CN102654503A - Method for detecting influence of attenuation bacillus calmette-guerin to asthma - Google Patents
Method for detecting influence of attenuation bacillus calmette-guerin to asthma Download PDFInfo
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- CN102654503A CN102654503A CN2011100517847A CN201110051784A CN102654503A CN 102654503 A CN102654503 A CN 102654503A CN 2011100517847 A CN2011100517847 A CN 2011100517847A CN 201110051784 A CN201110051784 A CN 201110051784A CN 102654503 A CN102654503 A CN 102654503A
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Abstract
The invention discloses a method for detecting influences of attenuation bacillus calmette-guerin to asthma. The method comprises the following steps of: establishing asthma models of animals, inoculating bacillus calmette-guerin many times, and detecting the expression of lung tissue TLR4 (toll-like receptor 4) of the small animals by western blotting. According to the method for detecting influences of attenuationbacillus calmette-guerin to asthma of the invention, the influences of attenuation bacillus calmette-guerin to asthma are as follows: the therapeutic effects of the bacillus calmette-guerin are relative to the enhancement of the TLR4 (toll-like receptor 4) of the lung tissue, so that the attenuation bacillus calmette-guerin can play an important function in treating the asthma.
Description
Technical field
The present invention relates to medical domain, be specifically related to the detection method of a kind of attenuated live BCG vaccine the asthma influence.
Background technology
Asthma has airway hyperreactivity, Ig-E regulates and the specific reaction gene.Toll appearance acceptor be in mammal, find with fruit bat the albumen of homology is arranged, called after TLR, the initial TLR4 that finds is that research is maximum at present.Research confirms; Its mycobacterium lipoprotein of attenuated live BCG vaccine can with Toll appearance receptors bind on the macrophage; Promote macrophage to produce IL-12, IFN-γ, suppress the generation of IL-4, IL-5, thus correct in the asthma Th1/Th2 subgroup number with (or) functional imbalance.
At present, the attenuated live BCG vaccine is also unknown to the influence that asthma TLR4 expresses.
Summary of the invention
The object of the present invention is to provide the detection method of a kind of attenuated live BCG vaccine to the asthma influence, having solved present attenuated live BCG vaccine influences the problem that does not also have corresponding detecting method to asthma.
A kind of attenuated live BCG vaccine of the present invention is to the detection method of asthma influence, and concrete steps are:
Step 1) preparing experiment animal and reagent: preparing experiment is with the mouse IgG antibody of toy, ovalbumin, attenuated live BCG vaccine, anti-TLR4 monoclonal antibody, anti-GAPDH monoclonal antibody and HRP mark;
Step 2) grouping of animal used as test and sensitization model preparation: the experiment toy is divided into three groups that the toy number of elements equates at random, is respectively blank group, asthma morbidity group and attenuated live BCG vaccine intervention group,
Asthma morbidity group: 1st, 7,14 days lumbar injection egg albumen sensitizations, with Al (OH)
3As immunologic adjuvant, began atomizing on the 15th day and excite, set up asthmatic model;
Attenuated live BCG vaccine intervention group: the foundation of asthmatic model is with asthma morbidity group, excites with last atomizing preceding 1 day of each sensitization and carries out the intracutaneous injection of attenuated live BCG vaccine in 1 hour;
The blank group: use the physiologic saline for substitute ovalbumin,
All toys obtain sample after exciting 24h the last time;
The Western Blotting of step 3) toy lung tissue TLR4 albumen detects: get each group experiment toy lung tissue and extract total protein, measure concentration.Behind each histone sample SDS-PAGE electrophoresis, electrotransfer is to pvdf membrane; Pvdf membrane is behind sealing 2h, with the TLR4 monoclonal antibody after the dilution, incubated overnight; Again further with two anti-hatching of horseradish peroxidase after, drip chemical luminous substrate, x-ray film sensitization detects protein signal;
The step 4) data statistics: all experimental results are carried out statistical study.
Preferably, said step 2) in, the concentration of the egg albumen sensitization of injection is 0.08%, injection volume is 0.2ml.
Preferably, said step 2) in, the each dosage of attenuated live BCG vaccine intracutaneous injection is 0.025mg.
Preferably, in the said step 3), the temperature of incubated overnight is 4 ℃.
Preferably, in the said step 4), what the statistical study of experimental result was adopted is SPSS10.0 software.。
Beneficial effect: through the detection method of attenuated live BCG vaccine of the present invention to the asthma influence; Analysis draws; Because TLR4 albumen only has the expression of trace in the lung tissue of blank group; The expression of TLR4 albumen slightly raises than control group in the asthma morbidity group, and the expression of TLR4 albumen all has significant enhancing for more preceding two groups in the attenuated live BCG vaccine intervention group, and the attenuated live BCG vaccine possibly be through stimulating the TLR4 up-regulated; And then regulate the Th1/Th2 balance, in the treatment of asthma morbidity, play a significant role.
Embodiment
Embodiment 1
1 materials and methods
1) preparation of animal used as test and reagent
The mouse IgG antibody of Kunming kind small white mouse, ovalbumin, attenuated live BCG vaccine, anti-TLR4 monoclonal antibody, anti-GAPDH monoclonal antibody, HRP mark etc.
2) grouping of animal used as test and sensitization model preparation
Animal used as test is divided into three groups at random, is respectively blank group, asthma morbidity group and attenuated live BCG vaccine intervention group, every group of 10 mouse.Asthma morbidity group: 1st, 7,14 days lumbar injection 0.08%OVA sensitization, each 0.2ml, Al (OH)
3As immunologic adjuvant.Beginning atomizing on the 15th day excites; Attenuated live BCG vaccine intervention group: the foundation of asthmatic model is with asthma morbidity group, excites in 1 hour the intracutaneous injection of attenuated live BCG vaccine to intervene once more in preceding 1 day of each sensitization and last atomizing, and each dosage is 0.025mg; Blank group: use physiologic saline for substitute OVA.All mouse obtain sample after exciting 24h the last time.
3) the Western Blotting of mouse lung tissue T LR4 albumen detects
Get and respectively organize experiment mice lung tissue extraction total protein, measure concentration.Behind each histone sample SDS-PAGE electrophoresis, electrotransfer is to pvdf membrane.Pvdf membrane is behind sealing 2h, with the TLR4 monoclonal antibody after the dilution, 4 ℃ of incubated overnight.Again further with two anti-hatching of horseradish peroxidase after, drip chemical luminous substrate, x-ray film sensitization detects protein signal.
4) data statistics
All experimental results adopt SPSS10.0 software to carry out statistical study, and the result representes with the form of
.Group difference adopts the t check.
2 results
TLR4 albumen only has the expression of trace in the lung tissue of blank group, the expression of TLR4 albumen slightly raises than control group in the asthma morbidity group, and the expression of TLR4 albumen all has significant enhancing for more preceding two groups in the attenuated live BCG vaccine intervention group.
3. conclusion
The attenuated live BCG vaccine is the attenuated live vaccine of tubercle bacillus, and its principal ingredient is a BCG vaccine RNA..Research confirms, after the part of TLR4 combines TLR4, through striding membrane structure the pathogen-associated molecular stimulus signal transduceed in the cell, produces complicated cascade signal reaction.
Find that through experiment the TLR4 gene expression amount is compared with blank group and asthma morbidity group all has remarkable rising after the attenuated live BCG vaccine is intervened.The attenuated live BCG vaccine possibly be through stimulation TLR4 up-regulated, and then regulates the Th1/Th2 balance, in the treatment of asthma morbidity, plays a significant role.
Claims (5)
1. an attenuated live BCG vaccine is characterized in that to the detection method that asthma influences concrete steps are:
1) preparing experiment animal and reagent: preparing experiment is with the mouse IgG antibody of toy, ovalbumin, attenuated live BCG vaccine, anti-TLR4 monoclonal antibody, anti-GAPDH monoclonal antibody and HRP mark;
2) grouping of animal used as test and sensitization model preparation: the experiment toy is divided into three groups that the toy number of elements equates at random, is respectively blank group, asthma morbidity group and attenuated live BCG vaccine intervention group,
Asthma morbidity group: 1st, 7,14 days lumbar injection egg albumen sensitizations, with Al (OH)
3As immunologic adjuvant, began atomizing on the 15th day and excite, set up asthmatic model;
Attenuated live BCG vaccine intervention group: the foundation of asthmatic model is with asthma morbidity group, excites with last atomizing preceding 1 day of each sensitization and carries out the intracutaneous injection of attenuated live BCG vaccine in 1 hour;
The blank group: use the physiologic saline for substitute ovalbumin,
All toys obtain sample after exciting 24h the last time;
3) the Western Blotting of toy lung tissue TLR4 albumen detects: get each group experiment toy lung tissue and extract total protein, measure concentration.Behind each histone sample SDS-PAGE electrophoresis, electrotransfer is to pvdf membrane; Pvdf membrane is behind sealing 2h, with the TLR4 monoclonal antibody after the dilution, incubated overnight; Again further with two anti-hatching of horseradish peroxidase after, drip chemical luminous substrate, x-ray film sensitization detects protein signal;
4) data statistics: all experimental results are carried out statistical study.
2. attenuated live BCG vaccine according to claim 1 is characterized in that said step 2 to the detection method of asthma influence) in, the concentration of the egg albumen sensitization of injection is 0.08%, injection volume is 0.2ml.
3. attenuated live BCG vaccine according to claim 1 is characterized in that said step 2 to the detection method of asthma influence) in, the each dosage of attenuated live BCG vaccine intracutaneous injection is 0.025mg.
4. attenuated live BCG vaccine according to claim 1 is characterized in that to the detection method of asthma influence in the said step 3), the temperature of incubated overnight is 4 ℃.
5. attenuated live BCG vaccine according to claim 1 is characterized in that the detection method of asthma influence, and in the said step 4), what the statistical study of experimental result was adopted is SPSS10.0 software.
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| CN2011100517847A CN102654503A (en) | 2011-03-04 | 2011-03-04 | Method for detecting influence of attenuation bacillus calmette-guerin to asthma |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030138436A1 (en) * | 1998-10-05 | 2003-07-24 | The Malaghan Institute Of Medical Research | Method of using a vaccine |
| CN101069743A (en) * | 2004-07-27 | 2007-11-14 | 张锦铭 | Use of inactive BCG vaccine in preparation of use for treatment of allergic action diseases |
| JP2009014626A (en) * | 2007-07-06 | 2009-01-22 | Medibic:Kk | Method of predicting bcg therapy effect |
| CN101813631A (en) * | 2010-03-26 | 2010-08-25 | 浙江万马药业有限公司 | BCG polyose nuclear acid injection polysaccharide rapid determination method |
-
2011
- 2011-03-04 CN CN2011100517847A patent/CN102654503A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030138436A1 (en) * | 1998-10-05 | 2003-07-24 | The Malaghan Institute Of Medical Research | Method of using a vaccine |
| CN101069743A (en) * | 2004-07-27 | 2007-11-14 | 张锦铭 | Use of inactive BCG vaccine in preparation of use for treatment of allergic action diseases |
| JP2009014626A (en) * | 2007-07-06 | 2009-01-22 | Medibic:Kk | Method of predicting bcg therapy effect |
| CN101813631A (en) * | 2010-03-26 | 2010-08-25 | 浙江万马药业有限公司 | BCG polyose nuclear acid injection polysaccharide rapid determination method |
Non-Patent Citations (3)
| Title |
|---|
| 张雅琴等: "减毒活菌卡介苗对哮喘TLR4表达的影响", 《中国实用医药》, vol. 35, no. 5, 31 December 2010 (2010-12-31) * |
| 易丽等: "活菌卡介苗对哮喘小鼠IL-17的调节作用", 《热带医学杂志》, vol. 11, no. 4, 30 April 2011 (2011-04-30) * |
| 王鹏等: "卵白蛋白致敏时减毒活菌卡介苗干预对哮喘小鼠气道炎性反应的影响", 《武警医学》, vol. 22, no. 7, 31 July 2011 (2011-07-31) * |
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Application publication date: 20120905 |