CN117426352A - Construction method and application of interstitial lung disease variable animal model - Google Patents
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- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 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
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- 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
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- 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
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- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a construction method of an animal model and application thereof, wherein periodontal pathogens are applied to induce a variable animal model of interstitial lung diseases, and further type II collagen and an adjuvant are applied to improve the severity of interstitial lung lesions. The pathological features of the animal model are highly similar to those of human beings, and better simulate the joint performance and lung performance of environmental factors (such as smoking patients and periodontitis patients) in RA-ILD.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a construction method and application of an interstitial lung disease variable animal model.
Background
Interstitial lung lesions (interstitial lung disease, ILD) are a general term for clinical-pathological entities consisting of groups of different kinds of diseases with diffuse lung parenchyma, alveolar inflammation and interstitial fibrosis as the pathological basis, with active dyspnea, diffuse infiltration shadows of X-ray chest, restrictive ventilation disorders, reduced Diffuse (DLCO) function and hypoxia as clinical manifestations.
ILD is not generally malignant nor considered to be caused by known infectious agents at present, but the most common extra-articular manifestations of rheumatoid arthritis (rheumatoid arthritis, RA) occur in about 40-80% of RA patients with mortality rates of 10-20% and average survival times of 5-8 years. ILD prognosis is extremely poor and is one of the leading causes of death in RA patients.
The establishment of an experimental animal model is an important basis for researching pathogenesis and developing innovative medicaments. Although RA animal models such as collagen-induced arthritis (CIA) and adjuvant-induced arthritis (AA) mimic the clinical symptoms and pathological features of arthritis very well, ILD morbidity is low. The tracheal injection of bleomycin is the most common method of ILD at present, but the method has simple operation and low cost, but the range of lung lesions is limited, and the method has differences with the diffuse distribution of human lesions and can cause irreversible organic injury to animals. It is therefore desirable to establish an animal model of RA-ILD that has both RA characteristics and high ILD incidence and significant ILD symptoms.
According to the prior literature, animal models of RA-ILD are mainly CIA, AA, collagen-induced bleomycin type (CIA+BLM), transgenic mice and zymosan-combined animal models of transgenic mice. However:
(1) The most used CIA model and AA model are common animal models of RA, and the incidence rate of the lung interstitial lesions of the CIA model and the AA model based on RA is low.
(2) The cia+blm model (e.g. CN110604098A, CN 116076438A) has a limited range of lung lesions, is different from the diffuse distribution of human lesions, and can cause irreversible organic injury to animals.
(3) Transgenic mice and transgenic mice combine with zymosan animal models, requiring a long time to develop, and are unable to replicate completely the associated pulmonary pathological manifestations of the major type UIP (interstitial pneumonia) in RA-ILD.
In addition, environmental factors including smoking and periodontitis are recognized environmental triggers that can cause inflammation in the lungs, and are also key risk factors for ILD occurrence in RA patients. It may induce the formation of autoantibodies in RA patients, associated with high titers of anti-Cyclic Citrullinated Peptide (CCP) antibodies. None of the animal models involved environmental factors, and therefore the model was different from the RA-ILD generation mechanism.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides an RA-ILD animal model which is established by combining periodontal pathogen infection with type II collagen and has RA characteristics, high ILD incidence rate and obvious ILD symptoms. Specific:
in a first aspect of the invention, there is provided a method of constructing an animal model comprising administration of periodontal pathogens, said animal model being a model of an altered interstitial lung disease.
Preferably, the periodontal pathogens include, but are not limited to, porphyromonas gingivalis @Porphyromonas gingivalisPg), fusarium, and the likeTannerella forsythia) Scale dense screw bodyTreponema denticola) Actinobacillus accompanied by actinobacillusActinobacillus actinomycetemcomitans) Fusobacterium nucleatumFusobacterium nucleatum) Prevotella intermediaPrevotella intermedia) Black-streaked Proteus spPrevotella nigrescens) Or actinomycetes viscosusActinomyces viscosus) One or a combination of two or more of them.
In one embodiment, the periodontal pathogen may be Porphyromonas gingivalis @, the periodontal pathogen isPorphyromonas gingivalis). Or porphyromonas gingivalis @, is preparedPorphyromonas gingivalis) With other periodontal pathogens (e.g. FusariumTannerella forsythia) Scale dense screw bodyTreponema denticola) Actinobacillus accompanied by actinobacillusActinobacillus actinomycetemcomitans) Fusobacterium nucleatumFusobacterium nucleatum) Prevotella intermediaPrevotella intermedia) Black-streaked Proteus spPrevotella nigrescens) Or actinomycetes viscosusActinomyces viscosus) One or a combination of two or more of them).
Preferably, the periodontal pathogen is administered every 1-7 (e.g., 1, 2, 3, 4, 5, 6, 7) days.
Preferably, the periodontal pathogen is administered at a rate of 1X 10 per administration 7 -1×10 10 (e.g. 1X 10) 7 、2×10 7 、3×10 7 、4×10 7 、5×10 7 、6×10 7 、7×10 7 、8×10 7 、9×10 7 、1×10 8 、2×10 8 、3×10 8 、4×10 8 、5×10 8 、6×10 8 、7×10 8 、8×10 8 、9×10 8 、1×10 9 、2×10 9 、3×10 9 、4×10 9 、5×10 9 、6×10 9 、7×10 9 、8×10 9 、9×10 9 、1×10 10 ）CFU。
Preferably, the total number of days of administration is 45-60 (e.g., 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60) days.
Preferably, the periodontal pathogen administration mode includes, but is not limited to, smearing administration, oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection or intravenous injection, and further preferably, the administration mode is smearing administration, more preferably, oral smearing, and even more preferably, smearing at the gingival margin of the upper and lower jaw.
Preferably, the method of construction further comprises administering type II collagen. Preferably, the type II collagen is administered at least once, for example 1, 2, 3 or more than 4 times; preferably, the interval between two administrations of type II collagen is 15-25 (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) days.
The period between the first administration of periodontal pathogen and the first administration of type II collagen is 8-20 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) days, and more preferably 14 days. Preferably, the period of time between the first administration of periodontal pathogens and the first administration of type II collagen and adjuvant is 8-20 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20) days.
Further preferred, the method further comprises administering an adjuvant. Even more preferred is the combined administration of type II collagen and an adjuvant. It is further preferred that the adjuvant is administered concurrently with the type II collagen. Preferably, the adjuvant includes, but is not limited to, one or a combination of two or more of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, alum, heat shock protein or cytokine.
In one embodiment, the method of construction is to administer only periodontal pathogens, type II collagen and an adjuvant.
Preferably, the method of construction comprises administration of periodontal pathogens followed by administration of type II collagen (preferably in combination with an adjuvant).
Preferably, the type II collagen and/or adjuvant is administered by a method including, but not limited to, a topical administration, an oral administration, an intradermal injection, a subcutaneous injection, an intramuscular injection, an intraperitoneal injection, or an intravenous injection.
Preferably, the type II collagen and the adjuvant are administered in equal volumes.
In one embodiment, the molding method comprises applying 1×10 daily to the gingival margin of the upper and lower jaws of the non-human animal from day 1 7 -1×10 10 CFU (e.g. 1X 10) 7 -1×10 10 Pg of CFU/mL of Porphyromonas gingivalis 10-1000. Mu.L of PBS with 2% CMC)Porphyromonas gingivalis) Continuing until the molding is finished; immunization with a combination of type II collagen and an adjuvant (wherein type II collagen and adjuvant are in equal volumes) was performed on days 8-20 and 30-40, respectively.
Preferably, the animal model is an animal model of arthritis combined with an animal model of interstitial lung disease, and more preferably, the arthritis is rheumatoid arthritis.
Preferably, the animal model may be any mammal other than a human, for example, a horse, sheep, pig, goat, camel, antelope, dog or rodent such as a mouse, rat, guinea pig and hamster. Particularly preferred are rodents such as mice, rats, guinea pigs and hamsters.
Preferably, the animal model is a rat or mouse having immune susceptibility.
In one embodiment, the species of mouse include, but are not limited to: ICR or DDY, BALB/cA, C57BL/6N, C H/HeN, DBA/1J, DBA/2N or CBA/N, BDF1 (C57 BL/6 x DBA/2), CDF1 (CBA/N x DBA/2) or B6C3F1 (C57 BL6 x C3H/HeN), anything used as an experimental mouse, such as BALB/C-nu or C, B-17SCID belonging to a mutant system, may be used.
Preferably, the method of construction further comprises administering an antibiotic prior to administration of periodontal pathogens, and more preferably, the antibiotic includes, but is not limited to, ampicillin, vancomycin, neomycin, metronidazole, or tetracycline.
In a second aspect of the present invention, there is provided an animal model obtained by the above-described construction method.
Preferably, the animal model develops symptoms of joint swelling, synovitis, pannus, cartilage destruction, bone destruction, and associated alveolar inflammatory lesions, interstitial inflammatory cell infiltration, and alveolar wall thickening.
In a third aspect, the invention provides an animal model obtained by the construction method or application of the animal model in screening or preparing medicines for treating interstitial lung lesions or arthritis combined interstitial lung lesions.
The application is realized by periodontal pathogen Porphyromonas gingivalisPorphyromonas gingivalisPg) in combination with type II collagen-induced arthritis (Pg+CIA) an RA-ILD animal model was established, together confirming RA occurrence by mouse arthritis index scoring, knee HE staining, anti-CCP antibody detection, and immunoglobulin IgG results. Meanwhile, the incidence and the incidence of mice are counted, and the occurrence of ILD is confirmed by combining HE staining and Masson staining of mouse tissues. With the prior RA-ILDCompared with the animal model of the application, the animal model has the following advantages:
(1) ILD incidence rate of Pg+CIA model is high.
(2) Since Pg is the main risk factor for RA caused by periodontitis, the pg+cia model better mimics joint and lung performance in RA-ILD for smokers and periodontitis patients.
(3) The lung HE and Masson staining pathology reports of mice in the Pg+CIA model group show that the pathological changes are high, and the pathological changes are similar to human beings in characteristics.
The terms "comprising" or "includes" are used in this specification to be open-ended, having the specified components or steps described, and other specified components or steps not materially affected.
Drawings
Fig. 1: clinical features of DBA/1J mice, A is a representative photograph of hind paws; b is an arthritis score. Wherein,P<0.01 vs Control group.
Fig. 2: DBA/1J mouse autoantibody detection, wherein,P<0.01 vs Control group; # P<0.01，#P<0.05 vs pg+cia group;&&P<0.01 vs CIA group.
Fig. 3: knee pathology staining (A) and pathology scoring (B), wherein,P<0.01 vs Control group; # P<0.01 vs pg+cia group;&&P<0.01 vs CIA group.
Fig. 4: micro-CT imaging detection and bone metrology parameter detection results, wherein,P<0.01 vs Control group; # P<0.01，#P<0.05 vs pg+cia group;&&P<0.01，&P<0.05 vs CIA group; BMD is knee joint bone density, BV/TV is bone volume fraction, tb.Th is bone trabecular thickness, tb.N is bone trabecularNumber of beams.
Fig. 5: lung tissue pathology staining and pathology scoring results,P<0.01 vs Control group, #P<0.01，#P<0.05 vs pg+cia group.
Fig. 6: lung tissue Masson staining and collagen area ratio,P<0.01 vs Control group; # P<0.01 vs pg+cia group.
Fig. 7: expression levels of lung tissue α -SMA, col I, FN protein and KL-6 mRNA expression levels (A) (B) expression levels of lung tissue α -SMA, col I, FN protein of each group of mice (immunohistochemistry, ×400); (C) expression levels of α -SMA, col I, FN, and KL-6 mRNA; wherein, P <0.01 vs Control group #)P<0.05 vs Pg+CIA group #)P <0.01 vs pg+cia group.
Fig. 8: expression levels of TNF-alpha, IL-6, IL-1β mRNA in lung tissue,P<0.01 vs Control group;&&P<0.01 vs CIA group; #P<0.05 vs Pg+CIA group; # P<0.01 vs pg+cia group.
Detailed Description
In order to better understand the technical solutions of the present invention, the following description will clearly and completely describe the technical solutions of the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Materials, reagents, instruments and the like used in the examples described below are commercially available unless otherwise specified.
Examples
1. Experimental methods and materials
1.1 Experimental animal
40 male 6 week old DBA/1J mice were purchased from Beijing Fukang Biotech Co., ltd. [ license number: SYXK (jing) 2019-0008], experimental unit use license number: SYXK (Beijing) 2021-0017. Feeding in environment of 22+ -2deg.C, and freely feeding and drinking water, and adapting to 7 days after feeding for subsequent experiment. Animal welfare and experimental procedures in this study both followed the relevant specifications of the ethical committee of animals of the chinese academy of medicine.
1.2 Medicine and reagent
Pg（Porphyromonas gingivalisPg) strain (ATCC # 33277) was purchased from North Naraxacum Biotechnology Co., ltd; columbia blood agar medium was purchased from Bikman Biotech Co.Ltd; bovine collagen two acetic acid solution (cat No. 2002-2) and complete Freund's adjuvant (cat No. 7001) were both purchased from Chondrex, U.S.A.; mouse Anti-cyclic citrullinated peptide antibody (Anti-CCP-Anti) ELISA kit (cat# CSB-EQ027743 MO) was purchased from CUSBIO company; the detection kit of the antibody of the porphyromonas gingivalis (goods number: ml 023665-J) and the detection kit of the immunoglobulin IgG of the mice (goods number: ml 057874) are all purchased from Shanghai enzyme-linked biotechnology Co., ltd; masson's trichromatic staining solution kit (lot number: C210601) was purchased from Pinctada martensii Biotechnology Co., ltd; DEPC enzyme-free water was purchased from Beijing blue Jiek technology Co., ltd (cat# BL 510B), reverse transcription kit (cat# AE 311) was purchased from full gold biotechnology Co., ltd, well as century Ultra SYBR Mixture (High ROX), absolute ethanol (cat# 100092683), environment-friendly dewaxing clear solution (cat# G1128), 20 Xcitric acid antigen retrieval solution (pH 6.0) (cat# G1202), 20 XTris-EDTA antigen retrieval solution (pH 9.0) (cat# G1203), 20 XTris-EDTA antigen retrieval solution (pH 8.0) (cat# G1206), hematoxylin dye solution (cat# G1004), hematoxylin differentiation solution (G1039), hematoxylin blue returning solution (cat# G1040), and DAB color developer (WW W1212) were purchased from Hansai Violet biotechnology Co., ltd.
1.3 Experimental instrument
Mitsubishi 2.5LMGC anaerobic tank, anaerobic gas bag (Mitsubishi chemical corporation, japan); multiskan MK3 microplate reader (Thermo Fisher Scientific). Sheet roasting machine (KPJ-1A), rotary slicing machine (QPJ-C), biological tissue embedding machine (BMJ-1B) (Tianjin Tianli aircraft electric Co., ltd.); BX50 type front-mounted microscope (olympus (japan)); microcomputer tomography Micro-CT (GE Healthcare Co., RS 0800604-0063), vortex mixing apparatus from Ningbo brand new Zhi Biotechnology Co., ltd., precooling centrifuge from Shanghai Chu Bai laboratory equipment Co., ltd., refiner from Ai Ka (Guangzhou) instruments Co., ltd., PCR apparatus (Yes, germany), simer FeqPCR apparatus.
1.4 Bacterial activation
About 0.3-0.5ml of sterile water or liquid culture medium is injected into the freeze-drying tube, gently blown, and fully dissolved into bacterial suspension. The bacterial suspension was aspirated, homogenized by pouring it onto the surface of 2 Columbia blood agar medium (about 200 uL/min), spread homogeneously, and equilibrated with a mixture of 90% nitrogen, 5% carbon dioxide and 5% hydrogen in an anaerobic tank and anaerobic gas-generating bag. After activation is successful, a correlation test can be performed. Bacterial cell count was determined by spectrophotometry at a spectrophotometry of 1.0 at 600nm, corresponding to 1X 10 9 CFU/ml. Then collect 10 9 CFU/ml bacteria were resuspended in PBS.
1.5 Modeling and grouping
The experimental animals were randomly divided into four groups, namely a normal Control group (Control), a collagen-induced group (CIA), a porphyromonas simplex infection group (Pg), and a collagen-induced combined porphyromonas gingivalis infection group (pg+cia). All mice were acclimatized for 7 days, and within three days prior to acclimatization, all groups were fed ampicillin (10 mg/500 mL) to eliminate endogenous bacteria that were detrimental to Pg colonization. The specific molding method of each group is as follows: (1) Control group: 200 mu L of PBS solution of 2% CMC (carboxymethyl cellulose) is smeared on the gingival margin of the upper jaw and the lower jaw of the mouse every day from the 1 st day, and the molding is finished; (2) CIA group: 200 mu L of PBS solution with 2% CMC is smeared on the gingival margin of the upper jaw and the lower jaw of the mouse every day from the 1 st day, and the operation is continued untilAnd finishing the molding. Immunization with equal volumes (100 μl each) of CII (collagen type II, 2 mg/ml) and CFA (complete Freund's adjuvant, 4 mg/ml) emulsion at day 14 and day 35, respectively, was injected subcutaneously in the tail of the mice; (3) Pg group: the gingival margin of the upper jaw and the lower jaw of the mouse is smeared with the composition containing 1 multiplied by 10 every day from the 1 st day 9 100 mu L of PBS solution of 2% CMC of Pg of CFU/mL is continued until the molding is finished; (4) Pg+CIA group: the gingival margin of the upper jaw and the lower jaw of the mouse is smeared with the composition containing 1 multiplied by 10 every day from the 1 st day 9 100. Mu.L of PBS solution of 2% CMC at Pg of CFU/mL was continued until the molding was completed. Equal volumes (100 μl each) of the emulsion were immunized with CII and CFA, respectively, on days 14 and 35, and injected subcutaneously in the tail of the mice. The number of molding days was 56. Wherein, the CII specifically uses bovine type II collagen acetic acid solution.
1.6 Morbidity and arthritis scoring
RA morbidity in mice was counted = number of mice per group/total number of mice per group x 100%. Arthritis score the mice were evaluated for 4 ankle joints, 4 midfoot joints, 12 toe joints with reference to table 1.
Table 1: arthritis scoring
1.7 ELISA detection
Mice were sacrificed, and eyeballs were harvested for blood collection, and serum was obtained after standing and centrifugation. Taking a 96-well plate of the kit, adding 50ul of diluted standard substance into the reaction well, and adding 50ul of sample to be detected into the reaction well. 50ul of biotin-labeled antibody was immediately added to the reaction well, and the well plate was attached with a sealing film, and incubated for 60 minutes with shaking at 37 ℃. The liquid in the 96-well plate was thrown off and patted dry, then 200ul of washing liquid was added to each well, and the plate was washed repeatedly 3 times. Plates were washed and then dried by patting, 80 ul affinity streptavidin-HRP was added to each well and incubated for 30 minutes with shaking at 37 ℃. The liquid in the 96-well plate was thrown off and patted dry, then 200ul of washing liquid was added to each well, and the plate was washed repeatedly 3 times. To the reaction wells, 100ul of an equal volume of a mixed solution of substrate A (color former A (hydrogen peroxide) in the kit is hydrogen donor (DH 2)) and substrate B (color former B (TMB) for color development) was added, and incubated for 10 minutes with shaking at 37℃and light was avoided. Finally, the 96-well plate is taken out, 50ul of stop solution is added into the reaction well, and absorbance is detected in a Multiskan MK3 microplate reader immediately after the addition is completed. OD values of each well were measured at the wavelength designated by the kit (mouse Anti-cyclic citrullinated peptide antibody (Anti-CCP-Anti) ELISA kit, mouse porphyromonas gingivalis antibody detection kit, mouse immunoglobulin IgG detection kit).
1.8 Knee joint HE staining
The knee joint is taken, paraformaldehyde is fixed, 10% EDTA is used for decalcification at a low speed, paraffin is used for embedding by using a biological tissue embedding machine, and a rotary slicing machine is used for slicing for standby. Placing the slices on a slice baking machine for baking at 60 ℃ for 30 min, dewaxing the slices in xylene and gradient ethanol to water, sequentially placing the carrier slices in hematoxylin dye solution and eosin dye solution for dyeing, washing with running water, returning to blue with 0.5% ammonia water after differentiation of the differentiation solution, and finally washing with running water again. After the sections are slightly dried, the sections are sealed by neutral resin, and observed and photographed under a BX50 type forward microscope. Specific observations and scoring criteria the higher score in table 2 below suggests a higher lesion level.
Table 2: knee joint pathology scoring table
1.9 Micro-CT scanning bone metrology analysis of inflammatory knee joint
The knee joint is placed in 10% formaldehyde solution for fixation for 48 hours, then Micro-CT scanning is carried out under the resolution of 25 mu m, the single time length of knee joint tissue scanning to be scanned is 72min, joint reconstruction is carried out after Micro-CT scanning shooting, and simultaneously bone metering parameter analysis is carried out. Bone metrology parameters such as bone density (bone mineral density, BMD), bone volume fraction (BV/TV), bone trabecular thickness (trabecular thickness, tb. Th), bone trabecular number (trabecular number, tb. N) were statistically analyzed for each group of mice.
1.10 HE staining of lung tissue
The rotary slicing machine is used for slicing, dewaxing is carried out until water is carried out, hematoxylin dye solution is 10min, tap water is fully washed, differentiation solution is 5s, tap water is fully washed, blue reflection liquid is reversely blue for 5s, tap water is fully washed, 0.1% eosin is 1min,95% ethanol is 2min,100% ethanol is 3min,100% ethanol is II 3min, xylene is 7min, xylene is II 7min, neutral gum is sealed, and observation and photographing are carried out under a BX50 type forward microscope. The pathological changes (degree of alveolar inflammatory lesions, interstitial inflammatory cell infiltration, degree of alveolar wall thickening) are marked as "+ + + +", the symptoms of slight, moderate and severe are respectively marked by 1-4 points, no lesions are marked as 0 points, and no deficiency is marked. The individual scores were the sum of the individual lesion scores (degree of alveolar inflammatory lesions, degree of interstitial inflammatory cell infiltration, degree of alveolar wall thickening) and specific scoring criteria are shown in table 3.
Table 3: lung tissue pathology scoring criteria
1.11 Masson staining
Placing the slices on a slice baking machine for baking at 65 ℃ for 1h, dewaxing the slices in xylene and gradient ethanol until the slices are water, sequentially placing the slices into hematoxylin dye solution, ponceau, phosphomolybdic acid and aniline blue for dyeing, washing with 1% glacial acetic acid solution, and dehydrating the xylene by the gradient ethanol until the xylene is transparent. After the sections are slightly dried, the sections are sealed by neutral resin, and observed and photographed under a BX50 type forward microscope. Under the light microscope (10×10) the same sample randomly selects 3 fields, calculates the collagen area ratio, adopts Image-Proplus to measure the collagen area and the total area of the whole Image, calculates the collagen area ratio, and calculates the collagen area ratio (%) = (collagen area/total area of the whole Image) ×100%. The experiments were specifically performed using the Masson trichromatic staining solution kit.
1.12 Immunohistochemistry of lung tissue
Dewaxing paraffin sections to water, then carrying out antigen retrieval, blocking endogenous peroxidase, sealing serum, dripping primary antibodies, dripping the primary antibodies prepared by PBS according to a certain proportion on the sections, and horizontally placing the sections in a kit for incubation at 4 ℃ overnight; dripping secondary antibody, incubating for 50min at room temperature, performing DAB color development, counterstaining cell nuclei, dehydrating and performing microscopic examination.
1.13 Detection of IL-1 beta, TNF-alpha and IL-6 of lung tissue by q-PCR
Extracting tissue RNA by TRIzol method, detecting concentration and purity, adjusting concentration, reversely transcribing into cDNA, using cDNA as template, detecting the expression of IL-1 beta, TNF-alpha, IL-6 mRNA by RT-qPCR. The upstream primer of GAPDH is AGTGGCAAAGTGGAGATT (SEQ ID NO: 1) and the downstream primer is GTGGAGTCATACTGGAACA (SEQ ID NO: 2); the IL-1β has an upstream primer of TCCTTGTGCAAGTGTCTGAAGC (SEQ ID NO: 3) and a downstream primer of ATGAGTGATACTGCCTGCCTGA (SEQ ID NO: 4); the TNF- α upstream primer was CACCACCATCAAGGACTCAA (SEQ ID NO: 5) and the downstream primer was AGGCAACCTGACCACTCTCC (SEQ ID NO: 6); the IL-6 has an upstream primer of CTCTGCAAGAGACTTCCATCCAGT (SEQ ID NO: 7) and a downstream primer of GAAGTAGGGAAGGCCGTGG (SEQ ID NO: 8).
1.14 Data processing and statistical analysis
Data statistics and plots were performed using GraphPad Prism 8.0.2 software and Image-Proplus software, and the results of the metering data are expressed as Mean (+ -Standard Deviation (SD).
2 experimental results
2.1 symptomatic manifestation after mouse modeling
The effect of Pg infection combined with collagen induction on RA occurrence in mice was evaluated, and first, the arthritis index, incidence and morbidity-related conditions were observed. The results show that the CIA group and the Pg+CIA group have obvious symptoms of joint redness, toe deformation, skin congestion, limited movement of part of joints and the like, the incidence rate is 100% (10/10), the clinical integral of arthritis is 70 and 68 respectively, and the clinical integral of the two groups has no statistical difference, and the specific see figure 1 and table 4.
Table 4: clinical characteristics of RA in different groups of DBA/1J mice
2.2 Serum antibody levels in model animals
The detection result of the mouse serum antibody shows that compared with the normal control group, the Pg groupThe expression level of the anti-Pg antibody is obviously increasedP<0.01 The content of IgG antibody of CIA group is obviously increasedP<0.01 The anti-Pg antibody and the IgG antibody of the Pg+CIA group are obviously increased and have statistical differenceP<0.01). Compared with CIA group, the IgG content of Pg group is obviously reducedP<0.01). Compared with Pg+CIA group, the expression level of anti-CCP antibody, anti-Pg antibody and IgG antibody in Pg group is obviously lessP<0.01 and 0.05) and the expression level of the CIA group anti-Pg antibody is reducedP<0.05, 0.01), as shown in particular in fig. 2.
2.3 Knee joint histopathological detection
The pathological characteristics of RA are chronic invasive arthritis, and histopathological observation of joints is the most important method for evaluating the morbidity characteristics and the disease severity of RA model animals in the current experimental study. The knee joint HE staining results show that the normal mice and the Pg mice have smooth joint surfaces, no pannus is formed, and the cells are orderly arranged. Both group CIA and pg+cia showed significant proliferation of synovial tissue around ankle cavity and protruding into joint cavity, joint gap was narrowed, the proliferated synovial tissue invaded subcutaneously, a fibrosclerotic focus was partially formed, massive inflammatory cell infiltration and granulation tissue proliferation in synovium, synovial pannus formation, and significant injury of bone and cartilage appeared, as shown in fig. 3. The pathological integral is carried out in 4 aspects of synovitis, pannus, cartilage destruction, bone destruction and the like.
2.4 Micro-CT imaging detection
The Micro-CT 3D reconstruction result shows that the knee joint of the Control group is smooth and complete, and the bone contour is clear. The Pg group showed slight bone erosion and joint cavity stenosis. The knee joint surfaces of the mice in the CIA group and the mice in the Pg+CIA group are loose and rough, the bone structure is eroded, and the bone tissue shows that a piece of honeycomb erosion points appear. Further bone metrology statistics showed that Pg+CIA groups BMD, BV/TV, tb.Th, tb.N were statistically different from Control and Pg groups (P < 0.05), as shown in FIG. 4.
2.5 Lung tissue HE staining pathology detection
The pathological characteristics of the lung interstitial lesions are alveolar and lung interstitial inflammatory cell infiltration, severe thickening of the alveolar wall, fibrosis and nodule formation, and histopathological observation of the lung are the most important methods for evaluating the disease characteristics and the disease severity of lung interstitial lesion model animals in the current experimental study. The lung tissue HE staining result shows that the lung tissue structure of the normal group mice is clear, the surface of the alveoli is smooth, no obvious deformation or rupture exists, the airway epithelial cells are orderly arranged, and the interstitial tissue is not infiltrated by inflammatory cells; group Pg mice have alveolar massive hemorrhage, edema, necrosis, moderate or severe thickening of alveolar septum, with mild or moderate inflammatory cell infiltration, predominantly lymphocytes and monocytes. The difference is obvious compared with the normal group; a small amount of fibrous tissue hyperplasia is visible in the interstitium and bronchi; the CIA mice have slightly or moderately thickened alveolar walls and slightly or slightly infiltrated inflammatory cells, mainly comprise lymphocytes and mononuclear macrophages, and have obvious differences compared with the normal group; the interstitial and bronchus have little fibrous tissue hyperplasia, and no statistical difference is seen compared with the normal group; the Pg+CIA group mice have large-area alveolar hemorrhage, edema and necrosis, the alveolar space is slightly to severely thickened, interstitial visible moderate or severe granulomatous inflammatory cell infiltration is distributed in a bulk shape, lymphocytes and monocytes are used as main materials, a small amount of neutrophil granulocyte is aggregated, interstitial and bronchus visible more fibrous tissue hyperplasia is obviously aggravated (P is less than 0.01) compared with the normal group, pg and model group. As shown in fig. 5, pathological integration was performed on the degree of alveolar inflammatory lesions, interstitial inflammatory cell infiltration, and the degree of alveolar wall thickening 3.
The Pg group can induce symptoms of interstitial lung disease, but after a certain period of administration of Pg, such as from the first immunization (day 14) or the second immunization (day 28), pg intervention cannot continue to significantly exacerbate lung lesions.
2.6 Masson staining of Lung tissue
Masson trichromatic staining is one of the most classical methods in connective tissue staining, also called Masson staining, is one of the main methods for showing fibers in tissues, and is an authoritative and classical technical method for staining collagen fibers. As shown in fig. 6, the lung tissue structure of the normal group mice is clear, the surface of the alveoli is smooth, no obvious deformation or rupture exists, the airway epithelial cells are orderly arranged, and the interstitial tissue is not infiltrated by inflammatory cells; large-area hemorrhage, edema and necrosis of alveoli, moderate or severe thickening of alveoli interval, mild or moderate inflammatory cell infiltration, mainly lymphocytes and monocytes are seen under Pg group microscope. The difference is obvious compared with the normal pair; a small amount of fibrous tissue hyperplasia is visible in the interstitium and bronchi; the CIA group can be used for observing the slight or moderate thickening of the alveolar wall, and the slight or slight inflammatory cell infiltration is carried out, and the lymphocyte and the mononuclear macrophage are mainly used, so that the difference is obvious compared with the normal group; the interstitial and bronchus have little fibrous tissue hyperplasia, and no statistical difference is seen compared with the normal group; the CIA+Pg group can be used for treating large-area alveolar hemorrhage, edema and necrosis, alveolar space is slightly-severely thickened, interstitial cells can be used for treating moderate or severe granulomatous inflammatory cell infiltration, the interstitial cells are distributed in a bulk shape, lymphocytes and monocytes are used as main components, a small amount of neutrophil granulocyte aggregates are also used for treating more fibrous tissue hyperplasia of the interstitial cells and bronchi, and the interstitial cells and bronchus are obviously aggravated (P is less than 0.01) compared with the normal group, the Pg model group and the CIA model group.
2.7 immunohistochemistry of Lung tissue
Compared with the normal control group, the expression levels of alpha-SMA, col I and FN protein in the lung tissues of the mice in the Pg group, the CIA group and the Pg+CIA group are obviously increased compared with the normal groupP<0.01 A) is provided; compared with Pg+CIA group, the expression level of ColI protein in Pg group is obviously reducedP<0.01 The expression level of alpha-SMA, col I and FN proteins in CIA group is obviously reducedP<0.05, 0.01); there was no significant difference in expression of the three proteins between the CIA and Pg groups. The result of the fibrosis related mRNA detection experiment shows that compared with the normal control group, the expression level of the Pg group alpha-SMA mRNA is obviously increasedP<0.01 The expression level of alpha-SMA and FN mRNA in CIA group is obviously increasedP<0.01 Pg+CIA group I Collagen (Collagen I, col I), alpha-SMA, FN and sialylated sugar chain antigen-6 (Correlation of Krebs von den Lungen-6, KL-6) mRNA expression levels were significantly increasedP<0.01 A) is provided; compared with Pg+CIA group, the expression quantity of alpha-SMA, FN and KL-6 mRNA in Pg group and CIA group is obviously reducedP<0.01, 0.05). The results are shown in FIG. 7, A, B, C.
2.8 Lung tissue q-PCR
The detection experimental result of inflammatory factor mRNA shows that compared with the normal control group, the Pg group tumor necrosis factor-alpha (tumor necrosis factor-alpha, TNF-alpha) mRNAThe expression quantity is obviously increasedP<0.01 The expression level of interleukin-1 beta (IL-1 beta) mRNA in CIA group is obviously increasedP<0.01 The expression level of Pg+CIA group TNF-alpha, interleukin-6 (IL-6) and IL-1 beta mRNA are obviously increasedP<0.01 A) is provided; compared with Pg+CIA group, the expression level of IL-1 beta mRNA in Pg group is obviously reducedP<0.01 IL-6 and TNF-alpha mRNA expression level in CIA group is obviously reducedP<0.05, 0.01); TNF-alpha mRNA expression level in Pg group is higher than that in CIA groupP<0.01). The results are shown in FIG. 8.
From the above results, it can be seen that Pg alone can induce only mild ILD, but not arthritis, CIA and Pg+CIA can both induce arthritis and ILD, and the pathological changes of the two groups of arthritis are similar; compared with the Pg and CIA groups alone, the ILD lesion degree of Pg+CIA, especially the fibrosis degree is obviously serious, the CCP resistant content is obviously increased, and the Pg+CIA can induce obvious RA-ILD, has high lesion degree of ILD and arthritis, has lesion characteristics similar to human beings, and better simulates joint performance and lung performance of smoking patients and periodontitis patients in RA-ILD.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications and substitutions may be made in the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and it is intended that all such modifications and substitutions be within the scope of the present invention/be within the scope of the present invention as defined by the appended claims.
Claims (10)
1. A construction method of an animal model is characterized by comprising the steps of applying periodontal pathogens, wherein the animal model is an interstitial lung disease variant model, and the periodontal pathogens comprise Porphyromonas gingivalis @Porphyromonas gingivalis）。
2. The construction method according to claim 1, wherein,the periodontal pathogen is administered once every 1-7 days, and the dosage of periodontal pathogen is 1×10 7 -1×10 10 CFU, the total number of days of administration is 45-60 days.
3. The method of claim 1, wherein the periodontal pathogen is administered by a method comprising a method selected from the group consisting of a smear, an oral method, an intradermal method, a subcutaneous method, an intramuscular method, an intraperitoneal method, and an intravenous method.
4. The method of claim 1, further comprising administering type II collagen; administering type II collagen at least once; the interval between two administrations of type II collagen is 15-25 days.
5. The method of claim 4, comprising administering an adjuvant concurrently with the type II collagen; the adjuvant comprises one or more than two of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide, alum, heat shock protein or cell factor.
6. The method of claim 5, wherein the type II collagen and/or adjuvant is administered by a method comprising administering, orally administering, intradermally injecting, subcutaneously injecting, intramuscularly injecting, intraperitoneally injecting, or intravenously injecting.
7. The method of claim 6, wherein the period between the first administration of periodontal pathogen and the first administration of type II collagen is 8-20 days; the interval between the first administration of periodontal pathogens and the first administration of type II collagen and adjuvant is 8-20 days.
8. The method of any one of claims 1-7, wherein the animal model is an arthritis combined interstitial lung disease variant model.
9. The method of claim 8, wherein the animal model is a non-human mammal.
10. Use of an animal model obtained by the construction method of any one of claims 1-9 for screening or preparing a medicament for treating interstitial lung lesions or arthritis combined with interstitial lung lesions.
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| US20170095573A1 (en) * | 2014-06-02 | 2017-04-06 | Baylor Research Institute | Methods and compositions for treating allergy and inflammatory diseases |
| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
| CN112924671A (en) * | 2021-01-28 | 2021-06-08 | 中国医学科学院北京协和医院 | Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof |
| CN115088676A (en) * | 2022-07-08 | 2022-09-23 | 中国中医科学院中药研究所 | Method for constructing ACPA positive bone erosion rheumatoid arthritis animal model |
| CN116076438A (en) * | 2023-03-21 | 2023-05-09 | 湖南中医药大学 | Animal model for rheumatoid arthritis combined with interstitial lung disease, construction method and application thereof |
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| US20170095573A1 (en) * | 2014-06-02 | 2017-04-06 | Baylor Research Institute | Methods and compositions for treating allergy and inflammatory diseases |
| CN110604098A (en) * | 2019-09-23 | 2019-12-24 | 广东省中医院（广州中医药大学第二附属医院、广州中医药大学第二临床医学院、广东省中医药科学院） | Method for constructing animal model of rheumatoid arthritis combined with interstitial lung disease |
| CN112924671A (en) * | 2021-01-28 | 2021-06-08 | 中国医学科学院北京协和医院 | Biomarker for diagnosing rheumatoid arthritis combined with pulmonary interstitial fibrosis and application thereof |
| CN115088676A (en) * | 2022-07-08 | 2022-09-23 | 中国中医科学院中药研究所 | Method for constructing ACPA positive bone erosion rheumatoid arthritis animal model |
| CN116076438A (en) * | 2023-03-21 | 2023-05-09 | 湖南中医药大学 | Animal model for rheumatoid arthritis combined with interstitial lung disease, construction method and application thereof |
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