KR101854909B1 - Mouse model and its manufacturing method having characterized the adjusted polarity of Macrophage - Google Patents

Abstract

The present invention is to propose a method of mouse model for studying the functions and characteristics of polarized macrophages in the body when diseases or infections are induced using macrophage activation or a polarized mouse model. More specifically, the M1 macrophage mouse model was stimulated with either Lipopolysaccharide (0.5-5 μg / kg) and IFN gamma (0.5-5 μg / kg) to induce the polarity of the papain macrophages in the mouse model, This is presented as a method of making a mouse model that can be applied to studies on the function and characteristics of macrophages acting upon induction.

Description

[0001] The present invention relates to a mouse model in which the polarity of macrophages is regulated,

The present invention relates to a mouse model in which the polarization of macrophages is regulated and a method for producing the same.

The present invention also relates to a mouse model applicable to in vivo experiments related to the polarity of macrophages and a method for producing the same.

In addition, the present invention relates to a mouse model and a method for preparing the same to examine the functions and characteristics of macrophages against Mycobacterium tuberculosis.

Tuberculosis is a chronic infectious disease caused by an infection of Mycobacterium species with Mycobacterium tuberculosis. Tuberculosis is a major disease in developing countries, with nearly 8 million new infections occurring each year, and 3 million deaths, and it is also a problem in developed countries. Infection may be asymptomatic for a considerable period of time, but tuberculosis is the most common symptom of acute inflammation of the lungs, resulting in fever and nonproductive cough. Untreated, usually causes serious complications and death.

Tuberculosis can be treated with long-term antibiotic therapy, but these therapies are not enough to prevent the spread of tuberculosis. Infected individuals do not exhibit symptoms, but may be contagious for a period of time. In addition, it is difficult to control the patient's behavior even though the treatment regimen is strictly followed. Some patients are unable to complete the treatment process, which can lead to ineffective treatment and development of drug resistance. Even if you complete the entire course of treatment, TB infection remains a latent infection that can not be eradicated from the infected individual and still be reactivated.

In order to control the spread of tuberculosis, effective vaccination and precise initial diagnosis of the disease are most important. Until now, live vaccine vaccination is the most effective way to induce protective immunity. The most common Mycobacterium used for this purpose is Bacillus Calmette-Guerin (BCG), a non-pathogenic species of M. bovis. However, the safety and efficacy of BCG is controversial, and some countries, such as the United States, are not vaccinating the public with the above products. Diagnosis of tuberculosis is generally performed using a skin test method, which involves intradermal exposure to tuberculin protein derivative (PPD). The antigen-specific T cell response results in a measurable degree of induration at the site of injection up to 48-72 hours post-injection, which means exposure to the Mycobacterium antigens. However, the above assay is problematic in sensitivity and specificity, and individuals vaccinated with BCG can not be distinguished from infected individuals.

Macrophages have been shown to play a major role in the mycobacterium immunity, and T cells are also potent inducers of this immunity. The essential role of T cells in protection against M. tuberculosis infection is explained by the frequent occurrence of Mycobacterium infections in AIDS patients due to the depletion of CD4 ⁺ T cells associated with human immunodeficiency virus (HIV) infection . Mycobacterium-sensitizing CD4 + T cells have been shown to be potential producers of γ-interferon (IFN-γ), which in turn has been proven to trigger anti-mycobacterial effects by macrophage cells in mice . Although the role of IFN-y in humans is less clear, the research has shown that 1,25-dihydroxy-vitamin D3 alone or in combination with IFN-y or tumor necrosis factor-alpha activates human macrophages, Infection. Furthermore, IFN-y is known to stimulate human macrophages to produce 1,25-dihydroxy-vitamin D3. Similarly, interleukin-12 (IL-12) has been shown to play a role in stimulating resistance to M. tuberculosis infection. (Chan & Kaufmann, Tuberculosis: Pathogenesis, Protection and Control (Bloom ed., 1994), Tuberculosis (2nd ed., Rom and Garay, eds., 2003) for a review of the immunology of Mycobacterium tuberculosis infection. , And Harrison's Principles of Internal Medicine, Chapter 150, pp. 953-966 (16th ed., Braunwald, et al, eds., 2005). There is still a need for an effective therapeutic strategy to prevent reactivation of M. tuberculosis infection in both active and latent infections.

Currently, more than 40% of patients treated with Mycobacterium tuberculosis treatment have no treatment effect. In addition, patients who show effects also take several weeks to several months. Accordingly, there is a growing need for the development of new therapeutic agents for Mycobacterium tuberculosis. Animal models are essential for the development of effective treatment for M. tuberculosis. However, to date, there has been no model that largely reflects the clinical characteristics of pulmonary disease.

The basic elements to be considered in animal models are operative proof of behavior and reactivity to reproducibility and treatment of lung disease. Considering this point, the development of an animal model of transplantation and chemical injection methods of the TB germ cell gene has great advantages in ensuring uniformity of symptoms and ensuring reproducible results. In addition, this can lead to a shortening of the evaluation period and development period of the new drug. In particular, when a new drug screening system is constructed using a mouse developed through gene transplantation, which is a cause of tubercle bacillus, it is expected that the efficacy of various substances can be extensively tested at an individual level in a short period of time.

The present inventors have made intensive efforts to develop a new mycobacterial animal model in order to solve the above technical problem. As a result, the present inventors have found that an overexpression of an overexpressed gene in an animal model results in uniform occurrence of mycobacterial symptoms, And completed the present invention.

The present invention relates to in vivo . A mouse model and its production for studying the function and characteristics of polarized macrophages in the body during disease or infection induction using macrophage activation or polarized mouse model And to provide a method for this.

According to an aspect of the present invention, there is provided a method of manufacturing a mouse model comprising: polarizing a macrophage of a mouse by injecting a cytokine into a nasal cavity of a mouse; And confirming the polarity of the macrophage after 12 to 72 hours from the polarizing step; And < RTI ID = 0.0 > M. tuberculosis < / RTI > And maintaining the polarity by injecting the cytokine into the nasal cavity after the infecting step

Wherein said pathogen comprises pathogens associated with Mycobacterium tuberculosis or inflammatory lung disease or pathogens associated with infectious pulmonary disease.

Wherein the polarization of the macrophages occurs in an in vivo state.

The cytokine is characterized by inhibiting the proliferation of pathogens by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis. The cytokine includes those composed of lipopolysaccharide and interferon gamma.

In particular, the composition used to activate the polarity of macrophages is characterized by the amount of lipopolysaccharide being 0.5-5 μg / kg and the amount of interferon gamma being 0.5-5 μg / kg.

The mouse model production method is characterized by maintaining the polarity of macrophages by injecting cytokine every 3, 8, and 14 days after infecting the Mycobacterium tuberculosis.

The above-mentioned tuberculosis can be diagnosed as tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, , Breast tuberculosis, and spinal tuberculosis.

In another aspect, the present invention includes a mouse model produced by the above method.

As described above, the mouse model newly established according to the present invention can be usefully used for developing a therapeutic agent for Mycobacterium tuberculosis.

FIG. 1A is a photograph of protein expression by Western blot showing STAT expression, M1 / M2 macrophage polar marker, in the lung tissue of M1 / M2 mouse model on days 1, 3, 6 and 10 after stimulation.
FIG. 1B is a schematic view showing a method of manufacturing a mouse model of M1 / M2 at the time of studying M. tuberculosis infection.
FIG. 1C is a photograph showing the expression of STAT / M1 marker M2 using the Weatern blotting method to confirm that the M1 / M2 macrophage polarity is maintained in the lung tissue of the M1 / M2 mouse model infected with Mycobacterium tuberculosis.
FIG. 2A is a graph showing the amount of cytokine secreted by the enzyme-linked immunosorbent assay (ELISA) assay in the serum of an M1 / M2 mouse model 20 days after infection with M. tuberculosis.
2B is a photograph showing the morphology of the lung tissue of the M1 / M2 mouse model after 20 days of TB infection.
FIG. 2C is a photograph showing the pathological results of Hematoxylin & Erosin (H & E) staining of the lung tissue of the M1 / M2 mouse model after 20 days of Mycobacterium tuberculosis infection.
FIG. 2d is a photograph of protein expression by Western blotting showing expression of CHOP and Caspase-3 protein induced by apoptosis in the lung tissue of a mouse model 20 days after infection with M. tuberculosis.
FIG. 2E is a photograph showing the number of tubercle bacilli surviving in the lung tissue of the M1 / M2 mouse model after 3, 7, and 20 days after the tuberculosis infection.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to the accompanying drawings. It should be understood, however, that the appended drawings illustrate only the contents and scope of technology of the present invention, and the technical scope of the present invention is not limited thereto. It will be apparent to those skilled in the art that various changes and modifications can be made within the scope of the technical idea of the present invention based on these examples.

The present invention relates to a mouse model and a method for producing the mouse model, and more particularly, to a method for producing a mouse model for studying the functions and characteristics of polarized macrophages in the body upon inducing disease or infection.

Hereinafter, a mouse model and a method of producing the mouse model according to the present invention will be described in detail with reference to embodiments and drawings.

A method of producing a mouse model according to an embodiment of the present invention comprises the steps of polarizing a mouse macrophage by injecting cytokine into the nasal cavity of a mouse; And confirming the polarity of the macrophage after 12 to 72 hours from the polarizing step; And < RTI ID = 0.0 > M. tuberculosis < / RTI > And maintaining the polarity by injecting the cytokine into the nasal cavity after the infecting step.

In order to induce the polarity of the macrophages in the alveoli of the mouse model according to one embodiment of the present invention, the M1 macrophage mouse model was prepared by mixing Lipopolysaccharide (0.5-5 μg / kg and IFNγ (0.5-5 μg / kg) The model stimulated intranasally with IL-4 (0.5-5 μg / kg) and IL-13 (0.5-5 μg / kg)

The monocyte is characterized in that the macrophage is polarized in vivo, and the cytokine inhibits proliferation of pathogens by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.

And then confirming the polarity of the macrophage within 12 to 72 hours after the step. 1A, phospho-STAT1 (p-STAT1), which is a marker for M1-phagocyte polarity in the lung tissue of the M1 / M2 mouse model on days 1, 3, 6 and 10 after stimulation, STAT3 (p-STAT3) and phospho-STAT6 (p-STAT6) by Western blotting. The M1 / M2 polarity of macrophages was induced from 12 hours to 72 hours after stimulation, and the trend of M1 / M2 marker disappeared after 6 days after stimulation. (STAT1, STAT3, STAT6 in the figure are the internal control, the same hereinafter)

And then infecting the mouse into the nasal cavity of the mouse with M. tuberculosis.

The above-mentioned tuberculosis can be diagnosed as tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, , Breast tuberculosis, and spinal tuberculosis.

Finally, the step of infecting the Mycobacterium tuberculosis with a cytokine is injected every 3, 8, and 14 days to maintain the polarity of the macrophage. More specifically, to maintain M1 / M2 macrophage polarity, the M1 macrophage mouse model is IFN gamma (0.5-5 mu g / kg) and the M2 macrophage mouse model is IL-4 (0.5 -5 [mu] g / kg) into the nasal cavity.

In yet another aspect, the present invention includes a mouse model produced by the method.

In addition, as a background concept of the present invention, interferon gamma has a stronger antigen presenting ability than the macrophage before the antigen, enhances complement-mediated cytotoxicity, and produces an proinflammatory cytokine and this is called a typical activated macrophage (M1) .

 Secondary T lymphocytes are classified into two different types depending on the cytokine secreted, and can be classified into Th1 and Th2 cells. The cytokines mainly secreted by Th2 cells, such as IL-4, increase macrophage expression in contrast to activation induced by interferon gamma, for example, the main histocompatibility antigen class II. The macrophage of this state is also called an alternative activated macrophage (M2).

Hereinafter, preferred embodiments of a method for inhibiting the survival and proliferation of intracellular Mycobacterium tuberculosis using macrophage polarization of the macrophage according to the present invention will be described in detail with reference to the accompanying drawings. It is to be understood that the present invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, It is provided to inform.

How to make a mouse model that can be applied to in vivo experiments related to the polarity of macrophages

1. Mouse model development to polarize macrophages to M1 / M2

In order to induce the polarity of the macrophage in the alveolar macrophage of the mouse model, the M1 macrophage mouse model was treated with Lipopolysaccharide (0.5-5 μg / kg and IFNγ 0.5-5 μg / kg), M2 macrophage mouse model IL- / kg) and IL-13 (0.5-5 [mu] g / kg) were intranasally stimulated

2. Identification of the polarity of the macrophage after 1 day of the step

1A, phospho-STAT1 (p-STAT1), which is a marker for M1-phagocyte polarity in the lung tissue of the M1 / M2 mouse model on days 1, 3, 6 and 10 after stimulation, STAT3 (p-STAT3) and phospho-STAT6 (p-STAT6) by Western blotting. The M1 / M2 polarity of macrophages was induced from day 1 after stimulation, and the trend of M1 / M2 marker disappeared after 6 days after stimulation. (STAT1, STAT3, STAT6 in the figure are the internal control, the same hereinafter)

3. Step of infecting the mouse with Mycobacterium tuberculosis

1B is a schematic diagram briefly illustrating a method for producing an M1 / M2 mouse model in the course of studying M. tuberculosis infection. Based on the results obtained in FIG. 1A, a macrophage polar mouse model was prepared and infected into the nasal cavity with a Mycobacterium tuberculosis (5X10 ⁶ ) on the first day after stimulation.

4. The step of maintaining the polarity by injecting interferon gamma into the nasal cavity after 3, 8, and 14 days after the step of inserting and confirming whether it is maintained.

 To maintain the M1 / M2 macrophage polarity in the lung model of the post-infection mouse model, the M1 macrophage mouse model is IFN gamma (0.5-5 mu g / kg) every 3, 8, and 14 days after infection and the M2 macrophage mouse model is IL -4 (0.5-5 μg / kg) was stimulated intranasally.

FIG. 1C shows the expression of the M1 / M2 marker STAT in the lung tissue of the M1 / M2 mouse model infected with Mycobacterium tuberculosis using Western blotting to confirm that M1 / M2 macrophage polarity is maintained. Protein expression of phospho-STAT1, a marker showing M1 macrophage polarity, and phospho-STAT3 and phospho-STAT6, a marker showing M2 macrophage polarity, in lung tissue of mouse model 3 days after infection with M. tuberculosis in M1 / M2 polar mouse model Were confirmed by Western blotting. After infection, the M1 / M2 polarity was maintained in the lung macrophages of the mouse model.

In the above example, M1 macrophages or M2 macrophage mouse models were prepared by confirming macrophage polarity of polarized macrophages, that is, mouse lungs in the alveoli of mice, and confirmed them. The macrophage polarity-regulated mouse model according to the present invention can also be applied to macrophage polarity control studies related to various pathogenic infections and diseases.

The method of confirming whether the polarization of macrophages is effective for the inhibition of mycobacteria by the prepared mouse model.

FIG. 2A is a graph showing the amount of cytokine secreted by the enzyme-linked immunosorbent assay (ELISA) analysis in the serum of the M1 / M2 mouse model 20 days after the infection with M. tuberculosis. IL-12p40, IL-6, and tumor necrosis factor (TNF) were measured by enzyme-linked immunosorbent assay (ELISA) after the isolation of serum from the blood of M1 / monocyte chemotactic protein-1 (MCP-1) and anti-inflammatory cytokine IL-10. The expression pattern of cytokine was different according to the M1 / M2 polarity of intracellular macrophages in the mouse model after infection with M. tuberculosis.

2B is a photograph showing the morphology of the lung tissue of the M1 / M2 mouse model after 20 days of TB infection. We observed that the number of inflammatory lesions was significantly increased in the lung tissue of the M2 mouse model than the M1 mouse model.

FIG. 2C is a photograph showing pathological results of H & E staining and microscopic observation of the lung tissue of the M1 / M2 mouse model after 20 days of TB infection. It was confirmed that granulocytes were formed in the lung tissue of the M2 mouse model than the M1 mouse model because of the concentration of immune cells due to inflammation.

FIG. 2D shows the results of Western blotting of CHOP and Caspase-3 protein expression induced by apoptosis in lung tissue of M1 / M2 mouse model 20 days after infection with M. tuberculosis. The activity of CHOP and Caspase-3 was significantly higher in the lung tissue of the M1 mouse model than the M2 mouse model.

FIG. 2E shows the number of Mycobacterium tuberculosis bacteria survived in the lung tissue of the M1 / M2 mouse model after 3, 7, and 20 days after the tuberculosis infection. It was confirmed that the survival of M. tuberculosis was favorable in the lung of the M2 mouse model than that of the M1 mouse model.

Claims (8)

A method of making a mouse model comprising the steps of:
(a) polarizing the macrophage of a mouse by injecting cytokine into the nasal cavity of a mouse;
(b) confirming the polarity of the macrophage after 12 to 72 hours from the polarizing step;
(c) infecting the nasal cavity of a mouse with Mycobacterium tuberculosis; And
(d) maintaining the polarity by injecting the cytokine into the nasal cavity after the infecting step. The method of claim 1, wherein
Wherein the polarization of the macrophage occurs in an in vivo state. The method according to claim 1,
Wherein the cytokine induces apoptosis of macrophages infected with Mycobacterium tuberculosis, thereby inhibiting proliferation of Mycobacterium tuberculosis. The method according to claim 1,
Wherein the cytokine comprises a lipopolysaccharide and interferon gamma. 5. The method of claim 4,
Wherein the amount of lipopolysaccharide is 0.5 to 5 μg / kg and the amount of interferon gamma is 0.5 to 5 μg / kg. The method according to claim 1,
Wherein the polarity of the macrophage is maintained by injecting cytokine every 3, 8, and 14 days after the step of infecting the Mycobacterium tuberculosis. The method according to claim 1,
The above-mentioned tuberculosis can be diagnosed as tuberculosis, skin tuberculosis, adrenal tuberculosis, renal tuberculosis, epidemic tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, , Breast tuberculosis or spinal tuberculosis. A mouse model produced by the method of any one of claims 1 to 7.

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