseptically, using an ampoule having a volume of 3 ml, 3 m
The physiological saline solution (1) was adjusted to contain 30 mg of purified human periodontal ligament cell adhesion factor, and 15 mg of human serum albumin was added to the resulting solution, which was sealed and used as an injection.

[0034]

EFFECTS OF THE INVENTION The purified human periodontal ligament cell adhesion factor of the present invention induces adhesion of periodontal ligament cells. Therefore, it is necessary to regenerate bone and periodontal ligament by local administration or systemic administration. It is effective in treating diseases or improving the implant survival rate of artificial dental roots. Further, the purified human periodontal ligament cell adhesion factor of the present invention can induce the adhesion of osteoblasts to bone, and is effective for bone regeneration in the treatment of osteoporosis, and also locally such as fracture. When bone formation is required, local administration can induce bone cell adhesion and promote bone formation.

[Brief description of drawings]

FIG. 1 shows the results of gel chromatography of HPLF-CM on a PO-60K column.

FIG. 2 is a view showing cell adhesion-inducing activity of HPLF-CM gel chromatography fractions.

FIG. 3 is a view showing a result of SDS-polyacrylamide electrophoresis of a fraction (HTP-0.5M). Lane A
Is a molecular weight marker, and Lane B is a fraction (HTP-0.5).
M), Lane C shows the fraction eluted with HPMF-CM by hydroxyapatite column chromatography 0.5 M phosphoric acid.

FIG. 4 is a diagram showing the results of human periodontal ligament cell adhesion-inducing activity. Blank is serum-free medium, HPLF-
CM is a culture supernatant of human periodontal ligament cells, GPC-Fr. III
Is a gel chromatography fraction III of HPLF-CM,
HTP-0.5M is 0.5M phosphoric acid elution fraction of hydroxyapatite column chromatography, HTP-P
“Ass” indicates a fraction having no affinity for the hydroxyapatite column.

FIG. 5 is a view showing an inhibitory effect (cell amount of adherent cells) on cell adhesion of GRDGS peptide. Blank is serum-free medium, HPLF-CM is culture supernatant of human periodontal ligament cells,
HTP-0.5M is a 0.5M phosphate elution fraction of hydroxyapatite column chromatography, FN is fibronectin, and VN is vitronectin.

FIG. 6 is a graph showing the inhibitory effect (% inhibition) on cell adhesion of GRDGS peptide. Blank is serum-free medium, HPLF-CM is culture supernatant of human periodontal ligament cells, H
TP-0.5M is a 0.5M phosphate elution fraction of hydroxyapatite column chromatography, FN is fibronectin, and VN is vitronectin.

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[Procedure amendment]

[Submission date] March 24, 1992

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] Full text

[Correction method] Change

[Correction content]

[Document name] Statement

Title: Human periodontal ligament cell adhesion factor

[Claims]

Detailed Description of the Invention

[0001]

The present invention relates to a novel human periodontal ligament cell adhesion factor BACKGROUND OF THE, a manufacturing method thereof, and to pharmaceutical compositions and 該Hi preparative periodontal ligament cell adhesion factor as an active ingredient.

[0002]

2. Description of the Related Art The adhesion between the periodontal ligament and the cementum / dentin is important for the regeneration of the periodontal ligament in the treatment of periodontal disease and the regeneration of teeth by implanting artificial dental roots (Inoue Takashi et al. Dental Outlook, 1986, 67, 1027-1
037). From such a point of view, experiments have been conducted on adhesion and implant engraftment of artificial material or hydroxyapatite, which is a constituent of cementum / dentin, and periodontal ligament cells.

However, fibronectin (Toru Takigami, Journal of Japan Periodontology Society, 1989, 31, 1989, 31) has been clarified as a factor inducing adhesion of periodontal ligament cells.
773-793), osteopontin (Somerm)
an MJ et al. Periodontol, 198
9, 60, 73-77), fibroblast growth factor (bF)
GF) (Terranova VP et al., J. Perio.
dontol, 1989, 60, 293-301).

On the other hand, as a protein produced from periodontal ligament cells, transforming growth factor β
(TGF-β) -like substance (Mitsuyoshi Kubota, Kanagawa Dental Science, 198
9, 24, 157-168), alkaline phosphatase (Ohshima M, et al., J. Nihon Uni.
v. Sch. Dent. , 1988, 30, 208-2
17; Kawase T et al., Adv. Den. Re
s. , 1988, 2, 234-239), osteonectin (Somerman MJ et al., Archs. Or).
al Biol. , 1990, 35, 241-247).
It has been known.

The aforementioned fibronectin is produced by various cells existing in vascular endothelial cells and other connective tissues and is involved in adhesion of almost all cells (Toshihiko Nagai, Biochemistry, 1991, 63, 507-512). Osteopontin is present in bone tissue but is also confirmed to be present in other tissues and has a cell adhesion signal (RGD sequence). The action is said to be related to general cell adhesion (Shimokawa et al., The Bone, 1989, 3, 79-88).
Fibroblast growth factor (bFGF) is produced by fibroblasts, acts on bone and cartilage progenitor cells, and is involved in differentiation and proliferation (Suzuki, protein nucleic acid enzyme, 1991, 36, 12).
59-1267).

[0006]

However, in normal periodontal tissues, adhesion between tooth dentin and periodontal ligament cells is important, and periodontal ligament cell adhesion factors that act locally are produced by periodontal ligament cells. It is presumed that the periodontal ligament cell adhesion factor produced by periodontal ligament cells has not been identified so far, and the isolation and purification thereof has not yet been performed.

A first object of the present invention is to provide a human periodontal ligament cell adhesion factor. The second object of the present invention is to
It is intended to provide a method for producing a human periodontal ligament cell adhesion factor. A third object of the present invention is to provide a pharmaceutical composition comprising, as an active ingredient a human periodontal ligament cell adhesion factor.

[0008]

Means for Solving the Problems The present inventors have accomplished the present invention as a result of intensive research for solving the above problems. That is, the gist of the present invention is (1) 6 by gel chromatography.
It shows a molecular weight of 0 ± 20 KD and (2) hydroxyapa
Has affinity for tight columns, 0.5 ± 0.1M phosphoric acid
Elute at (pH 7.4), (3) SDS-polyacry
Luamide electrophoresis and Comassie Brilliant Blue dyeing
A single band at an apparent molecular weight of 70 ± 5 KD in color
And a human periodontal ligament cell adhesion factor characterized by
Method for producing periodontal ligament cell adhesion factor, and the human periodontal ligament
The present invention relates to a pharmaceutical composition containing a cell adhesion factor as an active ingredient.

Further, human periodontal ligament cell adhesion factor of the present invention has the following biological characteristics. (1) human periodontal ligament cell adhesion factor of the present invention, is significantly enhanced adhesion to hydrophobic 96well microplate human periodontal ligament cells. (2) At that time, a significant development of adhered human periodontal ligament cells is observed by observation with a phase contrast microscope. (3) human periodontal ligament human periodontal ligament cell adhesion activity of inducing cell adhesion factor of the present invention is little affected by the addition of GRGDS synthetic peptide containing the RGD sequence which is a receptor binding site of fibronectin. This human periodontal ligament cell adhesion factor of the present invention shows that not to induce cell adhesion by cell adhesion signal represented by fibronectin (RGD sequence). (4) human periodontal ligament cell adhesion-inducing activity of human periodontal ligament cell adhesion factor of the present invention is not inhibited by addition of anti-fibronectin antibodies, anti-vitronectin antibodies or anti-fibronectin receptor antibodies. Human periodontal ligament cell adhesion factor of this is that the present invention is not a fibronectin and vitronectin-like substance, adhesion of human periodontal ligament cells by human periodontal ligament cell adhesion factor of the present invention, Hui pro vitronectin receptor and vitronectin receptor It indicates that it does not go through.

[0010] The human periodontal ligament cell adhesion factor of the present invention,
(A) The apparent molecular weight on SDS-polyacrylamide gel electrophoresis under reduction is 70 ± 5 KD,
(B) Since it is not involved in adhesion by the RGD sequence,
Fibronectin, vitronectin, laminin, ICA
Since it is not a known factor involved in cell adhesion such as M-1, and has an affinity for (C) hydroxyapatite, it has been conventionally known as a substance that induces adhesion of periodontal ligament cells. Fibronectin (~ 44
0KD), osteopontin (44KD), and fibroblast growth factor (bFGF) (16-32KD), which are physiologically active substances.

[0011] Human periodontal ligament cell adhesion factor of the present invention, with the culture supernatant of human periodontal ligament cells to gel chromatography and hydroxyapatite column chromatography
By doing so, they can be separated and acquired . here,
Human periodontal ligament cells used are medium in the presence of fetal bovine serum,
Alternatively, it can be cultured in a serum-free medium (for example, MCDB107 medium), but it is preferable to culture in a serum-free medium for the isolation of human periodontal ligament cell adhesion factor. As the human periodontal ligament cells, periodontal ligament cells collected from a dental treatment patient can be used, but it is also possible to use established human periodontal ligament cells.

The culture supernatant of human periodontal ligament cells is concentrated in advance to a content of 40 KD or more, for example, by ultrafiltration in order to be subjected to a separation step by gel chromatography. In this case, it is preferable to use a filtration membrane, such as Amicon Diaflow Membrane YM-10, which does not leak the contents of 40 KD or more. To urging gel chromatography, dialysis, to prepare a buffer by the method commonly ultrafiltration. During the concentration operation and the buffer preparation operation, various proteolytic enzyme inhibitors (eg, phenylmethylsulfonyl fluoride (PMSF), aprotinin, tranexamic acid, benzamide, etc.) can be added.

The concentrated culture supernatant is fractionated by gel chromatography using the above-prepared buffer to obtain a fraction having a molecular weight of 60 ± 20 KD. As a column for gel chromatogram, for example, PO-60K manufactured by JASCO Corporation can be used.

The fraction of 60 ± 20 KD is converted into a buffer of 0.01 M phosphoric acid (pH 7.4) by a method such as dialysis and ultrafiltration, and then separated by hydroxyapatite column chromatography. <br/> Used for the process. That is, chromatography was carried out with a phosphoric acid concentration gradient of 0.01 M to 1.0 M to obtain 0.5 ± 0.1
The fraction eluted with M is obtained. As the hydroxyapatite column, for example, Bio-Gel HTP (trade name) can be used. This fraction is SD under reducing
S-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue staining gives a single band at 70 ± 5 KD.

Further, unlike the above-mentioned manufacturing method, the order of the separation step by gel chromatography and the separation step by hydroxyapatite column chromatography may be exchanged. That is, the concentrated culture supernatant of human periodontal ligament cells containing substantially 40 KD or more of content was first fractionated by hydroxyapatite chromatography to obtain a fraction eluted with 0.5 M phosphoric acid, and then the gel. The human periodontal ligament cell adhesion factor of the present invention can also be obtained by a method of obtaining a 60 ± 20 KD fraction by chromatography.

The thus-obtained human periodontal ligament cell adhesion factor is further purified by known purification means such as ion exchange chromatography, hydrophobic groups such as 2 to 2 carbon atoms.
Further purification can be achieved by using hydrophobic chromatography using a silica gel-based carrier bearing 0 alkyl groups, phenyl groups, reverse phase chromatography, or the like, or a combination thereof.

The human periodontal ligament cell adhesion factor of the present invention, tooth regeneration of bone regeneration and the periodontal ligament in periodontal disease treatment, bone regeneration in osteoporosis therapy, promotion of implant engraftment of dental implants, or topically bone such as bone fractures It is effective for promoting bone formation when formation is necessary. Examples of osteoporosis include postmenopausal or senile osteoporosis, and secondary osteoporosis due to diabetes, renal dialysis, steroids and the like.

The growth of periodontal ligament cells is important for the regeneration of roots, and the growth of periodontal ligament cells promotes bone formation. In such the growth of periodontal ligament cells are essential adhesion to teeth and bone tissue periodontal ligament cells, human periodontal ligament cell adhesion factor of the present invention the purpose of inducing the adhesion of the periodontal ligament cells,
It is administered orally or parenterally to the periodontal region. Further, in the artificial tooth root implant, to含滲the human periodontal ligament cell adhesion factor of the present invention the inner surface or of the implant material, the artifact having supported thereon can be implanted, also human tooth root of the present invention The membrane cell adhesion factor can also be orally or parenterally administered to the periphery of the implant.

Activation of osteoblasts in bone formation is brought about by adhesion of osteoblasts to hydroxyapatite, collagen, proteoglycan, etc. which constitute the bone matrix. Human periodontal ligament cells are similar to osteoblasts, human periodontal ligament cell adhesion factor of the present invention may be involved in the adhesion of osteoblasts. Therefore, when local bone formation is required, such as healing of fracture ,
The human periodontal ligament cell adhesion factor for the purpose of adhesion induction of osteoblasts, administered fracture f orally or parenterally.

[0020] The pharmaceutical compositions of the present invention has as an active ingredient human periodontal ligament cell adhesion factor, pharmaceutically suitable carriers acceptable it is known, groups such as excipients One or more agents can be contained. The administration method can be local administration, or transmucosal administration, for example, nasal, rectal, orally, or parenterally, intramuscularly, subcutaneously, and intravenously. The pharmaceutical composition of the present invention can be prepared by a method known per se, for example, a conventional mixing,
Produced by dissolution, lyophilization, and about 0.1-10
0%, in particular containing human periodontal ligament cell adhesion factor of 1% to 50%. The form of the preparation is not particularly limited and may be, for example, an injection, a tablet, a dragee, an ampoule, a vial, a spray, or a suppository. The human periodontal ligament cell adhesion factor of the present invention depends on the body weight, symptoms, administration method, etc. of the patient, and is generally about 10 based on the judgment of a doctor.
0 μg to about 5000 μg / kg body weight / day is administered.

[0021]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. The abbreviations used in the examples have the following meanings. HPLF: human periodontal ligament cells, HPLF-CM: culture supernatant of human periodontal ligament cells,
BSA: bovine serum albumin, FCS: fetal bovine serum,
PBS (-): Phosph without Ca and Mg
ate buffer saline, manufactured by Nissui Pharmaceutical Co., Ltd.

Example 1 (Culture of human periodontal ligament cells) Human periodontal ligament was collected from a healthy and completely erupted first premolar extracted for the purpose of treatment from orthodontic patients aged 10 to 12 years. That is, the removed first premolar was stored in a medium containing an antibiotic prepared in advance. When collecting the periodontal ligament, in order to avoid mixing of tissues other than the periodontal ligament as much as possible, avoid the cervical part containing the gingiva and the apex part containing other connective tissues, and use a scalpel to remove the central part of the root. Peeled off. Peeling tissue pieces the river Serra method (Kawase T, et al., Adv.Dent.Re
s. , 1988, 22, 234-239),
Tissue culture 24 well plate (Falcon 304
5% fetal calf serum (FCS: Flo) in each well
w Laboratories), 50 μg / ml ascorbic acid (Wako Pure Chemical Industries, Ltd.) and antibiotics (10
Medium (Dulbecco, s Modified E) containing 0 units / ml penicillin and 100 μg / ml streptomycin, Gibco # 600-5 / 40)
It was suspended in a medium medium (DMEM, Nissui Pharmaceutical Co., Ltd.).

Thereafter, the tissue piece was allowed to stand still at the center of the bottom of the well, and a 15 mm round thermanox coverslip (round thermanox) was used.
coverslip) (Lux 5414).
This tissue piece was placed at 37 ° C., humidity of 95% or more, 5% carbon dioxide gas,
The cells were cultured under the condition of 95% air, the medium was exchanged every 3 to 4 days, cells were migrated from the tissue pieces, and the culture was continued for about 14 days until the migrated cells reached confluence. Confluent cells also adhere and grow on the bottom of the well and the surface of the coverslip.
ll and coverslips were both 0.25% trypsin (1
/ 250, Difco Laboratories) in PBS (-), the suspended cells were collected by centrifugation, seeded again, and passaged repeatedly. In addition,
In the present invention, HPLF used for 5-8 passages was used for the experiment.

(Collection of human periodontal ligament cell culture supernatant) The HPLF obtained by the above method was subcultured, and HPLF-CM was collected using the HPLF grown to confluence. That is, when the confluent HPLF medium was replaced, 50 μg / ml of ascorbic acid and antibiotics (100 units / ml penicillin and 100 μg / ml) were used.
ml streptomycin, Gibco # 600-5 / 4
The cell layer was washed twice with a serum-free medium MCDB107 (Far East Pharmaceutical Co., Ltd.) containing 0), and then cultured for 24 hours in the same medium. After that, the culture supernatant was collected, and 1,000 × g,
After centrifugation for 5 minutes to remove contaminating cells, the supernatant was frozen and stored at -20 ° C as HPLF-CM. After thawing, 10,000 × g, to remove insoluble components,
Centrifugation was performed at 4 ° C. for 10 minutes, and the supernatant was concentrated using an ultrafiltration membrane (Diaflow Membrane YM-10, Amicon Co., Ltd.).

(Separation of Human Periodontal ligament Cell Culture Supernatant by Gel Chromatography) HPLF-CM 0.1m concentrated 100 times by the ultrafiltration operation in the above method.
l was further passed through a filter having a pore size of 0.22 μm (Millex GV, manufactured by Nippon Millipore Industry Co., Ltd.) to give a sample for high performance liquid chromatography (HPLC). That is, gel chromatography (GPC) column PO-60K for HPLC (manufactured by JASCO Corporation)
PBS (-) containing 0.1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma Chemical Co., Ltd.)
Equilibration with a fraction collector (Gilson Model) every minute at a flow rate of 0.25 ml for 1 minute.
203, MS Equipment Co., Ltd.)
A separation operation was performed. That is, the amount of protein was monitored by absorption at 280 nm, and the whole was divided into 8 fractions. As a result, 1.2 was obtained as Fraction III corresponding to a molecular weight of 60 ± 20 KD.
ml was obtained (Fig. 1). In addition, HPLF-CM, each fraction by gel chromatography I to VIII, 50% FCS,
And Blank (serum-free medium) 100 μl each
Was added to a hydrophobic 96-well plate, and the number of human periodontal ligament cells adhered by the method described in Example 3 below was we.
It was measured by quantifying the amount of DNA per liter.
As a result, as shown in FIG. 2, high cell adhesion-inducing activity was observed in fraction III.

(Separation by hydroxyapatite chromatography) Fraction III5 having a molecular weight of 60 ± 20 KD obtained by gel chromatography in the above method
PB adjusted to pH 7.4 and 0.01M phosphoric acid concentration using ultrafiltration membrane YM-10 by collecting lots to 6 ml.
Replace the buffer with S (-), and then add a pore size of 0.2.
A 2 μm filter (Millex GV, manufactured by Nippon Millipore Industrial Co., Ltd.) was passed through to obtain a sample for high performance liquid chromatography (HPLC). That is, the HPLC column is a hydroxyapatite column (Bio-GelH).
TP) at pH 7.4 and a continuous concentration gradient (0.01) with PBS (-) from 0.01M to 1.0M phosphate concentration.
(M-1.0M / 40 minutes). Every 1 minute a fraction collector (Gilson Model 20
(3, MS Equipment Co., Ltd.) divided into test tubes and separated to obtain a fraction (HT) having a phosphoric acid concentration of 0.5M.
0.25 ml of P-0.5M) was obtained.

Example 2 (SDS-Polyacrylamide Electrophoresis) The fraction (HTP-0.5M) obtained in Example 1 and the HPLF-CM before fractionation obtained in Example 1 were similarly subjected to a hydroxyapatite column. The 0.5 M phosphoric acid elution fractions obtained by fractionating with (Bio-Gel HTP) were each concentrated about 10 times using Centricon 10 manufactured by Amicon. 20 μl of this concentrated solution was added to Selected Method in Cellular
Immunology (Selected Method)
Cellular Immunology (BB).
Michelland S. M. By Shiigi, p.
416, 1980, Freeman & Co. Published), the sample was treated with a sample buffer containing 2-mercaptoethanol, loaded on a 12.5% polyacrylamide gel, and 25 mM Tri as a buffer.
s, 190 mM glycine, 0.1% SDS was used, and electrophoresis was performed at a current of 25 mA for 3 hours. After the electrophoresis, the gel was stained with a solution of Coomassie PRILIANT BLUE ACETATE according to a conventional method. As a result, for the fraction (HTP-0.5M) (lane B), a single band was observed on 67KD molecular weight marker (lane A) of the Electrophoresis calibration kit manufactured by Pharmacia Co. (Fig. 3). Considering the protein separation ability of SDS-polyacrylamide electrophoresis, the molecular weight of the protein shown above 67 KD was determined to be 70 ± 5 KD.

[0028] was carried out according to the method of Example 3 Kawase et experiments fine 胞接 adhesion (human periodontal ligament adhesion inducing activity of cells) (tissue culture, see 1990,16,42-46). That is, HPLF is set to 1 × 10 ^6.
The cells were planted in cells / 100 mm dish and cultured for 3 days, and it was confirmed by a phase contrast microscope that HPLF was in the growth stage, and used for the experiment. HPLF-CM, fraction H having a molecular weight of 60 ± 20 KD after gel chromatography fractionation
I (GPC-Fr.III), a fraction eluted with 0.5M phosphoric acid by hydroxyapatite chromatography (HTP-0.5M), a fraction having no affinity for a hydroxyapatite column (HTP-Pass) 100 μl of each of each of the we in a hydrophobic 96-well plate
11 was coated at 37 ° C. for 1 hour. Then, the added sample was removed, and 1% BSA (Fraction V, Fraction V, deactivated by heat treatment at 80 ° C. for 15 minutes was used.
Mikes: Seikagaku Corporation 's PBS (-) solution
200 ml was added to each well and left in an incubator at 37 ° C. for 30 minutes. The BSA solution was removed, and at the same time, the growth stage HPLF was treated with PBS (−) containing 0.25% trypsin, and the released HPLF was inoculated with 2.5 × 10 ⁴ cells per well, and 50 μg /
ml ascorbic acid and antibiotics (100 units /
Culturing was further performed for 20 hours in a serum-free medium MCDB107 (Far East Pharmaceutical Co., Ltd.) containing ml penicillin, 100 μg / ml streptomycin, and Gibco # 600-5 / 40).

The number of cells was analyzed by measuring the amount of DNA, and the spread state of the cells was observed with a phase contrast microscope. D per cell
Since the NA amount was constant, the number of adherent cells was calculated from the DNA amount. DNA measurement method is Puzaus JE and Go
odman DBP method (Anal. Bioche
m. , 1978, 86, 50-55). That is, in order to measure the number of adherent cells, the cell layer of a hydrophobic 96-well plate was dried with a standard DNA amount at 60 ° C. for 1 hour. In the meantime, 2M diaminobenzoic acid (DABA, Aldrich) was mixed with activated carbon (Merck) for removing the fluorescence measurement inhibitor,
Centrifuge at 1,000 xg for 5 minutes at room temperature,
The supernatant was filtered with a filter having a pore size of 0.22 μm. 45 μl of the filtered 2M diaminobenzoic acid solution was added to each well in which the cells were dried, and the reaction was performed for 30 minutes in a dryer at 60 ° C. Then, 200 μl of 0.6 N perchloric acid was added to each well, and excitation was performed at 405 nm and fluorescence intensity measurement was performed at 500 nm using a fluorescence spectrophotometer for microplates (MRF-100, JASCO Corporation). However, the standard sample is DNA derived from salmon sperm.
(Sigma) was used. As a result, as shown in FIG. 4, HPLF-CM, GPC-Fr. III, HTP-
A high human periodontal ligament cell adhesion-inducing activity was observed at 0.5M.

Example 4 (Effect of Synthetic Peptide on Cell Adhesion) The basic adhesion signal of cell adhesion was determined by Hautenen A et al.
Since the sequence of GD (Arg-Gly-Asp) is the most important (J. Biol. Chem., 1989, 2).
64, 1437-1442), synthetic peptide GRG
The effect was examined using DS (Gly-Arg-Gly-Asp-Ser: Peptide Institute). That, B Lank Te experiments odor of cell adhesion Example 3
(Serum-free medium), HPLF-CM (culture supernatant of human periodontal ligament cells) and 100 μl each of HTP-0.5M (0.5M phosphate elution fraction of hydroxyapatite column chromatography), and FN (fibronectin) (Koken Co., Ltd., human plasma fibronectin) 1.2 μg / ml and VN (Vitronectin) (Koken Co., Ltd., human plasma vitronectin) 1.2 μg / ml, respectively, hydrophobic 96 well
Added to the plate and coated at 37 ° C for 1 hour
Otherwise, the procedure of Example 3, with the human periodontal ligament cell numbers adhered by quantifying the amount of DNA per well, measuring the respective cell adhesion-inducing activity, which
Were used as controls . On the other hand, in order to see the effect of the GRGDS peptide, the above experiment was performed in advance with the final concentration of GRGDS.
50 μg / ml ascorbine adjusted to 0.2 mM
Acid and antibiotics (100 units / ml penicillin and 1
00 μg / ml streptomycin, Gibco # 60
Serum-free medium MCDB107 (Far East)
HPLF processed at 37 ° C for 20 minutes
It was carried out using (the addition of GRGDS). The result is shown in FIG. Moreover, the inhibition rate of the GRGDS peptide was shown in FIG. 6 about those results. As is apparent from FIG. 6, G
It was found that the inhibition of human periodontal ligament cell adhesion by the RGDS peptide was strong in fibronectin, vitronectin, and serum-free medium, but was extremely weak in HTP-0.5M.

Example 5 (Effect of various antibodies on cell adhesion) Anti-fibronectin against human periodontal ligament cell adhesion-inducing activity of the fraction (HTP-0.5M) eluted with 0.5M phosphoric acid by hydroxyapatite chromatography Antibodies (Biomedical Technology, rabbit anti-human fibronectin), anti-vitronectin antibody (Transformation Research, rabbit anti-fibronectin receptor) and anti-fibronectin receptor antibody (Hail Chemical Pharmaceuticals, rabbit anti-human laminin) Considered the impact. That is, HTP-0.5
Experiment of cell adhesion of M was performed with HPLF2.5 × 10 ⁴ cells.
For l, each antibody was adjusted to a final concentration of 1 μg / ml, 50 μg / ml of ascorbic acid and antibiotics (100 units / ml penicillin and 100 μg / m).
l streptomycin, Gibco # 600-5 / 4
Example 3 except that the serum-free medium MCDB107 (Far East Pharmaceutical Co., Ltd.) containing 0) was used for 20 hours of culture.
I went in the same way . When the cell adhesion rate was measured by the amount of DNA in the same manner as in Example 3, the control HTP-0.5 was obtained.
In M, it was 285 ± 25 ng / well. On the other hand, 236 ± 45 ng / wel for anti-fibronectin antibody
l, 263 ± 37 ng / we for anti-vitronectin antibody
ll, 242 ± for anti-fibronectin receptor antibody
It was 42 ng / well. Also, in the same way as above
As a result of the experiment of cell adhesion of fibronectin, the adhesion-inducing action of fibronectin (1.2 μg / ml) (2
15 ± 23 ng / well), the anti-fibronectin antibody was 45 ± 5 ng / well, and the anti-fibronectin receptor antibody was 50 ± 6 ng / well, which greatly suppressed the adhesion.

Example 6 (Injection) Aseptically, using an ampoule having a volume of 3 ml, 3 m
Saline Gahi preparative periodontal ligament cell adhesion factor of l adjusted to contain 30mg, further resulting solution Human serum albumin 15mg addition to熔封, an injection.

[0033]

Human periodontal ligament cell adhesion factor of the present invention according to the present invention, since the induced adhesion of periodontal ligament cells by topical or systemic administration, of periodontal disease in need of regeneration of bone and periodontal ligament It is effective for treatment or for improving the implant survival rate of artificial dental roots. Also, human periodontal ligament cell adhesion factor of the present invention can induce the adhesion of osteoblasts to bone, for example, are effective in bone regeneration in osteoporosis therapy, also locally osteogenesis such fractures If you need
The local administration can induce the adhesion of bone cells and promote bone formation.

[Brief description of drawings]

FIG. 1 shows the results of gel chromatography of HPLF-CM on a PO-60K column.

FIG. 2 is a view showing cell adhesion-inducing activity of HPLF-CM gel chromatography fractions.

FIG. 3 is a view showing a result of SDS-polyacrylamide electrophoresis of a fraction (HTP-0.5M). Lane A
Is a molecular weight marker, and Lane B is a fraction (HTP-0.5).
M), Lane C shows the fraction eluted with HPMF-CM by hydroxyapatite column chromatography 0.5 M phosphoric acid.

FIG. 4 is a diagram showing the results of human periodontal ligament cell adhesion-inducing activity. Blank is serum-free medium, HPLF-
CM is a culture supernatant of human periodontal ligament cells, GPC-Fr. II
I is a gel chromatography fraction of HPLF-CM II
I, HTP-0.5M is 0.5M phosphoric acid elution fraction of hydroxyapatite column chromatography, HTP
-Pass indicates a fraction having no affinity for the hydroxyapatite column.

FIG. 5 is a view showing an inhibitory effect (cell amount of adherent cells) on cell adhesion of GRGDS peptide. Blank is serum-free medium, HPLF-CM is culture supernatant of human periodontal ligament cells,
HTP-0.5M is a 0.5M phosphate elution fraction of hydroxyapatite column chromatography, FN is fibronectin, and VN is vitronectin.

FIG. 6 is a view showing an inhibitory effect (inhibition rate%) of cell adhesion of GRGDS peptide. Blank is serum-free medium, HPLF-CM is culture supernatant of human periodontal ligament cells, H
TP-0.5M is a 0.5M phosphate elution fraction of hydroxyapatite column chromatography, FN is fibronectin, and VN is vitronectin.

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[Figure 6]

Claims (4)

[Claims] 1. A purified human periodontal ligament cell adhesion factor derived from a human periodontal ligament cell culture supernatant. 2. (1) 60 ± by gel chromatography
It has a molecular weight of 20 KD and has an affinity for (2) hydroxyapatite column, 0.5 ± 0.1 M phosphoric acid (p
H7.4), and (3) SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue shows a single band at an apparent molecular weight of 70 ± 5 KD. Human periodontal ligament cell adhesion factor. 3. A method for purifying a human periodontal ligament cell adhesion factor, which comprises a step of purifying a culture supernatant of human periodontal ligament cells by gel chromatography and hydroxyapatite column chromatography. 4. A pharmaceutical composition comprising the purified human periodontal ligament cell adhesion factor according to claim 1 as an active ingredient.