WO2019168328A1 - Nanovesicles derived from rhizobium sp. bacteria, and use thereof - Google Patents

Abstract

The present invention relates to vesicles derived from Rhizobium sp. bacteria, and a use thereof. The present inventors have experimentally ascertained that the vesicles in clinical samples from patients with stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease and dementia are significantly reduced in comparison to those from normal people, and when the vesicles derived from the strains are administered, the secretion of inflammatory mediators by pathogenic vesicles such as E. coli-derived vesicles is significantly inhibited. Therefore, the vesicles derived from Rhizobium sp. bacteria, according to the present invention, can be effectively used for the purpose of developing a method for diagnosis of stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia, and a composition for preventing, alleviating or treating the diseases.

Description

Blister-derived nanovesicles and uses thereof

The present invention relates to nanovesicles derived from bacteria of the genus Rizobium, and their use, and more particularly, to stomach cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma and diabetes using nanovesicles derived from bacteria of the genus Rizobium. And a method for diagnosing Parkinson's disease or dementia, and a composition for preventing, ameliorating or treating the disease, including the vesicle.

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 intractable chronic disease is characterized by chronic inflammation accompanied by abnormal immune function caused by the causative factor.

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 reactions, and 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 colitis and, when absorbed into blood vessels, promote systemic inflammatory and urine coagulation through vascular endothelial inflammatory responses. In addition, when insulin is absorbed into muscle cells, it causes 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.

A bacterium in rizobium is a Gram-negative bacterium that absorbs nitrogen from the soil, symbiotic with roots such as nuts, converts atmospheric nitrogen into ammonia, and provides nitrogen compounds such as glutamine to plants. However, it has not been reported that bacteria in rizobium secrete extracellular vesicles, and in particular, there have been no cases of application to diagnosis and treatment of intractable diseases such as cancer, neurological diseases, or metabolic diseases.

The present inventors earnestly researched to solve the above-mentioned problems, and as a result of metagenome analysis, the vesicle-derived vesicles of the genus Rizovium were found to have gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, Significant reductions in clinical samples of patients with diabetes, Parkinson's disease, and dementia confirmed that the disease could be diagnosed. Also, Rhizobium leguminosarum ) when the vesicles were isolated and treated with macrophages, it was confirmed that significantly inhibit the IL-6 and TNF-α secretion by pathogenic vesicles, the present invention was completed based on this.

Accordingly, an object of the present invention is to provide an information providing method for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia.

In addition, the present invention comprises a bacterial vesicle of the genus Rizobium as an active ingredient, gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and composition for the prevention, improvement or treatment of dementia To provide another purpose.

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, for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia Provide information methods:

(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) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, when the content of extracellular vesicles derived from bacteria in Rizobium is lower than that of normal people through quantitative analysis of the PCR products. Classify as Parkinson's disease, or dementia.

The present invention provides a method for diagnosing gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia, 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) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, when the content of extracellular vesicles derived from bacteria in Rizobium is lower than that of normal people through quantitative analysis of the PCR products. Determining Parkinson's disease or dementia.

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

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 another aspect, the present invention is a pharmaceutical composition for the prevention or treatment of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, including the bacteria-derived vesicles of the genus Rizobium To provide.

In addition, the present invention comprises a bacteria-derived vesicles of the genus Rizobium as an active ingredient. It provides a food composition for the prevention or improvement of stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia.

In addition, the present invention comprises the step of administering to the subject a pharmaceutical composition comprising a bacteria-derived vesicles of the genus Rizobium as an active ingredient, gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson Provided are methods for preventing or treating a disease or dementia.

The present invention also provides a prophylactic or therapeutic use of vesicles derived from bacteria of the genus Rizobium, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia.

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 bacteria of the genus Rizobium.

In another embodiment of the present invention, the bacteria-derived vesicles of the genus Rizobium may be vesicles derived from Rizium regyuminosarum.

The present inventors confirmed that the intestinal bacteria are not absorbed into the body, but when the bacteria-derived vesicles are absorbed into the body through epithelial cells, they are distributed systemically and excreted in vitro through the kidneys, liver, and lungs. Bacterial-derived vesicle metagenome analysis in blood or urine results in clinical samples of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and blood or urine in patients with dementia. It was confirmed that the vesicle-derived bacteria in rizobium were significantly reduced compared to normal people. In addition, it was observed that the vesicles were isolated by culturing Rizobium regyuminosarum, a species of bacteria in the genus Rizobium, and when administered to inflammatory cells in vitro, significantly inhibiting the secretion of inflammatory mediators by pathogenic vesicles. , Bacterial-derived vesicles of the genus Rizobium according to the present invention is a method for diagnosing gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, and preventing, improving or It is expected that the present invention may be usefully used in therapeutic 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, urine, 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 bacteria-derived vesicles of the genus Rizobium after the analysis of bacteria-derived vesicles metagenome present in gastric cancer patients and normal urine.

Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in colon cancer patients and normal urine.

Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in breast cancer patients and normal urine.

Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in ovarian cancer patients and normal urine.

Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacteria-derived vesicles metagenome present in bladder cancer patients and normal urine.

Figure 7 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analysis of bacteria-derived vesicles metagenome present in the prostate cancer patients and normal urine.

8 is a result of comparing the distribution of bacteria-derived vesicles of Rizobium after analysis of bacteria-derived vesicles metagenome present in asthmatic patients and normal blood.

Figure 9 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal urine.

Figure 10 is a result of comparing the distribution of bacteria-derived vesicles of the genus Rizobium after analyzing the bacterial-derived vesicles metagenome present in Parkinson's disease patients and normal urine.

11 is a result of comparing the distribution of bacteria-derived vesicles of Rizobium after analysis of bacteria-derived vesicles metagenome present in dementia patients and normal blood.

FIG. 12 is a pre-treatment of lysobium-derived vesicles prior to treatment with Escherichia coli vesicles ( E. coli EV) to evaluate the anti-inflammatory and immunomodulatory effects of lysodium regyuminosarum-derived vesicles. The results of the evaluation of the mediator IL-6 and TNF-α secretion was evaluated (NC: negative control; PC: positive control, E. coli EV 1 μg / ml; LP_1.0: Lactobacillus plantarum EV 1.0 μg / ml; RL_0.1, 1.0, 10: Rhizobium leguminosarum EV 0.1, 1.0, 10 μg / ml).

The present invention relates to vesicles derived from bacteria of the genus Rizobium and uses thereof.

We found that metagenome analysis significantly reduced vesicle-derived vesicles of Rizovium in clinical samples of patients with gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and dementia. It was confirmed that the disease can be diagnosed. In addition, the vesicles were isolated and analyzed from Rhizobium leguminosarum, and gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia. It was confirmed that it can be used as a composition for preventing, improving or treating diseases.

Accordingly, the present invention provides a method for providing information for the diagnosis of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, 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) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, when the content of extracellular vesicles derived from bacteria in Rizobium is lower than that of normal people through quantitative analysis of the PCR products. Classify as Parkinson's disease, or dementia.

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 the onset and level of disease, such as gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia.

As used herein, the term "nanovesicle" or "vesicle" refers to a structure of nanoscale membranes secreted by various bacteria. Gram-negative bacteria-derived vesicles or outer membrane vesicles (OMVs) contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, 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 from bacteria of the genus Rizobium, and have a spherical shape and have an average diameter of 10 to 100 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 or urine, but is not limited thereto.

As another embodiment of the present invention, the present invention comprises a bacterial vesicles of the genus Rizobium as an active ingredient, gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or prevention of dementia, It provides a composition for treatment or improvement. 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" means the inhibition of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia by administration of a composition according to the present invention. Or any action that delays onset.

As used herein, the term “treatment” refers to symptoms of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and / or dementia by administration of the composition according to the present invention. Means any action that improves or changes beneficial;

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 are centrifuged, ultra-fast centrifugation, autoclaving, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filtration by a culture medium containing bacteria in the riboseum 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, the bacteria and bacteria-derived vesicles orally administered to observe the body absorption, distribution, and excretion of the bacteria and vesicles in the body, in the case of bacteria is 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, bacterial metabolism using gastric, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, diabetes mellitus, and vesicles isolated from blood or urine of normal persons whose age and sex matched to Parkinson's disease patients. Genomic analysis was performed. As a result, compared to the normal sample, the bacteria-derived vesicles of Rizovium were significantly decreased in the clinical samples of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and dementia patients. It was confirmed (see Examples 3 to 12).

In another embodiment of the present invention, it was evaluated whether the vesicles secreted from the lysium regyuminosarum strain exhibited the immunomodulatory and anti-inflammatory effect, compared to the vesicles derived from various concentrations After treatment with phagocytes, E. coli-derived vesicles were treated to evaluate the secretion of inflammatory mediators. As a result, lysobium-dermiminosarum-derived vesicles efficiently secrete IL-6 and TNF-α secretion from E. coli-derived vesicles. Inhibition was confirmed (see Example 13).

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

EXAMPLE

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

In order to evaluate whether the intestinal bacteria and bacteria-derived vesicles are absorbed systemically through the gastrointestinal tract, experiments were performed as follows. Fluorescently labeled enterobacteriaceae and enteric bacteria-derived vesicles were administered to the gastrointestinal tract at doses of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the 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 fluoresced 3 hours after administration. 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 enteric bacteria-derived vesicles are absorbed systemically, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above in order to evaluate the infiltration into various organs. After 12 hours, urine, heart, lung, liver, kidney, 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 urine, heart, lung, liver, spleen, fat, muscle, kidney, it was found that the bacteria are not absorbed.

Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples

Clinical samples such as urine and blood were first placed in 10 ml tubes and the suspension was allowed to settle by centrifugation (3,500 x g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign substances using a 0.22㎛ filter, and transferred to centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes to discard the material smaller than 50 kD concentrated to 10 ml. Then, once again remove the bacteria and foreign substances by using a 0.22㎛ filter, discard the supernatant using ultra-fast centrifugation for 3 hours at 150,000 xg, 4 ℃ with a Type 90ti rotor and pellets ) Was dissolved in saline (PBS).

100 μl of the vesicles separated by the above method was boiled at 100 ° C. to allow DNA inside to come out of the lipid, cooled on ice for 5 minutes, and centrifuged at 10,000 × g and 4 ° C. for 30 minutes to remove any remaining suspended matter. DNA amount was quantified. Thereafter, PCR was performed with the 16s rDNA primer (primer) shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene is present in the extracted gene.

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

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

Example 3 Analysis of Bacterial-Derived Vesicular Metagenome in Urine of Gastric Cancer Patients

In the method of Example 2, the urine of 61 patients with gastric cancer and 120 urine of normal age matched with age and sex were extracted from vesicles present in urine and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of gastric cancer patients compared to normal urine (see Table 2 and FIG. 2).

Pee	Control		Stomach cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0060	0.0063	0.0001	0.0002	<0.0001	0.01

Example  4. Urine-derived vesicles from colon cancer patients Metagenome  analysis

In the method of Example 2, the genes were extracted from the vesicles in the urine of 38 colon cancers and the 38 urine of normal people, and the metagenome analysis was performed, and then the distribution of the bacterial vesicles of the genus Rizobium was evaluated. It was. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in urine of colorectal cancer patients compared to normal urine (see Table 3 and FIG. 3).

Pee	Control		Colorectal cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0056	0.0056	0.0000	0.0000	<0.0001	0.00

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

In the method of Example 2, the genes were extracted from vesicles of 127 breast cancer patients and 220 urine people of normal age who matched their age and sex. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of breast cancer patients compared to normal urine (see Table 4 and FIG. 4).

Pee	Control		Breast cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0041	0.0062	0.0000	0.0001	<0.0001	0.01

Example  6. Vesicle-derived vesicles from ovarian cancer patients Metagenome  analysis

The urine of 135 ovarian cancer patients and 136 normal urine of age and sex matched by the method of Example 2 were extracted from vesicles present in the urine, and then subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the lyzobiium significantly decreased in the urine of ovarian cancer patients compared to normal urine (see Table 5 and FIG. 5).

Pee	Control		Ovarian Cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0034	0.0036	0.0000	0.0001	<0.0001	0.00

Example  7. Bladder-derived vesicles from bladder cancer patients Metagenome  analysis

In the method of Example 2, the urine of bladder cancer patients and urine of 107 normal people whose age and sex were matched were extracted from vesicles present in the urine, followed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of bladder cancer patients compared to normal urine (see Table 6 and FIG. 6).

Pee	Control		Bladder cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0044	0.0068	0.0001	0.0003	<0.0001	0.01

Example  8. Urine-derived vesicles from prostate cancer patients Metagenome  analysis

The urine of 55 prostate cancer patients and the urine of 159 normal urine were extracted by the method of Example 2, and the genes were extracted from the vesicles present in the urine and subjected to metagenomic analysis. The distribution of was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of prostate cancer patients compared to normal urine (see Table 7 and FIG. 7).

Pee	Control		Prostate cancer		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0032	0.0050	0.0000	0.0000	<0.0001	0.00

Example  9. Blood-derived vesicles from asthma patients Metagenome  analysis

In the method of Example 2, blood was collected from 160 bloods of asthma patients and 114 normal bloods of age and sex, and genes were extracted from vesicles present in the blood, followed by metagenome analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the blood of asthma patients compared to normal blood (see Table 8 and FIG. 8).

blood	Control		asthma		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0001	0.0043	0.0000	0.0000	<0.0001	0.00

Example  10. Diabetic urine bacteria-derived vesicles Metagenome  analysis

In the method of Example 2, the urine of 60 diabetic patients and the urine of 134 normal persons whose age and sex were matched were extracted from the vesicles present in the urine and subjected to metagenomic analysis. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in urine of diabetic patients compared to normal urine (see Table 9 and FIG. 9).

Pee	Control		diabetes		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0030	0.0043	0.0000	0.0000	<0.0001	0.00

Example  11.Urine-derived vesicles from Parkinson's disease patients Metagenome  analysis

The urine of 39 patients with Parkinson's disease and 76 urine of age and sex matched by the method of Example 2 were extracted from vesicles present in the urine, followed by a metagenome analysis, followed by bacteria of the genus Rizovium. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the urine of Parkinson's disease patients compared to normal urine (see Table 10 and FIG. 10).

Pee	Control		Parkinson's disease		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0054	0.0061	0.0004	0.0014	<0.0001	0.07

Example  12. Blood Bacteria Derived from Dementia Patients Metagenome  analysis

In the blood of 60 asthma patients and 74 normal blood patients whose age and gender were matched by the method of Example 2, genes were extracted from vesicles present in the blood and metagenome analysis was performed, followed by the origin of bacteria of the genus Rizovium. The distribution of vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of Rizobium were significantly reduced in the blood of dementia patients compared to normal blood (see Table 11 and FIG. 11).

blood	Control		dementia		t-test
Taxon	Mean	SD	Mean	SD	p-value	Ratio
g__Rhizobium	0.0002	0.0043	0.0000	0.0000	0.01	0.07

Example  13. Rizobiium Regyu Minosa Room  Anti-inflammatory Effects of Derived Vesicles

Based on the results of the above example, after culturing the lysovium regyuminosarum strain belonging to the bacteria of the genus Rizobium, its vesicles were isolated. The rizobium regyuminosarum strain was subcultured after incubating in BHI (brain heart infusion) medium until the absorbance (OD ₆₀₀ ) was 1.0 to 1.5 in an aerobic chamber at 37 ° C. 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), and the Optiprep solution was prepared using HEPES-buffered saline (20 mM HEPES) for the preparation of low density solutions. , 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. Then, the protein was quantified using BCA (Bicinchoninic acid) assay, and the obtained vesicles were tested.

To investigate the effect of lysodium regyuminosalum-derived vesicles on inflammatory mediator secretion in inflammatory cells, the concentration of rizobium regyuminosalum-derived vesicles in raw 264.7 cells, a mouse macrophage, was measured at various concentrations (0.1, 1, 10 ㎍ / Ml) and then E. coli- derived vesicles from E. coli , an inflammatory disease pathogenic vesicle. EV) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF-α, etc.). More specifically, Raw 264.7 cells were dispensed into 24-well cell culture plates by 1 × 10 ⁵ cells and then cultured in Dulbeco's Modified Eagle's Medium (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, when pre-treatment of lysodium regyuminosarum-derived vesicles, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles is significantly suppressed (see Fig. 12). This means that Rizobium regyuminosarum-derived vesicles can effectively suppress the occurrence of 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.

Bacterial-derived vesicles of the genus Rizobium according to the present invention can be used for the prevention, improvement or treatment of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia as well as diagnostic methods for the disease. Since it can be used as a composition for the medical and food industry is expected to be usefully utilized.

Claims (13)

1. Informational methods for diagnosing gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, or dementia, 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) Gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's when the content of extracellular vesicles derived from bacteria of Rhizobium is lower than that of normal people through quantitative analysis of the PCR products. Classifying as disease, or dementia.
2. The method of claim 1,

   The sample in step (a) is characterized in that the blood or urine, information providing method.
3. Prevention of one or more diseases selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and dementia, including vesicles derived from bacteria of the genus Rhizobium , or Therapeutic pharmaceutical composition.
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 the bacteria of the genus Rhizobium , pharmaceutical composition.
6. The method of claim 3,

   Emptying the separation tank (Rhizobium) bacteria belonging to the genus Rhizopus-derived vesicles away LES gyumi industrial rooms (Rhizobium leguminosarum), pharmaceutical composition, it characterized in that the packet originated.
7. Prevention of one or more diseases selected from the group consisting of gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease, and dementia, including vesicles derived from bacteria of the genus Rhizobium , or Improvement food composition.
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 the bacteria of the genus Rhizobium , food composition.
10. The method of claim 7, wherein

    Emptying the separation tank (Rhizobium) bacteria belonging to the genus Rhizopus-derived vesicles away LES gyumi industrial rooms (Rhizobium leguminosarum), a food composition, characterized in that the resulting package.
11. A method of diagnosing gastric cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease or dementia, 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) Stomach cancer, colorectal cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, when the content of extracellular vesicles derived from bacteria in Rizobium is lower than that of normal people through quantitative analysis of the PCR products. Determining Parkinson's disease or dementia.
12. Gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease and dementia At least one disease prevention or treatment method selected from the group.
13. Use of a vesicle derived from bacteria of the genus Rizobium for the prevention or treatment of one or more diseases selected from the group consisting of gastric cancer, colon cancer, breast cancer, ovarian cancer, bladder cancer, prostate cancer, asthma, diabetes, Parkinson's disease and dementia.

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