WO2019172600A1 - Nanovesicles derived from enhydrobacter bacteria, and use thereof - Google Patents

Abstract

The present invention relates to vesicles derived from Enhydrobacter bacteria, and a use thereof. The present inventors experimentally confirmed that: compared to those of a normal person, the vesicles are significantly reduced in clinical samples from patients with pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, cirrhosis and diabetes; and when the vesicles isolated from a strain are administered, the secretion of inflammatory mediators by pathogenic vesicles, such as vesicles derived from Escherichia coli, is notably inhibited. Thus, the vesicles derived from Enhydrobacter bacteria, according to the present invention, may be usefully employed for the purpose of developing: a method for diagnosing pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, variant angina, cirrhosis or diabetes; and a composition for preventing, improving or treating said diseases.

Description

Enhydrobacter bacteria-derived nanovesicles and uses thereof

The present invention relates to nanovesicles derived from enhydrobacter bacteria and their use, and more specifically, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy and atrial fibrillation using nanovesicles derived from enhydrobacter bacteria. It relates to a diagnostic method such as angina, liver cirrhosis, or diabetes mellitus, and a composition for preventing, ameliorating or treating such diseases 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 the refractory chronic disease of the 21st century, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric disease are major diseases that determine human life and quality of life, which is a major problem in public health.

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

The symbiotic bacteria in the body and the bacteria present in the environment secrete nanometer-sized vesicles to exchange information such as genes, low molecular weight compounds, and proteins with other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacterial-derived vesicles are absorbed through the epithelial cells of the mucosa to induce local inflammatory responses, as well as vesicles passing through the epithelial cells are systemically absorbed through the lymphatic vessels and distributed to each organ. Regulates immune and inflammatory responses For example, vesicles derived from pathogenic gram-negative bacteria, such as Eshcherichia coli , locally cause inflammatory reactions and cancer, and systemic inflammatory and blood coagulation through vascular endothelial inflammatory responses when absorbed into blood vessels. It is also promoted, and insulin is absorbed in muscle cells, etc. cause insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control the disease by controlling the immune and metabolic 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), which is secreted from inflammatory cells such as macrophages in the process of inflammation, plays an important role. In charge.

Enhydrobacter bacteria are Gram-negative bacteria belonging to gamma proteobacteria. Enhydrobacter aerosaccus is the only species belonging to the genus Enhydrobacter genus, and is known as a catalase and oxidase positive bacterium. However, there has been no report on the secretion of extracellular vesicles from Enhydrobacter bacteria, and the application of Enhydrobacter vesicles for the diagnosis and treatment of intractable diseases such as cancer, cardiovascular disease and diabetes has not been reported. There is no bar.

The present inventors earnestly researched to solve the above-mentioned conventional problems, and compared with the normal person through pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina pectoris, cirrhosis It was confirmed that the contents of enhydrobacter bacteria-derived vesicles were significantly reduced in the samples derived from, and diabetic patients. In addition, when vesicles were isolated from enhydrobacter aerosacus bacteria belonging to the enhydrobacter bacterium and treated with macrophages, it was confirmed that significantly suppressed the secretion of IL-6 and TNF-α by pathogenic vesicles. The present invention has been completed.

Accordingly, an object of the present invention is to provide a method for providing information for the diagnosis of pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes mellitus.

In addition, the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, liver cirrhosis, or diabetes prevention, improvement or treatment containing vesicles derived from enhydrobacter bacteria as an active ingredient It is another object to provide a composition for use.

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

In order to achieve the object of the present invention as described above, the present invention comprises the following steps, pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes mellitus Provide informational methods for diagnosis:

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

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

(c) pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Classify as dysplastic angina, cirrhosis, or diabetes.

The present invention also provides a method for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, liver cirrhosis, or diabetes, comprising the following steps:

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

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

(c) pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Determining dysplastic angina, cirrhosis, or diabetes.

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

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 is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, It provides a pharmaceutical composition for the prevention or treatment of chronic liver disease or diabetes.

In addition, the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, Provided is a food composition for preventing or improving chronic liver disease or diabetes.

In addition, the present invention is an enhydrobacter bacteria-derived vesicles, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, Provided for the prevention or treatment of chronic liver disease or diabetes.

In addition, the present invention comprises administering to a subject a pharmaceutical composition comprising an enhydrobacter bacteria-derived vesicle as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, Provided are methods for preventing or treating dysplastic angina, chronic liver disease or diabetes.

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 in the enhydrobacterium bacteria.

In another embodiment of the present invention, the enhydrobacter bacteria-derived vesicles may be vesicles derived from enhydrobacter aeroscus.

The present inventors confirmed that the bacteria are not absorbed into the body, but the bacteria-derived vesicles are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs. Analysis of existing bacterial-derived vesicles metagenome revealed that vesicles derived from pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heterozygous angina, cirrhosis, and blood of diabetic patients It was confirmed that the significantly reduced compared to the normal person. In addition, it was observed that when vesicles were isolated by culturing enhydrobacter aerosacus, a species belonging to the enhydrobacter bacterium, and administered to inflammatory cells in vitro, it was observed that significantly inhibiting the secretion of inflammatory mediators by pathogenic vesicles. The vesicles derived from the enhydrobacter bacterium according to the present invention are pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, liver cirrhosis, or diagnostic method for diabetes, and prevention of the disease. It is expected that the present invention can be usefully used in improving or treating compositions.

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 Figure shows the distribution of bacteria, vesicles and vesicles in the body, liver and various organs.

Figure 2 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with pancreatic cancer and normal blood.

Figure 3 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with bile duct cancer.

Figure 4 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing the bacteria-derived vesicles metagenome analysis present in breast cancer patients and normal blood.

5 is a result of comparing the distribution of enhydrobacter bacterium-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in ovarian cancer patients and normal blood.

Figure 6 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in lymphoma patients and normal blood.

7 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in myocardial infarction patients and normal blood.

8 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after carrying out bacterial-derived vesicle metagenome analysis present in cardiomyopathy patients and normal blood.

9 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with atrial fibrillation and normal blood.

10 is a result of comparing the distribution of enhydrobacter bacterium-derived vesicles after performing a bacterial-derived vesicle metagenome analysis present in patients with heterozygous angina and normal blood.

11 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after performing the bacterial-derived vesicle metagenome analysis present in the liver cirrhosis transducer and normal blood.

12 is a result of comparing the distribution of enhydrobacter bacteria-derived vesicles after conducting a bacterial-derived vesicle metagenome analysis present in diabetic patients and normal blood.

13 is a pre-treatment of E. coli vesicles ( E. coli EV) before the treatment of Escherichia coli vesicles ( E. coli EV), in order to evaluate the anti-inflammatory and immunomodulatory effect of the endobacterial vesicles derived from E. hydrophilis, The results of the evaluation of the inflammatory mediators IL-6 and TNF-α secretion (PC: positive control; LP: Lactobacillus plantarum EVs; EA: Enhydrobacter aerosaccus EVs).

The present invention relates to vesicles derived from enhydrobacter bacteria and uses thereof.

The inventors noted that metagenome analysis of clinical samples of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, and diabetic patients compared to normal human It was confirmed that the disease can be diagnosed by the reduction. In addition, vesicles were isolated and characterized from enhydrobacter aerosacus, the only species belonging to the enhydrobacter bacterium, and found to be pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, It was confirmed that it can be used as a composition for preventing, improving or treating diseases such as chronic liver disease and diabetes.

Accordingly, the present invention provides a method for providing information for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, liver cirrhosis, or diabetes, comprising the following steps:

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

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

(c) pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Classify as dysplastic angina, cirrhosis, or diabetes.

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. Diagnosis in the present invention is to determine whether the pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, and / or diabetes, and the level of the disease.

As used herein, the term "nanovesicle" or "vesicle" refers to a structure of nanoscale membranes secreted by various bacteria. Vesicles derived from gram-negative bacteria, or outer membrane vesicles (OMVs), contain toxic proteins, bacterial DNA and RNA as well as endotoxins, and gram-positive bacteria-derived vesicles. In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls. In the present invention, nanovesicles or vesicles are naturally secreted or artificially produced by enhydrobacter bacteria, and have a spherical shape and have an average diameter of 10 to 200 nm.

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.

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

As another embodiment of the present invention, the present invention comprises an enhydrobacter bacteria-derived vesicles as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic liver disease, Or it provides a composition for preventing, treating or improving diabetes. The composition comprises a food composition and a pharmaceutical composition, in the present invention the food composition comprises a nutraceutical composition. The composition of the present invention may be a formulation of an oral nebulizer or inhalant.

As used herein, the term "prevention" refers to pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, chronic liver disease, and / or by administration of a composition according to the invention. It means any action that suppresses or delays the onset of diabetes.

As used herein, the term “treatment” means pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, dysplastic angina, chronic liver disease, and / or by administration of a composition according to the invention. Means any behavior that improves or beneficially changes the symptoms of diabetes.

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.

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

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

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

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

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

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

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

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

In one embodiment of the present invention by observing the bacteria and bacteria-derived vesicles orally by the mouse to observe the absorption, distribution, and excretion of the bacteria and vesicles in the body, when the bacteria are not absorbed through the intestinal membrane, the vesicles are administered 5 It was confirmed that it was absorbed within minutes and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).

In another embodiment of the present invention, the vesicles isolated from the blood of normal people matched age and sex to patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina with angina, liver cirrhosis, and diabetes Bacterial metagenome analysis was performed using. As a result, enhydrobacter bacterial-derived vesicles were significantly reduced in clinical samples of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, and diabetic patients compared to normal samples. (See Examples 3 to 13).

In another embodiment of the present invention, it was evaluated whether the vesicles secreted from the culturing enhydrobacter aerosakus exhibit immunomodulatory and anti-inflammatory effects, the various concentrations of the enhydrobacter aeroscus-derived vesicles macrophages After treatment with E. coli-derived vesicles, which are the causative agent of inflammatory diseases, the secretion of E. coli-derived vesicles was evaluated. (See Example 14).

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, the experiment was performed as follows. Fluorescently labeled bacteria and vesicle-derived vesicles were administered to the gastrointestinal tract at a dose of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the whole image of the mouse, as shown in FIG. 1A, the bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder fluorescence after 3 hours This was observed strongly, indicating that the vesicles were excreted by the urinary system. In addition, the vesicles were found to exist in the body until 12 hours of administration.

In addition, after the intestinal bacteria and bacteria-derived vesicles are systemically absorbed, in order to evaluate the invasion of various organs, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles are administered as described above. 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 Vesicles in Clinical Samples Metagenome  analysis

Blood was first placed in a 10 ml tube and the suspension was allowed to settle by centrifugation (3,500 × g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing 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 ultra-centrifugation for 3 hours at 150,000 xg and 4 ℃ using a Type 90ti rotor, and the lumped pellet was dissolved in physiological saline (PBS). .

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

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

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

Example  3. Blood Bacteria Derived from Pancreatic Cancer Patients Metagenome  analysis

In the method of Example 2, 176 blood of pancreatic cancer patients and 271 blood of normal people who matched age and sex were extracted from vesicles present in the blood and subjected to metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of pancreatic cancer patients compared to the normal blood (see Table 2 and FIG. 2).

blood	Control		Pancreatic cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0179	0.0228	0.0074	0.0075	<0.0001	0.41

Example  4. Bacterial-derived vesicles from bile duct cancer patients Metagenome  analysis

In the blood of 79 patients with cholangiocarcinoma patients and blood of 259 normal people whose age and sex were matched by the method of Example 2, genes were extracted from vesicles present in the blood, and a metagenomic analysis was performed. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived vesicles derived from the bacterium from the bile duct cancer significantly decreased compared to the normal blood (see Table 3 and Figure 3).

blood	Control		Bile duct cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0180	0.0229	0.0091	0.0158	0.0002	0.51

Example  5. Blood-derived vesicles from breast cancer patients Metagenome  analysis

In the method of Example 2, blood was extracted from vesicles present in the blood of 96 breast cancer patients and 192 normal blood patients whose age and sex were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 4 and FIG. 4).

blood	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0357	0.0445	0.0154	0.0122	<0.0001	0.43

Example  6. Blood Bacteria Derived from Ovarian Cancer Patients Metagenome  analysis

In the method of Example 2, 137 blood of ovarian cancer patients and 139 blood of normal and matched age and sex were extracted from the vesicles present in the blood to perform a metagenome analysis, followed by enhydrobacter The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of ovarian cancer patients compared to the normal blood (see Table 5 and FIG. 5).

blood	Control		Ovarian Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0283	0.0500	0.0160	0.0237	0.0097	0.57

Example  7. Blood Bacterium-Derived Vesicles for Lymphoma Patients Metagenome  analysis

In the method of Example 2, the blood of 63 lymphatic patients and 53 blood of normal people who matched age and sex were extracted from vesicles in the blood and subjected to metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium were significantly reduced in the blood of lymphoma patients compared to normal blood (see Table 6 and FIG. 6).

blood	Control		Lymphoma		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0229	0.0228	0.0119	0.0112	0.0023	0.52

Example  8. Vesicle-derived vesicles from myocardial infarction patients Metagenome  analysis

Gene extraction from vesicles present in the blood of 57 patients with myocardial infarction and 163 normal blood of age and sex matched by the method of Example 2, followed by metagenomic analysis, distribution of enhydrobacter bacterial vesicles Was evaluated. As a result, it was confirmed that the vesicles derived from the enhydrobacter bacterium significantly decreased in the blood of myocardial infarction patients compared to the normal blood (see Table 7 and FIG. 7).

blood	Control		Myocardial infarction		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0247	0.0524	0.0078	0.0234	0.0012	0.31

Example  9. Blood Bacteria Derived from Cardiomyopathy Patients Metagenome  analysis

Gene extraction from vesicles present in the blood of 72 cardiomyopathy patients and 163 normal blood of age and sex matched by the method of Example 2, followed by a metagenome analysis, distribution of vesicles derived from enhydrobacter bacteria Was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in the blood of cardiomyopathy patients compared to normal blood (see Table 8 and FIG. 8).

blood	Control		Cardiomyopathy		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0246	0.0524	0.0103	0.0142	0.0015	0.42

Example  10. Blood Bacteria Derived from Atrial Fibrillation Patients Metagenome  analysis

In the blood of 34 patients with atrial fibrillation and the blood of 62 normal persons whose age and sex were matched by the method of Example 2, the genes 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 vesicle-derived bacterium was significantly reduced in the blood of atrial fibrillation patients compared to normal blood (see Table 9 and FIG. 9).

blood	Control		Atrial fibrillation		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0164	0.0217	0.0054	0.0058	0.0003	0.33

Example  11. Patients with heterozygous angina  Blood Bacteria-Derived Vesicles Metagenome  analysis

In the method of Example 2, the blood of 80 patients with heteroangular angina and 80 normal blood whose age and sex were matched were extracted from the vesicles present in the blood and subjected to a metagenomic analysis, followed by enhydrobacter The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived vesicles derived from the bacterium from the angina pectoris significantly reduced compared to normal blood (see Table 10 and FIG. 10).

blood	Control		Angina		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0261	0.0579	0.0096	0.0164	0.0167	0.37

Example  12. Liver Cirrhosis Blood Bacteria-Derived Packets Metagenome  analysis

In the method of Example 2, the blood was extracted from vesicles of 130 livers and 145 normal blood persons whose ages and genders were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in the blood of liver cirrhosis compared to normal blood (see Table 11 and FIG. 11).

blood	Control		Cirrhosis		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0342	0.0781	0.0061	0.0164	0.0005	0.18

Example  13. Blood-derived vesicles from diabetic patients Metagenome  analysis

In the method of Example 2, the blood of 61 diabetic patients and 122 normal blood of age and sex matched were extracted from the vesicles present in the blood and subjected to a metagenomic analysis, followed by enhydrobacter bacteria. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that enhydrobacter bacteria-derived vesicles were significantly reduced in blood of diabetic patients compared to normal blood (see Table 12 and FIG. 12).

blood	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Enhydrobacter	0.0271	0.0286	0.0002	0.0002	<0.0001	0.01

Example  14. Enhydrobacter Arosas  Anti-inflammatory Effects of Derived Vesicles

Based on the results of the above example, the enhydrobacter aeroscus strain belonging to the enhydrobacter bacterium was isolated from the environmental sample, and then cultured to separate the vesicles thereof. Enhydrobacter aerosacus strains were incubated in BHI (brain heart infusion) medium until absorbance (OD ₆₀₀ ) was 1.0 to 1.5 in an aerobic chamber at 37 ° C. and then sub-cultured. Thereafter, the culture supernatant containing no strain was recovered, centrifuged at 10,000 g, 4 ° C. for 15 minutes, filtered through a 0.45 μm filter, and the filtered supernatant was used as a 100 kDa hollow filter membrane using a QuixStand benchtop system (GE Healthcare, UK). Concentrated to 200 ml volume by ultrafiltration. Then, the concentrated supernatant was once again filtered with a 0.22 μm filter, and the filtered supernatant was ultracentrifuged at 150,000 g, 4 ° C. for 3 hours, and the pellet was suspended in DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solution (Axis-Shield PoC AS, Norway) .Optiprep solution was prepared using HEPES-buffered saline (20 mM HEPES) to prepare low density solution. , 150 mM NaCl, pH 7.4) was used. After centrifugation at 200,000 g, 4 ° C for 2 hours, each solution fractionated with the same volume of 1 ml from the upper layer was further subjected to ultracentrifugation for 15 hours at 150,000 g, 4 ° C. Thereafter, the protein was quantified by BCA assay, and the obtained vesicles were tested.

To determine the effect of endobacterium-derived vesicles on inflammatory mediator secretion in inflammatory cells, endobacterial-derived vesicles from mouse macrophage Raw 264.7 cells were treated with various concentrations (0.1, 1, 10 ㎍ / Ml) and then E. coli- derived vesicles, E. coli , an inflammatory pathogenic vesicle. EV) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF-α, etc.). In addition, Lactobacillus plantarum derived vesicles (1 μg / ml) were used as beneficial control vesicles. More specifically, 1 × 10 ⁵ Raw 264.7 cells were dispensed into 24-well cell culture plates, and then cultured in DMEM complete medium for 24 hours. Then, the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatant was collected and stored at 4 ° C., followed by ELISA analysis.

As a result, as shown in Fig. 13, when pre-treatment with the endobacterial-derived vesicles or Lactobacillus plantarum-derived vesicles, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles is significantly suppressed . In particular, Enhydrobacter aerosac vesicles significantly inhibited TNF-α secretion compared to Lactobacillus plantarum vesicles. This means that the endobacterial-derived vesicle-derived vesicles can effectively suppress the occurrence of immune dysfunction and inflammation induced by pathogenic vesicles such as E. coli-derived vesicles.

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

The vesicles derived from the enhydrobacter bacterium according to the present invention include pancreatic cancer, cholangiocarcinoma, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heterogeneous angina pectoris, liver cirrhosis, or a diagnosis method for diabetes, as well as prevention of the above diseases, As it can be used as a composition for improvement or treatment, it is expected to be usefully used in the related medical and food industries.

Claims (13)

1. A method of providing information for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes, comprising the following steps:

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

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

   (c) pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal people through quantitative analysis of the PCR products. Classify as dysplastic angina, cirrhosis, or diabetes.
2. The method of claim 1,

   The sample in step (a) is characterized in that the blood, information providing method.
3. Selected from the group consisting of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, chronic liver disease, and diabetes, including vesicles derived from Enhydrobacter bacteria as an active ingredient Pharmaceutical compositions for the prevention or treatment of one or more diseases.
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 the natural or artificial secretion from Enhydrobacter bacteria, pharmaceutical composition.
6. The method of claim 3,

   The enhydrobacter bacteria-derived vesicles are characterized in that the vesicles derived from Enhydrobacter aerosaccus ( Enhydrobacter aerosaccus ), pharmaceutical composition.
7. Selected from the group consisting of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina pectoris, chronic liver disease, and diabetes, including vesicles derived from Enhydrobacter bacteria as an active ingredient Food composition for preventing or ameliorating one or more diseases.
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 the natural or artificial secretion from Enhydrobacter bacteria, food composition.
10. The method of claim 7, wherein

    The enhydrobacter bacteria-derived vesicles are characterized in that the vesicles derived from Enhydrobacter aerosaccus ( Enhydrobacter aerosaccus ), food composition.
11. A method of diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, angina, liver cirrhosis, or diabetes, comprising the following steps:

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

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

    (c) pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, if the content of extracellular vesicles derived from Enhydrobacter bacteria is lower than that of normal humans by quantitative analysis of the PCR products. Determining dysplastic angina, cirrhosis, or diabetes.
12. Pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, comprising administering to a subject a pharmaceutical composition comprising an enhydrobacter bacteria-derived vesicle as an active ingredient , Chronic liver disease and diabetes or the method of preventing or treating at least one disease selected from the group consisting of.
13. Of vesicles derived from Enhydrobacter bacteria, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, lymphoma, myocardial infarction, cardiomyopathy, atrial fibrillation, heteroangular angina, Use for the prevention or treatment of one or more diseases selected from the group consisting of chronic liver disease and diabetes.

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