CN113303285B - Method for constructing platelet-specific antigen-induced monkey ITP model - Google Patents

Abstract

The invention belongs to the field of biology, and particularly relates to a construction method of a platelet specific antigen (PAA) induced monkey ITP model. In particular, a method for obtaining an ITP model by immunizing monkeys with CD61 protein serving as a specific antigen is provided. The application of the CD61 protein in preparing an animal model of thrombocytopenic purpura is included, and the sequence of the CD61 protein is shown as SEQ ID NO: 2, respectively. The induction method can effectively induce the symptoms of thrombocytopenia and purpura and obtain a stable monkey ITP model.

Description

Method for constructing platelet-specific antigen-induced monkey ITP model

Technical Field

The invention belongs to the field of biology, and relates to a method for manufacturing an animal model of Idiopathic Thrombocytopenic Purpura (ITP), in particular to a method for manufacturing a monkey ITP model induced by a platelet-specific antigen.

Background

Immune Thrombocytopenic Purpura (ITP) is an autoimmune disease in which peripheral blood platelets are reduced, resulting in bleeding of the skin mucosa and internal organs. The cause is mainly increased platelet destruction mediated by anti-platelet autoantibodies or cytotoxic T cells in patients. When the platelet count is extremely low (Plt < 5X 10)⁹) Can cause fatal complications such as subarachnoid space, cerebral hemorrhage, digestive tract hemorrhage and other site hemorrhage. For clinical treatment of ITP patients, only a small fraction of patients can have complete remission (Plt > 100X 10)⁹And no bleeding), most patients undergo a process of chronic recurrent episodes that translates into chronic or refractory ITP. The pathogenesis of ITP is quite complex and is not fully elucidated at present. Harrington et al (Harrington WJ, Sprague CC, Minnich V, et al. immunological mecha [ 1)]nisms in idiopathic and neonatal thrombocytopenic purpura． Ann In[1]Tern Med, 1953; 38 (3): 433-. In recent years, it has been found that antiplatelet antibodies can be detected in 70-80% of patients, and some scholars have suggested that cellular immune abnormalities and antibodies can be detectedPlatelet destruction mediated by cytokine bias, such as Th 1/Th 2 cytokine bias, down-regulation of Treg cell levels, elevated CTL levels, and elevated Tfh cells.

In medical research, establishing an ideal ITP animal model is important for the research of ITP pathogenesis and pharmacodynamics and pharmaco-mechanistic research of leading-edge treatment drugs. The animal type of ITP is initially established by rats, and then, the ITP models of rabbits, dogs, mice and the like are successfully established. The ITP model in the prior art is mainly constructed in the following ways: antiplatelet serum (APS), modeling, monoclonal antibody modeling, NZW × BXSB, mouse modeling, antigen modeling, immune spleen cell transplantation model, transgenic model, and the like. At present, the experimental research on Idiopathic Thrombocytopenic Purpura (ITP) in China mainly adopts an immunization method to establish an animal model, or adopts the immunization method to combine with other methods to make the model. CN101480493A discloses a method for preparing an ITP animal model by immunizing a mouse with platelet antigen solution to obtain antiplatelet serum. CN106399364A discloses an ITP animal model for inhibiting thrombopoiesis by mutating thrombopoietin receptor in zebrafish.

Non-human primates, the animals most closely related to human genes and tissue structures, are undoubtedly one of the most ideal experimental animals selected by researchers in the pharmaceutical industry for preclinical studies. Cynomolgus monkeys (Macaca fascicularis) are non-human primates and are mainly distributed in southeast asia in countries such as indonesia, vietnam, laos, malaysia, and the like. The biological characteristics of the model are very similar to those of human beings in terms of tissue structure, physiology and metabolic function, the reaction of the organism to biological pathogens is also similar to that of human beings, the model has the characteristics of long life cycle and very similar biological characteristics to human beings, and the model is an ideal model for observing and researching chronic diseases and is also one of the most ideal experimental animals in laboratory pharmacological research. In the current research, a precedent that an ITP animal model is constructed by taking a primate, particularly a cynomolgus monkey, and an ITP specific antigen, particularly CD61 protein as immunogen does not appear, so that compared with other animal models, the ITP animal model can be more similar to the human pathogenesis, has the specificity of inducing pathogenesis, is favorable for deeply researching the ITP pathogenesis and is further applied to preparing a medicine for treating ITP.

Disclosure of Invention

The following is a summary of the subject matter described in detail herein. This summary is not intended to limit the scope of the claims.

According to the antigen simulation principle of immunology, the monkey is infused with a thrombocytopenic purpura (ITP) specific antigen to stimulate an immunoreaction aiming at self platelets in a monkey body so as to establish a more stable active immune type monkey ITP model.

The CD41/CD61 protein complex is a main specific glycoprotein on the surface of platelets, and the amino acid sequence of the complex is shown as SEQ ID No.: 1 (GenBank: NP-001075271.1), or a sequence which is identical to SEQ ID No.: 1 with at least 80% identity. Wherein, the CD41 antigen is an alpha II beta chain of the integrin, the CD61 antigen is a beta 2 chain, and the two together form a CD41/CD61 complex; the amino acid sequence of the CD61 antigen is shown as SEQ ID NO. 2 (GenBank: AAA 60122.1; Gene ID: 3690, updated on 7-Mar-2021), or is identical to SEQ ID NO.:2 with at least 80% identity. The research shows that Abciximab can be specifically combined with platelet glycoprotein CD61 receptor (alpha IIb beta 3 receptor) to block the combination of fibrinogen, VWD factor and other adhesive molecules with a receptor site, so that the platelet aggregation is inhibited to prevent the formation of thrombus; possible side effects may lead to a decrease in platelet count (Andrea Artoni et al, integral β 3 regions controlling binding of muscle mAb 7E3: abnormalities for the mechanism of integral α IIb β 3 activation, PNAS, September 7, 2004101 (36) 13114-13120). Based on this, the present application has intensively studied the role of CD61 in the establishment of a model in thrombocytopenia-associated diseases such as ITP. On one hand, the application provides the application of the CD61 protein in preparing an animal model of thrombocytopenic purpura, in particular to the application in preparing an ITP model of monkey; in another aspect, the present application provides a method for preparing an animal model of thrombocytopenic purpura, which comprises immunizing CD61 as a specific antigen to induce and obtain the animal model of thrombocytopenic purpura; in particular, the application takes cynomolgus monkeys as an animal model, and divides the cynomolgus monkeys into a control group and different immunization dose groups, and CD61 protein and complete Freund's adjuvant are prepared into an emulsifier to carry out animal immunization, so as to induce and obtain the thrombocytopenic purpura animal model with the symptoms of platelet quantity reduction, subcutaneous hemorrhage and the like.

Compared with the prior art, the invention has at least the following beneficial effects:

the immunized cynomolgus monkeys have the tendency of remarkably reducing blood platelets and have obvious subcutaneous bleeding, which shows that the induction method can successfully induce the blood platelet reduction and the purpura symptoms to obtain a stable monkey ITP animal model.

Drawings

FIG. 1: animal model weights of experimental group and control group.

FIG. 2: bleeding time of animal models of experimental group and control group.

FIG. 3: the number of platelets in animal models of experimental group and control group.

FIG. 4: animal model symptoms.

Detailed Description

The present invention is described in further detail below by way of examples to enable those skilled in the art to practice the present application. It is to be understood that other embodiments may be utilized and that changes may be made without departing from the spirit or scope of the present invention as appropriate. To avoid detail not necessary to enable those skilled in the art to practice the application, the description may omit certain information known to those skilled in the art. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims.

The following examples are presented to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention.

Example 1: induction of ITP animal models.

Experimental animals: cynomolgus monkey (Cynomolgus), male or female, age 2.5-4.0 years, weight 2.5-4.5 kg.

The experimental environment is as follows: the experimental monkeys were housed in stainless steel monkey cages (800 mm. times.700 mm. times.820 mm) in a setting up room for the biological animals, and the acclimation period was as follows: 2 per cage; the experimental period and postoperative care are as follows: each cage has 1 (with an equipped animal welfare implement). The room number where the animals were placed throughout the trial will be recorded in the experimental record. The room for placing the test monkey is located in the Pentium organism animal room, and the number of times of filtration, ventilation and air exchange in the area is 10-20 times per hour. The temperature will be maintained between 20-26 deg.C (68-79F) and the relative humidity between 40-70%. Temperature and humidity were continuously observed and recorded. The lighting conditions were 12 hours (08: 00-20: 00) day of daylight lamp illumination and 12 hours of no illumination. Animal welfare measures are performed according to the standard procedures set out to reduce and help sooth the stress of the experiment.

Grouping experiments: 4 cynomolgus monkeys were randomly divided into 4 groups according to body weight, 1 was used as a normal control group, and the remaining 3 were used as model treatment groups at different doses or in different modes.

Model induction: CD61 Protein (Human Integrin Beta III/CD61 (ITGB3) Protein (Abbexa # abx067294), amino acid sequence shown in SEQ ID NO: 2) was mixed with complete Freund's adjuvant to make 0.1-10 mg/mL of emulsifier, and about 100ul of each spot was immunized intradermally at the tail root with Day 0-Day 28. Specific grouping schemes and injection points are shown in table 1.

TABLE 1 grouping and Molding schemes

Grouping:	number of animals	CD61 emulsifier	Blank emulsifier
				G1: normal group	1	0	0
G2: model 1	1	1	9
				G3: model 2	1	3	7
G4: model 3	1	10	0

Example 3: and (4) detection of an ITP animal model.

Detection indexes are as follows:

1) weight: before the experiment, animal body weights were measured and recorded;

2) blood collection: before immunization, starting 7 days after immunization, performing intravenous blood collection twice a week, and performing EDTA anticoagulation for about 200ul to 28 days;

3) complete blood cell count: blood is taken frequently, the blood platelets are not obviously reduced within three weeks, the Day 7-28 animals are subjected to secondary antigen immunization, and the method is the same as the first time;

4) serum sample preparation: collecting blood frequently, separating serum to obtain about 400ul, and storing at-80 deg.C;

5) and (3) bleeding time measurement: prior to immunization and at several time points when significant reduction of platelets was found.

And (3) clinical observation: the health status of the animals and the comprehensive response to surgery and drugs were continuously observed during the experiment. Without any appearance or abnormal behavior.

Statistical analysis: experimental data were expressed using mean ± standard error (mean ± s.e.m.). Data were analyzed by Graphpad Prism or SPSS using the corresponding statistical methods. P <0.05 was considered a significant difference. Body weight, bleeding time, platelets are shown in tables 2-4 and FIGS. 1-3. The symptoms of the animal model are shown in figure 4. Compared with the control group, 3 immunized cynomolgus monkeys have a trend of remarkably reducing the blood platelets, wherein the bleeding time of the animals G2 and G4 is remarkably prolonged, and a remarkable subcutaneous bleeding phenomenon occurs, and the amplitude of the reduction of the blood platelets and the increase of the bleeding time of the animals G3 is slightly lower. The above results indicate that the current induction method can successfully induce the symptoms of thrombocytopenia and purpura.

TABLE 2 body weight

TABLE 3 bleeding time

TABLE 4 platelet count

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Claims (8)

1. The application of a CD61 protein in preparing an animal model of thrombocytopenic purpura is disclosed, wherein the sequence of the CD61 protein is shown as SEQ ID NO: 2, the animal is a cynomolgus monkey.

2. A method for preparing animal model of thrombocytopenic purpura, said method uses CD61 protein as specific antigen to carry on the immunization, induce and obtain animal model of thrombocytopenic purpura with the quantity reduction of platelet and subcutaneous hemorrhage symptom; the sequence of the CD61 protein is shown as SEQ ID NO: 2, the animal is a cynomolgus monkey.

3. The method of claim 2, wherein said CD61 protein is immunized with complete Freund's adjuvant in the form of an emulsion.

4. The method according to claim 3, wherein the concentration of CD61 protein in the emulsifier is 0.1-10 mg/ml.

5. The method of claim 4, wherein the concentration of CD61 protein in the emulsifier is 5 mg/ml.

6. The method of claim 2, wherein the immunization is performed intradermally at the tail root in two portions.

7. The method of claim 6, wherein the immunization is performed as multiple injections, each injection of about 100 ul.

8. The method of claim 6, wherein the second injection is administered within 7 to 28 days of the first injection.

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