WO2019139279A1 - Nanovesicles derived from morganella bacteria, and uses thereof - Google Patents

Abstract

The present invention relates to vesicles derived from Morganella bacteria and to uses thereof. It has been experimentally confirmed by the present inventors that the vesicles were significantly reduced in clinical samples derived from patients with malignant diseases such as gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, and lymphoma; cardiovascular diseases such as myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, and strokes; diabetes, and Parkinson's disease, in comparison with a normal person and that the vesicles inhibited carcinogenesis in cancer animal models while inhibiting the secretion of inflammatory mediators caused by pathogenic vesicles. The vesicles derived from Morganella bacteria according to the present invention will be usefully employed for the purposes of developing a method for diagnosing malignant diseases such as gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, and lymphoma; cardiovascular diseases such as myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, and strokes; diabetes, and Parkinson's disease, and a composition for preventing or treating said diseases or inflammatory diseases.

Description

Nano-vesicles derived from bacteria of Morganella and uses thereof

The present invention relates to a nano-vesicle derived from a bacterium belonging to the genus Morganella and a use thereof. More specifically, the present invention relates to a nano-vesicle derived from a bacterium belonging to the genus Morganella, A method for diagnosing cardiovascular diseases such as myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, etc., diabetes, and Parkinson's disease, and prevention or prevention of the above diseases or inflammatory diseases including the vesicles, ≪ / RTI >

In the 21st century, the importance of acute infectious diseases, which have been recognized as epidemic in the past, has become less important, while chronic diseases with immune dysfunction caused by incompatibility between human and microbiome have been linked to quality of life and life expectancy The disease pattern changed as a major disease. Cancer, cardiovascular disease, chronic pulmonary disease, metabolic disease, inflammatory disease, and neuropsychiatric disease are major diseases that determine human life and quality of life as a 21 st century intractable chronic disease.

Inflammation is a local or systemic defense mechanism against damage or infection of cells and tissues. It mainly affects the humoral mediator that is the immune system, or stimulates the local or systemic effector system It is caused by a series of biological reactions that occur. Major inflammatory diseases include gastrointestinal diseases such as gastritis and inflammatory bowel disease, oral diseases such as periodontitis, respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), rhinitis, skin diseases such as atopic dermatitis, hair loss, psoriasis, degenerative arthritis, Arthritis such as rheumatoid arthritis; And metabolic diseases such as obesity, diabetes, and liver cirrhosis. In addition, various studies have reported that sustained inflammation can cause cancer.

The number of microorganisms that are symbiotic to the human body is 10 times more than that of human cells, and the number of microorganisms is known to be over 100 times that of human genes. A microbiota or microbiome refers to a microbial community, including true bacteria, archaea, and eukarya in a given settlement.

Bacteria that coexist in our bodies and bacteria in the environment secrete nanometer-sized vesicles to exchange information such as genes, small molecules, and proteins into other cells. The mucous membrane forms a physical barrier that can not pass through particles of 200 nanometers (nm) or larger, and can not pass through the mucous membrane when the bacteria are symbiotic to the mucous membrane. However, since the bacterial-derived vesicles are less than 100 nanometers in size, It passes through epithelial cells through the mucosa and is absorbed by our body. The locally secreted bacterial-derived vesicles are absorbed through the mucosal epithelial cells or keratinocytes to induce a local inflammatory reaction, as well as being absorbed into our body and distributed in each organ to regulate immune and inflammatory responses in the organs absorbed do. For example, E. coli (Eshcherichia The vesicles derived from pathogenic Gram-negative bacteria such as E. coli , when absorbed into the blood vessels, promote the systemic inflammatory reaction and blood coagulation through the endothelial cell inflammatory reaction and are also absorbed into the muscle cells acting on the insulin, It triggers diabetes. On the other hand, beneficial bacteria-derived vesicles can regulate disease by controlling immune function and metabolic dysfunction by pathogenic vesicles (Choi YW et al., Gut microbe-derived extracellular vesicles induce insulin resistance, thereby impairing glucose metabolism in skeletal muscle. Scientific Reports, 2015.).

The immune response to bacterial-derived factors such as vesicles is a Th17 immune response characterized by IL-17 cytokine secretion, which upon exposure to bacterial-derived vesicles secretes IL-6, which induces a Th17 immune response. Th17 inflammation is characterized by neutrophil infiltration and TNF-alpha secreted by inflammatory cells such as macrophages plays an important role in the process of inflammation.

Morganella bacterium is an anaerobic gram-negative bacterium and is known to be a bacterium that symbiotes in the intestines of humans and animals. Among these bacteria, Morganella < RTI ID = 0.0 > morganii ) is known to be a pathogenic bacterium that causes postoperative infection or urinary tract infection. However, to date, there has been no report that bacterium Morganella secretes vesicles out of the cell, and applications for diagnosis and treatment of cancer, cardiovascular diseases, metabolic diseases, inflammatory diseases, and neuropsychiatric diseases have been reported There is no bar.

Accordingly, the present invention can diagnose disease by confirming that the vesicles derived from the genus Morganella are significantly reduced in clinical samples of patients suffering from cancer, cardiovascular diseases, metabolic diseases, inflammatory diseases, and neuropsychiatric disorders Respectively. In addition, the vesicles were isolated from Morganella Morgani and analyzed for their characteristics. As a result, it was confirmed that the vesicles could be used as a composition for preventing or treating malignant diseases, cardiovascular diseases, metabolic diseases, inflammatory diseases and neuropsychiatric diseases.

The present inventors have conducted intensive studies to solve the conventional problems as described above. As a result of the meta genome analysis, the present inventors have found that malignant diseases such as gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, , Myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes mellitus, and Parkinson's disease were significantly reduced in the clinical samples of patients with Morganella subspecies. Further, it was confirmed that when the vesicles were isolated from Morganella morganii belonging to the genus Morganella to treat macrophages, TNF-alpha secretion by the pathogenic vesicles was significantly inhibited, and thus the present invention was completed Respectively.

Accordingly, the present invention provides a method for diagnosis of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, And a method of providing the same.

The present invention also relates to a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, , Diabetes, Parkinson's disease, or inflammatory diseases.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can 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 provides a pharmaceutical composition for treating gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, Fibrinolysis, angina pectoris, stroke, diabetes, or Parkinson's disease:

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

(b) performing PCR on the extracted DNA using primer pairs prepared based on the gene sequences present in 16S rDNA to obtain respective PCR products; And

(c) Quantitative analysis of the PCR products revealed that the content of morganella bacteria-derived vesicles is lower than that of a normal person, and thus the gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, Cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, or Parkinson's disease.

The present invention also relates to a method for the treatment or prophylaxis of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, A method of diagnosing Parkinson's disease is provided:

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

(b) performing PCR on the extracted DNA using primer pairs prepared based on the gene sequences present in 16S rDNA to obtain respective PCR products; And

(c) Quantitative analysis of the PCR products revealed that the content of morganella bacteria-derived vesicles is lower than that of a normal person, and thus the gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, Cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, or Parkinson's disease.

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

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

The present invention also relates to a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, Angina pectoris, stroke, diabetes, Parkinson's disease, and inflammatory diseases. The present invention also provides a pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of angina pectoris, angina pectoris, stroke,

The present invention also relates to a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, Angina pectoris, stroke, diabetes, Parkinson's disease, and inflammatory diseases.

The present invention also relates to a method for treating cancer of the stomach, colon, pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, colon cancer, There is provided a method of preventing or treating at least one disease selected from the group consisting of lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, Parkinson's disease, and inflammatory diseases.

The present invention also relates to a method for the treatment and prophylaxis of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, , Parkinson's disease, and inflammatory diseases.

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 bowel disease, chronic obstructive pulmonary disease, sepsis, Inflammatory bowel disease, chronic inflammatory disease due to inflammatory osteolysis, viral or bacterial infection, colitis, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis Lyme disease, Borreliosis, Neurogenic-Borrelia, Tuberculosis, Sarcoidosis, Alzheimer's disease, Alzheimer's disease, Alzheimer's disease, Alzheimer's disease, Alzheimer's disease, Alzheimer's disease, Sarcoidosis, lupus, alopecia areata, tuberculosis lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis , 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 IL-6 or TNF-a.

In another 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 naturally or artificially secreted from Morganella spp.

In another embodiment of the present invention, the morgellella bacteria-derived vesicle may be secreted from Morganella Morgani.

The present inventors confirmed that intestinal bacteria are not absorbed into the body but they are absorbed into the body through epithelial cells in the case of bacterial-derived vesicles, and are excreted through the kidneys, liver, and lungs systemically, Colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke , Diabetes mellitus, and Parkinson's disease were significantly lower than those of normal persons. In addition, morganella morgani, a kind of bacterium belonging to the genus Morganella, was cultured in vitro to separate the vesicles and, when administered to the inflammatory cells in vitro, significantly suppressed the inflammatory mediator secretion by the pathogenic vesicles, The present inventors have found that the bacterium derived from Morganella bacterium according to the present invention is useful for the treatment of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, , Atrial fibrillation, angina pectoris, stroke, diabetes, and Parkinson's disease, and foods or medicines for the above diseases or inflammatory diseases.

FIG. 1A is a photograph showing distribution patterns of bacteria and vesicles by time after oral administration of bacteria and bacterial-derived vesicles (EV) to a mouse. FIG. 1B is a photograph of blood, kidney , Liver and various organs were extracted to evaluate the distribution patterns of bacteria and vesicles in the body.

FIGS. 2A to 2C show the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes existing in gastric cancer patients and the normal faeces (2a), blood (2b), and urine (2c) to be.

FIG. 3 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes present in colon cancer patients and normal urine.

FIG. 4 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes present in pancreatic cancer patients and normal human blood.

FIG. 5 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Morganella after carrying out the analysis of bacterium-derived vesicle metagenomes present in patients with biliary cancer and normal human blood.

FIG. 6 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Morganella after carrying out the analysis of microbial-derived vesicle metagenomes present in breast cancer patients and normal urine.

FIGS. 7A and 7B show the results of a comparison of the distribution of vesicles derived from Morganella subsp. Bacterium after the analysis of the bacterial-derived vesicle metagenomes existing in ovarian cancer patients and normal blood (7a) and urine (7b).

FIGS. 8A and 8B are the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of bacterial-derived vesicle metagenomes existing in bladder cancer patients and normal blood (8a) and urine (8b).

Fig. 9 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes present in prostate cancer patients and normal urine.

Fig. 10 shows the results of a comparison of the distribution of vesicles derived from Morganella subsp. Bacterium after the analysis of the bacterial-derived vesicle metagenomes in lymphoma patients and normal blood.

FIG. 11 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes existing in the myocardial infarction patient and the normal blood.

FIG. 12 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after carrying out the analysis of microbial-derived vesicle metagenomes present in blood of cardiomyopathy patients and normal subjects.

FIG. 13 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes existing in the blood of atrial fibrillation and normal human.

FIG. 14 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after carrying out the analysis of the bacterial-derived vesicle metagenomes present in blood of the patients with angina pectoris and normal.

15 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes present in the blood of a stroke patient and a normal person.

16A and 16B are the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes existing in the blood (16a) and the urine (16b) of diabetic patients and normal subjects.

Fig. 17 shows the results of a comparison of the distribution of vesicles derived from the genus Morganella after the analysis of the bacterial-derived vesicle metagenomes present in Parkinson's disease patients and normal urine.

18 is a result of evaluating the cell death effect of Morganella morgani-treated vesicles by treating macrophages derived from Morganella morgani to evaluate the apoptotic effect of vesicles derived from Morganella morgani.

19A and 19B show the results of pretreatment of Morganella Morgani vesicles prior to the treatment of E. coli EV, a pathogenic vesicle, to evaluate the anti-inflammatory effect of Morganella morganii-derived vesicles, (19a) and TNF-alpha (19b) secreted by the transfected cells.

Figure 20 is a know the Nella know going to compare the effect of each other, derived from different strains package for anti-inflammatory properties of the resulting package, separated from the other before the parcel pathogenic E. coli vesicles (E. coli EV) processing know (NC: negative control; PC: positive control; L. plantarum: Lactobacillus plantarum) was prepared by pretreating vesicles derived from Kanera morgani (MMR101, MMR201) and evaluating the effect on the secretion of TNF-α by E. coli vesicles.

21 is a graph showing the effect of heat treatment or acid treatment on the antiinflammatory effect of Morganella morgani-deficient vesicles in the presence of heat treated or acid treated Morganella Morgana before treatment with E. coli EV, a pathogenic vesicle, (NC: negative control; PC: positive control; L. plantarum: Lactobacillus plantarum) was pre-treated with vesicles derived from MMR101 and MMR201 and evaluated for TNF-α secretion by E. coli vesicles.

22 is a protocol in which a vesicle derived from Morganella morgani is administered to a mouse to evaluate the anticancer efficacy of Morganella morgani-derived vesicles.

23 shows the results of evaluating the effect of morganella morganis vesicles on the tumor development by cancer cells (IP) or oral administration (PO) in order to evaluate the anticancer efficacy of vesicles derived from Morganella morgani to be.

The present invention relates to vesicles derived from Morganella spp. And their use.

The present inventors have found that metagenomic analysis enables the diagnosis of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, It was confirmed that the content of Morganella microbes-derived vesicles was significantly reduced in the sample derived from the diseased patient, and the present invention was completed on the basis thereof.

Accordingly, the present invention provides a method of treating or preventing gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, Providing a method for providing information for diagnosis of Parkinson's disease.

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

(b) performing PCR on the extracted DNA using primer pairs prepared based on the gene sequences present in 16S rDNA to obtain respective PCR products; And

(c) Quantitative analysis of the PCR products revealed that the content of morganella bacteria-derived vesicles is lower than that of a normal person, and thus the gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, Cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, or Parkinson's disease.

The term " diagnosis " used in the present invention means, in a broad sense, judging the actual conditions of a patient in all aspects. The contents of the judgment are the pathology, etiology, pathology, severity, details of the disease, presence of complications, and prognosis. In the present invention, the diagnosis is made based on whether or not the disease is gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angelic angina, stroke, diabetes, And so on.

As used herein, the term "nanovesicle" or "vesicle" means a nano-sized membrane structure secreted by various bacteria. Gram-negative bacteria-derived vesicles or outer membrane vesicles (OMVs) have not only lipopolysaccharides but also toxic proteins and bacterial DNA and RNA, and gram-positive bacteria-derived vesicles Has peptidoglycan and lipoteichoic acid which are cell wall components of bacteria as well as protein and nucleic acid. In the present invention, nano-vesicles or vesicles are naturally secreted or artificially produced in bacteria belonging to the genus Morganella, and are spherical in shape and have an average diameter of 10 to 200 nm.

The vesicles can be obtained by culturing a culture containing Morganella bacterium by centrifugation, ultracentrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freezing-thawing, electroporation, mechanical degradation, May be separated using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. Further, it may further include processes such as washing for removal of impurities and concentration of the resulting vesicles.

The term "metagenome" as used in the present invention refers to the total of all genomes including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal field, etc., and refers to a microorganism It is used as a concept of a genome to explain the identification of many microorganisms at once using a sequencer for analysis. In particular, a metagenome is not a genome or a genome, but a kind of mixed genome as a genome of all species of an environmental unit. This is a term derived from the viewpoint that when defining a species in the course of omics biology development, it functions not only as an existing species but also as a species that interacts with various species to form a complete species. Technically, it is the subject of techniques that analyze all DNA and RNA regardless of species, identify all species in an environment, identify interactions, and metabolism using rapid sequencing.

In the present invention, the sample in step (a) may be blood, urine, or feces, but is not limited thereto.

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

In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating gastric cancer, colon cancer, pancreatic cancer, breast cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, , Atrial fibrillation, angina pectoris, stroke, diabetes, Parkinson's disease, and inflammatory diseases.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, There is provided a food composition for the prevention or amelioration of at least one disease selected from the group consisting of inflammatory diseases, pathologies, atrial fibrillation, angioedema, stroke, diabetes, Parkinson's disease, and inflammatory diseases.

In the present invention, the inflammatory diseases are selected from the group consisting of atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, sepsis, Inflammatory bowel disease, ulcerative colitis, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, arthritis, osteoarthritis, arthritis, osteoarthritis, , Lyme disease, Borreliosis, Neurogenic-Borrelia, Tuberculosis, Sarcoidosis (including, but not limited to, osteoporosis, atherosclerosis, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves disease, Sarcoidosis), lupus, albuminous lupus, tuberculous lupus, lupus nephritis, systemic lupus erythematosus, macular degeneration, uveitis, Atherosclerosis, Parkinson's disease, and multiple sclerosis, but the present invention is not limited thereto, and the present invention is not limited thereto. It does not.

In the present invention, the inflammatory disease may be a disease mediated by interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-a) Do not.

As used herein, the term " prevention " refers to all actions that inhibit or delay the onset of cancer, inflammatory disease, cardiovascular disease, metabolic disease, or neuropsychiatric disorder by administration of the food or pharmaceutical composition according to the present invention do.

The term " treatment " used in the present invention refers to any action that improves or alleviates symptoms of cancer, inflammatory disease, cardiovascular disease, metabolic disease, or neuropsychiatric disease by administration of the pharmaceutical composition according to the present invention .

The term " improvement " used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

In one embodiment of the present invention, bacterial and bacterial-derived vesicles were orally administered to mice to evaluate the absorption, distribution, and excretion of bacteria and vesicles in the body. In the case of bacteria, the vesicles were not absorbed through the intestinal membrane, And was excreted through kidneys, liver, and the like (see Example 1).

In another embodiment of the present invention there is provided a method of treating a patient suffering from gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, Were analyzed for bacterial metagenomes using vesicles isolated from the blood, urine, or feces of healthy individuals that matched age and gender. As a result, it was found that patients with gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, and Parkinson's disease It was confirmed that the sample contained morphologically reduced vesicles derived from Morganella (see Examples 3 to 15).

In another embodiment of the present invention, in order to evaluate the cytotoxic effect of M. morganii EV in inflammatory cells, the mouse macrophage cell line, Raw 264.7 cells, was inoculated with morganella morganii (MMR101, (MMR101, MMR201, MMR202) were treated with various concentrations (0.1, 1, 10 ㎍ / ㎖) and then evaluated for apoptosis. As a result, Was not observed (see Example 17).

In a further embodiment of the present invention, further studies were carried out to analyze the characteristics of Morganella Morgani bacterium-derived vesicles belonging to the genus Morganella based on the results of the above Examples. As a result, a strain of Morganella morgani And then examined for the anti-inflammatory effect of the vesicles secreted from the cells. After treating the vesicles derived from Morganella morgani with various concentrations of macrophages, the cells were treated with Escherichia coli-derived vesicles, As a result of the evaluation, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles effectively suppressed vesicles derived from Morganella morgani (see Example 18).

In another embodiment of the present invention, in order to evaluate the effect of the heat treatment or the acid treatment on the anti-inflammatory action of the mor- phanella morganis-derived vesicle, heat treatment or acid treatment of vesicles before treating the macrophages-derived vesicles is carried out . As a result, it was confirmed that the effect of suppressing the secretion of TNF-α by the vesicles derived from Morganella morganii was not changed by heat treatment or acid treatment (see Example 19).

In another embodiment of the present invention, a strain of Morganella morgani was cultured to evaluate whether the vesicles secreted therefrom had an anticancer therapeutic effect. For this purpose, a cancer model was prepared by subcutaneous injection of a cancer cell line, and the morphology of cancer tissues was measured for 20 days after oral administration of morganella morgani-derived vesicles administered orally or intraperitoneally to mice from 4 days before the cancer cell line treatment. When the vesicles were administered intraperitoneally and orally, the size of cancer tissues was decreased compared with the control group, and it was confirmed that the vesicles were remarkably decreased when administered orally (see Example 20).

The pharmaceutical composition according to the present invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the formulation and include, but are not limited to, saline, sterilized water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. In addition, it may be formulated into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into an injection, an inhalant, an external preparation for skin, or an oral ingestion agent.

The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenous, subcutaneous, skin, nasal, or airway) according to the desired method, The type of administration, the route of administration, and the time, but may be suitably 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, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type of disease, severity, activity of the drug, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal 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 body weight of the patient. Generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

The food composition of the present invention comprises a health functional food composition. The food composition according to the present invention can be used as it is or in combination with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range.

The food composition of the present invention has no particular limitation on the ingredients other than those containing the active ingredient as an essential ingredient in the indicated ratios 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 polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above . The ratio of the above-mentioned natural carbohydrate can be appropriately determined by a person skilled in the art.

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

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Analysis of intestinal absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles

Experiments were carried out in the following manner to evaluate whether intestinal bacteria and bacterial - derived vesicles were systemically absorbed through the gastrointestinal tract. Fluorescence was measured at 0 min, 5 min, 3 hr, 6 hr, and 12 hr after administration of fluorescein-labeled intestinal bacteria and intestinal bacterial-derived vesicles in the stomach of mice to the gastrointestinal tract at a dose of 50 μg, respectively. As a result of observing the whole image of the mouse, it was not systemically absorbed through the intestinal mucosa when it was a bacterium as shown in Fig. 1A, but when it was a bacterium-derived vesicle, it was systemically absorbed 5 minutes after administration, Fluorescence was observed intensely in the bladder, and vesicles were excreted in the urinary tract. It was also found that the vesicles were present in the body for up to 12 hours of administration (see FIG. 1A).

After intestinal bacteria and intestinal bacteria-derived vesicles were systemically absorbed, 50 μg of fluorescently labeled bacteria and bacterial-derived vesicles were administered as described above to evaluate the invasiveness of various organs. After 12 hours of administration Later, blood, heart, liver, kidney, spleen, fat, and muscle were collected. As a result of observing the fluorescence in the collected tissues, it was found that the bacterium-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, muscle and kidney as shown in Fig. (See FIG. 1B).

Example 2. Analysis of bacterial-derived vesicle metagenomes in clinical samples

Clinical samples such as blood, urine, and stool were first placed in a 10 ml tube and the supernatant was transferred to a new 10 ml tube by centrifugation (3,500 x g, 10 min, 4 ° C). Bacteria and foreign matter were removed using a 0.22 μm filter, transferred to centripreigugal filters (50 kD), centrifuged at 1500 x g for 15 minutes at 4 ° C to discard substances smaller than 50 kD and concentrated to 10 ml. After removing the bacteria and foreign matter by using a 0.22 μm filter, the supernatant was discarded by using a high speed centrifugation method at 150,000 × g and 4 ° C. for 3 hours using a Type 90ti rotor, and the lumpy pellet was dissolved in physiological saline (PBS) .

100 쨉 l of the vesicles isolated by the above method were boiled at 100 째 C to allow the internal DNA to come out of the lipid, and then cooled on ice for 5 minutes. The supernatant was collected by centrifugation at 10,000 x g for 30 minutes at 4 ° C to remove the remaining suspension. The amount of DNA was quantified using Nanodrop. Then, PCR was performed with the 16s rDNA primer shown in Table 1 below to confirm whether the DNA extracted from the bacterium was present in the extracted DNA to confirm that the gene derived from the bacterium existed 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

The DNA extracted by the above method was amplified using the 16S rDNA primer described above, followed by sequencing (Illumina MiSeq sequencer), and the result was output to a Standard Flowgram Format (SFF) file and analyzed using GS FLX software (v2.9) (.Fasta) and nucleotide quality score files, then check the reliability of the lead, and remove the portion of the window (20 bps) with an average base call accuracy of less than 99% (Phred score <20) Respectively. In order to analyze Operational Taxonomy Unit (OTU), clustering is performed according to sequence similarity using UCLUST and USEARCH. Genus is 94%, family is 90%, order is 85%, class is 80%, phylum is 75% Clustering based on the genomic sequence, classification of phylum, class, order, family, genus level of each OTU and sequence homology of 97% or more at the genome level using BLASTN and GreenGenes 16S RNA sequence database (108,453 sequence) Bacteria were profiled (QIIME).

Example 3. Analysis of vesicle metagenomes from gastric cancer stool, blood and urine bacteria

Gene samples were extracted from the faecal feces in the stool of 63 stomach cancer patients and 126 normal stools matched with age and gender by the method of Example 2 to perform metagenomic analysis, The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the stool of gastric cancer patients were significantly reduced compared to normal stools (see Table 2 and Fig. 2a).

credit	Control group		Gastric cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0001	0.0007	0.0000	0.0002	0.0472	0.20

Genomic DNA was extracted from the vesicles present in the blood of 66 gastric cancer patients and 198 healthy persons whose age and sex were matched by the method of Example 2, and the results were analyzed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of patients with gastric cancer were significantly reduced compared with normal blood (see Table 3 and Fig. 2b).

blood	Control group		Gastric cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0016	0.0096	0.0001	0.0003	0.0247	0.05

In addition, in a urine sample of 61 gastric cancer patients and a urine sample of 120 healthy control subjects whose sex and age were matched with each other, genes were extracted from the vesicles present in the urine and metagenomic analysis was carried out. The distribution of vesicles derived from bacteria in Nella was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of the gastric cancer patients were significantly decreased compared to the normal urine (see Table 4 and Fig. 2c).

Pee	Control group		Gastric cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0082	0.0217	0.0000	0.0001	0.0004	0.00

Example 4. Analysis of vesicle metagenomes derived from urine bacteria in patients with colorectal cancer

The urine samples were collected from 38 patients with colorectal cancer and 38 healthy adults who matched the age and sex by the method of Example 2. The genes were extracted from the vesicles present in the urine and analyzed by metagenome. The distribution of the resulting vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella were significantly reduced in the feces of colon cancer patients compared to normal human urine (see Table 5 and FIG. 3).

Pee	Control group		Colon cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0211	0.0295	0.0013	0.0060	0.0002	0.06

Example 5: Analysis of vesicle meta-genome derived from blood bacteria of pancreatic cancer patients

A total of 176 pancreatic cancer patients and 271 healthy persons whose age and gender matched each other were examined by the method of Example 2 were subjected to metagenome analysis by extracting genes from the vesicles present in the blood, The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of the pancreatic cancer patients were significantly reduced compared to the normal blood (see Table 6 and FIG. 4).

blood	Control group		Pancreatic cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0020	0.0088	0.0000	0.0001	0.0003	0.00

Example 6: Analysis of vesicle meta-genome from blood bacterium of patients with cholangiocarcinoma

Blood samples were collected from 79 cases of cholangiocarcinoma patients and 259 healthy persons whose age and gender matched with the method of Example 2, and the genes were extracted from the vesicles present in the blood, and the results were analyzed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the bacterium Morganella in the blood of the patients with biliary cancer were significantly reduced compared to the normal blood (see Table 7 and FIG. 5).

blood	Control group		Bile duct cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0021	0.0091	0.0000	0.0000	0.0442	0.00

Example 7. Analysis of vesicle metagenomes derived from urine bacteria of breast cancer patients

In a urine sample of 220 breast cancer patients whose sex and age were matched to 127 breast cancer patients by the method of Example 2, the genes were extracted from the vesicles present in the urine and analyzed by metagenome, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of the breast cancer patients were significantly decreased compared to the normal urine (see Table 8 and FIG. 6).

Pee	Control group		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0090	0.0238	0.0002	0.0007	0.0000	0.02

Example 8. Analysis of vesicle metagenomes derived from blood and urine bacteria of ovarian cancer patients

137 genes of ovarian cancer patients and 139 healthy individuals whose age and sex were matched by the method of Example 2 were subjected to the metagenome analysis by extracting genes from the vesicles present in the blood, The distribution of the resulting vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of the ovarian cancer patients were significantly reduced compared to the normal blood (see Table 9 and Fig. 7a).

blood	Control group		Ovarian cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0012	0.0032	0.0000	0.0002	0.0000	0.02

In addition, in the urine of 136 ovarian cancer patients and the urine of 136 normal control group matching sex and age, the gene was extracted from the vesicles present in the urine and analyzed by metagenome, The distribution of bacterial-derived vesicles in the genus Kanera was evaluated. As a result, it was confirmed that the vesicles derived from Morganella spp. Were significantly reduced in the urine of ovarian cancer patients compared to normal urine (see Table 10 and Fig. 7b).

Pee	Control group		Ovarian cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0091	0.0243	0.0000	0.0002	0.0000	0.00

Example 9. Analysis of vesicle metagenomes from bladder cancer patient blood and urine bacteria

Genes were extracted from the vesicles present in the blood of 184 blasts of 96 normal bladder cancer patients and sex-matched normal controls by the method of Example 2, and then metagenomic analysis was carried out. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the bladder cancer patients were significantly reduced compared with normal blood (see Table 11 and Fig. 8a).

blood	Control group		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0027	0.0128	0.0000	0.0000	0.0488	0.00

In addition, in a urine of a bladder cancer patient and urine of a normal control group that matched sex and age in the urine of a bladder cancer patient according to the method of Example 2, a gene was extracted from the vesicles present in the urine, The distribution of vesicles derived from bacteria in Nella was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of the bladder cancer patients were significantly reduced compared to the normal urine (see Table 12 and Fig. 8b).

Pee	Control group		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0079	0.0224	0.0003	0.0012	0.0009	0.04

Example 10. Analysis of vesicle metagenomes derived from urine bacteria in patients with prostate cancer

In the urine of 53 men of prostate cancer and the urine of 159 normal control group matched sex and age by the method of Example 2, the genes were extracted from the vesicles present in the urine and the metagenomic analysis was carried out. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of the prostate cancer patients were significantly reduced compared to the normal urine (see Table 13 and FIG. 9).

Pee	Control group		Prostate cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0042	0.0169	0.0004	0.0010	0.0050	0.09

Example 11. Analysis of vesicle meta-genomic DNA derived from blood microbes of lymphoma patients

The genomic DNA was extracted from the vesicles present in the blood of 63 lymphoma patients and 53 healthy persons whose age and sex were matched by the method of Example 2 to analyze the metagenome, The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of the lymphoma patients were significantly reduced compared to the normal blood (see Table 14 and FIG. 10).

blood	Control group		Lymphoma		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0005	0.0030	0.0000	0.0000	0.1967	0.00

Example 12. Analysis of vesicle meta genome derived from blood microbes in patients with heart disease

Genomic analysis was performed on the blood of 57 myocardial infarction patients and the blood of 163 normal controls matched with sex and age by the method of Example 2, and the genes were extracted from the vesicles present in the blood, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of patients suffering from myocardial infarction were significantly reduced compared to normal blood (see Table 15 and FIG. 11).

blood	Control group		Myocardial infarction		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0022	0.0103	0.0004	0.0022	0.0400	0.19

In addition, the genome was extracted from the vesicles present in the blood of blood of 72 patients with dilated cardiac myopathy according to the method of Example 2, and 163 blood of a normal control group matched with sex and age, The distribution of bacteria derived from Morganella was evaluated. As a result, it was confirmed that the vesicles derived from Morganella spp. Were significantly reduced in the blood of patients with dilated cardiac myopathy compared with normal blood (see Table 16 and Fig. 12).

blood	Control group		Cardiomyopathy		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0022	0.0104	0.0002	0.0006	0.0133	0.08

In addition, 32 normal control subjects whose blood and sex and age matched the blood of 32 patients with atrial fibrillation according to the method of Example 2 were subjected to metagenome analysis by extracting genes from the vesicles present in the blood, The distribution of vesicles derived from bacteria in Nella was evaluated. As a result, it was confirmed that the vesicles derived from Morganella spp. In the blood of patients with atrial fibrillation were significantly reduced compared with normal blood (see Table 17 and Fig. 13).

blood	Control group		Atrial fibrillation		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0002	0.0010	0.0000	0.0000	0.1774	0.00

In addition, 80 genes of dysmorphic angina pectoris and 80 healthy individuals matched with age and gender were analyzed by the method of Example 2, and the genes were extracted from the vesicles present in the blood to perform metagenome analysis. Then, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella were significantly reduced in the blood of the patients with diastolic myopathy compared with the normal blood (see Table 18 and Fig. 14).

blood	Control group		Angina pectoris		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0028	0.0146	0.0005	0.0020	0.1736	0.18

Example 13. Analysis of vesicle meta-genomes derived from blood microbes in stroke patients

Genes were extracted from vesicles present in the blood of blood samples of 115 stroke patients and normal 109 matched age and sex patients according to the method of Example 2 and the results were analyzed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of the stroke patients were significantly reduced compared to the normal blood (see Table 19 and FIG. 15).

blood	Control group		stroke		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0006	0.0028	0.0001	0.0014	0.1331	0.22

Example 14. Analysis of vesicle meta genome derived from blood bacteria of diabetic patients

The genomic DNA was extracted from the vesicles present in the blood of blood of the 73 diabetic patients and the blood of 146 normal control group matched with sex and age by the method of Example 2, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the blood of the diabetic patients were significantly reduced compared to the normal blood (see Table 20 and FIG. 16a).

blood	Control group		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0054	0.0177	0.0003	0.0003	0.0012	0.05

In the urine of a normal person who matched 60 persons with diabetes mellitus and normal persons matched with age and sex by the method of Example 2, the gene was extracted from the vesicles present in the urine and the result was analyzed by metagenome, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of the diabetic patients were significantly reduced compared to the normal urine (see Table 21 and Fig. 16b).

Pee	Control group		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0025	0.0119	0.0000	0.0000	0.0245	0.00

Example 15. Analysis of vesicle metagenomes from urine bacteria of Parkinson's disease patients

In a urine sample of 39 normal control subjects whose urine and sex matched the age and sex of 39 patients with Parkinson's disease by the method of Example 2, the genes were extracted from the vesicles present in the urine and analyzed by metagenome, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Morganella in the urine of patients with Parkinson's disease were significantly reduced compared to normal urine (see Table 22 and FIG. 17).

Pee	Control group		Parkinson's disease		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Morganella	0.0066	0.0182	0.0003	0.0008	0.0037	0.05

Example 16. Isolation of vesicles from a morganella Morgani culture

Based on the above examples, the M. morganii strain was isolated from one of the standard strains (MMR101) and two isolates (MMR201 and MMR202) isolated from humans in the Korean Microorganism Conservation Center (KCCM) After culturing, the vesicles were separated from the culture solution and analyzed for their characteristics. M. morganii was subcultured in an LB (Luria-Bertani) medium until the absorbance (OD 600) reached 1.0 ~ 1.5 in a 37 ° C incubator. Then, the culture containing the strain was recovered and centrifuged at 10,000 g at 4 ° C for 20 minutes to remove the strain, which was then filtered through a 0.22 μm filter. The filtered supernatant was concentrated to a volume of 50 ml or less through a microfiltration using a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US) using a MasterFlex pump system (Cole-Parmer, US). The concentrated supernatant was again filtered through a 0.22 [mu] m filter. Then, proteins were quantified by BCA assay, and the following experiments were carried out on the vesicles obtained.

Example 17. Cell death effect of vesicles derived from Morganella morganii

(MMR101, MMR201, MMR202) -deleted vesicles in the mouse macrophage cell line, Raw 264.7 cells, at various concentrations, to evaluate the cytotoxic effect of M. morganii EV in inflammatory cells (0.1, 1, 10 占 퐂 / ml), and the degree of apoptosis was evaluated. More specifically, Raw 264.7 cells were seeded at a density of 5 x 10 ⁴ cells in 48-well cell culture plates and treated with various concentrations of Morganella morgani (MMR101, MMR201, MMR202) vesicles diluted with DMEM serum- Lt; / RTI &gt; Cell death was measured using EZ-CYTOX (Dogen, Korea). As a result, cell death was not observed upon vesicle treatment from Morganella morgani (MMR101, MMR201, MMR202) (see Fig. 18).

Example 18. Anti-inflammatory effect of vesicles derived from Moganella morganis

In order to examine the effect of morganella morgani-derived vesicles on inflammatory mediator release in inflammatory cells, morphogranular mor- bane (MMR101) -derived vesicles were injected into mouse macrophages Raw 264.7 cells at various concentrations (0.1, 1, / Ml), and then E. coli- derived vesicle ( E. coli) EV) to measure the secretion amount of inflammatory mediators (IL-6, TNF-a, etc.). More specifically, Raw 264.7 cells were plated on 24-well cell culture plates at 1 × 10 ⁵ cells and then cultured in DMEM complete medium for 24 hours. The culture supernatant was collected in a 1.5 ml tube and centrifuged at 3000 g for 5 minutes. The supernatant was collected and stored at 4 ° C for ELISA analysis. As a result, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles was remarkably inhibited when the vesicles derived from Morganella morgani were pretreated (see FIGS. 19A and 19B). In particular, the TNF-a secretion in macrophages was markedly higher than that in Lactobacillus plantarum vesicles when the vesicles from Morganella morgani were pretreated (see Fig. 19B).

In order to evaluate the antiinflammatory effect of the vesicles isolated from the morganella morganii strains isolated from the various samples, morganella morganii (MMR101, MMR201, MMR202) at various concentrations (0.1, 1, 10 / / ) Was pretreated with mouse macrophage cells for 12 hours, treated with 1 μg / ml of E. coli-derived vesicle, which was a pathogenic vesicle, and the secretion of TNF-α, an inflammatory cytokine, was measured by ELISA after 12 hours. As a result, the suppressive effect of morpholino morgani (MMR101, MMR201, MMR202) -derived vesicles was higher than that of TNF-α secretion inhibition effected by pretreatment of the microbial control group Lactobacillus plantarum -derived vesicles (See Fig. 20). This means that irrespective of the source of Morgana Morgani, the parasite secreted by Morgana Morgani is anti-inflammatory.

Example  19. Morganella Morgani  Effect of heat or acid treatment on the anti-inflammatory action of the resulting vesicles

The antiinflammatory effect of the vesicles derived from the standard strains of Morganella morgani (MMR101) and the isolated strain (MMR201) was confirmed through Example 18, and furthermore, the stability of the vesicles and the characteristics of the active substances were examined in detail. For this purpose, two types of morganella morganis-derived vesicles (MMR101, MMR201) pretreated with macrophages (Raw 264.7) boiled at 100 ° C for 10 minutes or acid treated (pH 2.0) Respectively. As a result, it was confirmed that the anti-inflammatory effect of the vesicles derived from Morgana morgani was maintained even if the vesicles were boiled or treated with acid at 100 ° C (see FIG. 21). This suggests that the anti-inflammatory action of morganella morgana-derived vesicles is stable to temperature and acid.

Example  20. Morganella Morgani  Anticancer effect of derived parasites

Based on the above examples, further investigation was made on the anticancer effect of vesicles derived from Morgana morgani. For this, as shown in FIG. 22, vesicles derived from the isolate of Morganella morgani (MMR201) were intraperitoneally injected or injected intraperitoneally into C57BL / 6 male mice at 6 weeks of age and the cancer cells (CT26 cell) And subcutaneously injected into a cancer model. After administration of the cancer cell line, the vesicles derived from the isolate of Morganella morgani were intraperitoneally injected or injected daily, and the size of the cancer tissue was measured until day 24 (see FIG. 22). As a result, the size of cancer tissues was reduced in the size of cancer tissues in mice administered with intraperitoneal injection of the vesicles or oral administration of the vesicles compared with the control group of oral administration of physiological saline, and in particular, (See Fig. 23). This means that the morganella morgana-derived vesicles can effectively inhibit the growth of cancer tissues.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention. There will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

The vesicles derived from the genus Morganella according to the present invention can be used for the treatment of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, A diagnostic method for Parkinson's disease, and a composition for preventing or treating a food or a drug against the disease or inflammatory disease.

Claims (20)

1. The diagnosis of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, or Parkinson's disease How to provide information for:

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

   (b) performing PCR on the extracted DNA using primer pairs prepared based on the gene sequences present in 16S rDNA to obtain respective PCR products; And

   (c) Quantitative analysis of the PCR products revealed that the content of Morganella bacterium-derived vesicles is lower than that of a normal human, Myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, stroke, diabetes, or Parkinson's disease.
2. The method according to claim 1,

   Wherein the sample in step (a) is blood, urine, or sputum.
3. The method according to claim 1,

   Wherein the primer pair in the step (b) is a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
4. Know the Nella (Morganella) in bacterial-derived, gastric cancer, colon cancer comprising a package as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, A pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of stroke, diabetes, Parkinson's disease, and inflammatory diseases.
5. 5. The method of claim 4,

   The inflammatory disease may be atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, Inflammatory bowel disease, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, arthritis, osteoporosis, atherosclerosis, arthritis, arthritis, inflammatory bowel disease, chronic inflammatory disease due to inflammatory osteolysis, viral or bacterial infection, A disease selected from the group consisting of Crohn's disease, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borrelia, tuberculosis, sarcoidosis, Albuminous lupus, tuberculous 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.
6. 5. The method of claim 4,

   Wherein said inflammatory disease is a disease mediated by interleukin-6 (IL-6) or tumor necrosis factor-alpha (TNF-alpha).
7. 5. The method of claim 4,

   Wherein said vesicles have an average diameter of from 10 to 200 nm.
8. 5. The method of claim 4,

   Wherein said vesicles are secreted naturally or artificially in bacteria of the genus Morganella .
9. 5. The method of claim 4,

   Wherein the bacterium-derived vesicles of the genus Morganella are secreted in Morganella morganii .
10. Know the Nella (Morganella) in bacterial-derived, gastric cancer, colon cancer comprising a package as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, Stroke, diabetes, Parkinson's disease, and inflammatory diseases.
11. 11. The method of claim 10,

    The inflammatory disease may be atopic dermatitis, acne, psoriasis, sinusitis, rhinitis, conjunctivitis, asthma, dermatitis, inflammatory collagen vascular disease, glomerulonephritis, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease, sepsis, septic shock, pulmonary fibrosis, Inflammatory bowel disease, inflammatory bowel disease, arthritis, rheumatoid arthritis, reactive arthritis, osteoarthritis, arthritis, osteoporosis, atherosclerosis, arthritis, arthritis, inflammatory bowel disease, chronic inflammatory disease due to inflammatory osteolysis, viral or bacterial infection, A disease selected from the group consisting of Crohn's disease, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves disease, leprosy, syphilis, Lyme disease, Borreliosis, neurogenic-borrelia, tuberculosis, sarcoidosis, Albuminous lupus, tuberculous 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.
12. 11. The method of claim 10,

    Wherein the inflammatory disease is a disease mediated by Interleukin-6 (IL-6) or Tumor Necrosis Factor-alpha (TNF-alpha).
13. 11. The method of claim 10,

    Wherein said vesicles have an average diameter of 10 to 200 nm.
14. 11. The method of claim 10,

    Characterized in that said vesicles are secreted naturally or artificially in bacteria of the genus Morganella .
15. 11. The method of claim 10,

    The idea is Nella (Morganella) in bacterial-derived vesicles, a food composition characterized in that the idea is secreted from Nella know going (Morganella morganii).
16. Diagnostic methods of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes or Parkinson's disease :

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

    (b) performing PCR on the extracted DNA using primer pairs prepared based on the gene sequences present in 16S rDNA to obtain respective PCR products; And

    (c) Quantitative analysis of the PCR products revealed that the content of Morganella bacterium-derived vesicles is lower than that of a normal human, Myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, stroke, diabetes, or Parkinson's disease.
17. 17. The method of claim 16,

    Wherein the sample in step (a) is blood, urine, or sputum.
18. 17. The method of claim 16,

    Wherein the primer pair in step (b) is a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
19. Colon cancer, cholangiocarcinoma, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, lymphoma, liver cancer and the like, which comprises administering to a subject a pharmaceutical composition comprising an effective amount of a bacterium-derived vesicle of the genus Morganella . Myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, diabetes, Parkinson's disease, and inflammatory diseases.
20. The present invention relates to a method for the treatment and prophylaxis of Morganella genus-derived vesicles of gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, For the prevention or treatment of one or more diseases selected from the group consisting of inflammatory diseases, diseases, and inflammatory diseases.

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