CN102727887B - Application of chitosan serving as immuno-adjuvant in preparing mouse allergic asthma model - Google Patents
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Abstract
The invention belongs to the technical field of biology, and relates to an allergic asthma model, in particular to application of chitosan serving as immuno-adjuvant in preparing a mouse allergic asthma model. According to the application, the mouse allergic asthma model prepared by applying chitosan serving as immuno-adjuvant is subjected to airway responsiveness, lung tissue pathological examination and inflammation marker inspection, and results indicate that the mouse allergic asthma model prepared by applying chitosan serving as immuno-adjuvant is remarkably superior to a conventional immuno-adjuvant prepared asthma model in airway eosinophilic inflammation, airway responsiveness and airway inflammatory cytolines and other aspects. The chitosan has low price and rich sources; and the mouse asthma model by using chitosan as immuno-adjuvant is remarkably superior to a traditional adjuvant, and has the advantages of simple method, low cost, good reproducibility and the like.
Description
Technical field
The invention belongs to biological technical field, relate to allergic asthma model, relate in particular to chitosan as immunological adjuvant the purposes in preparing mouse allelgic asthma model.
Background technology
Bronchial asthma (asthma) is common clinical and frequently-occurring disease, and sickness rate is in rising trend in recent years.Asthma is a kind of Flavonoids disease being participated in by inflammation cells such as eosinophilic granulocyte, mastocyte and T lymphocytes, its main manifestations is airway hyperreactivity, reversible airflow limitation and mucus hypersecretion, and also can occur Airway Remodeling late period.Its pathogenesis is illustrated not yet completely.Think at present, it is main immunoreation that the overactivity of Th2 cell has caused Th2 reaction, and the secretion increasings such as Th2 cytokines IL-4, IL-5, IL-13, finally cause the inflammatory cells such as eosinophilic granulocyte to be assembled at air flue, cause airway inflammation.In view of the limitation of human trial, at present to asthma, the research because of aspects such as, pathogenesis and treatments needs to be undertaken by animal model to a great extent, sets up good bronchial asthma animal model significant.
The principal character of asthma mouse model is: airway reactivity increases, and air flue be take eosinophilic granulocyte and is main inflammatory cell infiltration, air flue viscous secretion showed increased, and serum IgE level increases, and in bronchoalveolar lavage fluid, Th2 cytokine levels increases etc.Existing comparatively ripe asthma mouse model has following features: antigen systems sensitization and local excitation combine, during sensitization, need share immunological adjuvant, many employing lumbar injection, subcutaneous injection or both combinations, the most common with independent lumbar injection, excite and adopt the even mode such as intratracheal instillation of atomization suction, collunarium, with atomization, suck the most frequently used.Both at home and abroad the most frequently used asthmatic model carries out sensitization and builds mouse asthmatic model for take chicken ovalbumin (OVA) that liquified hydrogen alumina gel is adjuvant, adopts lumbar injection sensitization more, can set up significant acidophilia's airway inflammation.The mechanism that aluminium hydroxide carries out sensitization as the auxiliary OVA antigen of immunological adjuvant is illustrated not yet completely.Think at present, aluminum hydroxide adjuvant major function is slow release, but has the activation to immunocyte simultaneously, by protein and aluminium hydroxide hybrid injection, has antigen slow release and nonspecific immunity stimulation.Gel aluminum hydroxide has good adsorption to protein molecule, to promoting the absorption of antigen to have important function.A large amount of clinical immunity tests thinks that alumine hydroxide colloid adjuvant has very strong excitation to the humoral immune reaction of Th2 mediation, and to a little less than the effect of the cell immune response of Thl mediation.Therefore, gel aluminum hydroxide preparation for asthmatic model as conventional immunological adjuvant.
But there are two problems in the method: the liquified hydrogen alumina gel of (1) import is expensive, adopts common aluminium hydroxide solution poor as adjuvant effect, and air flue acidophilia inflammation is not obvious; (2) take airway reactivity, the air flue inflammatory cytokine of asthma mouse model of the OVA induction that gel aluminum hydroxide is adjuvant increases not remarkable; and unstable; often there will be with normal mouse not remarkable without significant difference or difference; cause model construction failure, affect medicine therapeutic evaluation.Whether effectively it is the key character of mouse asthmatic model that airway hyperreactivity, air flue inflammatory cytokine increase, be also to evaluate asthmatic medicament critical index.Therefore, set up that airway hyperreactivity is significantly more stable, the more significant asthma mouse model of airway inflammation is all significant to the screening of the research of asthma disease and treatment asthmatic medicament.
Summary of the invention
For overcoming the deficiencies in the prior art, the object of this invention is to provide a kind of chitosan as immunological adjuvant the purposes in preparing mouse allelgic asthma model.
The purposes that the object of this invention is to provide a kind of chitosan, for the preparation of the immunological adjuvant of allergic asthma animal model.
Chitosan is a kind of biodegradability polysaccharide that extracts preparation from Crustacean, is the amino cationic polysaccharide of the unique positively charged alkalescence of nature, and nontoxic, without causing prominent property, toxic and side effects is little.Chitosan has following three advantages.The first, chitosan promotes body to the absorption of antigen, picked-up and utilization rate.Chitosan has high viscosity and positively charged after being dissolved in weakly acidic aqueous solution; can as interacting, the pharmaceutical grade proteins such as antigen form nanoparticle with electronegative macromolecular substances; can interact and stick to cell surface with surface of cell membrane negative charge; be conducive to antigen more effectively by cell endocytic; and can protect antigen and medicine not acid, alkali and the protease in body destroy, improve body to the uptake ratio of antigen and utilization rate.Chitosan can adhere to mucomembranous surface, is conducive to mucosa to the absorption of antigen and picked-up.The second, chitosan has the effect of slow release antigen.Much research all finds that chitosan bag carries the external sustainable release of protein drug more than 7 days.Our research also finds that the OVA release in vitro of chitosan nanoparticles parcel is sustainable more than 6 days.Three, chitosan has very strong immunostimulation.Abroad studies show that, chitosan stimulates the effect of immunity of organism, as activation antigen is delivery cell, strengthens Delayed onset allergy and cytotoxic T cell reaction.
It is mouse allelgic asthma model prepared by immunological adjuvant that a further object of the present invention is to provide application chitosan.
Mouse allelgic asthma model of the present invention is set up by the following method, said method comprising the steps of:
1) prepare chitosan-acetic acid solution;
2) prepare the protein solution of chitosan adjuvant parcel;
3) the protein solution sensitized mice wrapping up with chitosan adjuvant;
4) mice is carried out to one or many respiratory tract protein solution atomization suction and excite, obtain allergic asthma model.
In the present invention's experiment, described mice is BALB/c mouse.
In the present invention experiment, described chitosan-acetic acid solution preparation method is as follows: chitosan is dissolved in to 1% aqueous acetic acid, and to be prepared into concentration be 1.5mg/ml chitosan-acetic acid solution, and regulate pH to 5.5-5.7,0.22 μ m membrane filtration with NaOH.
In the present invention's experiment, the protein solution of described chitosan adjuvant parcel is prepared by following steps: under vortex oscillating condition, in 1:1 ratio, in the chitosan-acetic acid solution of 1.5mg/ml, slowly add 200 μ g/ml OVA aqueous solutions, and continue slowly to add 2.0mg/ml sodium tripolyphosphate solution, make the mass ratio of chitosan and sodium tripolyphosphate reach 10:1.
In the present invention's experiment, described OVA aqueous solution and described sodium tripolyphosphate solution are all by 0.22 μ m membrane filtration.
In the present invention's experiment, the method for described sensitized mice is lumbar injection.
In the present invention's experiment, mice is carried out to three respiratory tract protein solution Compressed air nebulizations and suck.
In the present invention's experiment, mice is carried out to the atomization of respiratory tract protein solution and suck each 35 minutes.
The invention has the advantages that: (1) be take chitosan and as immunological adjuvant, prepared mouse asthmatic model and be all significantly better than asthmatic model prepared by existing conventional adjuvant in various aspects such as air flue acidophilia inflammation, airway reactivity and air flue inflammatory cytokines.(2) chitosan is cheap, and source is abundant.Therefore, the preparation that chitosan is used for asthmatic model as immunological adjuvant is significantly better than traditional adjuvant, can overcome that traditional adjuvant is prepared the airway reactivity of model, air flue inflammatory cytokine does not increase or increase inapparent shortcoming, there is method simple, with low cost, high repeatability and other advantages, be applicable to many-sided researchs such as pathogenesis, medicine screening and mechanism of action of asthma.
Accompanying drawing explanation
The particle diameter testing result of Fig. 1 chitosan parcel OVA microgranule
The transmission electron microscope picture of Fig. 2 chitosan parcel OVA microgranule
The particle diameter testing result of Fig. 3 gel aluminum hydroxide parcel OVA microgranule
The transmission electron microscope picture of Fig. 4 gel aluminum hydroxide parcel OVA microgranule
The pathologic of the different immune mouse asthmatic models of Fig. 5
The specific embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.Unless otherwise defined, the same meaning that all specialties of using in literary composition and scientific words and one skilled in the art are familiar.In addition, any method similar or impartial to described content and material all can be applicable in the present invention.The use that better implementation method described in literary composition and material only present a demonstration.
One, materials and methods
1, set up the mouse allelgic asthma model (model 1) by chitosan adjuvant and chicken ovalbumin induction
Chitosan is from Qingdao Hai Hui company, and molecular weight is 100,000 dalton, deacetylation 95%.Chitosan is dissolved in to the chitosan-acetic acid solution that 1% aqueous acetic acid is formulated as concentration 1.5mg/ml, and with NaOH, regulating pH value is 5.5-5.7, aseptic 0.22 μ m membrane filtration.Under vortex oscillating condition, in 1:1 ratio, in the chitosan solution of 1.5mg/ml, slowly add 200 μ g/ml OVA aqueous solutions (aseptic 0.22 μ m membrane filtration), after standing approximately 10 minutes, under vortex oscillating condition, continue slowly to add 2.0mg/ml sodium tripolyphosphate solution (aseptic 0.22 μ m membrane filtration) to make the mass ratio of chitosan and sodium tripolyphosphate reach 10:1, maintain vibration 10-20 minute, after standing 1 hour, can be used for intraperitoneal injection of mice sensitization.Nano particle size instrument detects nanometer particle size and the Zeta potential of the OVA of chitosan parcel, the form of the OVA of transmission electron microscope observing chitosan parcel.
SPF level BALB/c male mice, in 5-6 week, at the OVA solution of the 0th, 7,14 days lumbar injection chitosan parcels, dosage is 200 μ l/ Mus, i.e. 20 μ g OVA/ Mus.28th, within 29,30 days, adopt the atomization of Compressed air nebulization device to suck 10mg/ml OVA solution (OVA is dissolved in normal saline) and excite mice, 35 minutes every days.Adopt same processing method, not contain the chitosan nano solution lumbar injection sensitized mice of the same volume of OVA, with normal saline atomization, suck and excite mice, as negative control.
2, set up the mouse allelgic asthma model (model 2) by gel aluminum hydroxide adjuvant and chicken ovalbumin induction
Liquified hydrogen alumina gel is from U.S. Sigma company.OVA is dissolved in normal saline, and compound concentration is the solution of 400 μ g/ml.400 μ g/ml OVA solution are mixed with 1:3 ratio with gel aluminum hydroxide, can be used for intraperitoneal injection of mice sensitization after mixing latter standing 1 hour.Nano particle size instrument detects particle diameter and the Zeta potential of absorption OVA gel aluminum hydroxide, the form of the OVA of transmission electron microscope observing gel aluminum hydroxide parcel.
SPF level BALB/c male mice, 5-6 week, the 0th, the OVA that 7,14 days lumbar injections mix with gel aluminum hydroxide, dosage is 200 μ l/ Mus, i.e. 20 μ g OVA/ Mus.28th, within 29,30 days, adopt the atomization of Compressed air nebulization device to suck 10mg/ml OVA solution (OVA is dissolved in normal saline) and excite mice, 35 minutes every days.Adopt same processing method, not contain the gel aluminum hydroxide solution lumbar injection sensitized mice of the same volume of OVA, with normal saline atomization, suck and excite mice, as negative control.
3, set up the mouse allelgic asthma model (model 3) of being induced by chicken ovalbumin
OVA is dissolved in normal saline, and compound concentration is the solution of 100 μ g/ml.After mixing latter standing 1 hour, can be used for intraperitoneal injection of mice sensitization.
SPF level BALB/c male mice, 5-6 week, the 0th, 7,14 days lumbar injection OVA solution, dosage is 200 μ l/ Mus, i.e. 20 μ g OVA/ Mus.28th, within 29,30 days, adopt the atomization of Compressed air nebulization device to suck 10mg/ml OVA solution (OVA is dissolved in normal saline) and excite mice, 35 minutes every days.
Above three kinds of models all, after exciting for the last time 48 hours, carry out model measurement.
Two, index determining
1, airway reactivity
Evaluate airway reactivity and comprise following two aspects: 1) compare the concentration that excites that can significantly reduce airway reactivity (P < 0.05) with asthma group; 2) Mch when airway reactivity raises as 2 times of base values excites concentration PC100, and PC100 value is less, and air flue sensitivity is higher.
Excite for the last time latter 48 hours, it is reactive that noinvasive lung function instrument detects airway of mice.With Buxco mice noinvasive lung function instrument, detect airway resistance, respiratory pause (Enhanced Pause, the Penh) expression of result to strengthen, the Penh value while exciting each concentration of mensuration by methacholine (Methacholine, Mch).Mch excite gradients concentration is respectively 3.12,6.25,12.5,25,50,100mg/ml.While take not Sucked medicine the Penh value of being surveyed as base value, by the percentage ratio of each concentration Penh (Penh/baseline%), as airway reactivity statistical indicator.
2, total cellular score and eosinophilic granulocyte, neutrophilic granulocyte, lymphocyte and macrophage proportion in bronchoalveolar lavage fluid (BALF)
After intraperitoneal injection of anesthesia mice, tracheostomize, intubate, PBS0.8ml/ time, lavation 3 times, irrigating solution response rate > 80%.Reclaim BALF, the centrifugal 10min of 1500r/min, cell precipitation is resuspended with lml PBS, gets a little in blood cell counting plate, and row total cellular score counting, calculates the cell number of every milliliter.Remaining cell solution, the centrifugal 10min of 1500r/min, abandons supernatant, with smear after 50 μ l PBS re-suspended cell precipitations, dries rear row HE dyeing.Under oil mirror, carry out classified counting of leucocyte, count 600 cells, calculate the percentage ratio that eosinophilic granulocyte, neutrophilic granulocyte, lymphocyte and macrophage respectively account for.
3, pathologic inspection:
Getting mouse lung tissue puts into 4% formaldehyde fixative and fixedly spends the night.After routine is drawn materials, carry out gradient alcohol dehydration, transparency of organization is processed, waxdip, specimens paraffin embedding slices, HE dyeing, om observation lung tissue inflammatory cell infiltration, edema and air flue epithelial damage situation.
4, inflammatory parameters:
Inflammatory parameters is BALF supernatant cytokine levels.Get BALF supernatant, adopt ELISA method to detect IL-4, IL-5 and IL-13 level in BALF, ELISA test kit is respectively purchased from eBioscience company and Shanghai Yi Kesai company, and by specification operates.
Three, result
1, the particle diameter of chitosan parcel OVA, aluminium hydroxide parcel OVA and Zeta potential detection, transmission electron microscope observing
As shown in accompanying drawing 1 and 3, through nano particle size instrument, detect, chitosan parcel OVA size is 249.9nm, and polydispersity is 0.175, and Zeta potential is+22.1mV.The size of aluminium hydroxide parcel OVA is 4366nm, polydispersity 0.091, and Zeta potential is-13.6mV.From above result, the particle diameter of chitosan parcel OVA is significantly less than aluminium hydroxide parcel OVA.And chitosan parcel OVA nanoparticle is positively charged, can, with surface of cell membrane negative charge by electrostatic interaction, be conducive to OVA and by body, absorbed and absorb.
The visible chitosan of transmission electron microscope picture shown in Fig. 2 and Fig. 4 parcel OVA size compared with homogeneous, be evenly distributed, without obvious coacervation, and the center dyeing of the chitosan particle of parcel OVA is darker, show that OVA is wrapped in chitosan inside, and the chitosan particle particle diameter that does not wrap up OVA is less, microgranule color even, without engrain district.And the obvious conglomerate of aluminium hydroxide parcel OVA causes particle diameter to increase.
2, airway reactivity result
As shown in table 1, at all methacholines (Mch), excite under concentration, all there were significant differences for the asthma group airway reactivity that the chitosan of take is adjuvant and two matched groups, and increase multiple and reach 2-4 doubly, be significantly and increase, and be also significantly higher than and take the asthma group that aluminium hydroxide is adjuvant in each concentration.And take asthma group that aluminium hydroxide is adjuvant only Mch concentration as 6.25 and during 12.5mg/ml airway reactivity be significantly higher than self corresponding matched group, and increase multiple and be all less than 2.As shown in table 2, PC100 result shows, the asthma group that the chitosan of take is adjuvant is at Mch inhaled concentration during as 5.14mg/ml, airway reactivity increases 2 times for base value, significantly lower than take aluminium hydroxide as adjuvant with without the asthma group of adjuvant, show to take that the air flue sensitivity of the asthma group that chitosan is adjuvant is significantly higher than two other asthma group.And without airway reactivity and the matched group zero difference of adjuvant asthma group.Based on the above results, take the airway reactivity of the asthmatic model that chitosan is adjuvant increases and is significantly better than take aluminium hydroxide as adjuvant with without the asthmatic model of adjuvant.
Each group of table 1 is at each concentration Mch(mg/ml) Penh/baseline (%) under exciting
Compare * p < 0.05 with chitosan-asthma group; Compare with corresponding matched group
aMP.AMp.Ampp < 0.05
Table 2 is respectively organized PC100 (mg/ml)
Compare * p < 0.05 with chitosan-asthma group; Compare with corresponding matched group
aMP.AMp.Ampp < 0.05
3, Bronchoalveolar Lavage Fluid Cells sum and classified counting of leucocyte result
As shown in table 3, in the bronchoalveolar lavage fluid of asthma group, total cellular score and eosinophilic granulocyte's ratio are all significantly higher than matched group.And the asthma group total cellular score that the chitosan of take is adjuvant and eosinophilic granulocyte's ratio are all significantly higher than take aluminium hydroxide as adjuvant with without the asthma group of adjuvant.Therefore, take asthma group airway allergy inflammation that chitosan is adjuvant is significantly better than take aluminium hydroxide as adjuvant with without the asthma group of adjuvant.
Table 3 is respectively organized the total cellular score (* lO of BALF cell smear
5/ ml) and classified counting of leucocyte (%)
Compare * p<0.05 with chitosan one asthma group; Compare with corresponding matched group
aMP.AMp.Ampp<0.05
4, pathologic inspection
Two matched group air flues and lung tissue are showed no obvious inflammation and change.And all there is air flue eosinophils in three groups of asthma group.As shown in Figure 5, the asthma group airway inflammation that the chitosan of take is adjuvant be take aluminium hydroxide as adjuvant and more remarkable without the asthma group of adjuvant, and main manifestations is that the inflammatory cell infiltrations such as air flue eosinophilic granulocyte, airway epithelia destroy, air flue edema is more remarkable.Without adjuvant asthma group airway inflammation, infiltrate the lightest.
5, inflammatory parameters
It is one of key character of asthma that Th2 cytokine levels increases.As known in table 4 result, to compare with matched group, the asthma group Th2 cytokine IL-4 that the aluminium hydroxide of take is adjuvant, IL-5 level are without significantly increasing.And take asthma group Th2 cytokine IL-4, IL-5, IL mono-13 levels that chitosan is adjuvant, be significantly higher than self corresponding matched group, be also significantly higher than and take aluminium hydroxide as adjuvant with without adjuvant asthma group.
Table 4 cytokines of bronchoalveolar lavage fluid level (pg/m1)
Compare * p<0.05 with chitosan one asthma group; Compare with corresponding matched group
aMP.AMp.Ampp<0.05
From above experimental result, the chitosan of take is all significantly better than take in various aspects such as airway reactivity, air flue acidophilia inflammation and air flue inflammatory cytokines asthmatic model, especially airway reactivity and the air flue inflammatory cytokine that aluminium hydroxide is prepared as immunological adjuvant as the auxiliary OVA of immunological adjuvant prepares mouse asthmatic model.Chitosan control group mice is without any asthmatic model feature, shows that chitosan itself can induced hypersensitivity asthma, main performance immunoadjuvant function in asthmatic model preparation.And chitosan is cheap, source is abundant.Therefore, chitosan is significantly better than tradition as adjuvant for the preparation of asthmatic model and usings aluminium hydroxide as adjuvant, have method simple, with low cost, high repeatability and other advantages, is applicable to many-sided researchs such as pathogenesis, medicine screening and mechanism of action of asthma.
Although embodiment of the present invention are open as above, but it is not restricted to listed utilization in description and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other modification, therefore do not deviating under the general concept that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend of describing.
Claims (6)
- Chitosan as immunological adjuvant the purposes in preparing mouse allelgic asthma model, it is characterized in that, comprising: utilize chitosan as immunological adjuvant, to prepare the construction method of mouse allelgic asthma model:1) prepare the chitosan-acetic acid solution that concentration is 1.5mg/ml, and regulate pH to 5.5-5.7 with NaOH;2) prepare the protein solution of chitosan adjuvant parcel: under vortex oscillating condition, in 1:1 ratio, in the chitosan-acetic acid solution of 1.5mg/ml, slowly add 200 μ g/ml OVA aqueous solutions, and continue slowly to add 2.0mg/ml sodium tripolyphosphate solution, make the mass ratio of chitosan and sodium tripolyphosphate reach 10:1;3) the protein solution sensitized mice wrapping up with chitosan adjuvant;4) mice is carried out to one or many respiratory tract protein solution atomization suction and excite, prepare allergic asthma model.
- 2. purposes as claimed in claim 1, is characterized in that, described mice is BALB/c mouse.
- 3. purposes as claimed in claim 2, is characterized in that, described chitosan-acetic acid solution, OVA aqueous solution and described sodium tripolyphosphate solution are all by aseptic 0.22 μ m membrane filtration.
- 4. purposes as claimed in claim 3, is characterized in that, the method for described sensitized mice is lumbar injection.
- 5. purposes as claimed in claim 4, is characterized in that, mice is carried out to three respiratory tract protein solution Compressed air nebulizations and suck to excite mice.
- 6. purposes as claimed in claim 5, is characterized in that, mice is carried out to the atomization of respiratory tract protein solution and suck each 35 minutes.
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| 国华等.粉尘螨2壳聚糖疫苗经鼻免疫治疗小鼠过敏性哮喘的实验研究.《现代免疫学》.2008,第28卷(第1期),21-25. |
| 粉尘螨2壳聚糖疫苗经鼻免疫治疗小鼠过敏性哮喘的实验研究;国华等;《现代免疫学》;20081231;第28卷(第1期);21-15 * |
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