CN102507707B - A kind of method detecting protein lyase content in level in gingival sulcus fluid - Google Patents

Abstract

The invention discloses a method for detecting orthodontic tooth root resorption, the steps are as follows: (1) extraction of gingival crevicular fluid; (2) measurement of the weight of gingival crevicular fluid; (3) protein extraction; (4) protein SDS-PAGE coagulation Gel electrophoresis; (5) Western blotting; (6) Data processing: Calculate the enzyme content per unit weight of gingival crevicular fluid based on the weight of gingival crevicular fluid measured above, and use SPSS 11.0 statistical package to make statistics on the weight of GCF and enzyme content academic analysis. The method of the present invention can be used to evaluate and screen the sensitive population of tooth root resorption; evaluate the occurrence state of tooth root resorption before orthodontic treatment; dynamically monitor the development and outcome of tooth root resorption in orthodontic treatment, so as to take effective measures to prevent the occurrence of tooth root resorption occur and develop.

Description

A method for detecting proteolytic enzyme content in gingival crevicular fluid

technical field

The present invention provides a method for detecting orthodontic root resorption.

Background technique

The incidence of orthodontic root resorption is high. Severe root resorption can cause irreversible shortening and dulling of the root apex, resulting in an imbalance of the crown-to-root ratio, loosening of the tooth, and even loss of the tooth. At present, there is no effective treatment for root resorption, and the only way to prevent its further development is by early detection and removal of the cause. Proteolytic enzymes are involved in degrading the extracellular matrix of various tissues throughout the body, including bones. Gingival crevicular fluid is the exudate in the gingival sulcus, and its composition changes with changes in surrounding tissues, including gingival crevicular fluid proteolytic enzymes . It has been widely used by stomatologists to diagnose diseases, monitor the course of diseases, and understand the effect of treatment by detecting the changes of certain substances in gingival crevicular fluid.

Contents of the invention

Aiming at the above-mentioned prior art, the inventors of the present invention have carried out related research, and the research shows that gingival crevicular fluid proteolytic enzyme is the main enzyme involved in the reconstruction of periodontal tissue, and is related to tooth root resorption. Therefore, the present invention provides a detection Methods of orthodontic root resorption.

The present invention is achieved through the following technical solutions:

A method for detecting orthodontic root resorption, the steps are as follows:

(1) Extraction of gingival crevicular fluid: clean the tooth surface of the subject to be tested, use cotton balls to insulate moisture, blow dry, and then stick the tip of 20# moisture-absorbing paper that has been sterilized by ultraviolet light to the gum of the buccal and lingual tooth surface of the tooth Leave the edge of the ditch for 30 seconds, take it out and immediately put it into an Ep tube to seal it, store it at -70°C, and wait for use; if you find blood contamination on the tip of the paper, discard it and take it again; take a sample every other week, at least twice;

(2) Measurement of the weight of gingival crevicular fluid: use an electronic analytical balance to weigh the weight of the moisture-absorbing paper tip and the Ep tube before extracting the gingival crevicular fluid and the weight after extracting the gingival crevicular fluid, and subtract the two to obtain the gingival crevicular fluid weight, that is: the weight of gingival crevicular fluid=extract the weight of (moisture-absorbing paper point+Ep tube)-(moisture-absorbing paper point+Ep tube) weight after gingival crevicular fluid;

(3) Protein extraction: Add 20 μl of protein extraction solution to the above-mentioned Ep tube containing gingival crevicular fluid, bathe in ice for 20-30 minutes, centrifuge at 15,000 r/min at 4°C for 10 minutes, extract 20 μl of supernatant, add an equal volume of Protein sample solution, cooked for 5 minutes to obtain protein solution, stored at -20°C;

(4) Protein SDS-PAGE gel electrophoresis: the above protein solution is subjected to SDS-PAGE gel electrophoresis, and then fixed and stained;

(5) Western blotting: After the above gel electrophoresis is completed, the gel is removed, and Western blotting is performed with a nitrocellulose membrane, and then the labeled secondary antibody is used to react with it and develop (a conventional technique). protein bands, measure the optical density and area of the bands, and calculate the content of the proteolytic enzymes by: the protein content of the proteolytic enzymes=the average optical density of the bands×the area of the bands;

(6) Data processing: Calculate the enzyme content of the corresponding unit weight of gingival crevicular fluid according to the gingival crevicular fluid weight measured above, use SPSS11.0 statistical package, carry out statistical analysis (paired T test) to GCF weight and enzyme content, The analysis results can be used as one of the intermediate parameters required by doctors for diagnosis or treatment (not a disease diagnosis method, which can only be used as a factor for reference by doctors), and there are three ways: A. Analyze the trend of data changes , if the GCF weight and enzyme content are on the rise, it means that the root resorption of the subject to be detected has a tendency to increase; if the GCF weight and enzyme content are on a downward trend, it means that the root resorption of the subject to be detected has a tendency to decrease; B. The analysis result is compared with the standard data (the standard data refers to the statistical data obtained from the detection of people without tooth root resorption). If the GCF weight and enzyme content are higher than the standard data (statistically significant), it means that the person to be tested has a tooth root. Absorption, if it is the same as the standard data (no significant difference), it means that the subject to be tested has no tooth root absorption; The obtained data are compared, if the GCF weight and enzyme content are higher than those of the control group (statistically significant), it means that the subject to be tested suffers from tooth root resorption, and if it is the same as the control group (no significant difference), it means that the subject to be tested has no Root resorption. The three methods can be used alone or in combination.

In the described step (3), the formula of the protein extract is: Tris-CL of 50mmol pH7.5, 150mmol NaCl, 1mmolEDTA, 1mmol Na VO _{4 (} sodium orthovanadate), 1% (volume ratio) NP-40 , 10% (volume ratio) Glycerol (glycerol), 50mmol NaF, 1mmol PMSF (protease inhibitor); each component can be mixed.

The method of the present invention can be used to evaluate and screen the sensitive population of tooth root resorption; evaluate the occurrence state of tooth root resorption before orthodontic treatment; dynamically monitor the development and outcome of tooth root resorption in orthodontic treatment, so as to take effective measures to prevent the occurrence of tooth root resorption occur and develop.

detailed description

The present invention will be further described below in conjunction with embodiment.

Embodiment 1 detects rat tooth root resorption experiment

The test method is:

1) Rat tooth root resorption model was established, 12 weeks old, wistar rats, divided into two groups of experimental side and control side (20 rats, using self-control mode, divided into control side and experimental side)).

1) The 20# moisture-absorbing paper tip is ultraviolet sterilized and used for later use.

2) Extraction of gingival crevicular fluid: extract maxillary and maxillary gingival crevicular fluid after the appliance is installed. Before sampling, clean the tooth surface, use cotton balls to prevent moisture, and blow dry. Put the tip of 20# moisture-absorbing paper close to the gingival sulcus edge of the buccal-lingual tooth surface of the experimental tooth, and place it for 30 seconds. After taking it out, immediately put it into an Ep tube to seal it, and store it at -70°C until use. If you find blood stains on the tip of the paper, discard it and take it again. Samples were taken every other week for a total of eight times.

3) Measurement of the weight of the gingival crevicular fluid: Weigh the weight of the moisture-absorbing paper tip and the Ep tube before the extraction of the gingival crevicular fluid with an electronic analytical balance and the weight after the extraction of the gingival crevicular fluid, and subtract the two to obtain the weight of the gingival crevicular fluid. weight. Weight of gingival crevicular fluid = weight of (moisture-absorbing paper tip + Ep tube) after extraction of gingival crevicular fluid - (moisture-absorbing paper tip + Ep tube) weight.

4) Protein extraction: add 20μl protein extract solution to each sample, ice bath for 20-30min, centrifuge at 15000r/min at 4°C for 10 minutes, extract 20μl supernatant, add an equal volume of protein sample solution, cook for about 5min, stored at -20°C. Protein extract formula: 50mmol Tris-CL pH7.5, 150mmol NaCl, 1mmol EDTA, 1mmol Na ₃ VO ₄ (sodium orthovanadate), 1% NP-40, 10% Glycerol (glycerol), 50mmol NaF, 1mmol PMSF (protease inhibitor). Protein sample solution is SDS buffer, the formula is: Tris-HCl pH6.8 (100mM), SDS (4%), bromophenol blue (0.2%), glycerol (20%) and β-mercaptoethanol or dithiothreo Sugar alcohol (200mM) is a commonly used reagent in this field.

5) Protein SDS-PAGE gel electrophoresis: (the formula of separating gel and stacking gel is shown in Table 1)

(1) Clean the glass plate and install the electrophoresis tank;

(2) Determine the gel volume, prepare 14% separating gel, mix gently, add between two layers of glass, and use 1-2ml double distilled water on the top layer to isolate the air to prevent oxidation;

(3) After the separation gel is polymerized, pour off the top water, insert the sample comb, prepare a 7.5% stacking gel, and add the stacking gel to the top of the separation gel. After the gel is solidified, gently pull out the sample comb;

(4) Mix the sample with an equal volume of 2× loading buffer, boil at 100°C for 5 minutes, and cool in an ice bath;

(5) Add a sufficient amount of electrophoresis buffer to the electrophoresis tank, gently add the processed samples into the sample wells with a sampler, and add 20 μl of gingival crevicular fluid samples to each well. The samples of each animal are added to the same electrophoresis tank to increase comparability;

(6) Electrophoresis: The voltage at the start of electrophoresis is 8V/cm (50V). After the phenol blue enters the separation gel, increase the voltage to 5V/cm (100V), and continue electrophoresis until the phenol blue reaches the bottom of the separation gel;

(7) Remove the gel, fix it, stain it (HE staining), and observe the result.

Table 1 Preparation of SDS-PAGE gel

Reagent Separating gel stacking gel 30% acrylamide solution 4.0ml 0.41ml 2×bufferA 4.0ml the 2×bufferB - 1.25ml water - 0.75ml TEMED 0.007ml 0.005ml 10% persulfate press 0.3ml 0.1ml

 6) Western blot:

(1) After the above SDS-PAGE gel electrophoresis is completed, carefully remove the gel and soak it in the transfer solution for 15 minutes;

(2) Cut two pieces of filter paper and one piece of nitrocellulose (NC) membrane with the same size as the gel, and soak in the transfer solution for 15 minutes;

(3) Membrane transfer: place filter paper, gel, NC membrane, and 2 layers of filter paper in sequence in the clamping pad, and remove air bubbles. Place the membrane towards the positive electrode and the glue towards the negative electrode, place it in the transfer tank (the tank is filled with transfer solution), and transfer overnight at 12v28mA;

(4) Take out the NC membrane, place it in 5% skimmed milk, and seal at room temperature for 1-2 hours;

(5) Wash the membrane three times with PBS containing 0.5% Tween-20, 5-10min each time;

(6) Put the NC membrane in a plate or a pouch, add 1:1000 MMP-1 primary antibody (10mlTTBS, 10ml deionized water, 20μl goat anti-mouse protein lyase polyclonal antibody, 20μl Thimerosal), immerse the NC membrane, Shake at room temperature for 1-1.5h;

(7) Take out the NC membrane, wash the membrane three times with PBS containing 0.5% Tween-20, 5-10min each time;

(8) Add 1:4000 enzyme-labeled secondary antibody (horse radish peroxidase-labeled horse anti-goat IgG), immerse the NC membrane, shake at room temperature for 1-1.5h;

(9) Wash the membrane three times with PBS containing 0.5% Tween-20, 5-10min each time;

(10) Put 1ml of components A and B of ECL (enhanced chemiluminesence, enhanced chemiluminescence reagent) into the reaction box, put the membrane into the box, and shake slowly for 5 minutes to make the membrane and the reagent fully contact and react;

(11) Take out the film, wrap it with plastic wrap, drive out the air bubbles, expose in the dark room for 5 minutes, put it into the developer, develop for 1-2 minutes, and fix for 10 minutes;

(12) The X-ray film was analyzed using the Gel-Doc ultraviolet gel imaging analysis system, and the protein content of the proteolytic enzyme was represented by the average optical density of the band × the area of the band.

7) Data processing: Calculate the corresponding enzyme content per unit weight of gingival crevicular fluid according to the measured weight of gingival crevicular fluid, and use SPSS11.0 statistical package to conduct statistical analysis on GCF weight and enzyme content.

8) The rats were killed, and tissue sections were made to observe the root resorption situation (to verify whether the root resorption model was successfully established; since the establishment of the tooth root resorption model in the prior art is very mature and belongs to common knowledge, no other information is provided in this embodiment. verification photo).

Statistical analysis: The results are shown in Table 2 and Table 3. Results analysis: As can be seen from Table 2 and Table 3, the GCF weight and enzyme content of the experimental side were significantly higher than that of the control side. Therefore, it can be confirmed that in the gingival crevicular fluid There is a certain relationship between the content of proteolytic enzyme and orthodontic root resorption. In practical application, it can be used as an intermediate data for doctors to refer to in diagnosis and treatment.

Table 2 GCF weight

Table 3 Enzyme content

Advantages: At present, X-ray film (curvature film or root tip film) is the only simple and feasible method for clinical detection of root resorption. Studies have confirmed that curved tomography may exaggerate the root resorption phenomenon by more than 20%; periapical tomography is more accurate in diagnosing root resorption than curved tomography, but there are still some limitations. First of all, the apical radiograph is not sensitive enough and has poor clarity, so it can only show obvious apical absorption, and the absorption in the mesio-distal root and root neck is not easy to detect, unless the root absorption range is large and the depth is deep. Secondly, due to the relationship of the projection angle, the absorption on the buccal and lingual surface of the tooth root cannot be observed on the apical X-ray film at all. When the tooth moves in all directions, especially when the rotation and tilt movement occurs, it is difficult to accurately estimate the amount of root loss on X-ray films. In addition, apical radiographs are susceptible to equipment and photographer's technique, poor comparability, and patients must endure the radiographic radiation. Therefore, it is of great significance to find a simple, easy, and sensitive indicator to diagnose root resorption early, because the extraction of gingival crevicular fluid is convenient, non-invasive, and fast, and it is of great significance to prevent root resorption in clinical orthodontic treatment.

The method of collecting gingival crevicular fluid with a hygroscopic paper tip used in this experiment is improved from the most commonly used filter paper strip adsorption method at present. However, general filter paper is thicker and troublesome to cut, so it is not suitable for the collection of gingival crevicular fluid. The 20# moisture-absorbing paper tip we used has a small tip, which is easy to operate in the oral cavity, and the moisture-absorbing paper tip has a strong adsorption capacity, which is beneficial to the collection of gingival crevicular fluid. In this experiment, the gingival crevicular fluid was collected by the extra-sulcus method. This method has good reliability and repeatability, and at the same time causes little damage to the gingival sulcus epithelium, reduces the interference of serum exudation, and the determination of gingival crevicular fluid is more accurate than other methods The method is more rapid and accurate.

Clinical significance: With the rapid development of molecular biology, many scholars focus on the synthesis and differentiation of dental hard tissue at the molecular level. Proteolytic enzymes are a family of proteases containing Zn ²⁺ that can degrade extracellular matrix, and are involved in the degradation of extracellular matrix in various tissues of the body including bone. By studying the changes in the composition and flow of gingival crevicular fluid after tooth stress, we can indirectly understand the remodeling process of periodontal tissue, which is a valuable in vivo research method. The research on gingival crevicular fluid during orthodontic tooth movement has achieved rich results. Our study found that the change of proteolytic enzyme content in gingival crevicular fluid is highly correlated with orthodontic tooth root resorption.

Clinical application: It can be used to evaluate and screen sensitive groups of tooth root resorption; evaluate the occurrence status of tooth root resorption before orthodontic treatment; dynamically monitor the development and outcome of tooth root resorption during orthodontic treatment, and take effective measures to prevent the occurrence and occurrence of tooth root resorption develop.

Claims (1)

1. A method for detecting proteolytic enzyme content in gingival crevicular fluid, characterized in that, the steps are as follows: (1) Extraction of gingival crevicular fluid: clean the tooth surface of the subject to be tested, use cotton balls to insulate moisture, blow dry, and then stick the tip of 20# moisture-absorbing paper that has been sterilized by ultraviolet light to the gum of the buccal and lingual tooth surface of the tooth Leave the edge of the ditch for 30 seconds, take it out and immediately put it into an Ep tube to seal it, store it at -70°C, and wait for use; take a sample every other week, at least twice; (2) Measurement of the weight of the gingival crevicular fluid: Weigh the weight of the moisture-absorbing paper tip and the Ep tube before the extraction of the gingival crevicular fluid and the weight after the extraction of the gingival crevicular fluid, and subtract the two to obtain the weight of the gingival crevicular fluid; (3) Protein extraction: add 20 μl of protein extract to the Ep tube containing gingival crevicular fluid, place in ice bath for 20-30 minutes, centrifuge at 15000 r/min at 4°C for 10 minutes, extract 20 μl of supernatant, add an equal volume of protein The sample solution was boiled for 5 minutes to obtain a protein solution, which was stored at -20°C; (4) Protein SDS-PAGE gel electrophoresis: the above protein solution is subjected to SDS-PAGE gel electrophoresis, and then fixed and stained; (5) Western blotting: After the above gel electrophoresis is completed, remove the gel, perform western blotting with a nitrocellulose membrane, and then use a labeled secondary antibody to react with it and develop it to display the protein bands on the membrane, and measure the strips. The optical density and area of the band are calculated by: the protein content of the proteolytic enzyme = the average optical density of the band × the area of the band, and the content of the proteolytic enzyme is calculated; The protein extract formula: 50mmol Tris-CL pH7.5, 150mmol NaCl, 1mmol EDTA, 1mmol Na ₃ VO ₄ , 1% NP-40, 10% glycerol, 50mmol NaF, 1mmol PMSF; the protein sample solution is SDS buffer Solution, the formula is: 100mM Tris-HCl pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200mM β-mercaptoethanol or dithiothreitol.