CN102669017A - Method for building in-vivo bacterial biofilm infected animal model - Google Patents
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- CN102669017A CN102669017A CN2011104279833A CN201110427983A CN102669017A CN 102669017 A CN102669017 A CN 102669017A CN 2011104279833 A CN2011104279833 A CN 2011104279833A CN 201110427983 A CN201110427983 A CN 201110427983A CN 102669017 A CN102669017 A CN 102669017A
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Abstract
The invention a method for building an in-vivo bacterial biofilm infected animal model. The method includes the steps of firstly, preparing bacterial suspension; secondly, performing anesthesia and back muscular infection to an experimental animal; and thirdly, identifying related indexes of the animal model. By the method, an in-vivo animal model with bacterial biofilms infected is built by performing back muscular infection of streptococcus suis to a zebra fish. Truth of the bacteria infected animal is simulated effectively. The condition that mass accumulation of streptococcus suis in back muscle of the zebra fish causes infection of the bacterial biofilms is found. The obtained in-vivo animal model is practical and highly stable. Therefore, the animal model is provided in order to know more about the truth of existence of bacteria in an animal body, know in-vivo forming mechanism of bacterial biofilms, understand pathogenesis of the bacteria in depth and develop related drugs. In addition, the method is simple to operate, low in technical and equipment requirement and high in success rate.
Description
Technical field
The present invention relates to the construction method of bacterial biofilm infected animal model in a kind of body, belong to medical faunae scale-model investigation field.
Background technology
(biofilm BF) is meant that single or multiple monoid bacterium in order to adapt to surrounding environment, is adsorbed in foreign matter or tissue surface to bacterial biofilm; Polysaccharide protein complexs such as a large amount of polysaccharide matrixes of breeding justacrine, fibrin, lipoprotein; Making bacterium gathering each other, adhesion, winding to form the film appearance thing with 3-D solid structure, is bacterial micro bacterium colony aggregation (Hall-Stoodley L; Costerton JW; Stoodley P.Bacterial biofilms:from the natural environment to infectious diseases.Nat Rev Microbiol, 2004,2:95-108).At occurring in nature; Any bacterium can form biofilm under maturation condition; And the microorganism more than 90% is (Costerton JW, Stewart PS, Greenberg EP.Bacterial biofilms:a common cause of persistent infections.Science with the form existence growth of biofilm; 1999,284:1318-1322).The expert of US Centers for Disease Control and Prevention estimates relevant (the Potera C with BF of the people's bacterial infection more than 65%; Forging a link between biofilms and disease.Science, 1999,283:1837; 1839), thus the research of relevant biofilm become focus day by day.Existing increasing experiment proof biofilm possibly be bacterium real existence form in host, if this prompting effectively prevention and control BF infect, in clinical infection is treated with significant.Bacterium character under the biofilm state has more different in the bacterium of floating state; Its immune response to antibiotic and host is insensitive; Even find that clinically test of many times proves effective medicine, can not thoroughly remove biofilm, this just causes infecting protracted course of disease; Waste great amount of manpower and material resources, formed public health problem.So, set up a kind of novel clinical practice situation that more meets, simple, bacterial biofilm animal infection modal in the body reliably, in order to the research bacterium in animal body necessary being form, thereby understand the pathogenesis of bacterium, in the hope of the control eqpidemic disease.
In recent years; The method for building up of biofilm model has a large amount of reports in the body, but major part is all to be artificial ectogenicly to be embedded in part apparatus (conduit is organized sleeve pipe etc.) in the animal body; Cause animal models infected in the body; These class methods all can not good bacterial infection that is virtually reality like reality approach, be colonizated in the position of tissue, certain difference is arranged with infecting in the desirable body.At present the body inner model of foundation such as heeling-in conduit mainly comprises infection model, urinary tract infection model, respiratory tract infection model and the osteomyelitis infection model etc. of allogenic material in the infection model, peritonaeum of CVC model, subcutaneous xenobiotic on the spinal animal model.The researcher utilizes rat model to study staphylococcus aureus (Ulphani JS first; Rupp ME; Model of Staphylococcus aureus central venous catheter-associated infection in rats.Lab Anim Sci, 1999,49:283-287) and MRSE (Rupp ME; Ulphani JS; Fey PD, Mack D, Characterization of Staphylococcus epidermidis polysaccharide intercellular adhesin/hemagglutinin in the pathogenesis of intravascular catheter-associated infection in a rat model.Infect Immun; 1999, the 67:2656-2659) formation of biofilm in animal body.On this model, silica gel catheter is buried on the ductus venosus as for rat, with the microbionation ductus venosus.Subcutaneous xenobiotic infection model is mainly used a kind of sleeve pipe of organizing by the polytetrafluoroethylene (PTFE) material, and the inside comprises bead and other materials, and the object of this external source is embedded in subcutaneous, makes bacterial biofilm to generate in the above.The major advantage of this model is that microbial cell can field planting in organizing sleeve pipe easily, need not shift out.In a lot of bodies in the biofilm research, bacterium can form biofilm imbedding on the intraperitoneal biomaterial of rabbit and mouse.This situation is a certain amount of bacterium of inoculation (Buret A after imbedding biomaterial; Ward KH; Olson ME, Costerton JW, An in vivo model to study the pathobiology of infectious biofilms on biomaterial surfaces.J Biomed Mater Res; 1991,25:865-874).The advantage of this model is to let body set up a kind of chronic infection; Make the host be in bacterial infection state (Gagnon RF, Richards GK, A mouse model of implant-associated infection.Int J Artif Organs for a long time; 1993,16:789-798).(Satoh M, Munakata K, Kitoh K such as Satoh; Takeuchi H, Yoshida O, A newly designed model for infection-induced bladder stone formation in the rat.J Urol; 1984,132:1247-1249) utilize this model to be imbedded at the zinc video disc in the bladder of rat, then through bladder inoculation proteus; Confirm subsequently bacterial biofilm in the urinary tract infection formation and matrix be created in the urinary tract infection play an important role (Nickel JC, Olson M, McLean RJ; Grant SK, Costerton JW, An ecological study of infected urinary stone genesis in an animal model.Br J Urol; 1987,59:21-30).After the tracheal catheter animal model mainly is the conduit insertion animal tracheae with sterilization; A certain amount of bacterium liquid is injected in the lung through conduit; This method is inconsistent with the actual forming process of tracheae infection biological tunicle clinically, and main reflection is not real situation in the tracheae.Above method all exists operation to place the infection that the external source foreign matter causes animal body; It or not the time of day that truly reflects the pathogen natural infection; Because operation is placed the external source foreign matter and has been brought bigger stimulation to animal body; And the infection of other bacteriums afterwards that might cause performing the operation, thereby interference experiment result.
Therefore, set up the body inner model that true reflection body biofilm infects, the mechanism that the further investigation bacterial biofilm forms in animal body just seems particularly important.Along with the continual renovation and the development of correlation technique, people will also just can control biofilm all the more deeply with thorough better to the understanding and the understanding of bacterial biofilm, benefit the society.
Summary of the invention
The object of the present invention is to provide a kind of simplely, low to researcher's specification requirement, the modelling success rate is high, can the real simulation bacterial body in the construction method of biofilm animal models infected.
To achieve these goals, technical scheme of the present invention has adopted the construction method of bacterial biofilm infected animal model in a kind of body, may further comprise the steps:
(1) preparation of bacterial suspension;
(2) anesthesia of laboratory animal and muscle of back infect;
(3) identify the animal model index of correlation.
Bacterial suspension is obtained by following method in the step (1): the bacterium of separating that is used to connect that preserves under-80 ℃ of temperature of general is drawn Colombia's blood plate, chooses single bacterium colony incubated overnight under 37 ℃ the temperature in medium; Get bacterium liquid then and add fresh THB medium, 37 ℃ of shaking tables continue overnight incubation, and after plate coating checking is pure culture, shakes bacterium with inoculation in 1: 100 and cultivate the thalline that obtained exponential phase in 4-6 hour, be between the 08-1.0 with spectrophotometric determination OD600 value; With thalline with aseptic THB clean 2-3 all over after; Regulating bacterial concentration with the THB medium is 10
6Individual/ml is subsequent use.
The said bacterium that is used to inoculate is the Streptococcus suis clinical separation strain.
Described laboratory animal is the pure lines zebra fish.
Anesthesia of laboratory animal and muscle of back infect for the MS-222 (MS-222) of concentration 95 μ g/mL zebra fish is anaesthetized; Back inoculation 5 * 10
4The bacterial number of/fish, control group injection THB medium infects back 24h, and aseptic position of taking the back to infect is fixed in muscle of back in 2% the paraformaldehyde.
After infecting 24 hours, the zebra fish of said experimental technique is put to death respectively, the zebra fish index of correlation is identified.
Described evaluation comprises muscle of back is prepared frozen tissue section, carries out HE dyeing, Gram and ESEM (SEM) and observes.
Adopt method of the present invention; Through zebra fish muscle of back injection Streptococcus suis, set up bacterium and formed the interior animal model of body that biofilm infects, simulated the truth of bacterial infection animal effectively; Find that Streptococcus suis gathers in a large number in the zebra fish muscle of back; Form the state that biofilm infects, animal model meets reality and good stability in the body of acquisition, thereby for further understanding the time of day that bacterium exists in animal body; Understand bacterial biofilm formation mechanism in vivo and reach the pathogenesis of deeply understanding bacterium, the exploitation related drugs provides animal model.Simultaneously, this construction method is simple to operate, and technology, equipment requirements is low, and success rate is high.
Description of drawings
Fig. 1 is a zebra fish muscle of back frozen section HE coloration result;
Fig. 2 is zebra fish muscle of back frozen section Gram result;
Fig. 3 is zebra fish muscle of back ESEM result.
Embodiment
Embodiment:
1. the preparation of bacterial suspension
The pig streptococcus bacterial strain of-80 ℃ of preservations is drawn Colombia's blood plate, choose single bacterium colony 37 ℃ of incubated overnight in medium.Get bacterium liquid then and add fresh THB medium (commercially available), 37 ℃ of shaking tables continue overnight incubation, and after plate coating checking is pure culture, shakes bacterium with inoculation in 1: 100 and cultivate the thalline that obtained exponential phase in 4-6 hour, be between the 08-1.0 with spectrophotometric determination OD600 value.With thalline with aseptic THB clean 2-3 all over after.Regulating bacterial concentration with the THB medium is 10
6Individual/ml is subsequent use.
2. anesthesia of laboratory animal and muscle of back infect
The raising method of zebra fish is raised by the raising method of general tropical fish.MS-222 (MS-222) (Hangzhou animal drug factory) with concentration 95 μ g/mL is anaesthetized zebra fish.The bacterium dosage that carries out preliminary experiment affirmation inoculation definite according to the laboratory report.Back inoculation 5 * 10
4The bacterial number of/fish, control group injection THB medium infects back 24h, and aseptic position of taking the back to infect is fixed in muscle of back in 2% the paraformaldehyde or in the liquid nitrogen.
3. the evaluation of animal model index of correlation
(1) preparation of frozen section
The big or small speed that the zebra fish muscle of back tissue for preparing is cut into about 24 * 24 * 2mm is put on the freezing stage, and is freezing.Mix up the thickness of desiring to cut, cut into slices.
(2) conventional H E staining procedure
Frozen section is fixed; (10～30s)--washing slightly; (1～2s)--the dyeing of bush seminal fluid; (30～60s)--flowing water flush away bush seminal fluid; (5～10s)--1% acidic alcohol; (1～3s)--washing slightly; (1～2s)--short blue liquid returns indigo plant; (5～10s)--the flowing water flushing; (15～30s)--the dyeing of 0.5% eosin liquid; (30～60s)--distilled water is washed slightly; (1～2s)--80% ethanol; (1～2s)--95% ethanol; (1～2s)--absolute ethyl alcohol; (1～2s)--carboxylol; (2～3s)--xylol; (I); (2～3s)--xylol; (II); (2～3s)--the neutral gum sealing.
(3) conventional Gram
The frozen section for preparing is carried out conventional Gram.Gram-negative bacteria takes on a red color, and gram-positive bacteria is possessed original purple.
(4) scanning electron microscopic observation
The aseptic zebra fish muscle of back tissue of taking is put in fixedly 6h of 4% glutaraldehyde; 0.1M phosphate buffer cleans 3 times, each 10min; 2% osmic acid is fixed to sample deceives thoroughly, cleans 3 times each 10min with the 0.1M phosphate buffer; Use 30%, 50%, 70% and 90% ethanol dehydration 15min more successively; Use 100% ethanol dehydration twice at last, each 15min; Put into dry basket, carry out drying with the critical point drying appearance; Take out drying sample, be bonded on the sample stage with conducting resinl, ion sputtering instrument carry out metal spraying handle (electric current 15mA, 2min).Regulate observation condition on demand, under ESEM, electromicroscopic photograph is observed and taken to the sample of handling well.
4. the evaluation result set up of model
(1) naked eyes analysis
The all visible muscle of back of all microbionation groups has necrosis region and diffusate is arranged, and shows and infects sign.Blank is formed face and is cleaned relatively, does not almost have and oozes out.
(2) HE dyeing optics microscopically is analyzed
Show that by Fig. 1 tangible colonization appears in Streptococcus suis in zebra fish muscular tissue.
A: the zebra fish muscle group is cut HE dyeing B: zebra fish muscle crosscut HE dyeing
(3) Gram result
Show that by Fig. 2 the phenomenon that tangible bacterium gathers a cluster appears in Streptococcus suis in zebra fish muscular tissue, the visible bacterium colony that obviously forms biofilm.
(4) scanning electron microscope analysis
Can be known that by Fig. 3 the surface of a wound that ESEM is presented at infection has the film spline structure that is similar to " biofilm " appearance of polysaccharide parcel, is wrapped to form cell cluster to bacterium, the film that is wrapped to form is different from host's cell membrane itself, and thickness is thicker than cell wall.
Claims (7)
1. the construction method of bacterial biofilm infected animal model in the body is characterized in that: may further comprise the steps,
(1) preparation of bacterial suspension;
(2) anesthesia of laboratory animal and muscle of back infect;
(3) identify the animal model index of correlation.
2. the construction method of bacterial biofilm infected animal model in the body according to claim 1; It is characterized in that: bacterial suspension is obtained by following method in the step (1): the bacterium of separating that is used to connect that preserves under-80 ℃ of temperature of general is drawn Colombia's blood plate, chooses single bacterium colony incubated overnight under 37 ℃ the temperature in medium; Get bacterium liquid then and add the THB medium, 37 ℃ of shaking tables continue overnight incubation, after plate coating checking is pure culture, shakes bacterium with inoculation in 1: 100 and cultivate the thalline that obtained exponential phase in 4-6 hour, and measuring the OD600 value is between the 08-1.0; With thalline clean 2-3 all over after; Regulating bacterial concentration is 10
6Individual/ml is subsequent use.
3. the construction method of bacterial biofilm infected animal model in the body according to claim 2, it is characterized in that: the said bacterium that is used to inoculate is the Streptococcus suis clinical separation strain.
4. according to the construction method of bacterial biofilm infected animal model in arbitrary described body among the claim 1-3, it is characterized in that: described laboratory animal is the pure lines zebra fishs.
5. according to the construction method of bacterial biofilm infected animal model in arbitrary described body among the claim 1-3, it is characterized in that: anesthesia of laboratory animal and muscle of back infect for the MS-222 (MS-222) of concentration 95 μ g/mL zebra fish is anaesthetized; Back inoculation 5 * 10
4The bacterial number of/fish, control group injection THB medium infects back 24h, and aseptic position of taking the back to infect is fixed in muscle of back in 2% the paraformaldehyde.
6. the construction method of bacterial biofilm infected animal model in the body according to claim 1 is characterized in that: after infecting 24 hours, the zebra fish of said experimental technique is put to death respectively, the zebra fish index of correlation is identified.
7. the construction method of bacterial biofilm infected animal model in the body according to claim 1; It is characterized in that: described evaluation comprises the muscle of back formaldehyde fixed is prepared histologic section, carries out HE dyeing, Gram and ESEM (SEM) and observes.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113189315A (en) * | 2021-04-13 | 2021-07-30 | 山东省医疗器械产品质量检验中心 | In-vitro dynamic model for evaluating antibacterial activity of antibacterial catheter and using method thereof |
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| CN1829732A (en) * | 2003-05-27 | 2006-09-06 | 抗癌公司 | Imageable animal model of SARS infection |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113189315A (en) * | 2021-04-13 | 2021-07-30 | 山东省医疗器械产品质量检验中心 | In-vitro dynamic model for evaluating antibacterial activity of antibacterial catheter and using method thereof |
| CN113189315B (en) * | 2021-04-13 | 2024-01-23 | 山东省医疗器械产品质量检验中心 | In vitro dynamic model for evaluating antibacterial activity of antibacterial catheter and application method thereof |
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