WO2005001484A1 - 歯周炎の発症を判定する方法 - Google Patents
歯周炎の発症を判定する方法 Download PDFInfo
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- WO2005001484A1 WO2005001484A1 PCT/JP2004/009117 JP2004009117W WO2005001484A1 WO 2005001484 A1 WO2005001484 A1 WO 2005001484A1 JP 2004009117 W JP2004009117 W JP 2004009117W WO 2005001484 A1 WO2005001484 A1 WO 2005001484A1
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- WIPO (PCT)
- Prior art keywords
- periodontitis
- gtp
- periodontal
- gingivitis
- gingival
- Prior art date
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/91—Transferases (2.)
- G01N2333/91045—Acyltransferases (2.3)
- G01N2333/91074—Aminoacyltransferases (general) (2.3.2)
- G01N2333/9108—Aminoacyltransferases (general) (2.3.2) with definite EC number (2.3.2.-)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/18—Dental and oral disorders
Definitions
- the present invention relates to a method for determining the onset of periodontitis and a method for determining the onset of peri-implantitis.
- the present invention relates to a method for determining the onset of periodontitis and a method for determining the onset of peri-implantitis, which comprises detecting or measuring ⁇ -GTP (Y-glutamyl transpeptidase) activity. —.
- Periodontal tissue is around a tooth and plays a role of supporting the tooth.
- the tissue is composed of gingiva, periodontal ligament, cementum, and alveolar bone strength.
- gingiva those in which the inflammation is localized to the gingiva are referred to as "gingivitis", and those in which the inflamed area extends beyond the gums and damages or destroys the periodontal ligament or alveolar bone is referred to as "periodontal periodontal" Flame. : "
- Periodontal disease is generally diagnosed by measuring periodontal pockets, attachment levels, and X-ray imaging.
- the term "periodontal pocket” refers to the gap between the teeth and the gingiva due to gingivitis or periodontitis and the formation of a groove (gingival sulcus) between the teeth.
- the degree of destruction of the periodontal tissue can be known to some extent.
- “Attachment level” is an index to measure the distance from the boundary between cementum and enamel to the bottom of the periodontal pocket, and to know the degree of destruction of periodontal support tissue.
- X-ray image diagnosis the resorption of alveolar bone and the like can be seen by X-ray photography.
- ALP alkaline phosphatase
- Patent Document 1 JP-A-60-222768
- Patent Document 2 Japanese Patent Publication No. 3-501447
- Patent Document 3 JP-A-5-176796
- the degree of inflammation of periodontal tissue is determined by measuring the increase in the activity of the enzyme peroxidase. Even in the case of inflammation, the enzyme activity of peroxidase increases, so it is difficult to clearly distinguish between gingivitis and periodontitis.
- inflammation or damage to the periodontal tissue is determined by measuring an increase in the concentration of the enzyme 'ALT, and thus, even in the case of In this case, ALT enzyme activity increases, so it is difficult to clearly distinguish gingivitis from periodontitis, even if the progress of periodontal disease symptoms can be predicted to some extent. .
- the enzyme released by the bacterium that causes periodontal disease is used as an index, so that it may be possible to predict the progress of periodontal disease to some extent. It is difficult to clearly distinguish between flame and periodontitis. In addition, because the correlation between the progression of periodontal disease and the growth of bacteria varies from individual to individual, even if the measured values of the enzymes released by the bacteria are the same in multiple patients, It is difficult to clearly distinguish between flame and periodontitis.
- an object of the present invention is to provide a method for easily and clearly distinguishing between gingivitis and periodontitis.
- the present invention provides the following means.
- O_GTP (o / _daltamyl transpeptidase) is an enzyme that is abundantly present in kidney, pancreas, liver and the like. It is used for the diagnosis of liver disease and the like because it emigrates into the blood.
- the inventor of the present application has proposed that the gingival sulcus of a patient with gingivitis does not contain ⁇ GTP, but the gingival sulcus of a patient with periodontitis that develops after the gingivitis. It was newly discovered that ⁇ -GTP was contained in the solution. In addition to this new discovery, we also confirmed that osteoclasts were induced in periodontal tissue when ⁇ GTP was expressed in periodontal tissue. These new findings clearly indicate that ⁇ -GTP is deeply involved in the development of periodontitis with alveolar bone resorption and destruction. Note that ⁇ GT When P is included, osteoclasts are induced into the periodontal tissue, so that alveolar bone resorption and destruction still begin.
- ⁇ GTP is useful as a marker for distinguishing between gingivitis and periodontitis.
- ⁇ GTP is useful as a marker for distinguishing between gingivitis and periodontitis.
- GTP ⁇ -daltamyl transpeptidase
- gingivitis ⁇ GTP hardly contains gingival crevicular fluid. In periodontitis, ⁇ GTP flows into gingival crevicular fluid. By detecting or measuring ⁇ GTP, it is possible to clearly determine whether periodontal disease is about to progress to gingivitis or periodontitis.
- gingival sulcus refers to a groove portion (periodontal pocket) between teeth and gingiva, which is caused by separation of teeth and gingiva due to gingival inflammation.
- gingival crevicular fluid refers to tissue fluid leaking into the gingival sulcus.
- the gingival sulcus can be formed when there is inflammation of the gingiva, so if the periodontal tissue is healthy, there is almost no clear gingival sulcus (periodontal pocket), and therefore almost all gingival sulcus exudate is collected. Can not.
- a method of collecting gingival crevicular fluid for example, a method of infiltrating the fluid with a paper point, a method of using a soccer (aspirator), and a method of collecting with a micropipette (capillary glass tube) There is a method of flowing a buffer solution into the gingival sulcus and collecting the solution.
- a method for detecting or measuring ⁇ GTP a known method generally used in laboratory diagnosis of liver disease or the like can be used, and a method capable of detecting or measuring ⁇ GTP can be used. If there is, it is not particularly limited. For example, a method for measuring the enzyme activity of ⁇ GTP, a direct quantification method using an antibody against ⁇ GTP, a gene detection method for confirming the expression of mRNA encoding ⁇ GTP by PCR, and the like can be employed. When detecting and measuring the enzyme activity of ⁇ -GTP using the enzyme activity measurement method, for example, the above-mentioned recommended method (see Clinical Chemistry, 24, 106-121, 1995) can be used.
- -GTP is a transferase that transfers the ⁇ -darcosyl group to the acceptor substrate, glycinoleglycine, etc. It has the effect of producing ⁇ -daltamylglycinoleglycine and 5-amino_2_nitrobenzoic acid.
- the enzymatic activity of ⁇ -GTP is detected and measured by measuring the change in the absorbance of 5_amino2_nitrobenzoic acid generated by this reaction.
- SSCC method synthetic substrate 'L- ⁇ daltamyl ⁇ -nitro-2-anilide
- the direct quantification method is a method for directly detecting and measuring ⁇ GTP using an anti- ⁇ GTP antibody (an antibody that specifically binds to ⁇ GTP).
- ⁇ GTP can be detected with high sensitivity by using a general method, Sandwich ELIsA method (Enzyme-Linked Immuno-Sorbent Assay).
- the present invention can also be applied to a method for determining the onset of peri-implantitis.
- Periodontitis is a type of periodontitis, and refers to the fact that implanting an implant in periodontal tissue causes inflammation around the implant in the periodontal tissue.
- An “implant” refers to a support that is embedded in the periodontal tissue. The lower part of the implant is embedded in the periodontal tissue, and the upper part of the implant is fitted to the denture, and the denture is attached to the periodontal tissue. .
- Peri-implantitis is a new periodontitis that is increasing in incidence due to the spread of dental implants and is not caused by the progression of the disease from gingivitis. When periodontitis develops, it has characteristics.
- the gingival crevicular fluid cannot be collected much.
- blood around the site where the implant is carried may be collected with a syringe or the like and used as a sample for detection and measurement of ⁇ -GT GT.
- Gingival sulcus exudate is lymph fluid exuded from the gingiva due to inflammation, and has almost the same content as serum. Therefore, ⁇ -GTP can be detected and measured even by sampling from blood. Note that the same method as described above can be used for detecting and measuring ⁇ -GTP.
- Example 1 an experiment for distinguishing gingivitis from periodontitis was performed by the following procedure, in which gingival crevicular exudate was collected and the enzyme activity of ⁇ -GTP was measured. Note that the present invention is not limited to the embodiments.
- gingival crevicular fluid was collected from three specimens (patients who had already cured periodontitis, gingivitis patients, and periodontitis patients).
- the gingival crevicular fluid was collected by inserting a paper pocket into the gingival sulcus of each sample for 1 minute and allowing the gingival crevicular fluid to soak into the paper point.
- gingival crevicular fluid was sampled at two points: a periodontal pocket with a shallow gingival sulcus and a deep periodontal pocket at another point.
- gingival sulcus area peripheral pocket
- gingival inflammation at other locations are suppressed.
- Gingival sulcus exudate was collected from three deep periodontal pockets in the affected area.
- the paper point that has infiltrated the gingival crevicular fluid is collected, and immediately the 70 ⁇ l of PBS buffer (or distilled water) is used to prevent the tip of the paper point (the part inserted into the periodontal pocket) from drying out. Water) for 1 minute. Then, the paper point was discarded, and only the PBS buffer in which the paper point was soaked for 1 minute was stored at 4 ° C. In this procedure, cut the tip of the paper point that soaked the gingival crevicular fluid, soak it in PBS buffer, etc., and keep one point of paper soaked in PBS buffer, etc., at 4 ° C. You can save it.
- PBS buffer or distilled water
- gingival crevicular fluid contains various bacteria, and when stored at room temperature, these bacteria grow and proteolytic enzymes Secretion, etc., and may degrade ⁇ -GTP. It also addresses these issues As a means to remove, 0.1 in PBS buffer etc. /.
- a method of adding BSA (for stabilizing a trace amount of ⁇ -GTP) and 0.2% sodium azide (preservative) may be used. By adopting any of the above methods, samples can be stored at 4 ° C (low temperature) or room temperature without breaking ⁇ -GTP.
- ⁇ -GTP was not detected in the gingival crevicular fluid collected from patients with gingivitis (Sample 3)
- ⁇ -GTP was detected in the gingival crevicular fluid of periodontal disease patients.
- Sample 4 This indicates that gingivitis and periodontitis can be clearly distinguished by detecting and measuring ⁇ -GTP.
- -GTP in gingival crevicular fluid in patients with gingivitis ⁇ -GTP is included in the periodontal disease patients in the gingival crevicular fluid.
- Samples 5 and 6 exhibit gingival crevicular fluid in patients whose gingival inflammation is suppressed by anti-inflammatory agents (such as steroids). Included ⁇ -Measured GTP.
- anti-inflammatory agents suppresses gingival inflammation (redness) on the surface.
- Periodontitis is not cured, and in fact, inflammation occurs deep in the periodontal tissue. ing. Therefore, in this experiment, it was examined whether or not the force having inflammation deep in the periodontal tissue can be determined by the method of the present invention.
- Table 1 ⁇ -GTP was detected in the patients of sample 5 and sample 6. Therefore, even if it appears that the periodontitis has healed on the surface with the anti-inflammatory agent, if the inflammation occurs deep in the periodontal tissue, the method of the present invention will The ability to judge the presence or absence S The ability to be able to S
- the gingival crevicular fluid (Sample 6) collected from the deep gingival sulcus (periodontal pocket) was found to have a shallow gingival sulcus (periodontal pocket) force.
- the ⁇ -GTP enzyme activity was higher than that of the sample (Sampnore 5). This suggests that ⁇ -GTP increases as the site of inflammation increases, and that ⁇ -GTP increases as the inflammation increases when gingival crevicular fluid is collected at the same depth of the gingival sulcus. ing. Therefore, it was found that the method of the present invention can also determine the degree of inflammation of the periodontal tissue when it is determined that the periodontitis can only distinguish between gingivitis and periodontitis. .
- Example 2 is an experiment in which gingival crevicular fluid of a periodontal disease patient was prepared using a gene detection method to confirm whether or not y-GTP mRNA was expressed.
- gingival crevicular fluid RNA of a patient with periodontal disease was extracted using an RNA extraction reagent “ISOGEN” (manufactured by Futatsu Gene Co., Ltd.).
- the desired DNA was synthesized and amplified by RT-PCR using RT-PCR high-Plus-kit (manufactured by TII).
- primers for RT-CR 5'_TCCCTTGACCTTCAGGAGAACGAG-3 'and 5,1-GTGTGGTGCTGTTGTAGATGGTGA-3' were used.
- RT-CR 5'_TCCCTTGACCTTCAGGAGAACGAG-3 'and 5,1-GTGTGGTGCTGTTGTAGATGGTGA-3' were used.
- the target DNA was amplified by the PCR method using this primer, it was confirmed that the mRNA of human ⁇ - GTP was expressed in the gingival crevicular fluid.
- the [0042] As a result, even by gene detection method, it was shown that it is possible to detect
- Example 3 is an experiment using an experimental periodontal disease model rat to show that ⁇ -GTP is involved in the destruction of alveolar bone. The procedure is shown below.
- LPS lipopolysaccharide (lipopolysaccharide), which is an important component of the outer membrane of Gram-negative bacteria. Since LPS has various biological activities as endotoxin, in this experiment, it was used to experimentally develop periodontitis by sensitizing rat molars by contacting them with LPS for a certain period of time. . E. coli-derived LPS was used.
- FIG. 1 the pathological section was stained with ⁇ -GTP (FIG. 1).
- a pathological section similarly prepared was stained with a non-specific antibody (FIG. 2).
- FIG. 1 it was found that a large amount of ⁇ -GTP was expressed around the alveolar bone of rats that had experimentally developed periodontitis.
- FIGS. 1 and 2 are obtained by staining a pathological section 3 days after LPS sensitization.
- TRAP staining is a staining method that uses sclerate-resistant acid phosphatase (a mature osteoclast marker), and can specifically stain osteoclasts.
- the results showed that a large number of osteoclasts were induced in the alveolar bone of rats that had experimentally developed periodontitis.
- the one shown in FIG. 3 is obtained by staining a pathological section 3 days after LPS sensitization. Light microscopy shows that pathological sections 3 days after LPS sensitization show induction of osteoclasts. Alveolar bone uptake and destruction are only partially observed.
- Gingivitis and periodontitis can be clearly distinguished by a simple method.
- the criteria for discriminating between gingivitis and periodontitis can be clarified, and the onset of periodontitis can be determined based on objective criteria rather than the respective criteria of the dentist. This can be useful for early diagnosis and early treatment of periodontitis.
- ⁇ -GTP occurs immediately before the absorption and destruction of the alveolar bone starts, it is also effective in determining the onset of periodontitis at an early stage.
- ⁇ -GTP For the detection and measurement of ⁇ -GTP, a known simple method used in laboratory diagnosis of liver disease and the like can be used. For this reason, the method of the present invention can be quickly and easily spread.
- the present invention provides a method for determining the onset of periodontitis using enzymes associated with inflammation or damage to periodontal tissue or enzymes released by bacteria that cause periodontal disease as markers. Since an enzyme directly related to the onset is used as a marker, it is possible to clearly and reliably distinguish between gingivitis and periodontitis and determine the onset of periodontal disease.
- the amount of detected ⁇ -GTP and the like are measured to determine the inflammation of periodontitis.
- the ability to judge the size of S is also possible.
- the present invention is also useful for determining the onset of peri-implantitis.
- FIG. 1 A substitute for a drawing in which rat alveolar bone was stained with ⁇ -GTP antibody 3 days after LPS sensitization.
- FIG. 2 Substitute drawing of rat alveolar bone stained with non-specific antibody 3 days after LPS sensitization
- FIG. 3 Substitute drawing drawing of rat alveolar bone TRAP stained 3 days after LPS sensitization
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP04746586A EP1640721A4 (en) | 2003-06-30 | 2004-06-28 | METHOD FOR ASSESSING THE BEGINNING OF PERIODONTITIS |
US10/561,967 US7432071B2 (en) | 2003-06-30 | 2004-06-28 | Method of diagnosing periodontitis by determining γ-glutamyltranspeptidase |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2003188247A JP3599283B1 (ja) | 2003-06-30 | 2003-06-30 | 歯周炎の発症を判定する方法。 |
JP2003-188247 | 2003-06-30 |
Publications (1)
Publication Number | Publication Date |
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WO2005001484A1 true WO2005001484A1 (ja) | 2005-01-06 |
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PCT/JP2004/009117 WO2005001484A1 (ja) | 2003-06-30 | 2004-06-28 | 歯周炎の発症を判定する方法 |
Country Status (5)
Country | Link |
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US (1) | US7432071B2 (ja) |
EP (1) | EP1640721A4 (ja) |
JP (1) | JP3599283B1 (ja) |
CN (1) | CN1813189A (ja) |
WO (1) | WO2005001484A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017018012A (ja) * | 2015-07-08 | 2017-01-26 | 長田産業株式会社 | 歯肉炎・歯周炎の簡易判定方法及びその簡易判定キット |
GB2566942A (en) * | 2017-09-22 | 2019-04-03 | Caperay Medical Pty Ltd | Multimodal imaging system and method |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006121956A (ja) * | 2004-10-28 | 2006-05-18 | Ortho Corp | 抗γ−GTP抗体を有効成分とする機能性食品 |
EP2414827A1 (en) * | 2009-04-01 | 2012-02-08 | Colgate-Palmolive Company | Metabolites for oral health and uses thereof |
JP5612269B2 (ja) | 2009-04-24 | 2014-10-22 | 花王株式会社 | 歯周病マーカー |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH04229198A (ja) * | 1990-12-26 | 1992-08-18 | Lion Corp | 歯周疾患検査方法 |
JPH06289020A (ja) * | 1993-04-06 | 1994-10-18 | Teijin Ltd | 歯槽骨由来タンパクの測定方法 |
JP2004129584A (ja) * | 2002-10-11 | 2004-04-30 | Applied Cell Biotechnologies Inc | 歯周病の検査方法及び検査診断薬 |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
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CA1252703A (en) | 1984-02-27 | 1989-04-18 | Nutan B. Shah | Diagnostic saliva test for detecting periodontal disease |
US4981787A (en) * | 1989-02-14 | 1991-01-01 | Xytronyx, Inc. | Method for diagnosis of periodontal disease by detection of L-alanine aminotransferase |
JPH05176796A (ja) | 1991-12-26 | 1993-07-20 | Lion Corp | 歯周疾患検査方法 |
AU8017594A (en) * | 1993-10-29 | 1995-05-22 | Institute Of Molecular Biology, Inc. | Osteocalcin as a marker of periodontal and peri-implant disease activity |
ATE203329T1 (de) * | 1995-04-12 | 2001-08-15 | Medix Biochemica Ab Oy | Verfahrens und testsätze zur diagnose periodentahlkrankheiten und zur vorhersage des risikos der progression davon |
JP3501447B2 (ja) | 2001-02-26 | 2004-03-02 | ジョプラックス株式会社 | 流水切換具 |
WO2002079780A1 (fr) * | 2001-03-28 | 2002-10-10 | Nihon University | Procede permettant de controler la parodontopathie |
-
2003
- 2003-06-30 JP JP2003188247A patent/JP3599283B1/ja not_active Expired - Fee Related
-
2004
- 2004-06-28 US US10/561,967 patent/US7432071B2/en not_active Expired - Fee Related
- 2004-06-28 CN CNA2004800183702A patent/CN1813189A/zh active Pending
- 2004-06-28 WO PCT/JP2004/009117 patent/WO2005001484A1/ja active Application Filing
- 2004-06-28 EP EP04746586A patent/EP1640721A4/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH04229198A (ja) * | 1990-12-26 | 1992-08-18 | Lion Corp | 歯周疾患検査方法 |
JPH06289020A (ja) * | 1993-04-06 | 1994-10-18 | Teijin Ltd | 歯槽骨由来タンパクの測定方法 |
JP2004129584A (ja) * | 2002-10-11 | 2004-04-30 | Applied Cell Biotechnologies Inc | 歯周病の検査方法及び検査診断薬 |
Non-Patent Citations (2)
Title |
---|
GAMMA-GLUTAMYLTRANSFERASE FROM THE OUTER CELL ENVELOPE OF TREPONEMA DENTICOLA ATCC 35405, INFECTION AND IMMUNITY, vol. 65, no. 2, 1997, pages 685 - 691, XP002903406 * |
See also references of EP1640721A4 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2017018012A (ja) * | 2015-07-08 | 2017-01-26 | 長田産業株式会社 | 歯肉炎・歯周炎の簡易判定方法及びその簡易判定キット |
GB2566942A (en) * | 2017-09-22 | 2019-04-03 | Caperay Medical Pty Ltd | Multimodal imaging system and method |
GB2566942B (en) * | 2017-09-22 | 2020-06-03 | Caperay Medical Pty Ltd | Multimodal imaging system and method |
Also Published As
Publication number | Publication date |
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JP2005024337A (ja) | 2005-01-27 |
CN1813189A (zh) | 2006-08-02 |
US7432071B2 (en) | 2008-10-07 |
US20060228768A1 (en) | 2006-10-12 |
EP1640721A4 (en) | 2009-06-17 |
EP1640721A1 (en) | 2006-03-29 |
JP3599283B1 (ja) | 2004-12-08 |
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