JP2003066039A - Examination method of periodontitis by quantitative determination of liver cell growth factor in sputum - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a determination method for the sputum of a periodontitis patient comprising measuring the liver cell growth factor concentration in a test sputum. SOLUTION: According to this method, since the progress of periodontitis can be easily and quickly judged without applying a load to the patient, a precise treatment can be performed.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for determining saliva of periodontitis patients, which is useful for diagnosing periodontitis.

[0002]

2. Description of the Related Art Early diagnosis of periodontitis is effective in enabling effective early treatment and, as a result, preventing the severity of lesions and complications. The conditions that the screening test for periodontitis should have are (1) accurate and accurate understanding of diseases and abnormalities, (2) simple and quick results, and (3) as little pain as possible thing,
(4) It is mentioned that the cost is low.

In the exudate from the periodontitis pocket, markers of cellular tissue degradation such as hydroxyproline and glycosaminoglycan, inflammatory mediators such as prostaglandin E ₂ and interleukin 1β, and enzymes such as aminotransferase and collagenase. It is known that the exudate is an excellent diagnostic material for periodontitis because it contains a group (CDA JOURNAL Vol. 21).
35-41 1993). However, periodontal tissue specificity cannot be confirmed by these indexes. In addition, it is necessary to remove saliva with a mouse rinse or perform a centrifugation operation or the like until the exudate is collected and adjusted to a sample to be used for measurement (J. Periodont. Res 25: 257-267, 1990). However, since it requires an expensive device and a plurality of operations, it has not yet been put into practical use as a screening test for periodontal disease that can be performed in general dental clinics and group examinations.

In addition, a method for separating and detecting pathogenic microorganisms of the collected periodontal pocket exudate and a method for measuring enzyme activity derived from pathogenic microorganisms have been reported (Journal of the Japanese Society of Periodontology, Volume 32). 249-260, 1990)
However, there is a drawback that it takes time to collect microorganisms from the periodontal pocket and measure enzyme activity.

[0005]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a new means for diagnosing periodontitis that targets saliva itself, not periodontal pocket exudate.

[0006]

The inventors of the present invention have variously studied the relationship between components in saliva and the progression of periodontitis. As a result, hepatocyte growth factor (hereinafter referred to as HG) in saliva.
F) There is a high correlation between the concentration and the degree of progression of periodontitis,
The present invention has been completed by finding that periodontitis can be diagnosed with high accuracy, simplicity and speed.

That is, the present invention relates to HGF in saliva to be tested.
The present invention provides a method for determining saliva of a patient with periodontitis, which comprises measuring the concentration.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, saliva is used as a subject. Further, saliva may be used directly, but mouthwash containing saliva may be used. It is unclear whether or not the components contained in periodontal pocket exudate are also contained in saliva, but the present inventor confirmed that HGF is present in saliva, and further confirmed that HGF contained in the saliva. The correlation between the HGF concentration and the degree of progression of periodontitis was found.

The amount of saliva used is usually 0.5 to 5.0 m
L, preferably 0.5-1.0 mL is sufficient.

The saliva may be directly used for the measurement of HGF, but it is also possible to use a solution prepared by adding a buffer solution or the like attached to a commercially available ELISA kit for measuring HGF and diluting it 2 to 5 times. Furthermore, it is more preferable to centrifuge the diluted solution to remove the precipitate and use the supernatant as the sample. In addition, saliva may be used after being frozen and thawed.

The means for measuring the concentration of HGF in saliva is not particularly limited, and examples include immunoassays, HPLC systems,
A protein chip + SELDI-TOF-MS system and the like can be mentioned, of which the immunological assay is more preferable. As an immunological measurement method, an ELISA method using a solid-phased anti-HGF antibody is more preferable. Commercial HGF
As an ELISA kit for measurement, a human HGF immunoassay kit (R & D Systems) can also be used.

In measuring the HGF concentration, it is preferable to measure in the presence of a protease inhibitor, since the correlation between the obtained measured value and periodontitis is further improved.
As protease inhibitors, aprotinin, A
EBSF, NEM, EDTA, E-64, bestatin,
One or more selected from leupeptin and pepstatin A, especially protease inhibitor cocktail P8
340 (Sigma, AEBSF, aprotinin, bestatin,
E-64, a mixture of leupeptin and pepstatin A), protease inhibitor cocktail P2714 (Si
gma, AEBSF, aprotinin, bestatin, EDTA,
A mixture of E-64, leupeptin and pepstatin A)
The protease inhibitor cocktail (Sigma) is particularly preferable. The content of these protease inhibitors in the measurement system varies depending on the type of protease inhibitor, but in the case of these protease inhibitor cocktails (Sigma), 10-20 U / mL saliva is preferable.

Further, among HGF, it is more preferable to measure the active HGF concentration. The active HGF concentration is preferably measured by, for example, an HGF “Otsuka” ELISA kit. When measuring the active HGF concentration by thawing frozen saliva, it is possible to obtain accurate active HGF by measuring in the presence of a protease inhibitor.
The concentration can be measured, which is particularly preferable.

The concentration of HGF in saliva and the probing depth (Ping depth: P) which is a clinical parameter of periodontitis patients.
D) and subjects → The number of PD per patient is 4 mm or more and the number of PD is 6 mm or more, and the deepest PD of the patient and the percentage of bleeding sites due to probing (BOP) are highly correlated. Have For example, the PD of saliva patients with high HGF concentration was 4 mm or more. The BOP of saliva having a high HGF concentration was 10% or more. Therefore, the measurement of HGF concentration in saliva is useful for the diagnosis of periodontitis.

[0015]

EXAMPLES Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

Example 1 (1) Collection and pretreatment of saliva Unstimulated whole saliva at rest is collected by the spit method. The saliva is centrifuged at 4 ° C. and the supernatant is collected. Dilute 2-fold by adding an equal volume of the dilution buffer included in the kit containing the protease inhibitor cocktail (Sigma).

(2) Method for measuring HGF concentration Using an HGF measurement ELISA kit (manufactured by R & D Systems, human HGF immunoassay kit), 150 μL of dilution buffer (RDIW) is added to each well of the plate. Then add 50 μL of HGF standard solution or saliva sample and incubate for 2 hours at room temperature. Wash plate 4 times. Add 200 μL of conjugate (enzyme antibody) solution and incubate for 1.75 hours. Wash plate 4 times. Add 200 μL of substrate solution and react for 30 minutes. After adding 50 μL of the stop solution, the absorbance is measured at a wavelength of 450 nm using a microplate reader. The HGF concentration in the saliva sample is determined from the calibration curve.

(3) Results Correlation coefficient r = between HGF amount in saliva and PD number of 4 mm or more
It was significantly correlated with 0.421 (Fig. 1). The number of PDs of 6 mm or more is r = 0.524, and the deepest PD is r = 0.49.
In 6 all were significantly correlated (Fig. 2, Fig. 3). In addition, the percentage of bleeding sites by probing (BOP)
Both were significantly correlated with r = 0.422 (Fig. 4).

[0019]

EFFECTS OF THE INVENTION According to the method of the present invention, the degree of progression of periodontitis can be determined simply and quickly without imposing a burden on the patient, so that an appropriate treatment can be performed.

[Brief description of drawings]

FIG. 1 is a diagram showing the correlation between HGF concentration in saliva and the number of PDs of 4 mm or more.

FIG. 2 is a diagram showing a correlation between HGF concentration in saliva and the number of PDs of 6 mm or more.

FIG. 3 is a diagram showing the correlation between the HGF concentration in saliva and the deepest PD.

FIG. 4 is a diagram showing the correlation between HGF concentration in saliva and BOP.

Claims (3)

[Claims] 1. A method for determining saliva of a periodontitis patient, which comprises measuring a hepatocyte growth factor concentration in a test saliva. 2. The method according to claim 1, wherein the hepatocyte growth factor is active hepatocyte growth factor. 3. The method according to claim 1, wherein the measurement is performed in the presence of a protease inhibitor.