WO2019164283A1 - Blautia sp. bacterium-derived nanovesicles and use thereof - Google Patents

Blautia sp. bacterium-derived nanovesicles and use thereof Download PDF

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Publication number
WO2019164283A1
WO2019164283A1 PCT/KR2019/002102 KR2019002102W WO2019164283A1 WO 2019164283 A1 WO2019164283 A1 WO 2019164283A1 KR 2019002102 W KR2019002102 W KR 2019002102W WO 2019164283 A1 WO2019164283 A1 WO 2019164283A1
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cancer
vesicles
disease
derived
blautia
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PCT/KR2019/002102
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French (fr)
Korean (ko)
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김윤근
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주식회사 엠디헬스케어
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Priority claimed from KR1020190019742A external-priority patent/KR102122898B1/en
Application filed by 주식회사 엠디헬스케어 filed Critical 주식회사 엠디헬스케어
Priority to CN201980015198.1A priority Critical patent/CN111936640A/en
Priority to US16/975,890 priority patent/US20210002707A1/en
Publication of WO2019164283A1 publication Critical patent/WO2019164283A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Blautia genus is an anaerobic Gram-positive bacterium found in the intestine, and it has been reported that mortality due to Graft versus Host disease is reduced when the bacteria increase in the intestine after bone marrow transplantation. However, it has not been reported that Blautia spp. Secrete extracellular vesicles, and there have been no reports of application to diagnosis and treatment of refractory diseases such as cancer or cardiovascular disease.
  • the present invention is to prevent colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina, graft versus host disease
  • Another object is to provide a composition for improvement, treatment or treatment.
  • the present invention comprises the following steps, information for the diagnosis of colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or heteroangular angina Provide a way to:
  • the present invention comprises administering to the subject a pharmaceutical composition
  • a pharmaceutical composition comprising a Blautia bacteria-derived vesicle as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction,
  • a pharmaceutical composition comprising a Blautia bacteria-derived vesicle as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction.
  • the vesicles may have an average diameter of 10 to 200 nm.
  • the vesicles may be secreted naturally or artificially from the Blautia genus bacteria.
  • the Blautia bacteria-derived vesicles may be Blautia cocoides-derived vesicles.
  • the colon disease may be colitis, irritable growth syndrome or colorectal cancer.
  • the pancreatic disease may be pancreatitis or pancreatic cancer.
  • the present inventors confirmed that intestinal bacteria are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs.
  • FIG. 6 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in ovarian cancer patients and normal blood.
  • 9 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in patients with atrial fibrillation and normal blood.
  • E. coli EV Escherichia coli vesicles
  • the present invention relates to vesicles derived from Blautia spp. And uses thereof.
  • the present inventors found that vesicles derived from Blautia bacteria were significantly reduced in clinical samples of patients with colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and angina in patients with a metagenome analysis. It was confirmed that the disease can be diagnosed.
  • 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 incidence and disease level of colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and / or dysplastic angina.
  • the present invention comprises a bladder-derived bacterial vesicles as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina or graft
  • a composition for preventing, treating or ameliorating host disease 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 an injection, oral nebulizer, or inhalant formulation.
  • the colon disease may be colitis, irritable growth syndrome or colorectal cancer, but is not limited thereto.
  • the liver disease may be hepatitis, cirrhosis or liver cancer, but is not limited thereto.
  • the pancreatic disease may be pancreatitis or pancreatic cancer, but is not limited thereto.
  • prevention means colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina, and / or graft formation by administration of a composition according to the present invention. It means any action that suppresses or delays the onset of a host disease.
  • treatment refers to colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina and / or graft versus host by administration of a composition according to the present invention. It means any action that improves or beneficially changes the symptoms of illness.
  • the term “improvement” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
  • the vesicles were centrifuged, ultra-fast centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, and filter.
  • the separation can be carried out using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
  • the pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • natural flavoring agents such as, tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
  • 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.
  • the bacteria and the bacteria-derived vesicles orally administered to evaluate the absorption, distribution, and excretion of the bacteria and vesicles in the body in the case of bacteria is not absorbed through the intestinal vesicles 5 minutes administration It was confirmed that it was absorbed within the body and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
  • a vesicle isolated from blood or stool of a normal person of age and sex matched in patients with colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and dysplastic angina pectoris Bacterial metagenome analysis was performed. As a result, compared to the normal sample, the Blautia bacteria-derived vesicles were significantly reduced in the clinical samples of colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and angina. It was confirmed (see Examples 3 to 11).
  • the Blautia cocoides strain was cultured to evaluate whether the secreted vesicles exhibit immunomodulatory and anti-inflammatory effects. After evaluating the secretion of inflammatory mediators by treating E. coli-derived vesicles, the inflammatory disease-causing factor, it was confirmed that Blautia cocoides-derived vesicles effectively suppressed IL-6 and TNF- ⁇ secretion by E. coli-derived vesicles. (See Example 13).
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • 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.
  • OFUTU operational taxonomy unit
  • 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 2 the genes were extracted from the vesicles in 29 stool patients and 358 stool patients in normal stool, followed by metagenomic analysis. Evaluated. As a result, it was confirmed that vesicles derived from Blautia bacteria were significantly reduced in the stool of colorectal cancer patients compared to normal stool (see Table 2 and FIG. 2).
  • Example 2 In the method of Example 2, 176 blood of pancreatic cancer patients and 271 blood of normal persons whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenome analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 4 and FIG. 4).
  • Example 2 In the method of Example 2, 137 blood of ovarian cancer patients and 139 normal blood of age and sex matched were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that vesicles derived from Blautia bacteria were significantly reduced in blood of ovarian cancer patients compared to normal blood (see Table 6 and FIG. 6).
  • Example 2 the blood of 91 bladder cancer patients and 176 bloods of normal age and gender matched were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 7 and FIG. 7).
  • Example 2 the blood of 80 patients with angina and 80 normal blood whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in blood of patients with heteroangular angina compared with normal blood (see Table 10 and FIG. 10).
  • Blautia cocoides-derived vesicles were collected in raw 264.7 cells. 0.1, 1, 10 ⁇ g / ml), followed by ELISA. More specifically, the Blautia cocooides-derived vesicles of various concentrations containing DMEM (Dulbeco's Modified Eagle's Medium) serum-free medium in 4 ⁇ 10 4 aliquots of 4 ⁇ 10 4 cells in 48-well cell culture plates were treated for 12 hours. Incubated. Then, the cell cultures were collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatants were collected, stored at -80 ° C, and then subjected to ELISA.
  • DMEM Dynamic microsomal growth factor
  • the capture antibody was diluted in phosphate buffered saline (PBS), and 50 ⁇ l was dispensed into 96 well polystyrene plates according to the working concentration, followed by reaction at 4 ° C. overnight. I was. After washing three times with 100 ⁇ l of PBST (phosphate buffered saline solution containing 0.05% tween-20), 100 ⁇ l of RD (phosphate buffered saline solution containing 1% BSA (bovine serum albumin) solution was dispensed. Blocking for 1 hour at room temperature.
  • PBS phosphate buffered saline
  • RD phosphate buffered saline solution containing 1% BSA (bovine serum albumin
  • Dispense the sample and standard by 50 ⁇ l according to the concentration react for 2 hours at room temperature, wash three times with 100 ⁇ l of PBST, and then dilute the detection antibody in RD and dispense 50 ⁇ l according to the working concentration. The reaction was carried out at room temperature for 2 hours.
  • Blautia cocoides-derived vesicles were treated with a macrophage line ( After pretreatment with Raw 264.7) for 12 hours, 1 ⁇ g / ml of E. coli vesicles derived from Escherichia coli (E. coli EV) were treated, and after 12 hours, the secretion of inflammatory cytokines was measured by ELISA. Lactobacillus plantarum- derived vesicles were used.
  • the extracellular vesicles derived from the Blautia spp. Bacterium according to the present invention are for preventing, improving or treating colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or angina, as well as the above diseases. Since it can be used as a composition for the medical and food industry is expected to be usefully utilized.

Abstract

The present invention relates to Blautia sp. bacterium-derived vesicles and a use thereof. The present inventors experimentally found that significantly reduced levels of the vesicles are in clinical samples from patients with colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and variant angina, compared to normal persons and that administration of vesicles isolated from the strain remarkably repressed the inflammatory mediator secretion induced by pathogenic vesicles such as vesicles derived from E. coli. The Blautia sp. bacterium-derived vesicles according to the present invention can be effectively used to develop a method for diagnosis of colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or variant angina and a composition for prevention, alleviation, or treatment of colorectal disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, variant angina, or graft-versus-host disease.

Description

블라우티아 속 세균 유래 나노소포 및 이의 용도Nanovesicles derived from Blautia spp. And uses thereof
본 발명은 블라우티아 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 블라우티아 속 세균에서 유래하는 나노소포를 이용한 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증 등의 진단방법, 및 상기 소포를 포함하는 상기 질병 등의 예방 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to nano-vesicles derived from Blautia spp. Bacteria and their use, and more particularly to colon cancer, liver cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, and the like. A method for diagnosing atrial fibrillation or heteroangular angina, and a composition for improving or treating such diseases including the vesicles.
21세기에 들어서면서 과거 전염병으로 인식되던 급성 감염성질환의 중요성이 덜해지는 반면, 인간과 마이크로바이옴과의 부조화에 의해 발생하는 면역기능 이상을 동반한 만성질환이 삶의 질과 인간 수명을 결정하는 주요 질환으로 질병패턴이 바뀌었다. 21세기 난치성 만성질환으로서, 암, 심혈관질환, 만성폐질환, 대사질환, 및 신경-정신질환이 인간 수명과 삶의 질을 결정하는 주요 질환으로서 국민보건에 큰 문제가 되고 있으며 상기 난치성 만성질환은 원인인자에 의한 면역기능 이상을 동반한 만성염증을 특징으로 한다.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. In the 21st century, intractable chronic diseases, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric diseases are major diseases that determine human life and quality of life. It is characterized by chronic inflammation accompanied by immune dysfunction by the causative factor.
한편, 인체에 공생하는 미생물은 100조에 이르러 인간 세포보다 10배 많으며, 미생물의 유전자수는 인간 유전자수의 100배가 넘는 것으로 알려지고 있다. 미생물총(microbiota 혹은 microbiome)은 주어진 거주지에 존재하는 진정세균(bacteria), 고세균(archaea), 진핵생물(eukarya)을 포함한 미생물 군집(microbial community)을 말한다. 우리 몸에 공생하는 세균 및 주변 환경에 존재하는 세균은 다른 세포로의 유전자, 저분자화합물, 단백질 등의 정보를 교환하기 위하여 나노미터 크기의 소포(vesicle)를 분비한다. 점막은 200 나노미터(nm) 크기 이상의 입자는 통과할 수 없는 물리적인 방어막을 형성하여 점막에 공생하는 세균인 경우에는 점막을 통과하지 못하지만, 세균 유래 소포는 크기가 100 나노미터 크기 이하라서 비교적 자유롭게 점막을 통하여 상피세포를 통과하여 우리 몸에 흡수된다. 국소적으로 분비된 세균 유래 소포는 점막의 상피세포를 통해 흡수되어 국소 염증반응을 유도할 뿐만 아니라, 상피세포를 통과한 소포는 림프관을 통해 전신적으로 흡수되어 각 장기로 분포하고, 분포된 장기에서 면역 및 염증반응을 조절한다. 예를 들어, 대장균( Eshcherichia coli)와 같은 병원성 그람음성세균에서 유래하는 소포는 국소적으로 대장염을 일으키고, 혈관으로 흡수된 경우에 혈관 내피세포 염증반응을 통해 전신적인 염증반응 및 혈액응고를 촉진시키고, 또한 인슐린이 작용하는 근육세포 등에 흡수되어선 인슐린저항성과 당뇨병을 유발한다. 반면, 유익한 세균에서 유래하는 소포는 병원성 소포에 의한 면역기능 및 대사기능 이상을 조절하여 질병을 조절할 수 있다. On the other hand, the microorganisms symbiotic to the human body is 100 trillion times more than human cells, 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 mucous membranes cannot pass through the mucosa. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacterial-derived vesicles are absorbed through the epithelial cells of the mucosa to induce local inflammatory responses, as well as vesicles passing through the epithelial cells are systemically absorbed through the lymphatic vessels and distributed to each organ. Regulates immune and inflammatory responses For example, vesicles derived from pathogenic Gram-negative bacteria, such as Eshcherichia coli , locally cause colitis and, when absorbed into blood vessels, promote systemic inflammatory and blood 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 immune and metabolic abnormalities caused by pathogenic vesicles.
세균에서 유래하는 소포 등의 인자에 대한 면역반응은 인터루킨(Interleukin, 이하 IL)-17 사이토카인 분비를 특징으로 하는 Th17 면역반응이 발생하는데, 이는 세균 유래 소포에 노출 시 IL-6가 분비되고, 이는 Th17 면역반응을 유도한다. Th17 면역반응에 의한 염증은 호중구 침윤을 특징으로 하고, 염증이 발생하는 과정에서 대식세포 등과 같은 염증세포에서 분비되는 종양괴사인자-알파(tumor necrosis factor-alpha, 이하 TNF-α)가 중요한 역할을 담당한다. 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.
블라우티아 속 세균은 장에서 발견되는 혐기성 그람양성세균으로서 골수이식 후 장에 상기 세균이 증가한 경우에 이식편대숙주병(Graft versus Host disease)에 의한 사망률이 줄어든다고 보고되었다. 그러나 아직까지 블라우티아 속 세균이 세포밖으로 소포를 분비한다는 사실이 보고되지 않았고, 특히 암 또는 심혈관질환 등과 같은 난치성 질환의 진단 및 치료에 응용한 사례는 보고된 바가 없다.Blautia genus is an anaerobic Gram-positive bacterium found in the intestine, and it has been reported that mortality due to Graft versus Host disease is reduced when the bacteria increase in the intestine after bone marrow transplantation. However, it has not been reported that Blautia spp. Secrete extracellular vesicles, and there have been no reports of application to diagnosis and treatment of refractory diseases such as cancer or cardiovascular disease.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 메타게놈 분석을 통해 정상인에 비하여 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 이형협심증 환자 유래 샘플에서 블라우티아 속 세균 유래 소포의 함량이 유의하게 감소되어 있음 확인하였다. 또한, 블라우티아 속 세균에 속하는 블라우티아 코코이데스( B. coccoides) 균에서 소포를 분리하여 대식세포에 처리하였을 때, 병원성 소포에 의한 IL-6 및 TNF-α의 분비를 현저히 억제함을 확인한 바, 이에 기초하여 본 발명을 완성하였다. The present inventors earnestly researched to solve the above-mentioned conventional problems. As a result of meta-genomic analysis, the present inventors have compared colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and dysplastic angina patients In the derived samples, it was confirmed that the content of the vesicles derived from the Blautia genus was significantly reduced. In addition, when the vesicles were isolated from B. coccoides bacteria belonging to the Blautia genus and treated with macrophages, they significantly suppressed the secretion of IL-6 and TNF-α by pathogenic vesicles. As a result, the present invention was completed based on this finding.
이에, 본 발명은 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a method for providing information for the diagnosis of colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or dysplastic angina.
또한, 본 발명은 블라우티아 속 세균 유래 소포를 유효성분으로 포함하는 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증, 또는 이식편대숙주병의 예방, 개선 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다. In addition, the present invention is to prevent colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina, graft versus host disease Another object is to provide a composition for improvement, treatment or treatment.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.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.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 단계를 포함하는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증의 진단을 위한 정보제공방법을 제공한다:In order to achieve the object of the present invention as described above, the present invention comprises the following steps, information for the diagnosis of colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or heteroangular angina Provide a way to:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(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) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 블라우티아 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증으로 분류하는 단계.(c) colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or heteromorphism Classifying as angina.
또한, 본 발명은 하기의 단계를 포함하는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증의 진단 방법을 제공한다:The present invention also provides a method of diagnosing colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or heteroangular angina, comprising the following steps:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(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) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 블라우티아 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증으로 판정하는 단계.(c) colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or heteroangular angina when the content of extracellular vesicles derived from Blautia bacteria is lower than that of normal people through quantitative analysis of the PCR product The step of determining.
본 발명의 일 구현예로, 상기 (a) 단계에서의 샘플은 혈액 또는 대변 일 수 있다. In one embodiment of the invention, the sample in step (a) may be blood or feces.
본 발명의 다른 구현예로, 상기 (b) 단계에서의 프라이머쌍은 서열번호 1 및 서열번호 2의 프라이머 일 수 있다. 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.
또한, 본 발명은 블라우티아 속 세균 유래 소포를 유효성분으로 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention includes a blotia-derived vesicle-derived vesicles as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina or graft-versus-host disease Or it provides a pharmaceutical composition for treatment.
또한, 본 발명은 블라우티아 속 세균 유래 소포를 유효성분으로 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병의 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention includes a blotia-derived vesicle-derived vesicles as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina or graft-versus-host disease Or it provides a food composition for improvement.
또한, 본 발명은 블라우티아 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병의 예방 또는 치료 방법을 제공한다. In addition, the present invention comprises administering to the subject a pharmaceutical composition comprising a Blautia bacteria-derived vesicle as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, Provided are methods for the prevention or treatment of atrial fibrillation, atypical angina or graft-versus-host disease.
또한, 본 발명은 블라우티아 속 세균 유래 소포의, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병의 예방 또는 치료 용도를 제공한다. The present invention also provides a bleeding-derived bleeding-derived blouse, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina or graft-versus-host disease. do.
본 발명의 일 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm인 것일 수 있다. In one embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.
본 발명의 다른 구현예로, 상기 소포는 블라우티아 속 세균에서 자연적 또는 인공적으로 분비되는 것일 수 있다. In another embodiment of the present invention, the vesicles may be secreted naturally or artificially from the Blautia genus bacteria.
본 발명의 또 다른 구현예로, 상기 블라우티아 속 세균 유래 소포는 블라우티아 코코이데스 유래 소포일 수 있다.In another embodiment of the present invention, the Blautia bacteria-derived vesicles may be Blautia cocoides-derived vesicles.
본 발명의 또 다른 구현예로, 상기 대장질환은 대장염, 과민성장증후군 또는 대장암 일 수 있다.In another embodiment of the present invention, the colon disease may be colitis, irritable growth syndrome or colorectal cancer.
본 발명의 또 다른 구현예로, 상기 간질환은 간염, 간경변 또는 간암 일 수 있다.In another embodiment of the present invention, the liver disease may be hepatitis, cirrhosis or liver cancer.
본 발명의 또 다른 구현예로, 상기 췌장질환은 췌장염 또는 췌장암 일 수 있다.In another embodiment of the present invention, the pancreatic disease may be pancreatitis or pancreatic cancer.
본 발명자들은 장내 세균인 경우에는 체내에 흡수되지 않지만, 세균 유래 소포인 경우에는 상피세포를 통해 체내에 흡수되어, 전신적으로 분포하고, 신장, 간, 폐를 통해 체외로 배설됨을 확인하였고, 환자 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 통해 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 이형협심증 환자의 혈액 또는 대변에 존재하는 블라우티아 속 세균 유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. 또한, 블라우티아 속 세균의 한 종인 블라우티아 코코이데스를 체외에서 배양하여 소포를 분리하여, 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 블라우티아 속 세균 유래 소포는 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증에 대한 진단방법, 및 상기 질환 등에 대한 예방, 개선 또는 치료용 조성물에 유용하게 이용될 수 있을 것으로 기대된다.The present inventors confirmed that intestinal bacteria are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs. Bacterial-derived vesicles from Blautia bacteria present in colon or liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and blood or stool in patients with dysplastic angina. It was confirmed that the significantly reduced compared to the normal person. In addition, when Blautia cocooides, a species of Blautia spp., Was cultured in vitro and isolated from vesicles and administered to inflammatory cells, it was observed that significantly inhibiting the secretion of inflammatory mediators by pathogenic vesicles. Blautia bacteria-derived vesicles according to the invention is a method for diagnosing colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or angina, and preventing, improving or treating the above diseases. It is expected to be usefully used.
도 1a는 마우스에 세균과 세균 유래 소포 (EV)를 구강으로 투여한 후, 시간별로 세균과 소포의 분포양상을 촬영한 사진이고, 도 1b는 구강으로 투여한 후 12시간째에, 혈액, 신장, 간, 및 여러 장기를 적출하여, 세균과 소포의 체내 분포양상을 평가한 결과이다.Figure 1a is a picture of the distribution of bacteria and vesicles by time after oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse, Figure 1b is 12 hours after oral administration, blood, kidney The results of evaluating the distribution of bacteria and vesicles in the liver, liver, and various organs were evaluated.
도 2는 대장암 환자 및 정상인 대변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. Figure 2 is a result of comparing the distribution of the bacteria-derived vesicles derived from Blautia after performing a bacterial-derived vesicle metagenome analysis present in colorectal cancer patients and normal feces.
도 3은 간암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. Figure 3 is a result of comparing the distribution of the bacteria-derived vesicles derived from Blautia after performing a bacterial-derived vesicle metagenome analysis present in liver cancer patients and normal blood.
도 4는 췌장암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. 4 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in pancreatic cancer patients and normal blood.
도 5는 담관암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. Figure 5 is a result of comparing the distribution of the bacteria-derived vesicles derived from Blautia after performing a bacterial-derived vesicle metagenome analysis present in patients with bile duct cancer.
도 6은 난소암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. 6 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in ovarian cancer patients and normal blood.
도 7은 방광암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. 7 is a result of comparing the distribution of vesicles derived from Blautia spp. After carrying out a bacterial-derived vesicle metagenome analysis present in bladder cancer patients and normal blood.
도 8은 심근경색환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. 8 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in myocardial infarction patients and normal blood.
도 9는 심방세동 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다. 9 is a result of comparing the distribution of vesicles derived from Blautia spp. After performing a bacterial-derived vesicle metagenome analysis present in patients with atrial fibrillation and normal blood.
도 10은 이형협심증 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 블라우티아 속 세균 유래 소포의 분포를 비교한 결과이다.10 is a result of comparing the distribution of vesicles derived from Blautia spp. After carrying out the analysis of bacteria-derived vesicles metagenome present in patients with heteroangular angina and normal blood.
도 11은 블라우티아 코코이데스 유래 소포의 염증유발 효과를 평가하기 위하여, 블라우티아 코코이데스 유래 소포를 대식세포(Raw264.7 cell)에 처리하여 염증매개체인 IL-6 및 TNF-α 분비 정도를 병원성 소포인 대장균 소포 (E. coli EV)와 비교한 결과이다.11 is to evaluate the inflammation-inducing effect of the Blautia cocoides-derived vesicles, Blautia Cocoides-derived vesicles treated with macrophage (Raw264.7 cells) secretion of IL-6 and TNF-α secretion Is compared with Escherichia coli vesicles (E. coli EV).
도 12는 블라우티아 코코이데스 유래 소포의 항염증 및 면역조절 효과를 평가하기 위하여, 병원성 소포인 대장균 소포 ( E. coli EV) 처리 전에 블라우티아균 유래 소포를 전처리하여, 대장균 소포에 의한 염증매개체인 IL-6 및 TNF-α 분비에 미치는 영향을 평가한 결과이다.12 is a pre-treatment of the Blautia bacteria-derived vesicles before treatment with Escherichia coli vesicles ( E. coli EV), in order to evaluate the anti-inflammatory and immunomodulatory effects of Blautia cocoides-derived vesicles. The results of the evaluation of the mediators on the secretion of IL-6 and TNF-α.
본 발명은 블라우티아 속 세균 유래 소포 및 이의 용도에 관한 것이다. The present invention relates to vesicles derived from Blautia spp. And uses thereof.
본 발명자들은 메타게놈 분석을 통해 블라우티아 속 세균 유래 소포가 정상인에 비하여 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 이형협심증 환자의 임상 샘플에 유의하게 감소되어 있음을 확인하여 상기 질병을 진단할 수 있음을 확인하였다. 또한, 블라우티아 속 세균에 속하는 블라우티아 코코이데스으로 부터 소포를 분리하고 특성을 분석한 결과, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병 등의 질환에 대한 예방, 개선 또는 치료용 조성물로 이용할 수 있음을 확인하였다.The present inventors found that vesicles derived from Blautia bacteria were significantly reduced in clinical samples of patients with colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and angina in patients with a metagenome analysis. It was confirmed that the disease can be diagnosed. In addition, the vesicles were isolated and characterized from Blautia cocooides belonging to the Blautia genus bacterium, and as a result, colorectal disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation and heteroangular angina Or it was confirmed that it can be used as a composition for preventing, improving or treating diseases such as graft-versus-host disease.
이에, 본 발명은 하기의 단계를 포함하는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증의 진단을 위한 정보제공방법을 제공한다:Accordingly, the present invention provides a method for providing information for diagnosing colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or heteroangular angina.
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(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) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 블라우티아 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증으로 분류하는 단계.(c) colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or heteromorphism Classifying as angina.
본 발명에서 사용되는 용어, “진단”이란 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 합병증의 유무, 및 예후 등이다. 본 발명에서 진단은 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및/또는 이형협심증 등의 발병 여부 및 질환의 수준 등을 판단하는 것이다. 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 incidence and disease level of colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and / or dysplastic angina.
본 발명에서 사용되는 용어, “나노소포(Nanovesicle)”혹은 “소포(Vesicle)”란, 다양한 세균에서 분비되는 나노크기의 막으로 된 구조물을 의미한다. 그람음성균(gram-negative bacteria) 유래 소포, 또는 외막 소포체(outer membrane vesicles, OMVs)는 내독소(lipopolysaccharide) 뿐만 아니라 독성 단백질 및 세균 DNA와 RNA도 가지고 있고, 그람양성균(gram-positive bacteria) 유래 소포는 단백질과 핵산 외에도 세균의 세포벽 구성성분인 펩티도글리칸(peptidoglycan)과 리포테이코산(lipoteichoic acid)도 가지고 있다. 본 발명에 있어서, 나노소포 혹은 소포는 블라우티아 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.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 by Blautia spp., And have a spherical shape and have an average diameter of 10 to 200 nm.
본 발명에서 사용되는 용어, “메타게놈”이란 “군유전체”라고도 하며, 흙, 동물의 장 등 고립된 지역 내의 모든 바이러스, 세균, 곰팡이 등을 포함하는 유전체의 총합을 의미하는 것으로, 주로 배양이 되지 않는 미생물을 분석하기 위해서 서열분석기를 사용하여 한꺼번에 많은 미생물을 동정하는 것을 설명하는 유전체의 개념으로 쓰인다. 특히, 메타게놈은 한 종의 게놈, 유전체를 말하는 것이 아니라, 한 환경단위의 모든 종의 유전체로서 일종의 혼합유전체를 말한다. 이는 오믹스적으로 생물학이 발전하는 과정에서 한 종을 정의할 때 기능적으로 기존의 한 종뿐만 아니라, 다양한 종이 서로 상호작용하여 완전한 종을 만든다는 관점에서 나온 용어이다. 기술적으로는 빠른 서열분석법을 이용해서, 종에 관계없이 모든 DNA, RNA를 분석하여, 한 환경 내에서의 모든 종을 동정하고, 상호작용, 대사작용을 규명하는 기법의 대상이다.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.
본 발명에 있어서, 상기 샘플은 혈액 또는 대변일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the sample may be blood or feces, but is not limited thereto.
본 발명의 다른 양태로서, 본 발명은 블라우티아 속 세균 유래 소포를 유효성분으로 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 또는 이식편대숙주병의 예방, 치료 또는 개선용 조성물을 제공한다. 상기 조성물은 식품 조성물 및 약학적 조성물을 포함하며, 본 발명에서 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명의 조성물은 주사제, 구강분무제, 또는 흡입제 제형일 수 있다.As another embodiment of the present invention, the present invention comprises a bladder-derived bacterial vesicles as an active ingredient, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina or graft Provided is a composition for preventing, treating or ameliorating host disease. 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 an injection, oral nebulizer, or inhalant formulation.
본 발명에서 상기 대장질환은 대장염, 과민성장증후군 또는 대장암 일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the colon disease may be colitis, irritable growth syndrome or colorectal cancer, but is not limited thereto.
본 발명에서 상기 간질환은 간염, 간경변 또는 간암 일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the liver disease may be hepatitis, cirrhosis or liver cancer, but is not limited thereto.
본 발명에서 상기 췌장질환은 췌장염 또는 췌장암 일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the pancreatic disease may be pancreatitis or pancreatic cancer, but is not limited thereto.
본 발명에서 사용되는 용어, “예방”이란 본 발명에 따른 조성물의 투여에 의해 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증, 및/또는 이식편대숙주병 등을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" means colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina, and / or graft formation by administration of a composition according to the present invention. It means any action that suppresses or delays the onset of a host disease.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 조성물의 투여에 의해 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 및/또는 이식편대숙주병 등에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used herein, the term "treatment" refers to colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, heteroangular angina and / or graft versus host by administration of a composition according to the present invention. It means any action that improves or beneficially changes the symptoms of illness.
본 발명에서 사용되는 용어, “개선”이란 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. 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.
상기 소포는 블라우티아 속 세균을 포함하는 배양액을 원심분리, 초고속 원심분리, 고압처리, 압출, 초음파분해, 세포 용해, 균질화, 냉동-해동, 전기천공, 기계적 분해, 화학물질 처리, 필터에 의한 여과, 겔 여과 크로마토그래피, 프리-플로우 전기영동, 및 모세관 전기영동으로 이루어진 군에서 선택된 하나 이상의 방법을 사용하여 분리할 수 있다. 또한, 불순물의 제거를 위한 세척, 수득된 소포의 농축 등의 과정을 추가로 포함할 수 있다. The vesicles were centrifuged, ultra-fast centrifugation, high pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, and filter. The separation can be carried out using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
본 발명에 따른 약학적 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다. 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.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.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.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/ 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.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.
구체적으로, 본 발명에 따른 약학적 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.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.
본 발명의 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명에 따른식품 조성물은 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.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.
본 발명의 식품 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물, 예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.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.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. 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.
본 발명의 일 실시예에서는 세균 및 세균 유래 소포를 마우스 경구로 투여하여 세균 및 소포의 체내 흡수, 분포, 및 배설 양상을 평가하여, 세균인 경우에는 장점막을 통해 흡수되지 않는데 비해 소포는 투여 5분 이내에 흡수되어 전신적으로 분포하고, 신장, 간 등을 통해 배설됨을 확인하였다(실시예 1 참조).In one embodiment of the present invention, the bacteria and the bacteria-derived vesicles orally administered to evaluate the absorption, distribution, and excretion of the bacteria and vesicles in the body, in the case of bacteria is not absorbed through the intestinal vesicles 5 minutes administration It was confirmed that it was absorbed within the body and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
본 발명의 일 실시예에서는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 이형협심증 환자에 연령과 성별을 매칭한 정상인의 혈액 또는 대변에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 이형협심증 환자의 임상샘플에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3 내지 11 참조).In one embodiment of the present invention, using a vesicle isolated from blood or stool of a normal person of age and sex matched in patients with colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and dysplastic angina pectoris Bacterial metagenome analysis was performed. As a result, compared to the normal sample, the Blautia bacteria-derived vesicles were significantly reduced in the clinical samples of colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and angina. It was confirmed (see Examples 3 to 11).
본 발명의 다른 실시예에서는, 블라우티아 코코이데스 균주를 배양하여 이로부터 분비된 소포가 면역조절 및 항염증 효과를 나타내는지를 평가하였는데, 다양한 농도의 블라우티아 코코이데스 유래 소포를 대식세포에 처리한 후, 염증질환 원인인자인 대장균 유래 소포를 처리하여 염증매개체 분비를 평가한 결과, 대장균 유래 소포에 의한 IL-6 및 TNF-α 분비를 블라우티아 코코이데스 유래 소포가 효율적으로 억제함을 확인하였다(실시예 13 참조).In another embodiment of the present invention, the Blautia cocoides strain was cultured to evaluate whether the secreted vesicles exhibit immunomodulatory and anti-inflammatory effects. After evaluating the secretion of inflammatory mediators by treating E. coli-derived vesicles, the inflammatory disease-causing factor, it was confirmed that Blautia cocoides-derived vesicles effectively suppressed IL-6 and TNF-α secretion by E. coli-derived vesicles. (See Example 13).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.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.
[[ 실시예Example ]]
실시예Example 1. 장내 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석 1. Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
장내 세균과 세균 유래 소포가 위장관을 통해 전신적으로 흡수되는 지를 평가하기 위하여 다음과 같은 방법으로 실험을 수행하였다. 마우스의 위장에 형광으로 표지한 장내세균과 장내 세균 유래 소포를 각각 50 μg의 용량으로 위장관으로 투여하고 0분, 5분, 3시간, 6시간, 12시간 후에 형광을 측정하였다. 마우스 전체 이미지를 관찰한 결과, 도 1a에 나타낸 바와 같이, 세균인 경우에는 전신적으로 흡수되지 않았지만, 세균 유래 소포인 경우에는, 투여 후 5분에 전신적으로 흡수되었고, 투여 3시간 후에는 방광에 형광이 진하게 관찰되어, 소포가 비뇨기계로 배설됨을 알 수 있었다. 또한, 소포는 투여 12시간까지 체내에 존재함을 알 수 있었다. 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.
또한, 장내세균과 장내 세균 유래 소포가 전신적으로 흡수된 후, 여러 장기로 침윤된 양상을 평가하기 위하여, 형광으로 표지한 50 μg의 세균과 세균 유래 소포를 상기의 방법과 같이 투여한 후, 투여 12시간 후에 혈액, 심장, 폐, 간, 신장, 비장, 지방, 근육을 채취하였다. 채취한 조직에서 형광을 관찰한 결과, 도 1b에 나타낸 바와 같이, 세균 유래 소포가 혈액, 심장, 폐, 간, 비장, 지방, 근육, 신장에 분포하였으나, 세균은 흡수되지 않음을 알 수 있었다.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, blood, heart, lungs, liver, kidneys, spleen, fat and muscle were collected. As a result of fluorescence observed in the collected tissue, as shown in Figure 1b, the bacteria-derived vesicles were distributed in the blood, heart, lung, liver, spleen, fat, muscle, kidney, it can be seen that the bacteria are not absorbed.
실시예Example 2. 임상샘플에서 세균 유래 소포  2. Bacterial-Derived Vesicles in Clinical Samples 메타게놈Metagenome 분석 analysis
혈액, 대변 등의 임상샘플을 먼저 10 ml 튜브에 넣고 원심분리법(3,500 x g, 10min, 4℃)으로 부유물을 가라앉히고 상등액만을 새로운 10 ml 튜브에 옮겼다. 0.22㎛ 필터를 사용하여 세균 및 이물질을 제거한 후, 센트리프랩튜브 (centrifugal filters 50 kD)에 옮겨서 1500 x g, 4℃에서 15분간 원심분리하여 50 kD 보다 작은 물질은 버리며 10 ml 까지 농축 시켰으며 다시 한 번 0.22㎛ 필터(filter)를 사용하여 박테리아 및 이물질을 제거한 후, Type 90ti 로터로 150,000 x g, 4℃에서 3시간동안 초고속원심분리방법을 사용하여 상등액을 버리고 덩어리진 펠렛(pellet)을 생리식염수(PBS)로 녹였다. Clinical samples of blood, feces, etc. 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 bacteria and foreign substances by using 0.22㎛ filter, it was transferred to centrifugal filters (50 kD) and centrifuged at 1500 xg and 4 ℃ for 15 minutes to discard the material smaller than 50 kD and concentrated to 10 ml again. Once the bacteria and foreign substances were removed using a 0.22㎛ filter, the supernatant was discarded by using a super fast centrifugation method at 150,000 xg for 3 hours at 4 ℃ with a Type 90ti rotor, and the lumped pellet was physiological saline. Dissolved in (PBS).
상기 방법으로 분리한 소포 100㎕를 100℃에서 끓여서 내부의 DNA를 지질 밖으로 나오게 하고 그 후 얼음에 5분 동안 식혔다. 그리고 남은 부유물을 제거하기 위하여 10,000 x g, 4℃에서 30분간 원심분리하고 상등액만을 모은 다음 Nanodrop을 이용하여 DNA 양을 정량하였다. 이후, 상기 추출된 DNA에 세균 유래 DNA가 존재하는지 확인하기 위하여 하기 표 1에 나타낸 16s rDNA 프라이머(primer)로 PCR을 수행하여 상기 추출된 유전자에 세균 유래 유전자가 존재하는 것을 확인하였다.100 μl of the vesicles separated by the above method was boiled at 100 ° C. to let the internal DNA come out of the lipid and then cooled on ice for 5 minutes. In order to remove the remaining suspended matter, centrifugation was performed at 10,000 x g and 4 ° C for 30 minutes, only the supernatant was collected, and the DNA amount was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rDNA primer (primer) shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene is present in the extracted gene.
primerprimer 서열order 서열번호SEQ ID NO:
16S rDNA16S rDNA 16S_V3_F16S_V3_F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 ' 1One
16S_V4_R16S_V4_R 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-35'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 22
상기 방법으로 추출한 DNA를 상기의 16S rDNA 프라이머를 사용하여 증폭을 한 다음 시퀀싱을 수행하고 (Illumina MiSeq sequencer), 결과를 Standard Flowgram Format (SFF) 파일로 출력하고 GS FLX software (v2.9)를 이용하여 SFF 파일을 sequence 파일 (.fasta)과 nucleotide qualityscore파일로 변환한 다음 리드의 신용도 평가를 확인하고, window (20 bps) 평균 base call accuracy가 99% 미만 (Phred score <20)인 부분을 제거하였다. Operational Taxonomy Unit (OTU) 분석을 위해서는 UCLUST와 USEARCH를 이용하여 시퀀스 유사도에 따라 클러스터링을 수행하고, 속(genus)은 94%, 과(family)는 90%, 목(order)은 85%, 강(class)은 80%, 문(phylum)은 75% 시퀀스 유사도를 기준으로 클러스터링을 하고 각 OTU의 문(phylum), 강(class), 목(order), 과(family), 속(genus) 레벨의 분류를 수행하고, BLASTN와 GreenGenes의 16S RNA 시퀀스 데이터베이스 (108,453 시퀀스)를 이용하여 속 수준에서 97% 이상의 시퀀스 유사도 갖는 세균을 프로파일링 하였다 (QIIME).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).
실시예Example 3. 대장암환자의 대변 내 세균 유래 소포  3. Bacterial-derived Vesicles in Feces of Colon Cancer Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 대장암환자 29명의 대변과, 정상인 358명의 대변을 대상으로, 대변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 대변에 비하여 대장암환자의 대변에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 2 및 도 2 참조).In the method of Example 2, the genes were extracted from the vesicles in 29 stool patients and 358 stool patients in normal stool, followed by metagenomic analysis. Evaluated. As a result, it was confirmed that vesicles derived from Blautia bacteria were significantly reduced in the stool of colorectal cancer patients compared to normal stool (see Table 2 and FIG. 2).
대변credit 대조군Control 대장암Colorectal cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00370.0037 0.01000.0100 0.00170.0017 0.00170.0017 0.00090.0009 0.450.45
실시예Example 4. 간암환자 혈액 세균 유래 소포  4. Blood-derived vesicles derived from liver cancer patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 간암환자 86명의 혈액과, 나이와 성별을 매칭한 정상인 331명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 간암 환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 3 및 도 3 참조).In the method of Example 2, genes were extracted from vesicles in the liver and 331 normal blood patients whose age and sex were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in blood of liver cancer patients compared to normal blood (see Table 3 and FIG. 3).
혈액blood 대조군Control 간암Liver cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.0095 0.0095 0.0134 0.0134 0.0043 0.0043 0.0043 0.0043 <0.0001 <0.0001 0.450.45
실시예Example 5. 췌장암환자 혈액 세균 유래 소포  5. Blood Bacteria Derived from Pancreatic Cancer Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 췌장암환자 176명의 혈액과, 나이와 성별을 매칭한 정상인 271명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 췌장암환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 4 및 도 4 참조).In the method of Example 2, 176 blood of pancreatic cancer patients and 271 blood of normal persons whose age and sex were matched were extracted from the vesicles present in the blood and subjected to metagenome analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 4 and FIG. 4).
혈액blood 대조군Control 췌장암Pancreatic cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00860.0086 0.01640.0164 0.00450.0045 0.00710.0071 0.00030.0003 0.520.52
실시예Example 6. 담관암환자 혈액 세균 유래 소포  6. Blood Bacteria Derived from Bile Duct Cancer Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 담관암환자 79명의 혈액과, 나이와 성별을 매칭한 정상인 259명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 담관암환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 5 및 도 5 참조).In the blood of 79 patients with cholangiocarcinoma patients and blood of 259 normal people whose age and sex were matched by the method of Example 2, genes were extracted from vesicles present in the blood and subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in the blood of bile duct cancer patients compared to normal blood (see Table 5 and FIG. 5).
혈액blood 대조군Control 담관암Bile duct cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00880.0088 0.01690.0169 0.00190.0019 0.00470.0047 <0.0001<0.0001 0.220.22
실시예Example 7. 난소암환자 혈액 세균 유래 소포  7. Blood Bacteria Derived from Ovarian Cancer Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 난소암환자 137명의 혈액과, 나이와 성별을 매칭한 정상인 139명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 난소암환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 6 및 도 6 참조).In the method of Example 2, 137 blood of ovarian cancer patients and 139 normal blood of age and sex matched were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that vesicles derived from Blautia bacteria were significantly reduced in blood of ovarian cancer patients compared to normal blood (see Table 6 and FIG. 6).
혈액blood 대조군Control 난소암Ovarian Cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00840.0084 0.00960.0096 0.00250.0025 0.00340.0034 <0.0001 <0.0001 0.30 0.30
실시예Example 8. 방광암환자 혈액 세균 유래 소포  8. Vesicle-derived vesicles from bladder cancer patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 방광암환자 91명의 혈액과, 나이와 성별을 매칭한 정상인 176명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 방광암환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 7 및 도 7 참조).In the method of Example 2, the blood of 91 bladder cancer patients and 176 bloods of normal age and gender matched were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 7 and FIG. 7).
혈액blood 대조군Control 방광암Bladder cancer t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.01290.0129 0.02090.0209 0.00050.0005 0.00050.0005 <0.0001 <0.0001 0.04 0.04
실시예Example 9. 심근경색환자 혈액 세균 유래 소포  9. Blood Bacteria Derived from Myocardial Infarction Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 심근경색 환자 57명의 혈액과, 나이와 성별을 매칭한 정상인 163명의 혈액의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심근경색환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 8 및 도 8 참조).In the blood of 57 patients with myocardial infarction and blood of 163 normal blood patients whose age and sex were matched, the genes were extracted from the vesicles present in the blood and subjected to metagenome analysis. The distribution of vesicles derived from the genus Tia was evaluated. As a result, it was confirmed that vesicles derived from Blautia bacteria were significantly reduced in blood of myocardial infarction patients compared to normal blood (see Table 8 and FIG. 8).
혈액blood 대조군Control 심근경색Myocardial infarction t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00610.0061 0.00850.0085 0.00120.0012 0.00530.0053 <0.0001<0.0001 0.19 0.19
실시예Example 10. 심방세동환자 혈액 세균 유래 소포  10. Blood Bacteria Derived from Atrial Fibrillation Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 심방세동 환자 34명의 혈액과, 나이와 성별을 매칭한 정상인 62명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심방세동환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 9 및 도 9 참조).In the blood of 34 patients with atrial fibrillation and 62 blood of normal people whose age and sex were matched by the method of Example 2, genes were extracted from the vesicles present in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in blood of atrial fibrillation patients compared to normal blood (see Table 9 and FIG. 9).
혈액blood 대조군Control 심방세동Atrial fibrillation t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.01390.0139 0.02590.0259 0.00200.0020 0.00280.0028 0.00060.0006 0.140.14
실시예Example 11.  11. 이형협심증환자Patients with heterozygous angina 혈액 세균 유래 소포  Blood Bacteria-Derived Vesicles 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 이형협심증 환자 80명의 혈액과, 나이와 성별을 매칭한 정상인 80명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 블라우티아 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 이형협심증환자의 혈액에 블라우티아 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 10 및 도 10 참조).In the method of Example 2, the blood of 80 patients with angina and 80 normal blood whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from Blautia bacteria were significantly reduced in blood of patients with heteroangular angina compared with normal blood (see Table 10 and FIG. 10).
혈액blood 대조군Control 이형협심증Angina t-testt-test
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio
g__Blautiag__Blautia 0.00780.0078 0.01170.0117 0.00410.0041 0.00490.0049 0.010.01 0.530.53
실시예Example 12.  12. 블라우티아Blautia 코코이데스Cocoides 유래 소포의 염증유발 효과 Inflammatory Effects of Derived Vesicles
염증세포에서 블라우티아 코코이데스 유래 소포의 염증매개체(IL-6, TNF-α) 분비에 대한 영향을 알아보기 위해, 마우스 대식세포주인 Raw 264.7 세포에 블라우티아 코코이데스 유래 소포를 다양한 농도(0.1, 1, 10 ㎍/㎖)로 처리한 후, ELISA를 진행하였다. 보다 구체적으로, 48-well 세포 배양 플레이트 안에 4 x 10 4 개씩 분주한 Raw 264.7 세포에 DMEM(Dulbeco’s Modified Eagle’s Medium) 무혈청 배지를 넣은 다양한 농도의 블라우티아 코코이데스 유래 소포를 처리하여 12시간 동안 배양하였다. 이후 세포 배양액을 1.5 ml 튜브에 모아 3000 g에서 5분간 원심분리하고 상층액을 모아 -80 ℃에 보관해두었다가 ELISA를 진행하였다. To investigate the effect of inflammatory mediators (IL-6, TNF-α) on inflammatory cells in inflammatory cells, Blautia cocoides-derived vesicles were collected in raw 264.7 cells. 0.1, 1, 10 μg / ml), followed by ELISA. More specifically, the Blautia cocooides-derived vesicles of various concentrations containing DMEM (Dulbeco's Modified Eagle's Medium) serum-free medium in 4 × 10 4 aliquots of 4 × 10 4 cells in 48-well cell culture plates were treated for 12 hours. Incubated. Then, the cell cultures were collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatants were collected, stored at -80 ° C, and then subjected to ELISA.
ELISA를 수행하기 위해, 캡쳐(Capture) 항체를 인산완충생리식염수(phosphate buffered saline, PBS)에 희석시켜 96 well 폴리스틸렌(polystyrene) 플레이트에 작용 농도에 맞게 50 μl 씩 분주한 후 4 ℃에서 하룻밤 동안 반응시켰다. 이후 PBST(0.05 % tween-20이 들어있는 인산완충생리식염수) 용액 100 μl로 세 번씩 씻어준 후, RD(1 % BSA(bovine serum albumin)가 들어있는 인산완충생리식염수) 용액 100 μl을 분주하여 상온에서 1시간 동안 블로킹(blocking) 하였다. 샘플 및 스텐다드(standard)를 농도에 맞게 50 μl씩 분주하고 상온에서 2시간 동안 반응시키고 PBST 100 μl로 세 번 씻어준 후, 검출(detection) 항체를 RD에 희석시켜 작용 농도에 맞게 50 μl씩 분주하여 상온에서 2시간 동안 반응시켰다. In order to perform ELISA, the capture antibody was diluted in phosphate buffered saline (PBS), and 50 μl was dispensed into 96 well polystyrene plates according to the working concentration, followed by reaction at 4 ° C. overnight. I was. After washing three times with 100 μl of PBST (phosphate buffered saline solution containing 0.05% tween-20), 100 μl of RD (phosphate buffered saline solution containing 1% BSA (bovine serum albumin) solution was dispensed. Blocking for 1 hour at room temperature. Dispense the sample and standard by 50 μl according to the concentration, react for 2 hours at room temperature, wash three times with 100 μl of PBST, and then dilute the detection antibody in RD and dispense 50 μl according to the working concentration. The reaction was carried out at room temperature for 2 hours.
그 다음, PBST 100 μl로 세 번 씻어준 후, Strpetavidin-HRP (R&D system, USA)를 RD에 1/40으로 희석시켜 50 μl씩 분주하여 상온에서 20분간 반응시켰으며, 마지막으로 PBST 100 μl로 세 번 씻어준 후, TMB 기질 (SurModics, USA) 50 μl를 분주하고 5분에서 20분 후 발색이 진행되었을 때, 1 M 황산용액을 50 μl씩 분주해 반응을 멈추고 SpectraMax M3 microplate reader (Molecular Devices, USA)를 이용해 450 nm에서 흡광도를 측정하였다.Then, after washing three times with 100 μl of PBST, Strpetavidin-HRP (R & D system, USA) was diluted 1/40 in RD, 50 μl were dispensed and reacted at room temperature for 20 minutes, and finally with 100 μl of PBST. After washing three times, 50 μl of TMB substrate (SurModics, USA) was dispensed and when color development progressed after 5 to 20 minutes, 50 μl of 1 M sulfuric acid solution was dispensed to stop the reaction and the SpectraMax M3 microplate reader (Molecular Devices , USA) was used to measure the absorbance at 450 nm.
그 결과, 도 11에 나타난 바와 같이 블라우티아 코코이데스 유래 소포를 대식세포주에 처리하였을 때, 염증매개체 분비는 병원성 소포인 대장균 유래 소포 (E. coli EV) 보다 현저히 낮음을 확인하였다.As a result, as shown in Figure 11, when treated with a macrophage line Blautia cocoides-derived, the secretion of inflammatory mediators was confirmed to be significantly lower than Escherichia coli-derived vesicles (E. coli EV).
실시예Example 13.  13. 블라우티아Blautia 코코이데스Cocoides 유래 소포의 항염증 효과 Anti-inflammatory Effects of Derived Vesicles
상기 실시예 12의 결과를 바탕으로, 블라우티아 코코이데스 유래 소포의 항염증 효과를 평가하기 위하여, 다양한 농도(0.1, 1, 10 ㎍/㎖)의 블라우티아 코코이데스 유래 소포를 대식세포주(Raw 264.7)에 12시간 전처리한 후, 병원성 소포인 대장균 유래 소포(E. coli EV) 1㎍/㎖을 처리하고 12시간 뒤 염증성 사이토카인의 분비를 ELISA로 측정하였으며 유용미생물 대조군으로는 락토바실러스 플란타룸( Lactobacillus plantarum) 유래 소포를 사용하였다. Based on the results of Example 12, in order to evaluate the anti-inflammatory effects of the Blautia cocoides-derived vesicles, Blautia cocoides-derived vesicles of various concentrations (0.1, 1, 10 μg / ml) were treated with a macrophage line ( After pretreatment with Raw 264.7) for 12 hours, 1 μg / ml of E. coli vesicles derived from Escherichia coli (E. coli EV) were treated, and after 12 hours, the secretion of inflammatory cytokines was measured by ELISA. Lactobacillus plantarum- derived vesicles were used.
그 결과, 도 12에 나타난 바와 같이 블라우티아 코코이데스 유래 소포를 전처리한 경우 대장균 유래 소포에 의한 IL-6 및 TNF-α의 분비가 현저히 억제됨을 확인하였으며 특히, 블라우티아 코코이데스 유래 소포의 전처리에 의한 TNF-α의 분비 억제효과가 유용미생물 대조군인 락토바실러스 플란타룸( Lactobacillus plantarum) 유래 소포의 전처리에 의한 TNF-α의 분비 억제효과보다 현저하게 큼을 확인하였다. 상기 결과는 대장균 유래 소포와 같은 병원성 소포에 의해 유도되는 염증반응을 블라우티아 유래 소포가 효율적으로 억제할 수 있음을 의미한다.As a result, as shown in FIG. 12, when pre-treatment of the Blautia cocoides-derived vesicles, the secretion of IL-6 and TNF-α by E. coli-derived vesicles was remarkably suppressed. The inhibitory effect of TNF-α secretion by pretreatment was significantly greater than the inhibitory effect of TNF-α secretion by pretreatment of vesicles derived from Lactobacillus plantarum , a useful microorganism control group. The above results indicate that the Blautia-derived vesicles can effectively suppress the inflammatory response induced by pathogenic vesicles such as E. coli-derived vesicles.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.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.
본 발명에 따른 블라우티아 속 세균 유래 세포밖 소포는 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증의 진단방법뿐만 아니라 상기 질환 등에 대한 예방, 개선 또는 치료용 조성물로 이용될 수 있으므로 관련 의료 및 식품 산업 분야에서 유용하게 활용 가능할 것으로 기대된다.The extracellular vesicles derived from the Blautia spp. Bacterium according to the present invention are for preventing, improving or treating colon cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or angina, as well as the above diseases. Since it can be used as a composition for the medical and food industry is expected to be usefully utilized.

Claims (19)

  1. 하기의 단계를 포함하는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증의 진단을 위한 정보제공 방법:Method for providing information for diagnosis of colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or angina, comprising the following steps:
    (a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
    (b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(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) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 블라우티아(Blautia) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증으로 분류하는 단계.(c) colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation when the content of extracellular vesicles derived from Blautia bacteria is lower than that of normal people through quantitative analysis of the PCR products , Or classifying dysplastic angina.
  2. 제1항에 있어서,The method of claim 1,
    상기 (a) 단계에서의 샘플은 혈액 또는 대변인 것을 특징으로 하는, 정보제공 방법.And the sample in step (a) is blood or feces.
  3. 블라우티아(Blautia) 속 세균 유래 소포를 유효성분으로 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 및 이식편대숙주병으로 이루어진 군으로부터 선택된 하나 이상의 질병의 예방 또는 치료용 약학적 조성물.From the group consisting of colorectal disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, angina with angina and graft-versus-host disease, including Blautia-derived bacterial vesicles as an active ingredient A pharmaceutical composition for the prophylaxis or treatment of one or more diseases selected.
  4. 제3항에 있어서,The method of claim 3,
    상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 약학적 조성물.The vesicles are characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
  5. 제3항에 있어서,The method of claim 3,
    상기 소포는 블라우티아 속 세균에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 약학적 조성물.The vesicles are characterized in that they are secreted naturally or artificially from the bacteria of the genus Blautia, pharmaceutical composition.
  6. 제3항에 있어서,The method of claim 3,
    상기 블라우티아 속 세균 유래 소포는 블라우티아 코코이데스( Blautia coccoides) 유래 소포인 것을 특징으로 하는, 약학적 조성물.The Blautia bacteria-derived vesicles are characterized in that the vesicles derived from Blautia coccoides .
  7. 제3항에 있어서,The method of claim 3,
    상기 대장질환은 대장염, 과민성장증후군, 또는 대장암인 것을 특징으로 하는, 약학적 조성물.The colon disease is colitis, irritable growth syndrome, or colorectal cancer, characterized in that the pharmaceutical composition.
  8. 제3항에 있어서,The method of claim 3,
    상기 간질환은 간염, 간경변, 또는 간암인 것을 특징으로 하는, 약학적 조성물.The liver disease is characterized in that hepatitis, cirrhosis, or liver cancer, pharmaceutical composition.
  9. 제3항에 있어서,The method of claim 3,
    상기 췌장질환은 췌장염 또는 췌장암인 것을 특징으로 하는, 약학적 조성물.The pancreatic disease is characterized in that pancreatitis or pancreatic cancer, pharmaceutical composition.
  10. 블라우티아(Blautia) 속 세균 유래 소포를 유효성분으로 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 및 이식편대숙주병으로 이루어진 군으로부터 선택된 하나 이상의 질병의 예방 또는 개선용 식품 조성물. From the group consisting of colorectal disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, angina with angina and graft-versus-host disease, including Blautia-derived bacterial vesicles as an active ingredient Food composition for the prevention or amelioration of one or more diseases selected.
  11. 제10항에 있어서,The method of claim 10,
    상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 식품 조성물.The vesicles are characterized in that the average diameter of 10 to 200 nm, food composition.
  12. 제10항에 있어서,The method of claim 10,
    상기 소포는 블라우티아 속 세균에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 식품 조성물.The vesicles are characterized in that the natural or artificial secretion from the Blautia genus bacteria, food composition.
  13. 제10항에 있어서,The method of claim 10,
    상기 블라우티아 속 세균 유래 소포는 블라우티아 코코이데스( Blautia coccoides) 유래 소포인 것을 특징으로 하는, 식품 조성물.The Blautia bacteria-derived vesicles are characterized in that the vesicles derived from Blautia coccoides , food composition.
  14. 제10항에 있어서,The method of claim 10,
    상기 대장질환은 대장염, 과민성장증후군, 또는 대장암인 것을 특징으로 하는, 식품 조성물.The colon disease is characterized in that colitis, irritable growth syndrome, or colorectal cancer, food composition.
  15. 제10항에 있어서,The method of claim 10,
    상기 간질환은 간염, 간경변, 또는 간암인 것을 특징으로 하는, 식품 조성물.The liver disease is characterized in that hepatitis, cirrhosis, or liver cancer, food composition.
  16. 제10항에 있어서,The method of claim 10,
    상기 췌장질환은 췌장염 또는 췌장암인 것을 특징으로 하는, 식품 조성물.The pancreatic disease is characterized in that the pancreatitis or pancreatic cancer, food composition.
  17. 하기의 단계를 포함하는, 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동 또는 이형협심증의 진단방법:A method of diagnosing colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation or angina, comprising the following steps:
    (a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
    (b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(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) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 블라우티아(Blautia) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 간암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 이형협심증으로 판정하는 단계.(c) colorectal cancer, liver cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation when the content of extracellular vesicles derived from Blautia bacteria is lower than that of normal people through quantitative analysis of the PCR products Or judging as heteromorphic angina.
  18. 블라우티아(Blautia) 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 및 이식편대숙주병으로 이루어진 군으로부터 선택된 하나 이상의 질병의 예방 또는 치료방법.Colorectal disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, comprising administering to the subject a pharmaceutical composition comprising a bleeding-derived vesicle of Blautia as an active ingredient , Method for preventing or treating at least one disease selected from the group consisting of angina and graft-versus-host disease.
  19. 블라우티아(Blautia) 속 세균 유래 소포의, 대장질환, 간질환, 췌장질환, 담관암, 난소암, 방광암, 심근경색, 심방세동, 이형협심증 및 이식편대숙주병으로 이루어진 군으로부터 선택된 하나 이상의 질병의 예방 또는 치료용도.Of at least one disease selected from the group consisting of bladder-derived vesicles of the Blautia genus, colon disease, liver disease, pancreatic disease, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, dysplastic angina and graft-versus-host disease For prophylactic or therapeutic purposes.
PCT/KR2019/002102 2018-02-26 2019-02-21 Blautia sp. bacterium-derived nanovesicles and use thereof WO2019164283A1 (en)

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