CN101658709A - Construction method of infected animal model using catheter with bacterial biofilm - Google Patents
Construction method of infected animal model using catheter with bacterial biofilm Download PDFInfo
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- CN101658709A CN101658709A CN200910190813A CN200910190813A CN101658709A CN 101658709 A CN101658709 A CN 101658709A CN 200910190813 A CN200910190813 A CN 200910190813A CN 200910190813 A CN200910190813 A CN 200910190813A CN 101658709 A CN101658709 A CN 101658709A
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Abstract
The method provides a construction method of an infected animal model using a catheter with a bacterial biofilm, which comprises the steps of: constructing a catheter adhered with a bacterial biofilmin vitro, securing the catheter in a trachea of an experimental animal, and identifying relevant indexes of an animal model. The construction method effectives simulates a micro-environment of an in-vivo trachea, facilitating the bacterial biofilm adhered on the catheter to be stably formed and developed in the micro-environment of the trachea, wherein the physiological form of the obtained bacterial biofilm is similar to that of the actual one with good stability, thereby providing a reliable source of the bacterial biofilm for the further research and development of drugs for the resistanceof relevant bacterial biofilms of tracheal catheter and for the selection of the drugs. Meanwhile, the inventive construction method has the advantages of simple operation, low requirements of both technique and equipment, as well as high success rate.
Description
Technical field
The present invention relates to a kind of construction method of infected animal model using catheter with bacterial biofilm, particularly a kind of construction method of endotracheal tube bacterial biofilm infected animal model belongs to medical science model field.
Background technology
In recent years, the treatment through MV via intubation has become the critical treatment means of rescuing the critical patient.Respirator dependency pneumonia is the severe complication that Failure Treated with Mechanical Ventilation is brought, and incidence rate and mortality rate are higher, the state of an illness repeatedly, the antibiotic weak curative effect is the clinical thorny problem that faces.The existence of patients with mechanical ventilation endotracheal tube bacterial biofilm be studies confirm that in a large number, in the pathogenesis research of respirator dependency pneumonia, finds to have closely related property between endotracheal tube bacterial biofilm and the respirator dependency pneumonia in recent years.To be bacterial adhesion form in activity or inert material surface bacterial biofilm and the corresponding growth pattern of the cell that swims, form by antibacterial and self excretory extracellular matrix, bacterial drug resistance is increased, infection is difficult to remove, thereby becoming a source of infection that continues to exist, is to cause a chronically infected major reason.The infection of bacterial biofilm dependency has become clinically very stubborn problem, so, set up a kind of novel the clinical practice situation that more meets, simple, reliable endotracheal tube dependency bacterial biofilm infection model, study in the body that endotracheal tube dependency bacterial biofilm infects in order to carrying out, thereby further develop anti-endotracheal tube dependency bacterial biofilm medicine and drug screening, therapeutic evaluation is to need the urgent problem that solves clinically.
In recent years, a large amount of in vitro studies has been carried out in the formation and development of directed toward bacteria biofilm and intervention, but less relatively at research in the body of endotracheal tube dependency biofilm.And the first step of carrying out research in the body is set up endotracheal tube dependency bacterial biofilm animal models infected exactly.At present, endotracheal tube dependency biofilm animal models infected mainly contains following two kinds: the one, behind the conduit insertion trachea with sterilization, a certain amount of bacterium liquid is injected in the lung by conduit; Another kind is that germ-carrying conduit per os under the guiding of conduit inner needle tube is inserted trachea, and needle tubing is extracted the back inlying catheter in trachea.The former is inconsistent with the actual forming process that endotracheal tube dependency biofilm clinically infects, and main reflection is not that bacterial biofilm forms situation in the trachea but in the lung; Latter's complicated operation, specification requirement height, the supravasal biomembrane of destructible in the process of operation influences the experiment accuracy simultaneously, and conduit is fixing in trachea, easily causes conduit to come off, lose, and makes the failure of an experiment.Therefore, the result who draws based on institute in the body of above model also is affected for the using value of clinical treatment.
Summary of the invention
The objective of the invention is to set up a kind of simple, low to the researcher specification requirement, modelling success rate height, the effectively construction method of the conduit dependency bacterial biofilm infected animal model of air flue microenvironment in the analogue body.
For achieving the above object, the present invention takes following technical scheme: a kind of construction method of infected animal model using catheter with bacterial biofilm, comprise: the conduit of external structure particle-bound bacteria biofilm, conduit is fixed in the laboratory animal trachea, the animal model index of correlation is identified.
Described laboratory animal is the SD rat.
Specifically, the external structure method of described particle-bound bacteria biofilm conduit is that the picking microbionation is cultivated in culture fluid, and inoculation bacterium liquid is cultivated on the sterility cover slide, obtains ripe bacterial biofilm.
More particularly, in the external structure of particle-bound bacteria biofilm conduit, the described antibacterial that is used to inoculate is a Pseudomonas aeruginosa clinical separation strain list bacterium colony; Culture fluid is the L-Broth culture fluid; Condition of culture is 37 ℃, 200rpm, incubated overnight; The bacterium liquid that is inoculated in the sterility cover slide is that spectrophotometric determination OD600 value is 0.5 bacterium liquid; Described sterility cover slide is as the carrier of bacterial biofilm growth, has 24 porocyte culture plates, and size is 8mm * 8mm, and its Tissue Culture Plate holds bacterium liquid; Change liquid next day that the biofilm training method being, cultivation cycle is 7 days.
Above-mentioned conduit at the endotracheal fixing means of laboratory animal is: cut the trachea of laboratory animal, the conduit of particle-bound bacteria biofilm is inserted and is sewn in the trachea.
The present invention put to death the SD rat in the 4th day and the 7th day through postoperative respectively, SD rat index of correlation is carried out routine identify that comprise: the SEM of Pseudomonas aeruginosa biofilm form detection, conduit colony counting, pulmonary's pathological change and/or pulmonary's colony counting are identified on the conduit.
The present invention is fixed in the trachea of laboratory animal such as rat by the conduit with the particle-bound bacteria biofilm of In vitro culture, simulated air flue microenvironment in the body effectively, make that supravasal bacterial biofilm can formation and development stably in the trachea microenvironment, the physiology and appearance of the bacterial biofilm that obtains and actual similar and good stability, thereby for further the medicine of the anti-endotracheal tube dependency of developmental research bacterial biofilm and the screening of medicine thereof provide reliable bacterial biofilm source.Simultaneously, this construction method is simple to operate, and technology, equipment requirements are low, the success rate height.
Description of drawings
Fig. 1 respectively organizes Pseudomonas aeruginosa biofilm form sem photograph on the conduit among the embodiment.
Fig. 2 is each group rat pulmonary's pathological change sem photograph (x 200).
The specific embodiment:
1. the external structure of particle-bound bacteria biofilm conduit
Picking Pseudomonas aeruginosa clinical separation strain list colony inoculation is in the L-Broth culture fluid, 37 ℃, the 200rpm shaken cultivation is spent the night, spectrophotometric determination bacterium liquid OD600 value, adjust OD600 value to 0.5, inoculation bacterium liquid is placed sterility cover slide (8mm * 8mm in advance, carrier as the biofilm growth) in the 24 porocyte culture plate, every hole 1ml, the next day change liquid, obtain the coverslip that ripe biofilm adheres to after 7 days, get two culture tubes at every turn at random and adopt plate dilution method to carry out colony counting.
2. the preparation of animal model
(1) animal grouping
40 SD rats (body weight 130-160g/ only) are divided into 2 groups, 20 every group at random;
First group is the bacterial biofilm infected group, inserts by tracheotomy and is fixed in the trachea with the conduit of ripe biofilm what In vitro culture obtained after 7 days, only gives normal saline 1ml/ every day;
Second group is blank conduit matched group, sterile catheter is inserted by tracheotomy and is fixed in the trachea, only gives normal saline 1ml/ every day;
(2) animal model is set up process
1. get 10% chloral hydrate 0.3ml/100g anesthetized rat;
2. fixedly rat extremity and head are cut jaw down to the fur between two forelegs, and with iodine tincture outwards week sterilization from the centre, reuse ethanol takes off iodine;
3. mention skin with tweezers, cut off an osculum, in the center of sterilization scope, cut an osculum that is about 1.5cm from jaw down to chest again with little shears;
4. cut off superficial fascia with shears, until exposing muscle, with curved forceps from central passivity separating muscle until exposing trachea;
5. separate the trachea surrounding tissue, little pipe pad is put under the trachea;
6. vertically cut off osculum that is about 0.5cm of trachea with little shears;
7. have the conduit of biofilm to put into trachea in blank conduit or length, reuse crack line pin passes muscle between the nearest cartilage of incision of trachea far-end, and passes conduit, pass from muscle between the nearly section of otch cartilage farthest, the tension suture is observed the rats breathing situation, as steadily getting final product ligation earlier.
8. subcutaneous tissue is made in order and built, will disinfect wound behind the skin closure in alcohol.
Each group was all put to death 10 rats in the 4th day and the 7th day.
3. modelling evaluation methodology
(1) SEM of Pseudomonas aeruginosa biofilm form detects on the conduit
In each time period, after putting to death rat, every group of conduit that takes out 2 rats at random, rip cutting is separately got half immediately with in the 2.5% glutaraldehyde PBS solution fixedly behind the 2h, through 2 times (pH=7.2) of 0.1molPBS flushing, after 30%～100% series concentration ethanol dehydration, replace again through 50%～100% tert-butyl alcohol, dry, ion sputtering instrument metal spraying are observed Pseudomonas aeruginosa biofilm microscopic pattern through SEM;
(2) conduit colony counting
In each time period, put to death rat after, every group of conduit that takes out 8 rats, rip cutting is separately got half and is placed the 1ml physiological saline solution, vibrate to get 100ml after 30 minutes and be tiled on the LB agar plate the dull and stereotyped upward clump count of counting after 37 ℃ of overnight incubation;
(3) pulmonary's pathological change
After putting to death rat, each time point takes out lung, getting one puts 4% paraformaldehyde and fixes, paraffin section, conventional hematoxylin-eosin staining, light microscopic is observed lung pathology down and is changed, the pulmonary pathological change changes index with pulmonary's general pathology, and (Lung indexof macroscopic pathology LIMP) represents, calculates the ratio of inflammatory cell infiltration and necrotic zone area and the area of whole visual field in each visual field;
(4) pulmonary's colony counting
Aseptic taking-up lung after each time point is put to death rat takes off a lung tissue and carries out homogenate (every 10mg lung tissue adds 6 μ l physiological saline solution).Get homogenate 100ml and be tiled on the LB agar plate, the dull and stereotyped clump count of going up of counting after 37 ℃ of overnight incubation.
4. the evaluation result of modelling
(1) Pseudomonas aeruginosa biofilm form on the conduit
As shown in Figure 1,
A represents 4 days biological tunicle infected group of postoperative; B represents the blank conduit matched group of postoperative;
C represents 7 days biological tunicle infected group of postoperative; D represents 7 days blank conduit matched groups of postoperative.
Operation back the 4th day, biofilm slabbing on the endotracheal tube of visible bacterial biofilm infected group rat, there have the mucus shape material of became uneven to connect on every side to be a sheet of, and antibacterial is wrapped up in it; The antibacterial that the visible inside pipe wall of blank conduit matched group has a small amount of shape to differ assembles.And after operation the 7th day, biofilm mixes with mucus materials in a large number on the endotracheal tube of visible bacterial biofilm infected group rat, and antibacterial is buried in it; At blank conduit matched group, visible accumulative antibacterial has cluster sample material to hold outward.
(2) conduit colony counting
Table 1 is respectively organized the colony counting of rat endotracheal tube (lgCFU, x ± s)
Table1?Bacterial?count?in?biofilm?on?the?tube(lgCFU，x±s)
| The biofilm infected group | Blank conduit matched group | |
| The 4th day | ??8.5669±0.11 ★ | ??6.2425±0.023 |
| The 7th day | ??8.5009±0.013 ★ | ??6.3933±0.065 |
Annotate: ★ represents and compares F=87.6 and 507.58, p＜0.05 with time period biofilm infected group.
The result shows that in each time period, the antibacterial that blank conduit matched group detects obviously is less than the bacterial biofilm infected group.
(3) pulmonary's pathological change
As shown in Figure 2,
A is a not intervention group of 4 days biological tunicles of postoperative; B is a not intervention group of 7 days biological tunicles of postoperative;
C is 4 days blank conduit matched groups of postoperative; D is 7 days blank conduit matched groups of postoperative.
Table 2 is respectively organized the LIMP of rat pulmonary integration, x ± s
Table2?LIMP?score?of?lungs，x±s
| The biofilm infected group | Blank conduit matched group | |
| The 4th day | ??0.1929±0.025 ★ | ??0.0286±0.005 |
| The 7th day | ??0.2357±0.031 ★ | ??0.04±0.009 |
Annotate: ★ represents and compares F=4.9, p＜0.05 with time period biofilm infected group.
In each time period, blank conduit set all is lower than the bacterial biofilm infected group LIMP integration (p＜0.05) with the time.
(4) pulmonary's colony counting
Table 3 is respectively organized the colony counting of rat pulmonary (lgCFU, x ± s)
Table3?Bacterial?count?in?lungs(lgCFU，x±s)
| The biofilm infected group | Blank conduit matched group | |
| The 4th day | ??6.3216±0.11 ★ | ??5.2483±0.061 |
| The 7th day | ??6.1729±0.013 ★ | ??5.1283±0.075 |
Annotate: ★ represents and compares F=52.3, p＜0.05 with time period biofilm infected group.
The result shows that the antibacterial that blank conduit matched group detects obviously is less than the bacterial biofilm infected group
Claims (7)
1. the construction method of an infected animal model using catheter with bacterial biofilm, it is characterized in that: it may further comprise the steps, and the conduit of external structure particle-bound bacteria biofilm is fixed on conduit in the laboratory animal trachea, and the animal model index of correlation is identified.
2. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 1, it is characterized in that: described laboratory animal is the SD rat.
3. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 1 or 2, it is characterized in that: the external structure method of particle-bound bacteria biofilm conduit is that the picking microbionation is cultivated in culture fluid, inoculation bacterium liquid is cultivated on the sterility cover slide, obtains ripe bacterial biofilm.
4. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 3, it is characterized in that: in the external structure of particle-bound bacteria biofilm conduit, the described antibacterial that is used to inoculate is a Pseudomonas aeruginosa clinical separation strain list bacterium colony; Culture fluid is the L-Broth culture fluid; Condition of culture is 37 ℃, 200rpm, incubated overnight; The bacterium liquid that is inoculated in the sterility cover slide is that spectrophotometric determination OD600 value is 0.5 bacterium liquid; Described sterility cover slide has 24 porocyte culture plates, and size is 8mm * 8mm, and bacterium liquid is seeded in the Tissue Culture Plate of this sterility cover slide; Change liquid next day that the biofilm training method being, cultivation cycle is 7 days.
5. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 1 or 2, it is characterized in that: described conduit is the trachea that cuts laboratory animal at the endotracheal fixing means of laboratory animal, and the conduit of particle-bound bacteria biofilm is inserted and is sewn in the trachea.
6. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 4, it is characterized in that: described conduit is the trachea that cuts laboratory animal at the endotracheal fixing means of laboratory animal, and the conduit of particle-bound bacteria biofilm is inserted and is sewn in the trachea.
7. the construction method of infected animal model using catheter with bacterial biofilm as claimed in claim 6, it is characterized in that: the 4th day after surgery and the rat execution with described experimental technique in the 7th day respectively, the rat index of correlation is carried out routine identify that comprise: the SEM of Pseudomonas aeruginosa biofilm form detection, conduit colony counting, pulmonary's pathological change and/or pulmonary's colony counting are identified on the conduit.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102274086A (en) * | 2011-05-05 | 2011-12-14 | 中国人民解放军第三军医大学第二附属医院 | Implant and method for building bacterial biofilm-infected animal model |
| CN102669017A (en) * | 2011-12-19 | 2012-09-19 | 河南科技大学 | Method for building in-vivo bacterial biofilm infected animal model |
| CN111248153A (en) * | 2020-03-27 | 2020-06-09 | 山东中医药高等专科学校 | Rat lung pseudomonas aeruginosa biofilm infection model and construction method thereof |
-
2009
- 2009-09-10 CN CN200910190813A patent/CN101658709A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102274086A (en) * | 2011-05-05 | 2011-12-14 | 中国人民解放军第三军医大学第二附属医院 | Implant and method for building bacterial biofilm-infected animal model |
| CN102669017A (en) * | 2011-12-19 | 2012-09-19 | 河南科技大学 | Method for building in-vivo bacterial biofilm infected animal model |
| CN111248153A (en) * | 2020-03-27 | 2020-06-09 | 山东中医药高等专科学校 | Rat lung pseudomonas aeruginosa biofilm infection model and construction method thereof |
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Open date: 20100303 |