WO2019168329A1 - Nanovesicles derived from acinetobacter bacteria and use thereof - Google Patents

Abstract

The present invention relates to vesicles derived from Acinetobacter bacteria and a use thereof. The present inventors have confirmed experimentally that vesicles are significantly reduced in clinical samples derived from patients with malignant diseases such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, and bladder cancer, cardiovascular diseases such as myocardial infarction, cardiomyopathy, and stroke, diabetes mellitus, dementia, and Parkinson disease compared to a normal person, and the vesicles significantly inhibit secretion of an inflammatory mediator by pathogenic vesicles. The present inventors have confirmed that the vesicles have an anticancer effect in a mouse cancer model. Therefore, vesicles derived from Acinetobacter bacteria according to the present invention may be effectively used for developing a diagnostic method for malignant diseases such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, and bladder cancer, cardiovascular diseases such as myocardial infarction, cardiomyopathy, and stroke, diabetes mellitus, dementia, and Parkinson disease, and for developing a composition for preventing or treating the above-mentioned diseases or inflammatory diseases.

Description

Nanovesicles derived from bacteria of the genus Acinetobacter and their use

The present invention relates to nanovesicles derived from the genus Acinetobacter and its use. More specifically, malignant diseases such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer and bladder cancer using nanovesicles derived from the genus Acinetobacter, myocardium The present invention relates to a method for diagnosing cardiovascular diseases such as infarction, cardiomyopathy and stroke, diabetes, dementia, and Parkinson's disease, and a composition for preventing or treating the disease or inflammatory disease.

In the 21st century, acute infectious diseases, which were previously recognized as infectious diseases, have become less important, while chronic diseases with immune dysfunctions caused by incompatibility between humans and microbiomes determine the quality of life and human life. As a major disease, the disease pattern has changed. As intractable chronic diseases of the 21st century, cancer, cardiovascular disease, metabolic diseases, and neuro-psychiatric diseases have become a major problem in public health. The intractable chronic disease is characterized by chronic inflammation accompanied by immune dysfunction.

Inflammation is a local or systemic defense against damage or infection of cells and tissues, primarily by the direct response of humoral mediators that make up the immune system, or by stimulating local or systemic effector systems. It is caused by a cascade of biological reactions that take place. Major inflammatory diseases include gastroenteritis, digestive diseases such as inflammatory bowelitis, oral diseases such as periodontitis, asthma, chronic obstructive pulmonary disease (COPD), respiratory diseases such as rhinitis, atopic dermatitis, hair loss, skin diseases such as psoriasis, degenerative arthritis, Arthritis, such as rheumatoid arthritis; And metabolic diseases such as obesity, diabetes, cirrhosis of the liver.

The microorganisms symbiotic to the human body reaches 100 trillion times more than human cells, and the number of genes of microorganisms is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) is a microbial community including microbes, archaea and eukarya that exist in a given settlement.

The symbiotic bacteria in the body and the bacteria present in the environment secrete nanometer-sized vesicles to exchange information such as genes, low molecular weight compounds, and proteins with other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacteria-derived vesicles are absorbed through epithelial cells or keratinocytes of the mucosa to induce local inflammatory responses, as well as to regulate immune and inflammatory responses in the organs absorbed by the body and distributed to each organ. do. For example, vesicles derived from pathogenic Gram-negative bacteria, such as Eshcherichia coli , promote systemic inflammatory and blood coagulation through vascular endothelial inflammatory reactions when absorbed into blood vessels, Absorption of cells causes insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control the disease by controlling immune and metabolic abnormalities caused by pathogenic vesicles.

Immune responses to factors such as vesicles derived from bacteria result in a Th17 immune response characterized by the secretion of IL-17 cytokines, which secrete IL-6 upon exposure to bacterial derived vesicles, which induce a Th17 immune response. do. Inflammation by the Th17 immune response is characterized by neutrophil infiltration, and TNF-alpha secreted from inflammatory cells such as macrophages plays an important role in the process of inflammation.

On the other hand, Acinetobacter spp. Is an aerobic Gram-negative bacillus, which is widely distributed in nature and is isolated from soil, water, and clinical specimens. Among them, Acinetobacter baumannii is known as a major causative agent of infection in the hospital. However, no application has been reported for the diagnosis and treatment of intractable diseases such as cancer, cardiovascular diseases, metabolic diseases, and neuro-psychiatric diseases through extracellular vesicles derived from bacteria of the genus Acinetobacter.

The present inventors earnestly researched to solve the above-mentioned conventional problems. As a result of meta-genome analysis, the present inventors compared gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, And it was confirmed that the content of the bacterial vesicles of the genus Acinetobacter in the sample derived from Parkinson's disease patients significantly decreased. In addition, when vesicles were isolated from Acinetobacter Baumani bacteria belonging to the genus Acinetobacter and treated with macrophages, TNF-alpha secretion by pathogenic vesicles was significantly inhibited, and anticancer effects were confirmed in mouse cancer models. To this end, the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for providing information for the diagnosis of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease.

In addition, the present invention from the group consisting of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases comprising an acinetobacter-derived vesicles as an active ingredient Another object is to provide a composition for the prevention, treatment or amelioration of one or more diseases selected.

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.

In order to achieve the object of the present invention as described above, the present invention comprises the following steps, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease Provide informational methods for diagnosis:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And

(c) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes mellitus when the content of bacteria-derived vesicles in Acinetobacter is lower than that of normal people through quantitative analysis of the PCR products Classifying as dementia, dementia, or Parkinson's disease.

The present invention also provides a method for diagnosing gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease, comprising the following steps:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And

(c) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes mellitus when the content of bacteria-derived vesicles in Acinetobacter is lower than that of normal people through quantitative analysis of the PCR products , Dementia, or Parkinson's disease.

In one embodiment of the invention, the sample in step (a) may be feces, blood, or urine.

In another embodiment of the present invention, the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the present invention includes a cine vesicles of the genus Acinetobacter as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and It provides a pharmaceutical composition for the prevention or treatment of one or more diseases selected from the group consisting of inflammatory diseases.

In addition, the present invention includes a cine vesicles of the genus Acinetobacter as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and It provides a food composition for the prevention or amelioration of one or more diseases selected from the group consisting of inflammatory diseases.

In addition, the present invention includes a cine vesicles of the genus Acinetobacter as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and An inhalant composition for preventing or treating at least one disease selected from the group consisting of inflammatory diseases is provided.

In one embodiment of the present invention, the inflammatory disease is atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, plaque Blood shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, chronic inflammatory diseases caused by viral or bacterial infections, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis , Scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, neurotic-borelia, tuberculosis, sarcoid Sarcoidosis, lupus, alumni lupus, tuberculosis lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveal , May be at least one selected from irritable bowel syndrome, Crohn's disease, it shows Gran syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, muscular encephalomyelitis, amyotrophic lateral sclerosis, Parkinson seunbyeong, and the group consisting of multiple sclerosis.

In another embodiment of the present invention, the inflammatory disease may be a disease mediated by Interleukin-6 (IL-6) or Tumor necrosis factor alpha (TNF-α).

The present invention also provides a cosmetic composition for the prevention or improvement of an inflammatory disease comprising a vesicle-derived vesicles of the genus Acinetobacter as an active ingredient.

 In one embodiment of the present invention, the inflammatory disease may be at least one selected from the group consisting of atopic dermatitis, acne, and psoriasis.

In addition, the present invention comprises the step of administering to the subject a pharmaceutical composition comprising an Acinetobacter genus bacteria-derived vesicles as an active ingredient, gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, myocardium A method of preventing or treating one or more diseases selected from the group consisting of conditions, stroke, diabetes, dementia, Parkinson's disease, and inflammatory disease.

In addition, the present invention is a group of vesicles derived from Acinetobacter genus, stomach cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases It provides a prophylactic or therapeutic use of one or more diseases selected from.

In one embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.

In another embodiment of the present invention, the vesicles may be secreted naturally or artificially from bacteria of the genus Acinetobacter.

In another embodiment, the vesicle-derived vesicles of the genus Acinetobacter may be vesicles derived from Acinetobacter baumannii .

The present inventors confirmed that the intestinal bacteria are not absorbed into the body, but when the bacteria-derived vesicles are absorbed into the body through epithelial cells, they are distributed systemically and excreted in vitro through the kidneys, liver, and lungs. Bacterial-derived vesicle metagenome analysis in the blood, blood, or urine may be used to detect feces, blood, or patients with gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, and Parkinson's disease. It was confirmed that the vesicle-derived bacteria of the genus Acinetobacter in urine were significantly reduced compared to normal individuals. In addition, it was observed that the vesicles were isolated by culturing Acinetobacter Baumani, a species of the genus Acinetobacter, in vitro and significantly inhibiting the secretion of inflammatory mediators by pathogenic vesicles when administered to inflammatory cells in vitro. Ascertaining anticancer effect in mouse cancer model, the bacterium-derived vesicles of the genus Acinetobacter according to the present invention are gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, and Parkinson's disease. It is expected that the present invention can be usefully used in diagnostic methods, and compositions for preventing or treating such diseases or inflammatory diseases.

Figure 1a is a photograph of the distribution of bacteria and vesicles by time after administration of bacteria and bacteria-derived vesicles (EV) to the mouth, Figure 1b is 12 hours after oral administration, blood, kidneys The results were obtained by evaluating the distribution of bacteria, vesicles, and organs in the liver, liver, and various organs.

Figure 2 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacteria-derived vesicles metagenome present in gastric cancer patients and normal feces and urine.

Figure 3 is a result of comparing the distribution of the bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacteria-derived vesicles metagenome present in colon cancer patients and normal urine.

Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacteria-derived vesicle metagenome present in the breast cancer patients and normal blood and urine.

5 is a result of comparing the distribution of vesicle-derived bacteria of the genus Acinetobacter after analysis of bacterial-derived vesicles metagenome present in ovarian cancer patients and normal urine.

Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacteria-derived vesicles metagenome present in bladder cancer patients and normal urine.

Figure 7 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analysis of bacterial-derived vesicles metagenome present in patients with myocardial infarction and normal blood.

Figure 8 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacterial-derived vesicles metagenome present in cardiomyopathy patients and normal blood.

9 is a result of comparing the distribution of vesicle-derived bacteria belonging to the genus Acinetobacter after analyzing the bacteria-derived vesicle metagenome present in blood and urine of diabetic patients and normal people.

Figure 10 is a result of comparing the distribution of bacteria-derived vesicles of the genus Acinetobacter after analyzing the bacteria-derived vesicles metagenome present in the stroke patients and normal blood.

11 is a result of comparing the distribution of vesicle-derived bacteria belonging to the genus Acinetobacter after analysis of bacterial-derived vesicles metagenome present in Parkinson's disease patients and normal urine.

12 is a result of comparing the distribution of bacterial-derived vesicles of the genus Acinetobacter after analyzing the bacterial-derived vesicles metagenome present in dementia patients and normal blood.

13 is a result of evaluating the degree of cell death by administering the vesicles to macrophages (Raw264.7) in order to evaluate the apoptosis effect of Acinetobacter Baumani-derived vesicles.

14 is a pre-treatment of Acinetobacter bacteria-derived vesicles prior to treatment with Escherichia coli vesicles (E. coli EV) to evaluate the anti-inflammatory and immunomodulatory effects of Acinetobacter Baumani-derived vesicles. The results of the evaluation of the mediators on the secretion of IL-6 and TNF-α.

Fig. 15 is an experimental protocol in which an acinetobacter Baumani-derived vesicle was administered to a mouse in order to evaluate the anticancer efficacy of the acinetobacter Baumani-derived vesicles.

FIG. 16 is a graph evaluating the effect on tumorigenesis by cancer cells by administering Acinetobacter Baumani vesicles intraperitoneally (IP) or orally (PO) in order to evaluate the anticancer efficacy of Acinetobacter Baumani vesicles. to be.

Figure 17 is a tumor to evaluate the antitumor effect of Acinetobacter Baumani-derived vesicles, by administering Acinetobacter Baumani vesicles intraperitoneally (IP) or oral (PO), to evaluate the effect on tumor development by cancer cells It is a photograph.

The present invention relates to vesicles derived from the genus Acinetobacter and to their use.

The present inventors have found that the metagenome analysis shows that the bacterial vesicles of the genus Acinetobacter are significantly reduced in clinical samples of patients with cancer, cardiovascular disease, metabolic disease, and neuropsychiatric disease compared to normal people. Confirmed that it can. In addition, as a result of separating and characterizing the vesicles from the Acinetobacter Baumani strain, it was confirmed that the strain-derived vesicles can regulate immune function abnormalities caused by pathogenic vesicles.

Accordingly, the present invention provides an information providing method for diagnosing gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease, comprising the following steps:

(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And

(c) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes mellitus when the content of bacteria-derived vesicles in Acinetobacter is lower than that of normal people through quantitative analysis of the PCR products Classifying as dementia, dementia, or Parkinson's disease.

As used herein, the term "diagnosis" means, in a broad sense, to determine the actual condition of a patient in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the onset of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, and Parkinson's disease and the level of the disease.

In the present invention, the sample may be feces, blood, or urine, but is not limited thereto.

In the present invention, the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.

The term used in the present invention, metagenome, also referred to as a military genome, refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil and animal intestine. It is used as a concept of genome to explain the identification of many microorganisms at once using sequencer for analysis. In particular, the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species. Technically, rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.

As another embodiment of the present invention, the present invention comprises a vesicle derived from the genus Acinetobacter as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease And it provides a composition for the prevention or treatment of one or more diseases selected from the group consisting of inflammatory diseases. In the present invention, the composition includes a pharmaceutical composition, an oral composition, or an inhalant composition, and may be a formulation of an oral spray or a nasal spray.

As used herein, the term “inflammatory disease” refers to a disease caused by an inflammatory response in a mammalian body. In the present invention, the inflammatory disease is atopic dermatitis, acne, psoriasis, sinusitis, rhinitis. , Conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathy, undifferentiated arthritis, arthritis, inflammatory osteolysis, virus Or bacterial inflammatory diseases, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, aneurysm- Borreliasis, Nucleus, Sarcoidosis, Lupus, Alumni Lupus, Tuberculosis Lupus, Lupus Nephritis, Systemic Lupus Erythematosus, Macular Degeneration, Uveitis, Irritable Bowel Syndrome, Crohn's Disease, Sjogren's Syndrome, Fibromyalgia, Chronic Fatigue Syndrome, Chronic Fatigue Immunity At least one selected from the group consisting of dysfunction syndrome, myalgia encephalomyelitis, amyotrophic lateral sclerosis, Parkinson's disease, and multiple sclerosis, but is not limited thereto.

As used herein, the term prophylaxis means any action that inhibits or retards the disease by administration of a composition according to the present invention.

As used herein, the term improvement means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.

As used herein, the term "treatment" refers to any action in which symptoms for the disease are improved or beneficially altered by administration of a composition according to the present invention.

The term used in the present invention, nanovesicles (Nanovesicle) or vesicles (Vesicles), refers to the structure of the nano-size membrane secreted by various bacteria. Vesicles or outer membrane vesicles (OMVs) derived from gram-negative bacteria have proteins, low molecular weight compounds, and bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria. In addition to proteins, low molecular weight compounds and nucleic acids, the vesicles also contain peptidoglycan and lipoteichoic acid. In the present invention, the nano vesicles or vesicles are naturally secreted or artificially produced from bacteria of the genus Acinetobacter, and have an average diameter of 20 to 100 nm.

The vesicle is centrifuged, ultra-fast centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filter by the culture medium containing bacteria of the genus Acinetobacter The separation can be carried out using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.

The pharmaceutical composition according to the invention may comprise 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 injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.

The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug and the drug. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg daily or every other day, per kg of body weight Or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

As another aspect of the present invention, the present invention comprises a vesicle derived from the genus Acinetobacter as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson Provided is a food composition for preventing or ameliorating at least one disease selected from the group consisting of a disease and an inflammatory disease.

The food composition of the present invention includes a nutraceutical composition. The food composition according to the present invention may be used as it is, or may be used in combination with other foods or food ingredients, or may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.

The food composition of the present invention, in addition to containing the active ingredient as an essential ingredient in the indicated ratio, there are no particular restrictions on other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.

In the inhalant composition of the present invention, the active ingredient may be added to the inhalant as it is, or may be used together with other ingredients, and may be appropriately used according to conventional methods. The amount of the active ingredient to be mixed may be suitably determined depending on the purpose of use (prevention or treatment).

In the present invention, the inflammatory disease may be a disease mediated by IL-6 or TNF-α, but is not limited thereto.

As another aspect of the present invention, the present invention provides a cosmetic composition for preventing or ameliorating an inflammatory disease comprising a vesicle-derived bacteria of the genus Acinetobacter as an active ingredient.

In the present invention, the cosmetic composition may be used for preventing or ameliorating an inflammatory disease selected from the group consisting of atopic dermatitis, acne, and psoriasis, but is not limited thereto.

Cosmetic compositions of the present invention may include components commonly used in cosmetic compositions, as well as vesicles derived from the genus Acinetobacter, and include, for example, conventional agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and perfumes. Adjuvants, and carriers.

In addition, the composition of the present invention may be used in addition to the acetobacter bacteria-derived vesicles, and a combination of organic sunscreens that have been conventionally used insofar as they do not impair the skin protection effect by reacting with the acetobacter bacteria-derived vesicles. Examples of the organic sunscreen include glyceryl pava, drometrizole trisiloxane, drometrizole, digaloyltrioleate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexyl butamidotriazone, diethylamino Hydroxybenzoylhexylbenzoate, die-methoxycinnamate, a mixture of lowson and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranilate, benzophenone -3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxyphenbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyldihydroxypropylpava, Octocrylene, ethylhexyldimethylpava, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltrizone, isoamyl-p-methoxycinnamate, polysilicon-15 (dimethicodiethylbenzalmal Ronate), terephthalylidene dicamphor sulfonic acid and salts thereof, thy-salicylate and aminobenzoic acid (Pava) can be used.

Examples of products to which the cosmetic composition of the present invention may be added include, for example, cosmetics such as astringent cosmetics, soft cosmetics, nourishing cosmetics, various creams, essences, packs, foundations, and the like, cleansing agents, soaps, treatments, and essences. Etc. Specific formulations of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, Formulations such as soaps, shampoos, cleansing foams, cleansing lotions, cleansing creams, body lotions, body cleansers, emulsions, lipsticks, makeup bases, foundations, press powders, loose powders, eye shadows and the like.

In an embodiment of the present invention, the bacteria and bacterial-derived vesicles were orally administered to mice to observe the absorption, distribution, and excretion of the bacteria and vesicles in the body. It was confirmed that it was absorbed within 5 minutes of administration and distributed systemically and excreted through the kidneys, liver, and the like (see Example 1).

In another embodiment of the present invention, the stool, blood of a normal person matched age and sex to patients with gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, Parkinson's disease, and dementia Bacterial metagenome analysis was performed using vesicles isolated from or urine. As a result, compared to normal samples, vesicles derived from Acinetobacter bacteria were more significant in samples of patients with gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, Parkinson's disease, and dementia. It was confirmed that it is reduced (see Examples 3 to 13).

In another embodiment of the present invention, based on the results of the above example further studies to analyze the characteristics of the acinetobacter Baumani-derived vesicle belonging to the genus Acinetobacter bacteria, by culturing the Acinetobacter Baumani strain The vesicles secreted from these samples were evaluated for anti-inflammatory effects. After treating various concentrations of Acinetobacter Baumani-derived vesicles with macrophages, E. coli-derived vesicles, the causative agent of inflammatory diseases, were evaluated for inflammatory mediator secretion. As a result, it was confirmed that the acinetobacter Baumani-derived vesicles effectively suppressed IL-6 and TNF-α secretion by E. coli-derived vesicles, which are pathogenic vesicles (see Example 15).

In another embodiment of the present invention, the culture of the Acinetobacter Baumani culture was evaluated to evaluate whether the vesicles secreted therefrom exhibit an anticancer therapeutic effect. To this end, a cancer model was made by subcutaneously injecting a cancer cell line, and the size of the cancer tissue was measured for 20 days after oral or intraperitoneal administration of the acinetobacter Baumani-derived vesicles to the mouse 4 days before the cancer cell line treatment. When the vesicles were administered orally, the size of the cancerous tissue was reduced compared to the control group (see Example 16).

Hereinafter, preferred examples are provided to aid in understanding 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.

EXAMPLE

Example 1 Analysis of Uptake, Distribution, and Excretion of Intestinal Bacteria and Bacterial-Derived Vesicle

In order to evaluate whether the intestinal bacteria and bacteria-derived vesicles are absorbed systemically through the gastrointestinal tract, experiments were performed as follows. Fluorescently labeled enterobacteriaceae and enteric bacteria-derived vesicles were administered to the gastrointestinal tract at doses of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the entire image of the mouse, as shown in Fig. 1A, the bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder fluorescence after 3 hours after administration. This was observed strongly, indicating that the vesicles were excreted by the urinary system. In addition, the vesicles were found to exist in the body up to 12 hours after administration (see FIG. 1A).

After the intestinal bacteria and enteric bacteria-derived vesicles were absorbed systemically, in order to evaluate the invasion of various organs, 50 μg of fluorescently labeled bacteria and vesicles derived from the fluorescein were administered as described above for 12 hours. Blood, heart, liver, kidneys, spleen, fat and muscle were collected later. As a result of fluorescence observation in the collected tissue, as shown in FIG. 1B, the vesicle-derived bacteria were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle and kidney, but bacteria were not absorbed. (See FIG. 1B).

Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples

Clinical samples of feces, blood, and urine were first placed in 10 ml tubes and the suspension was allowed to settle by centrifugation (3,500 × g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign substances using a 0.22㎛ filter, it was transferred to centripreigugal filters (50 kD) and centrifuged at 1500 x g and 4 ° C for 15 minutes to discard materials smaller than 50 kD and concentrated to 10 ml. Once again, the bacteria and foreign substances were removed using a 0.22㎛ filter, and the supernatant was discarded using ultra-centrifugation for 3 hours at 150,000 xg and 4 ℃ using a Type 90ti rotor, and the lumped pellet was dissolved in physiological saline (PBS). .

100 μl of the vesicles separated by the above method was boiled at 100 ° C. to let the internal DNA come out of the lipid and then cooled on ice for 5 minutes. And centrifuged at 10,000 x g, 4 ℃ 30 minutes to remove the remaining suspended solids and collected only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rDNA primer shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene exists in the extracted gene.

primer		order	SEQ ID NO:
16S rDNA	16S_V3_F	5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 '	One
	16S_V4_R	5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3	2

DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9). After converting the SFF file into a sequence file (.fasta) and a nucleotide qualityscore file, the credit rating of the lead was confirmed, and the window (20 bps) average base call accuracy was less than 99% (Phred score <20). . For operational taxonomy unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level. Was profiled (QIIME).

Example 3 Analysis of Vesicle Metagenome Derived from Stool and Urine Bacteria of Gastric Cancer Patients

In the method of Example 2, the genes were extracted from the vesicles in the stool and 67 feces of gastric cancer patients and 198 normal controls matched with sex and age were subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles of the genus Acinetobacter were significantly reduced in the stool of gastric cancer patients compared to the normal stool (see Table 2 and FIG. 2).

credit	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0134	0.0222	0.0063	0.0073	<0.0001	0.47

In the method of Example 2, the urine of 61 patients with gastric cancer and the urine of 120 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the urine of gastric cancer patients compared to normal urine (see Table 3 and FIG. 2).

Pee	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0776	0.1128	0.0338	0.0407	0.0007	0.44

Example 4 Analysis of Vesicle Metagenome Derived from Urine Bacteria of Colorectal Cancer Patients

In the method of Example 2, the urine of 52 colon cancer patients and the urine of 83 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenome analysis. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the urine of colorectal cancer patients compared to normal urine (see Table 4 and FIG. 3).

Pee	Control		Colorectal cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.1683	0.1396	0.0172	0.0138	<0.0001	0.10

Example 5 Analysis of Vesicular Metagenome Derived from Blood and Urine Bacteria of Breast Cancer Patients

In the method of Example 2, the genes were extracted from vesicles in the blood of 96 breast cancer patients and 192 blood of the normal control group matched with sex and age, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 5 and FIG. 4).

blood	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0390	0.0328	0.0160	0.0228	<0.0001	0.41

In the method of Example 2, the genes were extracted from vesicles in urine of 127 breast cancer patients and 220 urine control groups matched with sex and age, and then subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the urine of breast cancer patients compared to normal urine (see Table 6 and FIG. 4).

Pee	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0761	0.1046	0.0198	0.0264	<0.0001	0.26

Example 6 Analysis of Vesicle Metagenome Derived from Urine Bacteria in Ovarian Cancer Patients

In the method of Example 2, 136 urine patients with ovarian cancer and 136 urine in the normal control group matched with sex and age were extracted from the vesicles present in the urine, followed by metagenome analysis. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the bacterial vesicles of the genus Acinetobacter were significantly reduced in the urine of ovarian cancer patients compared to normal urine (see Table 7 and FIG. 5).

Pee	Control		Ovarian Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0816	0.1097	0.0338	0.0223	<0.0001	0.41

Example 7 Analysis of Vesicle Metagenome Derived from Urinary Bacteria in Bladder Cancer Patients

The urine of 95 patients with bladder cancer and the urine of 157 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the urine of bladder cancer patients compared to normal urine (see Table 8 and FIG. 6).

Pee	Control		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0583	0.1003	0.0162	0.0132	0.0001	0.28

Example 8. Vesicular Metagenome Analysis of Blood Bacteria from Myocardial Infarction Patients

The blood of 57 patients with myocardial infarction and the blood of 163 normal controls matched with sex and age were extracted from the vesicles present in the blood and subjected to metagenome analysis, followed by acinetobacter. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicles derived from Acinetobacter bacteria were significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 9 and FIG. 7).

blood	Control		Myocardial infarction		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0343	0.0391	0.0109	0.0254	<0.0001	0.32

Example 9 Vesicular Metagenome Analysis of Blood Bacteria from Cardiomyopathy Patients

In the method of Example 2, the blood of 72 patients with cardiomyopathy and the blood of 163 normal controls matched with sex and age were extracted from the vesicles present in the blood, followed by metagenome analysis, followed by acinetobacter. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter was significantly reduced in the blood of cardiomyopathy patients compared to normal blood (see Table 10 and FIG. 8).

blood	Control		Cardiomyopathy		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0343	0.0391	0.0162	0.0208	<0.0001	0.47

Example 10 Analysis of Vesicular Metagenome Derived from Diabetic Blood and Urine Bacteria

In the method of Example 2, blood was extracted from vesicles in the blood of 61 diabetic patients and 122 blood of the normal control group which matched gender and age. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the blood of diabetic patients compared to normal blood (see Table 11 and FIG. 9).

blood	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0369	0.0442	0.0003	0.0003	<0.0001	0.01

The urine of 60 diabetic patients and the urine of 134 normal controls matched with gender and age were extracted by the method of Example 2, and the genes were extracted from the vesicles present in the urine, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in urine of diabetic patients compared to normal urine (see Table 12 and FIG. 9).

Pee	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0564	0.0928	0.0157	0.0157	<0.0001	0.28

Example 11 Parcel Metagenome Analysis from Blood Bacteria from Stroke Patients

In the method of Example 2, the genes were extracted from the vesicles in the blood of 115 stroke patients and 109 blood of the normal control group matched with sex and age, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the blood of stroke patients compared to normal blood (see Table 13 and FIG. 10).

blood	Control		stroke		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0594	0.0803	0.0280	0.0573	<0.0001	0.47

Example 12 Analysis of Vesicular Metagenome Derived from Urinary Bacteria in Parkinson's Disease Patients

The urine of 39 Parkinson's disease patients and urine of 76 normal controls matched to gender and age was extracted by extracting genes from vesicles present in urine, followed by metagenome analysis. Distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the urine of Parkinson's disease patients compared to normal urine (see Table 14 and FIG. 11).

Pee	Control		Parkinson's disease		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0690	0.1047	0.0020	0.0022	<0.0001	0.03

Example 13 Analysis of Vesicular Metagenome Derived from Demented Patients with Blood Bacteria

In the method of Example 2, blood was collected from 67 blood of dementia patients and 70 blood of the normal control group which matched gender and age, and the genes were extracted from the vesicles present in the blood, followed by metagenome analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Acinetobacter were significantly reduced in the blood of dementia patients compared to normal blood (see Table 15 and FIG. 12).

blood	Control		dementia		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Acinetobacter	0.0400	0.0380	0.0190	0.0190	<0.0001	0.48

Example 14. Acinetobacter Baumani Culture and Vesicle Separation

After culturing A. baumannii strains, the vesicles were isolated and characterized. The Acinetobacter Baumani strain was cultured in NB (Nutrient broth) medium until the absorbance (OD 600) was 1.0-1.5 in an aerobic chamber at 37 ° C. and then sub-cultured to LB (Luria Bertani broth) medium. Then, the culture medium supernatant containing the strain was recovered and centrifuged at 10,000 g, 4 ° C. for 20 minutes, and then the strain was removed and filtered through a 0.22 μm filter. The filtered supernatant was concentrated to 50 ml volume by microfiltration using a MasterFlex pumpsystem (Cole-Parmer, US) with a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US). The concentrated supernatant was once again filtered through a 0.22 μm filter. Thereafter, the protein was quantified by BCA assay, and the following experiments were performed on the obtained vesicles.

Example 15 Anti-inflammatory Effect of Acinetobacter Baumani Derived Vesicles

To investigate the effects of acinetobacter Baumani-derived vesicles on apoptosis in inflammatory cells, A. baumannii EVs ( A. baumannii EV) were collected in raw 264.7 cells of mouse macrophage. 10 μg / ml), followed by Cell viability test. More specifically, the raw 264.7 cells divided into 4 x 10 ⁴ cells in 48-well cell culture plates were treated with various concentrations of Acinetobacter Baumani-derived vesicles loaded with DMEM serum-free medium and cultured for 12 hours. After the cells were treated with EZ-CYTOX (Dogen, Korea) for 4 hours, the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devies, USA). As a result, as shown in Fig. 12, when acinetobacter Baumani-derived vesicles were treated with macrophages, apoptosis was not induced at concentrations of 0.1 and 1 µg / ml, but at about 10% at concentration of 10 µg / ml. It was confirmed that apoptosis was induced (see FIG. 13).

Based on the above results, in order to evaluate the anti-inflammatory effect of Acinetobacter Baumani-derived vesicles, various concentrations (0.1, 1, 10 μg / ml) of Acinetobacter Baumani-derived vesicles were pretreated in macrophage lines for 12 hours. Subsequently, the secretion of inflammatory cytokines was measured by ELISA 12 hours after treatment with 1 μg / ml of Escherichia coli-derived vesicles, a pathogenic vesicle. In order to perform ELISA, Capture antibody was diluted in PBS, and 50 μl was dispensed into 96 well polystyrene plates according to the working concentration, followed by overnight reaction at 4 ° C. After washing three times with 100 μl PBST (PBS containing 0.05% tween-20) solution, 100 μl of RD (PBS containing 1% BSA) solution was dispensed and blocked for 1 hour at room temperature. Samples and standards were dispensed 50 μl according to the concentration and reacted at room temperature for 2 hours. After washing three times with 100 μl of PBST, the detection antibody was diluted in RD, and 50 μl was dispensed at a working concentration for 2 hours at room temperature. After washing three times with 100 μl of PBST, Streptavidin-HRP (R & D system, USA) was diluted to 1/40 in RD, 50 μl were dispensed and reacted at room temperature for 20 minutes. Finally, after washing three times with 100 μl of PBST, 50 μl of TMB substrate (SurModics, USA) is dispensed, and when color development proceeds after 5-20 minutes, 50 μl of 1 M sulfuric acid solution is stopped to stop the reaction and SpectraMax. Absorbance was measured at 450 nm using an M3 microplate reader (Molecular Devices, USA). As a result, when pre-treated with acinetobacter Baumani-derived vesicles, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles is significantly inhibited (see Fig. 14). In particular, the effect of inhibiting the secretion of TNF-α by the pretreatment of Acinetobacter Baumani-derived vesicles was significantly greater than the effect of inhibiting the secretion of TNF-α by the pretreatment of Lactobacillus plantarum- derived vesicles. It was confirmed (see FIG. 14). The above results indicate that acinetobacter Baumani-derived vesicles can effectively inhibit inflammatory responses induced by pathogenic vesicles such as E. coli-derived vesicles.

Example  16. Acinetobacter Baumani  Anticancer Effect of Derived Vesicles

Based on the above examples, the anticancer effect of Acinetobacter Baumani-derived vesicles was examined. To this end, as shown in Fig. 15, vesicles derived from the Acinetobacter Baumani strain were administered intraperitoneally or orally to 6-week-old C57BL / 6 male mice, and injected subcutaneously with a cancer cell line (CT26 cell) on day 4 of administration. A cancer model was created. After administration of the cancer cell line, vesicles derived from the Acinetobacter Baumani isolate were administered daily by intraperitoneal injection or orally, and the size of the cancer tissue was measured until the 24th day (see FIG. 15). As a result, the size of the cancer tissue was significantly decreased in the mouse orally administered vesicles compared to the control group oral saline administration group, and the size of the cancer tissue was increased intraperitoneally (Fig. 16 and FIG. 17). This means that administration of acinetobacter Baumani-derived vesicles to the mucosa can effectively inhibit the growth of cancerous tissues.

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.

The vesicles derived from the genus Acinetobacter according to the present invention are gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or a method for diagnosing Parkinson's disease, and the disease or inflammatory disease. It is expected that the present invention can be usefully used in the composition for preventing, treating or improving food or drugs.

Claims (21)

1. Methods for providing information for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease, comprising the following steps:

   (a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

   (b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And

   (c) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes mellitus when the content of bacteria-derived vesicles in Acinetobacter is lower than that of normal people through quantitative analysis of the PCR products Classifying as dementia, dementia, or Parkinson's disease.
2. The method of claim 1,

   The sample in step (a) is characterized in that the blood, stool, or urine, information providing method.
3. Group consisting of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases, including vesicles derived from bacteria of the genus Acinetobacter A pharmaceutical composition for the prophylaxis or treatment of one or more diseases selected from.
4. The method of claim 3,

   The inflammatory diseases include atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, undifferentiated Spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis Sclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, anxiety-borelia, tuberculosis, Sarcoidosis, lupus, Alumni lupus, tuberculosis lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Disease, show Gran syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, muscular encephalomyelitis, amyotrophic lateral sclerosis, Parkinson seunbyeong, and that at least one selected from the group consisting of multiple sclerosis, characterized in, the pharmaceutical compositions.
5. The method of claim 3,

   The inflammatory disease is characterized in that the disease mediated by Interleukin-6 (IL-6) or tumor necrosis factor alpha (TNF-α), pharmaceutical composition.
6. The method of claim 3,

   The vesicles are characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
7. The method of claim 3,

   The vesicles are characterized in that the natural or artificial secretion from the bacteria of the genus Acinetobacter , pharmaceutical composition.
8. The method of claim 3,

   The Acinetobacter (Acinetobacter) in bacterial-derived vesicles Acinetobacter baumannii (Acinetobacter baumannii), pharmaceutical composition, characterized in that the packet originated.
9. Group consisting of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases, including vesicles derived from bacteria of the genus Acinetobacter Food composition for the prevention or amelioration of one or more diseases selected from.
10. The method of claim 9,

    The inflammatory diseases include atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory enteritis, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, undifferentiated Spondyloarthropathy, undifferentiated arthrosis, arthritis, inflammatory osteolysis, chronic inflammatory disease caused by viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, scleroderma, osteoporosis, atherosclerosis Sclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, syphilis, Lyme disease, Borreliosis, anxiety-borelia, tuberculosis, Sarcoidosis, lupus, Alumni lupus, tuberculosis lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, irritable bowel syndrome, Disease, show Gran syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immune dysfunction syndrome, muscular encephalomyelitis, amyotrophic lateral sclerosis, Parkinson seunbyeong, and that at least one selected from the group consisting of multiple sclerosis, characterized in, a food composition.
11. The method of claim 9,

    The inflammatory disease is characterized in that the disease mediated by Interleukin-6 (IL-6) or tumor necrosis factor alpha (TNF-α), food composition.
12. The method of claim 9,

    The vesicles are characterized in that the average diameter of 10 to 200 nm, food composition.
13. The method of claim 9,

    The vesicles are characterized in that the natural or artificial secretion from the bacteria of the genus Acinetobacter , food composition.
14. The method of claim 9,

    The Acinetobacter (Acinetobacter) in bacterial-derived vesicles Acinetobacter baumannii (Acinetobacter baumannii), a food composition, characterized in that the resulting package.
15. Group consisting of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases, including vesicles derived from bacteria of the genus Acinetobacter Inhalant compositions for the prophylaxis or treatment of one or more diseases selected from.
16. Acinetobacter ( Acinetobacter ) comprising a vesicle derived from the bacteria as an active ingredient, a cosmetic composition for preventing or improving inflammatory diseases.
17. The method of claim 16,

    The inflammatory disease is characterized in that at least one selected from the group consisting of atopic dermatitis, acne, and psoriasis, cosmetic composition.
18. A method of diagnosing gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, or Parkinson's disease, comprising the following steps:

    (a) extracting DNA from extracellular vesicles isolated from normal and subject samples;

    (b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And

    (c) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes mellitus when the content of bacteria-derived vesicles in Acinetobacter is lower than that of normal people through quantitative analysis of the PCR products , Dementia, or Parkinson's disease.
19. The method of claim 18,

    The sample in step (a) is characterized in that the blood, stool, or urine, diagnostic method.
20. Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, comprising administering to the subject a pharmaceutical composition comprising a vesicle derived from bacteria of the genus Acinetobacter as an active ingredient A method of preventing or treating at least one disease selected from the group consisting of dementia, Parkinson's disease, and inflammatory disease.
21. One or more diseases selected from the group consisting of vesicles derived from bacteria of the genus Acinetobacter , gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, myocardial infarction, cardiomyopathy, stroke, diabetes, dementia, Parkinson's disease, and inflammatory diseases For prophylactic or therapeutic purposes.

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