WO2021194294A1 - Avatar model platform for evaluating autoimmune diseases - Google Patents

Abstract

The present invention relates to an avatar model platform for evaluating autoimmune diseases. An autoimmune disease model according to the present invention reflects the immune status of patients with autoimmune diseases well, and thus not only can be used as a preclinical model for monitoring the immune status of patients, but can also be used to screen for therapeutic substances for autoimmune diseases. Thus, the model can overcome limitations such as the absence of therapeutic drugs and the lack of clinical success of monitoring drugs with the current level of animal testing, which have been problems in the past, and thus can be used not only to evaluate drugs for autoimmune diseases but also to understand pathological mechanisms and develop tailored disease diagnosis and control systems. Therefore, the model can be effectively used in autoimmune disease-related fields or industries.

Description

Autoimmune disease evaluation avatar model platform

The present invention relates to an autoimmune disease evaluation avatar model platform.

Autoimmune disease is when the body's immune system malfunctions and its own cells attack their own cells. The human immune system basically recognizes foreign antigens against invading microorganisms and cancer cells, and has strong power to attack and remove them, but it does not attack its own cells because of its self-tolerance. This phenomenon is called self-tolerance of the human body. However, when the self-tolerance of the immune system is disrupted, autoreactive T cells that respond to self-cells (or autoantigens) are activated and autoantibodies are generated, constantly destroying self-cells, causing inflammation and immune responses. .

Cells that specifically respond to antigens in the immune system include T cells and B cells. When T cells meet a specific antigen presented by an antigen presenting cell, they respond according to the antigen. When the antigen presented by the antigen presenting cell is recognized as 'non-self', an immune response to remove it , and if recognized as 'self', the immune response is ignored and tolerated. When T cells are activated against an antigen, most B cells are activated one after another, and the B cells turn into plasma cells to produce antibodies that specifically react to the recognized antigen. Therefore, when autoimmunity occurs due to the breakdown of tolerance in the body, T cells are activated by abnormal recognition of autoantigens, and B cells are also activated to produce autoantibodies that respond to autoantigens. an immune response occurs.

On the other hand, in general, tailored medicine is also called order-made medicine or personalized medicine. means how to treat it. In order to implement customized medicine based on genetic information, biomarkers for genomic diagnosis, various genomic test methods, and medical informatics that can analyze/integrate genomic information derived using the biomarkers or obtained by the test method Analysis methods and targeted treatment technologies to which the results analyzed by the above analysis methods can be applied should be developed in advance and complementary to each other. That is, it is necessary not only to develop a technology for selecting a treatment suitable for a patient, but also to develop a technology for verifying the selected treatment.

As a method for realizing personalized medicine, it is necessary to develop a method for screening a drug suitable for a patient or a method for selecting a treatment method. Therefore, it is necessary to develop an animal model for preclinical research that can create an in vivo environment that reproduces biological characteristics in individuals.

On the other hand, an organoid is an organ-specific cell aggregate made by three-dimensionally culturing, aggregating, or recombination of stem cells, capable of self-renewal, and is also called a 'mini organ' or 'similar organ'. Organoids are very useful for basic research as they can implement in vitro studies that are difficult to implement in animal models such as molecular signal regulation in a form similar to that of real organs, as well as human development, disease model establishment, drug efficacy evaluation screening, It is a technology that can be very usefully used in various fields such as drug toxicity evaluation platform development and cell therapy drug development.

It is an object of the present invention to develop a humanized animal model of systemic lupus erythematosus in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from lupus disease patients.

An object of the present invention is to develop a method for producing a humanized lupus disease animal model, comprising injecting PBMC isolated from a lupus disease patient into immunodeficient mice.

It is an object of the present invention to develop an animal model of rheumatoid arthritis accompanied by pulmonary fibrosis, in which pulmonary fibrosis occurred by administering beta-glucan or a fungicide to an animal with rheumatoid arthritis.

An object of the present invention is to develop a method for producing an animal model of rheumatoid arthritis accompanied by lung fibrosis, comprising the step of inducing lung fibrosis by administering beta-glucan or a fungicide to a mouse with rheumatoid arthritis.

An object of the present invention is to develop an animal model of osteoarthritis accompanied by gout, in which gout is generated by administering potassium oxonate (PO) or hypoxanthine (HX) to an animal with osteoarthritis.

An object of the present invention is to develop a method for producing an animal model of osteoarthritis accompanied by gout, including the step of inducing gout by administering potassium oxonate or hypoxanthine to an animal with osteoarthritis.

An object of the present invention is a mouse To develop an organoid model of Sjogren's syndrome in which Interleukin-17 is administered to an organoid obtained by culturing the derived salivary gland stem cells.

It is an object of the present invention to develop a Sjogren's syndrome organoid model, including Patient-derived salivary gland epithelial cells.

An object of the present invention is a mouse culturing and obtaining derived salivary gland stem cells; and

To develop an organoid model production method for Sjogren's syndrome, which includes the step of culturing the cells to generate an organoid and then administering Interleukin-17.

It is an object of the present invention to develop a method for manufacturing a Sjogren's syndrome organoid model, which includes culturing Sjogren's patient-derived salivary gland epithelial cells.

In order to solve the above problems, the present invention provides a humanized lupus (systemic lupus erythematosus) disease animal model in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from lupus disease patients.

In order to solve the above problems, the present invention provides a method for manufacturing a humanized lupus disease animal model, comprising injecting PBMC isolated from a lupus disease patient into an immunodeficient mouse.

In order to solve the above problems, the present invention provides an animal model of rheumatoid arthritis accompanied by pulmonary fibrosis, in which pulmonary fibrosis is generated by administering beta-glucan or a fungicide to an animal with rheumatoid arthritis.

In order to solve the above problems, the present invention provides a method for preparing an animal model of rheumatoid arthritis accompanied by lung fibrosis, comprising the step of inducing lung fibrosis by administering beta-glucan or a fungicide to a mouse with rheumatoid arthritis.

In order to solve the above problems, the present invention provides an animal model of osteoarthritis accompanied by gout, in which gout is generated by administering potassium oxonate (PO) or hypoxanthine (HX) to an animal with osteoarthritis.

In order to solve the above problems, the present invention provides a method for producing an animal model of osteoarthritis accompanied by gout, comprising the step of inducing gout by administering potassium oxonate or hypoxanthine to an animal with osteoarthritis.

In order to solve the above problems, the present invention is a mouse Interleukin-17 (Interleukin-17) is administered to an organoid obtained by culturing the derived salivary gland stem cells, Sjogren's syndrome provides an organoid model.

In order to solve the above problems, the present invention provides a Sjogren's syndrome organoid model, including a Sjogren patient-derived salivary gland epithelial cell (Patient-derived salivary gland epithelial cell).

In order to solve the above problems, the present invention is a mouse culturing and obtaining derived salivary gland stem cells; And it provides a method for producing an organoid model of Sjogren's syndrome, comprising the step of culturing the cells to generate an organoid and then administering Interleukin-17.

In order to solve the above problems, the present invention provides a method for manufacturing a Sjogren's syndrome organoid model, comprising the step of culturing Sjogren patient-derived salivary gland epithelial cells.

Since the autoimmune disease model according to the present invention reflects the immune status of the autoimmune disease patient well, it can be used not only as a preclinical model for monitoring the patient's immune status, but also can be used for screening of therapeutic substances for autoimmune diseases. can Therefore, there is no treatment drug, which was a problem in the past, and monitoring drugs at the current animal test level can overcome the limitations of not being successful in clinical practice. It can be used in the field or industry related to autoimmune disease.

1 is a diagram schematically illustrating the lupus patient imitation avatar model construction protocol of the present invention.

2 is a diagram showing the results of reacting with human CD4, CD8 and CD19 antibodies to confirm human cell engraftment in a lupus patient mimic avatar model.

3 is a diagram illustrating the degree of infiltration of human immune cell subtypes (CD3, CD4, CD8, CD19) in tissues in order to confirm human cell engraftment in a lupus patient mimic avatar model.

4A, 4B and 4C are diagrams showing the results of measuring the dsDNA level measurement method, the urine human albumin concentration method, and the creatine concentration in order to confirm the autoantibody and proteinuria changes in the simulated avatar model of a lupus patient.

5 is a view confirming the infiltration of inflammatory cells in order to confirm the histological change of the kidney in the lupus patient simulated avatar model.

6 is a diagram comparing the arthritis scores for the curdlan-injected rheumatoid arthritis SKG animal model with lung fibrosis and the control group.

7 is a diagram comparing the weight change of the curdlan-injected rheumatoid arthritis SKG animal model with lung fibrosis and the control group.

8 is a view confirming the biochemical changes of each organ and tissue in the curdlan-injected rheumatoid arthritis SKG animal model and control group injected with pulmonary fibrosis by Pet-CT.

9A, 9B, 9C, 9D, 9E and 9F show the curdlan-injected rheumatoid arthritis SKG animal model and control group of GDF (Growth heart, lung, liver, muscle, joint and spine SUVs ( It is a diagram showing the results of evaluating the degree of inflammation increase according to the uptake level of GDF (Growth differentiation factor) according to the Standardized Uptake Value.

10 is a diagram confirming lung damage due to pulmonary fibrosis in the rheumatoid arthritis SKG animal model and control group injected with curdlan.

11A, 11B, and 11C are rheumatoid arthritis mice with PHMG lung fibrosis and control, confirming the arthritis index (A), alveolar bronchiolization (B), lung tissue and weight change (C) is a confirmation of

12A and 12B are diagrams confirming the degree of increase in macrophages, neutrophils and lymphocytes, which are immune macrophages, in rheumatoid arthritis mice with PHMG lung fibrosis and in the control group.

13 is a diagram confirming immune cell regulation according to the levels of Th1, Th2, Treg, and Th17 cells in rheumatoid arthritis mice with PHMG lung fibrosis and a control group.

14 is a graph showing the pain level (Procimal metatasal) for the animal model of osteoarthritis accompanied by gout (MIA+PO+HX) of the present invention, the time (sec) and the applied force ( g) is a diagram showing the measurement result.

15 is a result of measuring and confirming the weight bearing of the gout-accompanied osteoarthritis animal model (MIA+PO+HX) over time (Weight on right hind rimb) (%) of the present invention. is a diagram showing

16A, 16B, 16C, and 16D are views illustrating the deepening of osteoporosis according to the comparison of cartilage destruction and cartilage volume in the animal model of gout accompanying osteoarthritis (MIA+PO+HX) of the present invention, Trabecular bone (TB). ) (%), bone mineral density (BMD) (mg/cm3), and structure separation (St.sp) (U) were measured and confirmed.

17 is a diagram showing the results of observing the morphology of salivary gland epithelial cells derived from a patient with Sjogren's syndrome (passage-0).

18 is a diagram showing the results of observing the morphology of organoids using salivary gland epithelial cells (passage-1) derived from a patient with Sjogren's syndrome.

19 shows the results of treatment with isoproterenol and calcium chloride when culturing organoids using salivary gland epithelial cells derived from a Sjogren's syndrome patient, and FIG. 20 is a diagram confirming the increase in amylase of the organoid. am.

21 is a result of analysis by confocal microscopy of aquaporin-5, cytokeratin-19, and amylase A expression of organoids using salivary gland epithelial cells derived from a Sjogren's syndrome patient. is a diagram showing

22 is a view showing the result of confirming the degree of damage after treatment with IL-17 in order to prepare an organoid of mouse-derived salivary gland stem cells.

23 is a diagram showing the results of comparing the formation rate of salivary gland stem cells of the mouse-derived salivary gland stem cells organoids and the production level of alpha amylase, which is the main component of saliva, with the control group.

Figure 24a-d is a diagram confirming the expression level of aquaporin-5 (Aquaporin-5), nanog (Nanog), amylase 1 (Amylase 1), keratin 18 (Keratin 18) for organoids of mouse-derived salivary gland stem cells am.

The present invention provides a humanized animal model of systemic lupus erythematosus in which immunodeficient mice are administered with PBMCs (peripheral blood mononuclear cells) derived from lupus disease patients.

As used herein, the term "patient" means any single individual in need of treatment, including humans, apes, monkeys, cattle, dogs, guinea pigs, rabbits, mice, rats, chickens, insects, and the like. Also included in the subject are any subjects who participated in a clinical study trial without any clinical manifestations of any disease or subjects who participated in epidemiological studies or subjects used as controls.

As used herein, the term "immunodeficient mouse" means a mouse characterized by one or more of the following lists: defects in functional immune cells such as T cells and B cells; DNA repair defects; defects in the rearrangement of genes encoding antigen-specific receptors in lymphocytes; and defects in immune function molecules such as IgM, IgG1, IgG2a, IgG2b, IgG3 and IgA. In one embodiment, immunodeficient mice can be characterized by a deficiency in one or more genes involved in immune function, such as Rag1 and Rag2 (Oettinger et al, Science, 248:1517-1523, 1990; and Schatz et al, Cell, 59:1035-1048, 1989), immunodeficient mice may have these or other defects that result in abnormal immune function in the mouse.

Particularly useful immunodeficient mouse strains include NOD, Cg-PrkdcscidIl2rgtml Wjl/SzJ, commonly referred to as NOD scid gamma (NSG) mice, as detailed in Shultz et al,, J Immunol, 174: 6477-6489, 2005. and NOD.Cg-Rag1tmlMoml12rgtml Wjl/SzJ, which generally refers to NRG mice (Shultz et al, Clin Exp Immunol, 154(2):270-284, 2008). In an embodiment of the present invention, NSG mice were used.

As used herein, the term "severe combined immune deficiency (SCID)" refers to a symptom characterized by the absence of T cells and the absence of B cell function. SCID mutations refer to mutations that result in the deletion of functional B cells and T cells. For example, SCID mice (CB-17-Prkdcscid) are defective in rearrangement of B cell receptor (BCR) and T cell receptor (TCR), resulting in functional B and T cell deficits. this is caused The NSG (NOD scid gamma) mouse is also referred to as a NOD/SCID γnull mouse or a NOD/SCID IL-2RγKO mouse. NSG mice were developed in the laboratory of Dr Leonard Shultz at The Jackson Laboratory. The NSG mice can be purchased commercially from Jackson Laboratories, or prepared according to a known method (Shultz LD, et al, J Immunol 2005; 169: 204-209). For example, NSG mice can be obtained by backcross mating C57BL/6J-γ null mice and NOD/SCID mice 9 times. It is known that NSG mice lack functional T cells and functional B cells, have reduced macrophage function, abolished NK cells or NK activity, and reduced dendritic cell function. NSG mice are known to have a higher engraftment level of xenografts compared to NOD/SCID mice or γ2-microglobulin-deficient NOD/LtSz-SCID (NOD/SCID/γ2m null).

In the present invention, the terms "NOD scid gamma" and "NSG" may be used interchangeably. Cg-Prkdcscid NSG mice have multiple immunodeficiency based on NOD/ShiLtJ, severe combined immunodeficiency, and interleukin-2 receptor gamma chain. As a result, NSG mice lack cytokine signaling.NSG mice lack IL2R-γ (gamma c) expression, no detectable serum immunoglobulin, no hemolytic complement. , characterized by the absence of mature T lymphocytes and the absence of mature NK cells.

By using the humanized mouse model for preclinical studies, patient-specific lupus can be studied in the humanized mouse model. By administering any test compound or drug to the humanized mouse model according to one or more dosing regimens, the effectiveness, safety (e.g., any relevant adverse effects on the immune system) and efficacy of these test compounds or drugs can be studied. have. These methods are particularly effective for studies involving the interaction between lupus or immunomodulators, or diseased tissues (eg, cancer, autoimmune diseases) and the immune system.

This humanized mouse model can also be used to study any patient-derived xenograft in the presence of an engrafted human hematopoietic system. This includes tumor histology studies, omic studies or profiling (proteomic, genomic, metablomic, etc.). In certain embodiments, the patient-derived xenograft under study is Information on this can be obtained from the Mouse Tumor Biology Database (MTB), for areas such as selecting experimental models, examining patterns of mutations in specific cancers, and identifying genes in which mutations commonly occur across the cancer spectrum. designed to help researchers in

The present invention provides a method for preparing a humanized lupus disease animal model, comprising injecting PBMC isolated from a lupus disease patient into an immunodeficient mouse.

The present invention provides a method for screening a lupus therapeutic agent, comprising the step of treating a candidate substance in a humanized lupus animal model.

The present invention provides an animal model of rheumatoid arthritis accompanied by pulmonary fibrosis, in which pulmonary fibrosis occurs by administering beta-glucan or a fungicide to an animal with rheumatoid arthritis.

As used herein, the term "rheumatoid arthritis-inducing animal" may use SKG mice used as an animal model for rheumatoid arthritis because there is a genetic defect in the T-cell immune system and thus T-cell-induced chronic autoimmune disease is induced. In addition, a collagen-in-duced arthritis (CIA) model, a genetically modified naturally occurring arthritis model, an antibody-induced arthritis model, and a humanized severe combined immunodeficiency (SCID)-HuRAg mouse can be used. In addition, the types of laboratory mice that can be used for the preparation of the animal model according to the present invention include, but are not limited to, ICR or DDY belonging to a closed colony according to phylogenetic classification; BALB/cA, C57BL/6N, C3H/HeN, DBA/2N or CBA/N belonging to Inbred; BDF1 (C57BL/6 x DBA/2), CDF1 (CBA/N x DBA/2) or B6C3F1 (C57BL6 x C3H/HeN) belonging to the hybrid; Any one used as an experimental mouse, such as BALB/c-nu or C,B-17SCID belonging to the mutant family, can be used.

For the purpose of the present invention, the rheumatoid arthritis animal model has a limitation in that only some lung fibrosis occurs, and the incidence rate cannot be arbitrarily controlled. got to build

The beta glucan is curdlan, and the beta glucan may be administered at a concentration of 1 to 500 mg/kg, but is not limited thereto.

The disinfectant is polyhexamethylene guanidine (PHMG), hydrochloric acid polyhexamethylene biguanide (PHMB), methyl isothiazolinone (MIT), ethoxyethyl guanidine chloride (Chloride Ethoxyethyl Guanidine, PGH) , naphthalene, phthalate, didecyl methyl ammonium chloride (DDAC), benzisocyazolinone (BIT), chloroxylenol, titanium dioxide, zinc oxide and At least one selected from the group consisting of acetone, preferably polyhexamethylene guanidine (PHMG), which is a component of guanine series, polyhexamethylene biguanide hydrochloride (PHMB), ethoxyethyl chloride Adinine (Chloride Ethoxyethyl Guanidine, PGH), more preferably polyhexamethylene guanidine (Polyhexamethylene guanidine, PHMG), but is not limited thereto. The disinfectant may be administered at a concentration of 1-500 mg/kg, but is not limited thereto.

In the present invention, the term "polyhexamethylene guanidine (PHMG)" has been used as a major component of a humidifier disinfectant, and when exposed to the lungs at high concentrations, cold or pneumonia symptoms occur and develop into interstitial pneumonia, causing the lungs to harden. It may also cause shortness of breath. Lung damage cannot be repaired, and if a lung is not transplanted due to the fixed deterioration of lung function, it leads to death.

The administration may be selected from the group consisting of oral, intravenous, intramuscular, subcutaneous and intraperitoneal injections, and preferably, when administering the curdlan, it may be administered by oral administration and intraperitoneal injection. Curdlan may be administered in a weight ratio of 1:2, but is not limited thereto.

The administration may be administered 1-5 times for 1-5 weeks, preferably 1-3 times for 1-3 weeks, but is not limited thereto.

The animal is one or more selected from the group consisting of mice, rats and hamsters, rabbits, horses, cattle, dogs, cats, monkeys and guinea pigs, but preferably mice, but not limited thereto.

The lung fibrosis may cause one or more lung diseases selected from the group consisting of pulmonary fibrosis accompanying rheumatoid arthritis, common interstitial pneumonia, non-specific interstitial pneumonia, inflammatory airway disease, and organized pneumonia.

In the constructed animal model of rheumatoid arthritis with lung fibrosis, the arthritis index was higher than that of the control group, and it was confirmed that the mouse weight decreased. In addition, it was confirmed that inflammation was increased in the heart, lung, liver, muscle, joint and spine of mice, and damage to the lungs due to pulmonary fibrosis appeared. In addition, alveolar bronchiolization occurred significantly compared to the control group, macrophages, neutrophils, and lymphocytes were increased to increase immune response, and inflammatory cells, Th17 It was confirmed that the cells were increased and the Treg cells controlling the immune response were decreased.

The present invention also provides a method for preparing an animal model of rheumatoid arthritis accompanied by lung fibrosis, comprising the step of inducing pulmonary fibrosis by administering beta-glucan or a fungicide to a mouse with rheumatoid arthritis.

The present invention also provides a method for screening a rheumatoid arthritis therapeutic agent with lung fibrosis, comprising the step of treating the candidate substance to the rheumatoid arthritis animal accompanied by pulmonary fibrosis.

The present invention provides an animal model of osteoarthritis accompanied by gout, in which gout is developed by administering potassium oxonate (PO) or hypoxanthine (HX) to an animal with osteoarthritis.

In the present invention, the term "potassium oxonate (PO)" is a uric acid lyase inhibitor, which can induce hyperuricemia.

As used herein, the term "hypoxanthine (HX)" is a naturally occurring purine derivative. Hypoxanthin is often found as a component of nucleic acids and is present in the form of inosine, a nucleoside, in the anticodon of tRNA. Hypoxanthin is an additive necessary for the culture of certain cells, bacteria, and parasites as a substrate and nitrogen source.

As used herein, the term "osteoarthritis-inducing animal" refers to an animal that has developed osteoarthritis by an anterior cruciate ligament resection method, a meniscus resection method, a transgenic/knockout method, a chemical injection method, a naturally occurring mouse, or the like.

For the purposes of the present invention, sheep, dogs, rabbits, and rat animals using the anterior cruciate ligament resection method may be included, and may include meniscus-resected sheep, dogs, rabbits, guinea pigs, rats, mice, and the like. In addition, it may include ADAM-15 KO, MMP-14 KO, MMP-13 transgenic mice constructed by the transgenic/knockout method, and include mice, rabbits, and rats by collagenase constructed by the chemical injection method. can do. MIA (Monosodium iodoacetate) injection may include mice, rats, rabbits, and guinea pigs. Also includes naturally occurring DBA/1 (mouse), STR/ORT (mouse), C57BL6 (mouse), Hartley guinea pig, rhesus macaque (monkey), Cynomolgus macaque (monkey) and, if it is to construct an animal model of osteoarthritis accompanied by gout, it is not limited thereto.

For the purpose of the present invention, gout promotes bone destruction in osteoarthritis patients, and the animal model of osteoarthritis accompanied by gout of the present invention overcomes the limitation that some gout occurs in the conventional animal model of osteoarthritis, but the incidence rate cannot be arbitrarily controlled. , established an animal model of osteoarthritis with clear gout.

The administration may be selected from the group consisting of oral, intravenous, intramuscular, subcutaneous and intraperitoneal injection, oral administration may be administered with potassium oxonate, and intraperitoneal administration may be administered with hypoxanthine, but is not limited thereto.

The potassium oxonate and hypoxanthine may be administered in a weight ratio of 1:1, may be administered 1-10 times for 1-5 weeks, preferably 1-7 times for 1-3 weeks, but , but not limited thereto.

The present invention also provides a method for producing an animal model of osteoarthritis accompanied by gout, comprising the step of inducing gout by administering potassium oxonate or hypoxanthine to an animal with osteoarthritis.

In addition, the present invention provides a method for screening a treatment material for osteoarthritis accompanied by gout, comprising the step of treating the candidate material to the animal model of osteoarthritis accompanied by gout.

The present invention provides an organoid model of Sjogren's syndrome in which Interleukin-17 is administered to an organoid obtained by culturing mouse-derived salivary gland stem cells.

As used herein, the term "organoid" means that cells derived from tissues or embryonic stem cells can be cultured in a 3D form to form an artificial organ. Organoid is a suffix that has the same meaning as 'organ' of an organ, and has the word 'organ-like'. Organoids have a more well-arranged cell and cell function through a three-dimensional culture method, and have a functional organ-like form and function. Organoids are attracting attention along with research on optimization of growth and differentiation factors that can differentiate into various tissues, with stem cell research and 3D cell culture being developed.

As used herein, the term "saliva" is a mixed solution secreted from the parotid, submandibular, sublingual, and mucous glands present in the oral mucosa. Saliva is a key component of the human body, produced in the salivary glands and discharged into the oral cavity. Saliva is an essential component of the human body and contains bioactive proteins, digestive enzymes, mucus, immunoglobulins, and various salts. Saliva plays a very important role in maintaining the homeostasis of the human body as well as oral health. For example, the main components of saliva, mucin, immunoglobulin, etc., play a primary defense role against external infection, and protect the oral mucosa and teeth through lubrication and moisture maintenance of the oral cavity and teeth, and neutralizing pH. do. In addition, saliva contains digestive enzymes such as amylase, such as ptyalin, which promotes digestion by decomposing starch to maltose units. In addition, the body's water metabolism and body temperature control can be achieved by the secretion of saliva, and toxic substances (I, Hg, Pb, etc.) are excreted.

As used herein, the term "salivary gland" refers to an organ that produces and secretes saliva, such as parotid gland, submandibular gland, and sublingual gland. It is a minor salivary gland distributed in various parts of the oral mucosa, such as major salivary glands and mucous glands existing in the mucous membrane of oral cavity, such as mucous glands. classified.

In the present invention, the derived mouse is not limited, and general laboratory mice, immunodeficient mice, and the like may be used.

In addition, the present invention provides an organoid model of Sjogren's syndrome, including Patient-derived salivary gland epithelial cells.

When culturing Sjogren's patient-derived salivary gland epithelial cells as a single layer, it is difficult to ascertain the exact functions of acinar cells functioning in a 3D structure - for example, secretory protein production ability, water channel - and acinar cell polarization. When making organoids with salivary gland cells from normal individuals and Sjogren patients, no distinct characteristics appear, and the number of salivary gland stem cells decreases and salivation-related genes (Amy1, Aquaporin5, etc.) decrease.

In addition, the present invention is a mouse culturing and obtaining derived salivary gland stem cells; And it provides a method for producing an organoid model of Sjogren's syndrome, comprising the step of culturing the cells to generate an organoid and then administering Interleukin-17.

The present invention also provides a method for producing a Sjogren's syndrome organoid model, comprising culturing the salivary gland epithelial cells derived from a Sjogren patient.

In addition, the present invention provides a method for screening a Sjogren's syndrome therapeutic material, including; treating a candidate material in the Sjogren's syndrome organoid model.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and when it is determined that detailed descriptions of well-known techniques or configurations known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted, and , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of equivalents interpreted therefrom and the description of the claims to be described later.

The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the contents of the present invention, and the present invention is not limited thereto.

<Example 1> Lupus patient imitation avatar model evaluation platform

<Example 1-1> Establishment of an evaluation platform for lupus patient imitation avatar model

A humanized mouse model of lupus (avatar model) equipped with a human immune system

In order to establish liposomes, PBMCs (peripheral blood mononuclear cells) derived from normal or lupus patients were intraperitoneally injected into 8-10 week old NSG mice at 5x106/mice, and then 3 weeks

After confirming the engraftment of normal or lupus patient cells in the blood, serum and urine

it was Thereafter, the mice were sacrificed 4 weeks after cell transplantation to confirm the infiltration of human cells and histological changes in the tissue ( FIG. 1 ).

<Example 1-2> Confirmation of engraftment of human cells in blood of lupus patient imitation avatar model

In order to confirm that the lupus patient mimic avatar model was properly constructed in Example 1-1, engraftment of human cells was confirmed by analyzing T cells and B cells in the blood. Specifically, blood was obtained from the lupus humanized mice of Example 1-1 (normal PBMC-injected group and lupus patient PBMC-injected group), and cells that reacted with human CD4, CD8 and CD19 antibodies were analyzed through flow cytometry. .

As a result, human blood in mouse blood by PBMC injection of normal subjects and lupus patients

CD4+T, CD8+T, and Cd19+T cells were detected, confirming that human cells were well engrafted ( FIG. 2 ).

<Example 1-3> Confirmation of infiltration of human immune cell subtypes in a simulated avatar model of a lupus patient

In order to confirm the engraftment of human cells in the tissue in the humanized lupus mouse model established in Example 1-1, the humanized lupus mice were sacrificed 4 weeks after Example 1 and the kidneys were removed, and human immune cells in the tissue Subtypes (CD3, CD4, CD8, CD19) were stained with IHC to infiltrate. The excised tissue was fixed with formalin and then embedded in paraffin to create a 5 μm thick section. In order to observe the immune cells in the tissue, immunohistochemical analysis was performed by reacting with human CD3, CD4, CD8, and CD19 antibodies on the section slides.

As a result, human CD3+T, CD4+T, CD8+T, and Cd19+T cells were detected in the mouse kidney tissue injected with the PBMCs of normal persons of Example 1-1 and lupus patients, indicating that human cells were well engrafted. could be (Fig. 3).

<Example 1-4> Observation of changes in autoantibodies and proteinuria in a simulated avatar model of a lupus patient

According to the above series of examples, it was confirmed that the humanized mouse system was well established by confirming the engraftment of human cells in the lupus humanized mouse model established in Example 1-1. Therefore, in order to confirm whether there is a difference in the lupus phenotype according to the cells of normal people and lupus patients, changes in autoantibody and proteinuria were observed.

For dsDNA, the amount of dsDNA in the serum was measured using a human anti-double stranded DNA (dsDNA) antibody (IgG) ELISA Kit after diluting mouse serum to 1/20. In addition, the concentration of human albumin and creatine from urine isolated from mice was measured.

It was measured using mouse albumin ELISA Quantitation kit and creatinine kit.

Specifically, as a result of confirming the expression level of the dsDNA level in the humanized lupus mouse model and the normal human model, it was confirmed that the anti-dsDNA level was increased in the humanized lupus mouse model by PBMC injection of the patient. In addition, as a result of confirming the concentrations of human albumin and creatine in the urine, it was confirmed that the concentrations were increased in the humanized lupus mouse model.

In addition, as a result of comparing human creatine (creatine) levels and comparing the levels of albumin / creatine, it was confirmed that all of them were increased in the lupus humanized mouse model (Fig. 4).

<Example 1-5> Confirmation of histological changes in the kidney in the lupus patient simulated avatar model

In order to histologically analyze the kidney tissue of the humanized lupus mouse model established in Example 1-1, the kidney tissue of the humanized lupus mouse of Example 1-1 was stained with H&E (haematoxylin and eosin). Specifically, the excised tissue was fixed with formalin and then embedded in paraffin to generate a 5 μm thick section, and the section slide was deparaffinized with xylene for H&E staining. Thereafter, the cell nucleus was stained with hematoxylin, the cytoplasm with eosin, and periodic acid-Schiff (PAS) was stained to compare and analyze the pathology of the kidney tissue.

As a result, infiltration of inflammatory cells compared to normal in the lupus mouse model injected with PBMC.

It was confirmed that this increased (FIG. 5).

<Example 2> Model evaluation platform for pulmonary fibrosis comorbidity in rheumatoid arthritis patients

<Example 2-1> Preparation of rheumatoid arthritis model with lung fibrosis through Curdlan injection

2-1-1 Production of an animal model for rheumatoid arthritis with lung fibrosis through Curdlan injection (IL-1Ra KO model)

To construct an animal model of rheumatoid arthritis with lung fibrosis, SKG mice were used. Specifically, it is known that the SKG mouse has a genetic defect in the T-cell immune system and causes T-cell-induced chronic autoimmune disease. The following control and experimental groups were constructed in SKG male mice (within 8 weeks of age). The control group was treated with PBS, and the experimental group was injected with curdlan at 6 mg/kg intraperitoneal injection and 3 mg/kg oral injection once a week before the start of the experiment. built.

<Example 2-2> Effect of rheumatoid arthritis animal model with lung fibrosis through Curdlan injection

2-2-1 Confirmation of Arthritis Index of Curdlan-Injected Rheumatoid Arthritis Mice with Lung Fibrosis

Arthritis scores were confirmed for the SKG animal model of rheumatoid arthritis with lung fibrosis constructed in Example 2-1 and the control group. The scores and criteria for the evaluation of arthritis are as follows.

-Evaluation standard-

0 points: No edema or swelling.

Score 1: Mild swelling and redness confined to the foot or ankle joint

Score 2: Mild swelling and redness from the ankle joint to the metatarsal.

Score 3: Moderate swelling and redness from the ankle joint to the tarsal bone.

4 points: swelling and redness from the ankle to the entire leg

The best arthritic index per mouse is 4 points, so the best disease index per mouse is 16.

As a result, in the SKG animal model of rheumatoid arthritis with lung fibrosis, a value of 375 was

As the value of 20 appeared in the control group, the animal model constructed in the present invention

By confirming that the arthritis index was high in the , it was confirmed that an animal model in which rheumatoid arthritis was properly expressed was constructed ( FIG. 6 ).

Confirmation of body weight change in rheumatoid arthritis mice with 2-2-2 Curdlan-injected pulmonary fibrosis

The mouse weight changes were compared to the SKG animal model of rheumatoid arthritis with lung fibrosis constructed in Example 2-1 (24 weeks old, 17 weeks after curdlan injection) and control SKG mice (24 weeks old).

As a result, it was confirmed that the control SKG mouse weighed 277 g and the rheumatoid arthritis SKG animal model with lung fibrosis weighed 158 g, confirming that the weight of the rheumatoid arthritis SKG animal model with lung fibrosis decreased ( FIG. 7 ).

Confirmation of Inflammatory Response in Rheumatoid Arthritis Mice with 2-2-3 Curdlan Injection with Lung Fibrosis

Inflammatory responses were confirmed in the organs of the mouse compared to the SKG animal model of rheumatoid arthritis with lung fibrosis constructed in Example 2-1 (24 weeks old, 17 weeks after curdlan injection) and the control SKG mouse (24 weeks old).

Specifically, by Pet-CT imaging of the experimental group and control mice, biochemical and functional changes of each organ and tissue inside the living body were observed as images, and ROI (region of interest) values were obtained according to specific regions of interest for each organ. did. Standardized Uptake Value (SUV) values were obtained for inflammation of the heart, lung, liver, muscle, joint (articulation) and spine (vertebra) of each mouse, and the degree of growth differentiation factor (GDF) uptake The degree of increase in inflammation was evaluated.

As a result, it was confirmed that inflammation in the SKG animal model of rheumatoid arthritis with lung fibrosis was increased in the heart, lung, liver, muscle, joint and spine compared to the control SKG mouse. Therefore, it was confirmed that the animal model of the present invention showed an inflammatory response and properly exhibited rheumatoid arthritis accompanied by pulmonary fibrosis ( FIGS. 8 and 9a-f ).

Confirmation of damage caused by lung fibrosis in rheumatoid arthritis mice with 2-2-4 curdlan-injected lung fibrosis

Damage caused by lung fibrosis was confirmed in the SKG animal model of rheumatoid arthritis with lung fibrosis constructed in Example 2-1 (24 weeks old, 17 weeks after curdlan injection) and control SKG mice (24 weeks old).

As a result, as compared to control SKG mice, lung damage due to lung fibrosis was confirmed in the SKG animal model of rheumatoid arthritis with pulmonary fibrosis, and the animal model of the present invention clearly expressed lung fibrosis, resulting in rheumatoid arthritis with lung fibrosis. It is suggested to construct an arthritic animal model ( FIG. 10 ).

<Example 2-3> Production of an animal model of rheumatoid arthritis accompanied by lung fibrosis through polyhexamethylene guanidine (PHMG) injection

Collagen-induced rheumatoid arthritis animal model (CIA) was injected with polyhexamethylene guandinine (PHMG). Specifically, in order to increase the sensitivity, after immunization at week 0, 1% of 100ul polyhexamethylene guanine (PHMG) was intratracheal instillation at week 6, and administered only once at week 6 Then, the mice were sacrificed and the analysis was performed.

<Example 2-4> Effect of an animal model of rheumatoid arthritis with lung fibrosis through polyhexamethylene guandinine (PHMG) injection

2-4-1 Confirmation of Arthritis Index of Rheumatoid Arthritis Mice with PHMG Lung Fibrosis

Arthritis index for the PHMG-injected rheumatoid arthritis animal model with lung fibrosis and control mice constructed in Example 2-3 was confirmed. Specifically, the arthritis index was measured by the method described in 2-2-1 above, and the arthritis index from week 0 to week 7 when rheumatoid arthritis occurred was confirmed.

As a result, it was confirmed that the PHMG-injected rheumatoid arthritis animal model with lung fibrosis constructed in the present invention reduces the swelling of arthritis, but as the inflammatory response is induced while the arthritis index is maintained compared to the rheumatoid arthritis animal model, lung fibrosis is induced. (Fig. 11a)

2-4-2 Confirmation of lung tissue and weight change in rheumatoid arthritis mice with PHMG lung fibrosis

The effect of PHMG injection constructed in Example 2-3 on the lung tissue in the animal model of rheumatoid arthritis accompanied by lung fibrosis and the control mouse was confirmed.

Specifically, through alveolar bronchiolization, it is a dysplastic lesion characterized by cells similar to the bronchial epithelium covering the normal or thickened alveolar wall, in pathological conditions exposed to inflammation, chemical stimuli, etc. It is characterized by occurrence.

It was confirmed that tribronchialization of the alveoli of the PHMG-injected rheumatoid arthritis animal model with lung fibrosis constructed in the present invention occurred significantly compared to the control group (Fig. 11b). As a result, it was confirmed that the weight of the lungs increased compared to the control group, and the mass of the lungs compared to the body weight also increased. Therefore, the PHMG-injected rheumatoid arthritis animal model constructed in the present invention confirmed that lung fibrosis progressed and affected the lung tissue (FIG. 11c).

2-4-3 Immune Cell Identification in Lung Tissues from Rheumatoid Arthritis Mice with PHMG Lung Fibrosis

In the lung tissue of the PHMG-injected rheumatoid arthritis animal model with lung fibrosis and control mice constructed in Example 2-3, the degree of increase in immune cells, macrophages, neutrophils, and lymphocytes, was confirmed. did.

As a result, it was confirmed that macrophages, neutrophils and lymphocytes all significantly increased in the lung tissue of the PHMG-injected rheumatoid arthritis animal model with lung fibrosis constructed in the present invention (Fig. 12a). As a result of confirming the infiltration score, it was confirmed that the immune response was induced as it significantly increased in the animal model constructed in the present invention ( FIG. 12b ).

2-4-4 PHMG Confirmation of Immune Cell Regulation in Rheumatoid Arthritis Mice with Lung Fibrosis

T cells that have completed differentiation are largely divided into type 1 helper cells (Th1) and type 2 helper cells (Th2) according to their functions. The main function of Th1 cells is cell-mediated immunity, and Th2 cells are humoral. involved in immunity. Treg cells have the property of controlling the inflammatory response by inhibiting the function of abnormally activated cotton cells, and unlike Treg cells, Th17 cells are involved in the forefront of the inflammatory response seen in immune diseases and maximize the signal of the inflammatory response to disease. accelerate the progress of

For the PHMG-injected rheumatoid arthritis animal model with lung fibrosis and control mice constructed in Example 2-1-2, Th17 cells were IL-17+in CD4+, Treg cells were CD25+Foxp+inCD4+, and Th1 cells were IFN-γ +in CD4+ cells were identified, and Th2 cells were identified as IL-4+in CD4+ cells.

As a result, it was confirmed that Th17, an inflammatory cell, was increased, and the level of Treg cells controlling the immune response decreased (FIG. 13).

In the constructed animal model of rheumatoid arthritis with lung fibrosis, the arthritis index was higher than that of the control group, and it was confirmed that the mouse weight decreased. In addition, it was confirmed that inflammation was increased in the heart, lung, liver, muscle, joint and spine of mice, and damage to the lungs due to pulmonary fibrosis appeared. In addition, alveolar bronchiolization occurred significantly compared to the control group, macrophages, neutrophils, and lymphocytes were increased to increase immune response, and inflammatory cells, Th17 It was confirmed that the cells were increased and the Treg cells controlling the immune response were decreased.

<Example 3> Gout-accompanied arthritis model evaluation platform

<Example 3-1> Preparation of osteoarthritis model with gout simulated in osteoarthritis patients

Monosodium Iodoacetate (MIA, I2512, Sigma, Poole, UK) was dissolved in saline for injection at a concentration of 60 mg/ml, and prepared on the day of the start of the experiment (day 0). After respiratory anesthesia using isoflurane to rats (Wistar rats, 6 weeks old, 180g~220g) on the day of the start of the experiment, 50ul (MIA 3mg/body) MIA was injected into the right knee joint with a 26.5 gauge needle through the infrapatellar ligament. was injected to induce osteoarthritis (MIA).

300 mg/kg potassium oxonate (PO) was intraperitoneally injected into the induced osteoarthritis mice, and 300 mg/kg hypoxanthine (HX) was orally injected, which was injected 7 times a week until the end of the experiment Thus, an animal model of osteoarthritis accompanied by gout was constructed.

<Example 3-2> Confirmation of pain level (Procimal metatasal) of osteoarthritis patient-simulated gout-accompanied arthritis model

In order to measure the pain level of the animal model of osteoarthritis accompanied by gout constructed in Example 3-1, a Dynamic planter esthesiometer (UgoBasile, Comerio, Itaily) of the animal model of the control group and the present invention was used. Place the stimulator on the underside of the rat, adjust a 0.5mm thick plastic stimulation needle (Stimulating microfilament) to be positioned on the hind leg, and operate the machine. The time (sec) and applied force (g) until the rat could not withstand the stimulation and released the foot were measured by increasing it. Each measurement was performed three times and averaged.

As a result, compared to the normal group, the time (sec) and force (g) until taking off the foot without enduring the stimulus showed a significant difference in pain level (Procimal metatasal) in the animal model of osteoarthritis accompanied by gout. It was confirmed that the animal model of osteoarthritis accompanied by gout of the present invention was constructed to accompany gout while maintaining the pain characteristic of the animal model of osteoarthritis (MIA) (FIG. 14).

<Example 3-3> Confirmation of weight bearing degree of osteoarthritis patient simulated gout-accompanied arthritis model

In order to measure the pain level for the osteoarthritis animal model with gout constructed in Example 3-1, using an Incapacitance Meter (IITC, Victory Blvd Woodland Hills, CA, USA), the weight on the right hind limb (Weight on right hind rimb) ) (%) was measured.

As a result, it is determined that there is a significant difference in weight on right hind rimb (%) in the animal model of osteoarthritis accompanied by gout, and that there is a difference in weight bearing, and the animal model of osteoarthritis accompanied by gout of the present invention is It was confirmed that the animal model was constructed to accompany gout while maintaining the difference in weight bearing, which is a characteristic of the osteoarthritis animal model (MIA) (FIG. 15).

<Example 3-4> Confirmation of deepening osteoporosis according to comparison of cartilage destruction and cartilage volume in an osteoarthritis patient simulated gout-accompanied arthritis model

By confirming the cartilage destruction and cartilage volume in the animal model of osteoarthritis accompanied by gout constructed in Example 3-1, the degree of deepening of osteoporosis was confirmed.

Specifically, the femur (Femur) and tibia (Tibia) of the control group (WT), osteoarthritis animal model (MIA), and gout-accompanied osteoarthritis animal model (MIA+PO+HX) were collected and stained with India ink and then microcomputerized. The degree of destruction of cartilage and cartilage volume were analyzed by tomography (micro-CT image). Trabecular bone (TB) (%) decreases as osteoporosis intensifies, and bone mineral density (BMD) (mg/cm ³ ) is an indicator that decreases as osteoporosis intensifies. In addition, the structure separation (St.sp) (U) is the gap between the cancellous bones, and when osteoporosis occurs, the decrease in the cancellous bone (TB) occurs. The degree of osteoporosis was measured by increasing structural separation (St.sp).

As a result, compared with the control group (WT) and the animal model of osteoarthritis (MIA), the trabecular bone (TB) (%) and bone mineral density (BMD) ( mg/cm ³ ) was decreased, and the spacing between cancellous bones was increased, confirming that the structural separation (St.sp) was increased. Therefore, the animal model of osteoarthritis accompanied by gout (MIA+PO+HX) of the present invention confirmed that osteoporosis was aggravated as it was accompanied by gout, compared with the animal model of osteoarthritis (MIA).

<Example 4> Sjogren's disease salivary gland organoid model evaluation platform

<Example 4-1> Sjogren's disease salivary gland organoid model platform construction

4-1-1. Preparation and culture of Sjogren patient-derived salivary gland epithelial cells

Patient-derived salivary gland epithelial cells from Sjogren's patients were prepared. To obtain a culture solution of the cells, the medium was 2.5% Fetal bovine serum (Young In Frontier #US-FBS-500), DMEM/F12 (1:3 mixture, Gibco#11330-032), 1% Prepared with penicillin-streptomycin (Gibco#15140-122), additionally 0.4 μg/mL hydrocortisone (Sigma#H0888), 10 ng/mL mouse EGF (human/mouse common, Biolegend#585608), 0.5 μg/mL insulin (Gibco#12585-014). Using the medium, Sjogren's patient-derived salivary gland epithelial cells were diluted 20-fold in PureCol (Advanced biomatrix #5005) distilled water, put in a Petri dish, and left at 37°C for 1 hour or more, then removed and washed with PBS or medium before use. 1U/mL dispase II solution (Stemcell#07923) and 2mg/mL collagenase IV (Gibco#17104-019) were prepared. Thereafter, 3 mL of the washed thing was put in a 60 mm Petri dish, and the tissue was placed on it and cut as finely as possible. Incubated at 37°C, taken out every 15 minutes and resuspended. When the tissue became almost limp, it was transferred to a tube, centrifuged at 1500 rpm, 5 minutes, and 4°C, resuspended in medium, and incubated at 37°C. The salivary gland epithelial cells derived from the Sjogren patient were resuspended in 1 mL of 90% fetal bovine serum and 10% dimethyl sulfoxide solution and freeze-dried.

As a result, it can be confirmed that the salivary gland epithelial cells derived from Sjogren's syndrome patients form an epithelial-like morphology and grow as a single layer (FIG. 17).

In addition, subculture was performed to maintain Sjogren's patient-derived salivary gland epithelial cells, and the cells were injected at a ^{concentration of 1 x 10 5} cells/24 well plate and 5 x 10 ⁵ cells/6 well plate. PureCol (Advanced biomatrix #5005) was diluted 20 times in distilled water, put in a Petri dish, left at 37°C for 1 hour or more, then removed and washed with PBS or medium before use. After removing the media and washing with PBS, 2mL of 0.25% trypsin-EDTA was placed in a Petri dish and incubated at 37°C for 10 minutes. The resuspended cells were transferred to a 50 mL tube and centrifuged at 1000 rpm at 4°C for 5 minutes. The supernatant was discarded, resuspended in culture medium, and cultured in a Petri dish.

4-1-2. Production of organoids derived from salivary gland epithelial cells derived from Sjogren patients

Organoid culture medium includes Keratinocyte-Serum Free Media with EGF, bovine pituitary extract (Gibco#37010-022), 0.09mM Calcium chloride (Amresco) #E506-1) was used. The organoid culture method is to dilute Matrigel matrix (BD#354234) with refrigerated K-SFM medium at a ratio of 2:1 and place it on a 48 well-plate placed on ice, which is then placed in a 37 degree cell incubator. Transfer and coating for 1 hour. The salivary gland epithelial cells derived from Sjogren's patient cultured in 4-1-1 were resuspended at 37°C, and 5 x 10 ⁴ cells/500 μL/well were dispensed to the coated ones. Organoids were observed while changing the medium every 3 days.

As a result, it was confirmed that organoids using salivary gland epithelial cells derived from patients with Sjogren's syndrome were constructed in the form of spheroids after 1 to 2 days of culture, and duct organoids and acinar organoids after 7 to 8 days. (Fig. 18).

<Example 4-2> Analysis of amylase secretion ability of organoids derived from salivary gland epithelial cells derived from Sjogren patients

In order to analyze the amylase secretion ability of the organoids derived from salivary gland epithelial cells derived from Sjogren's patients prepared in Example 4-1-2 of the present invention, different stimuli were used.

Specifically, the patient-derived salivary gland organoids were cultured in a medium supplemented with 0.09 mM calcium chloride to obtain spiroids, and then with 10 mM isoproterenol and 2 mM calcium chloride. Stimulated for 18 hours. Thereafter, the supernatant was removed after microscopy, and amylase activity was measured using the cells (amylase activity assay kit, abcam#102523).

As a result, it was confirmed that when the cultured organoids were stimulated with isoproterenol and calcium chloride, proteins were observed around the organoids (FIG. 19). In addition, it was confirmed that amylase was very significantly increased in the organoid culture supernatant (FIG. 20).

<Example 4-3> Confocal microscopic analysis of aquaporin-5, cytokeratin-19, and amylase A expression of organoids derived from salivary gland epithelial cells derived from Sjogren's patient

As primary antibodies, rabbit anti-human aquaporin-5 antibody (Abcam#ab92320, diluted 1:100) and mouse anti-human amylase A antibody (Abcam#ab201450, diluted 1:100) were used. Secondary antibodies were Alexa488 donated goat anti-rabbit IgG (H+L) (Invitrogen# A32731, 1:500 dilution), Alexa594 donated goat anti-rabbit-mouse IgG (H+L) (Invitrogen#A32742, 1: 500 dilution) was used. After that, DAPI (4',6-Diamidino-2-Phenylindole, Dilactate, Invitrogen#D3571) was stained, and images were taken with a confocal microscope using Zeiss LSM700 equipment, Aquaporin-5 (Aquaporin-5), Cytokeratin Cell nuclei of -19 (Cytokeratin-19) and amylase A were observed.

As a result, it was confirmed that aquaporin-5, a cell-specific protein, was expressed in the organoids, and it was confirmed that the presence of amylase A secreted between the organoids by calcium chloride added to the organoid culture medium (FIG. 21) ).

<Example 4-4> Organoid production of mouse-derived salivary gland stem cells

Cells were isolated from the submandibular gland in mice, and the chopping operation was performed with calcium, magnesium, HBSS (Hank's balanced salt solution), hyaluronic acid degrading enzyme (hyaluronidase), and type II collagen. After that, the first strain was 100 μm and the second strain was 40 μm, and sorting was performed using a strainer to isolate single cells. Thereafter, salivary gland stem cells were obtained by culturing under the same conditions as in Example 4-1. This was cultured with Y-27632 for a long time to obtain organoids.

The organoid was treated with IL-17, and the salivary gland stem cells were damaged by treatment with IL-17 at a concentration of 10, 20, or 50 ng/ml. After that, the formation rate of salivary gland stem cells and the production level of alpha amylase, which is the main component of saliva, were confirmed.

As a result, it was also confirmed that when the organoid culture medium was mixed with Y-27632, a ROCK inhibitor, and cultured, the size of the spheroids was increased (FIG. 22). In addition, it was confirmed that the salivary gland stem cells were damaged by the IL-17 treatment (FIG. 22). In addition, it was confirmed that the formation rate of the IL-17-treated salivary gland stem cells was significantly reduced more than two-fold compared to the control group, and it was confirmed that alpha amylase was also significantly reduced ( FIG. 23 ). Therefore, it was confirmed that the mouse salivary gland stem cell organoid of the present invention is excellent as a model for Sjogren's syndrome.

<Example 4-5> Confirmation of expression of aquaporin-5, nanog, amylase 1, and keratin 18 in organoids of mouse-derived salivary gland stem cells

In order to confirm that the organoid of the mouse-derived salivary gland stem cells prepared in Example 4-4 was properly constructed as a model for Sjogren's syndrome, aquaporin-5, keratin 18 (genetic markers of salivary gland stem cells) ( Keratin 18), the expression of nanog (Nanog) was confirmed. In addition, the expression of amylase 1 involved in salivary secretion was confirmed. It confirmed the expression of each mRNA through quantitative PCR.

As a result, it was confirmed that the expression of aquaporin-5, keratin 18, and nanog was significantly reduced in mouse-derived salivary gland stem cell organoids. In addition, as it was confirmed that the expression of amylase 1 was reduced, it was confirmed that the mouse salivary gland stem cell organoid of the present invention was excellent as a model for Sjogren's syndrome (FIGS. 24a-d).

Claims (29)

1. A humanized animal model of systemic lupus erythematosus, in which immunodeficient mice were administered with PBMCs (peripheral blood mononuclear cells) derived from lupus disease patients.
2. The animal model according to claim 1, wherein the animal model has increased dsDNA in serum and increased concentrations of albumin and creatine in urine.
3. A method for producing a humanized lupus disease animal model, comprising injecting PBMC isolated from a lupus disease patient into an immunodeficient mouse.
4. A method of screening a lupus therapeutic agent, comprising; treating a candidate substance to the humanized lupus animal model of claim 1 .
5. An animal model of rheumatoid arthritis with lung fibrosis, in which pulmonary fibrosis was developed by administration of beta-glucan or a fungicide to an animal with rheumatoid arthritis.
6. The animal model of claim 5, wherein the beta glucan is Curdlan.
7. According to claim 5, wherein the disinfectant is polyhexamethylene guanidine (Polyhexamethylene guanidine, PHMG), hydrochloric acid polyhexamethylene biguanide (PHMB), methyl isothiazolinone (MIT), ethoxyethyl guanidine chloride ( Chloride Ethoxyethyl Guanidine, PGH), naphthalene, phthalate, Didecyl methyl ammonium chloride (DDAC), benzisocyazolinone (BIT), chloroxylenol, titanium At least one selected from the group consisting of dioxide, zinc oxide and acetone, an animal model.
8. The animal model of claim 5, wherein the administration is selected from the group consisting of oral, intravenous, intramuscular, subcutaneous and intraperitoneal injection.
9. The animal model according to claim 8, wherein the oral administration and intraperitoneal injection are administered in a weight ratio of 1:2 when beta-glucan is administered.
10. The animal model of claim 5, wherein the animal model has an increased immune response by increasing macrophages, neutrophils, and lymphocytes.
11. The animal model according to claim 5, wherein in the animal model, Th17 cells, which are inflammatory cells, are increased, and Treg cells that control the immune response are decreased.
12. The method according to claim 5, wherein the lung fibrosis is at least one lung disease selected from the group consisting of pulmonary fibrosis accompanying rheumatoid arthritis, common interstitial pneumonia, non-specific interstitial pneumonia, inflammatory airway disease, and organized pneumonia. , animal models.
13. Inducing lung fibrosis by administering beta-glucan or a fungicide to a mouse with rheumatoid arthritis;
14. The method of screening a rheumatoid arthritis treatment material with lung fibrosis, comprising the; treating the candidate substance to the rheumatoid arthritis animal accompanied by pulmonary fibrosis of claim 5.
15. An animal model of osteoarthritis with gout in which gout was developed by administration of potassium oxonate (PO) or hypoxanthine (HX) to an animal with osteoarthritis.
16. The animal model of claim 15 , wherein the administration is selected from the group consisting of oral, intravenous, intramuscular, subcutaneous and intraperitoneal injection.
17. The animal model according to claim 16, wherein the oral administration is administered with potassium oxonate, and the intraperitoneal administration is hypoxanthine administration.
18. The animal model according to claim 17, wherein the potassium oxonate and hypoxanthine are administered in a weight ratio of 1:1.
19. 16. The method of claim 15, wherein in the animal model, Trabecular bone (TB) and Bone Mineral Density (BMD) are decreased, and the gap between the cancellous bones is increased to increase structural separation (St.sp) so that osteoporosis is a control group Compared to and intensified, the animal model.
20. Inducing gout by administering potassium oxonate or hypoxanthine to an animal with osteoarthritis; A method for producing an animal model of osteoarthritis accompanied by gout.
21. A method of screening for a treatment material for osteoarthritis accompanied by gout, comprising the; treatment of a candidate material to the animal model of osteoarthritis accompanied by gout of claim 15.
22. mouse Interleukin-17 (Interleukin-17) is administered to the organoid obtained by culturing the derived salivary gland stem cells, Sjogren's syndrome organoid model.
23. The animal model of claim 22, wherein the Interleukin-17 is administered at a concentration of 0-100 ng/ml.
24. 23. The method of claim 22, wherein the organoid model aquaporin-5 (Aquaporin-5), nanog (Nanog), amylase 1 (Amylase 1) and keratin 18 (Keratin 18) expression of the inhibited, the organoid Model.
25. 23. The organoid model of claim 22, wherein said administering treats IL-17 for 1-20 days.
26. Sjogren's syndrome organoid model, including Patient-derived salivary gland epithelial cells.
27. mouse culturing and obtaining derived salivary gland stem cells; and

    After culturing the cells to generate an organoid, interleukin-17 (Interleukin-17) is administered, Sjogren's syndrome organoid model production method.
28. Sjogren's syndrome (Sjogren's syndrome) organoid model production method comprising the step of culturing the salivary gland epithelial cells derived from the patient.
29. The method of claim 22 or claim 26, wherein the Sjogren's syndrome (Sjogren's syndrome) comprising the step of processing a candidate material in the organoid model;

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