WO2019004668A1 - Nanovesicle derived from proteus genus bacteria, and use thereof - Google Patents

Abstract

The present invention relates to a vesicle derived from Proteus genus bacteria, and a use thereof. The inventors experimentally confirmed that, compared to normal people, the vesicle is significantly reduced in samples from cancer patients suffering from stomach cancer, colorectal cancer, liver cancer, biliary duct cancer, pancreatic cancer, lung cancer, breast cancer, or ovarian cancer and the like, and patients with allergic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), or atopic dermatitis and the like, cardiovascular diseases such as myocardial infarction, cardiomyopathy, variant angina pectoris, or atrial fibrillation and the like, metabolic diseases such as diabetes and the like and neuropsychiatric diseases such as Parkinson's disease or depression and the like, and the vesicle inhibits the secretion of inflammatory mediators by pathogenic vesicles, while also exhibiting an anticancer effect. It is anticipated that the vesicle derived from Proteus genus bacteria can be usefully utilized to develop diagnostic methods and compositions for preventing, treating, and/or improving cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric diseases, allergic respiratory diseases, and inflammatory intestinal diseases.

Description

Nano-vesicles derived from bacteria in the genus Proteus and their uses

The present invention relates to a nano-vesicle derived from a bacterium belonging to the genus Proteus, and to its use. More specifically, the present invention relates to a nano-vesicle derived from a bacterium of the genus Proteus, Respiratory diseases, and inflammatory bowel disease, and compositions for preventing or treating such vesicles.

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. In the 21st century, intractable chronic diseases are characterized by a chronic inflammatory reaction caused by immune dysfunction due to repetitive exposure to causal factors, which are caused by changes in lifestyle, including cancer, cardiovascular disease, metabolic disease, Allergic-respiratory diseases, and inflammatory bowel diseases are becoming serious problems in the public health.

Th17 (T helper 17) immune response, which is characterized by the secretion of interleukin-17 cytokine, is important for the immune response to the causative agent derived from bacteria. Interleukin-6, IL-6) secretion plays an important role in the differentiation of Th17 cells into innate immune responses. In addition, inflammation occurs when exposed to bacterial causative factors. In this process, inflammatory mediators such as tumor necrosis factor-alpha (TNF-α) are secreted to treat cancer, cardiovascular disease, - Mental illness, allergic - respiratory disease, and inflammatory bowel disease are known to occur.

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. Bacteria and archaea that coexist in our body secrete nanometer-sized vesicles to exchange information about genes, proteins, and so on 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 is absorbed into the epithelium through the mucosa to induce an inflammatory reaction, and is also absorbed into the body through the lymphatic vessels. Bacterial-derived vesicles, which are secreted locally from bacteria that coexist in our body, are absorbed into the epithelial cells of the mucosa to induce a local inflammatory reaction, and the vesicles derived from the pathogenic bacteria that are absorbed by our bodies are distributed in the organs through the blood and absorbed Increases inflammatory response in organs and exacerbates disease.

The genus Proteus bacterium is an anaerobic gram-negative bacterium that converts urea to ammonia via urease, which converts urine into ammonia, converting the urea to ammonia. Of these bacteria, Proteus mirabilis is known to be a pathogenic bacterium, accounting for 90% of human bacterial infections in the genus Proteus. The bacterium of the above-mentioned Proteus is not only symbiotic with human digestive organs, but also widely known as an environment such as soil and water.

On the other hand, an invention for treating diseases by using an extracellular endoplasmic reticulum derived from a Bacillus genus or a lactic acid bacterium has been disclosed (KR 10-1862507, KR 10-1726488). However, the fact that proteases secrete out vesicles Have not been reported, and there have been no reports of applications in the diagnosis and treatment of diseases such as cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric diseases, allergic-respiratory diseases, and inflammatory bowel diseases.

In the present invention, it was confirmed that the vesicles derived from the bacterium of the genus Proteus were significantly reduced in clinical samples of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disease, allergic-respiratory disease, and inflammatory bowel disease patients, Of the patients. In addition, it has been confirmed that the vesicles isolated from the culture broth of the Proteus bacterium can be used as a composition for preventing or treating cancer, cardiovascular diseases, metabolic diseases, neurological diseases, allergic-respiratory diseases, and inflammatory bowel diseases.

The inventors of the present invention have conducted intensive studies to solve the above conventional problems and have found that metagenomic analysis can be used to diagnose gastric cancer, colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, Cardiovascular diseases such as cardiovascular diseases such as cardiomyopathy such as cardiomyopathy, cardiomyopathy, atrial fibrillation, angina pectoris and stroke, diabetic and other metabolic diseases, neuropsychiatric diseases such as Parkinson's disease and depression, atopic dermatitis, asthma, chronic It has been confirmed that the contents of proteas-derived bacterial vesicles are significantly reduced in samples derived from inflammatory bowel disease patients such as allergic-respiratory diseases such as chronic obstructive pulmonary disease (COPD), irritable growth syndrome and inflammatory bowel disease.

Further, when the vesicle is isolated from Proteus mirabilis belonging to the bacterium of the genus Proteus and treated with the inflammatory cells, not only the TNF-α secretion by the pathogenic vesicles is remarkably suppressed, but also the anti-cancer efficacy As a result, it was confirmed that cancer development was significantly inhibited when the vesicles derived from Proteus mirabilis were orally administered. Based on this finding, the present invention was completed.

Accordingly, the present invention provides a method for treating or preventing asthma, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, A method for providing information for diagnosis of one or more diseases selected from the group consisting of myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression.

The present invention also relates to a pharmaceutical composition for preventing or treating gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, For the prevention, treatment and / or amelioration of one or more diseases selected from the group consisting of atopic dermatitis, hypersensitivity syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression And to provide a composition.

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 accomplish the object of the present invention, the present invention provides a method for the treatment and / or prophylaxis of cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric diseases, allergic-respiratory diseases and inflammatory bowel diseases, Provide a method of providing information for the diagnosis of the above diseases:

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

(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a pair of primers prepared based on the gene sequence existing in 16S rDNA to obtain respective PCR products; And

(c) Quantitative analysis of the PCR products reveals that the content of proteas-derived bacterial vesicles is lower than that of normal individuals, resulting in a decrease in the amount of vesicles derived from the group consisting of cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric disorders, allergic- respiratory diseases, Determining the selected one or more diseases.

The present invention also provides a method of diagnosing one or more diseases selected from the group consisting of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease, comprising the steps of:

(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 reveals that the content of proteas-derived bacterial vesicles is lower than that of normal individuals, resulting in a decrease in the amount of vesicles derived from the group consisting of cancer, cardiovascular diseases, metabolic diseases, neuropsychiatric disorders, allergic- respiratory diseases, Determining the selected one or more diseases.

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

The present invention also provides a method for preventing or treating at least one disease selected from the group consisting of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease, A pharmaceutical composition for therapeutic use is provided.

The present invention also provides a method for preventing or treating at least one disease selected from the group consisting of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease, There is provided a therapeutic inhaler composition.

The present invention also provides a method for preventing or treating at least one disease selected from the group consisting of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease, A composition for improvement is provided.

In one embodiment of the present invention, the composition for preventing or ameliorating the disease may be a food composition or a cosmetic composition.

In another embodiment of the present invention, the food composition may be a health functional food composition.

The present invention also relates to a method for treating cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease, comprising administering to a subject a pharmaceutical composition comprising as an active ingredient a bacterium- A disease, a disease, a disease or a disease.

The present invention also provides a prophylactic or therapeutic use of at least one disease selected from the group consisting of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease .

In one embodiment of the present invention, the cancer may be a gastric cancer, a colon cancer, a liver cancer, a bile duct cancer, a pancreatic cancer, a lung cancer, a breast cancer, an ovarian cancer, a bladder cancer, a prostate cancer, a lymphoma or a brain tumor.

In another embodiment of the present invention, the cardiovascular disease may be myocardial infarction, cardiomyopathy, atrial fibrillation, dysenteric angina, or stroke.

In another embodiment of the present invention, the metabolic disease may be diabetes.

In another embodiment of the present invention, the neuropsychiatric disorder may be Parkinson ' s disease or depression.

In another embodiment of the present invention, the allergic-respiratory disease may be atopic dermatitis, asthma, or chronic obstructive pulmonary disease.

In another embodiment of the present invention, the inflammatory bowel disease may be hypersensitivity syndrome, or inflammatory bowel disease.

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 vesicle may be secreted naturally or artificially in the bacterium of the genus Proteus.

In another embodiment of the present invention, the proteas-derived bacterium-derived vesicle may be secreted from Proteus mirabilis.

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, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, myocardial infarction, Allergic diseases such as cardiovascular diseases such as cardiomyopathy, atrial fibrillation, angina pectoris and stroke, metabolic diseases such as diabetes, neuropsychiatric diseases such as Parkinson's disease and depression, atopic dermatitis, asthma and chronic obstructive pulmonary disease (COPD) , Hypersensitivity growth syndrome, and inflammatory bowel disease, are present in the blood, urine, or feces of a patient suffering from inflammatory bowel disease. It was OK. In addition, when Proteus Mirabilis, a kind of bacterium of the genus Proteus, was cultured in vitro to separate vesicles and was administered to inflammatory cells in vitro, significant suppression of inflammatory mediator release by pathogenic vesicles was observed. The present inventors have found that the bacterium derived from the bacterium belonging to the genus Proteus according to the present invention is useful for the treatment of gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, Cardiovascular diseases such as cardiovascular diseases such as cardiomyopathy such as cardiomyopathy, cardiomyopathy, atrial fibrillation, angina pectoris, stroke, etc., neuropsychiatric diseases such as Parkinson's disease and depression, atopic dermatitis, asthma, chronic obstructive lung Disease, etc. - Diagnosis method for inflammatory bowel disease such as respiratory disease, hypersensitivity syndrome, inflammatory bowel disease, and foods, inhalants, cosmetics, medicines, etc. It is expected to be useful for use in for preventing, treating, and / or improve the composition.

FIG. 1A is a picture of distribution patterns of bacteria and vesicles after oral administration of bacteria and bacterial-derived vesicles (EVs) to a mouse.

FIG. 1B is a picture showing the distribution of bacteria and vesicles in the body by extracting blood, kidney, liver, and various organs at 12 hours after oral administration of bacteria and bacterial-derived vesicles (EV) to the mouse.

FIG. 2 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes in stool cancer patients, normal stool, blood, and urine.

Fig. 3 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of bacterial-derived vesicle metagenomes existing in colon cancer and normal human stool and urine.

FIG. 4 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes present in liver cancer, bile duct cancer, pancreatic cancer patients and normal human blood.

FIG. 5 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes existing in lung cancer patients and normal blood.

FIG. 6 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes existing in breast cancer, ovarian cancer patients and normal human urine.

FIG. 7 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes present in bladder cancer patients and normal human blood and urine.

Fig. 8 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of bacterial-derived vesicle metagenomes present in prostate cancer patients and normal urine.

FIG. 9 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of bacterial-derived vesicle metagenomes existing in lymphoma, brain tumor patients and normal human blood.

FIG. 10 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of bacterial-derived vesicle metagenomes existing in diabetic patients and normal blood.

Fig. 11 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle meta-genome present in myocardial infarction, cardiomyopathy, atrial fibrillation, dysmorphic angina and normal human blood.

FIG. 12 shows the results of a comparison of the distribution of vesicles derived from bacteria in the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes existing in the blood of a stroke patient and a normal person.

Fig. 13 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes in Parkinson's disease, depression patients and normal human urine.

Fig. 14 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes present in atopic dermatitis patients and normal human blood.

Fig. 15 shows the results of a comparison of the distribution of vesicles derived from bacteria belonging to the genus Proteus after the analysis of bacterial-derived vesicle metagenomes present in asthma, COPD patients and normal human blood.

FIG. 16 shows the results of a comparison of the distribution of vesicles derived from the bacteria belonging to the genus Proteus after the analysis of the bacterial-derived vesicle metagenomes present in hypersensitivity syndrome, inflammatory bowel disease (IBD), and normal feces.

FIG. 17A shows the results of observing the shape of vesicles by electron microscopy after culturing Proteus mirabilis in vitro, separating the vesicles from the culture solution.

17B shows the result of measurement of the size of vesicles isolated from the culture medium of Proteus mirabilis by dynamic light scattering.

18A and 18B show the results of pretreatment of the vesicles derived from the Proteus fungus before the treatment of the E. coli EV, which is a pathogenic vesicle, to evaluate the anti-inflammatory effect of the vesicles derived from the Proteus mirabilis, -6 secretion (Fig. 18A) and TNF-a secretion (Fig. 18B).

FIG. 19 is a graph showing the effect of vesicles derived from various strains on the anti-inflammatory effect of the proteasum-labile vesicles. In order to compare the effect of vesicles derived from various strains, (NC: negative control; PC: positive control), L. pantalum (positive control), and the control group (plantarum): Lactobacillus plantarum).

FIG. 20 is a graph showing the effect of heat treatment or acid treatment on the anti-inflammatory effect of the proteasum bilberry-derived vesicles in the presence of protease mirabilis (PMR) treated with heat treatment or acid treatment before the E. coli EV treatment, which is a pathogenic vesicle, (NC: negative control; PC: positive control; L. plantarum). The results are shown in FIG. : Lactobacillus plantarum).

21A is a protocol in which a vesicle derived from Proteus mirabilis is administered to a mouse to evaluate the anticancer efficacy of the parasite derived from Proteus mirabilis.

Fig. 21B shows the results of evaluating the effects of administration of a parasite derived from Proteus mirabilis on mice orally to tumorigenesis by cancer cells.

The present invention relates to a vesicle derived from bacteria belonging to the genus Proteus and a use thereof.

The present inventors have found that metagenomic analysis enables the diagnosis of cancers such as gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma and brain tumor, myocardial infarction, cardiomyopathy, Allergic-respiratory diseases such as atopic dermatitis, asthma or chronic obstructive pulmonary disease, hypersensitivity syndromes or inflammatory bowel disease, such as cardiovascular diseases such as atrial fibrillation and stroke, metabolic diseases such as diabetes, Parkinson's disease, The inventors of the present invention have completed the present invention based on this finding.

Accordingly, the present invention relates to a pharmaceutical composition for the treatment of gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, The present invention provides a method of providing information for diagnosis of at least one disease selected from the group consisting of inflammatory bowel disease, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression.

(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 protease-derived microbes is lower than that of a normal person, and thus, the amount of parasites from gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, ovarian cancer, bladder cancer, At least one disease selected from the group consisting of brain tumors, chronic obstructive pulmonary disease, atopic dermatitis, hypersensitivity syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression step.

The term " diagnosis " as used in the present invention means, in a broad sense, judging the actual condition 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 to judge whether or not the cancer, cardiovascular disease, metabolic disease, neuropsychiatric disease, allergic-respiratory disease, or inflammatory bowel disease occurs and the level of disease.

&Quot; Cancer " as a diagnostic target disease of the present invention means a malignant tumor that rapidly grows while infiltrating into surrounding tissues and diffuses or transitions to each part of the body and is life-threatening. Cells, the smallest unit of the body, normally divide and grow by the regulatory function of the cells themselves, and when they reach the end of their life or become damaged, they self-destruct themselves and maintain an overall balance of numbers. If there is a problem with the regulation of the cell itself, the abnormally normal cells that are to die are over-proliferated, invading the surrounding tissues and organs, forming a mass, destroying or deforming the existing structure. In the present invention, the cancer may preferably be gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma or brain tumor.

As used herein, the term " cardiovascular disease " means that a disease occurs in the cardiovascular system of a mammal, including, for example, heart disease such as myocardial infarction, cardiomyopathy, angina pectoris, Vasculitis; Dementia, stroke, etc. In the present invention, the cardiovascular diseases include, but are not limited to, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, or stroke.

The term " metabolic disease " used in the present invention means a disease in which complications occur in various organs due to metabolic disorders in the mammalian body. Examples of the metabolic diseases include metabolic disorders such as hyperlipidemia and diabetes, And complications. In the present invention, the metabolic diseases preferably include, but are not limited to, diabetes.

The term " neuropsychiatric disease " as used in the present invention means a disease occurring in the nervous system and the brain of mammals such as brain diseases such as Parkinson's disease and dementia; Depression, obsessive compulsive disorder, asthma and the like. In the present invention, the neuropsychiatric disorder preferably includes, but is not limited to, Parkinson's disease and depression.

The term " allergic disease " used in the present invention means a disease caused by an allergic mechanism in a mammal, and includes, for example, atopic dermatitis of the skin, allergic rhinitis of the respiratory system, asthma, In the present invention, the allergic diseases preferably include, but are not limited to, atopic dermatitis and asthma.

The term " respiratory disease " used in the present invention means a disease occurring in the respiratory system of a mammal such as rhinitis, asthma, chronic obstructive pulmonary disease, etc. In the present invention, Respiratory diseases preferably include, but are not limited to, asthma, chronic obstructive pulmonary disease.

The term " inflammatory bowel disease " of the present invention means chronic inflammation of unknown origin occurring in the intestine, and broadly refers to infectious enteritis such as bacterial, viral, amoebic, tuberculous enteritis, Inflammatory bowel disease, ischemic bowel disease, and inflammatory bowel disease. In the present invention, the inflammatory bowel disease preferably includes, but is not limited to, hypersensitivity syndrome, inflammatory bowel disease.

The term " Nanovesicle " or " Vesicle " as used in the present invention means a structure composed of nano-sized membranes secreted from 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, as well as various metabolites, gram-positive bacteria -positive bacterial cells have protein and nucleic acid as well as peptidoglycan and lipoteichoic acid, which are bacterial cell wall components. In the present invention, nano-vesicles or vesicles are naturally secreted or artificially produced in the bacterium of the genus Proteus, and have an average diameter of 10 to 200 nm.

The vesicles can be obtained by centrifugation, ultracentrifugation, extrusion, sonication, cell lysis, homogenization, freezing-thawing, electroporation, mechanical degradation, chemical treatment, filtration by filtration, gel filtration chromatography And may be separated using one or more methods selected from the group consisting of electrophoresis, electrophoresis, 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 is also referred to as " meta genome ", which means the total of all genomes including viruses, bacteria, fungi, etc. in an isolated region such as soil, It is used as a concept of a genome to explain the identification of many microorganisms at a time using a sequencer to analyze the microorganisms that are not. 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 may be blood, urine, or feces, but is not limited thereto.

In another aspect of the present invention, the present invention provides a method for the treatment of gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, For the prevention or treatment of one or more diseases selected from the group consisting of chronic obstructive pulmonary disease, atopic dermatitis, hypersensitivity syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression A pharmaceutical composition is provided.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, Prevention or treatment of one or more diseases selected from the group consisting of chronic obstructive pulmonary disease, atopic dermatitis, hypersensitivity syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression Thereby providing a drag inhaler composition.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating gastric cancer, colon cancer, liver cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, Prevention or amelioration of one or more diseases selected from the group consisting of chronic obstructive pulmonary disease, atopic dermatitis, hypersensitivity syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression ≪ / RTI > The composition comprises a food composition and a cosmetic composition.

As used herein, the term " prevention " refers to the administration of a pharmaceutical composition according to the present invention to inhibit or prevent cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease It means all actions that delay.

The term " treatment " used in the present invention means that the symptoms of cancer, cardiovascular disease, metabolic disease, neuropsychiatric disorder, allergic-respiratory disease, and inflammatory bowel disease are improved by administration of the pharmaceutical composition according to the present invention It means all acts that benefitfully change.

The term " improvement " as 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, blood of a normal control matched age in gastric cancer, colorectal cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor patients, , Urine, or feces were used to analyze bacterial metagenomes. As a result, the samples from patients with gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma and brain tumor were significantly reduced (See Example 3).

In another embodiment of the present invention, bacterial meta-genomic analysis is performed using vesicles isolated from samples of patients with diabetes, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, and stroke patients, Respectively. As a result, it was confirmed that the samples of patients suffering from diabetes, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, and stroke were significantly reduced in vesicles derived from bacteria in the genus Proteus (see Example 4).

In another embodiment of the present invention, bacterial metagenomic analysis was performed using parcels isolated from samples of patients with Parkinson ' s disease and depression, and with age matched to the patient. As a result, it was confirmed that the samples of patients suffering from Parkinson's disease and depression were significantly reduced in vesicles derived from bacteria in the genus Proteus (see Example 5).

In another embodiment of the present invention, bacterial metagenomics were performed using vesicles isolated from samples of atopic dermatitis, asthma, and chronic obstructive pulmonary disease, and normal persons matched with the age and sex of the patient. As a result, it was confirmed that the samples of patients suffering from atopic dermatitis, asthma, and chronic obstructive pulmonary disease had significantly decreased vesicles derived from the bacteria in the genus Proteus, compared with normal samples (see Example 6).

In another embodiment of the present invention, the bacterial metagenomic analysis was performed using vesicles isolated from samples of patients with hypersensitivity syndrome and inflammatory bowel disease and normal age matched patients. As a result, it was confirmed that the samples from the patients with hypersensitivity growth syndrome and inflammatory bowel disease were significantly reduced in the vesicles derived from the bacteria of the genus Proteus in comparison with the normal samples (see Example 7).

In another embodiment of the present invention, further studies have been made to analyze the characteristics of the microbial cells derived from the Proteus mirabilis species belonging to the genus Proteus, based on the results of the above Examples, , Preferably 41.40 ± 5.03 nm (see Example 8).

In another embodiment of the present invention, it was evaluated whether the parasites secreted from the culture of the Proteus mirabilis strain exhibited an anti-inflammatory effect. After the culture of the Proteus mirabilis strain isolated from various persons, After treating the parasites derived from Proteus mirabilis with macrophages, the inflammatory diseases, metabolic diseases, and cancer caused by Escherichia coli-derived vesicles were treated to evaluate inflammatory mediator secretion. As a result, It was confirmed that all of the vesicles derived from Mirabileis efficiently inhibited the secretion of TNF-? By Escherichia coli-derived vesicles (see Example 9).

In another embodiment of the present invention, in order to evaluate whether the active substance exhibiting the anti-inflammatory effect of Proteus mirabilis is a low-molecular compound or protein contained in the vesicles, the vesicles derived from Proteus mirabilis were subjected to heat treatment or acid treatment. Thereafter, the effect on the TNF-α secretion was evaluated by administering heat-treated or acid-treated vesicles to the macrophages before treating them with E. coli-derived vesicles. As a result, it was found that all of the vesicles derived from the proteasum- TNF-a < / RTI > secretion (see Example 10).

In another embodiment of the present invention, a cancer disease model was prepared by subcutaneously injecting a cancer cell line in order to evaluate whether or not the parasite secreted from the culture of the Proteus mirabilis strain exhibits an anticancer treatment effect, Derived vesicles were orally administered to mice 4 days prior to treatment with cancer cells and the size of cancer tissues was measured for 20 days. As a result, it was confirmed that the size of cancer tissues was significantly reduced when the vesicles were administered (See Example 11).

The pharmaceutical composition according to the present invention comprises a bacterium-derived vesicle of the genus Proteus as an active ingredient, and may include a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile 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 composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level is determined depending on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of 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 composition according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.001 to 150 mg, preferably 0.01 to 100 mg, It may be administered 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.

In the food composition of the present invention, the active ingredient can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to a conventional method. 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.

The cosmetic composition of the present invention may contain components commonly used in cosmetic compositions as well as microcapsules derived from bacteria belonging to the genus Proteus, and may contain conventional additives such as antioxidants, stabilizers, solubilizers, vitamins, pigments, , And a carrier.

In addition, the composition of the present invention may be used in combination with an organic ultraviolet screening agent that has been used in the past so long as it does not impair the skin protecting effect by reacting with the microbial virus derived from the bacteria belonging to the genus Proteus. Examples of the organic UV blocking agent include glyceryl paraben, drometrizol trisiloxane, drometrizol, dipaloyyl triolate, disodium phenyldibenzimidazole tetrasulfonate, diethylhexylbutamidotriazone, diethylamino Hydroxybenzoylhexyl benzoate, di-methoxycinnamate, a mixture of Rawson and dihydroxyacetone, methylene bis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidene camphor, menthyl anthranylate, benzophenone (Benzophenone-4), benzophenone-8 (dioxyphenylbenzone), butylmethoxydibenzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, synoxate, ethyl dihydroxypropylparaben, Ethylhexyldimethylpivalate, ethylhexylmethoxycinnamate, ethylhexylsalicylate, ethylhexyltriazone, isoamyl-p-methoxy cinnamate, polysilicon-15 (dimethicodimethylbenzalmate, At least one selected from the group consisting of terephthalylidene dicam peresophilic acid and its salts, thiai-salicylate and aminobenzoic acid (parabe) can be used.

Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotion, softening longevity lotion, nutrition lotion, various creams, essences, packs, foundation and the like, cleansing, cleanser, soap, . Specific formulations of the cosmetic composition of the present invention include skin lotions, skin softeners, skin toners, astringents, lotions, milk lotions, moisturizing lotions, nutritional lotions, massage creams, nutritional creams, moisturizing creams, hand creams, essences, It includes formulations such as soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, latex, lipstick, makeup base, foundation, press powder, loose powder, eye shadow and the like.

According to a preferred embodiment of the present invention, the content of proteasome-derived vesicles of the present invention is 0.00001-30 wt%, preferably 0.5-20 wt%, more preferably 1.0-10 wt% to be.

In the inhalant composition of the present invention, the active ingredient may be directly added to the inhalant or may be used together with other ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or treatment).

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 parasites

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 observation of the whole image of the mouse, as shown in Fig. 1A, when the bacterium was not absorbed systemically, the bacterium-derived vesicles were systemically absorbed at 5 minutes after the administration, And the 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 bacteria and foreign matter by using a 0.22 μm filter again, the supernatant was discarded using a high speed centrifugation method at 150,000 xg for 3 hours at 150 ° C. with a Type 90ti rotor, and the lumped pellet was washed with phosphate buffered saline.

100 μl of the vesicles isolated by the above method was boiled at 100 ° C. to expel the DNA outside the lipid. After cooling for 5 minutes on ice, the supernatant was centrifuged at 10,000 × g for 30 minutes at 4 ° C. to remove the remaining suspended matter . After the amount of DNA was quantified using Nanodrop, 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, It was confirmed that the gene derived from bacteria was present.

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), outputting the result as a Standard Flowgram Format (SFF) file, and using GS FLX software (v2 .9) to convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file, then verify the creditworthiness of the lead, and use the window (20 bps) average base call and the accuracy was less than 99% (Phred score <20). For the analysis of Operational Taxonomy Unit (OTU), clustering is performed according to sequence similarity using UCLUST and USEARCH. The genus is 94%, the family is 90 Clustering is performed based on the sequence similarity of 85% for order, 80% for class, 75% for phylum, and the phylum, class, order sequence, more than 97% sequence level at the genome level using the 16S RNA sequence database (108,453 sequence) of BLASTN and GreenGenes, Were profiled (QIIME).

Example 3. Analysis of bacterial-derived vesicle metagenomes in clinical samples of cancer patients

Genes were extracted from feces in stools of feces of 55 gastric cancer patients and feces of 99 normal control patients by the method of Example 2, and the distribution of the microbial cells derived from the bacteria of the genus Proteus was evaluated after performing the metagenome analysis. As a result, it was confirmed that the vesicles derived from the bacteria in the stomach cancer were significantly decreased in the stool of the stomach cancer patients compared with the normal stool (1.1% vs. 0.05%, p <0.001).

In addition, the blood of 67 stomach cancer patients and the blood of 198 normal control patients were subjected to metagenome analysis by extracting genes from the vesicles present in the blood, and then the distribution of the vesicles derived from the bacteria in the proteas was evaluated. As a result, it was confirmed that the vesicles derived from bacteria in the stomach cancer patients were significantly decreased (normal vs vs stomach cancer: 1.3% vs. 0.2%, p = 0.001) (see FIG. 2).

In the urine of 61 gastric cancer patients and 120 urine of the normal control group, the gene was extracted from the vesicles present in the urine and the metagenomic analysis was carried out to evaluate the distribution of the vesicles derived from the bacteria in the proteas. As a result, it was found that the vesicles derived from the bacteria in the stomach cancer were significantly decreased in the urine of the gastric cancer patients (normal vs vs stomach cancer: 1.8% vs 0.1%, p <0.0001, p <0.0001).

In addition, in the method of Example 2, the gene was extracted from the vesicles present in the stool of 38 stools of colorectal cancer patients and 55 stools of normal control cells, and the results of the metagenomic analysis were analyzed. Respectively. As a result, it was confirmed that the vesicles derived from the bacteria in the genus Proteus were significantly decreased in the stool of the colon cancer patients compared with the normal stool (1.2% vs 0.05%, p <0.001 in the normal vs. colorectal cancer patients, see FIG.

In addition, in the urine of 38 normal control subjects who matched the urine, sex, and age of 38 patients with colorectal cancer, the genes were extracted from the vesicles present in the urine and analyzed by metagenome. Respectively. As a result, it was confirmed that the vesicles derived from the bacteria in the genus Proteus were significantly decreased in the feces of the colon cancer patients (normal vs. vs colorectal cancer patients: 1.3% vs 0.06%, p <0.001) (see FIG.

In addition, by performing the metagenome analysis by extracting genes from the vesicles present in the blood of the blood of 94 liver cancer patients and the blood of 152 normal control patients by the method of Example 2, the distribution of the vesicles derived from the bacteria of the genus Proteus was evaluated Respectively. As a result, it was found that the vesicles derived from the proteasome in the blood of the liver cancer patients were significantly decreased (normal vs non-liver cancer patients: 0.7% vs 0.01%, p <0.001) (FIG.

In addition, the genomic DNA was extracted from the vesicles present in the blood of 84 biliary tract blood and 132 normal control blood, and the distribution of the vesicles derived from the bacteria in the proteus was evaluated. As a result, it was confirmed that the bacterium-derived vesicles in the blood of the biliary cancer patients were significantly decreased (0.7% vs 0.06%, p <0.001) in normal patients and biliary cancer patients (see FIG. 4).

In addition, the gene was extracted from the vesicles present in the blood of 191 pancreatic cancer patients and 291 normal control blood samples, and then the metagenomic analysis was performed to evaluate the distribution of the vesicles derived from the bacteria in the proteas. As a result, it was confirmed that the blood of the pancreatic cancer patient was significantly reduced in the proteasome-derived vesicles compared to the normal blood (0.7 vs 0.06% in normal vs. pancreatic cancer patients, p <0.001, p <0.001).

In addition, the gene was extracted from the vesicles present in the blood of 318 lung cancer patients and 234 normal control patients by the method of Example 2, and the metagenomic analysis was performed. Then, the distribution of the vesicles derived from the bacteria of the genus Proteus was evaluated Respectively. As a result, it was confirmed that the blood of the lung cancer patients was significantly decreased in the proteasome-derived vesicles compared with the normal blood (0.7 vs 0.1%, p <0.001).

In addition, in the case of the urine of 127 breast cancer patients and the urine of 220 normal breast cancer patients by the method of Example 2, the gene was extracted from the vesicles present in the urine and the metagenomic analysis was carried out. Then, the distribution of the vesicles derived from the bacteria in the Proteus was evaluated Respectively. As a result, it was found that the vesicles derived from the bacteria in the genus Proteus were significantly decreased in the urine of the breast cancer patients (normal vs. vs breast cancer: 1.4% vs 0.3%, p <0.001, p <0.001).

In the urine of 136 patients with ovarian cancer and the urine of 136 normal controls, genes were extracted from the vesicles present in the urine and the metagenomic analysis was performed to evaluate the distribution of the vesicles derived from the bacteria in the proteas. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Proteus were significantly decreased in the urine of the ovarian cancer patients compared with the normal urine (1.1% vs 0.1%, p <0.001).

In addition, the gene was extracted from the vesicles present in the blood of 96 bladder cancer patients and 184 normal control blood samples by the method of Example 2, followed by the metagenome analysis, and then the distribution of the vesicles derived from the bacteria of the genus Proteus was evaluated . As a result, it was confirmed that the vesicles derived from the bacterium in the bladder cancer patients were significantly decreased (normal vs. vs bladder cancer patients: 0.8% vs. 0.1%, p <0.001) (see FIG. 7).

In the urine of 95 bladder cancer patients and the urine of 157 normal controls, the gene was extracted from the vesicles present in the urine and the metagenomic analysis was performed to evaluate the distribution of the vesicles derived from the bacteria in the proteus. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Proteus were significantly decreased in the urine of the bladder cancer patients compared to the normal urine (1.9% vs 0.1%, p <0.000001).

In the urine of 53 men with prostate cancer and 159 men of normal control group, the gene was extracted from the vesicles present in the urine by the method of Example 2 and the metagenome was analyzed. The distribution of the vesicles derived from the bacteria in the proteus Respectively. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Proteus were significantly decreased in the urine of the prostate cancer patients compared to the normal urine (1.1% vs. 0.09%, p <0.000001).

In addition, in the method of Example 2, the genome was extracted from the vesicles present in the blood of 93 blood samples from lymphoma patients and 109 blood samples from a normal control sample, and the results of the metagenome analysis were analyzed. The distribution of the vesicles derived from the bacteria in the proteas Respectively. As a result, it was confirmed that the vesicles derived from the bacteria of the genus Proteus were significantly reduced (0.09% vs 0.00%, p <0.001) in the blood of the lymphoma patients (normal vs non-lymphoma patients, p <0.001).

In addition, the genomic DNA was extracted from the vesicles present in the blood of 84 brain samples and 92 normal control samples, and the distribution of the vesicles derived from the bacteria in the proteas was evaluated. As a result, it was confirmed that the blood of the brain tumor patients was significantly decreased in the proteasome-derived vesicles compared to the normal blood (0.09% vs 0.01%, p <0.01 in the normal vs.

Example  In patients with diabetes and cardiovascular disease, Meta genome  analysis

The gene was extracted from the vesicles present in the blood of the blood of 73 diabetic patients and the blood of 146 normal control patients by the method of Example 2, and the distribution of the vesicles derived from the bacteria of the genus Proteus was evaluated after performing the metagenome analysis. As a result, it was confirmed that the blood of the diabetic patients had a significant decrease in the vesicles derived from the bacteria in the proteasome (normal vs vs diabetic patients: 0.4% vs 0.01%, p <0.01) (see FIG. 10).

In addition, the gene was extracted from the vesicles present in the blood of blood of 57 myocardial infarction patients and 163 normal control patients by the method of Example 2, and the metagenome was analyzed. Then, the distribution of the vesicles derived from the bacteria in the proteas Respectively. As a result, it was confirmed that the blood of the myocardial infarction patient was significantly decreased in the blood of the proteus in the blood of the normal person (0.4% vs 0.07%, p <0.01 in the normal persons vs myocardial infarction patients, p <0.01).

In addition, the distribution of the vesicles derived from the bacteria in the genus Proteus was evaluated after extracting the genes from the vesicles present in the blood of 72 blood samples from cardiomyopathy patients and 163 blood samples from normal control samples. As a result, it was confirmed that the blood of the patient with cardiomyopathy was significantly decreased in the blood of the proteus in the blood of the normal person (0.4% vs. 0.08% in normal persons vs. cardiomyopathy, p <0.01) (see FIG. 11).

In addition, the genomic DNA was extracted from the vesicles present in the blood of 69 blood samples from atrial fibrillation patients and 103 blood samples from normal control samples, and the distribution of the microorganisms derived from the proteasome was evaluated. As a result, it was confirmed that the blood of the atrial fibrillation patients was significantly decreased in the blood of the proteus from the normal human blood (normal vs. Atrial fibrillation: 0.1% vs 0.01%, p <0.01).

In addition, the distribution of the vesicles derived from the bacteria in the genus Proteus was evaluated by extracting genes from the vesicles present in the blood of 32 blood samples from patients with angina pectoris and blood from 32 normal control samples. As a result, it was confirmed that the vesicles derived from the genus Proteus were significantly reduced in the blood of the patients with angiotension (0.6% vs 0.1%, p <0.05).

In addition, the blood samples from 87 stroke patients and 92 normal control samples were subjected to metagenome analysis by extracting genes from the vesicles present in the blood, and then evaluated for the distribution of the vesicles derived from the bacteria in the genus Proteus. As a result, it was confirmed that the blood of the stroke patients had a significant decrease in the vesicles derived from the bacteria in the proteasome (normal vs. vs stroke patients: 0.3% vs 0.00%, p <0.05).

Example 5: Analysis of bacterial-derived vesicle metagenomes in clinical samples of neuropsychiatric patients

The urine of 39 patients with Parkinson's disease and the urine of 79 normal controls were extracted from the vesicles present in the urine by the method of Example 2 to perform metagenome analysis and the distribution of the vesicles derived from the bacteria of the genus Proteus was evaluated . As a result, it was confirmed that the vesicles derived from the bacteria of the genus Proteus were significantly reduced in the urine of patients with Parkinson's disease (normal vs. vs Parkinson's disease: 0.6% vs 0.02%, p <0.0001, p <0.0001).

In addition, in the urine of 20 depressed patients and the urine of 20 normal control patients by the method of Example 2, the gene was extracted from the vesicles present in the urine and the metagenome was analyzed. The distribution of the vesicles derived from the bacteria in the Proteus was evaluated Respectively. As a result, it was found that the vesicles derived from the bacteria of the genus Proteus were significantly decreased in the urine of the depressed patients (normal vs non-depressed patients: 1.7% vs. 0.06%, p <0.01).

Example  6. Clinical samples of allergy and respiratory disease patients Bacterial-derived vesicles Meta genome  analysis

The gene was extracted from the vesicles present in the blood of the blood of 27 patients with atopic dermatitis and the blood of 138 normal control patients by the method of Example 2 and the metagenomic analysis was performed to evaluate the distribution of vesicles derived from the bacteria of the genus Proteus . As a result, it was confirmed that the blood of the atypical dermatitis patients had a significant decrease in the vesicles derived from the bacteria of the genus Proteus (2.0% versus 0.06%, p <0.00001 in normal persons vs atopic dermatitis patients, p <0.00001).

In addition, the gene was extracted from the vesicles present in the blood of 291 asthmatic patients, 207 COPD patients, and 291 normal control patients by the method of Example 2, and meta genome analysis was performed , And then the distribution of the microbial-derived vesicles in the Proteus was evaluated. As a result, it was found that the vesicles derived from bacteria in the genus Proteus were significantly decreased in the blood of patients with asthma and chronic obstructive pulmonary disease (normal vs non - asthmatic patients: 0.7% vs 0.08%, p <0.01; Patients with chronic obstructive pulmonary disease: 0.7% vs 0.07%, p <0.01) (see FIG. 15).

Example 7. Analysis of bacterial-derived vesicle metagenomes in clinical samples of inflammatory bowel disease patients

In the method of Example 2, genes were extracted from the vesicles present in the stool in the blood of 57 patients with irritable bowel syndrome (IBS) and 58 stools of the normal control, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from bacteria in the proteasome were significantly decreased in the feces of the patients with hypersensitivity syndromes compared with the normal feces (1.9% vs 0.00%, p <0.00001 in the normal persons vs hypersensitive growth syndrome patients, p <0.00001) ).

In addition, in a method of Example 2, genomic DNA was extracted from vesicles present in the stool of 91 stools of inflammatory bowel disease (IBD) and 99 normal control, The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from bacteria in the genus Proteus were significantly decreased in the feces of the patients with inflammatory bowel disease (normal vs. non-inflammatory bowel disease: 1.9% vs 0.00%, p <0.00001).

Example 8. Separation and characterization of vesicles in the culture medium of Proteus mirabilis

Based on the results of Examples 2 to 7, in order to evaluate the function of the microbial cells derived from the microorganism belonging to the genus Proteus, the microbial cells of the Proteus mummillus strain, which is one of the bacteria belonging to the genus Proteus, were cultured.

Proteus mirabilis strains isolated from clinical samples of various persons were cultured in an anaerobic chamber at 37 ° C in a brain heart infusion (BHI) medium until the absorbance (OD600) reached 1.0 to 1.5 Sub-culture. Subsequently, the supernatant containing the strain was recovered, centrifuged at 10,000 g for 15 minutes at 4 ° C, and filtered through a 0.45 μm filter. The supernatant was filtered through a 100 kDa hollow filter membrane with a QuixStand benchtop system system, GE Healthcare, UK). The supernatant, which had been concentrated, was filtered once again with a 0.22 μm filter. Then, proteins were quantified using BCA assay. The following experiments were carried out on the vesicles obtained. The vesicles were separated from the culture medium of the Proteus mirabilis cultured according to the above method, and then the shape and size were evaluated by an electron microscope Respectively.

As a result, as shown in Fig. 17A, it was observed that the shape of the vesicles isolated from the culture medium of Proteus mirabilis was spherical and the size was smaller than 200 nm, and the size of the vesicle Was found to be 37.8 ± 13.5 nm.

Example 9. Anti-inflammatory effect of parasites derived from Proteus mirabilis

In order to examine the effect of the proteasum mirabilis-derived vesicles on the inflammatory mediator secretion in inflammatory cells, the mouse macrophage cell line, Raw 264.7 cells, was infected with the parasites isolated from the proteus mirabilis strain ( P. mirabilis EV) was treated with various concentrations (0.1, 1, 10 占 퐂 / ml) and E. coli- derived vesicles EV) to measure the secretion amount of inflammatory mediators (IL-6, TNF-a, etc.).

More specifically, Raw 264.7 cells were plated at 1 × 10 ^{5 in} 24-well cell culture plates and cultured in DMEM (Dulbeco's Modified Eagle's Media) complete medium for 24 hours. Then, 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.

For ELISA analysis, the capture antibody was diluted in PBS (phosphate buffered saline) and dispensed in a 96-well polystyrene plate in an amount of 50 μl to the working concentration, followed by overnight reaction at 4 ° C. After washing with 100 μl of PBST (PBS containing 0.05% tween-20) twice, 100 μl of RD (PBST containing 1% bovine serum albumin (BSA)) solution was added and blocked for 1 hour at room temperature. After blocking, the cells were washed twice with 100 μl of PBST, and then 50 μl of each sample and standard was dispensed at a concentration of 1 μg / ml and reacted at room temperature for 2 hours. After washing twice with 100 μl of PBST, the detection antibody was diluted with RD and dispensed in a volume of 50 μl according to the concentration. The reaction was carried out at room temperature for 2 hours. After washing twice with 100 μl of PBST, streptavidin-horseradish peroxidase (Streptavidin-horseradish peroxidase) was diluted to 1/200 in the RD, and the cells were reacted at room temperature for 30 minutes. After washing, 50 μl of a 1: 1 mixture of TMB (3,3,5,5-tetramethylbenzidine) substrate and 0.04% hydrogen peroxide solution was dispensed, followed by color development after 5 minutes to 20 minutes The reaction was stopped by adding 50 μl of 1 M sulfuric acid solution and the absorbance was measured at 450 nm using Synergy ™ HT multi-detection microplate reader (BioTek, USA).

As a result, it was confirmed that the IL-6 and TNF-α secretion by the Escherichia coli-derived vesicles was remarkably inhibited when the vesicle derived from Proteus mirabilis was pretreated (see FIGS. 18A and 18B).

In order to evaluate the effect of the subtype of Proteus mirabilis on the anti-inflammatory effect of the parasites derived from the Proteus mirabilis, five parasites derived from Proteus mirabilis isolated strains (PMR201, PMR202, PMR203, PMR204 , PMR205) were pretreated for 12 hours at various concentrations, and 1 μg / ml of the E. coli-derived vesicle, a pathogenic vesicle, was treated for 12 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, followed by ELISA.

As a result, it was confirmed that the pretreatment of the parasite derived from the isolate of Proteus mirabilis significantly inhibited the secretion of TNF-? By the Escherichia coli-derived parasite as in the case of the parasite (PMR101) derived from the Proteus mirabilis premodern strain (see Fig. 19 ). In particular, it was confirmed that the effect of suppressing the secretion of TNF- [alpha] by pretreatment of the microbial control-derived vesicles derived from Lactobacillus plantarum was greater than that of the vesicles derived from Proteus mirabilis (see Fig. 19).

This means that the inflammatory reaction induced by pathogenic vesicles such as Escherichia coli-derived vesicles can effectively suppress the inflammatory reaction regardless of the subtype from the isolate derived from the Proteus mirabilis isolate.

Example  10. The anti-inflammatory action of parasites derived from Proteus mirabilis Acid-treated  effect

The anti-inflammatory effect of the standard strains of Proteus mirabilis and the isolates derived from the isolate strain was confirmed through Example 9, and further the stability of the vesicles and the characteristics of the active substances were investigated concretely. For this purpose, three kinds of proteasum-labile vesicles (PMR101, PMR202 and PMR205) were boiled for 10 minutes at 100 ° C for 10 minutes and then subjected to acid treatment (pH 2.0) for 10 minutes to pretreat the macrophages (Raw 264.7) Respectively.

As a result, it was confirmed that the anti-inflammatory effect of the parasites derived from Proteus mirabilis was maintained even if the parasites were boiled or acid-treated at 100 ° C (see FIG. 20). This means that the vesicles derived from Proteus mirabilis are stable at high temperatures and acids, and that exhibiting an anti-inflammatory effect in the vesicles derived from Proteus mirabilis is not a protein component.

Example 11. Anticancer effect of parasites derived from Proteus mirabilis

In the above examples, it was confirmed that the parasites derived from bacteria in the genus Proteus were significantly reduced in the clinical samples of the cancer patients as compared with the normal persons. Further, the anticancer effect of the parasites was investigated in detail. To this end, as shown in Fig. 21A, the parasites derived from Proteus mirabilis were orally administered to 6-week-old C57BL / 6 male mice and subcutaneously injected with a cancer cell line (CT26 cell) . The size of cancer tissues was measured until 20 days after the administration of cancer cells, and the cancer treatment effect was evaluated. As a result, the size of cancer tissue was remarkably reduced in the mice to which the vesicle was orally administered compared with the group administered with the physiological saline orally (see Fig. 21B).

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 bacterium-derived vesicles derived from the genus Proteus according to the present invention can be used for the treatment of cancer such as stomach cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, myocardial infarction, Allergic-respiratory diseases such as atopic dermatitis, asthma, and chronic obstructive pulmonary disease, hypersensitivity syndrome, inflammatory bowel disease, etc., which are caused by atopic dermatitis, cardiovascular diseases such as angina pectoris, stroke, metabolic diseases such as diabetes, Inflammatory bowel disease, and it is expected to be economical and applicable to a wide range of compositions for prevention, treatment, and / or improvement of foods, inhalants, cosmetics, and drugs, Functional foods, cosmetics industry, and the like.

Claims (17)

The present invention relates to a method for the treatment of gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, Methods for providing information for the diagnosis of one or more diseases selected from the group consisting of asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression: (a) extracting DNA from vesicles isolated from normal and subject samples; (b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a pair of primers prepared based on the gene sequence existing in 16S rDNA to obtain respective PCR products; And (c) Quantitative analysis of the above PCR products reveals that the content of microbes derived from bacteria in Proteus is lower than that of a normal human, , At least one disease selected from the group consisting of lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, irritable bowel syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression &Lt; / RTI &gt; The method according to claim 1, Wherein the sample in step (a) is blood, urine, or sputum. The present invention relates to a method for the treatment of gastric cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, Diagnosis of one or more diseases selected from the group consisting of asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression: (a) extracting DNA from vesicles isolated from normal and subject samples; (b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a pair of primers prepared based on the gene sequence existing in 16S rDNA to obtain respective PCR products; And (c) Quantitative analysis of the above PCR products reveals that the content of microbes derived from bacteria in Proteus is lower than that of a normal human, , At least one disease selected from the group consisting of lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, irritable bowel syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression &Lt; / RTI &gt; The method of claim 3, Wherein the sample in step (a) is blood, urine, or sputum. Cancer of the lungs, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, gastrointestinal cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, , At least one disease selected from the group consisting of hyperinsulinemic syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression. 6. The method of claim 5, Wherein said vesicles have an average diameter of from 10 to 200 nm. 6. The method of claim 5, Wherein said vesicles are secreted naturally or artificially in the bacterium of the genus Proteus. 6. The method of claim 5, Wherein the microbial virus derived from the genus Proteus is secreted from Proteus mirabilis. Cancer of the lungs, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, gastrointestinal cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, At least one disease selected from the group consisting of hyperinsulinemic syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression. 10. The method of claim 9, Characterized in that the vesicles are secreted naturally or artificially in the bacterium of the genus Proteus. 10. The method of claim 9, Wherein said microbial-derived vesicle is secreted from Proteus mirabilis. Cancer of the lungs, breast cancer, ovarian cancer, bladder cancer, prostate cancer, lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, gastrointestinal cancer, colon cancer, liver cancer, biliary cancer, pancreatic cancer, , At least one disease selected from the group consisting of hyperinsulinemic syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression. 13. The method of claim 12, Wherein the composition is a food composition or a cosmetic composition. 13. The method of claim 12, Characterized in that the vesicles are secreted naturally or artificially in the bacterium of the genus Proteus. 13. The method of claim 12, Wherein the proteasome-derived vesicle is secreted from Proteus mirabilis. Colon cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, colon cancer, colon cancer, colon cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer and prostate cancer, which comprises administering to a subject a pharmaceutical composition comprising an effective amount of a bacterium- , At least one disease selected from the group consisting of lymphoma, brain tumor, chronic obstructive pulmonary disease, atopic dermatitis, irritable bowel syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression &Lt; / RTI &gt; The present invention relates to a pharmaceutical composition comprising as an active ingredient a bacterium-derived vesicle of the genus Proteus in the genus Escherichia coli, which is useful as a therapeutic agent for gastric cancer, colon cancer, hepatic cancer, bile duct cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, bladder cancer, At least one disease selected from the group consisting of atopic dermatitis, atopic dermatitis, irritable bowel syndrome, inflammatory bowel disease, asthma, myocardial infarction, cardiomyopathy, angina pectoris, atrial fibrillation, stroke, diabetes, Parkinson's disease and depression.

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