WO2019164230A1 - Cupriavidus sp. bacterium-derived nanovesicles and use thereof - Google Patents

Abstract

The present invention relates to Cupriavidus sp. bacterium-derived nanovesicles and a use thereof. The present inventors experimentally found that significantly reduced levels of the vesicles are in samples from patients with malignant diseases, such as stomach cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, etc., cardiac diseases, such as cardiomyopathy, atrial fibrillation, variant angina, etc., chronic obstructive pulmonary disease, stroke, diabetes mellitus, renal insufficiency, dementia, Parkinson's disease, and depression, compared to normal persons and the vesicles significantly inhibit the pathogenic vesicle-induced secretion of inflammation mediators as well as suppressing oncogenesis. The Cupriavidus sp. bacterium-derived nanovesicles according to the present invention can be effectively used to develop a method for diagnosis of malignant diseases, such as stomach cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, etc., cardiac diseases, such as cardiomyopathy, atrial fibrillation, variant angina, etc., chronic obstructive pulmonary disease, stroke, diabetes mellitus, renal insufficiency, dementia, Parkinson's disease, or depression, and a composition for prevention or treatment of the diseases.

Description

Nanovesicles derived from bacteria of Cupri avidus and uses thereof

The present invention relates to nanovesicles derived from the genus Cupriavidus and its use, and more specifically, cancer, cardiovascular disease, lung disease, metabolic disease, neuro-psychiatric diseases, etc. It relates to a diagnostic method, and compositions for the prevention, improvement or treatment of the disease, including the vesicles.

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

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

The symbiotic bacteria in the body and the bacteria present in the environment secrete nanometer-sized vesicles to exchange information such as genes, low molecular weight compounds, and proteins with other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. After passing through epithelial cells through the mucous membrane, it is absorbed by our body. Locally secreted bacteria-derived vesicles are absorbed through epithelial cells or keratinocytes of the mucosa to induce local inflammatory responses, as well as to regulate immune and inflammatory responses in the organs absorbed by the body and distributed to each organ. do. For example, Eshcherichia vesicles derived from pathogenic Gram-negative bacteria such as coli ) are inhaled through the airways to induce emphysema, which leads to chronic obstructive pulmonary disease (COPD), and when absorbed through the intestine, causes colitis locally, Through vascular endothelial inflammatory response, it promotes systemic inflammatory response and blood coagulation, and is also absorbed into the muscle cells in which insulin acts, causing insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control disease by controlling immune function abnormalities caused by pathogenic vesicles.

The immune response to factors such as vesicles derived from bacteria causes a Th17 immune response characterized by the secretion of Interleukin (IL) -17 cytokines, which secrete IL-6 upon exposure to bacterial vesicles, This induces a Th17 immune response. Inflammation by the Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF-α) is secreted from inflammatory cells such as macrophages and neutrophils during inflammation. Plays an important role in the occurrence of.

Cupriavidus spp. Is an aerobic Gram-negative bacillus isolated from soil and clinical specimens. Among these, Cupriavidus metallidurans is characterized by resistance to toxic heavy metals, producing energy through phosphorylation through oxygen, and generally not pathogenic. Cupriavidus species, such as Cupriavidus necator and Cupriavidus taiwanensis, are known as bacteria that fix nitrogen in legumes. However, it has not been reported that bacteria in cupri avidus secrete extracellular vesicles, and in particular, have been applied to the diagnosis and treatment of refractory diseases such as cancer, cardiovascular disease, lung disease, metabolic disease and neuropsychiatric disease. It has not been done.

The present inventors earnestly researched to solve the above-mentioned conventional problems, and compared to the normal person through metagenomic analysis, stomach cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, etc. The contents of bacteria-derived vesicles from Cupri abidus were significantly increased in samples from patients with malignant diseases, cardiomyopathy, atrial fibrillation, heteroangular angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression. It was confirmed that it is reduced. In addition, when vesicles were isolated from C. metallidurans bacteria belonging to the genus of Cupriaviders and treated with macrophages, IL-6 and TNF-α secretion was significantly suppressed by pathogenic vesicles. At the same time confirmed that the anti-cancer effect in the mouse model, the present invention was completed based on this.

Accordingly, the present invention is gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease, stroke, diabetes, kidney failure, dementia To provide informational methods for diagnosing Parkinson's disease or depression.

In addition, the present invention is a gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, heteroangular angina, Another object is to provide a composition for preventing, ameliorating or treating chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention as described above, the present invention includes the following steps, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrium Provides information on the diagnosis of fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, kidney failure, dementia, Parkinson's disease, or depression:

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

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

(c) Gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, Classification of lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

In addition, the present invention includes the following steps, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease, Provides diagnostic methods for stroke, diabetes, kidney failure, dementia, Parkinson's disease, or depression:

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

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

(c) Gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, Classification of lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

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

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

In addition, the present invention includes a vesicle derived from the bacteria of the genus Cupriavidus as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, heterogeneity Provided is a pharmaceutical composition for preventing or treating angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

In addition, the present invention includes a vesicle derived from the bacteria of the genus Cupriavidus as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, heterogeneity Provided is a food composition for preventing or ameliorating angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

In addition, the present invention is a gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial chamber containing the cubic vesicles derived from the genus Cupriavidus as an active ingredient Provided is an inhalant composition for the prevention or treatment of fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease or depression.

In addition, the present invention comprises the step of administering to the subject a pharmaceutical composition comprising the cubic vesicles bacteria-derived vesicles as an active ingredient, gastric cancer, colon cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, Provided are methods for the prevention or treatment of head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, heterogeneous angina, chronic obstructive pulmonary disease, stroke, diabetes, kidney failure, dementia, Parkinson's disease, or depression.

In addition, the present invention is a vesicle derived from the genus Cupriavidus, stomach cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary Provided for the prophylaxis or treatment of a disease, stroke, diabetes, kidney failure, dementia, Parkinson's disease, or depression.

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

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

In another embodiment of the present invention, the cupria-derived bacteria-derived vesicles may be cupria-derived metallidurance-derived vesicles.

The present inventors confirmed that intestinal bacteria are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs. Stomach cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation , The presence of bacteria-derived vesicles in cupriavidus present in feces, blood, urine or saliva of patients with chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, and depression It was. In addition, the cuprous avidus metallidurans, a species of the bacterium of the cupri avidus, was cultured in vitro to separate the vesicles, and when administered to inflammatory cells in vitro, it significantly inhibited the secretion of inflammatory mediators caused by pathogenic vesicles. As confirmed, the bacterium-derived vesicles of the genus cupriavidus according to the present invention are gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, heteroangular angina, chronic It is expected that the present invention can be usefully used in the diagnosis of obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression, and compositions for preventing, improving or treating such diseases.

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

Figures 2a to 2c is a result of comparing the distribution of bacteria-derived vesicles of Cupri avidus after the bacteria-derived vesicle metagenome analysis present in gastric cancer patients and normal people, Figure 2a is feces, Figure 2b is blood, Figure 2c is urine The result is a sample.

Figure 3a and 3b is a result of comparing the distribution of bacteria-derived vesicles in cupri avidus after analyzing the bacteria-derived vesicle metagenome present in colorectal cancer patients and normal people, Figure 3a is a stool, Figure 3b is a urine sample The result is.

Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Cupri avids after analyzing the bacteria-derived vesicles metagenome present in the pancreatic cancer patients and normal blood.

Figure 5 is a result of comparing the distribution of the bacteria-derived vesicles of the genus Cupri avids after analyzing the bacteria-derived vesicles metagenome present in patients with bile duct cancer.

Figure 6a and 6b is a result of comparing the distribution of bacteria-derived vesicles of Cupri Avidus after the bacteria-derived vesicle metagenome analysis present in breast cancer patients and normal people, Figure 6a is a blood, Figure 6b is a result of the sample as urine to be.

7A and 7B show the distribution of bacteria-derived vesicles from cupriavidus after bacterial vesicle metagenome analysis in ovarian cancer patients and normal people. FIG. 7A is blood and FIG. 7B is urine as a sample. The result is.

Figures 8a and 8b is a result of comparing the distribution of bacteria-derived vesicles in cupri avids after analyzing the bacteria-derived vesicle metagenome analysis present in bladder cancer patients and normal people, Figure 8a is the blood, Figure 8b is the result of the sample as urine to be.

9 is a result of comparing the distribution of bacteria-derived vesicles of Cupri avidus after analyzing the bacteria-derived vesicles metagenome present in prostate cancer patients and normal urine.

Figure 10 is a result of comparing the distribution of the bacteria-derived vesicles of the genus Cupriavidus after analyzing the bacteria-derived vesicles metagenome present in head and neck cancer patients and normal saliva.

11 is a result of comparing the distribution of the bacteria-derived vesicles of Cupri avidus after analyzing the bacteria-derived vesicles metagenome present in lymphoma patients and normal blood.

12a to 12c is a result of comparing the distribution of bacteria-derived vesicles derived from cupriavidus after bacterial-derived vesicle metagenome analysis present in patients with heart disease and normal blood, Figure 12a is a cardiomyopathy, Figure 12b is atrial fibrillation, Figure 12c shows the case of heteroangular angina.

Fig. 13 shows the results of comparing the distribution of bacterial-derived vesicles of Cupri avidus after analyzing the bacterial-derived vesicles metagenome that are present in stroke patients and normal blood.

14 is a result of comparing the distribution of bacteria-derived vesicles of Cupri avidus after performing bacterial-derived vesicle metagenome analysis present in patients with chronic obstructive pulmonary disease (COPD) and normal blood.

Figure 15a to 15c is a result of comparing the distribution of bacteria-derived vesicles in cupri avidus after analyzing the bacteria-derived vesicle metagenome analysis present in diabetic patients and normal people, Figure 15a is blood, Figure 15b is urine, Figure 15c is saliva The result is a sample.

Fig. 16 shows the results of comparison of distribution of bacteria-derived vesicles belonging to Cupriaviders after analysis of bacterial-derived vesicles metagenome present in renal failure patients and normal blood.

Fig. 17 shows the results of comparing the distribution of bacterial-derived vesicles of Cupri avidus after the analysis of bacterial-derived vesicles metagenome present in dementia patients and normal blood.

Fig. 18 shows the results of comparing the distribution of bacterial-derived vesicles belonging to Cupriaviders after the bacterial-derived vesicle metagenome analysis present in Parkinson's disease patients and normal urine.

19 is a result of comparing the distribution of bacteria-derived vesicles of Cupri avidus after the analysis of bacteria-derived vesicles metagenome in depressed patients and normal urine.

20 is a result of evaluating the degree of apoptosis by administering the vesicles to macrophages (Raw264.7) in order to evaluate the apoptosis effect of cupri avidus metallidurans-derived vesicles.

Figure 21 is a pre-treatment of the cuprividus bacteria-derived vesicles before the treatment of E. coli EV, a pathogenic vesicle (E. coli EV) in order to evaluate the anti-inflammatory and immunomodulatory effect of the cupri-aviders metallidurans-derived vesicles, Is a result of evaluating the effect on the secretion of IL-6 and TNF-α.

FIG. 22 is a protocol in which cupriavidose metallidurans-derived vesicles were administered to mice in order to evaluate the anticancer efficacy of cupriavidus metallidurans-derived vesicles.

23 is to evaluate the anti-cancer efficacy of the cupri avids metallidurans-derived vesicles, administration of cupri avids metallidurans vesicles intraperitoneal (IP) or oral (PO), the effect on tumor development by cancer cells Is the result of evaluation.

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

The present inventors have found that, through metagenome analysis, the vesicle-derived vesicles of Cupri avidus are significantly reduced in clinical samples of patients with cancer, cardiovascular disease, lung disease, metabolic disease, and neuro-psychiatric disease compared to normal people. It was confirmed that can be diagnosed. In addition, the first separation and characterization of the vesicles from C. metallidurans strains, it was confirmed that the strain-derived vesicles can control immune function abnormalities, inflammation and cancer caused by pathogenic vesicles. .

Thus, the present invention includes the following steps, gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease, Provides information on how to diagnose stroke, diabetes, kidney failure, dementia, Parkinson's disease, or depression:

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

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

(c) Gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, Classification of lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.

As used herein, the term "diagnosis" in the broad sense means to determine the actual condition of the patient's disease in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. In the present invention, the diagnosis is gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease, stroke, diabetes, kidney failure, dementia , Parkinson's disease, and / or depression, and the level of disease.

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

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

As another aspect of the present invention, the present invention comprises a stomach-derived bacterium-derived vesicles, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy It provides a composition for the prevention, treatment or amelioration of atrial fibrillation, heteroangular angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression. The composition includes a food composition, an inhalant composition, and a pharmaceutical composition, in which the food composition comprises a nutraceutical composition. The composition of the present invention may also be a formulation of an oral spray, nasal spray or inhalant.

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

As used herein, the term "treatment" means any action that improves or advantageously changes the condition for the disease by administration of the composition according to the present invention.

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

As used herein, the term "nanovesicle" or "vesicle" refers to a structure of nanoscale membranes secreted by various bacteria. Vesicles or outer membrane vesicles (OMVs) derived from gram-negative bacteria have proteins, low molecular weight compounds, and bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria. In addition to proteins, low molecular weight compounds and nucleic acids, the vesicles also contain peptidoglycan and lipoteichoic acid. In the present invention, the nano-vesicles or vesicles are naturally secreted or artificially produced by the bacteria of the cupri avidus, and has an average diameter of 10 to 200 nm.

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

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

In another embodiment of the present invention, gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, Bacterial metagenomic analysis was performed using vesicles isolated from feces, blood, urine, or saliva of normal persons with age and gender matching to patients with renal failure, dementia, Parkinson's disease, and depression. Colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic obstructive pulmonary disease, stroke, diabetes, kidney failure, dementia, Parkinson's disease, and depression It was confirmed that the vesicles derived from the bacterium of Cupri avidus were significantly reduced in the sample of patients (see Examples 3 to 20).

In another embodiment of the present invention, based on the results of the above embodiment, further studies were conducted to analyze the characteristics of the cuprous-derived metalliduranus bacteria-derived vesicles belonging to the genus of the cupri-avider genus. To evaluate whether the vesicles secreted therefrom exhibited anti-inflammatory effects by culturing Durans strains, and treated the macrophage-derived vesicles derived from cupriavidus metallidurans at various concentrations, As a result of evaluating the secretion of inflammatory mediators, it was confirmed that the cupria-derived metallidurans-derived vesicles effectively suppressed IL-6 and TNF-α secretion by E. coli-derived vesicles (see Example 22).

In another embodiment of the present invention, the cupri avids metalliduran strains were cultured to evaluate whether the vesicles secreted therefrom have anti-cancer therapeutic effects. To this end, a cancer model was made by subcutaneously injecting a cancer cell line, and the size of the cancer tissue was measured for 20 days after oral or intraperitoneal administration of the cupriavidus metallidurans-derived vesicles to the mouse 4 days prior to treatment with the cancer cell line. As a result, it was confirmed that when the vesicles were administered intraperitoneally and orally, the size of the cancer tissues was reduced compared to the control group, and particularly, when the vesicles were administered orally (see Example 23).

The pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.

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

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

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

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

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

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

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

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

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

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

Furthermore, after the intestinal bacteria and enteric bacteria-derived vesicles are absorbed systemically, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above to evaluate the infiltration into various organs. After 12 hours, blood, heart, lungs, liver, kidneys, spleen, fat and muscle were collected. As a result of fluorescence observed in the collected tissue, as shown in Figure 1b, the bacteria-derived vesicles were distributed in the blood, heart, lung, liver, spleen, fat, muscle, kidney, it can be seen that the bacteria are not absorbed.

Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples

Clinical samples such as blood, urine, feces, or saliva were first placed in a 10 ml tube and the suspension was allowed to settle by centrifugation (3,500 x g, 10 min, 4 ° C) and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign substances using a 0.22㎛ filter, and transferred to centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes to discard the material smaller than 50 kD concentrated to 10 ml. Once again, the bacteria and foreign substances were removed using a 0.22㎛ filter, and the supernatant was discarded using ultrafast centrifugation for 3 hours at 150,000 xg and 4 ℃ with a Type 90ti rotor, and the pulverized pellet was physiologically It was dissolved in saline (PBS).

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

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

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

Example 3 Analysis of Vesicle Metagenome Derived from Stool, Blood, and Urine Bacteria of Stomach Cancer Patients

In the method of Example 2, the genes were extracted from the vesicles in the stool and 63 stools of gastric cancer patients and 126 normal controls matched with sex and age. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the Cupri avidus bacteria significantly reduced in the stool of gastric cancer patients compared to the normal stool (see Table 2 and Figure 2a).

credit	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0054	0.0308	0.0000	0.0001	0.001	0.01

In the method of Example 2, the blood of 67 gastric cancer patients and 198 blood of the normal control group matched with sex and age were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of gastric cancer patients compared to normal blood (see Table 3 and FIG. 2B).

blood	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0094	0.0158	0.0013	0.0026	<0.0001	0.13

In addition, in the method of Example 2, the genes were extracted from the vesicles present in the urine in the urine of 61 gastric cancer patients and 120 urine control groups matched with sex and age, followed by metagenomic analysis. The distribution of vesicles derived from bacteria in Biders was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders significantly decreased in the urine of gastric cancer patients compared to normal urine (see Table 4 and FIG. 2C).

Pee	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0139	0.0687	0.0045	0.0071	0.01	0.33

Example 4 Analysis of Vesicle Metagenome Derived from Stool and Urine Bacterial Patients

In the method of Example 2, the gene was extracted from the vesicles in the stool and subjected to metagenomic analysis of the feces of 52 colon cancer patients and 83 of the normal control group matched with sex and age, and then cupri-aviders. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of cupriavidus significantly decreased in the stool of colorectal cancer patients compared to the normal stool (see Table 5 and FIG. 3A).

credit	Control		Colorectal cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0052	0.0306	0.0021	0.0082	0.01	0.40

In addition, in the method of Example 2, the urine of 38 patients with colorectal cancer and the urine of 38 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenomic analysis. The distribution of vesicles derived from the genus Priaviders was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders significantly decreased in urine of colorectal cancer patients compared to normal urine (see Table 6 and FIG. 3B).

Pee	Control		Colorectal cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0249	0.1064	0.0062	0.0039	0.04	0.25

Example 5 Analysis of Vesicular Metagenome Derived from Blood Bacteria of Pancreatic Cancer Patients

In the method of Example 2, 291 blood of the pancreatic cancer patient and 291 blood of the normal control group having matched gender and age were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of pancreatic cancer patients compared to normal blood (see Table 7 and FIG. 4).

blood	Control		Pancreatic cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0056	0.0132	0.0002	0.0010	<0.0001	0.03

Example 6 Analysis of Blood Bacteria Derived from Bacterial Cancer Patients

In the blood of 79 patients with cholangiocarcinoma patients and blood of 259 normal controls matched with sex and age, the genes were extracted from the vesicles in the blood and metagenome analysis was performed. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in the blood of bile duct cancer patients compared to normal blood (see Table 8 and FIG. 5).

blood	Control		Bile duct cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0058	0.0135	0.0000	0.0002	<0.0001	0.01

Example 7 Vesicular Metagenome Analysis of Blood and Urine Bacteria from Breast Cancer Patients

In the method of Example 2, the genes were extracted from the vesicles in the blood of 96 breast cancer patients and 192 blood of the normal control group matched with sex and age, and then subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 9 and FIG. 6A).

blood	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0158	0.0347	0.0065	0.0059	0.0004	0.41

In the method of Example 2, the genes were extracted from the vesicles in the urine of 127 breast cancer patients and 220 urine in the normal control group matched with sex and age. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the urine of breast cancer patients compared to normal urine (see Table 10 and FIG. 6B).

Pee	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0249	0.0865	0.0072	0.0065	0.002	0.29

Example 8. Vesicular metagenome analysis from blood and urine bacteria of ovarian cancer patients

In the method of Example 2, 136 blood of ovarian cancer patients and 136 blood of a normal control group matched with sex and age were extracted from vesicles present in the blood and subjected to metagenomic analysis, followed by cupri avids. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in blood of ovarian cancer patients compared to normal blood (see Table 11 and FIG. 7A).

blood	Control		Ovarian Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0242	0.0432	0.0011	0.0017	<0.0001	0.04

In the method of Example 2, the genes were extracted from the vesicles in the urine of 136 ovarian cancer patients and 136 urine control groups matched with sex and age, and then subjected to metagenomic analysis. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders significantly decreased in urine of ovarian cancer patients compared to normal urine (see Table 12 and FIG. 7B).

Pee	Control		Ovarian Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0333	0.0988	0.0016	0.0028	0.0002	0.05

Example 9 Analysis of Vesicular Metagenome Derived from Blood and Urine Bacteria of Bladder Cancer Patients

In the method of Example 2, genes were extracted from vesicles in the bladder cancer patients and blood of 184 normal controls matched with sex and age. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of bladder cancer patients compared to normal blood (see Table 13 and FIG. 8A).

blood	Control		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0085	0.0202	0.0002	0.0002	<0.0001	0.02

In addition, in the method of Example 2, the urine of 95 patients with bladder cancer and the urine of 157 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenomic analysis. The distribution of vesicles derived from bacteria in Biders was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders significantly decreased in urine of bladder cancer patients compared to normal urine (see Table 14 and FIG. 8B).

Pee	Control		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0020	0.0039	0.0008	0.0013	0.008	0.42

Example 10 Analysis of Vesicle Metagenome Derived from Urine Bacteria in Patients with Prostate Cancer

In the method of Example 2, the urine of 53 patients with prostate cancer and the urine of 159 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenomic analysis. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the urine of prostate cancer patients compared to normal urine (see Table 15 and FIG. 9).

Pee	Control		Prostate cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0117	0.0530	0.0051	0.0054	0.01	0.44

Example 11 Analysis of Vesicular Metagenome Derived from Saliva Bacteria of Head and Neck Cancer

In the method of Example 2, the saliva of 57 head and neck cancer patients and 277 saliva of the normal control group were extracted from the vesicles present in the saliva, and the metagenome analysis was performed. Was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the saliva of head and neck cancer patients compared to normal saliva (see Table 16 and FIG. 10).

saliva	Control		Head and Neck Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0038	0.0105	0.0001	0.0003	<0.0001	0.02

Example 12 Analysis of Blood Bacteria Derived from Lymphoma Patients with Vesicular Metagenome

In the method of Example 2, blood was collected from 57 patients with lymphoma and 163 blood of a normal control group matched with sex and age, and a gene was extracted from vesicles present in blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders was significantly reduced in the blood of lymphoma patients compared to normal blood (see Table 17 and FIG. 11).

blood	Control		Lymphoma		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0048	0.0105	0.0003	0.0011	0.003	0.05

Example 13. Vesicular Metagenome Analysis from Blood Bacteria in Heart Disease Patients

In the blood of 72 patients with cardiomyopathy and blood of 163 normal controls matched with sex and age, the genes were extracted from the vesicles present in the blood and metagenome analysis was performed. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of cupri avidus significantly decreased in the blood of cardiomyopathy patients compared to normal blood (see Table 18 and FIG. 12A).

blood	Control		Cardiomyopathy		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0077	0.012	0.0006	0.0011	0.0002	0.07

In the blood of 34 patients with atrial fibrillation in the method of Example 2 and 63 blood of the normal control group which matched sex and age, the genes were extracted from the vesicles present in the blood and metagenome analysis was performed. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in blood of atrial fibrillation patients compared to normal blood (see Table 19 and FIG. 12B).

blood	Control		Atrial fibrillation		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0011	0.0027	0.0000	0.0000	0.01	0.00

In the method of Example 2, the blood of 80 patients with heterozygous angina, and the blood of 80 normal controls matched with sex and age were extracted from vesicles present in the blood, and then subjected to metagenomic analysis. The distribution of vesicles derived from the genus bacteria was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of patients with heterophagy compared to normal blood (see Table 20 and FIG. 12C).

blood	Control		Angina		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0242	0.0492	0.0024	0.004	0.0002	0.10

Example 14 Parcel Metagenome Analysis from Blood Bacteria from Stroke Patients

In the method of Example 2, the genes were extracted from the vesicles in the blood of 115 patients with stroke and 109 normal controls matched with sex and age, and then subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of stroke patients compared to normal blood (see Table 21 and FIG. 13).

blood	Control		stroke		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0099	0.031	0.0001	0.0006	0.001	0.01

Example  15. Patients with Chronic Obstructive Pulmonary Disease (COPD)  Blood Bacteria-Derived Vesicles Metagenome  analysis

In the method of Example 2, 205 blood of COPD patients and 231 blood of normal control group matched with sex and age were extracted from vesicles present in the blood, and then subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in blood of COPD patients compared to normal blood (see Table 22 and FIG. 14).

blood	Control		COPD		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0107	0.0237	0.0049	0.0052	<0.0001	0.45

Example 16 Analysis of Vesicular Metagenome Derived from Blood, Urine, and Saliva Bacteria of Diabetic Patients

In the method of Example 2, the blood of 61 diabetic patients and 122 blood of the normal control group matched with sex and age were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in blood of diabetic patients compared to normal blood (see Table 23 and FIG. 15A).

blood	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0172	0.0367	0.0001	0.0001	<0.0001	0.01

In the method of Example 2, the urine of 60 diabetic patients and the urine of 134 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders significantly decreased in urine of diabetic patients compared to normal urine (see Table 24 and FIG. 15B).

Pee	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0137	0.0551	0.0007	0.0007	0.007	0.05

In the method of Example 2, the salivary saline of 37 diabetic patients and salivary salivary control group of 277 age-matched normal control groups were extracted from the vesicles present in the saliva, and then subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders were significantly reduced in the saliva of diabetic patients compared to normal saliva (see Table 25 and FIG. 15C).

saliva	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0038	0.0105	0.0004	0.0010	<0.0001	0.12

Example 17 Analysis of Vesicular Metagenome Derived from Renal Transitioner Blood Bacteria

In the method of Example 2, the blood of 32 renal convertors and the blood of the normal control group matched with sex and age were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in the blood of the renal converter compared to the normal blood (see Table 26 and FIG. 16).

blood	Control		Kidney failure		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0084	0.0073	0.0001	0.0002	0.0001	0.01

Example 18 Vesicular Metagenome Analysis of Blood Bacteria Derived from Patients with Dementia

In the method of Example 2, blood was collected from 67 blood of dementia patients and 70 blood of the normal control group which matched gender and age, and the genes were extracted from the vesicles present in the blood, followed by metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupri avidus significantly decreased in blood of dementia patients compared to normal blood (see Table 27 and FIG. 17).

blood	Control		dementia		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0008	0.0023	0.0000	0.0002	<0.0001	0.02

Example 19 Analysis of Vesicular Metagenome Derived from Urinary Bacteria in Parkinson's Disease Patients

The urine of 39 patients with Parkinson's disease and the urine of 76 normal controls matched with sex and age were extracted by extracting genes from the vesicles present in the urine, followed by metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders was significantly reduced in the urine of Parkinson's disease patients compared to normal urine (see Table 28 and FIG. 18).

Pee	Control		Parkinson's disease		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0287	0.1006	0.0000	0.0002	0.01	0.00

Example 20 Analysis of Vesicular Metagenome Derived from Urinary Bacteria with Depression

In the method of Example 2, the urine of 20 depressive patients and the urine of 21 normal controls matched with sex and age were extracted from the vesicles present in the urine, followed by metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Cupriaviders was significantly reduced in the urine of depressed patients compared to normal urine (see Table 29 and FIG. 19).

Pee	Control		depression		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Cupriavidus	0.0361	0.0783	0.0001	0.0002	0.04	0.00

Example 21.Cupriavidus Metallidurans Culture and Vesicle Separation

C. metallidurans strains were cultured and their vesicles were isolated and characterized. The cupriavidus metalliduran strain was incubated in TSB (Tryptic soy broth) medium until absorbance (OD ₆₀₀ ) was 1.0 to 1.5 in an aerobic chamber at 28 ° C. and then subcultured to Luria Bertani broth (LB) medium. -culture). Then, the culture medium supernatant containing the strain was recovered and centrifuged at 10,000 g, 4 ° C. for 20 minutes, and the strain was removed and filtered through a 0.22 μm filter. The filtered supernatant was concentrated to a volume of 50 ml by microfiltration using a MasterFlex pumpsystem (Cole-Parmer, US) with a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US). The concentrated supernatant was once again filtered through a 0.22 μm filter. Then, the protein was quantified using BCA (Bicinchoninic acid) assay, and the following experiment was performed on the obtained vesicles.

Example 22. Anti-inflammatory Effects of Cupria Avids Metallidurans-derived Vesicles

To investigate the effect of cupriavidus metallidurance- derived vesicles on apoptosis in inflammatory cells, C. metallidurans EV ( C. metallidurans EV) was applied to raw 264.7 cells of mouse macrophage. 0.1, 1, 10 μg / ml) and then cell viability test was performed. More specifically, by treating the vesicles derived from cupriaviders metalliduranseum containing various concentrations of DMEM (Dulbeco's Modified Eagle's Medium) serum-free medium into raw 264.7 cells dispensed 4 x 10 ⁴ in 48-well cell culture plates. Incubated for hours. The cells were then treated with EZ-CYTOX (Dogen, Korea) for 4 hours, and then absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devies, USA). As a result, as shown in Fig. 20, when a cuprimaids-derived vesicle-derived vesicles treated macrophages, it was confirmed that apoptosis was not induced.

Based on the results, in order to evaluate the anti-inflammatory effect of cupriavidus metallidurans-derived vesicles, cupriavidose metallidurans-derived vesicles of various concentrations (0.1, 1, 10 μg / ml) were applied to macrophage lines. After 12 hours of pretreatment, 1 μg / ml of Escherichia coli-derived vesicles, which were pathogenic vesicles, were treated and 12 hours later, the secretion of inflammatory cytokines was measured by ELISA. In order to perform ELISA, the capture antibody was diluted in phosphate buffered saline (PBS), and 50 μl was dispensed into 96 well polystyrene plates according to the working concentration, followed by reaction at 4 ° C. overnight. I was.

Next, wash three times with 100 μl of PBST (phosphate buffered saline solution containing 0.05% tween-20), and then dispense 100 μl of RD (phosphate buffered saline solution containing 1% BSA) at room temperature. Blocking for a time (blocking) and the sample and standard (50 μl) was dispensed in accordance with the concentration and reacted for 2 hours at room temperature. Then, after washing three times with 100 μl of PBST, the detection antibody was diluted in RD and 50 μl was dispensed according to the working concentration for 2 hours at room temperature, and washed three times with 100 μl of PBST. Streptavidin-HRP (R & D system, USA) was diluted 1/40 in RD, and 50 μl was dispensed for 20 minutes at room temperature.

Finally, after washing three times with 100 μl of PBST, 50 μl of TMB substrate (SurModics, USA) was dispensed, and when the color development progressed after 5 to 20 minutes, 50 μl of 1 M sulfuric acid solution was stopped to stop the reaction. Absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA).

As a result, as shown in Fig. 21, when pre-treated with the cupri avidus metalliduran derived vesicles, it was confirmed that the secretion of IL-6 and TNF-α by E. coli-derived vesicles is significantly suppressed. In particular, the effect of inhibiting the secretion of TNF-α by pretreatment of cupriavidus metalliduran-derived vesicles was more pronounced than the effect of inhibiting the secretion of TNF-α by pretreatment of vesicles derived from Lactobacillus plantarum, a useful microorganism control group. Confirmed large. The above results indicate that cupriavidus metallidurans-derived vesicles can effectively inhibit inflammatory responses induced by pathogenic vesicles such as E. coli-derived vesicles.

Example 23. Anticancer Effect of Cupria Aviders Metallidurans-derived Vesicles

Based on the above examples, the anti-cancer effect of cupria vidders metallidurans-derived vesicles was to be examined. To this end, as shown in Figure 22, the vesicles derived from Cuprividus metalliduran strain (CMT101) is administered intraperitoneally or orally to 6-week-old C57BL / 6 male mice, the cancer cell line (CT26 cell) on day 4 Was injected subcutaneously to make a cancer model. After administration of the cancer cell line, the vesicles derived from cupriavidus metallidurans strain were administered daily by intraperitoneal injection or orally and the size of the cancer tissue was measured until the 24th day. As a result, as shown in Figure 23, the size of the cancer tissue was reduced in the size of the cancer tissue in mice orally administered mice and the oral administration of the vesicles compared to the physiological saline oral administration group as a control group, in particular, orally When administered, the size was further reduced. The results indicate that the administration of cupriavidus metallidurance-derived vesicles can effectively inhibit the growth of cancer tissues.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

The vesicle-derived vesicles of the genus Cupriavides according to the present invention are gastric cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina pectoris, chronic obstructive pulmonary disease, As a diagnostic method for stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression, it can be used as a composition for preventing, ameliorating or treating the disease, and is expected to be useful in related medical and food industries. do.

Claims (14)

1. Gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, comprising the following steps To provide information for diagnosing diabetes, dementia, Parkinson's disease, or depression:

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

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

   (c) Gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, when the content of extracellular vesicles derived from bacteria of Cupriavidus is lower than that of normal people through quantitative analysis of the PCR products. Classify as head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.
2. The method of claim 1,

   The sample in step (a) is characterized in that the feces, blood, urine, or saliva, information providing method.
3. Stomach cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, containing the vesicles derived from bacteria of Cupriavidus A pharmaceutical composition for preventing or treating at least one disease selected from the group consisting of chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, and depression.
4. The method of claim 3,

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

   The vesicles are characterized in that they are secreted naturally or artificially from the bacteria of the genus cupriavidose, pharmaceutical composition.
6. The method of claim 3,

   The cuprividus genus bacteria-derived vesicles are characterized in that the secretion from Cupriavidus metallidurans , pharmaceutical composition.
7. Stomach cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, containing the vesicles derived from bacteria of Cupriavidus Food composition for preventing or ameliorating at least one disease selected from the group consisting of chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, and depression.
8. The method of claim 7, wherein

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

   The vesicles are characterized in that they are secreted naturally or artificially from the bacteria of the genus cupriavidus, food composition.
10. The method of claim 7, wherein

    The cupria vesicles bacteria-derived vesicles are characterized in that the secretion from Cupriavidus metallidurans , food composition.
11. Stomach cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina, containing bacterial vesicles of Cupriavidus genus as an active ingredient Inhalation composition for preventing or treating at least one disease selected from the group consisting of chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, and depression.
12. Gastric cancer, colorectal cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, comprising the following steps To diagnose, dementia, Parkinson's disease, or depression:

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

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

    (c) Gastric cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, when the content of extracellular vesicles derived from bacteria of Cupriavidus is lower than that of normal people through quantitative analysis of the PCR products. Classify as head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease, or depression.
13. Stomach cancer, colorectal cancer, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, comprising administering to the subject a pharmaceutical composition comprising the cupria-derived bacteria-derived vesicles as an active ingredient. A method of preventing or treating one or more diseases selected from the group consisting of cardiomyopathy, atrial fibrillation, angina, chronic obstructive pulmonary disease, stroke, diabetes, renal failure, dementia, Parkinson's disease and depression.
14. Stomach cancer, colon cancer, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, bladder cancer, prostate cancer, head and neck cancer, lymphoma, cardiomyopathy, atrial fibrillation, dysplastic angina pectoris, chronic obstructive pulmonary disease, stroke, diabetes Or for the prevention or treatment of one or more diseases selected from the group consisting of renal failure, dementia, Parkinson's disease and depression.

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