CN102507707B - A kind of method detecting protein lyase content in level in gingival sulcus fluid - Google Patents
A kind of method detecting protein lyase content in level in gingival sulcus fluid Download PDFInfo
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- CN102507707B CN102507707B CN201110308602.XA CN201110308602A CN102507707B CN 102507707 B CN102507707 B CN 102507707B CN 201110308602 A CN201110308602 A CN 201110308602A CN 102507707 B CN102507707 B CN 102507707B
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Abstract
The invention discloses a kind of method detecting orthodontic tooth root resorption, step is as follows: the extraction of (1) level in gingival sulcus fluid; (2) measurement of level in gingival sulcus fluid weight; (3) Protein Extraction; (4) protein s DS-PAGE gel electrophoresis; (5) Western blot; (6) data processing: according to the enzyme content of the corresponding unit weight level in gingival sulcus fluid of the above-mentioned level in gingival sulcus fluid Weight computation recorded, uses SPSS 11.0 to add up bag, carries out statistical analysis to GCF weight and enzyme content.Method of the present invention can be used for the sensitive group evaluating and screen External apical root resorption; Evaluate the generation state of the External apical root resorption before orthodontic treatment; The development of dynamic monitoring root resorption during orthodontic treatment with lapse to, to take effective measures the generation and development that prevent External apical root resorption.
Description
Technical field
The invention provides a kind of method detecting orthodontic tooth root resorption.
Background technology
The incidence of disease of orthodontic tooth root resorption is high, serious External apical root resorption can cause apex radicis dentis not recoverability to shorten rust, thus cause tooth crown root than imbalance, tooth mobility, even comes off.At present effective methods for the treatment of be there is no for External apical root resorption, early detection can only be passed through, remove the cause of disease, thus prevent it from further developing.Protein lyase participates in the extracellular matrix that degraded comprises the various tissue of whole body of bone, and level in gingival sulcus fluid is the transudate in gingival sulcus, and its composition changes along with the change of surrounding tissue, comprising level in gingival sulcus fluid protein lyase.Diagnosed the illness by the change detecting Cucumber in level in gingival sulcus fluid, monitor the course of disease, understand result for the treatment of by stomatologist's widespread use.
Summary of the invention
For above-mentioned prior art, the research that the present inventor is correlated with, research shows that level in gingival sulcus fluid protein lyase is the main enzyme participating in Periodontium remodeling, relevant with External apical root resorption, therefore, the invention provides a kind of method detecting orthodontic tooth root resorption.
The present invention is achieved by the following technical solutions:
Detect a method for orthodontic tooth root resorption, step is as follows:
(1) extraction of level in gingival sulcus fluid: the facing of clean person to be detected, cotton balls, every wet, dries up, then the prior 20# hygroscopic paper point through disinfection by ultraviolet light is close to the gum furrow rim of the cheek-tongue side facing of tooth, places 30 seconds, after taking-up, put into the Ep seal of tube immediately,-70 DEG C of preservations, stand-by; If find that paper point has blood stains to contaminate, then that abandons refetches; Sample week about once, at least get twice;
(2) measurement of level in gingival sulcus fluid weight: weigh the weight that level in gingival sulcus fluid extracts front hygroscopic paper point and Ep pipe and the weight extracted after level in gingival sulcus fluid thereof respectively with electronic analytical balance, both subtract each other and obtain the weight of level in gingival sulcus fluid, that is: the weight of weight-(hygroscopic paper point+Ep manages) of (hygroscopic paper point+Ep manages) after the weight=extraction level in gingival sulcus fluid of level in gingival sulcus fluid;
(3) Protein Extraction: add 20 μ l protein extracts, ice bath 20-30min to above-mentioned being equipped with in the Ep pipe of level in gingival sulcus fluid, at 4 DEG C, 15000r/min is after centrifugal 10 minutes, extract 20 μ l supernatants, add isopyknic albumen sample solution, boil 5min, obtain protein liquid ,-20 DEG C of preservations;
(4) protein s DS-PAGE gel electrophoresis: above-mentioned protein liquid carries out SDS-PAGE gel electrophoresis, then fixing dyeing;
(5) Western blot: after above-mentioned gel electrophoresis, take off gel, Western blot is carried out with nitrocellulose filter, then resist with two of mark and react with it and carry out develop (for routine techniques routine operation), protein band on display film, measure optical density and the area of band, pass through: the area of the average optical × band of the protein content=band of protein lyase, calculate the content of protein lyase;
(6) data processing: according to the enzyme content of the corresponding unit weight level in gingival sulcus fluid of the above-mentioned level in gingival sulcus fluid Weight computation recorded, use SPSS11.0 statistics bag, statistical analysis (paired-samples T-test) is carried out to GCF weight and enzyme content, this analysis result can (be not the diagnostic method of disease as one of intermediate parameters needed for diagnosis or treatment, it can only as a factor, reference is carried out) by doctor, following three kinds of mode: A. are had to analyze its data variation trend, if GCF weight and enzyme content are ascendant trend, the External apical root resorption then representing person to be detected has the trend increased the weight of, if GCF weight and enzyme content are downtrending, the External apical root resorption then representing person to be detected has the trend alleviated, B. this analysis result is compared with normal data (normal data refers to the statistical data detecting and obtain without External apical root resorption person), if GCF weight and enzyme content is normal data high (having statistical significance) comparatively, then represent person to be detected and suffer from External apical root resorption, identical with normal data if (without significant difference), then represent person to be detected without External apical root resorption, C: simultaneously establish control group in step (1), control group is selected without External apical root resorption person, the data that experimental group and control group obtain are compared, if GCF weight and enzyme content is control group high (having statistical significance) comparatively, then represent person to be detected and suffer from External apical root resorption, identical with control group if (without significant difference), then represent person to be detected without External apical root resorption.Three kinds of modes can be used alone, and also can be combined.
In described step (3), the formula of protein extract is: the Tris-CL of 50mmol pH7.5,150mmol NaCl, 1mmolEDTA, 1mmol Na
3vO
4(sodium vanadate), 1% (volume ratio) NP-40,10% (volume ratio) Glycerol (glycerine), 50mmol NaF, 1mmol PMSF (protease inhibitors); Each component mixes.
Method of the present invention can be used for the sensitive group evaluating and screen External apical root resorption; Evaluate the generation state of the External apical root resorption before orthodontic treatment; The development of dynamic monitoring root resorption during orthodontic treatment with lapse to, to take effective measures the generation and development that prevent External apical root resorption.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
Embodiment 1 detects the experiment of rat External apical root resorption
Test method is:
1) rat External apical root resorption model is set up, 12 week age, wistar rat, point experimental side and control sides two groups (20 rats, adopt the mode of own control, point control sides and experimental side)).
1) for subsequent use after 20# hygroscopic paper point disinfection by ultraviolet light.
2) extraction of level in gingival sulcus fluid: extract lower jaw level in gingival sulcus fluid after installation appliance.Before sampling, clean facing, cotton balls, every wet, dries up.20# hygroscopic paper point is close to the gum furrow rim of the cheek-tongue side facing of experiment tooth, place 30 seconds, put into the Ep seal of tube immediately after taking-up ,-70 DEG C of preservations are stand-by.If find that paper point has blood stains to contaminate, then that abandons refetches.Sample week about once, get eight times altogether.
3) measurement of level in gingival sulcus fluid weight: weigh the weight that level in gingival sulcus fluid extracts front hygroscopic paper point and Ep pipe and the weight extracted after level in gingival sulcus fluid thereof respectively with electronic analytical balance, both subtract each other and obtain the weight of level in gingival sulcus fluid.The weight of weight-(hygroscopic paper point+Ep manages) of (hygroscopic paper point+Ep manages) after the weight=extraction level in gingival sulcus fluid of level in gingival sulcus fluid.
4) Protein Extraction: add 20 μ l protein extracts in each sample, ice bath 20-30min, at 4 DEG C, 15000r/min is after centrifugal 10 minutes, extracts 20 μ l supernatants, adds isopyknic albumen sample solution, boil about 5min ,-20 DEG C of preservations.Protein extraction formula of liquid: 50mmol Tris-CL pH7.5,150mmol NaCl, 1mmol EDTA, 1mmol Na
3vO
4(sodium vanadate), 1%NP-40,10%Glycerol (glycerine), 50mmol NaF, 1mmol PMSF (protease inhibitors).Albumen sample solution is SDS damping fluid, formula is: Tris-HCl pH6.8 (100mM), SDS (4%), bromjophenol blue (0.2%), glycerine (20%) and beta-mercaptoethanol or dithiothreitol (DTT) (200mM) are this area common agents.
5) protein s DS-PAGE gel electrophoresis: (formula of separation gel and concentrated glue is as shown in table 1)
(1) clean glass plate, install electrophoresis tank;
(2) determine gel volume, the separation gel of preparation 14%, mix gently, add between layer glass, top layer 1-2ml distilled water completely cuts off the anti-oxidation of air;
(3), after glue polymerization to be separated, outwell the water at top, insert sample comb, the concentrated glue of preparation 7.5%, adds to concentrated glue above separation gel.After gelling is solid, pull out sample comb gently;
(4) mixed with equal-volume 2 × loading buffer by sample, 100 DEG C are boiled 5min, and ice bath cools;
(5) electrophoresis tank is added enough electrophoretic buffers, slowly join in loading hole gently with injector by the sample handled well, every hole adds 20 μ l level in gingival sulcus fluid samples.The sample of every animal is added in same electrophoresis tank, increases comparability;
(6) electrophoresis: during beginning electrophoresis, voltage is 8V/cm (50V), after smelling phenol orchid and entering separation gel, voltage is increased to 5V/cm (100V), continues electrophoresis and arrives at bottom separation gel to smelling phenol orchid;
(7) gel is taken off, fixing, dyeing (HE dyeing), observations.
The preparation of table 1SDS-PAGE gel
| Reagent | Separation gel | Concentrated glue |
| 30% propylene phthalein amine aqueous solution | 4.0ml | 0.41ml |
| 2×bufferA | 4.0ml | |
| 2×bufferB | - | 1.25ml |
| Water | - | 0.75ml |
| TEMED | 0.007ml | 0.005ml |
| 10% persulfuric acid is pressed | 0.3ml | 0.1ml |
6) Western blot:
(1) above-mentioned SDS-PAGE gel electrophoresis is complete, carefully takes off gel, in transferring film liquid, soak 15min;
(2) cut the 2 piece filter paper identical with gel size and one piece of cellulose nitrate (NC) film, in transferring film liquid, soak 15min;
(3) transferring film: place filter paper, gel, NC film, 2 metafiltration paper successively in pad, and remove bubble.By film towards positive pole, glue is towards negative pole, and be placed in transfer groove (groove fills transfer liquid), 12v28mA transfer is spent the night;
(4) take out NC film, be placed in 5% skim milk, room temperature closes 1-2h;
(5) film is washed three times, each 5-10min with the PBS containing 0.5%Tween-20;
(6) NC film is placed in plate or pouch, adds MMP-1 primary antibodie (10mlTTBS, the 10ml deionized water of 1: 1000,20 μ l sheep anti mouse protein lyase polyclonal antibodies, 20 μ l Thimerosal), submergence NC film, shaken at room temperature 1-1.5h;
(7) NC film is taken out, wash film three times, each 5-10min with the PBS containing 0.5%Tween-20;
(8) ELIAS secondary antibody (the Ma Kangyang IgG of horseradish peroxidase-labeled) of 1: 4000 is added, submergence NC film, shaken at room temperature 1-1.5h;
(9) film is washed three times, each 5-10min with the PBS containing 0.5%Tween-20;
(10) put into the A of ECL (enhanced chemiluminesence, enhanced chemiluminescence reagent), each 1ml of B component in reaction box, film puts into box, slowly shakes 5min, makes film and the abundant contact reaction of reagent;
(11) take out film, wrap with preservative film, drive bubble out of, after darkroom exposure 5min, put into developer solution, development 1-2min, fixing 10min;
(12) X-ray uses Gel-Doc ultraviolet Labworks image acquisition and analysis software to analyze, the cartographic represenation of area of the average optical × band of the protein content band of protein lyase.
7) data processing: according to the enzyme content of the corresponding unit weight level in gingival sulcus fluid of the level in gingival sulcus fluid Weight computation recorded, uses SPSS11.0 statistics bag, carries out statistical analysis to GCF weight and enzyme content.
8) rat is put to death, do histotomy and observe External apical root resorption situation (to verify whether External apical root resorption model is successfully established; Because the foundation of External apical root resorption model in prior art is very ripe, belong to common practise, in the present embodiment, do not reoffer its checking photo).
Statistical analysis: result is as shown in table 2, table 3, interpretation of result: as can be seen from table 2 and table 3, the GCF weight of experimental side and enzyme content are apparently higher than control sides, therefore, can confirm, there is certain relation in the content of the protein lyase in level in gingival sulcus fluid and orthodontic tooth root resorption, in actual applications, can as an intermediate data, for doctor diagnosing, treatment time carry out reference.
Table 2GCF weight
Table 3 enzyme content
Advantage: at present, X-ray film (Orhopantomography or Dental X-ray film) is the uniquely simple and feasible means of clinical detection root absorption.Have research to confirm, Orhopantomography may exaggerate root absorption phenomenon more than 20%; Dental X-ray film comparatively Orhopantomography is more accurate in diagnosis External apical root resorption, but still has some limitations.First, Dental X-ray film is responsive not, poor definition, can only manifest the obvious tip of a root and absorb, and in nearly distal surface root, the absorption in collar portion not easily detects, unless just can manifest when root absorption scope is comparatively large and the degree of depth is darker.Secondly, due to the relation of projection angle, for the absorption on root of the tooth cheek lingual surface, tip of a root X-ray film cannot be observed at all.The movement of tooth in all directions, when particularly rotating with inclination movement, X-ray film is difficult to the loss amount accurately estimating root of the tooth.In addition, tip of a root X-ray film is subject to equipment and takes the photograph the impact of sheet people technology, and comparability is poor, and patient must stand x-ray radiation.Therefore, find a kind of simple, easy, responsive index, accomplish early diagnosis External apical root resorption, because level in gingival sulcus fluid is extracted conveniently, without wound, fast, prevented External apical root resorption to have a very big significance in correction clinical treatment.
The method that the hygroscopic paper point that this experiment adopts gathers level in gingival sulcus fluid improves according to filter paper bar sorption method the most frequently used at present.And general filter paper thickness is thicker, and cutting trouble, be not suitable for the collection of level in gingival sulcus fluid.The tip of the 20# hygroscopic paper point that we adopt is tiny, is easy to operate in the oral cavity, and the high adsorption capacity of hygroscopic paper point, be conducive to the collection of level in gingival sulcus fluid.What this experiment adopted is that the outer method of ditch gathers level in gingival sulcus fluid, and this method has good reliability and repeatability, simultaneously little to the damage of epithelium of gingival sulcus, decreases the interference that serum oozes out, and the mensuration of level in gingival sulcus fluid comparatively additive method is more rapid accurate.
Clinical meaning: along with molecular biological fast development, many scholars concentrate on research direction the synthesis differentiation on hard tooth tissue molecular level.Protein lyase is one group and contains Zn
2+can the protease family of degradation of cell epimatrix, participate in the extracellular matrix that degraded comprises the various tissue of whole body of bone.By the research to composition and fluctuations in discharge in the stressed rear level in gingival sulcus fluid of tooth, indirectly can understand the reconstruction process of periodontium, be a kind of valuable viviperception method.Achieved abundant achievement to the research of level in gingival sulcus fluid in Orthodontic movement process, our research finds that in level in gingival sulcus fluid, protein lyase content and orthodontic tooth root resorption have high correlation.
Clinical practice: can be used for the sensitive group evaluating and screen External apical root resorption; Evaluate the generation state of the External apical root resorption before orthodontic treatment; The development of dynamic monitoring root resorption during orthodontic treatment with lapse to, take effective measures the generation and development that prevent External apical root resorption.
Claims (1)
1. detect a method for protein lyase content in level in gingival sulcus fluid, it is characterized in that, step is as follows:
(1) extraction of level in gingival sulcus fluid: the facing of clean person to be detected, cotton balls, every wet, dries up, then the prior 20# hygroscopic paper point through disinfection by ultraviolet light is close to the gum furrow rim of the cheek-tongue side facing of tooth, places 30 seconds, after taking-up, put into the Ep seal of tube immediately,-70 DEG C of preservations, stand-by; Sample week about once, at least get twice;
(2) measurement of level in gingival sulcus fluid weight: weigh level in gingival sulcus fluid respectively and extract the weight of front hygroscopic paper point and Ep pipe and the weight after extracting level in gingival sulcus fluid thereof, both subtract each other and obtain the weight of level in gingival sulcus fluid;
(3) Protein Extraction: add 20 μ l protein extracts, ice bath 20-30min in the Ep pipe that level in gingival sulcus fluid is housed, at 4 DEG C, 15000r/min is after centrifugal 10 minutes, extract 20 μ l supernatants, add isopyknic albumen sample solution, boil 5min, obtain protein liquid ,-20 DEG C of preservations;
(4) protein s DS-PAGE gel electrophoresis: above-mentioned protein liquid carries out SDS-PAGE gel electrophoresis, then fixing dyeing;
(5) Western blot: after above-mentioned gel electrophoresis, take off gel, Western blot is carried out with nitrocellulose filter, then resist with two of mark and react with it and develop, protein band on display film, measure optical density and the area of band, pass through: the area of the average optical × band of the protein content=band of protein lyase, calculate the content of protein lyase;
Described protein extraction formula of liquid: 50mmol Tris-CL pH7.5,150mmol NaCl, 1mmol EDTA, 1mmol Na
3vO
4, 1%NP-40,10% glycerine, 50mmol NaF, 1mmol PMSF; Albumen sample solution is SDS damping fluid, fills a prescription to be: 100mM Tris-HCl pH6.8,4%SDS, 0.2% bromophenol blue, 20% glycerine and 200mM beta-mercaptoethanol or dithiothreitol (DTT).
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