CN111757944A - Nanovesicles derived from bacteria of the genus streptomyces and uses thereof - Google Patents
Nanovesicles derived from bacteria of the genus streptomyces and uses thereof Download PDFInfo
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- CN111757944A CN111757944A CN201980014391.3A CN201980014391A CN111757944A CN 111757944 A CN111757944 A CN 111757944A CN 201980014391 A CN201980014391 A CN 201980014391A CN 111757944 A CN111757944 A CN 111757944A
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Abstract
The present invention relates to vesicles derived from bacteria of the genus streptomyces and uses thereof, and the present inventors have experimentally confirmed that vesicles in a sample of a patient suffering from a malignant disease such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer or lymphoma, a cardiovascular disease such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease or depression are significantly reduced compared to normal individuals, and secretion of inflammatory mediators caused by pathogenic vesicles (e.g., vesicles derived from escherichia coli) is significantly inhibited when the vesicles isolated from a strain are administered. Therefore, it is expected that the vesicles derived from bacteria of the genus streptomyces according to the present invention can be effectively used in a method of diagnosing malignant diseases such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, and depression, and in developing a composition for preventing or treating the diseases.
Description
Technical Field
The present invention relates to nanovesicles derived from bacteria of the genus streptomyces (genus Micrococcus) and uses thereof, and more particularly, to a method of diagnosing malignant diseases such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease or depression by using nanovesicles derived from bacteria of the genus streptomyces, and a composition for preventing, alleviating or treating the diseases comprising the same.
Background
Since the beginning of the 21 st century, acute infectious diseases, which have been considered epidemic in the past, have become less important, and chronic inflammatory diseases accompanied by immune dysfunction due to disharmony between human and microbial communities have changed disease patterns, becoming major diseases that determine quality of life and human longevity. As intractable chronic diseases in the 21 st century, cancer, cardiovascular diseases, chronic pulmonary diseases, metabolic diseases, and neuropsychiatric diseases have become important problems of national public health, and have become major diseases that determine the life span and quality of life of human beings. In particular, these intractable chronic diseases are characterized by chronic inflammation accompanied by abnormal immune function caused by etiology.
As is well known, the number of coexisting microorganisms in the human body has reached 100 trillion, which is 10 times of the number of human cells, and the number of microbial genes is more than 100 times of the number of human genes. A microbiota or group of microorganisms refers to a community of microorganisms, including bacteria, archaea and eukaryotes present in a given habitat.
Bacteria present in our body and bacteria present in the surrounding environment secrete nano-sized vesicles to exchange information about genes, low-molecular compounds, proteins, and the like with other cells. The mucosa forms a physical defense membrane through which particles having a size of 200 nanometers (nm) or more cannot pass, and thus bacteria coexisting in the mucosa cannot pass, but vesicles derived from the bacteria have a size of 100 nm or less and are relatively freely passed through epithelial cells through the mucosa and then absorbed into our body. Vesicles derived from bacteria, which are locally secreted by bacteria, are absorbed through mucosal epithelial cells to cause local inflammatory reactions, while vesicles passing through epithelial cells are absorbed through lymphatic system to be distributed in various organs, and immune and inflammatory reactions are regulated in the organs where the vesicles are distributed. For example, vesicles derived from pathogenic gram-negative bacteria such as escherichia coli locally cause colitis, and when taken up into blood vessels, promote systemic inflammatory reactions and blood coagulation through an intravascular dermatitis reaction, and cause insulin resistance and diabetes when taken up into insulin-acting muscle cells. On the other hand, vesicles derived from beneficial bacteria can control diseases by controlling immune and metabolic dysfunctions caused by pathogenic vesicles.
As an immune response to factors such as bacteria-derived vesicles, a Th17 immune response, which is characterized by secretion of interleukin (hereinafter, IL) -17 cytokines, occurs, and IL-6 is secreted when exposed to bacteria-derived vesicles, thereby inducing a Th17 immune response. Inflammation caused by the Th17 immune response is characterized by neutrophil infiltration, and in the process of inflammation, tumor necrosis factor- α (hereinafter referred to as TNF- α) secreted from inflammatory cells such as macrophages plays an important role. Bacteria of the genus streptomyces are anaerobic gram-positive bacteria known to symbiotic in the intestinal tract. However, there have been no reports of extracellular secretion of vesicles by bacteria of the genus streptomyces, particularly no reports of the use of vesicles for diagnosis and treatment of cancer, cardiovascular diseases, metabolic diseases, and neuropsychiatric diseases.
Disclosure of Invention
[ problem ] to provide a method for producing a semiconductor device
As a result of earnest studies to solve the above conventional problems, the inventors confirmed that the content of vesicles derived from bacteria of the genus streptomyces in a sample derived from a patient suffering from a malignant disease such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer or lymphoma, a cardiovascular disease such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease or depression is significantly reduced as compared to a normal individual by metagenomic analysis. In addition, it has been confirmed that when vesicles are isolated from streptomyces triandrae (Catenibacterium mitookai) included in bacteria of the genus streptomyces to treat macrophages, IL-6 and TNF- α secretion by pathogenic vesicles is significantly inhibited, and canceration is significantly inhibited when vesicles derived from streptomyces triandrae are administered according to an evaluation of anticancer efficacy in a mouse cancer model. Thus, the present invention has been completed.
Accordingly, it is an object of the present invention to provide a method for providing information for diagnosing malignant diseases such as colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease or depression.
Further, it is another object of the present invention to provide a composition for preventing, alleviating or treating a malignant disease such as colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lymphoma, a cardiovascular disease such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease or depression, which comprises vesicles derived from bacteria of the genus streptomyces as an active ingredient.
However, the technical problems to be achieved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned may be clearly understood by those skilled in the art from the following description.
[ technical solution ] A
In order to achieve the above object of the present invention, the present invention provides a method for providing information for diagnosing colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease, or depression, the method comprising the steps of:
(a) extracting DNA from extracellular vesicles isolated from a sample of a normal individual and a sample of a subject;
(b) performing Polymerase Chain Reaction (PCR) on the extracted DNA using paired primers prepared based on a gene sequence present in 16S rDNA to obtain each PCR product; and
(c) classifying a case in which the content of extracellular vesicles derived from the bacteria of the genus streptomyces is lower than that of the normal individual sample into colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression by quantitative analysis of the PCR product.
In addition, the present invention provides a method for diagnosing colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease or depression, which comprises the steps of:
(a) extracting DNA from extracellular vesicles isolated from a sample of a normal individual and a sample of a subject;
(b) performing Polymerase Chain Reaction (PCR) on the extracted DNA using paired primers prepared based on a gene sequence present in 16S rDNA to obtain each PCR product; and
(c) classifying a case in which the content of extracellular vesicles derived from the bacteria of the genus streptomyces is lower than that of the normal individual sample into colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression by quantitative analysis of the PCR product.
As an exemplary embodiment of the present invention, the sample in step (a) may be blood, urine or feces.
As another embodiment of the present invention, the pair of primers in step (b) may be primers of SEQ ID No.1 and SEQ ID No. 2.
Further, the present invention provides a pharmaceutical composition for preventing or treating colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease or depression, comprising vesicles derived from the bacteria of the streptomyces genus as an active ingredient.
In addition, the present invention provides a food composition for preventing or alleviating colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease or depression, comprising vesicles derived from the bacteria of the genus streptomyces as an active ingredient.
In addition, the present invention provides a pharmaceutical composition for preventing or treating colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease or depression, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising vesicles derived from bacteria of the genus streptomyces as an active ingredient.
Further, the present invention provides use of vesicles derived from the bacterium of the genus streptomyces for preventing or treating colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression.
In yet another embodiment of the present invention, the average diameter of the vesicles may be 10 to 200 nm.
In another embodiment of the invention, the vesicles may be naturally or artificially secreted from bacteria of the genus streptomyces.
In another embodiment of the present invention, the vesicle derived from a bacterium of the genus streptomyces may be a vesicle derived from a bacterium of the genus streptococcus.
[ PROBLEMS ] the present invention
The inventors confirmed that intestinal bacteria are not absorbed into the body through epithelial cells, but vesicles derived from the bacteria are absorbed, distributed systemically, and then excreted out of the body through the kidney, liver, and lung, and that vesicles derived from bacteria present in the blood, urine, or feces of a patient are significantly reduced compared to normal individuals, from bacteria of the genus streptomyces present in the blood, urine, or feces of patients suffering from malignant diseases such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina or stroke, diabetes, parkinson's disease, or depression. In addition, when a triple-well streptomyces, which is one of bacteria of the genus streptomyces, is cultured in vitro to isolate vesicles, and then the vesicles are administered to inflammatory cells in vitro, it was confirmed that secretion of inflammatory mediators mediated by pathogenic vesicles is significantly inhibited. Therefore, it is expected that the vesicles according to the bacteria of the genus streptomyces of the present invention can be effectively used in a method for diagnosing malignant diseases such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina pectoris or stroke, diabetes, parkinson's disease and depression, and a composition for preventing, alleviating or treating these diseases.
Drawings
Fig. 1A is a series of photographs of distribution patterns of bacteria and bacteria-derived vesicles (EV) captured by time after oral administration of the bacteria and the bacteria-derived vesicles (EV) to mice, and fig. 1B is a result of evaluating the in vivo distribution patterns of the bacteria and the vesicles by collecting blood, kidneys, liver, and various organs 12 hours after oral administration of the bacteria and the vesicles.
FIG. 2 is a result of comparing the distribution of vesicles derived from bacteria of the genus Streptomyces after metagenomic analysis of vesicles derived from bacteria present in feces of colon cancer patients and normal individuals.
Fig. 3 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of pancreatic cancer patients and normal individuals.
Fig. 4 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of cholangiocarcinoma patients and normal individuals.
Fig. 5A and 5B are results of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis was performed on vesicles derived from bacteria present in ovarian cancer patients and normal individuals, wherein fig. 5A is a result obtained with a blood sample. Figure 5B is the results obtained with a urine sample.
Fig. 6 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of bladder cancer patients and normal individuals.
Fig. 7 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of lymphoma patients and normal individuals.
Fig. 8 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of patients with myocardial infarction and normal individuals.
Fig. 9 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of patients with atrial fibrillation and normal individuals.
Fig. 10 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of patients with variant angina and normal individuals.
Fig. 11 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of stroke patients and normal individuals.
Fig. 12 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in blood of diabetic patients and normal individuals.
Fig. 13 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in urine of patients with parkinson's disease and normal individuals.
Fig. 14 is a result of comparing the distribution of vesicles derived from bacteria of the genus streptomyces after metagenomic analysis of vesicles derived from bacteria present in urine of depression patients and normal individuals.
FIG. 15 is a result of evaluating the effect on secretion of IL-6 and TNF- α, inflammatory mediators by E.coli EV, by pretreatment of vesicles derived from bacteria of the genus Streptomyces before treatment of pathogenic vesicles such as E.coli EV, to evaluate the anti-inflammatory and immunomodulatory effects of vesicles derived from triple-well Streptomyces (NC: negative control; PC: positive control, E.coli EV 1. mu.g/ml; LP-1.0: Lactobacillus plantarum EV 1.0. mu.g/ml; GS-0.1, 1.0, 10: CM: triple-well Streptomyces EV0.1, 1.0, 10. mu.g/ml).
Detailed Description
The present invention relates to vesicles derived from bacteria of the genus streptomyces and uses thereof.
In the present invention, it was confirmed by metagenomic analysis that vesicles derived from bacteria of the genus streptomyces are significantly reduced in clinical samples obtained from patients with cancer, cardiovascular diseases, metabolic diseases, and neuropsychiatric diseases, as compared to normal individuals, and thus the diseases can be diagnosed. In addition, it was confirmed that the vesicle can be used for a composition for preventing or treating malignant diseases, cardiovascular diseases, metabolic diseases and neuropsychiatric diseases by isolating the vesicle from the triple-well streptomyces and analyzing the characteristics thereof.
Accordingly, the present invention provides a method of providing information for diagnosing colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression, the method comprising the steps of:
(a) extracting DNA from extracellular vesicles isolated from a sample of a normal individual and a sample of a subject;
(b) performing Polymerase Chain Reaction (PCR) on the extracted DNA using paired primers prepared based on a gene sequence present in 16S rDNA to obtain each PCR product; and
(c) determining a case in which the content of extracellular vesicles derived from the bacteria of the genus streptomyces is lower than that of the normal individual sample as colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression by quantitative analysis of the PCR product.
As used herein, the term "diagnosis" refers to the determination of a disease condition of a patient in a broad sense and in all aspects. The determination is made as to the entity, cause, pathogenesis, severity, detailed aspect of the disease, presence or absence of complications, prognosis, etc. of the disease. Diagnosis in the present invention refers to determining whether cancer, cardiovascular disease, metabolic disease and/or neuropsychiatric disease occur, the extent of the disease, etc.
As used herein, the term "nanovesicle" or "vesicle" refers to a structure composed of nano-sized membranes secreted from various bacteria. Vesicles or Outer Membrane Vesicles (OMVs) derived from gram-negative bacteria have endotoxins (lipopolysaccharides), toxic proteins, bacterial DNA and RNA, and vesicles derived from gram-positive bacteria have peptidoglycans and lipoteichoic acids, which are cell wall components of the bacteria, in addition to proteins and nucleic acids. In the present invention, the nanovesicles or vesicles are naturally secreted or artificially produced by bacteria of the genus streptomyces, are spherical, and have an average diameter of 10 to 200 nm.
Vesicles can be isolated from a culture fluid comprising bacteria of the genus streptomyces by using one or more methods selected from centrifugation, ultracentrifugation, high pressure processing, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical disintegration, chemical processing, filtration through a filter, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. In addition, processes such as washing for removing impurities and concentrating the obtained vesicles may be further included.
As used herein, the term "metagenome" also refers to microbiome, and refers to the entire genome including all viruses, bacteria, fungi, etc., in independent areas such as soil and animal intestines, and is generally used as a concept of genome explaining analysis of uncultured microorganisms by identifying a large number of microorganisms at a time using a sequence analyzer. In particular, metagenome does not refer to the genome of one species, but refers to a mixed genome, the genome of all species as one environmental unit. When a species is defined in the course of the development of omics biology, the metagenome is a term derived from the point of view of forming the complete species, which is formed by various species interacting with each other as well as a functionally present species. Technically, metagenome is the target of technology that recognizes all species in one environment by analyzing all DNA and RNA through a rapid sequence analysis method, and studies interaction and metabolism regardless of species.
In the present invention, the sample derived from the subject may be blood, urine or feces, but is not limited thereto.
As another aspect of the present invention, there is provided a pharmaceutical composition for preventing, treating or alleviating a malignant disease such as colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer or lymphoma, a cardiovascular disease such as myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease or depression, comprising vesicles derived from the bacteria of the genus streptomyces as an active ingredient. The composition includes a food composition and a pharmaceutical composition, and in the present invention, the food composition includes a health functional food composition. The compositions of the present invention may be oral sprays or inhalants.
As used herein, the term "prevention" refers to all actions that inhibit cancer, cardiovascular diseases, metabolic diseases and/or neuropsychiatric diseases or delay their onset by administering a composition according to the invention.
As used herein, the term "treatment" refers to all actions that alleviate or beneficially alter the symptoms of cancer, cardiovascular disease, metabolic disease, and/or neuropsychiatric disease by administering a composition according to the present invention.
As used herein, the term "alleviating" refers to all actions that at least reduce a parameter (e.g., the extent of symptoms) associated with the condition being treated.
The pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are generally used in the formulation, and include, but are not limited to, physiological saline (saline), sterile water, ringer's solution, buffered saline, cyclodextrin, glucose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like, and may further include other typical additives such as antioxidants and buffers, if necessary. In addition, the composition may be formulated into injectable preparations such as aqueous solutions, suspensions and emulsions, pills, capsules, granules or tablets by additionally adding diluents, dispersants, surfactants, binders, lubricants and the like. With respect to suitable pharmaceutically acceptable carriers and formulations, the compositions can be formulated according to each ingredient, preferably by using the method disclosed in Remington's medicament (the ramington literature). The formulation of the pharmaceutical composition of the present invention is not particularly limited, but may be formulated into injections, inhalants, external preparations for skin, oral preparations, and the like.
The pharmaceutical composition of the present invention may be orally administered or parenterally administered (e.g., intravenous, subcutaneous, intradermal, intranasal or intratracheal administration) according to a desired method, and the dosage may vary according to the condition and body weight of a patient, the severity of a disease, a pharmaceutical preparation, and the administration route and duration, but may be appropriately selected by one of ordinary skill in the art.
The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and an effective dosage level may be determined depending on factors including the type of disease of a patient, the severity of the disease, pharmaceutical activity, sensitivity to a drug, administration time, administration route, excretion rate, treatment period, and concurrent use of a drug, and other factors well known in the medical field. The composition according to the present invention may be administered as a therapeutic agent alone or in combination with other therapeutic agents, may be administered sequentially or simultaneously with the therapeutic agents in the related art, and may be administered in a single dose or multiple doses. In view of all the above factors, it is important to administer the composition in the minimum amount that can achieve the maximum effect without any side effects, which can be readily determined by one of ordinary skill in the art.
Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex and body weight of a patient, and is usually 0.001 to 150mg of the composition per 1 kg of body weight, preferably 0.01 to 100mg per 1 kg of body weight, and may be administered daily or every other day, or once to three times daily. However, the above dose is not intended to limit the scope of the present invention in any way, since the dose may be increased or decreased depending on the administration route, severity of obesity, sex, body weight or age.
The food composition of the present invention includes a health functional food composition. The food composition according to the present invention may be used by adding the active ingredient to food as it is, or may be used together with other food or food ingredients, but may be used as appropriate according to typical methods. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or alleviation). Generally, when preparing a food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, for long-term intake for health and hygiene purposes or for health control purposes, the amount may be less than the above range.
Other ingredients are not particularly limited except that the food composition of the present invention contains the active ingredient in a designated ratio as an essential ingredient, and the food composition of the present invention may contain various flavors, natural carbohydrates, etc. as additional ingredients, as in a typical beverage. Examples of the above natural carbohydrates include conventional sugars such as monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, and the like; and polysaccharides such as dextrin, cyclodextrin and the like; and sugar alcohols such as xylitol, sorbitol and erythritol. As a flavoring agent other than the above-described flavoring agents, natural flavoring agents (thaumatin, stevia extracts such as rebaudioside a, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
In addition to the additives, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents (e.g., synthetic flavoring agents and natural flavoring agents), coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. These ingredients may be used alone or in combination thereof. The proportion of these additives may also be appropriately selected by those of ordinary skill in the art.
In one embodiment of the present invention, by orally administering bacteria and vesicles derived from bacteria to mice and observing the in vivo absorption, distribution and excretion patterns of the bacteria and vesicles, it was confirmed that, although bacteria are not absorbed through the intestinal mucosa, the vesicles derived from bacteria are absorbed and distributed systemically within 5 minutes after administration and are excreted through the kidney, liver, and the like (see example 1).
In another embodiment of the present invention, the bacterial metagenomic analysis is performed by using vesicles isolated from blood, urine or feces of normal individuals whose age and sex are matched with patients suffering from colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease and depression. The results confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in clinical samples of patients with colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, and depression, as compared with samples of normal individuals (see examples 3 to 15).
In another embodiment of the present invention, streptococcus trialis was cultured to evaluate whether vesicles secreted therefrom have immunomodulatory and anti-inflammatory effects, and by evaluating secretion of inflammatory mediators via treatment of escherichia coli (e.coli EV) as a pathogenic factor of inflammatory diseases after treating macrophages with different concentrations of vesicles derived from streptococcus trialis, it was confirmed that secretion of IL-6 and TNF- α by escherichia coli vesicles (e.coli EV) was effectively inhibited by vesicles derived from streptococcus trialis (see example 15).
Hereinafter, preferred embodiments will be presented to aid in understanding the present invention. However, the following examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.
Examples
Example 1 in vivo absorption of enteric bacteria and vesicles derived from bacteria,Analysis of distribution and drainage patterns
To assess whether intestinal bacteria and vesicles derived from bacteria are absorbed systemically through the gastrointestinal tract, experiments were performed in the following manner. A 50 μ g dose of each of enteric bacteria with fluorescent labeling and vesicles derived from enteric bacteria was administered to the stomach of a mouse through the gastrointestinal tract, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, 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 vesicles derived from the bacteria were absorbed systemically 5 minutes after the administration, and intense fluorescence was observed in the bladder 3 hours after the administration, so that it can be seen that the vesicles were excreted to the urinary tract. Furthermore, it can be seen that vesicles are present in vivo until 12 hours post-administration (see figure 1A).
In order to evaluate the mode in which enteric bacteria and vesicles derived from enteric bacteria were permeated into each organ after systemic absorption, 50 μ g of bacteria carrying a fluorescent label and vesicles derived from bacteria were administered in the same manner as described above, and then blood, heart, lung, liver, kidney, spleen, fat, and muscle were collected 12 hours after the administration. As a result of the observed fluorescence in the collected tissue, as shown in fig. 1B, it can be seen that vesicles derived from bacteria are distributed in blood, heart, lung, liver, kidney, spleen, muscle and fat, but the bacteria are not absorbed (see fig. 1B).
Example 2 metagenomic analysis of vesicles derived from bacteria in clinical samples
After the clinical samples (e.g. blood, urine, faeces etc.) were first placed in 10ml tubes and the suspension was allowed to settle by centrifugation (3,500 Xg, 10 min, 4 ℃), only the supernatant was transferred to a new 10ml tube. After removing bacteria and impurities using a 0.22- μm filter, they were transferred to a Centriprep tube (centrifugal filter 50kD) and centrifuged at 1,500 Xg and 4 ℃ for 15 minutes, and substances less than 50kD were discarded, and the residue was concentrated to 10 ml. After removing bacteria and impurities again using a 0.22- μm filter, the supernatant was discarded by performing ultra-high speed centrifugation using a 90Ti type rotor at 150,000 Xg and 4 ℃ for 3 hours, and the aggregated pellet (pellet) was dissolved in physiological saline (PBS).
Mu.l of vesicles isolated by the above method were boiled at 100 ℃ to extract internal DNA from lipids, and then cooled on ice for 5 minutes. Then, in order to remove the remaining suspended matter, the DNA was centrifuged at 10,000 Xg at 4 ℃ for 30 minutes, and only the supernatant was collected. Also, the amount of DNA was quantified by using Nanodrop. Thereafter, in order to confirm the presence or absence of the bacteria-derived DNA in the extracted DNA, PCR was performed using 16s rDNA primers shown in table 1 below, and the presence of the bacteria-derived gene in the extracted gene was confirmed.
[ Table 1]
DNA extracted by the above method was amplified using 16S rDNA primers and then sequenced (illumina miseq sequencer), the results were exported as a Standard Flowsheet Format (SFF) file, which was converted into a sequence file (. fasta) and a nucleotide quality score file using GS FLX software (v2.9), and then confirmed for reliable estimates of reading, and where the window (20bps) of average base detection accuracy was less than 99% (Phred score <20) was deleted. For the Operational Taxonomy Unit (OTU) analysis, the classification was performed on phyla, classes, orders, families and genus levels per OUT by clustering according to sequence similarity using UCLUST and USEARCH based on sequence similarities of 94%, 90%, 85%, 80% and 75%, respectively, and the analysis was performed on bacteria having a sequence similarity of 97% or higher on the genus level using the 16S RNA sequence databases (108,453 sequences) of BLASTN and greengenes (qiime).
Example 3 metagenomic analysis of bacterially derived vesicles in stool from Colon cancer patients
Genes were extracted from vesicles present in stool samples from 38 colon cancer patients and 38 normal individuals (both age and sex matched) and the stool was metagenomically analyzed using the method of example 2, and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in feces from colon cancer patients compared with feces derived from normal individuals (see table 2 and fig. 2).
[ Table 2]
Example 4 metagenomic analysis of bacterially-derived vesicles in blood of pancreatic cancer patients
Genes were extracted from vesicles present in blood samples of 291 patients with pancreatic cancer and 291 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from pancreatic cancer patients compared with blood derived from normal individuals (see table 3 and fig. 3).
[ Table 3]
Example 5 metagenomic analysis of bacterially derived vesicles in blood of biliary tract cancer patients
Genes were extracted from vesicles present in blood samples of 121 cholangiocarcinoma patients and 131 normal individuals (both age and sex matched in both groups), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from a cholangiocarcinoma patient compared with blood derived from a normal individual (see table 4 and fig. 4).
[ Table 4]
Example 6 metagenomic analysis of bacterially derived vesicles in blood and urine of ovarian cancer patients
Genes were extracted from vesicles present in blood samples of 126 ovarian cancer patients and 131 normal individuals (both age and sex matched in both groups), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from ovarian cancer patients compared to blood derived from normal individuals (see table 5 and fig. 5A).
[ Table 5]
In addition, genes were extracted from vesicles present in urine samples of 136 ovarian cancer patients and 136 normal individuals (both groups matched in age and sex), and after metagenomic analysis of the urine using the method of example 2, the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in urine from ovarian cancer patients compared to urine derived from normal individuals (see table 6 and fig. 5B).
[ Table 6]
Example 7 metagenomic analysis of bacterially derived vesicles in blood of bladder cancer patients
Genes were extracted from vesicles present in blood samples of 96 bladder cancer patients and 184 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from patients with bladder cancer as compared with blood derived from normal individuals (see table 7 and fig. 6).
[ Table 7]
Example 8 metagenomic analysis of bacterially derived vesicles in blood of lymphoma patients
Genes were extracted from vesicles present in blood samples of 63 lymphoma patients and 53 normal individuals (both age and sex matched in groups), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from lymphoma patients as compared with blood derived from normal individuals (see table 8 and fig. 7).
[ Table 8]
Example 9 metagenomic analysis of bacterially-derived vesicles in blood of patients with myocardial infarction
Genes were extracted from vesicles present in blood samples of 57 patients with myocardial infarction and 163 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from patients with myocardial infarction compared to blood derived from normal individuals (see table 9 and fig. 8).
[ Table 9]
Example 10 metagenomic analysis of bacterially derived vesicles in the blood of patients with atrial fibrillation
Genes were extracted from vesicles present in blood samples of 32 patients with atrial fibrillation and 32 normal individuals (both age and sex matched in both groups), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from patients with atrial fibrillation compared to blood derived from normal individuals (see table 10 and fig. 9).
[ Table 10]
Example 11 metagenomic analysis of bacterially derived vesicles in blood of patients with variant angina pectoris
Genes were extracted from vesicles present in blood samples of 80 patients with variant angina pectoris and 80 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from patients with variant angina as compared with blood derived from normal individuals (see table 11 and fig. 10).
[ Table 11]
Example 12 metagenomic analysis of bacterially derived vesicles in blood of stroke patients
Genes were extracted from vesicles present in blood samples of 115 stroke patients and 109 normal individuals (both age and sex matched) and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from stroke patients compared with blood derived from normal individuals (see table 12 and fig. 11).
[ Table 12]
Example 13 metagenomic analysis of bacterially derived vesicles in blood of diabetic patients
Genes were extracted from vesicles present in blood samples of 73 diabetic patients and 146 normal individuals (both age and sex matched in both groups), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the blood using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in blood from diabetic patients as compared with blood derived from normal individuals (see table 13 and fig. 12).
[ Table 13]
Example 14 metagenomic analysis of bacterially derived vesicles in urine of Parkinson's disease patients
Genes were extracted from vesicles present in urine samples of 39 patients with Parkinson's disease and 79 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus Streptomyces was evaluated after metagenomic analysis of the urine using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in urine from parkinson's disease patients as compared with urine derived from normal individuals (see table 14 and fig. 13).
[ Table 14]
Example 15 metagenomic analysis of bacterially derived vesicles in urine of depression patients
Genes were extracted from vesicles present in urine samples of 20 depression patients and 20 normal individuals (both groups matched in age and sex), and the distribution of vesicles derived from bacteria of the genus streptomyces was evaluated after metagenomic analysis of the urine using the method of example 2. As a result, it was confirmed that vesicles derived from bacteria of the genus streptomyces were significantly reduced in urine from depression patients compared with urine derived from normal individuals (see table 15 and fig. 14).
[ Table 15]
Example 16 anti-inflammatory action of vesicles derived from Streptomyces trilobatus
According to the results of the above examples, a strain of triple-well streptomyces was cultured, and then vesicles thereof were isolated. Culturing a strain of Streptomyces trilobii in Brain Heart Infusion (BHI) medium until absorbance (OD) in an anaerobic chamber at 37 ℃600) To 1.0 to 1.5, and then subcultured. Thereafter, the culture supernatant without the strain was collected, centrifuged at 10,000g at 4 ℃ for 15 minutes, and then filtered through a 0.45- μm filter. The supernatant thus obtained was concentrated to an amount of 200mL by ultrafiltration using a quilx stand bench top system (GEHealthcare, uk) with a 100kDa hollow filter membrane. Subsequently, the concentrated supernatant was filtered again with a 0.22- μm filter and ultracentrifuged at 150,000g and 4 ℃ for 3 hours, and then the pellet was suspended in DPBS. Then, density gradient centrifugation was performed using 10%, 40% and 50% OptiPrep solution (Axis-Shield PoC AS, norway), and in order to prepare a low density solution, the OptiPrep solution was diluted with HEPES buffered saline (20mM HEPES, 150mM NaCl, ph7.4)) before use. After centrifugation at 200,000g and 4 ℃ for 2 hours, each solution fractionated from the top layer in 1mL of equal volume was ultracentrifuged at 150,000g and 4 ℃ for an additional 3 hours. Thereafter, a bicinchoninic acid (BCA) assay was usedThe protein was quantified and the vesicles obtained as described above were subjected to the experiment.
To examine the effect of the vesicles derived from triple-well streptomyces on the secretion of inflammatory mediators by inflammatory cells, mouse macrophage cell line (Raw 264.7 cells) was treated with the vesicles derived from triple-well streptomyces (c.mituokai EV) at various concentrations (0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL), and the secretion amounts of inflammatory mediators (IL-6 and TNF- α) were measured by treating the vesicles derived from escherichia coli (e.coli EV), which are vesicles that are the pathogenesis of inflammatory diseases5After collecting culture supernatant in 1.5mL tubes, centrifuging at 3000g for 5 minutes, thereby collecting supernatant, the supernatant was stored at 4 ℃, followed by elisa.
The above description of the present invention is provided for the purpose of illustration, and it will be understood by those skilled in the art to which the present invention pertains that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. It is therefore to be understood that the above described embodiments are illustrative in all respects only, and not restrictive.
[ INDUSTRIAL APPLICABILITY ]
Since the vesicles derived from bacteria of the genus streptomyces according to the present invention can be used in a method for diagnosing malignant diseases such as colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer or lymphoma, cardiovascular diseases such as myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease and depression, and a composition for preventing, alleviating or treating the diseases, it is expected that they will be effectively used in the related pharmaceutical and food industries.
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Claims (13)
1. A method of providing information useful in diagnosing colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression, the method comprising the steps of:
(a) extracting DNA from extracellular vesicles isolated from a sample of a normal individual and a sample of a subject;
(b) performing Polymerase Chain Reaction (PCR) on the extracted DNA using paired primers prepared based on a gene sequence present in 16S rDNA to obtain each PCR product; and
(c) the cases in which the content of extracellular vesicles derived from bacteria of the genus streptomyces is lower than that of the normal individual sample are classified into colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression by quantitative analysis of the PCR product.
2. The method of claim 1, wherein the sample in step (a) is blood, urine, or feces.
3. A pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, parkinson's disease, and depression, comprising vesicles derived from the bacteria of the genus streptomyces as an active ingredient.
4. The pharmaceutical composition of claim 3, wherein the average diameter of the vesicles is from 10 to 200 nm.
5. The pharmaceutical composition of claim 3, wherein the vesicle is naturally or artificially secreted from a bacterium of the genus Streptomyces.
6. The pharmaceutical composition according to claim 3, wherein the vesicle derived from the bacterium of the genus Streptomyces is a vesicle derived from Streptomyces trilobatus (Catenibacter mituokai).
7. A food composition for preventing or alleviating one or more diseases selected from the group consisting of colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina pectoris, stroke, diabetes, Parkinson's disease, and depression, comprising vesicles derived from bacteria of the genus Streptomyces as an active ingredient.
8. The food composition of claim 7, wherein the average diameter of the vesicles is from 10 to 200 nm.
9. The food composition of claim 7, wherein the vesicles are naturally or artificially secreted from a bacterium of the genus streptomyces.
10. The food composition of claim 7, wherein the vesicles derived from bacteria of the genus streptomyces are vesicles derived from streptomyces triandrus (Catenibacterium mitookai).
11. A method of diagnosing colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression, the method comprising the steps of:
(a) extracting DNA from extracellular vesicles isolated from a sample of a normal individual and a sample of a subject;
(b) performing Polymerase Chain Reaction (PCR) on the extracted DNA using paired primers prepared based on a gene sequence present in 16S rDNA to obtain each PCR product; and
(c) the cases in which the content of extracellular vesicles derived from bacteria of the genus streptomyces is lower than that of the normal individual sample are classified into colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression by quantitative analysis of the PCR product.
12. A method of preventing or treating colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease, or depression, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising vesicles derived from bacteria of the genus streptomyces as an active ingredient.
13. Use of vesicles derived from bacteria of the genus streptomyces for the prevention or treatment of colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, atrial fibrillation, variant angina, stroke, diabetes, parkinson's disease or depression.
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