CN104189020B - Construction method of post-transplantant candida albicans pulmonary infection mouse model - Google Patents
Construction method of post-transplantant candida albicans pulmonary infection mouse model Download PDFInfo
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Abstract
The invention relates to the technical field of preparation of animal models, and specifically discloses a construction method of a post-transplantant candida albicans pulmonary infection mouse model. The construction method comprises the following steps: selecting a male C57BL/6J mouse at the age of six to eight weeks as a donator, a male Balb/c mouse at the age of six to eight weeks as a receptor, and ciclosporin A as an immunosuppressor to construct a mouse skin transplantation model; and dropping 200 mu l of 5*10<5> CFU/ml candida albicans suspension into the body of the mouse suffering from lower back skin transplantation operation 7 days after the skin transplantation operation to obtain the post-transplantant candida albicans pulmonary infection mouse model. According to the construction method of the post-transplantant candida albicans pulmonary infection mouse model, the prepared mouse model is closer to the actual occurrence condition of post-transplantant fungal infection of a clinical patient; in addition, the model is stable; moreover, the method is simple and fast; a large number of stable mouse infection models can be obtained at a time for researchers to use in relevant study on clinical problems.
Description
Technical field
The present invention relates to animal model preparing technical field, specifically discloses the candida albicans bacterium infection of post-transplantation pulmonary
The construction method of mouse model.
Background technology
Infection, rejection and cardiovascular and cerebrovascular disease are the main causes for currently affecting transplant recipient prognosis.Organ transplantation
Patient because operating time length, intraoperative hemorrhage it is many, it is postoperative take the factors such as immunosuppressant, cause postoperative patient hypoimmunity,
Infection rate is high, has had a strong impact on long-term survival rate and the quality of life of patient.
Transplant recipient immunity of organisms has significantly different from general population, its pattern of infection, infection character, clinical table
There is larger difference in existing, diagnosis and treatment, it is concurrent that these factors greatly restrict China organ graft recipient infection
The therapeutic effect of disease.It would therefore be desirable to the animal infection modal of corresponding strain is set up, it is concurrent so as to studying transplant recipient infection
The early diagnosiss technology of disease and its prevention and controls, are that solid foundation is established in the prognosis for further improving transplant recipient.
Study according to experts such as Yang Chunhua, the fungal infection rate of patients with liver transplantation is 13.5% (120/886), and
Its mortality rate is up to 70.8%.It can be seen that, the diagnosis of organ transplantation postoperative patient person's fungal infection, treatment, preventative strategies can research work have
Extremely important practical significance.Candida albicans bacterium is the common opportunistic fungus of human body, about 2/3rds organ
Transplant recipient fungal infection is induced by it, and its topmost infection site is pulmonary（Account for 73.3%）.Therefore, organ shifting is set up
The animal model of postoperative pulmonary's candida albicans bacterium infection is planted, correlational study is most important for carrying out.
Mice is the model animal of the research human diseasess of current comparative maturity.MOUSE REPRODUCTION is fast, and genotype is clear and definite, filial generation
It is many.The mankind are quite ripe to the research of mouse genome, and its genome and the mankind 90% are homologous, and its Physiology and biochemistry and
Growth course is similar with the mankind, so the mouse model of human diseasess substantially can truly simulate the morbidity of human diseasess
Journey.Therefore, select mice that there is irreplaceable advantage as the model organism for setting up the model.
At present, the route of infection that common candida albicans bacterium mouse nuclei is adopted is for tail vein injection, genitals
Injection and gavage.Although such infection method is ripe, its common infection approach and infection with organ transplantation postoperative patient person
There is obvious difference at position so that the infection model can not simulation clinical infection patient very well.Different routes of infection, machine
Body participates in the cell mass of immunne response and has differences, and which has limited the clinical conversion of above-mentioned infection model experimental result.Additionally, existing
The bacterium amount that connects for having animal infection modal is remained necessarily disputing on, and connects that bacterium amount is too little, and infection model is not sufficiently stable, and it is too many to connect bacterium amount,
A situation arises does not meet with clinical practice infection, therefore, bacterium amount is connect for animal infection modal, in addition it is also necessary to further grope and
Balance.
Additionally, normal adult mice immunity of organisms is stronger, it is more difficult to infect candida albicans bacterium, therefore, set up corresponding
Mouse nuclei need to use immunosuppressant, and the immunosuppressant for being usually used in modeling at present be cyclophosphamide and can
Pine.But, cyclophosphamide will be caused little as the most frequently used alkylating agents antitumor drug, the side effect with bone marrow depression
Mus number of white blood cells is remarkably decreased.And cortisone acetate is used as adrenal cortex hormones drug, because its side effect it is more, mesh
Front clinic is less to be individually used for antirejection therapy.Therefore, the mice sense set up as immunosuppressant using this two classes medicine
Dye model has a certain distance with clinical practice.Therefore, the research direction needs foundation one kind and the more adjunction of clinical practice situation badly
Near mouse nuclei, further to promote the development of transplant recipient infectious disease correlational study.
The content of the invention
The technical problem to be solved in the present invention be in order to overcome prior art in candida albicans bacterium infecting mouse model not
It is too close to clinical practice infection conditions, do not consider the defect of transplanting factor etc., there is provided a kind of post-transplantation pulmonary candida albicans
The construction method of bacterium infecting mouse model.
The purpose of the present invention is achieved by the following technical programs：
The construction method of post-transplantation pulmonary candida albicans bacterium infecting mouse model, comprises the steps：
S1. from the male C 57 BL/6 J mouse of 6 ~ 8 weeks as donor, the male Balb/c mices of 6 ~ 8 weeks as receptor,
Using ciclosporin A as immunosuppressant, skin transplantation mouse model is built；
S2. by the 1 × 10 of 200ul by way of the instillation of nostril5~1×106CFU/ml candida albicans bacteria suspensions are added dropwise to
In the skin transplantation mice body that step S1 is successfully constructed, post-transplantation pulmonary candida albicans bacterium infecting mouse model is obtained.
There is certain difference in different lines, week old, the immune response of sex mice, it is for same graft
Also there is difference the repulsion time.Therefore, we set up the mouse nuclei, need ensure graft can survive suitable one section when
Between so that we have the sufficient time to carry out correlational study.Therefore, we are from 6-8 all male C 57 BL/6 J mouse conducts
Donor, the male Balb/c mices of 6 ~ 8 weeks are used as receptor.
Setting up corresponding mouse nuclei needs to use immunosuppressant, and is usually used in the immunosuppressant for modeling at present
For cyclophosphamide and cortisone, and the present invention selects use ciclosporin A as immunosuppressant, and it belongs to calcium and adjusts neuroprotein to press down
Preparation, by suppressing lymphopoiesis and various lymphokines to produce Selective depression immunne response is realized, main to suppress T to drench
Bar cell, has significant inhibitory action to cell immune response, also has certain inhibitory action to humoral immune reaction, can prevent
The only generation of rejection, while also suppressing body to resist and remove function to pathogen to a certain extent.Its advantage is
Small side effects, closer to clinical practice.The consumption of the ciclosporin A is calculated according to the body weight of mice, gives the milli of mice 15 ~ 30
G kg/day, till mouse skin repulsion or experiment terminate.
Different according to the research purpose or technical characterstic of research worker, the present invention can adopt lower back skin transplantation, tail
Portion's skin transplantation or ear skin are transplanted to build mouse model.But the donor dermal of different parts, what its rejection occurred
Time, the technological difficulties of operation technique etc. are not consistent.
Carry out transplant recipient infectious disease correlational study, it is desirable to be able to obtain sufficient amount of infection in the same time dynamic
Thing model, to reduce the interference that animal batch, time, body weight, state etc. are caused to experiment as far as possible.Accordingly, as preferred behaviour
Make, we are selected based on Mouse Lumbar Back skin grafting model, and this model has advantages below：1st, operating theater instruments, condition
Require simple, it is easy to carry out in each research center；2nd, operation technique is relatively easy, and operating time is short, is easy to obtain at short notice
More animal model；3rd, postoperative mice recovers fast, and graft survival situation is easy to observation；4th, 1 can only for body C57BL/6J mices
So that skin gives 6 ~ 9 Balb/c mices, the quantity of laboratory animal is saved, relatively reduced the individual variation of postoperative mice.
Preferably, when nostril described in S2 instills, simultaneously funguses suspension can not be instilled to both sides nostril, in order to avoid cause mice to stop up
Breath, additionally, funguses suspension is not instilled into mice lip and oral cavity.
The access amount of the funguses suspension is too many, and the lung volume of mice is inadequate, it is impossible to all instill, and the behaviour for instilling
Make process time oversize, mice easily moves, cause liquid to drip to position around.Access amount very little, needs to connect the concentration of bacterium solution just
Higher, the error that operation brings may be bigger, because often losing 1ul, the bacterium amount that it is contained within is more.In addition, accessing true
The less words of bacterium amount, it is possible to which model is unstable；Measure too greatly, but it is larger with clinical practice gap.It is therefore preferred that institute
The concentration for stating candida albicans bacteria suspension should be 1 × 105~1×106；Most preferably, candida albicans bacteria suspension concentration described in S2
For 5 × 105CFU/ml。
The invention has the beneficial effects as follows：
The present invention makes candida albicans bacterium infecting mouse pulmonary in the way of nostril instills, and the infection model for obtaining is closer
The practical situation of post-transplantation fungal infection, and model stability, disclosure satisfy that the needs that related science research is carried out.In addition,
The present invention selects ciclosporin A as immunosuppressant with growing with each passing hour in the selection of immunosuppressant, and its advantage is more can be true
The immune state of clinical infection patient is simulated on the spot, makes achievement in research be relatively easy to realize clinical conversion.
The present invention is using the postoperative pulmonary's candida albicans bacterium infecting mouse model of skin transplantation as transplant recipient infectivity disease
The basis that sick correlational study is carried out, can meet the demand that sufficient amount of infected animal model is obtained within the same time, with
The interference that animal batch, time, body weight, state etc. are caused to experiment is reduced as far as possible.
Description of the drawings
Fig. 1. donor skin graft picks up operation.
Fig. 2. donor skin graft shearing manipulation.
Fig. 3. the donor skin graft cut processes operation.
Fig. 4. receptor skin bed is sterilized.
Fig. 5. determine receptor skin bed region.
Fig. 6. shearing receptor skin a line circle.
Fig. 7. its excess-three bar border of shearing receptor skin.
Fig. 8. transplanting skin closure.
Fig. 9. transplanting skin wrapping.
Figure 10. postoperative 4th day situation.
Figure 11. postoperative rejection judges.
Figure 12. postoperative 7th day, the 21st day situation.
Figure 13. Mouse Weight changes.
Figure 14. leukocyte ratio change after toxin expelling.
Figure 15. neutrophilic granulocyte ratio change after toxin expelling.
Figure 16. percentage of lymphocyte change after toxin expelling.
Figure 17. mononuclear cell ratio change after toxin expelling.
Figure 18. eosinophilic granulocyte's ratio change after toxin expelling.
Figure 19. basophilic granulocyte ratio change after toxin expelling.
Figure 20. pulmonary's candida albicans bacterium DNA copy number change.
Figure 21. the candida albicans bacterium DNA copy number change of kidney portion.
Figure 22. lung tissue carries bacterium amount change.
Figure 23. nephridial tissue carries bacterium amount change.
Figure 24. 5 × 103CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 1st day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 25. 5 × 103CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 3rd day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 26. 5 × 104CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 1st day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 27. 5 × 104CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 3rd day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 28. 5 × 105CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 1st day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 29. 5 × 105CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 3rd day after poison（HE is dyeed, upper left：5
Times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 30. 5 × 105CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 14th day after poison（HE is dyeed, upper left：
5 times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 31. 5 × 105CFU/ml concentration group mice nasal cavities connect lung pathologies change in the 14th day after poison（Silver hexamine staining,
Upper left：5 times, upper right：10 times, lower-left：20 times, bottom right：40 times）.
Figure 32. each group murine skin graft survival curve.
Specific embodiment
The present invention is further described with reference to Figure of description and specific embodiment.Unless stated otherwise, this
The bright reagent for adopting, equipment are for the art conventional reagent and equipment.
Embodiment 1
First, the foundation of Mouse Lumbar Back skin grafting model
Below operation needs to be carried out in SPF level environment, and related experiment articles for use need Jing corresponding programs（High Temperature High Pressure, spray 75%
Ethanol and ultraviolet irradiate）Barrier environment is entered after process, laboratory animal need to shift to an earlier date performing check quarantine, treat that it adapts to experimental situation
After can use.
1st, article prepares：Eye scissorss, Smooth forceps, curved tweezer, slide gauge, marker pen, needle holder, glass dish, 10ml notes
Emitter, 1ml syringes, shaver, small size angular pin, silk thread, vaseline gauze, aseptic yarn, medical bandage, medical proof fabric, electronics day
Flat, 15ml sterile centrifugation tubes.
2nd, medicine prepares：Anesthetics, 75% medical alcohol, iodine tincture, physiological saline solution, ciclosporin A.
3rd, mice selects：From the male C 57 BL/6 J mouses of 6 ~ 8 weeks as donor, the male Balb/c mices of 6 ~ 8 weeks
As receptor.
4th, donor skin graft is prepared：
（1）With shaver preserved skin after death, is used at cervical dislocation method, scope is double between upper limb, double lower limb to donor Mus
Skin of back.Preserved skin scope is as far as possible big, and hair is cut short as far as possible, and althouging note that should not stave skin of back, uncomfortable if there is the person of scratching
In as skin grafts；
（2）With iodine tincture, 75% alcohol disinfecting skin donor site, sterilization scope should be greater than cropping scope, and dry confession with sterile gauze
The disinfectant solution in dermatotome；
（3）Level grips Smooth forceps, is picked up the skin level between homonymy upper and lower extremities using it, notes making partial skin
Smooth forceps upper limb is uniformly protruded from, is cut off the uniformly prominent skin of Smooth forceps upper limb with tissue shear, Fig. 1 is shown in concrete operations；
（4）The skin between opposite side upper and lower extremities, double upper limb, double lower limb is cut off with same method, at the edge of skin donor site
The incisxal edge of " mouth " word is formed, then one of corner is picked up with Smooth forceps, with eye scissorss its subcutaneous tissue, fascia are cut off,
It is careful not to damage and supplies skin, the confession skin for separating is put in the cut-and-dried plate containing physiological saline solution, concrete behaviour
Work is shown in Fig. 2；
（5）By the bulk skin of back cut upset, clamp for skin one jiao with Smooth forceps and fix, with curved tweezer by subcutaneous fat
Fat, connective tissue folder are removed, it is desirable to shave clean as far as possible, and the survival of skin graft, is typically seen with naked eyes after otherwise will affecting to transplant
The fat of noresidue, blood vessel, fascia are defined on donor skin graft, and the physiological saline solution in plate, concrete operations are changed if necessary
See Fig. 3；
（6）Donor dermal is cut into into the square skin graft of 0.8cm × 0.8cm sizes, is placed in flat containing physiological saline solution
It is standby in ware.
5th, receptor skin bed is prepared：
（1）Receptor Mus use anesthetics lumbar injection after weighing, after anesthesia success, lower back cropping on the right side of it with shaver
Preserved skin, scope about 2cm × 2cm；
（2）With iodine tincture, 75% alcohol disinfecting preserved skin area, sterilization scope should be greater than cropping scope, and be dried with sterile gauze standby
Fig. 4 is shown in the disinfectant solution in dermatotome, concrete operations；
（3）With slide gauge and marker pen in the predetermined square Pi Chuan areas of preserved skin area internal labeling, size about 1cm × 1cm,
Pi Chuan areas close proximity to back being advisable（It is easy to wrapping to fix）, less than double upper limb lines, inner side circle is or not the Dan Pichuan areas upper bound
More than dorsal midline, lateral border is less than right side posterior axillary line, and Fig. 5 is shown in concrete operations；
（4）Level grips Smooth forceps, and using its level the lateral boundaries skin of Pi Chuan areas one is picked up, and notes making partial skin uniform
Smooth forceps upper limb is protruded from, is cut off the shallow skin of the table for uniformly protruding from Smooth forceps upper limb with tissue shear, made sure to keep in mind to cut skin and want table shallow,
Typically it is advisable with cutting skin at 1mm under the skin edge of Smooth forceps upper limb skin, Fig. 6 is shown in concrete operations；
（5）The skin on its excess-three bar border of Pi Chuan areas is cut off with same method, in skin bed area edge " mouth " word is formed
Incisxal edge, then grip one of them corner shallow compared with table of Pi Chuan areas with tweezers, tweezers are assisted subcutaneous connective tissue with another
Peel off, note not damaging subcutaneous thin vessels（Never subcutaneous tissue is cut using shears, otherwise easy injured blood vessel）, it is seen that it is subcutaneous
Fig. 7 is shown in clearly vascular bed, concrete operations.
6th, skin transplantation and wrapping：
（1）The donor skin graft for preparing is placed in into Pi Chuan areas middle part against hair direction, four angles of skin graft with small size angular pins and
Whether silk thread respectively fix by one pin of seam, and note for receptor skin to connecting, and whether there is skin overlap or gap is excessive, and gives corresponding
Process；Fig. 8 is shown in concrete operations.
（2）One layer of 1.5cm × 1.5cm vaseline gauze is covered above skin grafts, then adds a cover one layer of sterile gauze, use one
Finger slightly presses fixed sterile gauze, and mice rostral is lifted so as to which the organ major part such as gastrointestinal hangs down to hypogastric region, Ran Houyong
Medicinal elastic bandage is around a circle, and pressure dressing is elastic appropriate（Neither to affect mice cardio-pulmonary function, it is unlikely to easily de- again
Fall to being advisable）, finally again with adhesive plaster around a circle fixation, Fig. 9 is shown in concrete operations.
（3）The mice supine position for bandaging is recovered on electric blanket or in calorstat, is typically advisable with 37 DEG C, treat it
After independently can standing up as ventricumbent position, it is transferred in clean mouse cage.7th, injecting immune inhibitor：Mice from skin transplantation day,
Daily lumbar injection ciclosporin A（CsA；Novartis Pharma Ltd；Germany；30mg/kg/day), until mouse skin
Repel or test till terminating, note before intraperitoneal injection of drugs with 75% alcohol disinfecting injection site skin.
8th, dressing and observation are removed：
（1）The active situation for noting observation mice in postoperative 24 hours, if mice occur eyes close, inertia, tail it is strong
It is straight to wait the uncomfortable performance of pain, give adjustment binder elasticity, lumbar injection analgesic drug product etc. and process, if mice binder comes off,
Should again sterilize and art mouth and wrap up again, otherwise not transplant skin easily because of new vesselses not and supply, moisture evaporation are excessive and dry and hard；
（2）Skin transplantation is postoperative 48 hours, removes mice art mouth dressing, if dressing has adhesion with art mouth, should not firmly tear
It is de-, after its art mouth healing, voluntarily can come off together with crust；
（3）Skin graft situation is observed before daily injecting immune inhibitor：Art mouth whether there is healing, and graft whether there is dry
Tie, come off, repel, whether there is new piliation etc.；The situation of postoperative 4th day is shown in Figure 10.
（4）Skin graft repels criterion：A:Color is in black；B:Cicatrix forms a scab；C:Necrosis comes off；D:Skin is moved
Plant is less than the 20% of raw hide piece.Postoperative rejection judges to see Figure 11.
（5）Post-transplantation 5 ~ 7 days, mice art mouth heals substantially, can reject operative failure（Because skin bed vascular injury is serious or
Donor skin graft still has fat, fascia etc. not to reject totally）The dry and hard person of coming off of transplanting skin is caused, the successful mice of skin transplantation is chosen
Proceeding to infectious laboratory carries out infection experiment.Postoperative 7th day, the situation of the 21st day see Figure 12.
2nd, the foundation of pulmonary's candida albicans bacterium infection model
The skin transplantation mice that above-mentioned steps one are successfully prepared proceeds to infectious laboratory, sets up post-transplantation pulmonary
Candida albicans bacterium infecting mouse model.Below operation is needed in infectious laboratory（P3）Inside carry out, concrete operations should be corresponding
Carry out in the Biohazard Safety Equipment of rank, related experiment articles for use need Jing corresponding programs in advance（High Temperature High Pressure, 75% ethanol of spray and purple
Outside line is irradiated）Barrier environment is entered after process, laboratory animal need to be proceeded in advance in corresponding isolation cage tool.The bacterium of fungi preservation liquid
Amount needs to determine in advance, is inoculated with first 2 hours and recovers, and makees gradient dilution with physiological saline solution, and concrete bacterium amount is determined, strain is multiple
Soviet Union and gradient method for resuscitation please investigate pertinent texts, document.
1st, apparatus and articles for use prepare：Anesthetics, 1ml syringes, 75% medical alcohol, 200ul pipette tips, 200ul pipettors, 5
×103CFU/ml、5×104CFU/ml、5×105CFU/ml candida albicans bacteria suspensions.
2nd, mouse weights, use anesthetics lumbar injection behind routine disinfection injection site, stand-by after it stands up areflexia；
3rd, test packet：The successful mice of skin transplantation that 20 above-mentioned steps one are prepared is taken, 5 one group, is divided into
Four groups, four groups are respectively 5 × 103CFU/ml concentration groups, 5 × 104CFU/ml concentration groups, 5 × 105CFU/ml concentration group and blank
Matched group（Blank control group is instilled to draw 200ul physiological saline solution by mice nostril）.Drawn respectively with pipettor
The 5 × 10 of 200ul3CFU/ml、5×104CFU/ml、5×105CFU/ml candida albicans bacteria suspensions（Note blowing before imbibition every time
Beat and shake up suspension）, it is in dorsal positions that left hand fixes mice, and the right hand is held pipettor, pipette tips gently leaned against on the nostril of mice side, is seen
Examine mice and whether there is and hide reaction, if any, then need to repeat operation after wait several minutes, or plus use anesthetics；
4th, mouse breathing frequency and rhythm are observed, when mice air-breathing, presses pipettor discharge part liquid（About 50ul）,
Observation liquid enters nostril with mice air-breathing, notices whether mice discharges nostril when exhaling by liquid, if without drain
Or bubble, then repeatedly aforesaid operations, until all suspensions instill mice nostril, if having drain or bubble, with original
Pipettor sucks back liquid, repetitive operation after waiting a moment, and notes that liquid can not be instilled to both sides nostril simultaneously, in order to avoid cause mice
Asphyxia, additionally, bacterium solution is not instilled into mice lip and oral cavity；
5th, keep mice head height tail low 30 ~ 60 seconds, by mice supine position in rewarming platform, be transferred to after its revival corresponding
In cage tool.
6th, model testing and relevant parameter：This experimental group is being groped to be provided with 5 × 10 during connecing bacterium amount3CFU/ml、5
×104CFU/ml、5×105Tri- concentration groups of CFU/ml, have monitored the change that each group Mouse Weight, peripheral blood are counted, and are connecing
Particular point in time collection mouse lung, kidney, peripheral blood are accordingly detected after bacterium, including Q-PCR, load bacterium amount and pathological section, together
When, we go back the time-to-live that comparative observation connects bacterium group and blank control group murine skin graft.The concrete method of sampling and
Related experiment Technical Reference the art conventional method.
3rd, experimental result
1st, blank control group and 5 × 105CFU/ml group Mouse Weight situations of change as shown in table 1 and Figure 13, * generations in Figure 13
Two groups of Mouse Weight t inspection p values of table are less than 0.05, illustrate that the difference of two groups of Mouse Weights is statistically significant.
The Mouse Weight of table 1. changes
From table 1 and Figure 13, mice is wrapped up after surgery in 48 hours of dressing, and weight loss is more apparent.May be with
Operation wound stimulates, dressing parcel limits mice feed, cause the reasons such as diarrhea of mouse relevant using immunosuppressant for the first time.With
That mice dressing dismounting, art mouth are progressively healed, mice is gradually adapted to immunosuppressant, mice starts normally to take food and drink water,
Body weight is progressively recovered.5×105After nasal cavity instills candida albicans bacterium, body weight increase is subject to obvious CFU/ml concentration group mices
Affect, body weight lags behind blank control group.
2nd, mouse peripheral blood change：The change that peripheral blood is counted mainly is determined and connects leukocyte after poison, neutrophilic granulocyte, pouring
The change of bar cell, mononuclear cell, eosinophilic granulocyte and basophilic granulocyte.Measurement result is shown in respectively Figure 14 ~ 19.Figure 14 ~
* represents two groups of mouse peripheral blood various types of cells t inspection p values less than 0.05 in 19, illustrates two groups of mouse peripheral blood various types of cells
Difference is statistically significant.
As seen from Figure 14,5 × 105CFU/ml concentration group mices connect after poison peripheral white blood cell mesh compared with blank
Group is significantly raised, wherein, the peripheral white blood cell mesh difference of the 1st, 4,14 days two groups of mices is statistically significant.
Observe the result of two groups of mouse peripheral blood differential blood counts（Figure 15 ~ 19）, it is seen that the 4th day after poison is connect,
Two groups of mouse peripheral blood mononuclear cell proportional differences are statistically significant, and 5 × 105CFU/ml concentration group mouse peripheral blood monokaryons are thin
Born of the same parents' ratio is significantly raised compared with blank control group, points out, in infection early stage, mainly to cause the peripheral blood mononuclear cell proliferation of mice, leads to
Cross phagocytosiss and remove candida albicans bacterium.
The 7th day after poison is connect, two groups of mouse peripheral blood neutrophilic granulocyte proportional differences are statistically significant, 5 ×
105CFU/ml concentration group mouse peripheral blood neutrophilic granulocyte ratios are significantly raised compared with blank control group, point out in infection mid-term, main
The peripheral blood neutrophil for causing mice is bred, and by phagocytosiss and release inflammatory mediator candida albicans bacterium is removed.
The 14th day after poison is connect, two groups of mouse peripheral blood eosinophilic granulocyte's proportional differences are statistically significant, 5 ×
105CFU/ml concentration group mouse peripheral blood eosinophilic granulocyte's ratios are significantly raised compared with blank control group, point out the phase after infection,
The periphery blood eosinophils for mainly causing mice breed, and this phenomenon may cause respiratory tract with the antigen of candida albicans bacterium
Local anaphylaxises are relevant.
3rd, each group mice Q-PCR results：Candida albicans bacterium DNA copy number in the lung and nephridial tissue of different disposal group
Change test result is shown in Figure 20 ~ 21.ND represents the sample detection that the concentration group time point is not carried out in test.
From Figure 20 ~ 21, the 1st, 4,7,14 days after nasal cavity instills candida albicans bacterium of three concentration groups exist respectively
Candida albicans bacterium DNA can be detected in lung and nephridial tissue.5×105The each time point lung of CFU/ml concentration group mices and kidney group
Candida albicans bacterium DNA copy number in knitting is high compared with remaining two groups.Detection lung and the reason for nephridial tissue it is：Pulmonary is the animal mould
The target infection organ of type, detects candida albicans bacterium DNA, and the inoculation method that prompting nasal cavity is instilled can actually infecting mouse
Lung tissue.It is the situation for entering blood if there is funguses and nephridial tissue is the target organ of candida albicans bacterium（Also known as bacterium blood
Disease）, then funguses can be through blood circulation, in being gathered in nephridial tissue, and it is different degrees of that the result points out each concentration group to may occur in which
Bacteremia.
4th, each group mice carries bacterium amount：Lung tissue, the load bacterium amount situation of change of nephridial tissue are determined respectively, and test result is shown in Table
2nd, table 3 and Figure 22 ~ 23.
The lung tissue of table 2 carries bacterium amount change
The nephridial tissue of table 3. carries bacterium amount change
The each time point blank space of each concentration group represents and can't detect that is, tissue culture does not go out candida albicans in Figure 22 ~ 23
Bacterium.By Figure 22 ~ 23 as can be seen that 5 × 105CFU/ml concentration groups nasal cavity is instilled the 1st, 3,7,14 days after candida albicans bacterium, lung
Tissue can turn out candida albicans bacterium bacterium colony, wherein, the clump count for meeting the 3rd day tissue culture after bacterium is most.And remaining
The lung tissue culture of two concentration groups is without candida albicans bacterium colony growth.
5×105CFU/ml concentration groups nasal cavity is instilled the 3rd, 7,14 days after candida albicans bacterium, and some animals nephridial tissue can be with
Candida albicans bacterium bacterium colony is turned out, wherein, the clump count for meeting the 3rd day tissue culture after bacterium is relatively more.And other two is dense
The nephridial tissue culture of degree group is without candida albicans bacterium colony growth.
5th, histopathology result：Test result is shown in Figure 24 ~ 31.From Figure 24 ~ 31,5 × 103CFU/ml、5×
104CFU/ml、5×105CFU/ml concentration group mice nasal cavities connect lung tissue after poison occur different degrees of hyperemia, ooze out, inflammation it is thin
Born of the same parents infiltrate.In same concentration group, the lesion degree that mice nasal cavity connects the 3rd day lung tissue after bacterium is serious compared with the 1st day, and extent of disease is more
Greatly.In variable concentrations group, 5 × 105The group mouse lung tissue pathological change of CFU/ml concentration is obvious compared with remaining two groups.
Additionally, 5 × 105CFU/ml concentration group mice nasal cavities are connect the 14th day after bacterium, and white vacation can be seen in lung tissue segment
Silk yeast mycelial growth（As shown in green box under silver hexamine staining group Figure 40 times mirror）.
Mice immunity of organisms is strong compared with the mankind, and pulmonary is studded with many phagocyte, and local resistance is strong, and, lungs
For gassiness hollow organ, organize slim, therefore, candida albicans bacterium is difficult to grow a large amount of mycelia in lung tissue.Nephridial tissue
Turn out candida albicans bacterium bacterium colony less, therefore corresponding histopathology image results are not provided.
6th, skin graft survival curve：Test result is shown in Figure 32.Known by Figure 32, nonimmune suppression group mice goes out earliest
Existing skin graft repels, skin graft survival median 15.10 ± 1.70 days；There is the latest skin in immunosuppressant group mice
Transplant rejection, skin graft survival median 24.10 ± 6.99 days；5×105CFU/ml concentration group skin graft survivals
Time occupy between the two, median 21.90 ± 5.65 days.Can be drawn by Log-rank (Mantel-Cox) inspections, immunity
Suppression group, 5 × 105CFU/ml concentration group murine skin graft time-to-live more nonimmune suppression group leader, its skin transplantation
Thing time-to-live difference statistically significant (p=0.0002, p=0.0010).And immunosuppressant group and 5 × 105CFU/ml is dense
Degree group murine skin graft time-to-live difference is not statistically significant (p=0.3027).
Show based on the above results：
Although 5 × 103CFU/ml concentration group and 5 × 104CFU/ml concentration group mices nasal cavity is instilled the after candida albicans bacterium
1st, 4,7 days, lung and nephridial tissue can detect that the candida albicans bacterium DNA of certain copy number, but its lung and nephridial tissue culture without
Candida albicans bacteria growing, pathological change is relatively less obvious, therefore, this two groups candida albicans bacterium senses for connecing bacteria concentration foundation
Dye model lacks necessary infection evidence and supports, relatively unstable.In this two groups, candida albicans bacterium spore is dripped by nasal cavity
After entering, most of spore is killed in lung tissue by the phagocytosis of local phagocyte；Fraction spore is entered after blood circulation in kidney
Dirty aggregation, is killed by the phagocyte phagocytosis in circulation；Additionally, the candida albicans bacterium DNA fragmentation after partial devitalization can pass through
Blood circulation, in being enriched in renal tissue.Therefore, we can necessarily be copied with detecting in two groups of mouse lungs of here and nephridial tissue
Several candida albicans bacterium DNA, but candida albicans bacterium bacterium colony cannot be turned out.
By Q-PCR, tissue culture and histopathologic examination, it can be found that proving 5 × 105CFU/ml concentration is set up vertical
Mouse nuclei infection evidence it is definite, point out this to connect the infection model of bacteria concentration foundation more stable.
Additionally, to 5 × 105CFU/ml concentration set up vertical mouse nuclei further studied it is found that
This group of mice is connect after bacterium, and body weight increase is significantly affected, and is shown as body weight increase and is limited, hence it is evident that lags behind blank control group.
Meanwhile, the prompting of mouse peripheral blood count results, it is significantly raised that this group of mice connects peripheral white blood cell mesh after poison.
Connect the 4th day after poison, 5 × 105CFU/ml concentration group mouse peripheral blood mononuclear cell ratios are significantly raised compared with blank control group, carry
Show that, in infection early stage, funguses mainly cause the peripheral blood mononuclear cell proliferation of mice, by phagocytosiss candida albicans are removed
Bacterium.
The 7th day after poison is connect, 5 × 105CFU/ml concentration group mouse peripheral blood neutrophilic granulocyte ratios are compared with blank
Group is significantly raised, points out in infection mid-term, and funguses mainly cause the peripheral blood neutrophil of mice to breed, by phagocytosiss
And release inflammatory mediator removes candida albicans bacterium.
And the 14th day after poison is connect, 5 × 105CFU/ml concentration group mouse peripheral blood eosinophilic granulocyte's ratios are more blank
Matched group is significantly raised, points out phase after infection, funguses mainly to cause the periphery blood eosinophils of mice to breed, this phenomenon
With the antigen of candida albicans bacterium respiratory tract local anaphylaxises may be caused relevant.
Finally, murine skin graft survival curve prompting：Although mice connects peripheral white blood cell mesh after poison and substantially rises
Height, its skin graft survival time is compared with the relative shortening of immunosuppressant group, but both difference are not statistically significant.
Claims (5)
1. the construction method of post-transplantation pulmonary candida albicans bacterium infecting mouse model, it is characterised in that comprise the steps：
S1. from the male C 57 BL/6 J mouse of 6 ~ 8 weeks as donor, the male Balb/c mices of 6 ~ 8 weeks as receptor, with ring
Spore element A builds skin transplantation mouse model as immunosuppressant；
S2. by the 1 × 10 of 200ul by way of the instillation of nostril5~1×106CFU/ml candida albicans bacteria suspensions are added dropwise to step
In the skin transplantation mice body that rapid S1 is successfully constructed, the mouse model of post-transplantation pulmonary candida albicans bacterium infection is obtained；
When nostril described in S2 instills, simultaneously funguses suspension can not be instilled to both sides nostril, in order to avoid cause mice to suffocate, additionally, not
Funguses suspension is instilled in mice lip and oral cavity.
2. the construction method of mouse model according to claim 1, it is characterised in that the consumption of ciclosporin A described in S1 is
15 ~ 30 mg/kg/days, till mouse skin repulsion or experiment terminate.
3. the construction method of mouse model according to claim 1, it is characterised in that skin transplantation described in S1 is lower back
The transplanting of skin transplantation, tail skin grafts or ear skin.
4. the construction method of mouse model according to claim 3, it is characterised in that skin transplantation described in S1 is lower back
Skin transplantation.
5. the construction method of mouse model according to claim 1, it is characterised in that candida albicans bacteria suspension described in S2
Concentration is 5 × 105CFU/ml。
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| 免疫低下肺部感染动物模型造模方法的探讨;宋夕元等;《中医学报》;20120331;第27卷(第3期);第325-327页 * |
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