CN115088676A - Method for constructing ACPA positive bone erosion rheumatoid arthritis animal model - Google Patents

Abstract

The invention relates to a method for constructing an ACPA positive combined bone erosion rheumatoid arthritis animal model, which comprises the following steps: smearing porphyromonas gingivalis suspension on a gum area of an oral cavity of a mouse every day in an animal model construction period; and mixing and emulsifying the bovine type II collagen solution and the complete Freund's adjuvant in equal volume to obtain emulsion, injecting the emulsion subcutaneously at the tail root of the mouse in a construction period for two times of immunization, and ending the modeling period to obtain the animal model. The animal model constructed by the method can stably and highly express the anti-citrullinated protein antibody (ACPA), and compared with the existing animal model, the method can effectively construct the RA animal model with higher morbidity and severity and accompanied with severe bone erosion. Thereby reducing the technical difficulty of the research related to the refractory rheumatoid arthritis and providing a solid technical foundation for further and widely developing the treatment means of RA.

Description

Method for constructing ACPA positive bone erosion rheumatoid arthritis combined animal model

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a method for constructing an ACPA positive bone erosion rheumatoid arthritis animal model.

Background

Rheumatoid Arthritis (RA) is a worldwide, refractory, systemic autoimmune disease, and its important immunological feature is the production of anti-citrullinated protein antibodies (ACPA) and the like. ACPA is a highly specific antibody present in 47-84% of RA patients, and is included in the serological diagnosis standard of RA in 2010 by the American college of rheumatology and the European Union of rheumatism. It can appear before the clinical symptoms of RA and is closely related to disease development, progression and prognosis. Research shows that compared with ACPA negative patients, ACPA positive RA patients have the most obvious clinical characteristics of severe joint and bone erosion, stronger disease activity, higher cardiovascular disease complication incidence rate, poorer prognosis and other clinical manifestations, and are also more difficult to treat. Even ACPA-positive and ACPA-negative RA have been considered as two diseases with different mechanisms. Therefore, the definition of the pathogenesis of ACPA-positive RA and the targeted ACPA therapy are hot spots for the study in the RA field.

The establishment of experimental animal models is an important basis for researching pathogenesis and developing innovative drugs. Although animal models of RA, such as collagen-induced arthritis and adjuvant-induced arthritis, mimic the clinical symptoms and pathological features of RA very well, ACPA is not a significant feature of these models. Therefore, the above RA animal model does not completely reflect the characteristics of human RA in terms of arthritis expression, immunology, pathology, etc., and further fails to elucidate the mechanism of autoantibody in the initiation and maintenance process of human RA immune inflammatory response, which seriously affects the pathogenesis of ACPA induction and the deep research of targeted ACPA therapeutic drugs, and therefore we need to establish an animal model with stable and high ACPA expression.

ACPA is highly cross-reactive, binding to a variety of citrullinated proteins, but the target antigens and their tissue localization associated in vivo are not known. Researchers build ACPA positive RA models by adopting citrullinated protein immunization, but the incidence rate and the severity degree are low, which indicates that single citrullinated protein can not effectively build ACPA positive RA models. And peptidyl arginine deiminase expressed by Porphyromonas gingivalis (pg) can citrullinate almost all of RA major antigenic proteins. The existing research shows that 8 months of oral cavity inoculation of sodium carboxymethyl cellulose gel containing pg to Lewis rats can induce spontaneous RA, and ACPA is obviously highly expressed in four months, but the molding method has long molding period, low morbidity and severity and is not beneficial to popularization and application. A researcher implants a coil chamber at the outer side of the back of a DBA mouse, bacteria are inoculated into an inner cavity after 2 weeks, CII emulsified by CFA is injected subcutaneously at the tail, and after 21 days, the CII with the same amount is used for strengthening immunity, so that an ACPA high-expression model can be established, but the model is high in modeling difficulty and animal mortality.

In addition, the inventor finds that the modeling mode and the animal species are extremely critical to establishing the model in the long-term research process, and the conventional animals are difficult to obviously express ACPA at a high level.

Disclosure of Invention

In order to solve at least one technical problem, better research on pathogenesis and medicines related to RA and relieve pain of patients, the invention provides a method for constructing an ACPA positive and bone erosion rheumatoid arthritis animal model.

In a first aspect, the invention provides a method for constructing an animal model of ACPA-positive combined bone erosion rheumatoid arthritis. The method comprises the following steps:

smearing porphyromonas gingivalis suspension on a gum area of an oral cavity of a mouse every day in an animal model construction period;

and (2) mixing and emulsifying the bovine type II collagen solution and the complete Freund's adjuvant in equal volume to obtain emulsion, injecting the emulsion subcutaneously at the tail root of the mouse in the construction period for two times of immunization, and ending the modeling period to obtain the animal model.

Preferably, the mouse is any one of DBA/1, B10, C57BL/6 and C3H/He, and more preferably C3H/He.

Preferably, the porphyromonas gingivalis suspension is prepared by suspending porphyromonas gingivalis in a 2% sterile sodium carboxymethyl cellulose solution, and more preferably, the concentration of the sterile sodium carboxymethyl cellulose solution is 0.2% to 2%, and more preferably 0.5% to 2%, for example: 0.5%, 0.75%, 0.10%, 0.12%, 0.15%, 0.18%, 2%.

Preferably, the bacterium content of the porphyromonas gingivalis suspension is 1.0-2.0 × 109CFU/mL, for example: 1.0X 109CFU/mL, 1.2X 109CFU/mL, 1.4X 109CFU/mL, 1.6X 109CFU/mL, 1.8X 109CFU/mL, 2.0X 109 CFU/mL.

Preferably, the smearing dosage of the porphyromonas gingivalis suspension is 50-200 mu L/piece, and more preferably 50-150 mu L/piece, such as: 50 μ L/only, 60 μ L/only, 70 μ L/only, 80 μ L/only, 90 μ L/only, 100 μ L/only, 110 μ L/only, 120 μ L/only, 130 μ L/only, 140 μ L/only, 150 μ L/only.

Preferably, the immunization time of the mice with the first emulsion is 0 to 15 days, more preferably 7 to 15 days, such as 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days after the start of the modeling.

Preferably, the construction period of the animal model is 6-9 weeks, and further preferably 6-8 weeks, for example: 6 weeks, 7 weeks, 8 weeks.

In order to ensure the construction effect of the mouse model, after the immunization of the mouse for the first time, a second immunization is required.

Preferably, the time interval between two immunizations is 7 to 21 days, more preferably 10 to 21 days, for example: 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days and 21 days.

In a specific embodiment of the present invention, the method for constructing the animal model may further include the steps of:

adaptively feeding the mice with antibiotics before the start of modeling; and/or

In the modeling process, detecting the content of anti-CCP antibody in the mouse serum, and detecting the ratio of the content of citrulline protein to the content of plant phosphoglycerate dehydrogenase in the inflammatory joint tissues of the mouse;

when the mouse arthritis score no longer increased, modeling was complete.

Preferably, the mouse arthritis score is not increased any more, namely the arthritis score is not increased any more for more than 10 consecutive days, and more preferably the arthritis score is not increased any more for more than 14 consecutive days.

Preferably, the mice are adapted for feeding for 7-10 days, such as 7 days, 8 days, 9 days, 10 days.

Preferably, the antibiotic is at least one of ofloxacin, metronidazole and compound sulfamethoxazole, and more preferably the compound sulfamethoxazole.

Preferably, the compound sulfamethoxazole is used in an amount of 5mg/kg to 50mg/kg, more preferably 5mg/kg to 40mg/kg, such as 5mg/kg, 7.5mg/kg, 10mg/kg, 12.5mg/kg, 15mg/kg, 17.5mg/kg, 20mg/kg, 22.5mg/kg, 25mg/kg, 27.5mg/kg, 30mg/kg, 32.5mg/kg, 35mg/kg, 37.5mg/kg, 40 mg/kg.

Preferably, the level of anti-CCP antibodies in the serum of said mouse is above a positive threshold.

Preferably, the positive threshold is determined with reference to clinical criteria, said positive threshold being equal to 3 times the average value of the anti-CCP content of the negative samples.

In a specific embodiment of the invention, the content of anti-CCP antibody in the serum of the mouse is more than 54.06U/mL.

Preferably, the ratio of the content of citrulline protein to the content of plant phosphoglycerate dehydrogenase is 0.55-0.90, more preferably 0.55-0.80, for example: 0.55, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80.

In a second aspect, the invention provides the use of a method according to the first aspect for the construction of an animal model.

Preferably, the animal model comprises an animal model of a rheumatoid arthritis-related study.

In a third aspect, the invention provides the use of the animal model of rheumatoid arthritis of the first aspect.

Preferably, the level of anti-CCP antibodies in the serum of said mouse is above a positive threshold.

Preferably, the positive threshold is determined with reference to clinical criteria, said positive threshold being equal to 3 times the average value of the anti-CCP content of the negative samples.

Preferably, the content of anti-CCP antibody in serum of the rheumatoid arthritis animal model is more than 54.06U/mL;

preferably, the ratio of the content of citrulline protein to the content of plant phosphoglycerate dehydrogenase in the inflammatory joint tissue of the mouse is 0.55-0.90, more preferably 0.55-0.80, such as: 0.55, 0.58, 0.60, 0.62, 0.64, 0.66, 0.68, 0.70, 0.72, 0.74, 0.76, 0.78, 0.80.

Preferably, the application comprises at least one of:

research related to the rheumatoid arthritis treatment mechanism;

research on a treatment method for rheumatoid arthritis;

screening drugs for preventing and/or treating rheumatoid arthritis;

the medicament optionally comprises a pharmaceutical excipient. Further preferably, the adjuvant comprises a pharmaceutically acceptable carrier, diluent and/or buffer, etc.

The invention has the beneficial effects that:

the invention firstly inoculates pg in an oral smearing mode two weeks before collagen immunization to ensure the colonization of bacteria.

Secondly, the C3H/He mouse is selected for the first time, and although the mouse is less used for model research related to rheumatoid arthritis, the IE beta k chain of the mouse is highly homologous with HLA-DRB1 allele of 'shared epitope' of the rheumatoid arthritis, and the mouse is an excellent animal species for researching RA and autoantibodies.

The animal model of the invention generates higher levels of citrullinated protein and ACPA than the normal group and the conventional type II collagen-induced group, has higher morbidity and morbidity severity, and accords with the clinical characteristics of ACPA-positive patients in terms of bone erosion and bone destruction.

The invention creates a brand-new construction method of the RA animal model, the animal model constructed by the method can stably and highly express ACPA, and compared with the existing animal model, the method can more quickly and effectively construct the animal model. Thereby reducing the technical difficulty of RA-related research and providing a solid technical foundation for further and widely developing the treatment means of RA.

Drawings

FIG. 1 shows a process of constructing a mouse animal model;

FIG. 2 shows arthritis scores in a mouse animal model;

FIG. 3 is a graph showing the incidence of disease in a mouse animal model;

FIG. 4 shows ACPA (anti-CCP) serum levels in mouse animal models;

FIG. 5 is a Western blot of citrullinated protein and GAPDH in knee tissue and spleen in a mouse animal model;

FIG. 6 is a graph showing the ratio of citrullinated protein to GAPDH in knee joints and spleen of a mouse animal model;

FIG. 7 shows TRAP osteoclast staining results in a mouse animal model;

FIG. 8 shows the results of Micro-CT scanning of knee joints in mouse animal models;

in each of the above figures, Control represents a normal Control group, pg represents a live pg infection group alone, CIA represents a two-type collagen + complete freund adjuvant induction group, and pg + CIA represents a live pg + two-type collagen + complete freund adjuvant infection group.

Detailed Description

The invention will be further described with reference to specific embodiments and drawings, the advantages and features of which will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

It should be noted that the reagents and experimental methods used in the present invention are all conventional in the art unless otherwise specified.

The experimental materials, reagents and manufacturers adopted in the invention are as follows:

porphyromonas gingivalis (pg) International Standard strain ATCC33277 from North Narco Biotech, Inc. C3H/He mice were purchased from Beijing Huafukang Biotech GmbH. Type II bovine collagen acetic acid solution (cat # 2002-2) and complete Freund's adjuvant (cat # 7001) were purchased from Chondrex, USA. The mouse anti-CCP antibody detection kit is purchased from Wuhan Huamei bioengineering Co., Ltd (the product number is CSB-EQ027743 MO). The citrullinated protein antibody primary antibody was purchased from Abcam corporation, USA (cat No. ab240908), the GAPDH antibody primary antibody was purchased from Cell Signaling Technology (cat No. 2118s), and the HRP goat anti-rabbit secondary antibody and the HRP goat anti-mouse secondary antibody were purchased from Beijing prilley Gene Technology, Inc. (cat nos.: C1308, C1309).

Example 1 construction of an anti-citrullinated protein antibody high expression RA animal model

A mouse animal model is constructed according to the construction process shown in figure 1, and the specific animal model construction process is as follows:

1.1 adaptive rearing of C3H/He mice

Male 6-week-old C3H/He mice 32 (body weight 20. + -.2 g) were randomized into 4 groups of 8 mice each. The four experimental groups are respectively a normal control group, a single live pg infection group, a type II collagen + complete Freund's adjuvant induction group, and a live pg + type II collagen + complete Freund's adjuvant infection group.

10mL of compound sulfamethoxazole suspension is added into 500mL of sterilized water to replace the daily drinking water of all mice to perform biological-resistant pretreatment on the mice for 3 days, and then all the mice are replaced by the daily drinking water and are continuously raised for 4 days. All mice were routinely managed.

1.2 model construction of C3H/He mice

1.2.1 mice were inoculated orally with Porphyromonas gingivalis suspension

After the adaptive feeding of all mice is finished, from day 1, the mice of the normal control group and the mice of the II type collagen + complete Freund's adjuvant induction group are coated with 100 mu L/mouse of 2% sterile sodium carboxymethylcellulose in the oral gingival area every day. The live pg bacteria were suspended in 2% sterile sodium carboxymethylcellulose solution to give a 1X 109CFU/mL suspension of pg. The mice of the single live pg infection group and the mice of the live pg + type II collagen + complete Freund adjuvant infection group are coated with 100 mu L/one of pg suspension in the oral gingival area every day and are coated with the oral cavity and the whole gingival margin, and in addition, the mice of the single live pg infection group and the mice of the live pg + type II collagen + complete Freund adjuvant infection group are injected with 100 mu L/one of the pg live PBS suspension in the gingival scattering point on the 1 st day.

1.2.2 type II collagen emulsion immunization

And (3) ultrasonically emulsifying the bovine type II collagen acetic acid solution into emulsion in the same volume of Freund's adjuvant until the emulsion is not dispersed when being dropped into water. On day 14, the tail and root hairs of the mice were shaved off, and the roots of the mice in the two groups were injected subcutaneously with 0.2 mL/mouse (containing 200. mu.g of bovine type II collagen) in addition to the normal control group and pg-alone infected group.

Repeating the steps on the 35 th day, strengthening the type II collagen emulsion immunity again, and then normally managing the mice in each group until the model construction is finished on the 56 th day to obtain the citrullinated protein antibody high expression RA animal model.

1.3 detection of C3H/He mouse model

1.3.1 determination of arthritis index

Starting on day 35, the arthritis score was recorded every second day for each mouse, according to the contents of table 1. Meanwhile, the incidence rate of arthritis of the mice is calculated according to the following formula.

Incidence rate is the number of mice per group/total number of mice per group x 100%.

TABLE 1 mouse arthritis Scoring standards

Note: each mouse was evaluated for 4 ankle, 4 midfoot, 12 toe joints, and 80 points

The results of arthritis scoring for each group of mice are shown in fig. 2. FIG. 2 shows that arthritis did not develop in normal control mice and live pg infection alone mice. The type II collagen and complete Freund's adjuvant induces mice to generate arthritis symptoms from 38-40 days, the mice reach inflammation peak period from 48-52 days, and then the inflammation of the joints is slightly relieved and continues to the 56 th day. The mice in the group infected by pg viable bacteria, type II collagen and complete Freund's adjuvant have arthritis symptoms from day 36 to day 38, reach the inflammation peak period from day 48 to day 50, and continue until day 56.

The results in figure 2 show that compared with the normal control group, RA of mice can be induced by live pg infection and type ii collagen + complete freund adjuvant infection, and the simultaneous treatment of live pg infection and type ii collagen + complete freund adjuvant infection can lead to better arthritis induction effect than any single infection treatment, and more prominent effect on the construction of mouse RA model.

The incidence of each group of mice is shown in figure 3. FIG. 3 shows that the incidence of the normal control group mice and the pg live bacteria infection group mice is zero in the whole model construction period, and the pg live bacteria infection alone does not cause arthritis of the mice. While the incidence of type II collagen and complete Freund's adjuvant induced mice was 25% on day 40, 62.5% on day 42, and no increase in late stage incidence was observed until day 56. The incidence of mice in the group infected by pg live bacteria, type II collagen and complete Freund's adjuvant is directly increased from zero to 37.5% on day 38, 87.5% on day 40 and 100% on day 42, and the incidence is continued until day 56.

The results in FIG. 3 show that infection with live pg alone did not cause arthritis in mice and that type II collagen + complete Freund's adjuvant induced arthritis in mice throughout the whole animal model construction cycle. Compared with the mice induced by type II collagen and complete Freund's adjuvant, after the mice are infected by the pg viable bacteria, the type II collagen and the complete Freund's adjuvant, arthritis of the mice can be induced more early, the incidence rate of the arthritis of the mice is higher, and all the experimental mice constructed by the model are attacked. This further demonstrates the effectiveness of live pgbacteria + collagen type ii + complete freund's adjuvant infection for mouse model construction.

Combining the results of fig. 2 and fig. 3, it can be seen that compared with mice infected with live pg bacteria alone and mice induced with type ii collagen and complete freund adjuvant, the severity of the arthritis animal model can be significantly increased by infecting the mice with live pg bacteria, type ii collagen and complete freund adjuvant, and the RA animal model can be constructed more rapidly and efficiently.

1.3.2 detection of ACPA expression level in mouse serum

Serum samples of each group of mice are collected according to a conventional method, anti-CCP antibody is used as the detection index of ACPA, a commercially available mouse anti-CCP antibody detection kit is adopted, the operation is carried out according to the instruction, and the content (U/mL) of the anti-CCP antibody in the serum of each group of mice is detected, and the result is shown in Table 2 and figure 4.

TABLE 2 anti-CCP antibody content in CH3/He mouse serum

Note: the Cut-off value was determined as 3 times the average value of the blank (negative sample) with reference to the clinical standard

As can be seen from the data in table 2 and fig. 4, compared to the normal control mice, the content of anti-CCP in the serum of the mice infected with live pg alone, the mice induced with type ii collagen + complete freund adjuvant alone, and the mice infected with live pg + type ii collagen + complete freund adjuvant was increased by 300%, 141%, 543%, respectively. The results show that the expression level of ACPA can be improved by a single live pg infection group, a type II collagen + complete Freund adjuvant induction group and a live pg + type II collagen + complete Freund adjuvant infection group, but the infection effects of the live pg + type II collagen + complete Freund adjuvant are more prominent, and the content of anti-CCP in serum reaches 115.9U/mL and is far higher than the Cut-off value of 54.06U/mL.

On the other hand, compared with the mice induced by type II collagen and complete Freund's adjuvant, the content of anti-CCP in the serum of the mice infected by pg live bacteria, type II collagen and complete Freund's adjuvant is improved by 167%; compared with a single live pg bacterium infected group mouse, the content of anti-CCP in the serum of the live pg bacterium + type II collagen + complete Freund adjuvant infected mouse is improved by 61%, which shows that in the construction process of a mouse RA model, the live pg bacterium and the type II collagen + complete Freund adjuvant are mutually cooperated to generate a unique technical effect, and the construction effect of the live pg bacterium + type II collagen + complete Freund adjuvant infected mouse model is far better than that of a mouse model induced by the live pg bacterium infection or the type II collagen + complete Freund adjuvant alone.

1.3.3 detection of citrullinated protein expression levels in mouse Knee Joint tissue and spleen tissue

Preparing protein samples of knee joint tissues and spleen tissues of each group of mice according to a conventional method, then performing Western blot experiment by adopting citrullinated protein antibody primary antibody, HRP goat anti-rabbit secondary antibody and HRP goat anti-mouse secondary antibody according to the conventional protein quantification, sealing, primary antibody incubation, secondary antibody incubation, exposure and other flows, and analyzing by using Image-Pro Plus 6.0 software. Citrullinated protein expression levels were analyzed using the ratio of citrullinated protein to plant phosphoglycerate dehydrogenase (GAPDH). The results are shown in Table 3, FIG. 5, and FIG. 6.

TABLE 3 citrullinated protein to GAPDH ratio

As can be seen from the data in table 3 and fig. 6, compared to the normal control mice, the citrullinated protein/GAPDH was increased by 148%, 349% and 598% in the knee joint tissues of the mice infected with live pg alone, the mice induced with type ii collagen + complete freund adjuvant, and the mice infected with live pg + type ii collagen + complete freund adjuvant. The results show that the citrullinated protein/GAPDH in the knee joint tissues of mice can be improved by a single live pg infection group, a type II collagen + complete Freund adjuvant induction group and a live pg + type II collagen + complete Freund adjuvant infection group, but the effects of live pg + type II collagen + complete Freund adjuvant infection are more prominent.

In addition, compared with the mice induced by type II collagen and complete Freund's adjuvant, the citrullinated protein/GAPDH content in the knee joint tissues of the mice infected by pg live bacteria, type II collagen and complete Freund's adjuvant is increased by 55%; compared with a single live pg infection group mouse, citrullinated protein/GAPDH in knee joint tissues of a mouse infected by live pg, type II collagen and complete Freund's adjuvant is improved by 181%, which indicates that in the construction process of a mouse RA model, live pg, type II collagen and complete Freund's adjuvant are mutually cooperated to generate a unique technical effect, and the construction effect of the mouse infected by live pg, type II collagen and complete Freund's adjuvant is far better than that of a mouse model infected by live pg or type II collagen and complete Freund's adjuvant.

On the other hand, the data in table 3 and fig. 6 show that compared to the normal control group mice, spleen tissues of pgh-infected mice, type ii collagen + complete freund adjuvant-induced mice, and pgh + type ii collagen + complete freund adjuvant-infected mice were increased by 184%, 202%, and 808%, respectively. The results show that the citrullinated protein/GAPDH in spleen tissues of mice can be improved by a single live pg infection group, a type II collagen + complete Freund adjuvant induction group and a live pg + type II collagen + complete Freund adjuvant infection group, but the effects of live pg + type II collagen + complete Freund adjuvant infection are more prominent.

On the other hand, compared with the mice induced by type II collagen and complete Freund's adjuvant, the citrullinated protein/GAPDH content in spleen tissues of mice infected by pg viable bacteria, type II collagen and complete Freund's adjuvant is increased by 201%; compared with a single live pg infection group mouse, citrullinated protein/GAPDH in spleen tissues of a live pg + type II collagen + complete Freund adjuvant infection mouse is improved by 219%, which indicates that in the construction process of a mouse RA model, live pg and type II collagen + complete Freund adjuvant are mutually cooperated to generate a unique technical effect, and the construction effect of the live pg + type II collagen + complete Freund adjuvant infection mouse model is far better than that of a mouse model induced by live pg infection or type II collagen + complete Freund adjuvant alone.

1.3.4 observation of mouse hind limb joint site

After the mouse model construction period is over, the knee joints of each group of mice are taken out, paraffin is embedded and fixed, the mice are sliced, H & E staining and pathological scoring are carried out, H & E staining and TRAP staining are carried out, the number of TRAP positive osteoclasts of each group is counted, and the results are shown in a table 4 and a figure 7.

TABLE 4 TRAP Positive osteoclast number

As can be seen from table 4, compared to the normal control mice, the TRAP positive osteoclast numbers of knee joints of mice infected with live pg alone, mice induced with type ii collagen + complete freund adjuvant, and mice infected with live pg + type ii collagen + complete freund adjuvant were respectively increased by 162%, 386%, and 748%. The results show that the number of TRAP positive osteoclasts of knee joints of mice can be increased by a single live pg infection group, a type II collagen + complete Freund adjuvant induction group and a live pg + type II collagen + complete Freund adjuvant infection group, but the infection effects of the live pg + type II collagen + complete Freund adjuvant are more prominent.

In addition, compared with mice induced by type II collagen and complete Freund's adjuvant, the number of TRAP positive osteoclasts of knee joints of mice infected by pg viable bacteria, type II collagen and complete Freund's adjuvant is increased by 75%; compared with a single live pg infection group mouse, the number of TRAP positive osteoclasts of the live pg + type II collagen + complete Freund adjuvant infected mouse is increased by 224%, which shows that in the construction process of a mouse RA model, the live pg and the type II collagen + complete Freund adjuvant are mutually cooperated to generate a unique technical effect, and the construction effect of the live pg + type II collagen + complete Freund adjuvant infected mouse model is far better than that of a mouse model induced by the live pg infection or the type II collagen + complete Freund adjuvant alone.

As can be seen from fig. 7, after the HE staining of the knee joint, synovial tissue around the knee joint cavity of mice in the group infected with pg viable bacteria, type ii collagen and complete freund's adjuvant significantly proliferated and protruded into the joint cavity, the joint space narrowed, the synovium pannus formed, significant damage to bone and cartilage and bone loss occurred, the proliferated synovial tissue invaded under the skin, and a fibrosclerosis focus was partially formed with a large amount of inflammatory cell infiltration and granulation tissue proliferation in the synovial membrane, compared to the other three groups. The result shows that compared with a type II collagen + complete Freund adjuvant induced mouse and a single live pg infection group mouse, the construction effect of the live pg + type II collagen + complete Freund adjuvant infected mouse model is better.

1.3.5 Micro-CT scan of inflammatory knee joints of mice

According to the conventional operation, the knee joints of each group of mice are fixed by 10% formaldehyde solution for 72h, and then the inflammatory knee joints of the mice are scanned by Micro-CT under the conditions that the resolution is 25 mu m and the single time is 72 min. The results are shown in FIG. 8.

As can be seen from FIG. 8, the joint of knee joint of mice infected with pg viable bacteria, type II collagen and Freund's complete adjuvant is damaged, the joint surface is loose and rough, the unevenness is uneven, and the bone structure is eroded. Compared with the other three groups, the mice infected by the pg viable bacteria, the type II collagen and the complete Freund's adjuvant have more obvious changes such as bone erosion, articular cavity stenosis and the like. The construction effect of the mouse model infected by the live pg bacteria, the type II collagen and the complete Freund adjuvant is better than that of the mouse model induced by the live pg bacteria alone or the type II collagen and the complete Freund adjuvant alone.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A method for constructing an ACPA positive bone erosion rheumatoid arthritis animal model is characterized by comprising the following steps: the method comprises the following steps:

smearing porphyromonas gingivalis suspension on a gum area of an oral cavity of a mouse every day in an animal model construction period;

and mixing and emulsifying the bovine type II collagen solution and the complete Freund's adjuvant in equal volume to obtain emulsion, subcutaneously injecting the emulsion at the tail root of a mouse in the animal model construction period to carry out twice immunization, and ending the modeling period to obtain the animal model.

2. The method of claim 1, wherein: the porphyromonas gingivalis suspension is prepared by suspending the porphyromonas gingivalis in 0.2-2% of sterile sodium carboxymethyl cellulose solution, and the bacterium content of the porphyromonas gingivalis suspension is 1.0-2.0 multiplied by 109 CFU/mL.

3. The method of claim 1, wherein: the smearing dosage of the porphyromonas gingivalis suspension is 50-200 mu L per unit.

4. The method of claim 1, wherein: the immunization time of the emulsion for the first time on the mouse is 0 to 15 days, preferably 7 to 15 days after the modeling starts; the construction period of the animal model is 6-9 weeks.

5. The method according to claim 1 or 4, characterized in that: the time interval between two immunizations is 7-21 days.

6. The method of claim 1, wherein: the method further comprises acclimatizing the mouse with an antibiotic prior to initiation of modeling.

7. The method of claim 1, wherein: the method also comprises the steps of detecting the content of anti-CCP antibody in the serum of the mouse and detecting the ratio of the content of citrulline protein to the content of plant phosphoglycerate dehydrogenase in the knee joint and spleen tissues of the inflammation of the mouse in the modeling process.

8. The method of claim 7, wherein: the content of the anti-CCP antibody is higher than a positive threshold value, and/or the ratio of the content of the citrulline protein to the content of the plant phosphoglycerate dehydrogenase is 0.55-0.90;

the positive threshold is determined by reference to clinical standards and is equal to 3 times of the average value of the content of anti-CCP antibodies in the negative samples.

9. The method according to any one of claims 1 to 8, wherein: the mice were C3H/He.

10. The use of the ACPA-positive bone erosion rheumatoid arthritis combined animal model of claim 9 in screening drugs for preventing and treating rheumatoid arthritis, characterized in that:

the content of anti-CCP antibody in serum of the ACPA positive bone erosion rheumatoid arthritis animal model is higher than a positive threshold value, and/or the ratio of the content of citrulline protein to the content of plant phosphoglycerate dehydrogenase is 0.55-0.90;

the positive threshold is determined by reference to clinical standards and is equal to 3 times of the average value of the content of anti-CCP antibodies in the negative samples.

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