CN117981712A - Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model - Google Patents
Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model Download PDFInfo
- Publication number
- CN117981712A CN117981712A CN202310907975.1A CN202310907975A CN117981712A CN 117981712 A CN117981712 A CN 117981712A CN 202310907975 A CN202310907975 A CN 202310907975A CN 117981712 A CN117981712 A CN 117981712A
- Authority
- CN
- China
- Prior art keywords
- mice
- pulmonary fibrosis
- mouse
- adenovirus
- acute exacerbation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000009798 acute exacerbation Effects 0.000 title claims abstract description 50
- 208000005069 pulmonary fibrosis Diseases 0.000 title claims abstract description 50
- 241000701161 unidentified adenovirus Species 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000010171 animal model Methods 0.000 title claims abstract description 31
- 241000699670 Mus sp. Species 0.000 claims abstract description 99
- 241000699666 Mus <mouse, genus> Species 0.000 claims abstract description 56
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 claims abstract description 56
- 108010006654 Bleomycin Proteins 0.000 claims abstract description 52
- 229960001561 bleomycin Drugs 0.000 claims abstract description 52
- 239000013598 vector Substances 0.000 claims abstract description 27
- 201000009794 Idiopathic Pulmonary Fibrosis Diseases 0.000 claims abstract description 17
- 230000000694 effects Effects 0.000 claims abstract description 17
- 230000004083 survival effect Effects 0.000 claims abstract description 12
- 239000007924 injection Substances 0.000 claims abstract description 11
- 238000002347 injection Methods 0.000 claims abstract description 11
- 239000003814 drug Substances 0.000 claims abstract description 8
- 230000010076 replication Effects 0.000 claims abstract description 5
- 210000003437 trachea Anatomy 0.000 claims abstract description 4
- 208000024891 symptom Diseases 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 22
- 210000004072 lung Anatomy 0.000 claims description 17
- 230000036387 respiratory rate Effects 0.000 claims description 12
- 210000004704 glottis Anatomy 0.000 claims description 9
- 239000008363 phosphate buffer Substances 0.000 claims description 8
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 claims description 7
- 229960002725 isoflurane Drugs 0.000 claims description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- 230000035611 feeding Effects 0.000 claims description 6
- 230000002618 waking effect Effects 0.000 claims description 6
- 210000000038 chest Anatomy 0.000 claims description 5
- 230000034994 death Effects 0.000 claims description 5
- 206010019030 Hair colour changes Diseases 0.000 claims description 4
- 206010067484 Adverse reaction Diseases 0.000 claims description 3
- 239000002390 adhesive tape Substances 0.000 claims description 3
- 230000006838 adverse reaction Effects 0.000 claims description 3
- 230000003444 anaesthetic effect Effects 0.000 claims description 3
- 238000000889 atomisation Methods 0.000 claims description 3
- 230000037396 body weight Effects 0.000 claims description 3
- 238000012377 drug delivery Methods 0.000 claims description 3
- 210000004283 incisor Anatomy 0.000 claims description 3
- 108700016226 indium-bleomycin Proteins 0.000 claims description 3
- 229910052742 iron Inorganic materials 0.000 claims description 3
- 210000000115 thoracic cavity Anatomy 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 238000009736 wetting Methods 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 2
- 210000004369 blood Anatomy 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 230000037308 hair color Effects 0.000 claims description 2
- 238000002663 nebulization Methods 0.000 claims 1
- 230000002265 prevention Effects 0.000 claims 1
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 230000006378 damage Effects 0.000 abstract description 4
- 238000003912 environmental pollution Methods 0.000 abstract description 3
- 230000036407 pain Effects 0.000 abstract description 2
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 230000009385 viral infection Effects 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 13
- 238000000465 moulding Methods 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 208000036971 interstitial lung disease 2 Diseases 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 4
- 206010061218 Inflammation Diseases 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 230000004761 fibrosis Effects 0.000 description 4
- 230000004054 inflammatory process Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 3
- 208000004852 Lung Injury Diseases 0.000 description 3
- 206010069363 Traumatic lung injury Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 3
- 229960002591 hydroxyproline Drugs 0.000 description 3
- 231100000515 lung injury Toxicity 0.000 description 3
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 3
- 241001068263 Replication competent viruses Species 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000007923 nasal drop Substances 0.000 description 2
- 229940100662 nasal drops Drugs 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- OOGFPFWTORZYAN-AQHGXSAQSA-N (2s,4r)-4-oxidanylpyrrolidine-2-carboxylic acid Chemical compound O[C@H]1CN[C@H](C(O)=O)C1.O[C@H]1CN[C@H](C(O)=O)C1 OOGFPFWTORZYAN-AQHGXSAQSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 208000000059 Dyspnea Diseases 0.000 description 1
- 206010013975 Dyspnoeas Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 238000012387 aerosolization Methods 0.000 description 1
- 210000002821 alveolar epithelial cell Anatomy 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000037319 collagen production Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 239000005338 frosted glass Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 238000012637 gene transfection Methods 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 238000003125 immunofluorescent labeling Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 208000017804 lesions in lung Diseases 0.000 description 1
- 230000004199 lung function Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003300 oropharynx Anatomy 0.000 description 1
- 230000036284 oxygen consumption Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 208000037821 progressive disease Diseases 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000009325 pulmonary function Effects 0.000 description 1
- 210000004879 pulmonary tissue Anatomy 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/30—Animals modified by surgical methods
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention belongs to the field of biological medicine animal models, and particularly discloses a method for establishing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model, which at least comprises the following steps: on the basis of building a mouse pulmonary fibrosis model by bleomycin, an adenovirus vector Ad without replication activity is administrated through a trachea to induce an acute exacerbation symptom AE, so that an adenovirus induced pulmonary fibrosis acute exacerbation animal model AE-IPF is formed; the invention adopts the intratracheal injection administration under the direct vision of the mouse laryngoscope, not only can ensure the reliability and the accuracy of the administration, but also can reduce the damage to the mouse and improve the survival rate and the humanization protection; the administration time is preferably at day 14 (mice pulmonary fibrosis stage) and is a single administration, highly mimicking clinical practice and alleviating mice pain; creatively selects and inactivates adenovirus, not only can simulate the possible AE of IPF patients after virus infection, but also can reduce environmental pollution and toxic and side effects and reduce the risk of infection of experimental personnel.
Description
Technical Field
The invention belongs to the field of biological medicine animal models, and particularly discloses a method for establishing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model.
Background
Idiopathic Pulmonary Fibrosis (IPF) is a chronic progressive disease of unknown etiology with an average survival of only 2-3 years after diagnosis. Some patients may develop Acute Exacerbation (AE) during the course of the disease, which is manifested by acute exacerbation of dyspnea with worsening of pulmonary function, a significant increase in oxygen consumption, and diffuse lung injury (DAD), which can lead to rapid exacerbation and even death of the patient during the course of the disease. At present, the treatment of IPF in the stabilization period is mainly to prevent fibrosis and slow down the decline of lung function, but when AE occurs, no effective treatment method exists. More importantly, the etiology and pathogenesis of AE-IPF is currently unclear. Histologically, AE-IPF appears as DAD, massive inflammatory cell exudation, acute non-characteristic inflammatory changes, and stromal collagen production. Research has found that infection, drug toxicity, surgery or operation, aspiration and other factors can induce AE, and immune-related lung injury and apoptosis of alveolar epithelial cells may be one of the important mechanisms of AE-IPF pathogenesis. Therefore, establishing a reasonable AE-IPF animal model is of great importance to study the mechanism.
In recent years, a plurality of AE-IPF animal models are reported, wherein the more common is that a mouse pulmonary fibrosis model is firstly established by induction of Bleomycin (BLM), fluorescein Isothiocyanate (FITC) or a transgenic method and the like; on the other hand, the AE is induced by stimulating with viruses, bacteria, other physical and chemical substances, and the like. However, the modeling method reported in the literature has a lot of disputes, mainly focusing on the mode of administration, time and frequency of administration and reagent selection of induced AE, and animal models of different AE-IPFs are quite different.
In terms of administration modes, current literature reports on nasal drops, oral pharyngeal inhalation, intratracheal injection, incisional administration by a syringe tube, subcutaneous injection and the like; the nasal drops and the inhalation through the oropharynx can have the problems that the medicine can not completely enter the lung, the medicine is lost, the molding effect is not uniform due to large individual difference, and the like; intratracheal injection is related to experience of experimenters, and the experience is insufficient and the experimenters can mistakenly enter the esophagus; the damage caused by incision of the organ pipe is large, and the infection rate and death rate of the mice are high; subcutaneous injections may present absorption problems.
In terms of administration time and administration times, the problem is that the administration time is too early, for example, the AE is induced by re-administration after the bleomycin is administered for less than 14 days, at this time, the mouse model is still in an acute inflammation stage, but not in a stage with obvious fibrosis, and the re-administration cannot accurately simulate the clinical actual situation. Part of the schemes adopt a method of multiple dosing, are complex and complicated to operate, easily cause the increase of the death rate of mice, and cannot embody a certain factor of 'exciting' AE, which is different from the 'sudden' and rapid progress condition when the clinical IPF patient generates AE.
As AE causes are various, the literature reports that stimulators inducing AE in animal models are also various, such as bacteria, replication-competent viruses, physicochemical agents and the like, and the bacteria and replication-competent viruses have the risks of high laboratory requirements, easy infection and the like; the physicochemical reagents are greatly different from the clinical actual conditions.
Disclosure of Invention
In order to solve the problems, the invention discloses a method for establishing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model, which has the advantages of accuracy, high efficiency, safety and easiness in implementation.
The technical scheme of the invention is as follows:
A method for establishing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model at least comprises the following steps: on the basis of establishing a mouse pulmonary fibrosis model by bleomycin, an acute exacerbation symptom AE is induced by tracheal administration of an adenovirus vector Ad without replication activity, and an adenovirus induced pulmonary fibrosis acute exacerbation animal model AE-IPF is formed. Replication-defective adenovirus (Ad) vectors, i.e., empty adenovirus vectors, refer to viral vectors that do not carry any foreign DNA or RNA, are now widely used in gene transfection and gene therapy research. Although Ad empty vector may cause some acute inflammatory reaction and lung injury, in normal mice empty virions do not cause chronic diseases such as pulmonary fibrosis, and only a slight inflammatory reaction may occur. The inventor finds that the application of a certain dose of replication-defective adenovirus (Ad) vector on the basis of building a mouse pulmonary fibrosis model by bleomycin can induce acute lung injury of a pulmonary fibrosis mouse, and the acute exacerbation is shown, so that compared with other active virus inducers, the risk of infection and experimental difficulty are reduced.
Furthermore, in the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model, the tracheal administration is that the mouse intrapulmonary aerosolization administration device is inserted into the mouse trachea to administer the replication-inactive Ad carrier solution. The tracheal injection is used for administration, thereby not only ensuring the accuracy of administration, but also reducing unnecessary injury to mice and external environmental pollution
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model is characterized in that the replication-inactive Ad vector solution is prepared by dissolving a certain amount of replication-inactive Ad in phosphate buffer.
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model is characterized in that the replication-inactive Ad vector solution is prepared by dissolving 0.5-2X 10 8 pfu of replication-inactive Ad vector in 25-100 mu L of phosphate buffer. Preferably, 1X 10 8 pfu of replication-inactive Ad vector is dissolved in 50. Mu.L of phosphate buffer.
Furthermore, in the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model, the bleomycin is used for establishing the mouse pulmonary fibrosis model, and the dosage of the bleomycin is 3-5mg/kg, preferably 5mg/kg.
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model is characterized in that the mice are C57BL/6J male mice of 6-8 weeks.
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model comprises the following steps of:
Air-tube injection of bleomycin induces pulmonary fibrosis in mice:
① Weighing the mice on day 0, and marking with ear marks; the isoflurane is inhaled by atomization to fully anesthetize the mice, and the temperature is kept;
② After the mice are anesthetized successfully, the mice are placed on a mouse plate in a supine position, upper incisors are fixed by iron wires, and limbs are fixed by adhesive tapes;
③ Adjusting the mouse plate to a proper angle, and gently pulling out the tongue of the mouse by forceps to deviate to one side;
④ The left hand mouse laryngoscope is inserted into the throat of the mouse, the tongue root is pressed, the mouse laryngoscope is lifted slightly, the glottis is fully exposed, and the glottis law can be seen to be unfolded and folded; gently inserting an intrapulmonary aerosolized drug delivery device into the glottis of a mouse, and injecting a corresponding volume of bleomycin solution;
⑤ After modeling, the respiratory rate of the mice is observed to be increased, the thoracic fluctuation of the mice is obvious, and the mice are moved to the auricle to be audible and the wetting sound is observed;
⑥ The assistant contacts and fixes, and places the prone position of the mouse back into the cage; observing the state of the mice after waking;
⑦ Mice were observed daily for activity, respiratory rate, feeding, changes in hair color, and survival, and mice were measured for body weight every other day for record.
Furthermore, in the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model, the time for the tracheal administration of the adenovirus vector Ad without replication activity is the pulmonary fibrosis period of the mice after the administration of bleomycin.
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model comprises the following steps of:
Acute exacerbation of pulmonary fibrosis in bleomycin mice is induced by tracheal injection of replication-inactive Ad vector solution:
1) The isoflurane is inhaled again in an atomized manner on the 14 th day after the bleomycin administration to fully anesthetize the mice, and the anesthetic effect and adverse reaction are observed;
2) After the mice are anesthetized successfully, the replication-inactive Ad vector solution is injected into the air pipe;
3) After modeling, observing whether the respiratory rate of the mice is increased or not, and observing the survival state and death rate of the mice;
4) The assistant contacts and fixes, and places the prone position of the mouse back into the cage; observing the state of the mice after waking;
5) Daily observing the activity, respiratory rate, feeding, hair color change and survival condition of the mice, measuring the weight of the mice every other day, and recording;
6) On day 20, mice were subjected to chest CT examination, on day 21, mice were sacrificed by cervical marrow disruption, and samples such as blood, lung tissue, and alveolar lavage fluid were collected, and modeling effect was evaluated.
Furthermore, the method for establishing the adenovirus-induced pulmonary fibrosis acute exacerbation animal model is applied to the development of medicaments for treating/preventing idiopathic pulmonary fibrosis diseases.
The invention has the following beneficial effects:
The method adopts the currently accepted bleomycin to establish a mouse pulmonary fibrosis model, and adopts the mouse laryngoscope to directly inject and administer under the air tube aiming at the administration mode, thereby not only ensuring the reliability and the accuracy of administration, but also reducing the damage to mice, improving the survival rate and humanization protection, the administration time is selected on the 14 th day (the mouse pulmonary fibrosis period), being single administration, highly simulating clinical practice, relieving the pain of the mice, creatively selecting and inactivating adenovirus, simulating the possible AE of IPF patients after virus infection, reducing the environmental pollution and the toxic and side effects, and reducing the risk of infection of experimental staff.
Drawings
FIG. 1 is a flow chart of modeling a mouse with acute exacerbation of pulmonary fibrosis;
Fig. 2 is a graph of post-molding mouse chest CT (20 days post BLM, 7 days post Ad;
FIG. 3 shows lung histopathological changes in mice after molding (day 20 post BLM, day 7 post Ad);
FIG. 4 shows the hydroxyproline content (Hydroxyproline) analysis of the lung tissue of mice after molding (day 20 post BLM, day 7 post Ad);
FIG. 5 is a graph showing apoptosis analysis of lung tissue of mice after molding (day 20 post BLM, day 7 post Ad);
FIG. 6 is a marker analysis of mouse lung tissue fibrosis after molding (day 20 post BLM, day 7 post Ad).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The reagents or instruments used in the examples of the present invention were not manufacturer-identified and were conventional reagent products commercially available.
Equipment, reagents, and mouse preparation:
mice:
c57BL/6J male mice were selected from 6-8 weeks.
An instrument:
ear tag, adjustable mouse plate, fixer (rubber band, sticky tape), blunt forceps, mouse laryngoscope, and device for atomizing and administering medicine in mouse lung.
Reagent:
isoflurane, bleomycin powder, replication-inactive Ad vectors (and Meta Biotechnology (Shanghai) Inc., cat# H201), phosphate buffer.
Bleomycin solution preparation and replication-inactive Ad carrier solution preparation:
Preparation of bleomycin solution:
the specification of the medicine is 15 mg/bottle.
15Mg of bleomycin was dissolved in 7.5ml of phosphate buffer (0.01M) and shaken well (bleomycin concentration 2. Mu.g/. Mu.l) in a sterile bench
Bleomycin concentration was 5mg/kg and corresponding volumes of solution were aspirated based on mouse body weight.
Replication-inactive Ad vector solution preparation:
The Ad vector without replication activity had a size of 1.58X10 11 pfu/ml, 50. Mu.l/branch.
Each mouse was intended to be used in an amount of 1X 10 8 pfu, dissolved in 50. Mu.l of phosphate buffer and injected intratracheally
Thus, the replication-inactive Ad vector stock was diluted 1580-fold (i.e., 7.9 ml) to give 1X 10 8 pfu/50. Mu.l of the target solution.
The modeling flow of the whole pulmonary fibrosis acute exacerbation mouse is shown in figure 1.
Example 1
Experiment 1: air-tube injection of bleomycin induces pulmonary fibrosis in mice:
① Weighing the weight of the mice on day 0, and marking with ear marks; the isoflurane is inhaled by atomization to fully anesthetize the mice, and the temperature is kept;
② After the mice are anesthetized successfully, the mice are placed on a mouse plate in a supine position, iron wires are used for fixing upper incisors, and adhesive tapes are used for fixing limbs;
③ Adjusting the mouse plate to a proper angle, and gently pulling out the tongue of the mouse with forceps to deflect to one side;
④ The left hand-held mouse laryngoscope is inserted into the throat of the mouse, the tongue root is pressed, the mouse is lifted slightly, the glottis is fully exposed, and the glottis law can be seen to be unfolded and folded; gently inserting an intrapulmonary aerosolized drug delivery device into the glottis of a mouse, and injecting a corresponding volume of bleomycin solution;
⑤ After modeling, the respiratory rate of the mice is observed to be increased, the thoracic fluctuation of the mice is obvious, and the mice are moved to the auricle to be audible and the wetting sound is observed;
⑥ Placing the mouse in a prone position back into a cage by the aid of contact fixation; the mice were observed for a state after waking.
⑦ The mice are observed daily for activity, respiratory rate, feeding, hair color change and survival, and the weight of the mice is measured every other day for recording.
Example 2
Experiment 2: acute exacerbation of pulmonary fibrosis in bleomycin mice is induced by tracheal injection of replication-inactive Ad vector solution:
① Re-atomizing and inhaling isoflurane again on day 14 to fully anesthetize the mice, and observing the anesthetic effect and whether adverse reaction exists;
② After successful anesthesia of the mice, 50 μl of replication-inactive Ad vector solution was injected intratracheally in the same manner as in scheme 3.1.
③ After modeling, it is observed whether the respiratory rate of the mice increases, the survival status and mortality of the mice;
④ Placing the mouse in a prone position back into a cage by the aid of contact fixation; the mice were observed for a state after waking.
⑤ The mice are observed daily for activity, respiratory rate, feeding, hair color change and survival, and the weight of the mice is measured every other day for recording.
⑥ On day 20, mice were subjected to chest CT examination, on day 21, mice were sacrificed by cervical marrow disruption method, lung tissue samples were collected, and modeling effect was evaluated.
Experimental results
As shown in fig. 2, post-molding mouse chest CT (20 days post BLM, 7 days post Ad) showed: the mice in the BLM+Ad co-intervention group showed diffuse double-lung frosted glass exudation, which was significantly worse than that of the NS group alone, the BLM group, the Ad group, and the BLM+BLM group.
As shown in fig. 3, mice after modeling had lung histopathological changes (day 20 post BLM, day 7 post Ad): HE staining results of A-E show that acute inflammatory reaction occurs in lung tissues of mice in the group of mice with BLM+Ad co-intervention, and the acute inflammatory reaction is obviously aggravated compared with other mice in each group; the results of F-J Marathon staining showed that the mice with the combined intervention of BLM+Ad showed more severe collagen deposition in lung tissue than in the other groups.
As shown in fig. 4, post-molding mice lung tissue hydroxyproline (Hydroxyproline) content analysis (day 20 post BLM, day 7 post Ad): the lung tissue hydroxyproline content of mice in the BLM+Ad co-intervention group is obviously increased compared with that of the mice in BLM and NS, the difference is significant (P value is less than 0.05), but compared with the BLM+BLM group, the difference is increased but the difference is not significant (P value is more than 0.05).
As shown in fig. 5, apoptosis analysis of lung tissue of mice after molding (day 20 post BLM, day 7 post Ad): TUNEL immunofluorescent staining showed a significant increase in apoptosis in lung tissue in mice from blm+ad co-intervention group compared to single BLM, two BLM and NS groups (×200 fold).
Marker analysis of pulmonary tissue fibrosis in mice after molding (day 20 post BLM, day 7 post Ad) as shown in fig. 6: the WB results showed that the protein expression levels of Collagen-1, α -SMA and TGF- β1 were significantly increased in mice lung tissue from the BLM+Ad co-intervention group compared to BLM and NS, and also increased compared to the two BLM model group.
From the above experimental results, it can be concluded that: we used BLM-induced pulmonary fibrosis mice, ad vector was given on day 14 after BLM molding, and then observed on day 21 that the BLM-induced pulmonary fibrosis mice showed diffuse lesions in lung tissue, significantly increased inflammatory and fibrotic responses, and significantly increased apoptosis compared to normal saline control and BLM group mice. The results suggest that one-time direct intratracheal injection of replication-inactive Ad vectors induces AE in BLM mice, which can successfully establish an animal model of acute exacerbation of pulmonary fibrosis.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be appreciated by persons skilled in the art that the above embodiments are not intended to limit the invention in any way, and that all technical solutions obtained by means of equivalent substitutions or equivalent transformations fall within the scope of the invention.
Claims (10)
1. A method for establishing an adenovirus-induced acute exacerbation animal model of pulmonary fibrosis, which is characterized by at least comprising the following steps: on the basis of establishing a mouse pulmonary fibrosis model by bleomycin, an acute exacerbation symptom AE is induced by tracheal administration of an adenovirus vector Ad without replication activity, and an adenovirus induced pulmonary fibrosis acute exacerbation animal model AE-IPF is formed.
2. The method of claim 1, wherein said tracheal administration is performed by inserting a mouse intrapulmonary nebulization administration device into a mouse trachea for administration of replication-inactive Ad carrier solution.
3. The method of claim 2, wherein the replication-inactive Ad carrier solution is prepared by dissolving a certain amount of replication-inactive Ad in phosphate buffer.
4. The method for constructing an animal model for acute exacerbation of adenovirus-induced pulmonary fibrosis according to claim 3, wherein the replication-inactive Ad vector solution is prepared by dissolving 0.5-2 x 10 8 pfu of replication-inactive Ad vector in 25-100 μl of phosphate buffer.
5. The method for constructing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model according to claim 1, wherein the bleomycin is used in an amount of 3-5mg/kg in the established mouse pulmonary fibrosis model.
6. The method of claim 5, wherein the mice are 6-8 week C57BL/6J male mice.
7. The method for constructing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model of claim 6, wherein the method for constructing a mouse pulmonary fibrosis model with bleomycin comprises the following steps:
Air-tube injection of bleomycin induces pulmonary fibrosis in mice:
① Weighing the mice on day 0, and marking with ear marks; the isoflurane is inhaled by atomization to fully anesthetize the mice, and the temperature is kept;
② After the mice are anesthetized successfully, the mice are placed on a mouse plate in a supine position, upper incisors are fixed by iron wires, and limbs are fixed by adhesive tapes;
③ Adjusting the mouse plate to a proper angle, and gently pulling out the tongue of the mouse by forceps to deviate to one side;
④ The left hand mouse laryngoscope is inserted into the throat of the mouse, the tongue root is pressed, the mouse laryngoscope is lifted slightly, the glottis is fully exposed, and the glottis law can be seen to be unfolded and folded; gently inserting an intrapulmonary aerosolized drug delivery device into the glottis of a mouse, and injecting a corresponding volume of bleomycin solution;
⑤ After modeling, the respiratory rate of the mice is observed to be increased, the thoracic fluctuation of the mice is obvious, and the mice are moved to the auricle to be audible and the wetting sound is observed;
⑥ The assistant contacts and fixes, and places the prone position of the mouse back into the cage; observing the state of the mice after waking;
⑦ Mice were observed daily for activity, respiratory rate, feeding, changes in hair color, and survival, and mice were measured for body weight every other day for record.
8. The method of claim 1, wherein the time for the administration of the replication-inactive adenovirus vector Ad to the trachea is the pulmonary fibrosis period of the mice after the administration of bleomycin.
9. The method for constructing an adenovirus-induced pulmonary fibrosis acute exacerbation animal model of claim 8, comprising the steps of:
Acute exacerbation of pulmonary fibrosis in bleomycin mice is induced by tracheal injection of replication-inactive Ad vector solution:
1) The isoflurane is inhaled again in an atomized manner on the 14 th day after the bleomycin administration to fully anesthetize the mice, and the anesthetic effect and adverse reaction are observed;
2) After the mice are anesthetized successfully, the replication-inactive Ad vector solution is injected into the air pipe;
3) After modeling, observing whether the respiratory rate of the mice is increased or not, and observing the survival state and death rate of the mice;
4) The assistant contacts and fixes, and places the prone position of the mouse back into the cage; observing the state of the mice after waking;
5) Daily observing the activity, respiratory rate, feeding, hair color change and survival condition of the mice, measuring the weight of the mice every other day, and recording;
6) On day 20, mice were subjected to chest CT examination, on day 21, mice were sacrificed by cervical marrow disruption, and samples such as blood, lung tissue, and alveolar lavage fluid were collected, and modeling effect was evaluated.
10. Use of the method for constructing an animal model for acute exacerbation of adenovirus-induced pulmonary fibrosis according to any one of claims 1-9 in the development of a medicament for the treatment/prevention of acute exacerbation of idiopathic pulmonary fibrosis.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310907975.1A CN117981712A (en) | 2023-07-24 | 2023-07-24 | Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310907975.1A CN117981712A (en) | 2023-07-24 | 2023-07-24 | Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117981712A true CN117981712A (en) | 2024-05-07 |
Family
ID=90887879
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310907975.1A Pending CN117981712A (en) | 2023-07-24 | 2023-07-24 | Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117981712A (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060195916A1 (en) * | 2005-02-22 | 2006-08-31 | Boehringer Ingelheim International Gmbh | Transgenic Animal as a Model for Fibrotic Diseases |
| CN103221423A (en) * | 2010-09-24 | 2013-07-24 | 昂克斯治疗有限公司 | Oncolytic adenoviral vectors and methods and uses related thereto |
| WO2020247815A1 (en) * | 2019-06-06 | 2020-12-10 | Cornell University | Transgenic rodent model for lung fibrosis and uses thereof |
| CN112437663A (en) * | 2018-05-17 | 2021-03-02 | 开动疗法公司 | Treatment of idiopathic pulmonary fibrosis with a beta-type glycogen synthase kinase 3 inhibitor |
-
2023
- 2023-07-24 CN CN202310907975.1A patent/CN117981712A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060195916A1 (en) * | 2005-02-22 | 2006-08-31 | Boehringer Ingelheim International Gmbh | Transgenic Animal as a Model for Fibrotic Diseases |
| CN103221423A (en) * | 2010-09-24 | 2013-07-24 | 昂克斯治疗有限公司 | Oncolytic adenoviral vectors and methods and uses related thereto |
| CN112437663A (en) * | 2018-05-17 | 2021-03-02 | 开动疗法公司 | Treatment of idiopathic pulmonary fibrosis with a beta-type glycogen synthase kinase 3 inhibitor |
| WO2020247815A1 (en) * | 2019-06-06 | 2020-12-10 | Cornell University | Transgenic rodent model for lung fibrosis and uses thereof |
Non-Patent Citations (2)
| Title |
|---|
| K.K.JAIN等: "《基因治疗学》", 31 October 2000, 世界图书出版西安公司, pages: 235 * |
| SOO JUNG CHO等: "GLUT1-dependentglycolysis regulates exacerbation of fibrosis via AIM2 inflammasome activation", 《THORAX》, 10 December 2019 (2019-12-10), pages 227 - 236 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ari et al. | Evaluation of aerosol generator devices at 3 locations in humidified and non-humidified circuits during adult mechanical ventilation | |
| Gunkel et al. | Current techniques in avian anesthesia | |
| JP5490683B2 (en) | Administration method of pulmonary surfactant | |
| Bertelsen | Squamates (snakes and lizards) | |
| SE506208C2 (en) | Device for collecting gas from the upper respiratory tract and delivering this gas to the inhalation air in a respirator | |
| CN105722543B (en) | Pass through the improved method and system of atomization application Curosurf | |
| CN113876457A (en) | Economical, quantitative and noninvasive method for establishing mouse model for occupational disease of pulmonary fibrosis caused by silicon dust and preparation device | |
| US20200268656A1 (en) | Nebulized Ethanol for Internal Disinfecting and Improvement | |
| US6537265B2 (en) | Method for nasal application of a medicinal substance | |
| Dargaville et al. | An authentic animal model of the very preterm infant on nasal continuous positive airway pressure | |
| CN114794015A (en) | Construction method and application of lung injury animal model | |
| Comolli et al. | Technological advances in exotic pet anesthesia and analgesia | |
| Gruntman et al. | Gene Transfer in the Lung Using Recombinant Adeno‐Associated Virus | |
| US20200197415A1 (en) | Methods and Compositions for the Treatment of Obesity | |
| CN117981712A (en) | Method for establishing adenovirus-induced pulmonary fibrosis acute exacerbation animal model | |
| CN111494392A (en) | Composition for treating acute lung injury and application thereof | |
| Ronaldson et al. | Anesthesia and anesthetic-related complications of 8 elegant-crested tinamous (Eudromia elegans) undergoing experimental surgery | |
| CA2982685A1 (en) | Device for facilitating the administration of a medicament to the lung by a catheter | |
| Gaddam et al. | Erythropoietin and cytoprotective cytokines in experimental traumatic brain injury | |
| Kretzschmar et al. | Bronchoconstriction induced by inhaled methacholine delays desflurane uptake and elimination in a piglet model | |
| Bujold et al. | Novel methods for the assessment of safety pharmacology and toxicology parameters in anesthetized and ventilated dogs receiving inhaled drugs | |
| CN108837283A (en) | Stem bronchi cell precise positioning slow-released system | |
| Mostafa et al. | Nebulized 10% lignocaine for awake fibreoptic intubation | |
| CN111714474B (en) | Tetracaine composite preparation and preparation method and application thereof | |
| AU2020104218A4 (en) | Application of interleukin-37 in treating idiopathic pulmonary fibrosis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |