CN111248153A - Rat lung pseudomonas aeruginosa biofilm infection model and construction method thereof - Google Patents
Rat lung pseudomonas aeruginosa biofilm infection model and construction method thereof Download PDFInfo
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- 208000015181 infectious disease Diseases 0.000 title claims abstract description 51
- 241000589517 Pseudomonas aeruginosa Species 0.000 title claims abstract description 42
- 210000004072 lung Anatomy 0.000 title claims abstract description 27
- 238000010276 construction Methods 0.000 title abstract description 7
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 210000000621 bronchi Anatomy 0.000 claims description 6
- 210000003928 nasal cavity Anatomy 0.000 claims description 6
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- 206010057190 Respiratory tract infections Diseases 0.000 claims description 4
- 238000002695 general anesthesia Methods 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
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- 206010033078 Otitis media Diseases 0.000 claims description 3
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- 238000012258 culturing Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of preparations for in vivo experiments, and discloses a rat lung pseudomonas aeruginosa biomembrane infection model and a construction method thereof. The in vitro cultured pseudomonas aeruginosa biomembrane structure is cultured by putting the adjusted bacterial suspension into a six-hole plate; scraping off with cells, vibrating to form a small biological membrane structure, and detecting and adjusting the bacterial concentration. The bacterial biofilm provided by the invention has a real and stable structure and good repeatability, the prepared pseudomonas aeruginosa biofilm suspension causes the lung infection of rats by adopting a nasal drip method, and then a pathogenic mechanism and an intervention treatment effect are determined; the animal model is closer to the pathological change of the infection of the lung part of the bacterial biofilm of the human body.
Description
Technical Field
The invention belongs to the technical field of preparations for in vivo experiments, and particularly relates to a rat lung pseudomonas aeruginosa biomembrane infection model and a construction method thereof.
Background
Currently, the current state of the art commonly used in the industry is such that: pseudomonas aeruginosa (P.a) is a common pathogenic bacterium infected in a refractory hospital, and various infections caused by the Pseudomonas aeruginosa are related to Biofilm (BF) formation, and related genes are all regulated and controlled by Quorum Sensing (QS). Clinically, diseases of a plurality of subjects such as chronic respiratory tract infection, chronic urogenital system infection, osteomyelitis, endocarditis, otitis media, prostatitis, periodontitis, infection of in-vivo indwelling catheters and the like are closely related to the chronic and refractory infection, and pseudomonas aeruginosa infection is one of the most extensive and serious problems in various nosocomial infections, and the nosocomial infection caused by the pseudomonas aeruginosa infection reaches more than 30 percent, wherein the respiratory tract infection is the first. Therefore, the research on the pseudomonas aeruginosa biofilm pneumonia has great significance for the prevention and treatment of the diseases clinically. The existing in vitro model preparation technology of the pseudomonas aeruginosa biomembrane is basically mature, the sensitivity detection research of related antibiotics, traditional Chinese medicines and the like is more, but the preparation of in vivo models is not ideal all the time. The preparation of a rat pneumonia model caused by different approaches is found in a lung infection model of planktonic bacteria, and compared with a nasal drip approach infection model-making mode, the operation is simple, the wound is small, the death rate of animals is obviously reduced, and the infection result has little difference. Clinically, pseudomonas aeruginosa biofilm infection is very common, the treatment effect is poor, the treatment course is long, the economic burden is heavy, the most common refractory infection is the infection in the lung, the respiratory function of a patient is influenced, and the life quality is obviously reduced. In the past, the carrier can not be implanted in the lung, the tracheotomy wound is large, the death rate of animals is very high, and the research on the aspect is limited. At present, two methods are mostly adopted for animal models infected by pseudomonas aeruginosa biomembranes, (1) the infection is simulated in a carrier transplant with the pseudomonas aeruginosa biomembranes prepared in vitro, the death rate of animals is extremely high in a molding method, the carrier can only be placed in abdominal cavities, tracheas and the like, and the infection of bacterial biomembranes without foreign matters can not be simulated; (2) alginate and other simulated biomembrane-wrapped bacteria are injected into the body, and the bacteria are not real biomembrane-wrapped structures and have larger pathogenicity difference.
In summary, the problems of the prior art are as follows:
(1) the carrier with the pseudomonas aeruginosa biomembrane prepared in vitro is transplanted in vivo to simulate infection, the death rate of animals is extremely high in the molding method, the carrier can only be placed in the abdominal cavity, trachea and the like, and the infection of the bacterial biomembrane without foreign matters can not be simulated.
(2) Alginate and other simulated biofilm-wrapped bacteria are injected into the body, the bacteria are not real biofilm-wrapped structures, the pathogenicity difference is large, and the pathogenicity of clinical diseases cannot be really explained.
The difficulty and significance for solving the technical problems are as follows: at present, no good animal model which is wider in clinical occurrence but difficult to treat respiratory system infection exists, basic research on the mechanism of the disease is accelerated, and the problem of clinical serious chronic difficult to treat infection is solved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a rat lung pseudomonas aeruginosa biomembrane infection model and a construction method thereof.
The rat lung pseudomonas aeruginosa biomembrane infection model is realized by that after a rat is subjected to conventional anesthesia, the nostril of the rat is inclined upwards at 45 degrees, 0.6mL of bacterial liquid is dripped into the deep part of the nasal cavity by a microsyringe, the position of the rat is kept in an upright position for 1min, and the bacterial liquid enters the trachea and the bronchus.
The invention also aims to provide a method for constructing the rat lung pseudomonas aeruginosa biomembrane infection model, which comprises the following steps:
step one, inoculating bacteria on an agar plate, selecting a single colony to inoculate 5mL of LB culture solution at 37 ℃, centrifuging the bacteria solution after shaking, discarding the supernatant, washing the bacterial precipitate for 2 times by using sterile normal saline, and adjusting the OD600 of the bacterial suspension by using the normal saline to be 0.05 bacteria solution for later use to obtain the planktonic bacteria of P.aeruginosa;
step two, adopting a 6-pore plate method to construct BF, adding 0.1mL of bacteria liquid +2mLLB broth culture medium into each pore of a 6-pore plate, placing the 6-pore plate into an incubator at 37 ℃ for standing culture for 3d, changing the liquid on day 2, forming an obvious membrane at the bottom of the 6-pore plate after culturing for 3d, discarding the supernatant, washing the membrane for 2 times by using physiological saline to remove planktonic bacteria, scraping the BF formed at the bottom of the pore by using a cell scraper, transferring the BF to a sterile centrifuge tube, fully oscillating the BF on an oscillator to disperse the BF, centrifuging the supernatant, and re-suspending and diluting the supernatant by using the sterile physiological saline to obtain the tunica of the bacterium P.aeruginosa;
smearing a smear, and dyeing and observing a pseudomonas aeruginosa biomembrane;
fourthly, dripping the bacteria liquid into the nose, after the rat is subjected to general anesthesia, enabling the nostril of the rat to be in an upward inclined state of 45 degrees, dripping 0.6mL of the bacteria liquid into the deep part of the nasal cavity by using a microsyringe, changing the body position of the rat, keeping the vertical position for 1min, and ensuring that the bacteria liquid enters the trachea and the bronchus; detecting the lung infection condition and pathological changes of the rats after 1d, 3d, 7d and 14d of infection.
Further, 200rmin in the first step-1Shaking for 20 hr, and mixing with bacterial solution 3000rmin-1Centrifuge for 10 min.
Further, in the second step, the supernatant was centrifuged off, and the supernatant was resuspended in a bacterial solution diluted to an OD600 of 0.05 with sterile physiological saline.
The invention also aims to provide a medicament for treating chronic respiratory tract infection, which is obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
The invention also aims to provide a medicament for treating chronic urogenital infection, which is obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
The invention also aims to provide a medicament for treating osteomyelitis, which is obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
The invention also aims to provide a medicament for treating periodontitis obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
The invention also aims to provide a medicament for treating otitis media obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
The invention also aims to provide a medicament for treating the indwelling catheter infection, which is obtained from the rat lung pseudomonas aeruginosa biomembrane infection model.
In summary, the advantages and positive effects of the invention are: the in vitro cultured pseudomonas aeruginosa biomembrane structure is cultured by putting the adjusted bacterial suspension into a six-hole plate; scraping off with cells, vibrating to form a small biological membrane structure, and detecting and adjusting the bacterial concentration. The bacterial biofilm provided by the invention has a real and stable structure and good repeatability, the prepared pseudomonas aeruginosa biofilm suspension causes the lung infection of rats by adopting a nasal drip method, and then a pathogenic mechanism and an intervention treatment effect are determined; the animal model is closer to the pathological change of the infection of the lung part of the bacterial biofilm of the human body.
Drawings
FIG. 1 is a flowchart of a method for constructing a model of Pseudomonas aeruginosa biofilm infection in rat lung according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The successful construction of the animal model provides good conditions for the observation of pathological process and intervention treatment effect of diseases.
The rat lung pseudomonas aeruginosa biomembrane infection model provided by the embodiment of the invention is characterized in that after a rat is subjected to conventional anesthesia, the nostril of the rat is in an upward inclined state of 45 degrees, 0.6mL of bacterial liquid is dripped into the deep part of the nasal cavity by a microsyringe, the body position of the rat is changed, the rat keeps an upright position for about 1min, and the bacterial liquid is ensured to enter the trachea and the bronchus.
As shown in fig. 1, the method for constructing a model of pseudomonas aeruginosa biofilm infection in rat lung provided by the embodiment of the present invention comprises the following steps:
s101: the bacteria were inoculated on agar plates, single colonies were picked and inoculated into 5mL LB medium at 37 ℃ with 200rmin-1After shaking for 20h, 3000rmin of bacterial liquid is added-1Centrifuging for 10min, removing supernatant, washing the bacterial precipitate with sterile normal saline for 2 times, adjusting OD600 of bacterial suspension with normal saline to 0.05 bacterial liquid for use, and obtaining the bacteria which are the planktonic bacteria of P.aeruginosa;
s102: obtaining construction of P.aeruginosa-BF and BF bacterial suspension, adopting a 6-pore plate method to construct BF, adding 0.1mL of bacterial liquid plus 2mLLB broth culture medium into each pore of a 6-pore plate, placing the 6-pore plate into an incubator at 37 ℃ for standing culture for 3d, carefully changing the liquid on day 2, forming an obvious membrane at the bottom of the 6-pore plate after culturing for 3d, discarding supernatant, washing for 2 times by using physiological saline to remove planktonic bacteria, scraping the BF formed at the bottom of the pore by using cell scraping, transferring the BF to a sterile centrifuge tube, fully oscillating the BF on an oscillator to disperse the BF, centrifuging the supernatant, diluting the supernatant into bacterial liquid with sterile physiological saline to obtain OD630 ═ 0.06, wherein the bacteria obtained by the method is the envelope bacteria of the P.aeruginosa;
s103: the smear can be carried out, and the condition of the pseudomonas aeruginosa biomembrane can be observed through staining;
s104: dripping into nose for infection, after general anesthesia, making nostril of rat in 45 degree upward inclined state, dripping 0.6mL bacterial liquid into deep of nasal cavity with microsyringe, changing rat body position, keeping it upright for about 1min, and ensuring bacterial liquid entering into trachea and bronchus. After 1d, 3d, 7d and 14d of infection, the lung infection condition, pathological changes and the effect of intervening treatment and observation of the rat can be detected.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (10)
1. The model is characterized in that after the rat lung pseudomonas aeruginosa biomembrane infection model is used for general anesthesia of the rat, the nostril of the rat is inclined upwards at 45 degrees, 0.6mL of bacterial liquid is dripped into the nasal cavity by a microsyringe, the position of the rat is kept in an upright position for 1min, and the bacterial liquid enters the trachea and the bronchus.
2. The method for constructing the model of Pseudomonas aeruginosa biofilm infection in the lung of rat as claimed in claim 1, wherein the method for constructing the model of Pseudomonas aeruginosa biofilm infection in the lung of rat comprises the following steps:
step one, inoculating bacteria on an agar plate, selecting a single colony to inoculate 5mL of LB culture solution at 37 ℃, centrifuging the bacteria solution after shaking, discarding the supernatant, washing the bacterial precipitate for 2 times by using sterile normal saline, and adjusting the OD600 of the bacterial suspension by using the normal saline to be 0.05 bacteria solution for later use to obtain the planktonic bacteria of P.aeruginosa;
step two, adopting a 6-pore plate method to construct BF, adding 0.1mL of bacteria liquid +2mLLB broth culture medium into each pore of a 6-pore plate, placing the 6-pore plate into an incubator at 37 ℃ for standing culture for 3d, changing the liquid on day 2, forming an obvious membrane at the bottom of the 6-pore plate after culturing for 3d, discarding the supernatant, washing the membrane for 2 times by using physiological saline to remove planktonic bacteria, scraping the BF formed at the bottom of the pore by using a cell scraper, transferring the BF to a sterile centrifuge tube, fully oscillating the BF on an oscillator to disperse the BF, centrifuging the supernatant, and re-suspending and diluting the supernatant by using the sterile physiological saline to obtain the tunica of the bacterium P.aeruginosa;
smearing a smear, and dyeing and observing a pseudomonas aeruginosa biomembrane;
step four, dripping nose infection, after the rat is subjected to general anesthesia, enabling the nostril of the rat to be in a 45-degree upward inclined state, dripping 0.6mL of bacterial liquid into the nasal cavity by using a microsyringe, changing the body position of the rat, keeping the vertical position for 1min, and ensuring that the bacterial liquid enters the trachea and the bronchus; detecting the lung infection condition and pathological changes of the rats after 1d, 3d, 7d and 14d of infection.
3. The method for constructing a model of Pseudomonas aeruginosa biofilm infection in rat lung according to claim 2, wherein 200rmin is used in the first step-1After shaking for 20h, 3000rmin of bacterial liquid is added-1Centrifuge for 10 min.
4. The method for constructing a model of Pseudomonas aeruginosa biofilm infection in rat lung according to claim 2, wherein the supernatant obtained by centrifugation in the second step is resuspended in a bacterial solution diluted to OD600 ═ 0.05 with sterile physiological saline.
5. A medicament for treating chronic respiratory tract infection, which is obtained from the rat lung Pseudomonas aeruginosa biofilm infection model in claim 1.
6. A medicament for treating chronic urogenital infection, which is obtained from the rat pulmonary Pseudomonas aeruginosa biofilm infection model of claim 1.
7. A medicament for treating osteomyelitis, which is obtained from the rat pulmonary Pseudomonas aeruginosa biofilm infection model according to claim 1.
8. A medicament for treating periodontitis obtained from the rat pulmonary Pseudomonas aeruginosa biofilm infection model of claim 1.
9. A medicament for treating otitis media derived from the rat pulmonary Pseudomonas aeruginosa biofilm infection model of claim 1.
10. A medicament for treating indwelling catheter infection obtained from the model of Pseudomonas aeruginosa biofilm infection in the lungs of rat as described in claim 1.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101658709A (en) * | 2009-09-10 | 2010-03-03 | 重庆医科大学附属儿童医院 | Construction method of infected animal model using catheter with bacterial biofilm |
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
| CN109833333A (en) * | 2019-02-16 | 2019-06-04 | 首都医科大学附属北京中医医院 | A kind of method preparing pneumonia rats model and model evaluation method |
| CN110156737A (en) * | 2019-04-19 | 2019-08-23 | 中国人民解放军第四军医大学 | The application of a kind of pyranone compounds and its resisting pseudomonas aeruginosa biomembrane |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101658709A (en) * | 2009-09-10 | 2010-03-03 | 重庆医科大学附属儿童医院 | Construction method of infected animal model using catheter with bacterial biofilm |
| CN104189898A (en) * | 2014-06-27 | 2014-12-10 | 四川大学 | Pseudomonas aeruginosa vaccine and preparation method thereof |
| CN109833333A (en) * | 2019-02-16 | 2019-06-04 | 首都医科大学附属北京中医医院 | A kind of method preparing pneumonia rats model and model evaluation method |
| CN110156737A (en) * | 2019-04-19 | 2019-08-23 | 中国人民解放军第四军医大学 | The application of a kind of pyranone compounds and its resisting pseudomonas aeruginosa biomembrane |
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