WO2012093754A1 - Composition comprising extracellular vesicles derived from inside a mammalian body and a use therefor - Google Patents
Composition comprising extracellular vesicles derived from inside a mammalian body and a use therefor Download PDFInfo
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- WO2012093754A1 WO2012093754A1 PCT/KR2011/002835 KR2011002835W WO2012093754A1 WO 2012093754 A1 WO2012093754 A1 WO 2012093754A1 KR 2011002835 W KR2011002835 W KR 2011002835W WO 2012093754 A1 WO2012093754 A1 WO 2012093754A1
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- Prior art keywords
- extracellular vesicles
- inflammatory
- disease
- isolated
- mammal
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- A—HUMAN NECESSITIES
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- A61K35/66—Microorganisms or materials therefrom
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/37—Digestive system
- A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
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- A—HUMAN NECESSITIES
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Definitions
- the present invention relates to a composition for the treatment or prevention of inflammatory diseases and cancer containing the extracellular vesicles isolated from the body of a mammal as an active ingredient, and to a method for diagnosing inflammatory diseases and cancers using the extracellular vesicles.
- Inflammation is a local or systemic defense mechanism against damage or infection of cells and tissues. Inflammation is primarily caused by a cascade of biological reactions that occur by the direct response of numerous humoral mediators that make up the immune system or by stimulating local or systemic effector systems. Mediators involved in this inflammatory response include immune cells, such as helper T lymphocytes (Th cells) and cytotoxic T lymphocytes (Tc cells), macrophage cells, neutrophils, eosinophils, and stroma cells. inflammatory cells such as (mast cell) and cytokines secreted from these cells. Inflammatory diseases are particularly characterized by tissue remodeling due to the secretion of inflammatory cytokines, resulting in tissue damage and imbalance of healing.
- Th cells helper T lymphocytes
- Tc cells cytotoxic T lymphocytes
- macrophage cells macrophage cells
- neutrophils neutrophils
- eosinophils eosinophils
- stroma cells stroma cells.
- inflammatory cells
- TNF- ⁇ tumor necrosis factor- ⁇
- IL-1 interleukin-1, interleukin-1
- IL-6 IL-8
- Major inflammatory diseases include asthma, chronic obstructive pulmonary disease, respiratory diseases such as rhinitis, skin diseases such as atopic dermatitis, gastroenteritis, gastritis, gastric ulcer, inflammatory growthitis, and other infectious rhinitis, allergic rhinitis, chronic rhinitis, Rhinitis and sinusitis, such as acute sinusitis and chronic sinusitis; Otitis media, such as acute purulent otitis media and chronic purulent otitis media; Pneumonia, such as bacterial pneumonia, bronchial pneumonia, lobar pneumonia, regoraella pneumonia and viral pneumonia; Acute or chronic gastritis; Enteritis, such as infectious enterocolitis, Crohn's disease, idiopathic ulcerative colitis, gastric colitis, and the like; Arthritis such as purulent arthritis, tuberculosis arthritis, degenerative arthritis and rheumatoid arthritis; And diabetes mellitus, arteriosclerosis, and the like.
- H. pylori Helicobacter pylori
- H. pylori infection is important in chronic gastritis, peptic ulcer disease, the occurrence of gastric cancer, it is also H. pylori is known to not penetrate the mucosa above a gram-negative bacteria, leading to inflammation .
- the risk of gastric cancer is significantly increased in the case of chronic gastritis caused by H. pylori infection, but the incidence of gastric cancer in patients with peptic ulcer caused by H.
- pylori infection is lower than that of chronic gastritis.
- This report that generated by strain which secretes the protein, such as a cytotoxin-associated antigen (CagA) in the case of H. pylori strain causing peptic ulcer as compared with H. pylori strain associated with the occurrence of chronic gastritis and gastric cancer by H. pylori Considering this, it means that the ability to cause disease depends on the strain.
- intestinal bacteria are known to be important for the development of inflammatory bowel disease occurring in the large intestine.
- Inflammatory growthitis includes Crohn's disease, which is characterized by a Th1 immune response, and ulcerative colitis, which is characterized by a Th17 immune response.
- Chronic inflammatory growth inflammation is attracting attention as a risk factor for colorectal cancer.
- the role of IL-6 is very important in the generation of Th17 immune response, which induces differentiation into Th17 cells that secrete IL-17 to naive T cells through signaling pathways through STAT3.
- Bactroides fragillus which secretes metalloprotease toxin from intestinal bacteria, induces IL-6 secretion, which induces a Th17 immune response through STAT3 signaling, resulting in colon cancer. have.
- Extracellular vesicles secreted from gram-negative bacteria contain lipopolysacharide (LPS) and protein derived from bacteria.
- LPS lipopolysacharide
- the present inventors have found that extracellular vesicles derived from enteric symbiotic gram-negative bacteria are absorbed into the blood to induce a systemic inflammatory response. I said.
- the present inventors recently reported that Gram-positive bacteria secrete extracellular vesicles, and proteome analysis includes protein causing disease in the endoplasmic reticulum, and reports that atopic dermatitis occurs when administered to the skin.
- extracellular vesicles derived from various cancer cells have been reported to contain proteins necessary for the growth and metastasis of cancer cells.
- extracellular vesicles have been found in various secretions, excreta, or tissue washes of human or laboratory animals.
- tissue-derived extracellular vesicles are known to reflect the state of tissues secreting vesicles, and it has been reported that they can be used to diagnose diseases.
- extracellular vesicles have been reported in the small intestine of experimental animals.
- the present invention seeks to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from the mammalian body.
- the present invention provides a composition for preventing and / or treating inflammatory diseases and cancer, a vaccine for treating or preventing inflammatory diseases and cancer, containing the extracellular vesicles as an active ingredient.
- the present invention provides a method for diagnosing the cause of a disease, the occurrence or course of a disease, etc. using an extracellular vesicle derived from the body of a mammal.
- the present invention provides a composition for the treatment or prevention of inflammatory diseases and cancer, containing as an active ingredient extracellular vesicles derived from the body of a mammal.
- the inflammatory diseases include respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, systemic diseases such as brain diseases, and complications thereof.
- the extracellular vesicles may be isolated from the small intestine, or the stool of a mammal, but are not limited thereto.
- the extracellular vesicles may be to inhibit the secretion of inflammatory mediators in cells of mammals, but is not limited thereto.
- the cells include epithelial cells and inflammatory cells.
- the inflammatory cells include macrophages and eosinophils.
- the inflammatory mediator may be interleukin 6 (IL-6), but is not limited thereto.
- IL-6 interleukin 6
- the extracellular vesicles may be, but is not limited to, extracellular vesicles derived from symbiotic bacteria in a mammal.
- the extracellular vesicles may be an extracellular vesicle derived from epithelial cells or inflammatory cells of the mammal, but is not limited thereto.
- the extracellular vesicles of the present invention may have an average diameter of 20 nm to 200 nm, but is not limited thereto.
- composition of the present invention may be a pharmaceutical composition, a food composition, a cosmetic composition and the like, but is not limited thereto.
- the present invention also provides a vaccine for the treatment or prophylaxis of inflammatory diseases and cancer, containing as an active ingredient extracellular vesicles derived from the body of a mammal.
- the vaccine may be used alone or in combination with the extracellular vesicles and the pathogenic vesicles.
- the pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.
- the present invention also provides a method for preventing or treating a disease, comprising administering to a mammal an extracellular vesicle derived from the body of the mammal at a less than lethal dose.
- the disease includes inflammatory disease, cancer and the like.
- the present invention also provides a method for diagnosing the cause of a disease, the occurrence or course of a disease by using an extracellular vesicle derived from the body of a mammal.
- the disease includes inflammatory disease, cancer and the like.
- the method may comprise the steps of: separating extracellular vesicles in the body of a mammal; Culturing the isolated extracellular vesicles in cells; And measuring the level of inflammatory mediator in said cell culture.
- the extracellular vesicles can be isolated from gastric juice of a mammal.
- the step of separating the extracellular vesicles from the body of the mammal may comprise the steps of: obtaining the body material (including secretion) of the mammal; Centrifuging the obtained material to remove impurities, animal cells, and bacteria; And separating the extracellular vesicles by performing ultracentrifugation.
- the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice
- compositions comprising extracellular vesicles isolated from in vitro culture of Helicobacter pylori.
- the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Provided is a method for diagnosing a causative agent of a disease, the occurrence or course of a disease using an extracellular vesicle.
- the disease includes gastritis, peptic ulcer, gastric cancer, and the like.
- the method may comprise the following steps: Helicobacter pylori isolated from human gastric tissue or gastric juice (Helicobacter pylori) origin Treating the cells with extracellular vesicles and culturing; And measuring the level of inflammatory mediator in said cell culture.
- Helicobacter pylori isolated from human gastric tissue or gastric juice (Helicobacter pylori) origin Treating the cells with extracellular vesicles and culturing And measuring the level of inflammatory mediator in said cell culture.
- the method comprises measuring a genetic material or protein in the extracellular vesicles, measuring an immune response to the extracellular vesicles, or measuring an antibody against the extracellular vesicles. Step and the like.
- the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Helicobacter pylori containing extracellular vesicles as an active ingredient (Helicobacter pylori)
- the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Helicobacter pylori, comprising administering an extracellular vesicle to a mammal at a sublethal dose (Helicobacter pylori) It provides a method for preventing or treating a disease by.
- the composition of the present invention containing the extracellular vesicles isolated in the body of a mammal as an active ingredient can be used as pharmaceutical compositions, food compositions, cosmetic compositions and the like for the treatment or prevention of inflammatory diseases including cancer.
- the extracellular vesicles isolated in the body of a mammal suffering from an inflammatory disease may cause the secretion of inflammatory mediators of inflammatory cells
- the present invention is to determine the causative agent of gastric disease and gastric disease by inflammatory mediator secretion of H. pylori- derived extracellular vesicles isolated from patients with gastric disease, analysis of genetic material or protein in endoplasmic reticulum, and measurement of vesicle-specific immune responses. It is possible to diagnose the occurrence or course.
- 1 is a diagram showing the result of separating extracellular vesicles through ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
- Figure 2 is an electron micrograph of the extracellular vesicles present in the F3 region when ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
- Figure 3 is an electron micrograph of the extracellular vesicles present in the F6 portion when ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
- Figure 4 shows the results of the protein contained only in the extracellular vesicles present in the F6 couple when the presence or absence of proteins in the extracellular vesicles isolated from gastric lavage fluid of peptic ulcer patients.
- FIG. 5 is a diagram showing the secretion of inflammatory mediators TNF- ⁇ when the extracellular vesicles isolated from gastric lavage fluid of peptic ulcer patients treated with various macrophages.
- Figure 6 shows the effect of polymyxin B and heat treatment on the secretion of inflammatory mediators (TNF- ⁇ ) by the endoplasmic reticulum in the F3 part when isolated from extracellular vesicles in gastric lavage fluid of peptic ulcer patients.
- Figure 7 shows the effect of polymyxin B and heat treatment on the secretion of inflammatory mediators (IL-6 and TNF- ⁇ ) by the endoplasmic reticulum in the F6 region when isolated extracellular vesicles from gastric lavage fluid of peptic ulcer patients to be.
- IL-6 and TNF- ⁇ inflammatory mediators
- FIG. 8 is an electron microscope photograph of extracellular vesicles isolated from the culture by H. pylori cultured from patients with gastritis, gastric ulcer, and gastric cancer.
- FIG. 10 is a diagram showing the results of separating the extracellular vesicles from the mouse small intestine, a separation protocol for separating the extracellular vesicles from the mouse small intestine (A), electron microscopy (TEM) image of the isolated extracellular vesicles (b), Quantitative results of the isolated extracellular vesicles (c) and bacterial culture results (d) confirming the bacterial contamination of the isolated extracellular vesicles.
- A results of separating the extracellular vesicles from the mouse small intestine
- A electron microscopy
- b electron microscopy
- c Quantitative results of the isolated extracellular vesicles
- bacterial culture results confirming the bacterial contamination of the isolated extracellular vesicles.
- 11 is a result of evaluating the in vitro immune response by extracellular vesicles isolated from the small intestine of normal mice, after treatment of macrophages with extracellular vesicles IL-6 (left) and TNF- inflammatory mediators in cell culture It is the result of measuring the secretion amount of (right) by ELISA.
- FIG. 13 shows the results of SDS-PAGE on extracellular vesicle proteins for comparing aspects of proteins contained in extracellular vesicles isolated from the small intestine of normal mice and inflammatory bowel disease mice.
- inflammatory mediator secretion by inflammatory bowel disease mouse small intestine-derived extracellular vesicles is induced by endoplasmic reticulum derived from intestinal symbiotic Gram-negative bacteria.
- the amount of secretion of inflammatory mediators IL-6 (left) and TNF- ⁇ (right) was measured in the macrophages (RAW 246.7 cells) culture medium by ELISA.
- FIG. 16 is a result of evaluating the preventive effect of pre-administration of normal mouse small intestine-derived extracellular vesicles on inflammatory mediator expression by inflammatory bowel disease mouse small intestine-derived extracellular vesicles.
- FIG. 16 shows normal mice in macrophages (RAW 246.7 cells). After pre-treatment of the small intestine-derived extracellular vesicles by time, the cell culture medium was removed after a certain period of time, and the inflammatory mediators were treated in the cell culture solution for 6 hours after mixing and treating the extracellular vesicles derived from the inflammatory bowel disease mouse small intestine. IL-6 (left) and TNF- ⁇ (right) were measured.
- Figure 17 shows the anti-inflammatory properties of extracellular vesicles isolated from the small intestine of normal state against the secretion of inflammatory mediators by LPS or gram-negative bacteria E. coli-derived extracellular vesicles, which are components of gram-negative bacterial cell walls known as inflammatory bowel diseases. Effect The evaluation results are shown.
- Figure 18 shows the results of measuring the endoplasmic reticulum-specific antibody in the serum after three subcutaneous injection of the vesicles isolated from the small intestine.
- 19 is a result of measuring the concentrations of gamma interferon and interleukin-10 after injecting vesicles isolated from the small intestine three times subcutaneously, separating immune cells from the spleen and treating the vesicles with immune cells.
- Figure 21 shows the results of measuring the endoplasmic reticulum-specific antibody in the serum after three subcutaneous injection of the vesicles isolated from the stool.
- 22 is a result of measuring the concentration of interleukin-10 after treatment of endoplasmic reticulum in immune cells by injecting vesicles isolated from feces three times subcutaneously and then separating immune cells from the spleen.
- the present inventors have tried to develop a composition for the prevention and / or treatment of inflammatory diseases, diagnostic methods, etc., when the extracellular vesicles are isolated from the gastric lavage fluid of peptic ulcer patients to evaluate the immune response, derived from Gram-negative bacteria
- One endoplasmic reticulum was found to induce the release of IL-6, which induces a Th17 immune response, and when the immune response was evaluated by separating extracellular vesicles from H. pylori isolated from patients with chronic gastritis, peptic ulcer, and gastric cancer, Extracellular vesicles isolated from peptic ulcer patients were found to have a significantly increased effect of secreting IL-6 compared to H. pylori- derived extracellular vesicles isolated from other disease patients.
- the present inventors found that the extracellular vesicles isolated from the small intestine of the inflammatory bowel disease state had significantly increased IL-6 secretion ability compared to the extracellular vesicles isolated from the small intestine of the normal mammal without disease.
- the extracellular vesicles present in the small intestine were found to be able to inhibit inflammatory responses caused by endotoxins or Gram-negative bacteria-derived extracellular vesicles, which cause inflammatory growth inflammation.
- the inventors have discovered that when injected subcutaneously with small intestine-derived extracellular vesicles, no immune response to the endoplasmic reticulum is induced.
- the present inventors have extracellular vesicles in feces of mammals, which are composed of gram-negative bacteria, gram-positive bacteria as well as extracellular vesicles derived from host cells, and when injected subcutaneously, IL-10 specific for vesicles is injected. It has been found that secreting regulatory T (Treg) cells are induced.
- the present invention provides a composition for the treatment or prophylaxis of inflammatory diseases and cancer, comprising as an active ingredient extracellular vesicles derived from the body of a mammal.
- intrabody is meant to include the inside of the mammal's body and the skin surface, and may be, for example, inside the organs of the mammal, including humans, inside the mouth, skin surface and the like.
- extracellular vesicles derived from the body of a mammal includes vesicles present in the body of a mammal or secreted into gastric juice, feces, urine, bronchoalveolar lavage fluid, etc., characterized in that the size is smaller than the original cells.
- the present invention is not limited thereto.
- the extracellular vesicles of the present invention may be symbiotic bacteria or extracellular vesicles secreted by epithelial cells of the host.
- the symbiotic bacteria in the body refers to the bacteria present in the body of a mammal, for example, enterobacteria and the like.
- any of the extracellular vesicles of the present invention can be used as long as it is separated from the body of a mammal, but depending on the site of the inflammatory disease to be treated or prevented, the extracellular vesicles alone or in combination with the pathogenic vesicles causing the disease can be used.
- the pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.
- the extracellular vesicles can be used alone to separate the extracellular vesicles present in the small intestine or feces of a mammal, and to prevent or prevent inflammatory lung disease.
- the extracellular vesicles can be used in combination with a pathogenic vesicle which causes inflammatory lung disease by separating extracellular vesicles present in the small intestine or feces of a mammal, and for the prevention or treatment of inflammatory skin diseases.
- the extracellular vesicles can be used in combination with pathogenic vesicles that cause inflammatory skin diseases by separating extracellular vesicles present in the small intestine or feces of a mammal, and systemic such as sepsis, arteriosclerosis, arthritis, brain diseases, etc.
- the cell Outer vesicles can be used in combination with pathogenic vesicles that cause systemic disease by separating extracellular vesicles present in the small intestine or feces of a mammal.
- the method for separating the extracellular vesicles from the body or secretion of the mammal is not particularly limited as long as the extracellular vesicles can be separated purely.
- methods such as centrifugation, ultracentrifugation, filtration by filters, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, and the like, and combinations thereof, for example in mammalian body material, body tissues, secretions, and the like.
- Extracellular vesicles can be isolated using.
- it may further include a process for washing to remove impurities, concentration of the obtained extracellular vesicles and the like.
- the extracellular vesicles separated by the above method may have an average diameter of 500 nm or less, and preferably 20 nm to 200 nm.
- the composition may be prepared as a pharmaceutical composition. While it may be possible to administer the extracellular vesicles of the present invention for use in treatment and / or prophylaxis, it is preferred that the extracellular vesicles be included as active ingredients of the pharmaceutical composition.
- the pharmaceutical composition may contain the isolated extracellular vesicles as an active ingredient, and may include a pharmaceutically acceptable carrier.
- pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included.
- diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
- Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.
- the pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.
- the method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be selected by intravenous, subcutaneous, intraperitoneal, inhalation, dermal application, sublingual administration, nasal administration, anal administration, oral administration, etc. .
- Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient.
- Daily dosage refers to the amount of therapeutic substance of the invention sufficient for treatment for a disease state alleviated by administration to a subject in need thereof. Effective amounts of therapeutic agents depend on the particular compound, disease state and severity thereof, and on the individual in need thereof, and can be routinely determined by one skilled in the art.
- the dosage of the composition according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient and may be based on an adult patient weighing 70 kg. At this time, it is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at regular time intervals.
- the composition of the present invention may be prepared as a cosmetic composition.
- Ingredients included in the cosmetic composition of the present invention, in addition to containing the extracellular vesicles as an active ingredient, include components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Conventional adjuvants such as, and carriers.
- the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, a flexible lotion, a lotion, an essence, a nourishing gel or a massage cream.
- the composition of the present invention may be prepared as a food composition.
- the composition of the present invention is prepared as a food composition, not only contains the extracellular vesicles as an active ingredient, but also includes components commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, Flavoring and flavoring agents may be included.
- citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and the like may be further included in addition to the extracellular vesicles of the present invention.
- treatment or prevention of an inflammatory disease is meant to include the alleviation, alleviation and improvement of symptoms of an inflammatory disease, and also to reduce the possibility of developing an inflammatory disease.
- inflammatory disease refers to a disease caused by an inflammatory response in a mammalian body, and representative examples thereof include respiratory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis; Skin diseases such as atopic dermatitis, psoriasis and contact dermatitis; Gastrointestinal diseases such as gastritis, peptic ulcer, and inflammatory bowel disease; Systemic diseases such as sepsis, arteriosclerosis, arthritis, and brain diseases, and complications thereof.
- the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, and includes, for example, lung cancer, stomach cancer, colon cancer and the like.
- the composition of the present invention can treat or prevent inflammatory diseases by inhibiting the secretion of inflammatory mediators or inducing immune tolerance in inflammatory cells of mammals.
- the extracellular vesicles isolated from the body of the mammal of the present invention not only inhibit the secretion of IL-6, an important biomarker associated with the development of inflammatory diseases and / or cancers, but also induce specific immunotolerance to pathogenic vesicles. Since it is possible, the composition of the present invention is capable of treating or preventing IL-mediated Th-17 inflammatory disease and cancer caused by IL-6 or Th-17 inflammatory response.
- the present invention also provides a method for diagnosing an inflammatory disease using extracellular vesicles derived from the body of a mammal.
- Extracellular vesicles isolated from the body of a mammal with an inflammatory disease can cause the secretion of inflammatory mediators of inflammatory cells.
- the diagnostic method comprises the steps of: separating the extracellular vesicles from the body of the mammal (including secretions); Culturing the isolated extracellular vesicles on inflammatory cells; And measuring the level of an inflammatory mediator in said cell culture.
- the separation method of the extracellular vesicles can be separated by the same method as the method of separating the extracellular vesicles in the body of the mammal described above, and the separation method is not particularly limited as long as the extracellular vesicles can be separated purely.
- the isolated extracellular vesicles are administered to inflammatory cells in vitro and cultured together, and by measuring the type and level of the inflammatory mediator secreted from the inflammatory cells, it is possible to predict the cause or progression of the causative agent or disease of the inflammatory disease. Do.
- the causative agent of an inflammatory disease by measuring the genetic material or protein in the endoplasmic reticulum to diagnose the causative agent using the isolated extracellular vesicles.
- the isolated extracellular vesicles are added to the macrophage culture medium and cultured together, and the occurrence and progression of the inflammatory disease can be diagnosed by measuring the level of IL-6 secreted by the macrophages.
- the level of inflammatory mediators eg, IL-6
- the present invention is cultured in vitro H. pylori in vitro, the vesicles are isolated from the culture medium, and then measured as genetic material, protein analysis, or vesicle-specific immune response in the endoplasmic reticulum as a causative agent of inflammatory diseases and cancer
- a method for diagnosing H. pylori- derived extracellular vesicles is provided.
- the present inventors have significantly increased the IL-6 secreted is compared with the extracellular vesicles separated from the culture medium by culturing H. pylori isolated from peptic ulcer patients in vitro, and in or out of gastritis H. pylori-derived cells isolated from patients with gastric cancer ER Confirmed.
- H. pylori- derived extracellular vesicles isolated from peptic ulcer patients have significantly higher activity causing tissue damage due to inflammation compared to H. pylori- derived extracellular vesicles isolated from gastritis or gastric cancer patients.
- Genetic and protein analysis in extracellular vesicles, or by measuring the immune response by extracellular vesicles makes it possible to diagnose specific H. pylori-derived extracellular vesicles.
- the diagnostic method comprises the steps of separating and culturing H. pylori in patients with gastritis, peptic ulcer, gastric cancer; Isolating extracellular vesicles from H. pylori culture; Culturing the isolated extracellular vesicles on inflammatory cells; And measuring the level of an inflammatory mediator in said cell culture.
- the method measures the immune response to the endoplasmic reticulum removed in step, or H. pylori culture media for measuring the protein in the endoplasmic reticulum in a separate step of measuring the dielectric material, H. pylori culture on the endoplasmic reticulum isolated from H. pylori culture medium It may include the step and the like.
- the precipitates of the separated portions were adsorbed on a glow-discharged carbon-coated copper grid for 3 minutes and washed with distilled water. After staining with 2% uranilacetate for 1 minute, it was observed with a JEM101 (Joel, Japan) electron microscope.
- 2 and 3 are electron micrographs of the extracellular vesicles present in the F3 and F6 portions, respectively, and were found to have extracellular vesicles of about 100-200 nm in only the F3 and F6 portions.
- Example 2 By extracellular vesicles isolated from gastric lavage fluid in vitro Inflammatory mediator secretion and effects of polymyxin B or heat treatment
- polymyxin B (10 ⁇ g / ml), or protein denaturation, that binds to lipopolysaccharide present in Gram-negative bacteria-derived extracellular vesicles to inhibit the activity of F3 and F6 in order to assess the mechanism of inducing inflammatory mediators
- the resulting heat 99 ° C., 20 min was treated in two parts F3 and F6 to confirm the expression of the inflammatory mediator TNF- ⁇ in this manner.
- Example 3 Isolation from Patients with Gastritis, Peptic Ulcer and Gastric Cancer H. pylori Isolation of extracellular vesicles from culture
- H. pylori a gram-negative bacterium in the stomach, was cultured in vitro to evaluate the secretion of extracellular vesicles.
- H. pylori was isolated from gastric lavage fluid of two patients with gastritis, peptic ulcer and gastric cancer. Each of the isolated H. pylori bacteria was incubated until the absorbance was 1.0, and then the cell culture was centrifuged at 3,000 xg for 20 minutes to separate the extracellular vesicles from 1 L of culture. After removing H. pylori and impurities, the supernatant was removed again using a 0.22 ⁇ m filter. The filtered supernatant was ultracentrifuged at 100,000 xg for 2 hours. The sunk extracellular vesicles were released with 200 ⁇ l of physiological saline, and the shape and size of the extracellular vesicles were confirmed by electron microscopy in the same manner as in Example 1.
- Example 2 In order to measure the secretion ability of the inflammatory mediator IL-6 by H.pylori- derived extracellular vesicles isolated from patients with gastritis, gastric ulcer and gastric cancer, the experiment of Example 2 was conducted.
- H. pylori-derived extracellular vesicles are important for the development of gastric disease caused by H. pylori infection, and ultimately, by measuring the genetic material, protein, or vesicle-specific immune response in the endoplasmic reticulum, It means that the progress can be diagnosed.
- mice To isolate extracellular vesicles from mouse intra-intestinal secretions, six-week old female BALB / c mice were isolated from the duodenum just below the stomach to the jejunum, including the jejunum.
- the small intestine separated into 3 parts was expanded and then cut into 1 cm size in a 50 ml tube containing 30 ml of physiological saline.
- the prepared tube was washed five times for 10 seconds using a stirrer.
- the small intestine was filtered out and the filtered small intestinal lavage was centrifuged at 500 x g for 20 minutes, 3000 x g for 20 minutes, and 10,000 x g for 2 minutes to remove impurities, small cells and bacteria.
- the supernatant from which impurities were removed was separated from the extracellular vesicle layer using ultra-high speed centrifugation at 100,000 ⁇ g for 2 hours using a sucrose cushion.
- the isolated extracellular vesicles were observed through a transmission electron microscope (TEM) to confirm that vesicles were present between 50 nm and 100 nm (see FIG. 10B).
- TEM transmission electron microscope
- bacteria were cultured in LB and MRS medium using extracellular vesicles in order to confirm whether the bacteria were contaminated in the obtained extracellular vesicles, but there was no bacterial contamination through the cultivation of bacteria. (See FIG. 10D).
- 3 x 10 5 cells were dispensed into 24 well cell culture plates and incubated for 24 hours. After incubation, physiological saline and endotoxin (lipopolysacharide) (10 ng / ml) were treated as a control group, and the extracellular vesicles isolated from the small intestine of normal mice were treated with concentrations of 1, 5 and 10 ⁇ g / ml, respectively. After 6 hours after treatment with extracellular vesicles, cell culture fluids were obtained, and IL-6 and TNF- ⁇ , which are inflammatory mediators, were measured in the cell culture by ELISA, and the results are shown in FIG. 11.
- the positive control group induced the expression of the inflammatory mediators IL-6 and TNF- ⁇ , whereas the experimental group of the extracellular vesicles did not induce the expression of IL-6 regardless of the concentration.
- TNF- ⁇ it was confirmed that the induction of a small amount, but compared to the positive test group was found to be insignificant. The results indicate that extracellular vesicles isolated from the small intestine of normal mice do not induce inflammation.
- DSS dextran sulfate sodium
- mice were dissected to separate extracellular vesicles from the small intestine according to the method of Example 1. In order to confirm the secretion of inflammatory mediators of the extracellular vesicles derived from the mouse in the condition of isolated inflammatory bowel disease was evaluated according to the method of Example 2.
- Proteins contained in 30 ⁇ g of the extracellular vesicles isolated from the small intestine of normal and inflammatory bowel disease mice were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacryl amide gel electrophoresis). Separation was based on molecular weight using the method.
- the isolated proteins were stained using coomasie brilliant blue, and then compared to those of proteins contained in extracellular vesicles isolated from the small intestine of normal (siEVs) and inflammatory bowel disease mice (DSS siEVs). Is shown in FIG. 13.
- the protein could not be identified, but the protein contained in the extracellular vesicles derived from normal and inflammatory bowel disease mice was confirmed to have a big difference.
- LPS-derived endotoxin (LPS) from symbiotic bacteria influences the secretion of inflammatory mediators of extracellular vesicles from mice with normal and enteric diseases. Evaluation was carried out according to the method of Example 2 using the antagonist polymixin B (10 ⁇ g / ml).
- Example 10 Anti-inflammatory Effect of Extracellular Vesicles Isolated from Normal Intestine on Secretion of Inflammatory Mediators by Extracellular Vesicles Isolated from Intestine with Inflammatory Bowel Disease
- Induction of inflammatory mediators of extracellular vesicles isolated from the small intestine of inflammatory bowel disease mice was evaluated according to the method of Example 2 to determine whether the extracellular vesicles isolated from the small intestine of normal mice were inhibited.
- mice can antagonize the expression of inflammatory mediators such as IL-6 by inflammatory bowel disease mouse small intestine-derived extracellular vesicles.
- mice have the effect of inhibiting the expression of inflammatory mediators such as IL-6 by inflammatory bowel disease mouse small intestine-derived extracellular vesicles.
- Extracellular vesicles derived from LPS or Escherichia coli, which are components of the cell wall of Gram-negative bacteria known to cause inflammatory bowel disease E.coli Anti-inflammatory effects of extracellular vesicles (siEVs) isolated from normal small intestine on inflammatory mediator secretion by C4 EVs)
- siEVs extracellular vesicles isolated from normal small intestine
- C4 EVs extracellular vesicles
- IL-6 an inflammatory mediator
- the small intestine-derived extracellular vesicles were isolated from the 5 week old male C57BL / 6 mice using the method of Example 5 to determine whether the vesicle-specific immune tolerance by the mouse small intestine-derived extracellular vesicles.
- cytokines secreted by stimulating splenocytes at 0.1 or 1 ⁇ g / ml to the splenocytes isolated from mice injected with the endoplasmic reticulum three times were confirmed.
- the expression of inflammatory mediators such as IFN- ⁇ was observed. Not only was there no change, but there was no change in the expression of inflammatory regulatory media such as IL-10 (see Figure 19).
- Example 13 Proteome analysis of extracellular vesicles isolated from mouse feces
- mice 25 g of feces obtained from 5-week-old male C57BL / 6 mice were placed in 2 L of phosphate buffered saline (PBS) and suspended at 4 ° C. for 16 hours. The suspension was centrifuged at 10,000 x g for 20 minutes at 4 ° C, then the supernatant was taken and passed through a filter with a 0.45 ⁇ m pore size. The bacteria-free passage solution was concentrated about 30-fold to 70 ml using a QuickStand Benchtop System equipped with a membrane capable of removing proteins below 100 kDa molecular weight. The concentrated solution was again centrifuged at 4 ° C.
- PBS phosphate buffered saline
- the precipitate was suspended in 2.2 ml of 50% Optiprep solution and placed in an ultracentrifuge tube, followed by 2 ml of 40% Optiprep solution followed by 0.8 ml of 10% Optiprep solution. After ultracentrifugation for 2 hours at 40,000 ° C, 200,000 ⁇ g, extracellular vesicles were obtained in the layer between 40% Optiprep solution and 10% Optiprep solution.
- In-solution trypsin proteolysis was used for proteome analysis of mouse stool-derived extracellular vesicles.
- 50 mg of extracellular vesicles isolated from mouse feces by the method of Example was dissolved in digestion solution (7 M urea, 2 M Thiourea, 100 mM NH 4 HCO 3 ), followed by 10 mM Dithiothreitol (DTT) at 60 ° C. Reduction was performed for 45 minutes. Thereafter, the sample was cooled to room temperature, 55 mM ioacetamide was added thereto, and the protein was alkylated for 30 minutes at room temperature while blocking light.
- digestion solution 7 M urea, 2 M Thiourea, 100 mM NH 4 HCO 3
- DTT Dithiothreitol
- Mass spectrometry was performed using nano ionization mass spectrometry (Nano-LC-ESI-MS / MS).
- Decomposed peptides of mouse stool-derived extracellular vesicles prepared by in-solution digestion were loaded onto a column (75 ⁇ m ⁇ 12 cm) filled with a 5 ⁇ m C18 resin (75 ⁇ m ⁇ 12 cm) and separated as follows: 3- 40% buffer B 70 min; Blood flow rate 0.3 ml / min (buffer A composition: 0.1% formic acid in H 2 O, buffer B composition: 0.1% formic acid in acetonitrile).
- the isolated peptides were analyzed using LTQ-ion-trap mass spectrometer (Thermo Finnigan).
- the voltage of the ionized electrospary was 1.9 kV, and mass spectrometry (MS / MS) was performed under a condition of 35% normalized collision energy. All spectra were acquired with a data-dependent scan.
- the parameters of the LTQ were the fragmentation of the five most abundant spectra in full MS scan, the repeat count of dynamic exclusion was 1, The repeat duration is set to 30 seconds, the dynamic exclusion duration is 180 seconds, the exclusion mass width is 1.5 Da, and the list size of the dynamic exclusion is 50.
- mice database which has a previously constructed amino acid sequence
- enteric bacteria we used a database of bacteria (Uniprot) of 10 species representing a large number of genus in the intestine. A total of 11 data analyzes were performed using each database. All spectra from the mass spectrometry (MS spectrum and MS / MS spectrum) were analyzed using the MASCOT protein analysis engine version 2.2 (http://www.matrixscience.com).
- Validation of the assay was performed by selecting peptides with more than 95% / 99% of the peptide prophet / protein prophet, and spectra of the individual peptides identified in each protein were analyzed directly on the amino acid sequence. validation) to increase reliability.
- Figure 20 shows the results of proteome analysis of extracellular vesicles present in the stool of the mouse by the above method, 73 proteins out of a total of 295 proteins were host cell-derived protein, 222 were bacteria-derived protein, double gram negative bacteria-derived protein 77 gram-positive bacteria-derived proteins were identified.
- Gram-negative bacteria were mainly derived from Bacteroides thetaiotaomicron, Escherichia coli K-12 and Klebsiella pneumonia .
- Gram-positive bacteria were mainly derived from Clostridium perfringens and Lactibacillus reuteri .
- the stool-derived extracellular vesicles were isolated using the method of Example 13 and evaluated according to the method of Example 12. At this time, the amount injected into the mouse was extracted by dissolving 50 and 100 ⁇ g of fecal-derived extracellular vesicles in 200 ⁇ l of saline, respectively.
- the present inventors first identified that the extracellular vesicles isolated from the small intestine of the normal state inhibits IL-6 secretion, which is an important biomarker for causing inflammatory diseases and cancer, and disease states such as inflammatory bowel disease.
- the extracellular vesicles isolated from the small intestine were found to be involved in disease by secreting inflammatory mediators through Gram-negative bacteria-derived extracellular vesicles containing endotoxins (LPS).
- extracellular vesicles isolated from the normal small intestine inhibit the inflammatory mediator secretion by LPS or E. coli-derived extracellular vesicles as well as inflammatory responses by the extracellular vesicles isolated from the diseased small intestine. .
- the extracellular vesicles of the present invention have been isolated from the small intestine and feces, but are not limited thereto.
- the extracellular vesicles derived from the small intestine of inflammatory bowel disease induced the secretion of inflammatory mediators compared to the extracellular vesicles isolated from the small intestine of the normal state, thereby predicting the occurrence or course of diseases mediated by IL-6. it means. This focuses on, but is not limited to, inflammatory bowel disease.
- the present invention seeks to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from the mammalian body.
- the composition of the present invention containing the extracellular vesicles isolated in the body of a mammal as an active ingredient can be used as pharmaceutical compositions, food compositions, cosmetic compositions and the like for the treatment or prevention of inflammatory diseases, including cancer.
- the present invention is to determine the causative agent of gastric disease and gastrointestinal diseases through inflammatory mediator secretion of H. pylori- derived extracellular vesicles isolated from patients with gastric disease, analysis of genetic material or protein in endoplasmic reticulum, and measurement of vesicle-specific immune responses. It is possible to diagnose the occurrence or course.
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Abstract
The present invention relates to a method or the like for treating or preventing inflammatory diseases and cancers by using extracellular vesicles isolated from inside the body of a mammal. More specifically, provided are: a composition for preventing and/or treating an inflammatory disease, which contains extracellular vesicles isolated from inside the body of a mammal as an active ingredient; and a vaccine for treating and/or preventing an inflammatory disease, which contains the extracellular vesicles as an active ingredient. Also provided is a method or the like for diagnosing the causative substance of a disease or the occurrence or progress of a disease by using the extracellular vesicles. In addition, in the present invention, extracellular vesicles derived from H. pylori isolated from stomach-disease patients can be subjected to inflammatory mediator secretion ability, intravesicular genetic material or protein analysis, vesicle and particularly immune response measurement and the like, thereby diagnosing the causative substance of the stomach disease and the occurrence or progress of the stomach disease.
Description
본 발명은 포유동물의 체내에서 분리한 세포밖 소포체를 유효성분으로 함유하는 염증성 질환 및 암의 치료 또는 예방용 조성물, 및 상기 세포밖 소포체를 이용한 염증성 질환 및 암의 진단 방법 등에 관한 것이다.The present invention relates to a composition for the treatment or prevention of inflammatory diseases and cancer containing the extracellular vesicles isolated from the body of a mammal as an active ingredient, and to a method for diagnosing inflammatory diseases and cancers using the extracellular vesicles.
염증(Inflammation)은 세포 및 조직의 손상이나 감염에 대한 국소적인 또는 전신적인 방어 기작이다. 염증은 주로 면역계를 이루는 수많은 체액성 매개체(humoral mediator)가 직접 반응하거나 국소적 또는 전신적 작동 시스템(effector system)을 자극함으로써 일어나는 연쇄적인 생체반응에 의해 유발된다. 이러한 염증반응에 관여하는 매개체들로는 조력 T 림프구 (helpher T lymphocyte, Th cell), cytotoxic T lymphocyte (Tc cell) 등과 같은 면역세포, 대식세포 (macrophage), 호중구 (neutrophil), 호산구 (eosinophil), 비반세포 (mast cell) 등과 같은 염증세포와 이들 세포에서 분비되는 사이토카인 등이 있다. 염증성 질환은 특히, 염증성 사이토카인의 분비, 이로 인한 조직손상과 치유의 불균형에 의한 조직개형을 특징으로 한다. 현재까지 알려진 주요한 염증성 사이토카인으로는 대식세포 및 단핵구 세포에 의해 생성되는 TNF-α(tumor necrosis factor-α), IL-1 (interleukin-1, 인터루킨-1), IL-6 및 IL-8 등이 있다. 이와 같은, 염증성 사이토카인의 불균형에 의한 염증성 질환을 치료하기 위하여 상기 염증성 사이토카인을 억제하고자 하는 연구가 다양하게 이루어지고 있다. Inflammation is a local or systemic defense mechanism against damage or infection of cells and tissues. Inflammation is primarily caused by a cascade of biological reactions that occur by the direct response of numerous humoral mediators that make up the immune system or by stimulating local or systemic effector systems. Mediators involved in this inflammatory response include immune cells, such as helper T lymphocytes (Th cells) and cytotoxic T lymphocytes (Tc cells), macrophage cells, neutrophils, eosinophils, and stroma cells. inflammatory cells such as (mast cell) and cytokines secreted from these cells. Inflammatory diseases are particularly characterized by tissue remodeling due to the secretion of inflammatory cytokines, resulting in tissue damage and imbalance of healing. The major inflammatory cytokines known to date include TNF-α (tumor necrosis factor-α), IL-1 (interleukin-1, interleukin-1), IL-6 and IL-8 produced by macrophages and monocytes There is this. In order to treat inflammatory diseases caused by imbalance of inflammatory cytokines, various studies have been made to suppress the inflammatory cytokines.
주요 염증성 질환으로는 천식, 만성폐쇄성폐질환, 비염 등의 호흡기질환, 아토피 피부염 등의 피부질환, 위염, 위궤양, 염증성장염 등의 소화기질환이 포함되며, 기타 감염성 비염, 알레르기성 비염, 만성 비염, 급성 부비동염 및 만성 부비동염 등과 같은 비염 및 부비동염; 급성화농성 중이염 및 만성화농성 중이염 등과 같은 중이염; 세균성 폐렴, 기관지 폐렴, 대엽성 폐렴, 레지오렐라 폐렴 및 바이러스성 폐렴 등과 같은 폐렴; 급성 또는 만성 위염; 감염성 소장결장염, 크론씨 병(Crohn's disease), 특발성 궤양성 대장염, 위막성 대장염 등과 같은 장염; 화농성 관절염, 결핵성 관절염, 퇴행성 관절염 및 류마티스 관절염 등과 같은 관절염; 및 당뇨병, 동맥경화증 등이 있다. Major inflammatory diseases include asthma, chronic obstructive pulmonary disease, respiratory diseases such as rhinitis, skin diseases such as atopic dermatitis, gastroenteritis, gastritis, gastric ulcer, inflammatory growthitis, and other infectious rhinitis, allergic rhinitis, chronic rhinitis, Rhinitis and sinusitis, such as acute sinusitis and chronic sinusitis; Otitis media, such as acute purulent otitis media and chronic purulent otitis media; Pneumonia, such as bacterial pneumonia, bronchial pneumonia, lobar pneumonia, regoraella pneumonia and viral pneumonia; Acute or chronic gastritis; Enteritis, such as infectious enterocolitis, Crohn's disease, idiopathic ulcerative colitis, gastric colitis, and the like; Arthritis such as purulent arthritis, tuberculosis arthritis, degenerative arthritis and rheumatoid arthritis; And diabetes mellitus, arteriosclerosis, and the like.
암의 발생에 염증이 중요하다는 가설은 수세기 이전부터 제기되어 왔었고, 만성 염증에 따른 조직손상과 이의 치유과정의 잘못으로 암이 발생한다고 제안되었다. 위장관에 발생하는 질환 중에서 Helicobacter pylori (헬리코박터 파일로리) 감염이 만성위염, 소화성궤양, 위암의 발생에 중요하다고 알려져 있으며, 또한 H. pylori 는 그람 음성균으로서 위점막에 침투하지 않고, 염증을 유도한다고 알려져 있다. H. pylori 감염에 의해 만성위염이 발생한 경우 위암 발생의 위험도가 현저히 증가하지만, H. pyrlori 감염에 의해 소화성궤양이 발생한 경우 위암의 발생빈도는 만성위염에 비해 그 위험도가 낮은 것으로 보고되었다. 이는 H. pylori 에 의한 만성위염과 위암의 발생에 관련된 H. pylori strain에 비하여 소화성궤양을 일으키는 H. pylori strain인 경우에 cytotoxin-associated antigen (CagA)과 같은 단백질을 분비하는 strain에 의해 발생한다는 보고를 감안하면, strain에 따라 질병을 일으키는 능력이 다름을 의미한다. The hypothesis that inflammation is important in the development of cancer has been suggested for centuries, and it has been suggested that cancer occurs due to tissue damage caused by chronic inflammation and a mistake in its healing process. It is known that in a disease of the gastrointestinal tract, Helicobacter pylori (H. pylori) infection is important in chronic gastritis, peptic ulcer disease, the occurrence of gastric cancer, it is also H. pylori is known to not penetrate the mucosa above a gram-negative bacteria, leading to inflammation . The risk of gastric cancer is significantly increased in the case of chronic gastritis caused by H. pylori infection, but the incidence of gastric cancer in patients with peptic ulcer caused by H. pylori infection is lower than that of chronic gastritis. This report that generated by strain which secretes the protein, such as a cytotoxin-associated antigen (CagA) in the case of H. pylori strain causing peptic ulcer as compared with H. pylori strain associated with the occurrence of chronic gastritis and gastric cancer by H. pylori Considering this, it means that the ability to cause disease depends on the strain.
한편, 대장에 발생하는 염증성장염의 발생에 장내 세균이 중요하다고 알려져 있다. 염증성장염에는 크게 Th1 면역반응을 특징으로 하는 크론병과 Th17 면역반응을 특징으로 하는 궤양성대장염이 있다. 만성염증성장염이 대장암의 위험인자로 주목을 받고 있는데, 특히 궤양성대장염 환자에서 대장암의 발생률이 현저히 증가한다고 알려져 있다. Th17 면역반응의 발생에는 IL-6의 역할이 매우 중요하고, 이는 STAT3를 통한 신호전달경로를 통해 naive T세포에 대하여 IL-17을 분비하는 Th17 세포로 분화를 유도한다. 또한, 최근 보고에 의하면 장내 세균중에서 metalloprotease toxin을 분비하는 Bactroides fragillus 균이 IL-6 분비를 유도하고, 이는 STAT3 신호를 통해 Th17 면역반응을 유도하여, 대장암을 발생시킨다는 동물실험결과가 주목을 받고 있다. On the other hand, intestinal bacteria are known to be important for the development of inflammatory bowel disease occurring in the large intestine. Inflammatory growthitis includes Crohn's disease, which is characterized by a Th1 immune response, and ulcerative colitis, which is characterized by a Th17 immune response. Chronic inflammatory growth inflammation is attracting attention as a risk factor for colorectal cancer. In particular, it is known that the incidence of colorectal cancer increases significantly in patients with ulcerative colitis. The role of IL-6 is very important in the generation of Th17 immune response, which induces differentiation into Th17 cells that secrete IL-17 to naive T cells through signaling pathways through STAT3. In addition, recent reports show that Bactroides fragillus, which secretes metalloprotease toxin from intestinal bacteria, induces IL-6 secretion, which induces a Th17 immune response through STAT3 signaling, resulting in colon cancer. have.
원핵세포 또는 진핵세포는 세포밖 소포체를 분비하고, 분비된 세포밖 소포체는 여러 기능들이 있음이 최근 보고 되었다. 그람 음성균에서 분비된 세포밖 소포체는 내독소(lipopolysacharide, LPS)와 세균 유래 단백질들을 함유하고 있고, 최근 본 발명자들은 장내 공생 그람 음성균에서 유래한 세포밖 소포체가 혈액으로 흡수되어 전신 염증반응을 유도함을 밝힌 바 있다. 또한, 최근 본 발명자들은 그람 양성균이 세포밖 소포체를 분비하고, 프로테옴 분석을 통해 소포체내에 질병을 일으키는 단백질이 포함되어 있음을 보고하였고, 이를 피부에 투여하였을 때 아토피피부염이 발생함을 보고하였다. Prokaryotic or eukaryotic cells secrete extracellular vesicles, and recently released extracellular vesicles have several functions. Extracellular vesicles secreted from gram-negative bacteria contain lipopolysacharide (LPS) and protein derived from bacteria. Recently, the present inventors have found that extracellular vesicles derived from enteric symbiotic gram-negative bacteria are absorbed into the blood to induce a systemic inflammatory response. I said. In addition, the present inventors recently reported that Gram-positive bacteria secrete extracellular vesicles, and proteome analysis includes protein causing disease in the endoplasmic reticulum, and reports that atopic dermatitis occurs when administered to the skin.
한편, 여러 종의 암세포에서 유래된 세포밖 소포체는 암세포의 성장 및 전이에 필요한 단백질들을 함유하고 있음이 보고되었다. 비슷한 맥락에서, 사람 또는 실험동물의 여러 분비물, 배설물 또는 조직 세척액 등에서 세포밖 소포체가 발견되었음이 보고 되었다. 또한, 조직 유래 세포밖 소포체는 소포체를 분비하는 조직의 상태를 반영하는 것으로 알려져 있으며, 질병의 진단에 이용 될 수 있음이 보고되었다. 최근에는 실험 동물의 소장에서 세포밖 소포체가 존재함이 보고된 바 있다. 그러나, 아직까지 H. pylori 가 생성하는 세포밖 소포체와 위염, 소화성궤양, 및 위암의 발생과의 연관성, 혹은 H. pylori 유래 소포체, 또는 H. pylori에 의해 숙주세포에서 유래된 세포밖 소포체를 이용하여 H. pylori 감염에 의해 발생하는 위질환에 대한 진단기술 및 예방 혹은 치료백신을 개발하고자 한 연구에 대한 보고는 전무한 상황이다. 또한, 소장 및 대장 공생세균에 의해 장 상피세포 등과 같은 숙주세포에서 분비되는 세포밖 소포체의 면역학적 중요성 및 이를 응용하여 진단 및 예방, 치료기술에 적용한 사례는 보고되지 않았다. On the other hand, extracellular vesicles derived from various cancer cells have been reported to contain proteins necessary for the growth and metastasis of cancer cells. In a similar context, it has been reported that extracellular vesicles have been found in various secretions, excreta, or tissue washes of human or laboratory animals. In addition, tissue-derived extracellular vesicles are known to reflect the state of tissues secreting vesicles, and it has been reported that they can be used to diagnose diseases. Recently, extracellular vesicles have been reported in the small intestine of experimental animals. However, the association between the extracellular vesicles produced by H. pylori and the development of gastritis, peptic ulcer, and gastric cancer, or H. pylori- derived vesicles, or extracellular vesicles derived from host cells by H. pylori Therefore, there are few reports on studies to develop diagnostic techniques and preventive or therapeutic vaccines for gastric diseases caused by H. pylori infection. In addition, the immunological significance of extracellular vesicles secreted from host cells such as intestinal epithelial cells by small and large intestine commensal bacteria, and the application of the same to the diagnosis, prevention, and treatment techniques have not been reported.
본 발명은 포유동물 체내에서 유래하는 세포밖 소포체를 이용하여 염증성 질환 및 암을 예방 및/또는 치료하는 방법을 제공하고자 한다. 구체적으로, 본 발명은 상기 세포밖 소포체를 유효성분으로 함유하는, 염증성 질환 및 암의 예방 및/또는 치료용 조성물, 염증성 질환 및 암의 치료 또는 예방용 백신 등을 제공한다. 이에 더하여, 본 발명은 포유동물의 체내에서 유래하는 세포밖 소포체를 이용하여 질환의 원인물질,질환의 발생 또는 경과를 진단하는 방법 등을 제공한다. The present invention seeks to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from the mammalian body. Specifically, the present invention provides a composition for preventing and / or treating inflammatory diseases and cancer, a vaccine for treating or preventing inflammatory diseases and cancer, containing the extracellular vesicles as an active ingredient. In addition, the present invention provides a method for diagnosing the cause of a disease, the occurrence or course of a disease, etc. using an extracellular vesicle derived from the body of a mammal.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은 포유동물의 체내에서 유래된 세포밖 소포체를 유효 성분으로 함유하는, 염증성 질환 및 암의 치료 또는 예방용 조성물을 제공한다. 상기 염증성 질환은 호흡기질환, 소화기질환, 피부질환, 패혈증, 동맥경화증, 관절염, 뇌질환 등의 전신질환 및 이의 합병증 등을 포함한다. The present invention provides a composition for the treatment or prevention of inflammatory diseases and cancer, containing as an active ingredient extracellular vesicles derived from the body of a mammal. The inflammatory diseases include respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, systemic diseases such as brain diseases, and complications thereof.
본 발명의 예시적 구현예에 있어서, 상기 세포밖 소포체는 포유동물의 소장 대장, 또는 대변에서 분리된 것일 수 있으나, 이제 제한되는 것은 아니다. In an exemplary embodiment of the invention, the extracellular vesicles may be isolated from the small intestine, or the stool of a mammal, but are not limited thereto.
본 발명의 예시적 구현예에 있어서, 상기 세포밖 소포체는 포유동물의 세포에서 염증성 매개체의 분비를 억제하는 것일 수 있으나, 이에 제한되는 것은 아니다. 상기 세포는 상피세포 및 염증세포를 포함한다. 상기 염증세포는 대식세포 및 호산구를 포함한다. In an exemplary embodiment of the invention, the extracellular vesicles may be to inhibit the secretion of inflammatory mediators in cells of mammals, but is not limited thereto. The cells include epithelial cells and inflammatory cells. The inflammatory cells include macrophages and eosinophils.
본 발명의 예시적 구현예에 있어서, 상기 염증성 매개체는 인터루킨 6(IL-6)일 수 있으나, 이에 제한되는 것은 아니다. In an exemplary embodiment of the present invention, the inflammatory mediator may be interleukin 6 (IL-6), but is not limited thereto.
본 발명의 예시적 구현예에 있어서, 상기 세포밖 소포체는 포유동물의 체내 공생 세균 유래의 세포밖 소포체일 수 있으나, 이에 제한되는 것은 아니다. In an exemplary embodiment of the present invention, the extracellular vesicles may be, but is not limited to, extracellular vesicles derived from symbiotic bacteria in a mammal.
본 발명의 예시적 구현예에 있어서, 상기 세포밖 소포체는 상기 포유동물의 상피세포 또는 염증세포에서 유래하는 세포밖 소포체일 수 있으나, 이에 제한되는 것은 아니다. In an exemplary embodiment of the present invention, the extracellular vesicles may be an extracellular vesicle derived from epithelial cells or inflammatory cells of the mammal, but is not limited thereto.
본 발명의 세포밖 소포체는 평균 직경이 20 nm 내지 200 nm 일 수 있으나, 이에 제한되는 것은 아니다. The extracellular vesicles of the present invention may have an average diameter of 20 nm to 200 nm, but is not limited thereto.
본 발명의 조성물은 약학적 조성물, 식품 조성물, 화장료 조성물 등일 수 있으나, 이에 제한되는 것은 아니다. The composition of the present invention may be a pharmaceutical composition, a food composition, a cosmetic composition and the like, but is not limited thereto.
또한, 본 발명은 포유동물의 체내에서 유래된 세포밖 소포체를 유효성분으로 함유하는, 염증성 질환 및 암의 치료 또는 예방용 백신을 제공한다. 상기 백신은 세포밖 소포체를 단독으로 사용하거나, 상기 세포밖 소포체와 병원성 소포체를 혼합하여 사용할 수 있다. 상기 병원성 소포체는 체내 공생 세균에서 유래한 병원성 소포체, 그람 양성 세균에서 유래한 병원성 소포체, 및 공기에 존재하는 병원성 소포체 등을 포함한다. The present invention also provides a vaccine for the treatment or prophylaxis of inflammatory diseases and cancer, containing as an active ingredient extracellular vesicles derived from the body of a mammal. The vaccine may be used alone or in combination with the extracellular vesicles and the pathogenic vesicles. The pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.
또한, 본 발명은 포유동물의 체내에서 유래된 세포밖 소포체를 치사량 미만으로 포유동물에게 투여하는 단계를 포함하는, 질환의 예방 또는 치료 방법을 제공한다. 상기 질환은 염증성 질환 및 암 등을 포함한다. The present invention also provides a method for preventing or treating a disease, comprising administering to a mammal an extracellular vesicle derived from the body of the mammal at a less than lethal dose. The disease includes inflammatory disease, cancer and the like.
또한, 본 발명은 포유동물의 체내에서 유래하는 세포밖 소포체를 이용하여 질환의 원인물질,질환의 발생 또는 경과를 진단하는 방법을 제공한다. 상기 질환은 염증성 질환 및 암 등을 포함한다. The present invention also provides a method for diagnosing the cause of a disease, the occurrence or course of a disease by using an extracellular vesicle derived from the body of a mammal. The disease includes inflammatory disease, cancer and the like.
본 발명의 예시적 구현예로, 상기 방법은 하기단계를 포함할 수 있다: 포유동물의 체내에서 세포밖 소포체를 분리하는 단계; 상기 분리된 세포밖 소포체를 세포에 처리하여 배양하는 단계; 및 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계. In an exemplary embodiment of the invention, the method may comprise the steps of: separating extracellular vesicles in the body of a mammal; Culturing the isolated extracellular vesicles in cells; And measuring the level of inflammatory mediator in said cell culture.
본 발명의 예시적 구현예로, 상기 세포밖 소포체는 포유동물의 위액에서 분리할 수 있다. In an exemplary embodiment of the invention, the extracellular vesicles can be isolated from gastric juice of a mammal.
본 발명의 예시적 구현예로, 상기 포유동물의 체내로부터 세포밖 소포체를 분리하는 단계는 하기 단계를 포함할 수 있다: 포유동물의 체내 물질(분비물 포함)을 수득하는 단계; 상기 수득된 물질을 원심분리하여 불순물, 동물세포, 및 세균을 제거하는 단계; 및 초원심분리를 수행하여 세포밖 소포체를 분리하는 단계. In an exemplary embodiment of the invention, the step of separating the extracellular vesicles from the body of the mammal may comprise the steps of: obtaining the body material (including secretion) of the mammal; Centrifuging the obtained material to remove impurities, animal cells, and bacteria; And separating the extracellular vesicles by performing ultracentrifugation.
또한, 본 발명은 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 의 체외 배양액에서 분리한 세포밖 소포체를 포함하는 조성물을 제공한다. In addition, the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice Provided are compositions comprising extracellular vesicles isolated from in vitro culture of Helicobacter pylori.
또한, 본 발명은 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 이용하여 질환의 원인물질, 질환의 발생 또는 경과를 진단하는 방법을 제공한다. 상기 질환은 위염, 소화성궤양, 및 위암 등을 포함한다. In addition, the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Provided is a method for diagnosing a causative agent of a disease, the occurrence or course of a disease using an extracellular vesicle. The disease includes gastritis, peptic ulcer, gastric cancer, and the like.
본 발명의 예시적 구현예로, 상기 방법은 하기 단계를 포함할 수 있다: 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 세포에 처리하여 배양하는 단계; 및 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계. In an exemplary embodiment of the invention, the method may comprise the following steps: Helicobacter pylori isolated from human gastric tissue or gastric juice(Helicobacter pylori)origin Treating the cells with extracellular vesicles and culturing; And measuring the level of inflammatory mediator in said cell culture.
본 발명의 예시적 구현예로, 상기 방법은 상기 세포밖 소포체 내 유전물질 또는 단백질을 측정하는 단계, 상기 세포밖 소포체에 대한 면역반응을 측정하는 단계, 또는 상기 세포밖 소포체에 대한 항체를 측정하는 단계 등을 포함할 수 있다. In an exemplary embodiment of the invention, the method comprises measuring a genetic material or protein in the extracellular vesicles, measuring an immune response to the extracellular vesicles, or measuring an antibody against the extracellular vesicles. Step and the like.
또한, 본 발명은 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 유효성분으로 함유하는, 헬리코박터 파일로리 (Helicobacter pylori) 에 의한 질환의 치료 또는 예방용 백신을 제공한다. In addition, the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Helicobacter pylori containing extracellular vesicles as an active ingredient(Helicobacter pylori)Provided are vaccines for the treatment or prevention of diseases.
또한, 본 발명은 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 치사량 미만으로 포유동물에게 투여하는 단계를 포함하는, 헬리코박터 파일로리 (Helicobacter pylori) 에 의한 질환의 예방 또는 치료 방법을 제공한다.In addition, the present invention is a Helicobacter pylori isolated from human gastric tissue or gastric juice From Helicobacter pylori Helicobacter pylori, comprising administering an extracellular vesicle to a mammal at a sublethal dose(Helicobacter pylori)It provides a method for preventing or treating a disease by.
본 발명자들은 정상 상태의 포유동물의 체내에서 분리된 세포밖 소포체는 염증성 질환과 관련된 염증성 매개체인 IL-6의 분비를 억제할 수 있으며, 이에 따라 염증반응을 차단시킬 수 있음을 발견하였다. 따라서, 포유동물의 체내에서 분리된 세포밖 소포체를 유효성분으로 함유하는 본 발명의 조성물은 암을 포함한 염증성 질환의 치료 또는 예방을 위한 약학적 조성물, 식품 조성물, 화장료 조성물 등으로 이용될 수 있다. The inventors have found that extracellular vesicles isolated in the body of a normal mammal can inhibit the secretion of IL-6, an inflammatory mediator associated with inflammatory diseases, thereby blocking the inflammatory response. Therefore, the composition of the present invention containing the extracellular vesicles isolated in the body of a mammal as an active ingredient can be used as pharmaceutical compositions, food compositions, cosmetic compositions and the like for the treatment or prevention of inflammatory diseases including cancer.
또한, 염증성 질환에 걸린 포유동물의 체내에서 분리된 세포밖 소포체는 염증세포의 염증성 매개체의 분비를 유발시킬 수 있기 때문에, 포유동물 체내에서 분리된 본 발명의 세포밖 소포체를 이용하여 염증성 질환의 발생 또는 경과를 진단할 수 있다. In addition, since the extracellular vesicles isolated in the body of a mammal suffering from an inflammatory disease may cause the secretion of inflammatory mediators of inflammatory cells, the development of an inflammatory disease using the extracellular vesicles of the present invention isolated in a mammalian body. Or diagnose progress.
이에 더하여, 본 발명은 위질환 환자에서 분리한 H. pylori 유래 세포밖 소포체에 대한 염증성 매개체 분비능, 소포체 내 유전물질 혹은 단백질 분석, 소포체 특이 면역반응 측정 등을 통해 위질환의 원인물질과 위질환의 발생 또는 경과를 진단하는 것을 가능하게 한다.In addition, the present invention is to determine the causative agent of gastric disease and gastric disease by inflammatory mediator secretion of H. pylori- derived extracellular vesicles isolated from patients with gastric disease, analysis of genetic material or protein in endoplasmic reticulum, and measurement of vesicle-specific immune responses. It is possible to diagnose the occurrence or course.
도 1은 소화성궤양환자의 위세척액에서 초원심분리를 통해 세포밖 소포체를 분리한 결과를 보여주는 그림이다. 1 is a diagram showing the result of separating extracellular vesicles through ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
도 2는 소화성궤양환자의 위세척액에서 초원심분리를 하였을 때, F3 부분에 존재하는 세포밖 소포체의 전자현미경 사진이다. Figure 2 is an electron micrograph of the extracellular vesicles present in the F3 region when ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
도 3는 소화성궤양환자의 위세척액에서 초원심분리를 하였을 때, F6 부분에 존재하는 세포밖 소포체의 전자현미경 사진이다. Figure 3 is an electron micrograph of the extracellular vesicles present in the F6 portion when ultracentrifugation in gastric lavage fluid of peptic ulcer patients.
도 4는 소화성궤양환자의 위세척액에서 분리한 세포밖 소포체 내 단백질의 존재유무를 Coommasie blue staining을 통해 평가하였을 때, F6 부부에 존재하는 세포밖 소포체에만 단백질이 함유된 결과를 나타낸 것이다. Figure 4 shows the results of the protein contained only in the extracellular vesicles present in the F6 couple when the presence or absence of proteins in the extracellular vesicles isolated from gastric lavage fluid of peptic ulcer patients.
도 5는 소화성궤양환자의 위세척액에서 분리한 세포밖 소포체를 여러 대식세포에 처리하였을 때, 염증성 매개체인 TNF-α 의 분비를 나타낸 그림이다.5 is a diagram showing the secretion of inflammatory mediators TNF-α when the extracellular vesicles isolated from gastric lavage fluid of peptic ulcer patients treated with various macrophages.
도 6은 소화성궤양환자의 위세척액에서 세포밖 소포체를 분리하였을 때, F3 부분에 존재하는 소포체에 의한 염증성 매개체 (TNF-α) 분비에 polymyxin B와 열처리의 효과를 보여주는 그림이다. Figure 6 shows the effect of polymyxin B and heat treatment on the secretion of inflammatory mediators (TNF-α) by the endoplasmic reticulum in the F3 part when isolated from extracellular vesicles in gastric lavage fluid of peptic ulcer patients.
도 7은 소화성궤양환자의 위세척액에서 분리한 세포밖 소포체를 분리하였을 때, F6 부분에 존재하는 소포체에 의한 염증성 매개체 (IL-6 및 TNF-α) 분비에 polymyxin B와 열처리의 효과를 보여주는 그림이다. Figure 7 shows the effect of polymyxin B and heat treatment on the secretion of inflammatory mediators (IL-6 and TNF-α) by the endoplasmic reticulum in the F6 region when isolated extracellular vesicles from gastric lavage fluid of peptic ulcer patients to be.
도 8은 위염, 위궤양, 및 위암 환자에서 분리한 H. pylori를 배양하여, 배양액에서 분리한 세포밖 소포체를 전자현미경으로 촬영한 사진이다. FIG. 8 is an electron microscope photograph of extracellular vesicles isolated from the culture by H. pylori cultured from patients with gastritis, gastric ulcer, and gastric cancer.
도 9는 만성위염, 위궤양, 위암 환자에서 분리한 H. pylori를 배양하여,배양액에서 분리한 세포밖 소포체를 대식세포에 처리하여 분비된 IL-6의 양을 측정하였을 때, 위궤양환자에서 분리한 H. pylroi 유래 소포체가 IL-6의 분비를 현저히 증가시킨 결과를 나타낸 것이다. 9 is isolated from gastric ulcer patients when cultured H. pylori isolated from patients with chronic gastritis, gastric ulcer, gastric cancer, and treated with extracellular vesicles isolated from the culture medium to the macrophages, the amount of IL-6 secreted H. pylroi-derived endoplasmic reticulum showed a significant increase in the secretion of IL-6.
도 10은 마우스 소장으로부터 세포밖 소포체를 분리한 결과를 보여주는 도면으로서, 마우스 소장으로부터 세포밖 소포체를 분리하기 위한 분리 프로토콜(a), 분리된 세포밖 소포체의 전자현미경(TEM) 이미지(b), 분리된 세포밖 소포체의 정량결과(c), 및 분리된 세포밖 소포체의 세균 오염 여부를 확인한 세균 배양 결과(d)를 나타낸 도면이다. 10 is a diagram showing the results of separating the extracellular vesicles from the mouse small intestine, a separation protocol for separating the extracellular vesicles from the mouse small intestine (A), electron microscopy (TEM) image of the isolated extracellular vesicles (b), Quantitative results of the isolated extracellular vesicles (c) and bacterial culture results (d) confirming the bacterial contamination of the isolated extracellular vesicles.
도 11은 정상 마우스의 소장에서 분리된 세포밖 소포체에 의한 in vitro 면역반응을 평가한 결과로서, 대식 세포에 세포밖 소포체를 처리한 뒤 세포배양액에서 염증성 매개체인 IL-6(좌)와 TNF-α(우)의 분비량을 ELISA를 통해 측정한 결과이다. 11 is a result of evaluating the in vitro immune response by extracellular vesicles isolated from the small intestine of normal mice, after treatment of macrophages with extracellular vesicles IL-6 (left) and TNF- inflammatory mediators in cell culture It is the result of measuring the secretion amount of (right) by ELISA.
도 12는 염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체에 의한 in vitro 면역반응을 평가한 결과로서, 대식 세포에 세포밖 소포체를 처리한 뒤 세포배양액에서 염증성 매개체인 IL-6(좌)와 TNF-α(우)의 분비량을 ELISA를 통해 측정한 결과이다. 12 is a result of evaluating the in vitro immune response by extracellular vesicles isolated from the small intestine of inflammatory bowel disease mice, and treated with extracellular vesicles in macrophages and IL-6 (left), an inflammatory mediator in cell culture. The secretion of TNF-α (right) was measured by ELISA.
도 13은 정상 마우스와 염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체에 함유된 단백질의 양상을 비교하기 위한 세포밖 소포체 단백질에 대한 SDS-PAGE의 결과를 나타낸 도면이다. FIG. 13 shows the results of SDS-PAGE on extracellular vesicle proteins for comparing aspects of proteins contained in extracellular vesicles isolated from the small intestine of normal mice and inflammatory bowel disease mice.
도 14는 염증성 장질환 마우스 소장 유래 세포밖 소포체에 의한 염증성 매개체 분비가, 장내 공생 그람 음성균 유래의 소포체에 의해 유도되는지를 평가한 결과로서, 리포폴리사카라이드(LPS) 길항체인 polymixin B를 소포체에 처리하여 염증성 매개체인 IL-6(좌)와 TNF-α(우)의 분비량을 대식세포 (RAW 246.7세포) 배양액에서 ELISA를 이용하여 측정한 결과를 나타낸 것이다. 14 is a result of evaluating whether inflammatory mediator secretion by inflammatory bowel disease mouse small intestine-derived extracellular vesicles is induced by endoplasmic reticulum derived from intestinal symbiotic Gram-negative bacteria. The amount of secretion of inflammatory mediators IL-6 (left) and TNF-α (right) was measured in the macrophages (RAW 246.7 cells) culture medium by ELISA.
도 15는 염증성 장질환 마우스 소장 유래 세포밖 소포체의 염증성 매개체 발현에 대한 정상 마우스 소장 유래 세포밖 소포체의 치료효과를 평가한 결과로서, 대식세포 (RAW 246.7 세포)에 염증성 장질환 마우스 소장 유래 세포밖 소포체와 정상 마우스 소장 유래 세포밖 소포체를 동시에 처리하을 때, 염증성 매개체 IL-6(좌)와 TNF-α(우)의 분비를 나타낸 결과이다. 15 is a result of evaluating the therapeutic effect of normal mouse small intestine-derived extracellular vesicles on the expression of inflammatory mediators of inflammatory bowel disease-derived extracellular vesicles derived from mouse small intestine. When the endoplasmic reticulum and extracellular vesicles derived from the normal mouse small intestine were simultaneously treated, the secretion of inflammatory mediators IL-6 (left) and TNF-α (right) was shown.
도 16은 염증성 장질환 마우스 소장 유래 세포밖 소포체에 의한 염증성 매개체 발현에 대해 정상 마우스 소장 유래 세포밖 소포체를 미리 투여하였을 때의 예방효과를 평가한 결과로서, 대식세포 (RAW 246.7 세포)에 정상 마우스 소장 유래 세포밖 소포체를 시간 별로 전 처리를 한 후 일정 시간이 지난 후 세포 배양액을 제거하고, 염증성 장질환 마우스 소장 유래 세포밖 소포체를 새로운 세포 배양액에 섞어 처리한 후 6 시간째 세포 배양액에서 염증성 매개체 IL-6(좌)와 TNF-α(우)를 측정한 결과이다. 16 is a result of evaluating the preventive effect of pre-administration of normal mouse small intestine-derived extracellular vesicles on inflammatory mediator expression by inflammatory bowel disease mouse small intestine-derived extracellular vesicles. FIG. 16 shows normal mice in macrophages (RAW 246.7 cells). After pre-treatment of the small intestine-derived extracellular vesicles by time, the cell culture medium was removed after a certain period of time, and the inflammatory mediators were treated in the cell culture solution for 6 hours after mixing and treating the extracellular vesicles derived from the inflammatory bowel disease mouse small intestine. IL-6 (left) and TNF-α (right) were measured.
도 17은 염증성 장질환의 원인물질로 알려져 있는 그람 음성균 세포벽의 구성물질인 LPS 또는 그람 음성균인 대장균 유래 세포밖 소포체에 의한 염증성 매개체 분비에 대하여, 정상 상태의 소장에서 분리한 세포밖 소포체의 항염증 효과를 평가한 결과를 나타낸 것이다. Figure 17 shows the anti-inflammatory properties of extracellular vesicles isolated from the small intestine of normal state against the secretion of inflammatory mediators by LPS or gram-negative bacteria E. coli-derived extracellular vesicles, which are components of gram-negative bacterial cell walls known as inflammatory bowel diseases. Effect The evaluation results are shown.
도 18은 소장에서 분리한 소포체를 3회 피하로 주사한 후 혈청에서 소포체 특이 항체를 측정한 결과이다. Figure 18 shows the results of measuring the endoplasmic reticulum-specific antibody in the serum after three subcutaneous injection of the vesicles isolated from the small intestine.
도 19은 소장에서 분리한 소포체를 3회 피하로 주사한 후 비장에서 면역세포를 분리하여 면역세포에 소포체를 처리한 후 감마인터페론과 인터루킨-10의 농도를 측정한 결과이다. 19 is a result of measuring the concentrations of gamma interferon and interleukin-10 after injecting vesicles isolated from the small intestine three times subcutaneously, separating immune cells from the spleen and treating the vesicles with immune cells.
도 20는 마우스 대변 유래 세포밖 소포체의 단백질 질량 분석을 통해 체내 공생 세균을 동정한 결과이다. 20 shows the results of identifying symbiotic bacteria in the body through protein mass spectrometry of mouse stool-derived extracellular vesicles.
도 21은 대변에서 분리한 소포체를 3회 피하로 주사한 후 혈청에서 소포체 특이 항체를 측정한 결과이다. Figure 21 shows the results of measuring the endoplasmic reticulum-specific antibody in the serum after three subcutaneous injection of the vesicles isolated from the stool.
도 22은 대변에서 분리한 소포체를 3회 피하로 주사한 후 비장에서 면역세포를 분리하여 면역세포에 소포체를 처리한 후 인터루킨-10의 농도를 측정한 결과이다. 22 is a result of measuring the concentration of interleukin-10 after treatment of endoplasmic reticulum in immune cells by injecting vesicles isolated from feces three times subcutaneously and then separating immune cells from the spleen.
본 발명자들은 염증성 질환의 예방 및/또는 치료를 위한 조성물과, 그 진단 방법 등을 개발하고자 노력한 결과, 소화성궤양 환자의 위세척액에서 세포밖 소포체를 분리하여 면역반응을 평가하였을 때, 그람 음성균에서 유래한 소포체가 Th17 면역반응을 유도하는 IL-6의 분비를 유도함을 발견하였고, 또한 만성위염, 소화성궤양, 및 위암 환자에서 분리한 H. pylori 에서 세포밖 소포체를 분리하여 면역반응을 평가하였을 때, 소화성궤양 환자에서 분리한 세포밖 소포체가 타 질환 환자에서 분리한 H. pylori 유래 세포밖 소포체에 비하여 IL-6를 분비하는 효과가 현저히 증가되어 있음을 발견하였다. The present inventors have tried to develop a composition for the prevention and / or treatment of inflammatory diseases, diagnostic methods, etc., when the extracellular vesicles are isolated from the gastric lavage fluid of peptic ulcer patients to evaluate the immune response, derived from Gram-negative bacteria One endoplasmic reticulum was found to induce the release of IL-6, which induces a Th17 immune response, and when the immune response was evaluated by separating extracellular vesicles from H. pylori isolated from patients with chronic gastritis, peptic ulcer, and gastric cancer, Extracellular vesicles isolated from peptic ulcer patients were found to have a significantly increased effect of secreting IL-6 compared to H. pylori- derived extracellular vesicles isolated from other disease patients.
또한, 본 발명자들은 염증성장염 상태의 포유동물의 소장에서 분리한 세포밖 소포체가 질병에 걸리지 않은 정상 상태의 포유동물의 소장에서 분리된 세포밖 소포체에 비하여 IL-6 분비능이 크게 증가되어 있었고, 정상 상태의 소장에 존재하는 세포밖 소포체는 염증성장염을 유도하는 원인물질인 내독소나 그람 음성균 유래 세포밖 소포체에 의한 염증 반응을 억제할 수 있음을 발견하였다. 이에 더하여, 본 발명자들은 소장유래 세포밖 소포체를 피하로 주사하였을 때 소포체에 대한 면역반응이 유도되지 않는다는 사실을 발견하였다. In addition, the present inventors found that the extracellular vesicles isolated from the small intestine of the inflammatory bowel disease state had significantly increased IL-6 secretion ability compared to the extracellular vesicles isolated from the small intestine of the normal mammal without disease. The extracellular vesicles present in the small intestine were found to be able to inhibit inflammatory responses caused by endotoxins or Gram-negative bacteria-derived extracellular vesicles, which cause inflammatory growth inflammation. In addition, the inventors have discovered that when injected subcutaneously with small intestine-derived extracellular vesicles, no immune response to the endoplasmic reticulum is induced.
본 발명자들은 포유동물의 대변에 세포밖 소포체가 존재하고, 이는 그람 음성균, 그람 양성균뿐만 아니라 숙주세포에서 유래하는 세포밖 소포체로 구성되어 있고, 이를 피하로 주사하였을 때 소포체에 특이적인 IL-10을 분비하는 regulatory T (Treg) 세포가 유도됨을 발견하였다. The present inventors have extracellular vesicles in feces of mammals, which are composed of gram-negative bacteria, gram-positive bacteria as well as extracellular vesicles derived from host cells, and when injected subcutaneously, IL-10 specific for vesicles is injected. It has been found that secreting regulatory T (Treg) cells are induced.
상기 발견들에 기초하여, 본 발명은 포유동물의 체내에서 유래된 세포밖 소포체를 유효 성분으로 함유하는, 염증성 질환 및 암의 치료 또는 예방용 조성물을 제공한다. Based on the above findings, the present invention provides a composition for the treatment or prophylaxis of inflammatory diseases and cancer, comprising as an active ingredient extracellular vesicles derived from the body of a mammal.
본 발명에서, "체내"란 포유동물의 신체 내부 및 피부 표면을 포함하는 의미하며, 예를 들어, 인간을 비롯한 포유동물의 장기 내부, 구강 내부, 피부 표면 등일 수 있다. In the present invention, "intrabody" is meant to include the inside of the mammal's body and the skin surface, and may be, for example, inside the organs of the mammal, including humans, inside the mouth, skin surface and the like.
본 발명에서, "포유동물의 체내에서 유래된 세포밖 소포체"란 포유동물의 체내에 존재하거나 위액, 대변, 소변, 기관지폐포세척액 등에 분비된 소포체를 포함하며, 크기가 원래의 세포보다 작은 것을 특징으로 하나, 이에 제한되는 것은 아니다. 예를 들면, 본 발명의 세포밖 소포체는 체내 공생 세균 혹은 숙주의 상피세포에 의하여 분비되는 세포밖 소포체 일 수 있다. 상기 체내 공생 세균은 포유동물의 체내에 존재하는 세균을 의미하며, 예를 들어, 장내세균 등이 포함된다. In the present invention, "extracellular vesicles derived from the body of a mammal" includes vesicles present in the body of a mammal or secreted into gastric juice, feces, urine, bronchoalveolar lavage fluid, etc., characterized in that the size is smaller than the original cells. However, the present invention is not limited thereto. For example, the extracellular vesicles of the present invention may be symbiotic bacteria or extracellular vesicles secreted by epithelial cells of the host. The symbiotic bacteria in the body refers to the bacteria present in the body of a mammal, for example, enterobacteria and the like.
또한 본 발명의 세포밖 소포체는, 포유동물의 체내에서 분리된 것이라면 어떤 것이라도 사용될 수 있으나, 치료 또는 예방하고자 하는 해당 염증성 질환의 부위에 따라 세포밖 소포체 단독 혹은 질병을 일으키는 병원성 소포체와 혼합하여 사용할 수 있다. 상기 병원성 소포체는 체내 공생 세균에서 유래한 병원성 소포체, 그람 양성 세균에서 유래한 병원성 소포체, 및 공기에 존재하는 병원성 소포체 등을 포함한다. In addition, any of the extracellular vesicles of the present invention can be used as long as it is separated from the body of a mammal, but depending on the site of the inflammatory disease to be treated or prevented, the extracellular vesicles alone or in combination with the pathogenic vesicles causing the disease can be used. Can be. The pathogenic vesicles include pathogenic vesicles derived from symbiotic bacteria in the body, pathogenic vesicles derived from Gram-positive bacteria, pathogenic vesicles present in the air, and the like.
예컨대, 상기 조성물이 염증성 장질환의 예방 또는 치료를 위한 조성물인 경우, 상기 세포밖 소포체는 포유동물의 소장 또는 대변 내에 존재하는 세포밖 소포체를 분리하여 단독으로 사용할 수 있으며, 염증성 폐질환의 예방 또는 치료를 위한 조성물인 경우, 상기 세포밖 소포체는 포유동물의 소장 또는 대변 내에 존재하는 세포밖 소포체를 분리하여 염증성 폐질환을 일으키는 병원성 소포체와 혼합하여 사용할 수 있으며, 염증성 피부질환의 예방 또는 치료를 위한 조성물인 경우, 상기 세포밖 소포체는 포유동물의 소장 또는 대변 내에 존재하는 세포밖 소포체를 분리하여 염증성 피부질환을 일으키는 병원성 소포체와 혼합하여 사용할 수 있으며, 패혈증, 동맥경화증, 관절염, 뇌질환 등의 전신질환의 예방 또는 치료를 위한 조성물인 경우, 상기 세포밖 소포체는 포유동물의 소장 또는 대변 내에 존재하는 세포밖 소포체를 분리하여 전신질환을 일으키는 병원성 소포체와 혼합하여 사용할 수 있다. For example, when the composition is a composition for the prevention or treatment of inflammatory bowel disease, the extracellular vesicles can be used alone to separate the extracellular vesicles present in the small intestine or feces of a mammal, and to prevent or prevent inflammatory lung disease. In the case of a composition for treatment, the extracellular vesicles can be used in combination with a pathogenic vesicle which causes inflammatory lung disease by separating extracellular vesicles present in the small intestine or feces of a mammal, and for the prevention or treatment of inflammatory skin diseases. In the case of the composition, the extracellular vesicles can be used in combination with pathogenic vesicles that cause inflammatory skin diseases by separating extracellular vesicles present in the small intestine or feces of a mammal, and systemic such as sepsis, arteriosclerosis, arthritis, brain diseases, etc. When the composition for the prevention or treatment of the disease, the cell Outer vesicles can be used in combination with pathogenic vesicles that cause systemic disease by separating extracellular vesicles present in the small intestine or feces of a mammal.
상기 포유동물의 체내 또는 분비물에서 상기 세포밖 소포체를 분리하는 방법은 세포밖 소포체를 순수하게 분리할 수 있다면 특별히 제한되지 않는다. 예를 들면, 예컨대 포유동물의 체내 물질, 체내 조직, 분비물 등에서, 원심분리, 초원심분리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 모세관 전기영동 등의 방법 및 이들의 조합을 이용하여 세포밖 소포체를 분리할 수 있다. 또한, 불순물의 제거를 위한 세척, 수득된 세포밖 소포체의 농축 등의 과정을 추가로 포함할 수 있다. The method for separating the extracellular vesicles from the body or secretion of the mammal is not particularly limited as long as the extracellular vesicles can be separated purely. For example, methods such as centrifugation, ultracentrifugation, filtration by filters, gel filtration chromatography, pre-flow electrophoresis, capillary electrophoresis, and the like, and combinations thereof, for example in mammalian body material, body tissues, secretions, and the like. Extracellular vesicles can be isolated using. In addition, it may further include a process for washing to remove impurities, concentration of the obtained extracellular vesicles and the like.
상기 방법에 의하여 분리된 세포밖 소포체는 평균 직경이 500 nm 이하일 수 있으며, 바람직하게는 20 nm 내지 200 nm 일 수 있다. The extracellular vesicles separated by the above method may have an average diameter of 500 nm or less, and preferably 20 nm to 200 nm.
본 발명의 일 구현예에 따르면, 상기 조성물은 약학적 조성물로 제조될 수 있다. 치료 및/또는 예방에 사용하기 위해 본 발명의 세포밖 소포체 자체를 투여하는 것이 가능할 수 있지만, 약학적 조성물의 활성 성분으로서 상기 세포밖 소포체가 포함되는 것이 바람직하다. According to one embodiment of the invention, the composition may be prepared as a pharmaceutical composition. While it may be possible to administer the extracellular vesicles of the present invention for use in treatment and / or prophylaxis, it is preferred that the extracellular vesicles be included as active ingredients of the pharmaceutical composition.
상기 약학적 조성물은 상기 분리된 세포밖 소포체를 유효 성분으로 함유하며, 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌 (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제 등으로 제제화할 수 있다. The pharmaceutical composition may contain the isolated extracellular vesicles as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, or external skin preparation.
본 발명의 약학적 조성물의 투여방법은 특별히 제한되는 것은 아니나, 목적하는 방법에 따라 정맥내, 피하, 복강 내, 흡입, 피부도포, 설하 투여, 비강투여, 항문 투여, 경구 복용 등을 선택할 수 있다. The method of administering the pharmaceutical composition of the present invention is not particularly limited, but may be selected by intravenous, subcutaneous, intraperitoneal, inhalation, dermal application, sublingual administration, nasal administration, anal administration, oral administration, etc. .
투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 일일 투여량은 치료를 필요로 하는 개체에 투여됨으로서 경감된 질병 상태에 대한 치료에 충분한 본 발명의 치료용 물질의 양을 의미한다. 치료용 물질의 효과적인 양은 특정 화합물, 질병 상태 및 그의 심각도, 치료를 필요로 하는 개체에 따라 달라지며, 이는 당업자에 의해 통상적으로 결정될 수 있다. 비제한적 예로서, 본 발명에 의한 조성물의 인체에 대한 투여량은 환자의 나이, 몸무게, 성별, 투여 형태, 건강 상태 및 질환 정도에 따라 달라질 수 있으며, 몸무게가 70 ㎏인 성인 환자를 기준으로 할 때, 일반적으로는 0.01 ~ 1000 ㎎/일, 바람직하게는 1 ~ 500 ㎎/일이며, 일정시간 간격으로 1일 1회 내지 수회에 분할 투여할 수도 있다. Dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Daily dosage refers to the amount of therapeutic substance of the invention sufficient for treatment for a disease state alleviated by administration to a subject in need thereof. Effective amounts of therapeutic agents depend on the particular compound, disease state and severity thereof, and on the individual in need thereof, and can be routinely determined by one skilled in the art. By way of non-limiting example, the dosage of the composition according to the present invention to the human body may vary depending on the age, weight, sex, dosage form, health condition and degree of disease of the patient and may be based on an adult patient weighing 70 kg. At this time, it is generally 0.01 to 1000 mg / day, preferably 1 to 500 mg / day, and may be dividedly administered once to several times a day at regular time intervals.
본 발명의 다른 구현예에 따르면, 본 발명의 조성물은 화장료 조성물로 제조될 수 있다. 본 발명의 화장료 조성물에 포함되는 성분은 유효 성분으로서의 상기 세포밖 소포체를 함유하는 것 이외에, 화장료 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제, 및 담체를 포함할 수 있다. According to another embodiment of the present invention, the composition of the present invention may be prepared as a cosmetic composition. Ingredients included in the cosmetic composition of the present invention, in addition to containing the extracellular vesicles as an active ingredient, include components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Conventional adjuvants such as, and carriers.
본 발명의 화장료 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 영양 크림, 수렴 화장수, 유연 화장수, 로션, 에센스, 영양젤 또는 마사지 크림의 제형으로 제조될 수 있다. The cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and sprays, and the like, but are not limited thereto. More specifically, it may be prepared in the form of a nourishing cream, astringent lotion, a flexible lotion, a lotion, an essence, a nourishing gel or a massage cream.
본 발명의 또 다른 구현예에 따르면, 본 발명의 조성물은 식품 조성물로 제조될 수 있다. 본 발명의 조성물이 식품 조성물로 제조되는 경우, 유효성분으로서 상기 세포밖 소포체를 함유할 뿐만 아니라, 식품 제조시에 통상적으로 첨가되는 성분을 포함하며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제를 포함할 수 있다. 예컨대, 본 발명의 식품 조성물이 드링크제로 제조되는 경우에는 본 발명의 세포밖 소포체 이외에 구연산, 액상과당, 설탕, 포도당, 초산, 사과산, 과즙 등을 추가로 포함시킬 수 있다. According to another embodiment of the present invention, the composition of the present invention may be prepared as a food composition. When the composition of the present invention is prepared as a food composition, not only contains the extracellular vesicles as an active ingredient, but also includes components commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, Flavoring and flavoring agents may be included. For example, when the food composition of the present invention is prepared with a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and the like may be further included in addition to the extracellular vesicles of the present invention.
본 발명에서 "염증성 질환의 치료 또는 예방"이란, 염증성 질환의 경감, 완화 및 증상의 개선을 포함하며, 또한 염증성 질환이 걸릴 가능성을 경감하는 것을 포함하는 의미이다. In the present invention, "treatment or prevention of an inflammatory disease" is meant to include the alleviation, alleviation and improvement of symptoms of an inflammatory disease, and also to reduce the possibility of developing an inflammatory disease.
본 발명에서 "염증성 질환"이란, 포유동물 체내의 염증 반응에 의하여 유발되는 질환을 의미하며, 대표적인 예로는, 천식, 만성폐쇄성폐질환, 비염 등의 호흡기질환; 아토피피부염, 건선, 접촉피부염 등의 피부질환; 위염, 소화기궤양, 염증성장염 등의 소화기질환; 패혈증, 동맥경화증, 관절염, 뇌질환 등의 전신질환 및 이의 합병증 등을 포함한다. 또한, 상기 염증성 질환은 일반적인 염증성 질환 이외에도, 염증 반응과 관련된 암을 포함하는 의미로 사용되며, 예컨대, 폐암, 위암, 대장암 등을 포함한다. In the present invention, "inflammatory disease" refers to a disease caused by an inflammatory response in a mammalian body, and representative examples thereof include respiratory diseases such as asthma, chronic obstructive pulmonary disease and rhinitis; Skin diseases such as atopic dermatitis, psoriasis and contact dermatitis; Gastrointestinal diseases such as gastritis, peptic ulcer, and inflammatory bowel disease; Systemic diseases such as sepsis, arteriosclerosis, arthritis, and brain diseases, and complications thereof. In addition, the inflammatory disease is used in the sense including cancer associated with the inflammatory response, in addition to the general inflammatory disease, and includes, for example, lung cancer, stomach cancer, colon cancer and the like.
본 발명의 조성물은 포유동물의 염증세포에서 염증성 매개체의 분비를 억제하거나 면역관용을 유도함으로써, 염증성 질환의 치료 또는 예방이 가능하다. 특히, 본 발명의 포유동물의 체내에서 분리한 세포밖 소포체가 염증성 질환 및/또는 암의 발생과 관련된 중요한 바이오마커인 IL-6의 분비를 억제할 뿐만 아니라 병원성 소포체에 대한 특이 면역관용을 유도하는 것이 가능하기 때문에, 본 발명의 조성물은 IL-6를 매개로 하는 Th-17 염증성 질환 및 IL-6 또는 Th-17 염증 반응에 의한 암을 치료 또는 예방하는 것이 가능하다. The composition of the present invention can treat or prevent inflammatory diseases by inhibiting the secretion of inflammatory mediators or inducing immune tolerance in inflammatory cells of mammals. In particular, the extracellular vesicles isolated from the body of the mammal of the present invention not only inhibit the secretion of IL-6, an important biomarker associated with the development of inflammatory diseases and / or cancers, but also induce specific immunotolerance to pathogenic vesicles. Since it is possible, the composition of the present invention is capable of treating or preventing IL-mediated Th-17 inflammatory disease and cancer caused by IL-6 or Th-17 inflammatory response.
또한, 본 발명은 포유동물의 체내에서 유래된 세포밖 소포체를 이용하여 염증성 질환을 진단하는 방법을 제공한다. The present invention also provides a method for diagnosing an inflammatory disease using extracellular vesicles derived from the body of a mammal.
염증성 질환에 걸린 포유동물의 체내로부터 분리된 세포밖 소포체는 염증세포의 염증성 매개체의 분비를 유발시킬 수 있다. 따라서, 상기 진단 방법은, 포유동물의 체내(분비물 포함)로부터 세포밖 소포체를 분리하는 단계; 상기 분리된 세포밖 소포체를 염증세포에 처리하여 배양하는 단계; 및 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계를 포함할 수 있다. Extracellular vesicles isolated from the body of a mammal with an inflammatory disease can cause the secretion of inflammatory mediators of inflammatory cells. Thus, the diagnostic method comprises the steps of: separating the extracellular vesicles from the body of the mammal (including secretions); Culturing the isolated extracellular vesicles on inflammatory cells; And measuring the level of an inflammatory mediator in said cell culture.
상기 세포밖 소포체의 분리 방법은 전술한 포유동물의 체내에서 세포밖 소포체를 분리하는 방법과 동일한 방법으로 분리가 가능하며, 세포밖 소포체를 순수하게 분리할 수 있다면 분리 방법은 특별히 제한되지 않는다. The separation method of the extracellular vesicles can be separated by the same method as the method of separating the extracellular vesicles in the body of the mammal described above, and the separation method is not particularly limited as long as the extracellular vesicles can be separated purely.
상기 분리된 세포밖 소포체를 체외의 염증세포에 투여하여 함께 배양하고, 상기 염증세포에서 분비되는 염증성 매개체의 종류 및 수준을 측정하여, 염증성 질환의 원인물질 혹은 질환의 발생 또는 진행을 예측하는 것이 가능하다. The isolated extracellular vesicles are administered to inflammatory cells in vitro and cultured together, and by measuring the type and level of the inflammatory mediator secreted from the inflammatory cells, it is possible to predict the cause or progression of the causative agent or disease of the inflammatory disease. Do.
예컨대, 상기 분리된 세포밖 소포체를 이용하여 원인물질을 진단하기 위하여소포체 내 유전물질 혹은 단백질을 측정함으로서 염증성 질환의 원인물질을 진단하는 것이 가능하다. 또한, 상기 분리된 세포밖 소포체를 대식세포 배양액 중에 첨가하여 함께 배양하고, 상기 대식세포가 분비하는 IL-6의 수준을 측정함으로써 염증성 질환의 발생 여부 및 진행 정도를 진단할 수 있다. 또한, 상기 분리된 세포밖 소포체를 실험 동물에 직접 투여하여, 실험 동물의 체내에 생성되는 염증성 매개체(예컨대, IL-6)의 수준을 측정함으로써, 세포밖 소포체를 분리한 대상 개체의 염증성 질환의 발생 여부 및 진행 정도를 예측하는 것이 가능하다. For example, it is possible to diagnose the causative agent of an inflammatory disease by measuring the genetic material or protein in the endoplasmic reticulum to diagnose the causative agent using the isolated extracellular vesicles. In addition, the isolated extracellular vesicles are added to the macrophage culture medium and cultured together, and the occurrence and progression of the inflammatory disease can be diagnosed by measuring the level of IL-6 secreted by the macrophages. In addition, by administering the isolated extracellular vesicles directly to the experimental animals, by measuring the level of inflammatory mediators (eg, IL-6) produced in the body of the experimental animals, It is possible to predict the occurrence and progression.
또한, 본 발명은 위에서 분리한 H. pylori 를 체외에서 배양하고, 그 배양액에서 소포체를 분리한 후, 소포체 내 유전물질, 단백질 분석, 또는 소포체 특이 면역반응을 측정함으로써 염증성 질환 및 암의 원인물질로서 H. pylori 유래 세포밖 소포체를 진단할 수 있는 방법을 제공한다. In addition, the present invention is cultured in vitro H. pylori in vitro, the vesicles are isolated from the culture medium, and then measured as genetic material, protein analysis, or vesicle-specific immune response in the endoplasmic reticulum as a causative agent of inflammatory diseases and cancer A method for diagnosing H. pylori- derived extracellular vesicles is provided.
본 발명자들은 소화성궤양 환자에서 분리한 H. pylori 를 체외에서 배양하고, 그 배양액에서 분리한 세포밖 소포체가 위염 혹은 위암 환자에서 분리한 H. pylori 유래 세포밖 소포체에 비하여 IL-6 분비를 현저히 증가시킴을 확인하였다. 이는 소화성궤양환자에서 분리한 H. pylori 유래 세포밖 소포체가 위염 혹은 위암 환자에서 분리한 H. pylori 유래 세포밖 소포체에 비하여 염증에 따른 조직손상을 일으키는 활성이 현저히 높음을 의미하며, 궁극적으로, 세포밖 소포체 내 유전물질과 단백질 분석, 혹은 세포밖 소포체에 의한 면역반응을 측정함으로서 특정 H. pylori 유래 세포밖 소포체를 진단하는 것을 가능하게 한다. The present inventors have significantly increased the IL-6 secreted is compared with the extracellular vesicles separated from the culture medium by culturing H. pylori isolated from peptic ulcer patients in vitro, and in or out of gastritis H. pylori-derived cells isolated from patients with gastric cancer ER Confirmed. This means that H. pylori- derived extracellular vesicles isolated from peptic ulcer patients have significantly higher activity causing tissue damage due to inflammation compared to H. pylori- derived extracellular vesicles isolated from gastritis or gastric cancer patients. Genetic and protein analysis in extracellular vesicles, or by measuring the immune response by extracellular vesicles, makes it possible to diagnose specific H. pylori-derived extracellular vesicles.
따라서, 상기 진단 방법은, 위염, 소화성궤양, 위암 환자에서 H. pylori 를 분리하여 배양하는 단계; H. pylori 배양액에서 세포밖 소포체를 분리하는 단계; 상기 분리된 세포밖 소포체를 염증세포에 처리하여 배양하는 단계; 및 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계 등을 포함할 수 있다. 또한 상기 방법은 H. pylori 배양액에서 분리한 소포체에서 유전물질을 측정하는 단계, H. pylori 배양액에서 분리한 소포체에서 단백질을 측정하는 단계, 또는 H. pylori 배양액에서 분리한 소포체에 대한 면역반응을 측정하는 단계 등을 포함할 수 있다. Therefore, the diagnostic method comprises the steps of separating and culturing H. pylori in patients with gastritis, peptic ulcer, gastric cancer; Isolating extracellular vesicles from H. pylori culture; Culturing the isolated extracellular vesicles on inflammatory cells; And measuring the level of an inflammatory mediator in said cell culture. In addition, the method measures the immune response to the endoplasmic reticulum removed in step, or H. pylori culture media for measuring the protein in the endoplasmic reticulum in a separate step of measuring the dielectric material, H. pylori culture on the endoplasmic reticulum isolated from H. pylori culture medium It may include the step and the like.
이하, 본 발명의 이해를 돕기 위하여 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다. Hereinafter, examples are provided to help understand the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예 1. 환자의 위세척액에서 세포밖 소포체 분리 및 구조 분석 Example 1.Isolation and Structure Analysis of Extracellular Vesicles from Gastric Lavage in Patients
소화성궤양 환자의 위세척액에서 세포밖 소포체를 분리하기 위해서 10ml의 위세척액을 3200 x g에서 15 분간 원심분리를 하여 불순물 또는 세포들을 제거한 뒤 상층액을 얻었다. 얻어진 상측액에서 세포밖 소포체를 분리하기 위해 200,000 x g에서 2시간 초원심분리를 수행하였다. 이후 생리식염수 400μl로 풀어내어 밀도 기울기 원심분리를 수행하였다. 밀도 기울기 원심분리를 수행하기 위해서, 세포밖 소포체와 50 % 농도의 옵티프렙 솔루션을 섞어 30% 농도로 4.8 ml을 만들었다. 이것을 10ml 초원심분리 튜브에 넣고 그 위에 20 % 옵티프렙 솔루션 3 ml, 5 % 옵티프렙 솔루션 2.5ml을 채워 넣고 200,000 g x에서 2시간 초원심분리를 하여 밀도에 따라 세포밖 소포체를 분리하였다. F3 (1.7㎍/ml), F6 (0.56㎍/ml ) 의 두 부분에서 눈으로 밴드를 확인 할 수 있었으며 각각의 부분에서(F1-10) 초원심분리를 통해 침전물을 분리하였다 (도 1 참조). In order to separate extracellular vesicles from gastric lavage fluid of patients with peptic ulcer, 10 ml of gastric lavage fluid was centrifuged at 3200 x g for 15 minutes to remove impurities or cells and supernatant was obtained. In order to separate the extracellular vesicles from the obtained supernatant, ultracentrifugation was performed for 2 hours at 200,000 × g. After releasing with 400μl saline and performing a density gradient centrifugation. To perform density gradient centrifugation, extracellular vesicles and 50% concentrations of Optiprep solution were mixed to make 4.8 ml at 30% concentrations. This was placed in a 10 ml ultracentrifuge tube, filled with 3 ml of 20% Optiprep solution and 2.5ml of 5% Optiprep solution, followed by ultracentrifugation at 200,000 g x for 2 hours to separate extracellular vesicles according to density. The bands were visually identified in two parts of F3 (1.7 μg / ml) and F6 (0.56 μg / ml), and the precipitates were separated by ultracentrifugation in each part (F1-10) (see FIG. 1). .
분리된 각 부분의 침전물을 글로방전 탄소 코팅 구리 그리드 (glow-discharged carbon-coated copper grid)에서 3분간 흡착시킨 후 증류수로 세척하였다. 2% 우라닐아세테이트(uranylacetate)로 1분간 염색 후 JEM101 (Joel, Japan) 전자현미경으로 관찰하였다. 도 2 및 도 3은 각각 F3 및 F6 부분에 존재하는 세포밖 소포체의 전자현미경 사진으로, F3, F6 두 부분에서만 약 100-200 nm의 세포밖 소포체를 가지고 있는 것으로 확인되었다. The precipitates of the separated portions were adsorbed on a glow-discharged carbon-coated copper grid for 3 minutes and washed with distilled water. After staining with 2% uranilacetate for 1 minute, it was observed with a JEM101 (Joel, Japan) electron microscope. 2 and 3 are electron micrographs of the extracellular vesicles present in the F3 and F6 portions, respectively, and were found to have extracellular vesicles of about 100-200 nm in only the F3 and F6 portions.
이에 더하여, 환자의 위 세척액에서 분리된 세포밖 소포체를 밀도 기울기 원심분리에 의해 분리한 뒤 각각의 부분에서 단백질의 구성 정도를 확인하기 위해 분리된 각 부분의 단백질 (10㎍)을 8 % poly-acryl amide gel을 이용하여 단백질의 분자량에 따라 분리하여 그 결과를 도 4에 나타내었다. 도 4에 나타난 바와 같이, F1-5에서는 단백질 성분을 확인할 수 없었으나, F6-10에서는 단백질 성분을 확인 할 수 있었다. In addition, the extracellular vesicles isolated from the gastric lavage of the patient were separated by density gradient centrifugation, and then the separated protein (10 μg) was separated by 8% poly- The acryl amide gel was used to separate the proteins according to the molecular weight. The results are shown in FIG. 4. As shown in FIG. 4, protein components could not be identified in F1-5, but protein components could be identified in F6-10.
실시예 2. 위세척액에서 분리한 세포밖 소포체에 의한 Example 2. By extracellular vesicles isolated from gastric lavage fluid
in vitroin vitro
염증성 매개체 분비 및 polymyxin B 혹은 열 처리의 효과 Inflammatory mediator secretion and effects of polymyxin B or heat treatment
소화성궤양 환자의 위세척액에서 분리한 세포밖 소포체가 염증성 매개체를 유도하는지 확인하기 위하여 마우스의 대식세포주인 RAW 264.7를 이용하여 in vitro에서 염증성 사이토카인 TNF-α의 발현 정도를 확인 하였다. To determine whether extracellular vesicles isolated from gastric lavage in patients with peptic ulcer induced inflammatory mediators, the expression level of inflammatory cytokine TNF-α was determined in vitro using RAW 264.7, a mouse macrophage.
구체적으로, 24웰(well)의 세포배양 플레이트에 3 x 106의 세포를 분주하고 24시간 동안 배양 하였다. 배양 후 대조군으로 생리식염수, 실험군으로 F3부분과 F6부분을 농도 의존적으로 처리한 후 6시간 뒤에 세포배양액에서 염증성 매개체인 TNF-α를 ELISA법으로 정량하고 그 결과를 도 5에 나타내었다. Specifically, 3 x 10 6 cells were dispensed into 24 well cell culture plates and incubated for 24 hours. After cultivation of physiological saline as a control group, and F3 and F6 sections as a control group, the inflammatory mediator TNF-α was quantified in the cell culture solution after 6 hours by ELISA and the results are shown in FIG. 5.
도 5에 나타난 바와 같이, F3, F6 두 부분 모두 농도의존적으로 TNF-α의 발현을 유도하는 것을 확인하였다. As shown in FIG. 5, it was confirmed that both parts of F3 and F6 induced TNF-α expression in a concentration-dependent manner.
이에 더하여, F3와 F6 두 부분이 염증성 매개체를 유도하는 기전을 평가 하기 위하여 그람 음성균 유래 세포밖 소포체에 존재하는 lipopolysaccharide에 결합하여 활성을 억제하는 polymyxin B(10㎍/ml), 혹은 단백질의 변성을 초래하는 열(99 ℃, 20 min)을 F3와 F6의 두 부분에 처리하여 상기 방법으로 염증성 매개체 TNF-α의 발현을 확인하였다. In addition, polymyxin B (10 µg / ml), or protein denaturation, that binds to lipopolysaccharide present in Gram-negative bacteria-derived extracellular vesicles to inhibit the activity of F3 and F6 in order to assess the mechanism of inducing inflammatory mediators The resulting heat (99 ° C., 20 min) was treated in two parts F3 and F6 to confirm the expression of the inflammatory mediator TNF-α in this manner.
그 결과 F3, F6 두 부분에서, 염증성 매개체 TNF-α의 발현은 polymyxin B 또는 열에 영향을 받지 않는 것으로 확인된 반면 (도 6 및 도 7 참조), IL-6의 분비는 F6 부분에 존재하는 세포밖 소포체에 의해 현저히 증가하고, 이는 소포체에 대한 polymyxin B 혹은 열 처리에 의해 현저히 억제되는 것으로 나타났다 (도 7 참조). As a result, in both F3 and F6, the expression of inflammatory mediator TNF-α was found not to be affected by polymyxin B or heat (see FIGS. 6 and 7), whereas IL-6 secretion was present in the F6 cell. It was significantly increased by the outer endoplasmic reticulum, which was shown to be significantly inhibited by polymyxin B or heat treatment of the endoplasmic reticulum (see FIG. 7).
상기 결과는 F6 부분에 존재하는 세포밖 소포체가 IL-6 분비를 유도하고, 이는 그람 음성균에서 유래되며, IL-6 분비를 유도함에 있어 소포체 내 단백질 성분이 중요한 역할을 담당함을 의미한다. The results indicate that extracellular vesicles present in the F6 moiety induce IL-6 secretion, which is derived from Gram-negative bacteria, and that protein components in the endoplasmic reticulum play an important role in inducing IL-6 secretion.
실시예 3. 위염, 소화성궤양, 위암 환자에서 분리한 Example 3 Isolation from Patients with Gastritis, Peptic Ulcer and Gastric Cancer
H. pyloriH. pylori
배양액에서 세포밖 소포체 분리 Isolation of extracellular vesicles from culture
위에 서식하는 그람 음성균인 H. pylori가 체외에서 배양하였을 때, 세포밖 소포체를 분비하는지를 평가하였다. H. pylori , a gram-negative bacterium in the stomach, was cultured in vitro to evaluate the secretion of extracellular vesicles.
구체적으로, 위염, 소화성궤양, 위암 환자 각각 2명의 위세척액에서 H. pylori를 분리하였다. 분리된 각각의 H.pylori균을 흡광도 1.0이 될 때까지 배양한 다음, 1L의 배양액에서 세포밖 소포체를 분리하기 위해 세포 배양액을 3,000 x g 에서 20분간 원심분리하였다. H. pylori 및 불순물을 제거한 후, 상층액을 0.22㎛의 필터를 이용하여 제거되지 못한 불순물을 다시 제거하였다. 필터 된 상층액을 100,000 x g 에서 2시간 동안 초원심분리를 하였다. 가라앉은 세포밖 소포체는 200μl의 생리식염수로 풀어내고, 실시예 1의 방법과 마찬가지로 전자 현미경을 통해서 세포밖 소포체의 모양 및 크기를 확인하였다. Specifically, H. pylori was isolated from gastric lavage fluid of two patients with gastritis, peptic ulcer and gastric cancer. Each of the isolated H. pylori bacteria was incubated until the absorbance was 1.0, and then the cell culture was centrifuged at 3,000 xg for 20 minutes to separate the extracellular vesicles from 1 L of culture. After removing H. pylori and impurities, the supernatant was removed again using a 0.22 μm filter. The filtered supernatant was ultracentrifuged at 100,000 xg for 2 hours. The sunk extracellular vesicles were released with 200 μl of physiological saline, and the shape and size of the extracellular vesicles were confirmed by electron microscopy in the same manner as in Example 1.
도 8에 나타난 바와 같이, 위염, 위궤양, 및 위암환자에서 분리된 H. pylori 모두가 세포밖 소포체를 분비함을 알 수 있었다. As shown in FIG. 8, all of H. pylori isolated from gastritis, gastric ulcer, and gastric cancer patients secrete extracellular vesicles.
실시예 4. 위염, 위궤양, 위암 환자에서 분리한 H. pylori 유래 세포밖 소포체에 의한 IL-6 분비능 Example 4 IL-6 Secretion by H. pylori-derived Extracellular Vesicles Isolated from Patients with Gastritis, Gastric Ulcer and Gastric Cancer
위염, 위궤양, 위암 환자에서 분리한 H.pylori 유래 세포밖 소포체에 의한 염증성 매개체 IL-6의 분비능을 측정하기 위해서 실시예 2의 방법으로 실험을 진행하였다. In order to measure the secretion ability of the inflammatory mediator IL-6 by H.pylori- derived extracellular vesicles isolated from patients with gastritis, gastric ulcer and gastric cancer, the experiment of Example 2 was conducted.
구체적으로, 24웰(well)의 세포배양 플레이트에 3 x 106의 RAW 264.7 세포를 분주하고 24시간 동안 배양 하였다. 배양 후 대조군으로 생리식염수, 실험군으로 각 질병에서 분리한 H. pylori에서 유래한 세포밖 소포체를 농도 의존적으로 처리한 후 6시간 뒤에 세포배양액에서 염증성 매개체인 IL-6를 ELISA법으로 정량하고 그 결과를 도 9에 나타내었다. Specifically, 3 x 10 6 RAW 264.7 cells were dispensed into 24 well cell culture plates and incubated for 24 hours. After incubation with normal saline control group, the IL-6 Determination of inflammatory mediators out of the cells derived from the endoplasmic reticulum H. pylori separated from each disease in the cell culture medium after 6 hours after treatment with a concentration dependent manner as experiment by ELISA and the results Is shown in FIG. 9.
도 9에 나타난 바와 같이, 100 ng/ml의 세포밖 소포체를 처리했을 때, 위궤양 환자에서 분리한 H. pylori 유래세포밖 소포체만이 IL-6의 분비를 유도하였고, 1㎛/ml 소포체의 농도로 처리한 경우, 위암환자에서 분리한 H. pylori 유래 세포밖 소포체도 IL-6의 분비를 유도하였으나, 분비 정도가 소화성궤양환자에서 분리한 H. pylori 유래 세포밖 소포체보다 현저히 낮음을 알 수 있다. As shown in Figure 9, when treated with 100 ng / ml extracellular vesicles, only H. pylori- derived extracellular vesicles isolated from patients with gastric ulcer induced IL-6 secretion, concentration of 1㎛ / ml vesicles When treated with, H. pylori-derived extracellular vesicles isolated from gastric cancer patients induced IL-6 secretion, but their secretion was significantly lower than that of H. pylori- derived extracellular vesicles isolated from peptic ulcer patients. .
상기 결과는 위염 및 위암 환자에서 분리한 H. pylori 유래 세포밖 소포체와 위궤양 환자에서 분리한 H. pylori 유래 세포밖 소포체의 활성이 전혀 다름을 의미한다. 또한 H. pylori 감염에 의한 위질환의 발생에 H. pylori 유래 세포밖 소포체가 중요하고, 궁극적으로 소포체 내 유전물질, 단백질, 혹은 소포체 특이 면역반응을 측정함으로서 위질환의 원인물질 혹은 질병의 발생 혹은 경과를 진단할 수 있음을 의미한다. The results indicate a difference at all, the activity of gastritis and outside derived from H. pylori was separated H. pylori-derived cells outside the vesicles and ulcers patient Isolated from patients with gastric cancer cell endoplasmic reticulum. In addition, H. pylori- derived extracellular vesicles are important for the development of gastric disease caused by H. pylori infection, and ultimately, by measuring the genetic material, protein, or vesicle-specific immune response in the endoplasmic reticulum, It means that the progress can be diagnosed.
실시예 5. 정상 마우스 소장내 분비물에서 세포밖 소포체 분리Example 5. Isolation of Extracellular Vesicles from Normal Mouse Intestinal Secretions
마우스 소장내 분비물에서 세포밖 소포체를 분리하기 위해 6 주령의 암컷(female) BALB/c 마우스에서 위장 바로 아래 십이지장부터 공장을 포함 회장까지를 분리하였다. To isolate extracellular vesicles from mouse intra-intestinal secretions, six-week old female BALB / c mice were isolated from the duodenum just below the stomach to the jejunum, including the jejunum.
도 10a에 나타낸 프로토콜에 따라 분리된 소장을 3등분 하고 펼쳐 낸 뒤 30 ml의 생리식염수가 담겨있는 50 ml 튜브에 1 cm 크기로 잘라 넣었다. 준비된 튜브는 교반기를 이용하여 10 초간 5번 세척하였다. 소장은 채반에 걸러내고 걸러진 소장 세척액은 500 x g에서 20 분, 3000 x g에서 20 분, 10,000 x g에서 2 분 동안 원심분리를 하여 불순물, 소장세포 및 세균을 제거하였다. According to the protocol shown in FIG. 10a, the small intestine separated into 3 parts was expanded and then cut into 1 cm size in a 50 ml tube containing 30 ml of physiological saline. The prepared tube was washed five times for 10 seconds using a stirrer. The small intestine was filtered out and the filtered small intestinal lavage was centrifuged at 500 x g for 20 minutes, 3000 x g for 20 minutes, and 10,000 x g for 2 minutes to remove impurities, small cells and bacteria.
불순물이 제거된 상층액을 수크로스 쿠션(sucrose cushion)을 이용해 100,000 x g 에서 2시간 동안 초고속 원심분리를 이용해 세포밖 소포체 층을 분리하였다. 분리된 세포밖 소포체를 전자현미경(Transmission electron microscope, TEM)을 통해 관찰한 결과 50 nm에서 100 nm 사이의 소포체가 존재하는 것을 확인 하였다 (도 10b 참조). The supernatant from which impurities were removed was separated from the extracellular vesicle layer using ultra-high speed centrifugation at 100,000 × g for 2 hours using a sucrose cushion. The isolated extracellular vesicles were observed through a transmission electron microscope (TEM) to confirm that vesicles were present between 50 nm and 100 nm (see FIG. 10B).
또한, 브래드포드(Bradford) 단백질 정량법을 이용하여 세포밖 소포체의 양을 확인한 결과 마우스 1마리당 0.81 μg의 세포밖 소포체를 획득하였음을 확인하였다 (도 10c 참조). In addition, it was confirmed that the amount of extracellular vesicles by using Bradford protein quantitative assay, 0.81 μg extracellular vesicles per mouse was obtained (see Fig. 10c).
이에 더하여, 획득한 세포밖 소포체에 세균의 오염여부를 확인하기 위하여 세포밖 소포체를 이용하여 LB와 MRS 배지에 각각 세균배양을 하였으나 세균의 배양이 일어나지 않은 것을 통해서 세균의 오염이 없었음을 확인하였다 (도 10d 참조).In addition, bacteria were cultured in LB and MRS medium using extracellular vesicles in order to confirm whether the bacteria were contaminated in the obtained extracellular vesicles, but there was no bacterial contamination through the cultivation of bacteria. (See FIG. 10D).
실시예 6. 정상 상태의 소장에서 분리한 세포밖 소포체의 염증유발 효과Example 6 Inflammatory Effect of Extracellular Vesicles Isolated from Normal Small Intestine
정상 마우스의 소장에서 분리해낸 세포밖 소포체의 염증 유도 여부를 확인하기 위해 골수 유래의 대식 세포인 Raw 264.7 세포에 정상 마우스의 소장에서 분리해낸 세포밖 소포체를 농도 의존적으로 처리하여 염증성 매개체인 IL-6 및 TNF-α의 분비를 확인하였다. To determine whether the extracellular vesicles isolated from the small intestine of normal mice induce inflammation, a concentration-dependent treatment of the extracellular vesicles isolated from the small intestine of bone marrow-derived macrophage-derived raw 264.7 cells was used to induce inflammation. And secretion of TNF-α was confirmed.
구체적으로, 24 웰(Well)의 세포배양 플레이트에 3 x 105의 세포를 분주하고 24 시간 동안 배양하였다. 배양 후 대조군으로 생리식염수와 내독소(lipopolysacharide) (10 ng/ml)를 처리하고 실험군으로 정상 마우스의 소장에서 분리해낸 세포밖 소포체를 각각 1, 5, 10 μg/ml의 농도로 처리하였다. 세포밖 소포체 처리 후 6 시간 뒤에 세포배양액을 획득하여, 세포배양에서 염증성 매개체인 IL-6와 TNF-α를 ELISA 방법으로 측정하고 그 결과를 도 11에 나타내었다. Specifically, 3 x 10 5 cells were dispensed into 24 well cell culture plates and incubated for 24 hours. After incubation, physiological saline and endotoxin (lipopolysacharide) (10 ng / ml) were treated as a control group, and the extracellular vesicles isolated from the small intestine of normal mice were treated with concentrations of 1, 5 and 10 μg / ml, respectively. After 6 hours after treatment with extracellular vesicles, cell culture fluids were obtained, and IL-6 and TNF-α, which are inflammatory mediators, were measured in the cell culture by ELISA, and the results are shown in FIG. 11.
도 11에 나타난 바와 같이, 양성 대조군(LPS)이 염증성 매개체 IL-6와 TNF-α의 발현을 유도하는 반면, 실험군인 세포밖 소포체는 농도에 관계없이 IL-6의 발현을 유도하지 않는 것을 확인 할 수 있었으며, TNF-α의 경우 소량 유도함을 확인하였으나 양성 실험군에 비해 미미한 정도임을 확인하였다. 상기 결과는 정상 마우스의 소장에서 분리한 세포밖 소포체는 염증을 유도하지 않음을 의미한다. As shown in FIG. 11, the positive control group (LPS) induced the expression of the inflammatory mediators IL-6 and TNF-α, whereas the experimental group of the extracellular vesicles did not induce the expression of IL-6 regardless of the concentration. In the case of TNF-α it was confirmed that the induction of a small amount, but compared to the positive test group was found to be insignificant. The results indicate that extracellular vesicles isolated from the small intestine of normal mice do not induce inflammation.
실시예 7. 염증성 장질환 상태의 소장에서 분리한 세포밖 소포체에 의한 염증성 매개체 분비 효과Example 7 Inflammatory Media Secretion Effect by Extracellular Vesicles Isolated from Small Intestine with Inflammatory Bowel Disease
염증성 장질환 상태의 마우스의 소장에서 분리해낸 세포밖 소포체의 염증성 매개체의 분비를 확인하기 위하여 덱스트란설페이트소듐(Dextran sulfate sodium, DSS)을 이용한 장질환 마우스 모델을 이용하였다. In order to confirm the secretion of inflammatory mediators of extracellular vesicles isolated from the small intestine of inflammatory bowel disease, enteric disease mouse model using dextran sulfate sodium (DSS) was used.
구체적으로, 장질환을 유도하기 위해 마우스의 배양에 1% 덱스트란설페이트소듐을 녹인 물을 5 일간 이용하였다. 5 일 후 마우스를 해부하여 실시예 1의 방법에 따라 소장에서 소포밖 세포체를 분리했다. 분리된 염증성 장질환 상태의 마우스 유래의 세포밖 소포체의 염증성 매개체 분비여부를 확인하기 위해 실시예 2의 방법을 따라 평가하였다. Specifically, water in which 1% dextransulfate sodium was dissolved in the culture of mice was used for 5 days to induce intestinal diseases. After 5 days, the mice were dissected to separate extracellular vesicles from the small intestine according to the method of Example 1. In order to confirm the secretion of inflammatory mediators of the extracellular vesicles derived from the mouse in the condition of isolated inflammatory bowel disease was evaluated according to the method of Example 2.
이 결과 염증성 장질환 상태의 세포밖 소포체의 농도 의존적으로 염증성 매개체인 IL-6와 TNF-α의 발현이 유도됨을 확인 할 수 있었다. 이를 통해 염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체가 염증을 유도함을 확인하였다 (도 12 참조). As a result, it was confirmed that the expression of inflammatory mediators IL-6 and TNF-α are induced depending on the concentration of extracellular vesicles in inflammatory bowel disease. This confirmed that the extracellular vesicles isolated from the small intestine of inflammatory bowel disease mice induce inflammation (see FIG. 12).
실시예 8. 정상 및 염증성 장질환 상태의 소장에서 분리한 세포밖 소포체에 함유된 단백질 양상Example 8. Protein Aspects Contained in Extracellular Vesicles Isolated from Small Intestine with Normal and Inflammatory Bowel Disease
정상 및 염증성 장질환 마우스의 소장에서 분리해낸 세포밖 소포체에 함유된 단백질의 발현 양상을 확인하기 위하여 분리된 세포밖 소포체 30 μg에 함유된 단백질을 SDS-PAGE(sodium dodecyl sulfate-polyacryl amide gel electrophoresis)법을 이용하여 분자량을 기준으로 분리하였다. Proteins contained in 30 μg of the extracellular vesicles isolated from the small intestine of normal and inflammatory bowel disease mice were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacryl amide gel electrophoresis). Separation was based on molecular weight using the method.
분리된 단백질을 쿠마시블루(coomasie brilliant blue)를 이용하여 염색 한 뒤 정상(siEVs) 및 염증성 장질환 마우스(DSS siEVs)의 소장에서 분리해낸 세포밖 소포체에 함유된 단백질의 양상을 비교하고 그 결과를 도 13에 나타내었다. The isolated proteins were stained using coomasie brilliant blue, and then compared to those of proteins contained in extracellular vesicles isolated from the small intestine of normal (siEVs) and inflammatory bowel disease mice (DSS siEVs). Is shown in FIG. 13.
도 13에 나타난 바와 같이, 단백질의 동정을 할 수는 없었으나 정상 및 염증성 장질환 마우스에서 유래된 세포밖 소포체에 함유된 단백질의 양상은 큰 차이가 있음을 확인 할 수 있었다. As shown in FIG. 13, the protein could not be identified, but the protein contained in the extracellular vesicles derived from normal and inflammatory bowel disease mice was confirmed to have a big difference.
실시예 9. 염증성 매개체 분비에 미치는 정상 및 염증성 장질환 상태의 소장에서 분리한 세포밖 소포체의 영향Example 9 Effect of Extracellular Vesicles Isolated from Small Intestine with Normal and Inflammatory Bowel Disease on Inflammatory Mediated Secretion
마우스 장내에는 많은 공생 세균이 살고 있다는 사실에 근거하여, 정상 및 장질환 상태의 마우스 유래의 세포밖 소포체의 염증성 매개체분비에 공생 세균에서 유래하는 내독소(LPS)가 영향을 미치는지 확인하기 위해 LPS의 길항체인 폴리믹신 B(polymixin B) (10 μg/ml)를 이용하여 실시예 2의 방법에 따라 평가하였다. Based on the fact that a large number of commensal bacteria live in the mouse intestine, LPS-derived endotoxin (LPS) from symbiotic bacteria influences the secretion of inflammatory mediators of extracellular vesicles from mice with normal and enteric diseases. Evaluation was carried out according to the method of Example 2 using the antagonist polymixin B (10 μg / ml).
그 결과, 염증성 장질환 마우스 유래의 세포밖 소포체에 의한 염증성 매개체 IL-6의 발현이 LPS의 길항체인 폴리믹신 B에 의해 억제되는 것을 확인하였다. 반면에 염증성 매개체인 TNF-α의 발현은 LPS의 길항체인 폴리믹신 B에 의한 영향이 없음을 확인하였다. 이를 통해 염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체(DSS siEVs)에 의한 IL-6 등의 염증성 매개체의 발현은 공생 그람 음성 세균 유래의 LPS에 의함을 확인 하였다 (도 14 참조). As a result, it was confirmed that the expression of the inflammatory mediator IL-6 by the extracellular vesicles derived from the inflammatory bowel disease mouse was inhibited by polymyxin B, an antagonist of LPS. On the other hand, the expression of inflammatory mediator TNF-α was confirmed that there is no effect by polymyxin B, an antagonist of LPS. Through this, expression of inflammatory mediators such as IL-6 by extracellular vesicles (DSS siEVs) isolated from the small intestine of inflammatory bowel disease mice was confirmed by LPS derived from symbiotic Gram-negative bacteria (see FIG. 14).
실시예 10. 염증성 장질환 상태의 소장에서 분리한 세포밖 소포체에 의한 염증성 매개체 분비에 대한 정상 상태의 소장에서 분리한 세포밖 소포체의 항염증 효과Example 10 Anti-inflammatory Effect of Extracellular Vesicles Isolated from Normal Intestine on Secretion of Inflammatory Mediators by Extracellular Vesicles Isolated from Intestine with Inflammatory Bowel Disease
염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체의 염증성 매개체의 유도에 정상 마우스의 소장에서 분리된 세포밖 소포체에 의한 억제 여부를 확인하기 위해 실시예 2 의 방법에 따라 평가하였다. Induction of inflammatory mediators of extracellular vesicles isolated from the small intestine of inflammatory bowel disease mice was evaluated according to the method of Example 2 to determine whether the extracellular vesicles isolated from the small intestine of normal mice were inhibited.
도 15에 나타난 바와 같이, 염증성 매개체 IL-6의 발현이 염증성 장질환 마우스의 소장에서 분리된 세포밖 소포체 10 μg/ml을 처리한 대조군(DSS siEVs)에 비교해 장질환 마우스의 소장에서 분리된 세포밖 소포체와 정상 마우스의 소장에서 분리된 세포밖 소포체 10 μg/ml을 동시에 처리한 실험군(DSS siEVs + siEVs)에서 억제되는 것을 확인 할 수 있었다. 하지만, 염증성 매개체 TNF-α의 발현에는 변화가 없음을 확인하였다. As shown in Figure 15, the expression of the inflammatory mediator IL-6 cells isolated from the small intestine of intestinal disease mice compared to the control (DSS siEVs) treated with 10 μg / ml of extracellular vesicles isolated from the small intestine of inflammatory bowel disease mice It was confirmed that the experimental group (DSS siEVs + siEVs) simultaneously treated with 10 μg / ml of the extracellular vesicles and the extracellular vesicles isolated from the small intestine of normal mice was confirmed. However, it was confirmed that there is no change in the expression of the inflammatory mediator TNF-α.
상기 결과는 정상 마우스의 소장 유래 세포밖 소포체는 염증성 장질환 마우스 소장 유래 세포밖 소포체에 의한 IL-6와 같은 염증성 매개체의 발현을 길항 할 수 있음을 의미한다. The results indicate that the small intestine-derived extracellular vesicles of normal mice can antagonize the expression of inflammatory mediators such as IL-6 by inflammatory bowel disease mouse small intestine-derived extracellular vesicles.
또한, 정상 마우스의 소장에서 분리한 세포밖 소포체의 염증 매개체 분비에 대한 예방 효과를 평가하기 위하여 골수 유래 대식세포인 RAW 264.7 세포에 정상 상태의 소장에서 분리한 세포밖 소포체를 5 μg/ml의 농도로 각각 1, 2, 3시간 전에 처리를 한 후, 세포 배양액을 제거하고, 새로운 세포 배양액에 염증성 장질환 상태의 소장에서 분리한 세포밖 소포체를 5 μg/ml의 농도로 6시간 동안 처리 후, 세포 배양액에서 염증성 매개체 IL-6, TNF-α의 발현 정도를 확인 하였다. In addition, in order to evaluate the prophylactic effect of inflammatory mediator secretion of extracellular vesicles isolated from the small intestine of normal mice, the concentration of 5 μg / ml of extracellular vesicles isolated from the small intestine in the normal state to RAW 264.7 cells, bone marrow-derived macrophages 1, 2, 3 hours before each treatment, the cell culture medium was removed, and the extracellular vesicles isolated from the small intestine of the inflammatory bowel disease state in a new cell culture for 6 hours at a concentration of 5 μg / ml, The expression level of the inflammatory mediators IL-6 and TNF-α in cell cultures was confirmed.
도 16에 나타난 바와 같이, 염증성 매개체인 IL-6의 발현량이 실험군에서 정상 상태 소장 유래 세포밖 소포체의 처리 시간에 관계없이 대조군에 비해 현저히 감소됨을 확인하였다. 반면에, TNF-α의 발현에는 의미있는 변화가 없는 것으로 확인되었다. As shown in FIG. 16, it was confirmed that the expression level of the inflammatory mediator IL-6 was significantly decreased in the experimental group compared to the control group regardless of the treatment time of the steady-state small intestine-derived extracellular vesicles. On the other hand, there was no significant change in the expression of TNF-α.
상기 결과는 정상 마우스의 소장 유래 세포밖 소포체는 염증성 장질환 마우스 소장 유래 세포밖 소포체에 의한 IL-6와 같은 염증성 매개체의 발현을 억제하는 효과가 있음을 의미한다. The results indicate that the small intestine-derived extracellular vesicles of normal mice have the effect of inhibiting the expression of inflammatory mediators such as IL-6 by inflammatory bowel disease mouse small intestine-derived extracellular vesicles.
실시예 11. 내독소(LPS) 혹은 대장균 유래 세포밖 소포체에 의한 염증성 매개체 분비에 대한 정상 상테의 소장에서 분리한 세포밖 소포체의 항염증 효과Example 11. Anti-inflammatory Effects of Extracellular Vesicles Isolated from Normal Small Intestine on Inflammatory Mediator Secretion by Endotoxin (LPS) or Escherichia Coli-derived Extracellular Vesicles
염증성 장질환의 원인으로 알려져 있는 그람 음성균의 세포벽의 구성물질인 LPS 또는 대장균 유래 세포밖 소포체(E.coli C4 EVs)에 의한 염증성 매개체 분비에 대한 정상 상태의 소장에서 분리한 세포밖 소포체(siEVs)의 항염증 효과를 평가하기 위하여 골수 유래 대식세포 RAW 246.7에 LPS(10 ng/ml) 또는 대장균 유래 세포밖 소포체(100 ng/ml)와 함께 정상 상태의 소장에서 분리한 세포밖 소포체(20 μg/ml)를 6시간 동안 처리한 뒤, 세포 배양액에서 염증성 매개체 IL-6의 발현 정도를 확인 하였다. Extracellular vesicles derived from LPS or Escherichia coli, which are components of the cell wall of Gram-negative bacteria known to cause inflammatory bowel disease (E.coli Anti-inflammatory effects of extracellular vesicles (siEVs) isolated from normal small intestine on inflammatory mediator secretion by C4 EVs) To evaluate, 6 hours of extracellular vesicles (20 μg / ml) isolated from normal small intestine with LPS (10 ng / ml) or E. coli-derived extracellular vesicles (100 ng / ml) in bone marrow-derived macrophages RAW 246.7 After the treatment, the expression level of the inflammatory mediator IL-6 was confirmed in the cell culture.
도 17에 나타난 바와 같이, 대조군에 비교하여 정상 상태의 소장에서 분리한 세포밖 소포체를 같이 처리해준 실험군에 염증성 매개체인 IL-6가 의미 있게 감소하는 것을 알 수 있다. As shown in FIG. 17, compared to the control group, IL-6, an inflammatory mediator, was significantly reduced in the experimental group treated with the extracellular vesicles isolated from the small intestine in the normal state.
상기 결과는 정상 마우스의 소장에 존재하는 세포밖 소포체는 염증성 장질환의 원인으로 알려져 있는 그람 음성균의 세포벽 구성물질인 LPS 또는 대장균 유래 세포밖 소포체에 의한 염증성 매개체의 발현을 억제 할 수 있음을 의미한다. The results indicate that the extracellular vesicles present in the small intestine of normal mice can inhibit the expression of inflammatory mediators by LPS or E. coli-derived extracellular vesicles, which are gram-negative bacterial cell walls known to cause inflammatory bowel disease. .
실시예 12. 소장에서 분리한 세포밖 소포체에 의한 소포체 특이 면역관용 유도Example 12 Induction of Vesicular Specific Immune Tolerance by Extracellular Vesicles Isolated from Small Intestine
마우스 소장 유래 세포밖 소포체에 의한 소포체 특이적 면역관용 여부를 확인해 보기 위하여 5 주령 수컷 C57BL/6 마우스로부터 실시예 5의 방법을 이용하여 소장 유래 세포밖 소포체를 분리하였다. The small intestine-derived extracellular vesicles were isolated from the 5 week old male C57BL / 6 mice using the method of Example 5 to determine whether the vesicle-specific immune tolerance by the mouse small intestine-derived extracellular vesicles.
소포체 10, 50, 100 μg을 각각 생리식염수 200㎕에 풀어내어 서로 다른 마우스에 피하 주사(subcutaneous injection)의 방법을 통해 5일 간격으로 3번 주사하였다. 3번째 주사 후, 마우스에서 피를 뽑아 800 x g에서 10 분 동안 원심분리를 하여 혈청(serum) 부분을 분리하였다. ELISA 플레이트에 웰(well)당 소장 유래 세포밖 소포체 100 ng을 생리식염수 100㎕에 녹여 넣어 붙이고(coating) 24시간 후 ELISA 방법으로 분리된 혈청 내에 소포체 특이적 항체 형성을 평가하였다. 그 결과, 소장 유래 세포밖 소포체에 특이적인 항체가 형성되지 않는 것을 확인하였다 (도 18 참조). 10, 50, and 100 μg of the endoplasmic reticulum were respectively extracted in 200 μl of saline, and injected into the mice three times at 5 day intervals by subcutaneous injection. After the third injection, blood was drawn from the mice and centrifuged at 800 × g for 10 minutes to separate serum portions. 100 ng of small intestine-derived extracellular vesicles per well were dissolved in 100 μl of saline and coated with ELISA plates to evaluate vesicle-specific antibody formation in serum isolated by ELISA. As a result, it was confirmed that no antibody specific to the small intestine-derived extracellular vesicles was formed (see FIG. 18).
또한, 소포체를 3번 주사 한 마우스에서 분리한 비장 세포에 동일한 소포체 0.1 혹은 1 μg/ml로 자극을 주어 분비하는 사이토카인(cytokine)의 양상을 확인한 결과, IFN-γ와 같은 염증성 매개체의 발현에 변화가 없을 뿐 아니라 IL-10과 같은 염증 조절 매개체의 발현에도 변화가 없음을 확인하였다 (도 19 참조). In addition, cytokines secreted by stimulating splenocytes at 0.1 or 1 μg / ml to the splenocytes isolated from mice injected with the endoplasmic reticulum three times were confirmed. As a result, the expression of inflammatory mediators such as IFN-γ was observed. Not only was there no change, but there was no change in the expression of inflammatory regulatory media such as IL-10 (see Figure 19).
상기 결과는 소장 유래 세포밖 소포체는 생체 내에서 T 세포 반응을 유도하지 않는 면역 관용이 일어남을 의미한다. The results indicate that the small intestine-derived extracellular vesicles undergo immune tolerance that does not induce T cell responses in vivo.
실시예 13. 마우스 대변에서 분리한 세포밖 소포체의 프로테옴 분석 Example 13. Proteome analysis of extracellular vesicles isolated from mouse feces
마우스 대변 유래 세포밖 소포체를 분리하기 위해 5 주령 수컷 C57BL/6 마우스로부터 얻은 대변 25 g을 생리식염수 (phosphate buffered saline, PBS) 2 L에 넣고, 4°C에서 16 시간 동안 현탁 (resuspension)하였다. 이 현탁액을 4°C, 10,000×g에서 20 분 간 원심분리한 후, 상층액을 취하여 0.45㎛ 구멍 크기를 가진 필터에 통과시켰다. 이렇게 박테리아가 제거된 통과액은 100 kDa 분자량 이하의 단백질을 제거할 수 있는 막 (membrane)을 장착한 퀵스탠드 시스템 (QuixStand Benchtop System)을 사용하여 70 ml로 약 30 배 농축하였다. 농축액을 재차 4°C, 10,000×g에서 20 분 간 원심분리하여 상층액을 얻었다. 이 상층액을 0.5 ml의 2.5 M 슈크로스 용액 (2.5 M sucrose/20 mM HEPES/150 mM NaCl, pH7.4)과 1 ml의 0.8 M 슈크로스 용액 (0.8 M sucrose/20 mM HEPES/150 mM NaCl, pH7.4)이 담긴 초원심분리 튜브 (ultracentrifuge tube)에 담고 4°C, 100,000×g에서 4 시간 동안 초원심분리한 후, 2.5 M 과 0.8 M 슈크로스 용액 사이에 위치한 세포밖 소포체 포함층을 취하였다. To isolate the mouse stool-derived extracellular vesicles, 25 g of feces obtained from 5-week-old male C57BL / 6 mice were placed in 2 L of phosphate buffered saline (PBS) and suspended at 4 ° C. for 16 hours. The suspension was centrifuged at 10,000 x g for 20 minutes at 4 ° C, then the supernatant was taken and passed through a filter with a 0.45 μm pore size. The bacteria-free passage solution was concentrated about 30-fold to 70 ml using a QuickStand Benchtop System equipped with a membrane capable of removing proteins below 100 kDa molecular weight. The concentrated solution was again centrifuged at 4 ° C. and 10,000 × g for 20 minutes to obtain a supernatant. This supernatant was mixed with 0.5 ml of 2.5 M sucrose solution (2.5 M sucrose / 20 mM HEPES / 150 mM NaCl, pH7.4) and 1 ml of 0.8 M sucrose solution (0.8 M sucrose / 20 mM HEPES / 150 mM NaCl). , ultracentrifuge tube containing pH 7.4), ultracentrifuged at 4 ° C and 100,000 × g for 4 hours, followed by extracellular vesicle-containing layer located between 2.5 M and 0.8 M sucrose solution Was taken.
이를 PBS로 10 배 희석한 후, 0.15 ml의 2.5 M 슈크로스 용액과 0.35 ml의 0.8 M 슈크로스 용액이 담긴 초원심분리 튜브에 담고 4°C, 200,000×g에서 2 시간 동안 초원심분리하였다. 2.5 M 과 0.8 M 슈크로스 용액 사이에 위치한 세포밖 소포체 포함층을 PBS로 10배 희석한 후, 4°C, 150,000×g에서 3 시간 동안 초원심분리하여 침전시켰다. After diluting it 10 times with PBS, it was placed in an ultracentrifuge tube containing 0.15 ml of 2.5 M sucrose solution and 0.35 ml of 0.8 M sucrose solution, and ultracentrifuged at 4 ° C., 200,000 × g for 2 hours. The extracellular vesicle-containing layer located between 2.5 M and 0.8 M sucrose solution was diluted 10-fold with PBS, and then precipitated by ultracentrifugation at 4 ° C. and 150,000 × g for 3 hours.
침전물을 2.2 ml의 50% 옵티프렙 (Optiprep) 용액에 현탁하고 초원심분리 튜브에 담은 후, 그 위에 2 ml의 40% 옵티프렙 용액과 0.8 ml의 10% 옵티프렙 용액을 차례로 담았다. 이후 4°C, 200,000×g에서 2 시간 동안 초원심분리하고, 40% 옵티프렙 용액과 10% 옵티프렙 용액 사이의 층에서 세포밖 소포체를 얻었다. The precipitate was suspended in 2.2 ml of 50% Optiprep solution and placed in an ultracentrifuge tube, followed by 2 ml of 40% Optiprep solution followed by 0.8 ml of 10% Optiprep solution. After ultracentrifugation for 2 hours at 40,000 ° C, 200,000 × g, extracellular vesicles were obtained in the layer between 40% Optiprep solution and 10% Optiprep solution.
마우스 대변 유래 세포밖 소포체의 프로테옴 분석을 위해 인-솔루션 (in-solution) 트립신 단백질 분해 방법을 이용하였다. 마우스 대변에서 상기 실시예의 방법으로 분리한 세포밖 소포체 50 mg을 분해용액 (7 M urea, 2 M Thiourea, 100 mM NH4HCO3)으로 녹인 후, 10 mM Dithiothreitol (DTT)를 이용하여 60℃에서 45 분 간 환원 (reduction)시켰다. 이후 샘플을 상온에서 식힌 후 55 mM 요오드아세트아미드 (iodoacetamide)를 넣고, 빛을 차단한 상태로 상온에서 30 분 간 단백질을 알킬화 (alkylation)시켰다. In-solution trypsin proteolysis was used for proteome analysis of mouse stool-derived extracellular vesicles. 50 mg of extracellular vesicles isolated from mouse feces by the method of Example was dissolved in digestion solution (7 M urea, 2 M Thiourea, 100 mM NH 4 HCO 3 ), followed by 10 mM Dithiothreitol (DTT) at 60 ° C. Reduction was performed for 45 minutes. Thereafter, the sample was cooled to room temperature, 55 mM ioacetamide was added thereto, and the protein was alkylated for 30 minutes at room temperature while blocking light.
이 후 10㎍의 트립신을 처리하고 초음파분해 (sonication)로 트립신의 활성을 높인 후, 37℃에서 12 시간 동안 반응시켰다. 분해된 펩타이드는 오프젤 분리 시스템 (OFFGEL fractionators system, Agilent)을 이용하여 분리하였다. 우선 24 cm 의 IPG strip (pH 3-10)을 IPG 가수화 (IPG-rehydration) 버퍼로 가수반응시켰다. 분해된 펩타이드를 2.8 ml의 오프젤 (off-gel) 버퍼에 녹이고, 그 중 150μl를 한 레인(lane)에 로딩한 후 50μA, 8000 V 전압으로 40 시간 동안 전기영동하여 펩타이드를 각각의 등전점 (pI)에 따라 분리하였다. 분리 후 얻은 샘플을 PepClean C18 spin column을 이용하여 제염(desalting)하였다. Thereafter, 10 μg of trypsin was treated, and the activity of trypsin was increased by sonication, followed by reaction at 37 ° C. for 12 hours. The digested peptides were separated using the OFFGEL fractionators system (Agilent). First, 24 cm of IPG strip (pH 3-10) was hydrolyzed with IPG-rehydration buffer. The digested peptide was dissolved in 2.8 ml of off-gel buffer, 150 μl of which was loaded in one lane, and then electrophoresed at 50 μA, 8000 V for 40 hours to separate peptides at their respective isoelectric points (pI). ). Samples obtained after separation were desalted using a PepClean C18 spin column.
나노 이온화 질량 분석 (Nano-LC-ESI-MS/MS)을 이용하여 질량 분석을 수행하였다. 인-솔루션 분해 방법으로 준비한 마우스 대변 유래 세포밖 소포체의 분해 펩타이드를 5㎛ 크기의 C18 레진 (resin)이 충전된 컬럼 (75㎛ x 12 cm)에 로딩하여 다음과 같은 방법으로 분리하였다: 3-40% 버퍼 B 70 분; 혈류 속도 0.3 ml/min (버퍼 A 조성: 0.1% formic acid in H2O, 버퍼 B 조성: 0.1% formic acid in acetonitrile). 분리된 펩타이드는 LTQ-ion-trap 질량 분석기 (Thermo Finnigan)을 이용하여 분석하였다. 이온화 전기분무 (electrospary)의 전압은 1.9 kV로 하고, 35%의 규격화된 충돌 에너지 (normalized collision energy) 조건으로 질량 분석 (MS/MS)을 수행하였다. 모든 스펙트럼은 데이터-종속 (data-dependent) 스캔 (scan)으로 획득하였다. LTQ의 매개변수 (parameter)는 풀 매스 스캔 (full MS scan)에서 5개의 최다 스펙트럼 (most abundant spectrum)을 단편화 (fragmentation)하였고, 동적 배제 (dynamic exclusion)의 반복 수 (repeat count)는 1로, 반복 시간 (repeat duration)은 30 초로, 동적 배제 시간 (dynamic exclusion duration)은 180 초로, 배제 질량 너비 (exclusion mass width)는 1.5 Da, 동적 배제의 리스트 크기 (list size)는 50으로 설정하였다. Mass spectrometry was performed using nano ionization mass spectrometry (Nano-LC-ESI-MS / MS). Decomposed peptides of mouse stool-derived extracellular vesicles prepared by in-solution digestion were loaded onto a column (75 μm × 12 cm) filled with a 5 μm C18 resin (75 μm × 12 cm) and separated as follows: 3- 40% buffer B 70 min; Blood flow rate 0.3 ml / min (buffer A composition: 0.1% formic acid in H 2 O, buffer B composition: 0.1% formic acid in acetonitrile). The isolated peptides were analyzed using LTQ-ion-trap mass spectrometer (Thermo Finnigan). The voltage of the ionized electrospary was 1.9 kV, and mass spectrometry (MS / MS) was performed under a condition of 35% normalized collision energy. All spectra were acquired with a data-dependent scan. The parameters of the LTQ were the fragmentation of the five most abundant spectra in full MS scan, the repeat count of dynamic exclusion was 1, The repeat duration is set to 30 seconds, the dynamic exclusion duration is 180 seconds, the exclusion mass width is 1.5 Da, and the list size of the dynamic exclusion is 50.
마우스 장 세포에서 유래된 세포밖 소포체에 존재하는 단백질의 분석을 위해 기존에 아미노산 염기서열이 구축된 마우스의 데이터베이스 (Uniprot)를 이용함과 동시에 장내 박테리아에서 유래된 세포밖 소포체에 존재하는 단백질의 분석을 위해서 장내에 다수로 존재하는 속 (genus)을 대표하는10 종 (species)의 박테리아 데이터베이스 (Uniprot)를 이용하였다. 각각의 데이터베이스를 이용하여 총 11 번의 데이터 분석을 수행하였다. MASCOT 단백질 분석 엔진 version 2.2 (http://www.matrixscience.com)를 이용하여 질량분석에서 나온 모든 스펙트럼 (MS spectrum and MS/MS spectrum)을 분석하였다. 분석에 대한 검증은 펩타이드 프로펫/단백질 프로펫 (peptide prophet/protein prophet) 95%/99% 이상으로 신뢰도 높은 단백질들을 선별하였고, 각각의 단백질에서 동정된 펩타이드가 한 개인 스펙트럼은 직접 아미노산 서열을 분석(manual validation)하여 신뢰도를 높였다. For the analysis of proteins present in extracellular vesicles derived from mouse intestinal cells, we use the mouse database (Uniprot), which has a previously constructed amino acid sequence, and at the same time analyze the proteins in extracellular vesicles derived from enteric bacteria. To this end, we used a database of bacteria (Uniprot) of 10 species representing a large number of genus in the intestine. A total of 11 data analyzes were performed using each database. All spectra from the mass spectrometry (MS spectrum and MS / MS spectrum) were analyzed using the MASCOT protein analysis engine version 2.2 (http://www.matrixscience.com). Validation of the assay was performed by selecting peptides with more than 95% / 99% of the peptide prophet / protein prophet, and spectra of the individual peptides identified in each protein were analyzed directly on the amino acid sequence. validation) to increase reliability.
도 20은 상기의 방법으로 마우스의 대변에 존재하는 세포밖 소포체의 프로테옴 분석 결과로서, 총 295 개의 단백질 중 73개의 단백질은 숙주세포 유래 단백질이었고, 222 개가 박테리아 유래 단백질이었으며 이중 그람 음성 세균유래 단백질이 77개, 그람 양성 세균 유래 단백질이 145 개 동정되었다. 그람 음성 세균의 경우 Bacteroides thetaiotaomicron, Escherichia coli K-12, Klebsiella pneumonia 유래 단백질이 주를 이루었으며, 그람 양성 세균의 경우 Clostridium perfringens, Lactibacillus reuteri 유래 단백질이 주를 이루었다. Figure 20 shows the results of proteome analysis of extracellular vesicles present in the stool of the mouse by the above method, 73 proteins out of a total of 295 proteins were host cell-derived protein, 222 were bacteria-derived protein, double gram negative bacteria-derived protein 77 gram-positive bacteria-derived proteins were identified. Gram-negative bacteria were mainly derived from Bacteroides thetaiotaomicron, Escherichia coli K-12 and Klebsiella pneumonia . Gram-positive bacteria were mainly derived from Clostridium perfringens and Lactibacillus reuteri .
실시예 14. 대변에서 분리한 세포밖 소포체에 의한 소포체 특이 regulatory T (Treg) 세포 유도Example 14 Induction of Vesicular Specific Regulatory T (Treg) Cells by Extracellular Vesicles Isolated from Stool
실시예 13의 방법을 이용하여 대변 유래 세포밖 소포체를 분리하고 실시예 12의 방법에 따라 평가하였다. 이때, 마우스에 주사하는 양은 대변 유래 세포밖 소포체 50, 100 μg을 각각 생리식염수 200 ㎕에 풀어내어 주사하였다. The stool-derived extracellular vesicles were isolated using the method of Example 13 and evaluated according to the method of Example 12. At this time, the amount injected into the mouse was extracted by dissolving 50 and 100 μg of fecal-derived extracellular vesicles in 200 μl of saline, respectively.
그 결과, 대변 유래 세포밖 소포체에 특이적인 항원이 농도 의존적으로 증가함을 확인하였다 (도 21 참조). 또한, 염증성 매개체로 작용하는 사이토카인의 발현은 유도되지 않으면서 염증 조절 매개체로 작용할 수 있는 IL-10의 발현은 농도 의존적으로 증가시키는 것을 확인하였다 (도 22 참조). As a result, it was confirmed that the antigen-specific antigen-specific increase in fecal-derived extracellular vesicles (see FIG. 21). In addition, it was confirmed that the expression of IL-10, which can act as an inflammatory mediator, was increased in a concentration-dependent manner without the induction of cytokines acting as an inflammatory mediator (see FIG. 22).
상기 결과는 대변 유래 세포밖 소포체에 의한 소포체 특이 T reg 세포를 유도할 수 있음을 의미한다. The results indicate that vesicle-derived extracellular vesicles can induce vesicle-specific T reg cells.
상기 실시예 등을 통하여, 본 발명자들은 정상 상태의 소장에서 분리한 세포밖 소포체가 염증성 질환 및 암을 일으키는데 중요한 바이오마커인 IL-6 분비를 억제함을 최초로 규명하였으며, 염증성 장질환 등의 질병 상태의 소장에서 분리한 세포밖 소포체는 내독소(LPS) 등을 소포체에 함유하는 그람 음성균 유래 세포밖 소포체를 통해 염증성 매개체를 분비하여 질병에 관여함을 밝혔다. Through the above examples, the present inventors first identified that the extracellular vesicles isolated from the small intestine of the normal state inhibits IL-6 secretion, which is an important biomarker for causing inflammatory diseases and cancer, and disease states such as inflammatory bowel disease. The extracellular vesicles isolated from the small intestine were found to be involved in disease by secreting inflammatory mediators through Gram-negative bacteria-derived extracellular vesicles containing endotoxins (LPS).
이에 더하여, 본 발명자들은 정상 상태의 소장에서 분리한 세포밖 소포체는 질병 상태의 소장에서 분리한 세포밖 소포체에 의한 염증반응 뿐만 아니라 LPS 혹은 대장균유래 세포밖 소포체에 의한 염증성 매개체 분비를 억제함을 밝혔다. 이는 장내 세균 혹은 유산균 등이 장 상피세포 등의 숙주세포와의 상호 작용에 의해 장 내에 세포밖 소포체를 분비하고, 따라서 IL-6 등에 의해 매개되는 염증성 질환 및 암 등의 발생 및 진행을 막을 수 있음을 의미한다. In addition, the inventors found that extracellular vesicles isolated from the normal small intestine inhibit the inflammatory mediator secretion by LPS or E. coli-derived extracellular vesicles as well as inflammatory responses by the extracellular vesicles isolated from the diseased small intestine. . This is because gut bacteria or lactic acid bacteria secrete extracellular vesicles in the intestine by interacting with host cells such as intestinal epithelial cells, thus preventing the development and progression of inflammatory diseases and cancers mediated by IL-6 and the like. Means.
본 발명의 세포밖 소포체는 소장 및 대변에서 분리했으나, 이에 제한하는 것은 아니다. 또한, 염증성 장질환 상태의 소장에서 유래한 세포밖 소포체가 정상 상태의 소장에서 분리한 세포밖 소포체에 비하여 염증성 매개체 분비를 유도함은 IL-6 등에 의해 매개되는 질환의 발생 혹은 경과를 예측할 수 있음을 의미한다. 이는 염증성 장질환에 초첨을 맞추었으나, 이에 제한하는 것은 아니다. The extracellular vesicles of the present invention have been isolated from the small intestine and feces, but are not limited thereto. In addition, the extracellular vesicles derived from the small intestine of inflammatory bowel disease induced the secretion of inflammatory mediators compared to the extracellular vesicles isolated from the small intestine of the normal state, thereby predicting the occurrence or course of diseases mediated by IL-6. it means. This focuses on, but is not limited to, inflammatory bowel disease.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명은 포유동물 체내에서 유래하는 세포밖 소포체를 이용하여 염증성 질환 및 암을 예방 및/또는 치료하는 방법을 제공하고자 한다. 또한, 포유동물의 체내에서 분리된 세포밖 소포체를 유효성분으로 함유하는 본 발명의 조성물은 암을 포함한 염증성 질환의 치료 또는 예방을 위한 약학적 조성물, 식품 조성물, 화장료 조성물 등으로 이용될 수 있다. 이에 더하여, 본 발명은 위질환 환자에서 분리한 H. pylori 유래 세포밖 소포체에 대한 염증성 매개체 분비능, 소포체 내 유전물질 혹은 단백질 분석, 소포체 특이 면역반응 측정 등을 통해 위질환의 원인물질과 위질환의 발생 또는 경과를 진단하는 것을 가능하게 한다.The present invention seeks to provide a method for preventing and / or treating inflammatory diseases and cancer using extracellular vesicles derived from the mammalian body. In addition, the composition of the present invention containing the extracellular vesicles isolated in the body of a mammal as an active ingredient can be used as pharmaceutical compositions, food compositions, cosmetic compositions and the like for the treatment or prevention of inflammatory diseases, including cancer. In addition, the present invention is to determine the causative agent of gastric disease and gastrointestinal diseases through inflammatory mediator secretion of H. pylori- derived extracellular vesicles isolated from patients with gastric disease, analysis of genetic material or protein in endoplasmic reticulum, and measurement of vesicle-specific immune responses. It is possible to diagnose the occurrence or course.
Claims (64)
- 포유동물의 체내에서 유래된 세포밖 소포체를 유효 성분으로 함유하는, 염증성 질환의 치료 또는 예방용 조성물. A composition for the treatment or prevention of inflammatory diseases, comprising as an active ingredient extracellular vesicles derived from the body of a mammal.
- 포유동물의 체내에서 유래된 세포밖 소포체를 유효 성분으로 함유하는, 암의 치료 또는 예방용 조성물. A composition for the treatment or prevention of cancer, comprising as an active ingredient extracellular vesicles derived from the body of a mammal.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체는 상기 포유동물의 소장 또는 대장에서 분리된 것인, 조성물. Wherein said extracellular vesicles are isolated from the small or large intestine of said mammal.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체는 상기 포유동물의 대변에서 분리된 것인, 조성물. Wherein said extracellular vesicles are isolated from feces of said mammal.
- 제1항에 있어서, The method of claim 1,상기 염증성 질환은 호흡기질환, 소화기질환, 피부질환, 패혈증, 동맥경화증, 관절염, 뇌질환 및 이의 합병증으로 이루어지는 군에서 선택되는 것인, 조성물. The inflammatory disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof.
- 제5항에 있어서, The method of claim 5,상기 호흡기 질환은 비염, 천식, 및 만성폐쇄성폐질환을 포함하는 것인, 조성물. Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
- 제5항에 있어서, The method of claim 5,상기 소화기 질환은 위염, 소화성궤양, 및 염증성대장염을 포함하는 것인, 조성물. Wherein the digestive disease comprises gastritis, peptic ulcer, and inflammatory colitis.
- 제5항에 있어서, The method of claim 5,상기 피부 질환은 아토피피부염, 건선, 및 접촉피부염을 포함하는 것인, 조성물. Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
- 제2항에 있어서, The method of claim 2,상기 암은 폐암, 위암, 및 대장암을 포함하는 것인, 조성물. Wherein the cancer comprises lung cancer, gastric cancer, and colorectal cancer.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체는 포유동물 세포에서 염증성 매개체의 분비를 억제하는 것인, 조성물. Wherein said extracellular vesicles inhibit the secretion of inflammatory mediators in mammalian cells.
- 제10항에 있어서, The method of claim 10,상기 염증성 매개체는 인터루킨 6(IL-6)인, 조성물. The inflammatory mediator is interleukin 6 (IL-6).
- 제10항에 있어서, The method of claim 10,상기 세포는 상피세포 또는 염증세포인, 조성물. The cell is an epithelial cell or an inflammatory cell.
- 제12항에 있어서, The method of claim 12,상기 염증세포는 대식세포 또는 호산구인, 조성물. Wherein said inflammatory cell is a macrophage or eosinophil.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체가 상기 포유동물의 체내 공생 세균 유래의 세포밖 소포체인, 조성물. Wherein said extracellular vesicles are extracellular vesicles derived from symbiotic bacteria in said mammal.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체가 상기 포유동물의 상피세포 또는 염증세포 유래의 세포밖 소포체인, 조성물. Wherein said extracellular vesicles are extracellular vesicles derived from epithelial cells or inflammatory cells of said mammal.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 세포밖 소포체는 그 평균 직경이 20 nm 내지 200 nm 인, 조성물. Wherein said extracellular vesicles have an average diameter of 20 nm to 200 nm.
- 제1항 또는 제2항에 있어서, The method according to claim 1 or 2,상기 조성물은 약학적 조성물, 식품 조성물, 또는 화장료 조성물인, 조성물. The composition is a pharmaceutical composition, a food composition, or a cosmetic composition.
- 포유동물의 체내에서 유래된 세포밖 소포체를 유효성분으로 함유하는, 염증성 질환의 치료 또는 예방용 백신. A vaccine for the treatment or prophylaxis of inflammatory diseases, which contains as an active ingredient extracellular vesicles derived from the body of a mammal.
- 포유동물의 체내에서 유래된 세포밖 소포체를 유효성분으로 함유하는, 암의 치료 또는 예방용 백신. A vaccine for the treatment or prevention of cancer, comprising an extracellular vesicle derived from the body of a mammal as an active ingredient.
- 제18항 또는 제19항에 있어서, The method of claim 18 or 19,상기 세포밖 소포체는 상기 포유동물의 소장 또는 대장에서 분리된 것인, 백신. The extracellular vesicles are isolated from the small or large intestine of the mammal.
- 제18항 또는 제19항에 있어서, The method of claim 18 or 19,상기 세포밖 소포체는 상기 포유동물의 대변에서 분리된 것인, 백신. Wherein said extracellular vesicles are isolated from feces of said mammal.
- 제18항에 있어서, The method of claim 18,상기 염증성 질환은 호흡기질환, 소화기질환, 피부질환, 패혈증, 동맥경화증, 관절염, 뇌질환 및 이의 합병증으로 이루어지는 군에서 선택되는 것인, 백신. The inflammatory disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof.
- 제22항에 있어서, The method of claim 22,상기 호흡기 질환은 비염, 천식, 및 만성폐쇄성폐질환을 포함하는 것인, 백신. Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
- 제22항에 있어서, The method of claim 22,상기 소화기 질환은 위염, 소화성궤양, 및 염증성대장염을 포함하는 것인, 백신. The gastrointestinal disease includes gastritis, peptic ulcer, and inflammatory colitis.
- 제22항에 있어서, The method of claim 22,상기 피부 질환은 아토피피부염, 건선, 및 접촉피부염을 포함하는 것인, 백신. Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
- 제19항에 있어서, The method of claim 19,상기 암은 폐암, 위암, 및 대장암을 포함하는 것인, 백신. Wherein the cancer comprises lung cancer, gastric cancer, and colon cancer.
- 제18항 또는 제19항에 있어서, The method of claim 18 or 19,상기 백신은 세포밖 소포체를 단독으로 사용하는 것인, 백신. The vaccine is to use extracellular vesicles alone.
- 제18항 또는 제19항에 있어서, The method of claim 18 or 19,상기 백신은 상기 세포밖 소포체와 병원성 소포체를 혼합하여 사용하는 것인, 백신. The vaccine is to use a mixture of the extracellular vesicles and pathogenic vesicles.
- 제28항에 있어서, The method of claim 28,상기 병원성 소포체는 체내 공생 세균에서 유래한 병원성 소포체인, 백신. The pathogenic vesicles are pathogenic vesicles derived from symbiotic bacteria in the body, vaccine.
- 제28항에 있어서, The method of claim 28,상기 병원성 소포체는 그람 양성 세균에서 유래한 병원성 소포체인, 백신. Wherein said pathogenic vesicles are pathogenic vesicles derived from Gram-positive bacteria.
- 제28항에 있어서, The method of claim 28,상기 병원성 소포체는 공기에 존재하는 병원성 소포체인, 백신. Wherein said pathogenic vesicles are pathogenic vesicles present in air.
- 제28항에 있어서, The method of claim 28,상기 세포밖 소포체는 그 평균 직경이 20 nm 내지 200 nm 인, 백신. The extracellular vesicles have a mean diameter of 20 nm to 200 nm.
- 포유동물의 체내에서 유래된 세포밖 소포체를 치사량 미만으로 포유동물에게 투여하는 단계를 포함하는, 질환의 예방 또는 치료 방법. A method of preventing or treating a disease comprising administering to a mammal an extracellular vesicle derived from the body of the mammal at a less than lethal dose.
- 제33항에 있어서, The method of claim 33, wherein상기 세포밖 소포체는 상기 포유동물의 소장 또는 대장에서 분리된 것인, 방법. Wherein said extracellular vesicles are isolated from the small or large intestine of said mammal.
- 제33항에 있어서, The method of claim 33, wherein상기 세포밖 소포체는 상기 포유동물의 대변에서 분리된 것인, 방법. Said extracellular vesicles are isolated from feces of said mammal.
- 제33항에 있어서, The method of claim 33, wherein상기 질환은 호흡기질환, 소화기질환, 피부질환, 패혈증, 동맥경화증, 관절염, 뇌질환 및 이의 합병증, 및 암으로 이루어지는 군에서 선택되는 것인, 방법. The disease is selected from the group consisting of respiratory diseases, digestive diseases, skin diseases, sepsis, arteriosclerosis, arthritis, brain diseases and complications thereof, and cancer.
- 제36항에 있어서, The method of claim 36,상기 호흡기 질환은 비염, 천식, 및 만성폐쇄성폐질환을 포함하는 것인, 방법. Wherein the respiratory disease comprises rhinitis, asthma, and chronic obstructive pulmonary disease.
- 제36항에 있어서, The method of claim 36,상기 소화기 질환은 위염, 소화성궤양, 및 염증성대장염을 포함하는 것인, 방법. Wherein said digestive disease comprises gastritis, peptic ulcer, and inflammatory colitis.
- 제36항에 있어서, The method of claim 36,상기 피부 질환은 아토피피부염, 건선, 및 접촉피부염을 포함하는 것인, 방법. Wherein said skin disease comprises atopic dermatitis, psoriasis, and contact dermatitis.
- 제36항에 있어서, The method of claim 36,상기 암은 폐암, 위암, 및 대장암을 포함하는 것인, 방법. Wherein the cancer comprises lung cancer, gastric cancer, and colorectal cancer.
- 제33항에 있어서, The method of claim 33, wherein상기 방법은 상기 세포밖 소포체를 단독으로 사용하는 것인, 방법. The method is to use the extracellular vesicles alone.
- 제33항에 있어서, The method of claim 33, wherein상기 방법은 상기 세포밖 소포체와 병원성 소포체를 혼합하여 사용하는 것인, 방법. The method is to use a mixture of the extracellular vesicles and pathogenic vesicles.
- 제42항에 있어서, The method of claim 42, wherein상기 병원성 소포체는 체내 공생 세균에서 유래한 병원성 소포체인, 방법. The pathogenic endoplasmic reticulum is a pathogenic endoplasmic reticulum derived from symbiotic bacteria in the body.
- 제42항에 있어서, The method of claim 42, wherein상기 병원성 소포체는 그람 양성 세균에서 유래한 병원성 소포체인, 방법. Said pathogenic endoplasmic reticulum is a pathogenic endoplasmic reticulum derived from gram positive bacteria.
- 제42항에 있어서, The method of claim 42, wherein상기 병원성 소포체는 공기에 존재하는 병원성 소포체인, 방법. Wherein said pathogenic vesicles are pathogenic vesicles present in air.
- 제33항에 있어서, The method of claim 33, wherein상기 세포밖 소포체는 상기 포유동물의 상피세포 또는 염증세포 유래의 세포밖 소포체인, 방법. Wherein said extracellular vesicles are extracellular vesicles derived from epithelial cells or inflammatory cells of said mammal.
- 제 33항에 있어서, The method of claim 33,상기 투여는 피하주사, 정맥주사, 비강투여, 설하투여, 흡입투여, 경구복용, 항문투여, 및 피부투여로 이루어진 군으로부터 선택되는 것인, 방법. Wherein said administration is selected from the group consisting of subcutaneous injection, intravenous injection, nasal administration, sublingual administration, inhalation administration, oral administration, anal administration, and skin administration.
- 포유동물의 체내에서 유래된 세포밖 소포체를 이용하여 질환의 원인물질, 질환의 발생 또는 경과를 진단하는 방법으로, 하기 단계를 포함하는 방법: A method of diagnosing a causative agent of a disease, the occurrence or course of a disease using extracellular vesicles derived from the body of a mammal, the method comprising the following steps:(a) 포유동물의 체내에서 세포밖 소포체를 분리하는 단계; (a) isolating extracellular vesicles in the body of a mammal;(b) 상기 분리된 세포밖 소포체를 세포에 처리하여 배양하는 단계; 및 (b) treating the isolated extracellular vesicles with cells and culturing the cells; And(c) 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계. (c) measuring the level of inflammatory mediator in said cell culture.
- 제48항에 있어서, The method of claim 48,상기 (a) 단계는 포유동물의 체내 물질을 수득하는 단계; 상기 수득된 물질을 원심분리하여 불순물, 동물세포, 및 세균을 제거하는 단계; 및 초원심분리를 수행하여 세포밖 소포체를 분리하는 단계를 포함하는, 방법. Step (a) is the step of obtaining a mammalian body material; Centrifuging the obtained material to remove impurities, animal cells, and bacteria; And performing ultracentrifugation to separate extracellular vesicles.
- 제48항에 있어서, The method of claim 48,상기 세포밖 소포체는 상기 포유동물의 위액에서 분리된 것인, 방법. Said extracellular vesicles are isolated from gastric juice of said mammal.
- 제48항에 있어서, The method of claim 48,상기 세포밖 소포체는 상기 포유동물의 소장 또는 대장에서 분리된 것인, 방법. Wherein said extracellular vesicles are isolated from the small or large intestine of said mammal.
- 제48항에 있어서, The method of claim 48,상기 세포밖 소포체는 상기 포유동물의 대변에서 분리된 것인, 방법. Said extracellular vesicles are isolated from feces of said mammal.
- 제48항에 있어서, The method of claim 48,상기 (b) 단계의 세포는 상피세포 또는 염증세포인, 방법. The cell of step (b) is epithelial cells or inflammatory cells.
- 제53항에 있어서, The method of claim 53,상기 염증세포는 대식세포 또는 호산구인 방법. The inflammatory cells are macrophages or eosinophils.
- 제48항에 있어서, The method of claim 48,상기 (c) 단계의 염증성 매개체는 인터루킨 6(IL-6)인, 방법. The inflammatory mediator of step (c) is interleukin 6 (IL-6).
- 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 의 체외 배양액에서 분리한 세포밖 소포체를 포함하는 조성물. A composition comprising extracellular vesicles isolated from in vitro culture of Helicobacter pylori isolated from human gastric tissue or gastric juice.
- 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 이용하여 질환의 원인물질, 질환의 발생 또는 경과를 진단하는 방법. Helicobacter pylori isolated from human gastric tissue or gastric juice(Helicobacter pylori)origin A method for diagnosing the cause of a disease, the occurrence or course of a disease by using extracellular vesicles.
- 제57항에 있어서, The method of claim 57,상기 질환은 위염, 소화성궤양, 및 위암을 포함하는, 방법. The disease includes gastritis, peptic ulcer, and gastric cancer.
- 제57항에 있어서, The method of claim 57,상기 방법은 상기 세포밖 소포체를 세포에 처리하여 배양하는 단계; 및 상기 세포 배양액 중의 염증성 매개체의 수준을 측정하는 단계를 포함하는, 방법. The method comprises the steps of culturing the extracellular vesicles to the cells; And measuring the level of an inflammatory mediator in said cell culture.
- 제57항에 있어서, The method of claim 57,상기 방법은 상기 세포밖 소포체 내 유전물질 또는 단백질을 측정하는 단계를 포함하는, 방법. The method comprises measuring genetic material or protein in the extracellular vesicles.
- 제57항에 있어서, The method of claim 57,상기 방법은 상기 세포밖 소포체에 대한 면역반응을 측정하는 단계를 포함하는, 방법. The method comprises measuring an immune response to the extracellular vesicles.
- 제57항에 있어서, The method of claim 57,상기 방법은 상기 세포밖 소포체에 대한 항체를 측정하는 단계를 포함하는, 방법. The method comprises measuring an antibody against the extracellular vesicles.
- 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 유효성분으로 함유하는, 헬리코박터 파일로리 (Helicobacter pylori) 에 의한 질환의 치료 또는 예방용 백신. Helicobacter pylori isolated from human gastric tissue or gastric juice(Helicobacter pylori)origin Helicobacter pylori containing extracellular vesicles as an active ingredient(Helicobacter pylori)Vaccines for the treatment or prevention of diseases by.
- 사람의 위조직 또는 위액에서 분리한 헬리코박터 파일로리 (Helicobacter pylori) 유래 세포밖 소포체를 치사량 미만으로 포유동물에게 투여하는 단계를 포함하는, 헬리코박터 파일로리 (Helicobacter pylori)에 의한 질환의 예방 또는 치료 방법. Helicobacter pylori isolated from human gastric tissue or gastric juice(Helicobacter pylori)origin Helicobacter pylori, comprising administering an extracellular vesicle to a mammal at a sublethal dose(Helicobacter pylori)A method for preventing or treating a disease by.
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| CN111249449A (en) * | 2020-02-28 | 2020-06-09 | 东南大学 | Extracellular vesicle-interleukin-10 nano targeted drug and preparation method and application thereof |
| CN111344391A (en) * | 2017-10-31 | 2020-06-26 | 株式会社爱茉莉太平洋 | Immunomodulating composition comprising extracellular vesicles derived from lactic acid bacteria |
| WO2024102969A1 (en) * | 2022-11-10 | 2024-05-16 | Board Of Regents, The University Of Texas System | Use of fecal derivatives and fecal derived extracellular vesicles to enhance immunity |
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| CN106177742A (en) * | 2016-09-08 | 2016-12-07 | 四川聚豪生物科技有限公司 | A kind of for Chinese medicine composition treating insufficiency of stomach and preparation method thereof |
| CN111344391A (en) * | 2017-10-31 | 2020-06-26 | 株式会社爱茉莉太平洋 | Immunomodulating composition comprising extracellular vesicles derived from lactic acid bacteria |
| CN111249449A (en) * | 2020-02-28 | 2020-06-09 | 东南大学 | Extracellular vesicle-interleukin-10 nano targeted drug and preparation method and application thereof |
| WO2024102969A1 (en) * | 2022-11-10 | 2024-05-16 | Board Of Regents, The University Of Texas System | Use of fecal derivatives and fecal derived extracellular vesicles to enhance immunity |
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