WO2021194294A1 - Plateforme de modèle d'avatar pour évaluer des maladies auto-immunes - Google Patents

Plateforme de modèle d'avatar pour évaluer des maladies auto-immunes Download PDF

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WO2021194294A1
WO2021194294A1 PCT/KR2021/003756 KR2021003756W WO2021194294A1 WO 2021194294 A1 WO2021194294 A1 WO 2021194294A1 KR 2021003756 W KR2021003756 W KR 2021003756W WO 2021194294 A1 WO2021194294 A1 WO 2021194294A1
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animal model
model
cells
animal
organoid
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Korean (ko)
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박성환
조미라
곽승기
이주하
이재선
문정현
황선희
박진실
박민정
백진아
최정원
나현식
이선영
조근형
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가톨릭대학교 산학협력단
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Priority claimed from KR1020200037763A external-priority patent/KR20210121366A/ko
Priority claimed from KR1020200037764A external-priority patent/KR20210120706A/ko
Priority claimed from KR1020200037761A external-priority patent/KR20210120705A/ko
Priority claimed from KR1020200037762A external-priority patent/KR20210121365A/ko
Application filed by 가톨릭대학교 산학협력단 filed Critical 가톨릭대학교 산학협력단
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • 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.
  • 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. .
  • T cells that specifically respond to antigens in the immune system include T cells and B cells.
  • T cells meet a specific antigen presented by an antigen presenting cell, they respond according to the antigen.
  • 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.
  • 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.
  • tailored medicine is also called order-made medicine or personalized medicine. means how to treat it.
  • 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.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • 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.
  • PO potassium oxonate
  • HX hypoxanthine
  • 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.
  • An object of the present invention is a mouse culturing and obtaining derived salivary gland stem cells
  • 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.
  • 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • 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.
  • 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.
  • 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.
  • gout is generated by administering potassium oxonate (PO) or hypoxanthine (HX) to an animal with osteoarthritis.
  • 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.
  • 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.
  • 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).
  • 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 provides a method for manufacturing a Sjogren's syndrome organoid model, comprising the step of culturing Sjogren patient-derived salivary gland epithelial cells.
  • 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.
  • FIG. 1 is a diagram schematically illustrating the lupus patient imitation avatar model construction protocol of the present invention.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 6 is a diagram comparing the arthritis scores for the curdlan-injected rheumatoid arthritis SKG animal model with lung fibrosis and the control group.
  • FIG. 7 is a diagram comparing the weight change of the curdlan-injected rheumatoid arthritis SKG animal model with lung fibrosis and the control group.
  • 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.
  • FIG. 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.
  • FIG. 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.
  • FIG. 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.
  • 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.
  • FIG. 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
  • 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.
  • FIG. 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.
  • PBMCs peripheral blood mononuclear cells
  • 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.
  • 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.
  • 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.
  • SCID severe combined immune deficiency
  • SCID mutations refer to mutations that result in the deletion of functional B cells and T cells.
  • SCID mice CB-17-Prkdcscid
  • BCR B cell receptor
  • TCR T cell receptor
  • the NSG (NOD scid gamma) mouse is also referred to as a NOD/SCID ⁇ null mouse or a NOD/SCID IL-2R ⁇ KO 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).
  • NSG mice can be obtained by backcross mating C57BL/6J- ⁇ 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 ⁇ 2-microglobulin-deficient NOD/LtSz-SCID (NOD/SCID/ ⁇ 2m null).
  • NSG mice 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- ⁇ (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.
  • patient-specific lupus can be studied in the humanized mouse model.
  • 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.).
  • 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 MTB.
  • MTB Mouse Tumor Biology Database
  • 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.
  • 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.
  • CIA collagen-in-duced arthritis
  • SCID humanized severe combined immunodeficiency
  • 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.
  • 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
  • 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.
  • the arthritis index was higher than that of the control group, and it was confirmed that the mouse weight decreased.
  • inflammation was increased in the heart, lung, liver, muscle, joint and spine of mice, and damage to the lungs due to pulmonary fibrosis appeared.
  • 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.
  • PO potassium oxonate
  • HX hypoxanthine
  • the term "potassium oxonate (PO)" is a uric acid lyase inhibitor, which can induce hyperuricemia.
  • hypoxanthine 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.
  • 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.
  • 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.
  • 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 Monitoringosodium iodoacetate
  • injection may include mice, rats, rabbits, and guinea pigs.
  • gout promotes bone destruction in osteoarthritis patients
  • 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.
  • 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.
  • 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.
  • 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.
  • saliva 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.
  • saliva contains digestive enzymes such as amylase, such as ptyalin, which promotes digestion by decomposing starch to maltose units.
  • amylase such as ptyalin
  • ptyalin ptyalin
  • 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.
  • saliva 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.
  • the derived mouse is not limited, and general laboratory mice, immunodeficient mice, and the like may be used.
  • the present invention provides an organoid model of Sjogren's syndrome, including Patient-derived salivary gland epithelial cells.
  • 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.
  • 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.
  • a humanized mouse model of lupus equipped with a human immune system
  • PBMCs peripheral blood mononuclear cells
  • mice were sacrificed 4 weeks after cell transplantation to confirm the infiltration of human cells and histological changes in the tissue ( FIG. 1 ).
  • Example 1-1 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. .
  • CD4+T, CD8+T, and Cd19+T cells were detected, confirming that human cells were well engrafted ( FIG. 2 ).
  • Example 1-1 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 ⁇ m 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.
  • tissue Subtypes CD3, CD4, CD8, CD19
  • dsDNA human anti-double stranded DNA
  • IgG human anti-double stranded DNA
  • 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 ⁇ m 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.
  • H&E haematoxylin and eosin
  • 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.
  • 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.
  • the best arthritic index per mouse is 4 points, so the best disease index per mouse is 16.
  • mice 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).
  • 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.
  • PHMG polyhexamethylene guandinine
  • Example 2-4 Effect of an animal model of rheumatoid arthritis with lung fibrosis through polyhexamethylene guandinine (PHMG) injection
  • 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.
  • Example 2-3 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.
  • alveolar bronchiolization 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.
  • Th1 cells type 1 helper cells
  • Th2 cells type 2 helper cells
  • 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
  • Th17 cells were IL-17+in CD4+
  • Treg cells were CD25+Foxp+inCD4+
  • Th1 cells were IFN- ⁇ +in CD4+ cells were identified
  • Th2 cells were identified as IL-4+in CD4+ cells.
  • the arthritis index was higher than that of the control group, and it was confirmed that the mouse weight decreased.
  • inflammation was increased in the heart, lung, liver, muscle, joint and spine of mice, and damage to the lungs due to pulmonary fibrosis appeared.
  • 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.
  • MIA Monosodium Iodoacetate
  • saline for injection at a concentration of 60 mg/ml, and prepared on the day of the start of the experiment (day 0).
  • 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).
  • Example 3-1 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.
  • UgoBasile, Comerio, Itaily 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
  • 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 3-1 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 Boulevard Woodland Hills, CA, USA), the weight on the right hind limb (Weight on right hind rimb) ) (%) was measured.
  • IITC Incapacitance Meter
  • 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
  • bone mineral density (BMD) mg/cm 3 ) is an indicator that decreases as osteoporosis intensifies.
  • 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).
  • 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).
  • 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.
  • tissue 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.
  • 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
  • PureCol 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.
  • 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 ⁇ L/well were dispensed to the coated ones. Organoids were observed while changing the medium every 3 days.
  • 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
  • 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).
  • 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
  • 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.
  • DAPI 4,6-Diamidino-2-Phenylindole, Dilactate, Invitrogen#D3571
  • DAPI DAPI-4',6-Diamidino-2-Phenylindole, Dilactate, Invitrogen#D3571
  • 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.
  • 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.
  • Example 4-4 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.

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Abstract

La présente invention concerne une plateforme de modèle d'avatar pour évaluer des maladies auto-immunes. Un modèle de maladie auto-immune selon la présente invention reflète bien l'état immunitaire de patients atteints de maladies auto-immunes, et peut donc non seulement être utilisé comme modèle préclinique pour surveiller l'état immunitaire de patients, mais également être utilisé pour dépister des substances thérapeutiques pour des maladies auto-immunes. Ainsi, le modèle peut surmonter les limitations telles que l'absence de médicaments thérapeutiques et le manque de succès clinique de surveillance de médicaments avec le niveau actuel d'expérimentation sur les animaux, qui ont été des problèmes dans le passé, et peut donc être utilisé non seulement pour évaluer des médicaments pour des maladies auto-immunes, mais également pour comprendre les mécanismes pathologiques et développer des systèmes de diagnostic et de contrôle de maladie adaptés. Par conséquent, le modèle peut être utilisé efficacement dans les domaines ou industries liés aux maladies auto-immunes.
PCT/KR2021/003756 2020-03-27 2021-03-26 Plateforme de modèle d'avatar pour évaluer des maladies auto-immunes WO2021194294A1 (fr)

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KR1020200037763A KR20210121366A (ko) 2020-03-27 2020-03-27 류마티스 관절염 환자 폐섬유화 동반 질환 모델 평가 플랫폼
KR1020200037764A KR20210120706A (ko) 2020-03-27 2020-03-27 통풍 동반 관절염 모델 평가 플랫폼
KR1020200037761A KR20210120705A (ko) 2020-03-27 2020-03-27 루푸스 환자 모사 아바타 모델 평가 플랫폼
KR1020200037762A KR20210121365A (ko) 2020-03-27 2020-03-27 쇼그렌 질환 침샘 오가노이드 모델 평가 플랫폼

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