CN108524912A - A kind of construction method of pulmonary fibrosis animal model - Google Patents
A kind of construction method of pulmonary fibrosis animal model Download PDFInfo
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- CN108524912A CN108524912A CN201810382567.8A CN201810382567A CN108524912A CN 108524912 A CN108524912 A CN 108524912A CN 201810382567 A CN201810382567 A CN 201810382567A CN 108524912 A CN108524912 A CN 108524912A
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- pulmonary fibrosis
- animal model
- construction method
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- fibrosis animal
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/14—Peptides containing saccharide radicals; Derivatives thereof, e.g. bleomycin, phleomycin, muramylpeptides or vancomycin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/195—Chemokines, e.g. RANTES
Abstract
A kind of construction method of pulmonary fibrosis animal model, using cause can the chemistry of pulmonary fibrosis or the mode construction pulmonary fibrosis animal model of biological agent and chemotactic factor (CF) administering drug combinations.Pulmonary fibrosis animal model constructed by the method for the present invention has apparent fibrosis lesion, it is similar to clinical truth height to mankind's study of pulmonary fibrosis feature, modeling is with obvious effects; lesion is evenly distributed; result is stablized; the death rate is low, can carry out clinical simulation to the pulmonary fibrosis disease of the different courses of disease.The construction method of pulmonary fibrosis animal model of the present invention is applied to teiology, pathology and the pharmaceutical research of pulmonary fibrosis disease.
Description
Technical field
The present invention relates to medical experiment fields, and in particular to a kind of construction method of fibrosis animal model especially relates to
And a kind of construction method of pulmonary fibrosis fibrosis animal model.
Background technology
Pulmonary fibrosis（IPF）It is that a kind of reason is unknown, scorching with Diffuse alveolar and alveolar structure is disorderly, and eventually leads to lung
The disease that interstitial fibrosis is characterized, Histopathology feature are plain edition interstitial pneumonias（UIP）, i.e. epithelial cell increasing
It is raw, basilar memebrane is exposed, alveolar consolidation, fibroblast stove occurs etc..Currently, clinically lack effective treatment means, spy's hair
The incidence of property pulmonary fibrosis, the death rate constantly rise, and with advancing age, morbidity and mortality also gradually rise
Height, after the onset 5 annual survival rates only 20%.Animal model is the important means of study of disease, and ideal animal model, which can simulate, is in
The various features of existing disease, pathogenesis and the effective therapeutic scheme of screening to study of disease play a crucial role, but current lung
Fibrosis animal model still has various limitations.Common pulmonary fibrosis animal model has drug, poisonous substance guidance model and turns base
Because of model.Although transgenic models are quickly grown, transgenic models can not fibrosis caused by simulated environment factor.
Bleomycin（BLM）It is a kind of multicomponent composite antibiotic with antitumor action, one of toxic side effect is
Cause pulmonary fibrosis, be closer to mankind's pulmonary fibrosis since its Histopathology changes, therefore is widely used in induction structure
Pulmonary fibrosis model.The approach that model preparation is carried out using BLM includes Intratracheal administration, and nose administration is injected intravenously, intraperitoneal injection
Deng wherein transtracheal administration has been generally acknowledged both at home and abroad maturation method.But the animal pulmonary fibrosis model built with BLM inductions
It derives mankind's pulmonary fibrosis, or research BLM is to the toxicity of mankind's lung, has clearly disadvantageous, clinical doctor cannot be met
Pathological study demand.
First, it is single administration structure pulmonary fibrosis animal model disease development time and be spatially different from
The occurrence and development process of human diseases.Human organ's tissue fibrosis disease be mostly for a long time it is repeated multiple times to tissue such as
Alveolar epithelial cells damages, and leads to the result that can not be repaired completely.It is fine with mankind's lung even single medicine multiple dosing model
The time of dimensionization morbidity is compared, still too short.
Secondly, the pathological characteristics of the pulmonary fibrosis animal model of single administration structure differ very with clinical truth
Far.The single administration model of pulmonary fibrosis often has apparent neutrophil infiltration, and patient's IPF neutrophil infiltration is unknown
It is aobvious；II type epithelial hyperplasia unobvious, but it is apparent in patient IPF；Lung tissue's fibrosis of pulmonary fibrosis model is unstable
It is fixed, often subside after 6 weeks, there is no fibroblast stove, and in patient IPF lung tissue fibrosis long-term existence, and
Fibroblast stove is fairly obvious.
Therefore, it is single administration structure pulmonary fibrosis animal model compared with mankind's pulmonary fibrosis in terms of pathological characters still
There are great drawbacks.
CN106214280A discloses a kind of minimally invasive method for making pulmonary fibrosis animal model.Specific steps include：It is dynamic
Object is anaesthetized, trachea cannula, drug infusion.With oblique lateral position Baoding after Animal Anesthesia, with tracheal inserting device from oral cavity through glottis
It is inserted into animal unilateral lung and dabbling drug.According to tracheae when animal lung, the anatomical structure of trachea and bronchus and Intratracheal administration
Intubaction device rests on depth and the position of side lung, and drug can be mostly evenly distributed in the lung of side, can also concentrate on side
In some lobe of the lung of lung.This modeling method pathomechanism is simple, and the fibroblast stove in model is not obvious, this method and people
The time of class morbidity is compared, still too short,
CN107006422A discloses a kind of smoke and causes the method for building up of tree shrew multiple organ fibrosis model.This method is will be wild
After raw tree shrew is tamed three months, in unaired room, tree shrew is allowed to absorb smoke from cigarette under general breathing state, daily two
Secondary, 30 minutes every time, 6 days weekly, exposure duration was 9 months；The smoke from cigarette is the smog that burning 100g tobacco leaves generate；With
The smoking of Masson staining evaluation tree shrews causes multiple organ pulmonary fibrosis animal model.This method is cumbersome, and easily causes model
Animal dead.
Invention content
The technical problem to be solved by the present invention is to overcome drawbacks described above of the existing technology, provide a kind of modeling effect
Fruit is apparent, and lesion is evenly distributed, and as a result stablizes, and the death rate is low, and the pulmonary fibrosis animal model of structure has apparent fibrosis
Lesion, it is similar to clinical truth height to mankind's study of pulmonary fibrosis feature, it can be to the pulmonary fibrosis disease of the different courses of disease
Disease carries out the construction method of the pulmonary fibrosis animal model of clinical simulation.
The technical solution adopted by the present invention to solve the technical problems is as follows：A kind of structure side of pulmonary fibrosis animal model
Method utilizes the mode construction pulmonary fibrosis animal for the chemistry or biological agent and chemotactic factor (CF) administering drug combinations that can cause pulmonary fibrosis
Model.
Further, described that the biological agent of pulmonary fibrosis can be caused to be bleomycin.
Further, the pattern of the administering drug combinations, chemotactic factor (CF) are the cell receptor for guiding fibroblast to be migrated
Corresponding ligand.
Further, the chemotactic factor (CF) include the ligand CXCL 12 of CXCR4 receptors and the ligand CCL19 of CCR7 receptors or
CCL21。
Further, the experimental animal used in the animal model includes rodent, rabbit, canine, porcine, sheep class or monkey class.
Further, the rodent includes mouse, rat, hamster or cavy.
Further, the administration frequency of the pattern of the administering drug combinations is：Bleomycin is administered 1~2 time every 2 weeks, continuous 4~
10 weeks；Daily 1~3 time of chemotactic factor (CF) stops administration bleomycin and stops that chemotactic factor (CF) is administered after 1~3 week.
Further, the dosage of the pattern of the bleomycin and chemotactic factor (CF) administering drug combinations is：By mouse weight 2~
Bleomycin is administered in 8 mg/kg or 0.1~2.0 U/kg；By mouse weight 0.1~1.0 mg/kg administration Chemokines CCs XCL
12 or CCL19 or CCL21.
Further, the administration route of the pattern of the administering drug combinations, which is medical experimental zoology, allows administration route, such as
Intratracheal instillation, intravenous injection and intraperitoneal injection etc..
Preferably, the administering mode of the construction method of the pulmonary fibrosis animal model is Intratracheal instillation.
Chemotactic factor (CF) (Chemokine) is generally made of 70~125 amino acid, and molecular weight is smaller（6~14KD）.According to
Chemotactic factor (CF) can be divided into 4 subtribes by level-one peptide chain structure feature, the position of N-terminal cysteine residues and number：CC、CXC、
C and CX3C（C is cysteine, and X is arbitrary amino acid）.According to the expression way of chemotactic factor (CF) and its in immune system
Effect, two classes can be divided into：Homeostasis chemotactic factor (CF) and inflammatory chemotactic factor (CF).Homeostasis chemotactic factor (CF) master
It to be expressed in place of going back to the nest, have the function of maintaining homeostasis, and have explicitly to lymphocyte homing and maturation
Effect.Inflammatory chemotactic factor (CF) is expressed by the cell that is stimulated, such as the induction of inflammatory cytokine, bacteriotoxin or other broken
The stimulation of the factor of bad homeostasis, major function is to raise effector cell, in coordinating natural and the acquired immune response
It plays an important role.Chemotactic factor (CF) will play biological action, it is necessary to be combined with corresponding receptor.Chemokine CXCL12 is
Stromal cell derived factor-1 (SDF-1), receptor are CXCR4, can attract the fiber finer that CXCL4 is expressed in peripheral blood
Born of the same parents.Chemokine CXCL12 has apparent promotion fibrocyte travel motion effect, especially accelerates fiber in peripheral blood
Cell promotes the formation of fiber stove to aggregation at disease damage.Concentration using CXCL12 in the mouse lung of bleomycin induction is apparent
Higher than the concentration in peripheral blood, to promote to express the fibrocyte of CXCL4 in peripheral blood, i.e. surface markers are CD45+
ColI+CXCR4+Cell is assembled to intrapulmonary injury region.In animal（Including people）Internal there is also another cells, i.e. CD45+
ColI+CCR7+Cell, their function and CD45+ColI+CXCR4+Cell is very much like.Its ligand is Chemokines CC XCL19
Or Chemokines CC XCL21.It is demonstrated experimentally that Chemokines CC XCL19 or Chemokines CC XCL21 can promote CD45+ColI+CCR7+Cell is assembled to intrapulmonary.Combine while the pulmonary fibrosis animal model transtracheal perfusion bleomycin of the method for the present invention structure
Chemokines CC XCL 12 or CCL19 or CCL21 are given, fibrocyte in peripheral blood can be accelerated while causing lung to lose
Assemble to injury region, promote the formation of fiber stove, is not easy to cause animal pattern dead in modeling process midway.
Beneficial effects of the present invention：
（1）There is the pulmonary fibrosis animal model of the method for the present invention structure obvious fibrosis lesion, lesion to be evenly distributed, as a result surely
Fixed, the death rate is low, the lung fiber animal mould highly similar with the clinical truth of mankind's pulmonary fibrosis on pathological characteristics
Type, it is closer with mankind's Development process in time, clinical simulation can be carried out to the pulmonary fibrosis disease of the different courses of disease.
（2）It is external not only to remain body by the pattern of bleomycin and chemotactic factor (CF) administering drug combinations for the method for the present invention
Factor causes alveolar epithelial cells to convert fibroblast the damaging action repeatedly of lung tissue, it is also creative using CXCR4 by
Fibroblast in the ligand CXCL 12 of body and ligand CXCL19 or the CXCL21 guide blood of CCR7 receptors is to pulmonary lesion
Place's aggregation promotes lung tissue's fibrosis, consistent with clinical patient's IPF pathogenesis, and people is imitated from molecular level height
Body fibrosis lesion process.
Description of the drawings
Fig. 1 is 12 administering drug combinations of ligand CXCL of 1 bleomycin of the embodiment of the present invention and Chemokines CC XCR4 receptors
The lung sections Masson dyeing of mode construction pulmonary fibrosis animal model（× 200）Light microscopic figure under microscope mirror.
Fig. 2 is the mould of the ligand CXCL19 administering drug combinations of 2 bleomycin of the embodiment of the present invention and Chemokines CC CR7 receptors
Formula builds the lung sections Masson dyeing of pulmonary fibrosis animal model（× 200）Light microscopic figure under microscope mirror.
Fig. 3 is the lung sections Masson of the isometric bleomycin structure pulmonary fibrosis animal model of the single administration of comparative example 1
Dyeing（× 200）Light microscopic figure under microscope mirror.
Fig. 4 is the lung sections Masson of the isometric physiological saline structure pulmonary fibrosis animal model of the single administration of comparative example 2
Dyeing（× 200）Light microscopic figure under microscope mirror.
Specific implementation mode
With reference to embodiment and attached drawing, the invention will be further described.
The ligand of ligand CXCL12, CCR7 receptor of Chemokines CC XCR4 receptors used in the embodiment of the present invention
CCL19 is purchased from Sigma-Aldrich (USA)；Bleocin HC1 vial（BLM）Purchased from Nippon Kayaku K. K；
Tri- color reagent boxes of Masson are purchased from Shanghai Rui Sai Bioisystech Co., Ltd（Pudong, Shanghai）.
Embodiment 1
Utilize the mode construction pulmonary fibrosis animal mould of bleomycin and 12 administering drug combinations of ligand CXCL of Chemokines CC XCR4
Type：
Administration frequency：Bleomycin is administered once every 2 weeks, continuous 8 weeks；CXCL12 is administered once a day；It is rich next mould to stop administration
Element stops that CXCL12 is administered after 1 week again.
Dosage：Bleomycin is administered by mouse weight 2 mg/kg or 0.1 U/kg；By 0.1 mg/kg of mouse weight
Chemokines CC XCL 12 is administered.
Administration route：Intratracheal instillation.
Embodiment 2
Utilize the mode construction pulmonary fibrosis animal of bleomycin and 19 administering drug combinations of ligand CXCL of Chemokines CC CR7 receptors
Model：
Administration frequency：Bleomycin is administered twice every 2 weeks, continuous 8 weeks；CXCL19 is administered once a day；It is rich next mould to stop administration
Element stops that CXCL19 is administered after 1 week again.
Dosage：Bleomycin is administered by mouse weight 4 mg/kg or 0.4 U/kg；By mouse weight 0.4mg/kg
Chemokines CC XCL 19 is administered.
Administration route：Tail vein injection.
The modeling effect assessment of embodiment 1 and embodiment 2：
Pulmonary fibrosis animal model pathological section is carried out to Example 1 and Example 2 of the present invention using random controls zoopery
Characterization and evaluation modeling effect.
Random controls zoopery described in the embodiment of the present invention are Central-South in Hunan Province in March, 2017 in March, 2018
The experimental animal department of the Chinese Academy of Sciences of university Animal Lab. is completed.
Experimental animal in the random controls zoopery：Male SPF grades of C57BL/6 mouse 200, age of mouse when buying
It it is 3~5 weeks, weight is 16~20 g, is provided by the experimental animal department of the Chinese Academy of Sciences of Central South University animal experimental center, Mouse feeder is in SPF
In animal house, give SPF grades of feeds, high pressure sterilization water is fed, bedding and padding are through high-temp sterilizing, 22~24 DEG C of animal house room temperature,
Humidity 40%~70%, sunshine and 12 h of night alternating.
Experiment packet in the random controls zoopery：Mouse adaptable fed is tested after 1 week.It is 200 small
Mouse is divided into four groups, i.e. embodiment 1, embodiment 2, comparative example 1, comparative example 2 at random.Implementation is randomly selected respectively after completing administration
10 mouse in example 1, embodiment 2, comparative example 1, comparative example 2, cervical dislocation put to death animal, left lung are then taken to be put into 10%（v/v）
It is fixed in formalin, through dehydration, embedding, slice, carry out Masson dyeing.Lung sections pathological identification after dyeing see Fig. 1~
4。
Fig. 1 is embodiment 1（12 administering drug combinations of ligand CXCL of bleomycin and Chemokines CC XCR4）The lung of structure is fine
The lung sections Masson dyeing of dimensionization animal model（× 200）Light microscopic figure under microscope mirror.It can be seen that there are big rule in lung tissue
The consolidation of mould, consolidation area alveolar wall thickening, interval fracture, fusion form pulmonary belb, there are a large amount of agglomerating fibrosis lesions and dissipate
In inflammatory cell infiltration, alveolar septum thickens and perivascular collagen proteinosis.
Fig. 2 is embodiment 2（19 administering drug combinations of ligand CXCL of bleomycin and Chemokines CC CR7 receptors）The lung of structure
The lung sections of fibrosis animal model, it is seen that a large amount of collagen depositions of alveolar septum, collagen deposition position are predominantly located at pleura
Interstitial lung around lower or blood vessel, in point stove sample, group's piece sample distribution, the lighter lung tissue phase with the lung tissue or lesion of surrounding normal
It is adjacent（Special heterogeneity）；Compared with comparative example 1, fibrosis patch shaded area obviously increases.
Fig. 3 is comparative example 1（It is single to give the bleomycin isometric with embodiment 1）, it is seen that lung tissue occurs different
The consolidation of degree, consolidation area alveolar wall thickening, interval fracture, fusion form pulmonary belb, and interstitial has more inflammation, cellular infiltration
It is formed with fibroid tissue, there is single agglomerating fibrosis lesion and is dispersed in inflammatory cell infiltration.
Fig. 4 is comparative example 2（Single administration and the isometric physiological saline of embodiment 1）, it is seen that alveolar form is normal, alveolar
Wall is very thin, there is minimal amount of inflammatory cell infiltration in interstitial.
In conclusion the method for the present invention using bleomycin multiple dosing and CXCL12 or bleomycin multiple dosing and
The animal model of the mode construction of CCL19 is compared to the mouse lung tissue fibrosis higher of single administration bleomycin, effect
Fruit is more preferable, and mouse does not occur death.
Claims (9)
1. a kind of construction method of pulmonary fibrosis animal model, which is characterized in that using can cause can pulmonary fibrosis chemistry or
The mode construction pulmonary fibrosis animal model of biological agent and chemotactic factor (CF) administering drug combinations.
2. the construction method of pulmonary fibrosis animal model according to claim 1, which is characterized in that the mould of the administering drug combinations
The biological agent of pulmonary fibrosis can be caused to be bleomycin in formula.
3. the construction method of pulmonary fibrosis animal model according to claim 1 or claim 2, which is characterized in that the administering drug combinations
Pattern in chemotactic factor (CF) be the ligand guided corresponding to the cell receptor that is migrated of fibroblast.
4. according to the construction method of one of claims 1 to 3 pulmonary fibrosis animal model, which is characterized in that the chemotactic
The factor includes the ligand CXCL 12 of CXCR4 receptors and the ligand CCL19 or CCL21 of CCR7 receptors.
5. according to the construction method of one of Claims 1 to 4 pulmonary fibrosis animal model, which is characterized in that the animal
Experimental animal used in model includes rodent, rabbit, canine, porcine, sheep class or monkey class.
6. according to the construction method of one of Claims 1 to 5 pulmonary fibrosis animal model, which is characterized in that the grinding tooth
Class includes mouse, rat, hamster or cavy.
7. according to the construction method of one of claim 1~6 pulmonary fibrosis animal model, which is characterized in that the joint
The administration frequency of the pattern of administration is：Bleomycin is administered 1~2 time every 2 weeks, continuous 4~10 weeks；Chemotactic factor (CF) daily 1~3
It is secondary, stop administration bleomycin and stops that chemotactic factor (CF) is administered after 1~3 week.
8. according to the construction method of one of claim 1~7 pulmonary fibrosis animal model, which is characterized in that the joint
The dosage of the pattern of administration is：Bleomycin is administered by mouse weight 2~8 mg/kg or 0.1~2.0 U/kg；By small
Mouse weight 0.1~1.0 mg/kg administration Chemokines CCs XCL 12 or CCL19 or CCL21.
9. according to the construction method of one of claim 1~8 pulmonary fibrosis animal model, which is characterized in that the joint
The administration route of the pattern of administration is Intratracheal instillation, intravenous injection or intraperitoneal injection.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810382567.8A CN108524912A (en) | 2018-04-26 | 2018-04-26 | A kind of construction method of pulmonary fibrosis animal model |
| HK19100380A HK1255800A2 (en) | 2018-04-26 | 2019-01-10 | Construction method of an animal model of lung fibrosis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810382567.8A CN108524912A (en) | 2018-04-26 | 2018-04-26 | A kind of construction method of pulmonary fibrosis animal model |
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| CN108524912A true CN108524912A (en) | 2018-09-14 |
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| CN201810382567.8A Withdrawn CN108524912A (en) | 2018-04-26 | 2018-04-26 | A kind of construction method of pulmonary fibrosis animal model |
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| HK (1) | HK1255800A2 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112868596A (en) * | 2020-10-30 | 2021-06-01 | 广州医科大学附属第一医院（广州呼吸中心） | Pulmonary fibrosis rat model, and establishment method and evaluation method thereof |
| CN115838684A (en) * | 2023-02-13 | 2023-03-24 | 淇嘉科技(天津)有限公司 | In-vitro accurate simulation method for pulmonary fibrosis |
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| CN101351545A (en) * | 2005-12-23 | 2009-01-21 | 密执安大学评议会 | Materials and methods for treating chronic fibrotic disease |
| US20090131359A1 (en) * | 2007-11-19 | 2009-05-21 | Atamas Sergei P | Antifibrotic therapy |
| CN107115343A (en) * | 2017-05-16 | 2017-09-01 | 温州市人民医院 | A kind of Rat Pulmonary fibrosis model |
| CN107469069A (en) * | 2017-08-18 | 2017-12-15 | 四川普莱美生物科技集团有限公司 | Modeling method, preparation, the preparation method of preparation, rhesus macaque pulmonary fibrosis model and its application of rhesus macaque pulmonary fibrosis model |
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2018
- 2018-04-26 CN CN201810382567.8A patent/CN108524912A/en not_active Withdrawn
-
2019
- 2019-01-10 HK HK19100380A patent/HK1255800A2/en unknown
Patent Citations (4)
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|---|---|---|---|---|
| CN101351545A (en) * | 2005-12-23 | 2009-01-21 | 密执安大学评议会 | Materials and methods for treating chronic fibrotic disease |
| US20090131359A1 (en) * | 2007-11-19 | 2009-05-21 | Atamas Sergei P | Antifibrotic therapy |
| CN107115343A (en) * | 2017-05-16 | 2017-09-01 | 温州市人民医院 | A kind of Rat Pulmonary fibrosis model |
| CN107469069A (en) * | 2017-08-18 | 2017-12-15 | 四川普莱美生物科技集团有限公司 | Modeling method, preparation, the preparation method of preparation, rhesus macaque pulmonary fibrosis model and its application of rhesus macaque pulmonary fibrosis model |
Non-Patent Citations (1)
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| 任锦: "趋化因子受体CXCR4在大鼠肺纤维化模型肺组织中的表达和意义", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112868596A (en) * | 2020-10-30 | 2021-06-01 | 广州医科大学附属第一医院（广州呼吸中心） | Pulmonary fibrosis rat model, and establishment method and evaluation method thereof |
| CN115838684A (en) * | 2023-02-13 | 2023-03-24 | 淇嘉科技(天津)有限公司 | In-vitro accurate simulation method for pulmonary fibrosis |
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| Publication number | Publication date |
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| HK1255800A2 (en) | 2019-08-23 |
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Application publication date: 20180914 |