JP2022512469A - Nanovesicles derived from Corynebacterium and their uses - Google Patents

Nanovesicles derived from Corynebacterium and their uses Download PDF

Info

Publication number
JP2022512469A
JP2022512469A JP2021532852A JP2021532852A JP2022512469A JP 2022512469 A JP2022512469 A JP 2022512469A JP 2021532852 A JP2021532852 A JP 2021532852A JP 2021532852 A JP2021532852 A JP 2021532852A JP 2022512469 A JP2022512469 A JP 2022512469A
Authority
JP
Japan
Prior art keywords
vesicles
corynebacterium
derived
inflammatory disease
preventing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP2021532852A
Other languages
Japanese (ja)
Other versions
JP7186470B2 (en
JPWO2020122449A5 (en
Inventor
キム、ユン-クン
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MD Healthcare Inc
Original Assignee
MD Healthcare Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020190132137A external-priority patent/KR102223406B1/en
Application filed by MD Healthcare Inc filed Critical MD Healthcare Inc
Publication of JP2022512469A publication Critical patent/JP2022512469A/en
Publication of JPWO2020122449A5 publication Critical patent/JPWO2020122449A5/ja
Application granted granted Critical
Publication of JP7186470B2 publication Critical patent/JP7186470B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, 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
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135Bacteria or derivatives thereof, e.g. probiotics
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66Microorganisms or materials therefrom
    • A61K35/74Bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Birds (AREA)
  • Food Science & Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Dermatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Nutrition Science (AREA)
  • Immunology (AREA)
  • Polymers & Plastics (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)

Abstract

本発明は、コリネバクテリウム属細菌由来の小胞及びその用途に関するもので、本発明らは、コリネバクテリウム属細菌由来の小胞が肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者の臨床サンプルで減少しており、炎症を誘導する病原性小胞による炎症メディエーターの分泌を効率的に抑制することを実験的に確認したところ、本発明によるコリネバクテリウム属細菌由来の小胞は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープなどを含む炎症疾患の予防又は治療用組成物として有用に用いられ得ると期待される。【選択図】図14The present invention relates to vesicles derived from a bacterium belonging to the genus Corinebacterium and uses thereof, and the present inventions describe vesicles derived from a bacterium belonging to the genus Corinebacterium to cause liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, and the like. It has been reduced in clinical samples of patients with asthma, dementia and nasal polyps, and it was experimentally confirmed that the secretion of inflammatory mediators by pathogenic vesicles that induce inflammation was effectively suppressed. The vesicles derived from the bacterium of the genus Um can be usefully used as a composition for the prevention or treatment of inflammatory diseases including liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. Be expected. [Selection diagram] FIG. 14

Description

本発明は、コリネバクテリウム属細菌由来のナノ小胞及びその用途に関し、より具体的には、コリネバクテリウム属細菌に由来するナノ小胞を用いた肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープの診断方法、及び前記小胞を含む前記疾患に対する予防、改善又は治療用組成物などに関する。 The present invention relates to nanovesicles derived from Corynebacterium bacteria and their uses, and more specifically, liver cirrhosis, stroke, diabetes, asthma, and atopic skin using nanovesicles derived from Corynebacterium bacteria. It relates to a method for diagnosing inflammation, depression, breast cancer, dementia and nasal polyps, and a composition for preventing, ameliorating or treating the disease including the follicles.

本出願は、2018年12月10日に出願された大韓民国特許出願第10-2018-0158622号及び2019年10月23日に出願された大韓民国特許出願第10-2019-0132137号に基づく優先権の利益を主張し、当該出願の明細書及び図面に開示されたすべての内容は本出願に援用される。 This application has priority based on the Republic of Korea Patent Application No. 10-2018-0158622 filed on December 10, 2018 and the Republic of Korea Patent Application No. 10-2019-0132137 filed on October 23, 2019. All content claimed in the application and disclosed in the specification and drawings of the application is incorporated herein by reference.

21世紀に入ってから、過去には伝染病と認識されていた急性感染性疾患の重要性が減る一方、ヒトとマイクロバイオーム(Microbiome)との不調和により発生する免疫機能の異常を伴った慢性炎症疾患が主要疾患になり疾病パターンが変わった。特に、食習慣の西欧化による肥満、糖尿病、心血管疾患、神経精神疾患及び癌などの慢性炎症疾患と家屋構造の変化による室内大気汚染と室内での生活時間の増加によって皮膚及び呼吸器炎症疾患が国民保健において大きな問題となっている。 Since the beginning of the 21st century, the importance of acute infectious diseases, which were previously recognized as infectious diseases, has diminished, while chronic with abnormalities in immune function caused by incongruity between humans and microbiomes. Inflammatory diseases have become the main disease and the disease pattern has changed. In particular, chronic inflammatory diseases such as obesity, diabetes, cardiovascular disease, neuropsychiatric disease and cancer due to westernized eating habits, indoor air pollution due to changes in the structure of the house, and skin and respiratory inflammation diseases due to increased living time indoors. Has become a major problem in national health.

前記炎症疾患の発生には、外部の原因因子に対する免疫機能に異常を伴っている。細菌に由来する原因因子に対する免疫反応は、インターロイキン(Interleukin、以下、IL)-17サイトカインを分泌するTh17免疫反応が重要であり、細菌性原因因子に露出するとき、Th17免疫反応による好中球性炎症が発生する。また、炎症が発生する過程で細菌性原因因子により分泌される腫瘍壊死因子-α(Tumor Necrosis Factor-alpha、以下、TNF-α)のような炎症性メディエーターが炎症及び癌発生において重要な役割を担う。炎症性メディエーターのうち細菌性原因因子により分泌されるIL-6は、Th17細胞への分化に重要な役割を担い、Th17免疫反応による慢性炎症は、慢性炎症疾患だけでなく癌発生とも密接な関連があると最近報告されている。 The development of the inflammatory disease is accompanied by abnormalities in immune function against external causative factors. The Th17 immune response, which secretes interleukin (IL) -17 cytokines, is important for the immune response to bacterial causative factors, and when exposed to bacterial causative factors, Th17 immune response neutrophils. Sexual inflammation occurs. Inflammatory mediators such as Tumor Necrosis Factor-alpha (TNF-α), which is secreted by bacterial causative factors during the development of inflammation, play an important role in inflammation and cancer development. Carry. Among the inflammatory mediators, IL-6 secreted by bacterial causative factors plays an important role in the differentiation into Th17 cells, and chronic inflammation due to the Th17 immune response is closely related not only to chronic inflammatory diseases but also to cancer development. It has recently been reported that there is.

人体に共生する微生物の個数は、100兆個に達し、ヒト細胞より約10倍多く、微生物の遺伝子数は、ヒト遺伝子数の100倍を超えることが知られている。微生物叢(microbiotaあるいはmicrobiome)は、与えられた居住地に存在する真正細菌、古細菌、真核生物を含む微生物群集(microbial community)を言い、腸内微生物叢は、ヒトの生理現象に重要な役割をし、人体細胞と相互作用を通じてヒトの健康と疾病に大きい影響を及ぼすものと知られている。 It is known that the number of microorganisms coexisting with the human body reaches 100 trillion, which is about 10 times larger than that of human cells, and the number of genes of microorganisms exceeds 100 times the number of human genes. The microbial flora (microbiota or microbiome) refers to a microbial community containing eubacteria, archaea, and eukaryotes present in a given place of residence, and the intestinal microbial flora is important for human physiological phenomena. It is known to play a role and have a great impact on human health and diseases through interaction with human body cells.

人体に共生する真正細菌及び古細菌は、他の細胞への遺伝子、タンパク質などの情報を交換するためにナノメートルサイズの小胞(vesicle)を分泌する。粘膜は、200ナノメートル(nm)サイズ以上の粒子は通過できない物理的な防御膜を形成して、粘膜に共生する細菌の場合には、粘膜を通過できないが、細菌由来の小胞は、サイズが100ナノメートルサイズ以下であるので、比較的自由に粘膜を通じて上皮細胞を通過して人体に吸収される。人体に吸収される病原性細菌由来の小胞は、最近、糖尿病、肥満など代謝疾患の病因において重要な役割を担うことが明らかにされた。 Symbiotic eubacteria and archaea in the human body secrete nanometer-sized vesicles to exchange information such as genes and proteins to other cells. The mucosa forms a physical protective membrane that particles larger than 200 nanometers (nm) cannot pass through, and in the case of bacteria that coexist with the mucosa, it cannot pass through the mucosa, but bacterial vesicles are sized. Is less than 100 nanometers in size, so it is relatively free to pass through epithelial cells through the mucosa and be absorbed by the human body. Vesicles derived from pathogenic bacteria absorbed by the human body have recently been shown to play an important role in the etiology of metabolic diseases such as diabetes and obesity.

コリネバクテリウム(Corynebacterium)属細菌は、好気性グラム陽性細菌であって、自然界に広がって真核生物と共生して生きて行く細菌として知られている。特に、コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)菌は、産業的にアミノ酸、核酸などの生産に広く用いられている菌である。しかし、現在までコリネバクテリウム属細菌の細胞外小胞を用いた治療技術については報告されたことがない。 Corynebacterium genus bacteria are aerobic gram-positive bacteria and are known as bacteria that spread in nature and live in symbiosis with eukaryotes. In particular, Corynebacterium glutamicum is a bacterium that is industrially widely used for the production of amino acids, nucleic acids and the like. However, to date, no therapeutic technique using extracellular vesicles of Corynebacterium spp. Has been reported.

そこで、本発明では、コリネバクテリウム属細菌から小胞を最初に分離し、その特性を確認することで、前記小胞を多様な炎症疾患の診断及び炎症疾患の予防、改善又は治療用組成物として用いられることを確認した。 Therefore, in the present invention, by first separating vesicles from Corynebacterium spp. And confirming their characteristics, the vesicles can be used for diagnosis of various inflammatory diseases and for prevention, improvement or therapeutic composition of inflammatory diseases. It was confirmed that it was used as.

本発明者らは、上記のような従来の問題点を解決するために鋭意研究した結果、メタゲノム分析を通じて正常ヒトに比べて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者由来のサンプルからコリネバクテリウム属細菌由来の小胞の含量が有意に減少していることを確認した。また、コリネバクテリウム属細菌由来の小胞が病原性小胞による炎症反応を効率的に抑制することを確認し、これに基づいて本発明を完成した。 As a result of diligent research to solve the above-mentioned conventional problems, the present inventors conducted liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, and cognition as compared with normal humans through metagenome analysis. It was confirmed that the content of vesicles derived from Corinebacterium spp. Was significantly reduced from the samples derived from patients with dementia and nasal polyps. In addition, it was confirmed that vesicles derived from Corynebacterium spp. Efficiently suppress the inflammatory reaction caused by pathogenic vesicles, and the present invention was completed based on this.

そこで、本発明は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法又は診断のための情報提供方法を提供することを目的とする。 Therefore, the present invention provides information for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. The purpose is to provide a method.

また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供することを他の目的とする。 Another object of the present invention is to provide a composition for preventing, ameliorating or treating an inflammatory disease containing vesicles derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

しかし、本発明が達成しようとする技術的課題は、上記で言及した課題に制限されず、言及しなかったまた他の課題は、下の記載から当業者に明確に理解されるべきである。 However, the technical issues to be achieved by the present invention are not limited to those mentioned above, and other issues not mentioned above should be clearly understood by those skilled in the art from the description below.

上記のような本発明の目的を達成するために、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断のための情報提供方法を提供する: In order to achieve the object of the present invention as described above, the present invention is selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps including the following steps. Providing informational methods for the diagnosis of one or more diseases that have been made:

(a)正常ヒト及び被検者のサンプルから分離した小胞からDNAを抽出するステップ; (A) Step of extracting DNA from vesicles isolated from normal human and subject samples;

(b)前記抽出したDNAに対して16S rDNAに存在する遺伝子配列に基づいて製作したプライマー対を用いてPCR(Polymerase Chain Reaction)を行った後、それぞれのPCR産物を収得するステップ;及び (B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and

(c)前記PCR産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。 (C) Liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and when the content of vesicles derived from Corinebacterium spp. A step of determining one or more diseases selected from the group consisting of nasal polyps.

また、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法を提供する: The present invention also provides a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, which include the following steps. do:

(a)正常ヒト及び被検者のサンプルから分離した小胞からDNAを抽出するステップ; (A) Step of extracting DNA from vesicles isolated from normal human and subject samples;

(b)前記抽出したDNAに対して16S rDNAに存在する遺伝子配列に基づいて製作したプライマー対を用いてPCRを行った後、それぞれのPCR産物を収得するステップ;及び (B) A step of obtaining each PCR product after performing PCR on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA; and

(c)前記PCR産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。 (C) Liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and when the content of vesicles derived from Corinebacterium spp. A step of determining one or more diseases selected from the group consisting of nasal polyps.

本発明の一具現例で、前記ステップ(a)でのサンプルは、血液、小便、大便、唾液又は鼻粘膜であってもよい。 In one embodiment of the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa.

本発明の他の具現例で、前記ステップ(b)でのプライマー対は、配列番号1及び配列番号2で表示される塩基配列を含むプライマー対であってもよい。 In another embodiment of the present invention, the primer pair in step (b) may be a primer pair containing the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2.

また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供する。 The present invention also provides a composition for preventing, ameliorating or treating an inflammatory disease containing vesicles derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

前記組成物は、薬学的組成物、食品組成物、化粧料組成物及び吸入剤組成物を含むことができる。 The composition can include a pharmaceutical composition, a food composition, a cosmetic composition and an inhalant composition.

また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物を個体に投与するステップを含む炎症疾患の予防又は治療方法を提供する。 The present invention also provides a method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

また、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患の予防又は治療用途を提供する。 The present invention also provides a prophylactic or therapeutic use for inflammatory diseases of vesicles derived from Corynebacterium spp.

また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物の炎症疾患の予防又は治療用途を提供する。 The present invention also provides a prophylactic or therapeutic use for an inflammatory disease of a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

また、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患に用いられる薬剤を生産するための用途を提供する。 The present invention also provides applications for producing agents used for inflammatory diseases of vesicles derived from Corynebacterium spp.

本発明の一具現例で、前記小胞は、コリネバクテリウム・グルタミクムから分泌されるものであってもよい。 In one embodiment of the present invention, the vesicle may be secreted from Corynebacterium glutamicum.

本発明の他の具現例で、前記小胞は、平均直径が10~200nmであってもよい。 In another embodiment of the invention, the vesicles may have an average diameter of 10-200 nm.

本発明のまた他の具現例で、前記小胞は、コリネバクテリウム属細菌から自然的又は人工的に分泌されるものであってもよい。 In yet another embodiment of the invention, the vesicles may be naturally or artificially secreted by a Corynebacterium bacterium.

本発明のまた他の具現例で、前記人工小胞は、細菌に熱処理、加圧処理などの方法で分泌されるものであってもよい。 In still another embodiment of the present invention, the artificial vesicle may be secreted by a bacterium by a method such as heat treatment or pressure treatment.

本発明のまた他の具現例で、前記炎症疾患は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であってもよい。 In yet another embodiment of the invention, the inflammatory disease is one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. There may be.

本発明者らは、細菌の場合には体内に吸収されないが、細菌由来の小胞の場合には粘膜の防御膜を通過して粘膜の上皮細胞に吸収されて全身的に分布し、腎臓、肝臓、肺を通じて体外に排泄されることを確認した。また、患者の血液に存在する細菌由来の小胞のメタゲノム分析を通じて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者の血液又は鼻粘膜に存在するコリネバクテリウム属細菌由来の小胞が正常ヒトに比べて有意に減少していることを確認した。また、コリネバクテリウム属細菌の一種であるコリネバクテリウム・グルタミクムを体外で培養して小胞を分離して炎症細胞に投与したとき、病原性小胞によるTNF-αなどの炎症メディエーターの分泌を有意に抑制したことを確認したところ、本発明によるコリネバクテリウム属細菌由来の小胞は、前記炎症性疾患の予防用あるいは治療用組成物に有用であり得ると期待される。 In the case of bacteria, the present inventors are not absorbed in the body, but in the case of bacterial vesicles, they pass through the protective membrane of the mucosa and are absorbed by the epithelial cells of the mucosa and distributed systemically, and the kidneys, It was confirmed that it was excreted outside the body through the liver and lungs. In addition, corine bacteria present in the blood or nasal mucosa of patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps through metagenome analysis of bacterial vesicles present in the patient's blood. It was confirmed that the number of vesicles derived from the genus Bacteria was significantly reduced compared to normal humans. In addition, when Corynebacterium glutamicum, which is a type of Corynebacterium genus, is cultured in vitro and the vesicles are isolated and administered to inflammatory cells, the pathogenic vesicles secrete inflammatory mediators such as TNF-α. After confirming that it was significantly suppressed, it is expected that the vesicles derived from Corynebacterium genus according to the present invention may be useful for the preventive or therapeutic composition of the inflammatory disease.

図1aは、マウスに細菌(bacteria)と細菌由来の小胞(EV)を口腔で投与した後、時間別に細菌と小胞の分布様相を撮影した写真であり、図1bは、口腔で投与した後12時間目に、血液、腎臓、肝臓及び様々な臓器を摘出して細菌と小胞の体内分布様相を評価した図である。FIG. 1a is a photograph of mice administered with bacteria (bacteria) and vesicles derived from the bacteria (EV) orally, and then the distribution aspect of the bacteria and vesicles was photographed by time, and FIG. 1b was administered with the oral cavity. 12 hours later, blood, kidneys, liver and various organs were removed to evaluate the distribution of bacteria and vesicles in the body.

図2は、マウスに細菌(bacteria)と細菌由来の小胞(EV)を投与した後、粘膜の上皮細胞で細菌と細菌由来の小胞の浸潤有無を評価した図である(Lu、lumen;LP、lamina propria)。FIG. 2 is a diagram evaluating the presence or absence of bacterial and bacterial vesicle infiltration in mucosal epithelial cells after administration of bacteria (bacteria) and bacterial vesicles (EV) to mice (Lu, lumen; LP, lamina bacterium).

図3は、肝硬変患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 3 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of patients with liver cirrhosis and normal humans.

図4は、脳卒中患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 4 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of stroke patients and normal humans.

図5は、糖尿病患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 5 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenomic analysis of vesicles derived from bacteria present in the blood of diabetic patients and normal humans.

図6は、喘息患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 6 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of asthmatic patients and normal humans.

図7は、アトピー性皮膚炎患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 7 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of patients with atopic dermatitis and normal humans.

図8は、鬱病患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 8 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of depressed patients and normal humans.

図9は、乳癌患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 9 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of breast cancer patients and normal humans.

図10は、認知症患者及び正常ヒトの血液に存在する細菌由来の小胞のメタゲノム分析を実施した後、コリネバクテリウム属細菌由来の小胞の分布を比較した結果である。FIG. 10 shows the results of comparing the distribution of vesicles derived from Corynebacterium spp. After performing metagenome analysis of vesicles derived from bacteria present in the blood of dementia patients and normal humans.

図11は、アレルギー性及び非アレルギー性鼻ポリープ患者及び正常対照群の鼻腔の粘膜組織のコリネバクテリウム属細菌由来の小胞の浸潤様相を、メタゲノム分析を通じて評価した図である。FIG. 11 is a diagram in which the infiltration aspect of vesicles derived from Corynebacterium spp. In the mucosal tissue of the nasal cavity of patients with allergic and non-allergic nasal polyps and the normal control group was evaluated through metagenome analysis.

図12は、コリネバクテリウム・グルタミクム由来の小胞の細胞死滅効果を評価するために、コリネバクテリウム・グルタミクム由来の小胞をマクロファージ(Raw264.7 cell)に処理して細胞死滅を評価した結果である(EV、extracellular vesicle;CGT101、Corynebacterium glutamicum EV)。FIG. 12 shows the results of evaluating cell killing by treating vesicles derived from Corynebacterium glutamicum with macrophages (Raw264.7 cell) in order to evaluate the cell killing effect of vesicles derived from Corynebacterium glutamicum. (EV, extracellular vesicle; CGT101, Corynebacterium glutamicum EV).

図13a及び図13bは、コリネバクテリウム・グルタミクム由来の小胞(CGT101)の炎症誘発効果を評価するために、コリネバクテリウム菌由来の小胞をマクロファージ(Raw264.7 cell)に処理して炎症メディエーターの分泌程度を病原性小胞である大腸菌小胞(E.coli EV)と比較した結果であって、図13aは、IL-6の分泌程度を比較した図であり、図13bは、TNF-αの分泌程度を比較した図である(EV、extracellular vesicle)。In FIGS. 13a and 13b, in order to evaluate the pro-inflammatory effect of vesicles derived from Corinebacterium glutamicum (CGT101), vesicles derived from Corinebacterium bacterium are treated with macrophages (Raw264.7 cell) for inflammation. The results of comparing the degree of secretion of the mediator with the pathogenic vesicle E. colli EV, FIG. 13a is a diagram comparing the degree of secretion of IL-6, and FIG. 13b is a diagram comparing the degree of secretion of IL-6. It is a figure comparing the degree of secretion of −α (EV, extracellular inflammation).

図14は、コリネバクテリウム・グルタミクム由来の小胞(CGT101)の抗炎症効果を評価するために、病原性小胞である大腸菌小胞(E.coli EV)の処理前にコリネバクテリウム菌由来の小胞を前処理して、大腸菌小胞による炎症メディエーターであるTNF-αの分泌に及ぶ影響を評価した結果である(EV、extracellular vesicle)。FIG. 14 is derived from Corinebacterium vesicles prior to treatment with pathogenic vesicles E. coli EV in order to evaluate the anti-inflammatory effect of vesicles derived from Corinebacterium glutamicum (CGT101). This is the result of pretreating the vesicles of Escherichia coli and evaluating the effect of E. coli vesicles on the secretion of the inflammation mediator TNF-α (EV, extracellular vesicle).

本発明は、コリネバクテリウム属細菌由来の小胞及びその用途に関する。 The present invention relates to vesicles derived from Corynebacterium spp. And their uses.

本発明者らは、メタゲノム分析を通じて正常ヒトに比べて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者由来のサンプルでコリネバクテリウム属由来の小胞の含量が顕著に減少していることを確認した。また、本発明者らは、病原性原因因子を投与する前にコリネバクテリウム・グルタミクム菌由来の小胞で炎症細胞に処理すると、病原性原因因子による炎症反応を効率的に抑制することを確認し、これに基づいて本発明を完成した。 We found that samples from patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps compared to normal humans through metagenome analysis of vesicles derived from the genus Corinebacterium. It was confirmed that the content was significantly reduced. In addition, the present inventors confirmed that treatment of inflammatory cells with vesicles derived from Corynebacterium glutamicum before administration of the pathogenic causative factor efficiently suppresses the inflammatory reaction caused by the pathogenic causative factor. Based on this, the present invention was completed.

これにより、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法又は診断のための情報提供方法を提供する: Accordingly, the present invention is a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, which include the following steps. Providing informational methods for diagnosis:

(a)正常ヒト及び被検者のサンプルから分離した小胞からDNAを抽出するステップ;
(b)前記抽出したDNAに対して16S rDNAに存在する遺伝子配列に基づいて製作したプライマー対を用いてPCR(Polymerase Chain Reaction)を行った後、それぞれのPCR産物を収得するステップ;及び
(c)前記PCR産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。
(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corinebacterium spp. Is lower than that in normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A step of determining one or more diseases selected from the group consisting of.

本発明で用いられる用語「診断」とは、広い意味では患者の病気の実態を全ての面にわたって判断することを意味する。判断の内容は、病名、病因、病型、軽重、病状の詳細な様態、合併症の有無及び予後などである。本発明で「診断」は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の発病有無及び疾患の程度などを判断することである。 The term "diagnosis" used in the present invention means to judge the actual condition of a patient's illness in all aspects in a broad sense. The contents of the judgment include the name of the disease, the etiology, the type of the disease, the severity of the disease, the detailed mode of the condition, the presence or absence of complications, and the prognosis. In the present invention, "diagnosis" refers to the presence or absence of onset of one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, and the degree of the disease. Is to judge.

本発明で用いられる用語「ナノ小胞(Nanovesicle)」あるいは「小胞(Vesicle)」とは、多様な細菌から分泌されるナノサイズの膜からなる構造物を意味する。コリネバクテリウムのようなグラム陽性菌(gram-positive bacteria)由来の小胞は、タンパク質と核酸外にも細菌の細胞壁の構成成分であるペプチドグリカン(peptidoglycan)とリポテイコ酸(lipoteichoic acid)、そして小胞内に多様な低分子化合物を有している。本発明において、ナノ小胞あるいは小胞は、コリネバクテリウム属の細菌から自然的に分泌されるか又は細菌に熱処理、加圧処理などを通じて人工的に生産するものであって、10~200nmの平均直径を有している。 As used in the present invention, the term "nanovesicle" or "vesicle" means a structure consisting of nano-sized membranes secreted by various bacteria. Gram-positive bacteria-derived vesicles, such as corynebacterium, include peptidoglycan, lipoteichoic acid, and vesicles, which are constituents of the cell wall of bacteria as well as proteins and nucleic acids. It has a variety of low molecular weight compounds within. In the present invention, nanovesicles or vesicles are naturally secreted from bacteria of the genus Corynebacterium or artificially produced by heat treatment, pressure treatment, etc. on the bacteria, and have a diameter of 10 to 200 nm. Has an average diameter.

本発明で用いられる用語「メタゲノム」とは、「群遺伝体」とも言い、土壌、動物の腸など孤立した地域内の全てのウイルス、細菌、カビなどを含む遺伝体の総合を意味するもので、主に培養されない微生物を分析するためにシーケンサーを用いて一度に多くの微生物を同定することを説明する遺伝体の概念として用いられる。特に、「メタゲノム」は、一種のゲノム、遺伝体を言うのではなく、一つの環境単位の全ての種の遺伝体として一種の混合遺伝体を言う。これは、オミックス的に生物学が発展する過程で一つの種を定義するとき、機能的に既存の一種だけではなく、多様な種が互いに相互作用して完全な種を作るという観点から生じた用語である。技術的には、迅速配列分析法を用いて種に関係なく全てのDNA、RNAを分析し、一つの環境内での全ての種を同定し、相互作用、代謝作用を解明する技法の対象である。 The term "metagenome" used in the present invention is also referred to as "group gene" and means a synthesis of all genes including all viruses, bacteria, molds, etc. in isolated areas such as soil and animal intestines. It is mainly used as a concept of genetics to explain the identification of many microorganisms at once using a sequencer to analyze uncultured microorganisms. In particular, "metagenomics" does not refer to a kind of genome or genetic body, but to a kind of mixed genetic body as a genetic body of all species in one environmental unit. This arose from the perspective that when defining a species in the process of omics-like development of biology, not only functionally existing species but also diverse species interact with each other to form a complete species. It is a term. Technically, it is the subject of a technique that analyzes all DNA and RNA regardless of species using rapid sequence analysis, identifies all species in one environment, and elucidates interactions and metabolic effects. be.

前記小胞は、コリネバクテリウム属の細菌を含む培養液を遠心分離、超高速遠心分離、押圧、超音波分解、細胞溶解、均質化、冷凍-解凍、電気穿孔、機械的分解、化学物質処理、フィルタによる濾過、ゲル濾過クロマトグラフィー、フリーフロー電気泳動及び毛細管電気泳動からなる群より選択された一つ以上の方法を用いて分離することができる。また、不純物の除去のための洗浄、収得された小胞の濃縮などの過程を追加で含むことができる。 The vesicles are obtained by centrifuging, ultrafast centrifuging, pressing, ultrasonically decomposing, cell lysing, homogenizing, freezing-thawing, electrophoresing, mechanically decomposing, and treating chemical substances in a culture solution containing bacteria of the genus Corinebacterium. Separation can be performed using one or more methods selected from the group consisting of filtration through a filter, gel filtration chromatography, free-flow electrophoresis and capillary electrophoresis. In addition, additional processes such as washing for removing impurities and concentration of obtained vesicles can be included.

本発明において、前記ステップ(a)でのサンプルは、血液、小便、大便、唾液又は鼻粘膜であってもよいが、これに制限されるものではない。 In the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa, but is not limited thereto.

本発明において、前記ステップ(b)でのプライマー対は、配列番号1及び配列番号2で表示される塩基配列を含むプライマー対であってもよいが、これに制限されるものではない。 In the present invention, the primer pair in the step (b) may be a primer pair containing the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, but is not limited thereto.

本発明の他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供する。 As another aspect of the present invention, the present invention provides a composition for preventing, ameliorating or treating an inflammatory disease containing a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.

前記組成物は、薬学的組成物、食品組成物、化粧料組成物及び吸入剤組成物を含む。 The compositions include pharmaceutical compositions, food compositions, cosmetic compositions and inhalant compositions.

本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物を個体に投与するステップを含む炎症疾患の予防又は治療方法を提供する。 As yet another aspect of the present invention, the present invention provides a method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium of the genus Corynebacterium as an active ingredient.

本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患の予防又は治療用途を提供する。 As yet another aspect of the invention, the invention provides for the prevention or treatment of inflammatory diseases of vesicles derived from Corynebacterium spp.

本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物の炎症疾患の予防又は治療用途を提供する。 As yet another aspect of the present invention, the present invention provides a prophylactic or therapeutic use for an inflammatory disease of a composition containing a vesicle derived from a Corynebacterium genus bacterium as an active ingredient.

本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患に用いられる薬剤を生産するための用途を提供する。 As yet another aspect of the invention, the invention provides applications for producing agents used in inflammatory diseases of vesicles derived from Corynebacterium spp.

本発明で用いられる用語「炎症疾患(Inflammatory disease)」とは、炎症を誘発する原因因子に露出されて皮膚あるいは腸の上皮細胞に損傷とその結果として炎症が発生して生ずる疾患を意味し、炎症の結果として発生する代謝疾患、心血管疾患、神経精神疾患、癌を含むが、これに制限されない。 The term "Inflammatory Disease" as used in the present invention means a disease caused by damage to skin or intestinal epithelial cells and consequent inflammation caused by exposure to a causative factor that induces inflammation. It includes, but is not limited to, metabolic disorders, cardiovascular disorders, neuropsychiatric disorders, and cancers that occur as a result of inflammation.

本発明による前記炎症疾患の例としては、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症、鼻ポリープなどがあるが、これに制限されるものではない。 Examples of the inflammatory disease according to the present invention include, but are not limited to, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia, and nasal polyps.

本発明で用いられる用語「予防」とは、本発明による組成物の投与によって炎症疾患を抑制させたり発病を遅延させる全ての行為を意味する。 As used in the present invention, the term "prevention" means all actions that suppress or delay the onset of inflammatory diseases by administration of the composition according to the present invention.

本発明で用いられる用語「治療」とは、本発明による組成物の投与によって炎症疾患に対する症状が好転したり有利に変更される全ての行為を意味する。 As used in the present invention, the term "treatment" means any act in which the administration of the composition according to the present invention improves or favorably changes the symptoms of an inflammatory disease.

本発明で用いられる用語「改善」とは、治療される状態と関連したパラメータ、例えば、症状の程度を少なくとも減少させる全ての行為を意味する。 As used in the present invention, the term "improvement" means any action that at least reduces the degree of symptoms, eg, parameters associated with the condition being treated.

本発明の一実施例では、細菌及び細菌由来の小胞をマウスに経口投与して細菌及び小胞の体内吸収、分布及び排泄様相を評価して、細菌の場合には、腸粘膜を介して吸収されないのに比べて、小胞は、投与5分以内に吸収されて全身的に分布し、腎臓、肝臓などを通じて排泄されることを確認した(実施例1参照)。 In one embodiment of the invention, bacteria and vesicles derived from the bacteria are orally administered to mice to evaluate the absorption, distribution and excretion aspects of the bacteria and vesicles, and in the case of bacteria, via the intestinal mucosa. It was confirmed that the vesicles were absorbed within 5 minutes of administration, distributed systemically, and excreted through the kidney, liver, etc., as compared with the non-absorbed vesicles (see Example 1).

本発明の他の実施例では、細菌と細菌由来の小胞を腸に直接投与して腸粘膜の防御膜を通過するかを評価した結果、細菌の場合には、腸粘膜の防御膜を通過しなかったが、細菌由来の小胞の場合には、粘膜の防御膜を通過することを確認した(実施例2参照)。 In another embodiment of the present invention, as a result of directly administering bacteria and vesicles derived from the bacteria to the intestine and evaluating whether or not the vesicles pass through the protective membrane of the intestinal mucosa, in the case of bacteria, they pass through the protective membrane of the intestinal mucosa. However, in the case of vesicles derived from bacteria, it was confirmed that they passed through the protective membrane of the mucosa (see Example 2).

本発明のまた他の実施例では、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者と正常対照群の臨床サンプルからメタゲノム分析を通じてコリネバクテリウム属細菌の分布を比較した結果、正常対照群に比べて前記疾患者の臨床サンプルにコリネバクテリウム属細菌由来の小胞の分布が有意に減少していることを確認した(実施例3~12参照)。 In yet another example of the invention, patients with corynebacterium spp. From clinical samples of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps and normal control groups. As a result of comparing the distributions, it was confirmed that the distribution of vesicles derived from Corynebacterium spp. Was significantly reduced in the clinical sample of the diseased person as compared with the normal control group (see Examples 3 to 12).

本発明のまた他の実施例では、コリネバクテリウム属細菌に属するコリネバクテリウム・グルタミクム菌株を培養し、それから分泌された小胞の炎症誘発効果を評価した。多様な濃度のコリネバクテリウム・グルタミクム由来の小胞をマクロファージに処理した後、代表的病原性小胞である大腸菌由来の小胞を処理して炎症メディエーターの分泌程度を比較した結果、大腸菌由来の小胞によるIL-6及びTNF-αの分泌と比較してコリネバクテリウム・グルタミクム由来の小胞による分泌能が顕著に減少していた(実施例14参照)。 In yet another example of the present invention, a strain of Corynebacterium glutamicum belonging to a bacterium belonging to the genus Corynebacterium was cultured, and the pro-inflammatory effect of vesicles secreted from the strain was evaluated. After treating vesicles derived from corinebacterium glutamicum at various concentrations into macrophages, vesicles derived from Escherichia coli, which is a typical pathogenic vesicle, were treated and the degree of secretion of inflammatory mediators was compared. The secretory capacity of the vesicles derived from corynebacterium glutamicum was significantly reduced as compared with the secretion of IL-6 and TNF-α by the vesicles (see Example 14).

本発明のまた他の実施例では、コリネバクテリウム・グルタミクム菌株由来の小胞の抗炎症効果を評価した。病原性小胞である大腸菌由来の小胞を処理する前に多様な濃度のコリネバクテリウム・グルタミクム由来の小胞をマクロファージに処理した後、炎症メディエーターの分泌を評価した結果、炎症誘発大腸菌由来の小胞によるTNF-αの分泌をコリネバクテリウム・グルタミクム由来の小胞が効率的に抑制することを確認した(実施例15参照)。 In yet another example of the present invention, the anti-inflammatory effect of vesicles derived from the Corynebacterium glutamicum strain was evaluated. Before treating vesicles derived from Escherichia coli, which are pathogenic vesicles, macrophages were treated with vesicles derived from corinebacterium glutamicum at various concentrations, and then the secretion of inflammation mediators was evaluated. It was confirmed that the vesicles derived from corynebacterium glutamicum efficiently suppressed the secretion of TNF-α by the vesicles (see Example 15).

本発明による薬学的組成物は、薬学的に許容可能な担体を含むことができる。前記薬学的に許容可能な担体は、製剤時に通常的に用いられるものであって、食塩水、滅菌水、リンゲル液、緩衝食塩水、シクロデキストリン、デキストロース溶液、マルトデキストリン溶液、グリセロール、エタノール、リポソームなどを含むが、これに限定されず、必要に応じて、抗酸化剤、緩衝液など他の通常の添加剤をさらに含むことができる。また、希釈剤、分散剤、界面活性剤、結合剤、潤滑剤などを付加的に添加して水溶液、懸濁液、乳濁液などのような注射用剤型、丸薬、カプセル、顆粒又は錠剤で製剤化することができる。適合した薬学的に許容される担体及び製剤化についてはレミントンの文献に開示されている方法を用いて各成分によって好ましく製剤化することができる。本発明の薬学的組成物は、剤型に特別な制限はないが、注射剤、吸入剤、皮膚外用剤又は経口摂取剤などに製剤化することができる。 The pharmaceutical composition according to the invention can include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is usually used at the time of preparation, and includes saline solution, sterile water, Ringer's solution, buffered saline solution, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes and the like. However, the present invention is not limited to this, and other usual additives such as antioxidants and buffers can be further included, if necessary. In addition, a diluent, a dispersant, a surfactant, a binder, a lubricant, etc. are additionally added to form an injection form such as an aqueous solution, a suspension, an emulsion, a pill, a capsule, a granule, or a tablet. Can be formulated with. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated with each component using the methods disclosed in the Remington literature. The pharmaceutical composition of the present invention can be formulated into an injection, an inhalant, an external skin preparation, an oral ingestion, or the like, although the dosage form is not particularly limited.

本発明の薬学的組成物は、目的とする方法によって経口投与するか非経口投与(例えば、静脈内、皮下、皮膚、鼻腔、気道に適用)でき、投与量は、患者の状態及び体重、疾病の程度、薬物形態、投与経路及び時間によって異なるが、当業者により適切に選択され得る。 The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, skin, nasal cavity, airway) according to the method of interest, and the dose is the patient's condition and weight, disease. Depending on the degree of the drug, the form of the drug, the route of administration and the time, it can be appropriately selected by those skilled in the art.

本発明による薬学的組成物は、薬学的に有効な量で投与する。本発明において、薬学的に有効な量は、医学的治療に適用可能な合理的なベネフィット/リスクの割合で疾患を治療するに十分な量を意味し、有効容量のレベルは、患者の疾患の種類、重症度、薬物の活性、薬物に対する敏感度、投与時間、投与経路及び排出の割合、治療期間、同時に用いられる薬物を含んだ要素及びその他医学分野によく知られた要素によって決定され得る。本発明による組成物は、個別治療剤で投与するか他の治療剤と併用して投与され得、従来の治療剤とは順次又は同時に投与され得、単一又は多重投与され得る。上記した要素を全て考慮して副作用なしに最小限の量で最大の効果を得ることができる量を投与することが重要であり、これは、当業者によって容易に決定され得る。 The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is a patient's disease. It can be determined by type, severity, activity of the drug, sensitivity to the drug, duration of administration, route of administration and rate of excretion, duration of treatment, factors containing the drug used simultaneously and other factors well known in the medical field. The compositions according to the invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. It is important to consider all of the above factors and administer an amount that will give the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

具体的に、本発明による薬学的組成物の有効量は、患者の年齢、性別、体重によって変わり得、投与経路、肥満の重症度、性別、体重、年齢などによって増減され得る。 Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, gender, body weight of the patient, and may be increased or decreased depending on the route of administration, severity of obesity, sex, body weight, age and the like.

本発明の吸入剤組成物で有効成分を吸入剤にそのまま添加するか他の成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的(予防又は治療用)によって適切に決定され得る。 In the inhaler composition of the present invention, the active ingredient may be added to the inhalant as it is or used in combination with other ingredients, or may be appropriately used by a usual method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (preventive or therapeutic).

本発明の食品組成物は、健康機能食品組成物を含む。本発明による食品組成物は、有効成分を食品にそのまま添加するか他の食品又は食品成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的(予防又は改善用)によって適切に決定され得る。一般的に、食品又は飲料の製造時に、本発明の組成物は、原料に対して15重量%以下、好ましくは、10重量%以下の量で添加される。しかし、健康及び衛生を目的とするか又は健康調節を目的とする長期間の摂取の場合には、前記量は前記範囲以下であってもよい。 The food composition of the present invention includes a health functional food composition. The food composition according to the present invention may be added to the food as it is or used in combination with other foods or food ingredients, or may be appropriately used by a usual method. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention or improvement). Generally, during the production of foods or beverages, the compositions of the present invention are added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials. However, in the case of long-term ingestion for the purpose of health and hygiene or for the purpose of health regulation, the amount may be less than or equal to the above range.

本発明の食品組成物は、指示された割合で必須成分として前記有効成分を含有すること以外に他の成分には特別な制限がなく、通常の飲料のように多様な香味剤又は天然炭水化物などを追加成分として含有できる。上述した天然炭水化物の例は、モノサッカライド、例えば、ブドウ糖、果糖など;ジサッカライド、例えば、マルトース、スクロースなど;及びポリサッカライド、例えば、デキストリン、シクロデキストリンなどのような通常的な糖、及びキシリトール、ソルビトール、エリスリトールなどの糖アルコールである。上述したもの以外の香味剤として天然香味剤(ソーマチン、ステビア抽出物、例えば、レバウディオサイドA、グリチルリチンなど)及び合成香味剤(サッカリン、アスパルテームなど)を有利に用いることができる。前記天然炭水化物の割合は、当業者の選択によって適切に決定され得る。 The food composition of the present invention has no special restrictions on other ingredients other than containing the active ingredient as an essential ingredient in an specified ratio, and has various flavoring agents or natural carbohydrates as in ordinary beverages. Can be contained as an additional ingredient. Examples of natural carbohydrates mentioned above include monosaccharides such as glucose, fructose; disaccharides such as maltose, sucrose; and polysaccharides such as dextrins, cyclodextrins and the like, and xylitol. It is a sugar alcohol such as sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrates can be appropriately determined by one of ordinary skill in the art.

上記外に、本発明の食品組成物は、様々な栄養剤、ビタミン、ミネラル(電解質)、合成風味剤及び天然風味剤などの風味剤、着色剤及び充填剤(チーズ、チョコレートなど)、ペクチン酸及びその塩、アルギン酸及びその塩、有機酸、保護性コロイド増粘剤、pH調節剤、安定化剤、防腐剤、グリセリン、アルコール、炭酸飲料に用いられる炭酸化剤などを含有することができる。このような成分は、独立的に又は組み合わせて用いることができる。このような添加剤の割合も当業者により適切に選択され得る。 In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), and pectic acids. And its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonizing agents used in carbonated beverages and the like. Such components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.

本発明の前記化粧料組成物は、コリネバクテリウム属細菌由来の小胞だけでなく、化粧料組成物に通常的に用いられる成分を含むことができ、例えば、抗酸化剤、安定化剤、溶解化剤、ビタミン、顔料及び香料のような通常的な補助剤、そして担体を含むことができる。 The cosmetic composition of the present invention may contain not only vesicles derived from Corynebacterium spp., But also components commonly used in cosmetic compositions, such as antioxidants, stabilizers, and the like. Common auxiliary agents such as solubilizers, vitamins, pigments and fragrances, and carriers can be included.

また、本発明の組成物は、コリネバクテリウム属細菌由来の小胞以外に、コリネバクテリウム属細菌由来の小胞と反応して皮膚保護効果を損傷させない限度で従来から用いられてきた有機紫外線遮断剤を混合して用いてもよい。前記有機紫外線遮断剤としては、グリセリルPABA、ドロメトリゾールトリシロキサン、ドロメトリゾール、ジガロイルトリオレエート、フェニルジベンズイミダゾールテトラスルホン酸2Na、イスコトリジノール、ジエチルアミノヒドロキシベンゾイルヘキシルベンゾエート、DEA-メトキシシンナメート、ローソンとジヒドロキシアセトンの混合物、メチレンビス-ベンゾトリアゾリルテトラメチルブチルフェノール、4-メチルベンジリデンカンファ、アントラニル酸メンチル、ベンゾフェノン-3(オキシベンゾン)、ベンゾフェノン-4、ベンゾフェノン-8(ジオキシベンゾン)、ブチルメトキシジベンゾイルメタン、ビスエチルヘキシルオキシフェノールメトキシフェニルトリアジン、シノキサート、エチルジヒドロキシプロピルPABA、オクトクリレン、エチルヘキシルジメチルPABA、エチルヘキシルメトキシシンナメート、エチルヘキシルサリシレート、エチルヘキシルトリアゾン、イソアミル-p-メトキシシンナメート、ポリシリコン-15(マロン酸ジメチコジエチルベンザル)、テレフタリデンジカンファスルホン酸及びその塩類、TEA-サリシレート及びアミノ安息香酸(PABA)からなる群より選択された1種以上を用いることができる。 Further, the composition of the present invention has been conventionally used for organic ultraviolet rays to the extent that it reacts with vesicles derived from Corynebacterium genus bacteria and does not damage the skin protective effect in addition to vesicles derived from Corynebacterium genus bacteria. A blocking agent may be mixed and used. Examples of the organic ultraviolet blocking agent include glyceryl PABA, drometrizoletrisiloxane, drometrizole, digalloyltrioleate, phenyldibenzimidazole tetrasulfonic acid 2Na, iscotridinol, diethylaminohydroxybenzoylhexylbenzoate, and DEA-methoxycinnamate. , Lawson and dihydroxyacetone mixture, Methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylenekanfa, menthyl anthranilate, benzophenone-3 (oxybenzon), benzophenone-4, benzophenone-8 (dioxybenzone), butylmethoxydi Benzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, cinoxate, ethyldihydroxypropyl PABA, octocrylene, ethylhexyldimethyl PABA, ethylhexylmethoxycinnamate, ethylhexyl salicylate, ethylhexyltriazone, isoamyl-p-methoxycinnamate, polysilicon-15 (malon) One or more selected from the group consisting of dimethylodiethylbenzalate), terephthalidenedidicanphasulfonic acid and its salts, TEA-salicylate and aminobenzoic acid (PABA) can be used.

本発明の化粧料組成物を添加できる製品としては、例えば、収れん化粧水、柔軟化粧水、栄養化粧水、各種クリーム、エッセンス、パック、ファンデーションなどのような化粧品類とクレンジング、洗顔剤、石鹸、トリートメント、美容液などがある。本発明の化粧料組成物の具体的な剤型としては、スキンローション、スキンソフナー、スキントナー、アストリンゼント、ローション、ミルクローション、モイスチャーローション、栄養ローション、マッサージクリーム、栄養クリーム、モイスチャークリーム、ハンドクリーム、エッセンス、栄養エッセンス、パック、石鹸、シャンプー、クレンジングフォーム、クレンジングローション、クレンジングクリーム、ボディーローション、ボディークレンザー、乳液、リップスティック、メイクアップベース、ファンデーション、プレスパウダー、ルーズパウダー、アイシャドウなどの剤型を含む。 Products to which the cosmetic composition of the present invention can be added include, for example, cosmetics such as astringent lotion, soft lotion, nutritional lotion, various creams, essences, packs, foundations, cleansing, facial cleansers, soaps, etc. There are treatments, beauty essences, etc. Specific dosage forms of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, moisture cream, hand cream, and the like. Essence, nutritional essence, pack, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, milky lotion, lipstick, makeup base, foundation, press powder, loose powder, eye shadow, etc. include.

以下、本発明の理解を助けるために好ましい実施例を提示する。しかしながら、下記の実施例は、本発明をより容易に理解するために提供されるものに過ぎず、下記実施例によって本発明の内容が限定されるものではない。 Hereinafter, preferred embodiments will be presented to aid in the understanding of the present invention. However, the following examples are provided only for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[実施例1.細菌及び細菌由来の小胞の体内吸収、分布及び排泄様相の分析]
細菌と細菌由来の小胞が粘膜を通じて全身的に吸収されるかを評価するために次のような方法で実験を行った。マウスの胃腸に蛍光で標識した細菌と細菌由来の小胞をそれぞれ50μgの用量で胃腸管に投与し、それぞれ0分、5分、3時間、6時間、12時間後に蛍光を測定した。マウスの全体イメージを観察した結果、図1aに示したように、細菌の場合には、全身的に吸収されなかったが、細菌由来の小胞の場合には、投与5分後に全身的に吸収され、投与3時間後には、膀胱で蛍光が濃く観察されて、小胞が泌尿器系に排泄されることが分かった。また、小胞は、投与12時間まで体内に存在することが分かった(図1a参照)。
[Example 1. Analysis of absorption, distribution and excretion aspects of bacteria and vesicles derived from bacteria]
Experiments were conducted by the following methods to evaluate whether bacteria and vesicles derived from bacteria are systemically absorbed through the mucosa. Bacteria labeled with fluorescence in the gastrointestinal tract of mice and vesicles derived from the bacteria were administered to the gastrointestinal tract at doses of 50 μg each, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours, respectively. As a result of observing the whole image of the mouse, as shown in FIG. 1a, it was not systemically absorbed in the case of bacteria, but in the case of vesicles derived from bacteria, it was systemically absorbed 5 minutes after administration. 3 hours after administration, a strong fluorescence was observed in the bladder, indicating that vesicles were excreted in the urinary system. It was also found that vesicles were present in the body up to 12 hours after administration (see FIG. 1a).

細菌と細菌由来の小胞が全身的に吸収された後、多くの臓器に浸潤した様相を評価するために、蛍光で標識した50μgの細菌と細菌由来の小胞を上記の方法のように投与した後、投与12時間後に血液、心臓、肺、肝臓、腎臓、脾臓、脂肪、筋肉を採取した。採取した組織で蛍光を観察した結果、図1bに示したように、細菌由来の小胞が血液、心臓、肺、肝臓、腎臓、脾臓、脂肪、筋肉に分布したが、細菌は吸収されないことが分かった(図1b参照)。 After systemic absorption of bacteria and bacterial vesicles, 50 μg of fluorescently labeled bacteria and bacterial vesicles were administered as described above to assess the appearance of invasion into many organs. Then, 12 hours after administration, blood, heart, lung, liver, kidney, spleen, fat, and muscle were collected. As a result of observing fluorescence in the collected tissue, as shown in FIG. 1b, bacterial vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, and muscle, but the bacteria were not absorbed. Okay (see Figure 1b).

[実施例2.細菌及び細菌由来の小胞の粘膜防御膜浸透有無の評価]
細菌と細菌由来の小胞が粘膜防御膜を通過して上皮組織に浸潤するかを評価するために、細菌と細菌由来の小胞を腸に直接投与した後、免疫組織染色(Immunohistochemistry)方法で粘膜防御膜を通過して上皮組織への浸潤を評価した。粘膜組織で細菌と小胞の存在を評価するために、細菌と小胞に対する抗体を作製してGFP(Green fluorescent protein)をつけて用い、DAPI(4,6-diamidino 2-phenylindole)染色をした後、顕微鏡で観察した。
[Example 2. Evaluation of the presence or absence of permeation of the mucosal protective membrane of bacteria and vesicles derived from bacteria]
In order to evaluate whether bacteria and vesicles derived from bacteria pass through the mucosal defense membrane and invade epithelial tissue, vesicles derived from bacteria and bacteria are directly administered to the intestine and then immunohistochemistry is used. Invasion into epithelial tissue through the mucosal defense membrane was evaluated. In order to evaluate the presence of bacteria and vesicles in mucosal tissue, antibodies against the bacteria and vesicles were prepared and used with GFP (Green Fluorescent protein) and stained with DAPI (4,6-diamidino 2-phenylindole). Later, it was observed under a microscope.

その結果、細菌の場合には、粘膜防御膜を通過しなかった一方、細菌由来の小胞は、粘膜で通過して上皮組織に浸潤することを確認した(図2参照)。 As a result, it was confirmed that in the case of bacteria, the vesicles derived from the bacteria did not pass through the mucosal protective membrane, while the vesicles derived from the bacteria passed through the mucosa and infiltrated the epithelial tissue (see FIG. 2).

[実施例3.臨床サンプルに存在する細菌由来の小胞のメタゲノム分析]
血液又は鼻粘膜組織などを先に10mlチューブに入れ、遠心分離機(3,500×g、10min、4℃)で浮遊物を沈めた後、上澄液のみを新しい10mlチューブに移した。0.22μmフィルタを用いて細菌及び異物を除去した後、セントリプレップチューブ(centripreigugal filters 50kD)に移し、1500×g、4℃で15分間遠心分離して50kDより小さい物質は捨て、10mlまで濃縮させた。もう一度、0.22μmフィルタを用いてバクテリア及び異物を除去した後、Type 90tiローターで150,000×g、4℃で3時間の間超高速遠心分離を行い、上澄液を除去した後、固まったペレットを生理食塩水(Phosphate buffered saline、PBS)でとかした。
[Example 3. Metagenomic analysis of bacterial-derived vesicles present in clinical samples]
Blood or nasal mucosa was first placed in a 10 ml tube, the suspended matter was submerged in a centrifuge (3,500 × g, 10 min, 4 ° C.), and then only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign matter using a 0.22 μm filter, transfer to a centriplegic filter (50 kD), centrifuge at 1500 × g at 4 ° C for 15 minutes, discard substances smaller than 50 kD, and concentrate to 10 ml. rice field. Once again, after removing bacteria and foreign matter using a 0.22 μm filter, ultrafast centrifugation was performed at 150,000 × g with a Type 90ti rotor at 4 ° C. for 3 hours to remove the supernatant, and then solidified. The pellet was dissolved in Phosphate buffered saline (PBS).

上記方法で分離した小胞100μlを100℃でボイルして内部のDNAを脂質外に出るようにし、その後、氷で5分間冷やした。その後、残った浮遊物を除去するために、10,000×g、4℃で30分間遠心分離し、上澄液のみを集めた後、ナノドロップ(Nanodrop)を用いてDNA量を定量した。その後、前記抽出されたDNAに細菌由来のDNAが存在するかを確認するために、下記表1に示した16s rDNAプライマーでPCRを行って、前記抽出された遺伝子に細菌由来の遺伝子が存在することを確認した。 100 μl of the vesicles separated by the above method were boiled at 100 ° C. to allow the internal DNA to come out of the lipid, and then cooled with ice for 5 minutes. Then, in order to remove the remaining suspended matter, the mixture was centrifuged at 10,000 × g at 4 ° C. for 30 minutes, only the supernatant was collected, and then the amount of DNA was quantified using Nanodrop. Then, in order to confirm whether or not bacterial-derived DNA is present in the extracted DNA, PCR is performed with the 16s rDNA primers shown in Table 1 below, and the bacterial-derived gene is present in the extracted gene. It was confirmed.

Figure 2022512469000002
Figure 2022512469000002

上記方法で抽出したDNAを上記の16S rDNAプライマーを用いて増幅した後にシークエンシングを行い(Illumina MiSeq sequencer)、結果をSFF(Standard Flowgram Format)ファイルで出力し、GS FLXソフトウェア(v2.9)を用いてSFFファイルをsequenceファイル(.fasta)とnucleotide quality scoreファイルに変換した後、リードの信用度評価を確認し、window(20 bps)平均base call accuracyが99%未満(Phred score<20)である部分を除去した。OUT(Operational Taxonomy Unit)分析のために、UCLUSTとUSEARCHを用いてシーケンス類似度によってクラスタリングを行い、属(genus)は94%、科(family)は90%、目(order)は85%、網(class)は80%、門(phylum)は75%のシーケンス類似度を基準にクラスタリングし、各OTUの門(phylum)、網(class)、目(order)、科(family)、属(genus)レベルの分類を行い、BLASTNとGreenGenesの16S RNAシーケンスデータベース(108,453シーケンス)を用いて属レベルで97%以上のシーケンス類似度を有する細菌をプロファイリングした(QIIME)。 The DNA extracted by the above method is amplified using the above 16S rDNA primer and then sequenced (Illumina MiSeq sequencer), the result is output as an SFF (Standard Flowgram Form) file, and GS FLX software (v2.9) is used. After converting the SFF file into a sequence file (.fasta) and a nucleice quality score file using, the credit rating of the lead was confirmed, and the window (20 bps) average base call currency was less than 99% (Phred score <20). The part was removed. For OUT (Operational Taxonomy Unit) analysis, clustering was performed by sequence similarity using UCLUST and USEARCH, 94% for genus, 90% for family, 85% for order, and net. (Class) is 80%, phylum (phylum) is clustered based on the sequence similarity of 75%, and each OTU phylum (phylum), net (class), order (order), family (family), genus (genus). ) Level classification was performed and bacteria with a sequence similarity of 97% or higher at the genus level were profiled using the 16S RNA sequence database (108,453 sequences) of BLASTN and GreenGenes (QIIME).

[実施例4.肝硬変患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で肝硬変患者97人と年齢と性別をマッチングした正常ヒト171人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて肝硬変及び肝臓癌患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図3参照)。
[Example 4. Metagenomic analysis of bacterial vesicles in blood of patients with cirrhosis]
In the blood of 97 patients with liver cirrhosis and 171 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of patients with liver cirrhosis and liver cancer as compared with the blood of normal humans (see FIG. 3).

[実施例5.脳卒中患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で脳卒中患者79人と年齢と性別をマッチングした正常ヒト158人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて脳卒中患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図4参照)。
[Example 5. Metagenomic analysis of bacterial vesicles in the blood of stroke patients]
In the blood of 79 stroke patients and 158 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of stroke patients as compared with the blood of normal humans (see FIG. 4).

[実施例6.糖尿病患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で糖尿病患者81人と年齢と性別をマッチングした正常ヒト126人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて糖尿病患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図5参照)。
[Example 6. Metagenomic analysis of bacterial vesicles in the blood of diabetic patients]
In the blood of 81 diabetic patients and 126 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles present in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of diabetic patients as compared with the blood of normal humans (see FIG. 5).

[実施例7.喘息患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で喘息患者182人と年齢と性別をマッチングした正常ヒト180人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて喘息患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図6参照)。
[Example 7. Metagenomic analysis of bacterial vesicles in the blood of asthmatic patients]
In the blood of 182 asthma patients and 180 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of asthma patients as compared with the blood of normal humans (see FIG. 6).

[実施例8.アトピー性皮膚炎患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法でアトピー性皮膚炎患者42人と年齢と性別をマッチングした正常ヒト40人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べてアトピー性皮膚炎患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図7参照)。
[Example 8. Metagenome analysis of bacterial vesicles in the blood of patients with atopic dermatitis]
After extracting genes from vesicles present in the blood and performing metagenome analysis on the blood of 42 patients with atopic dermatitis and 40 normal humans whose age and gender were matched by the method of Example 3 above. , The distribution of vesicles derived from Corynebacterium spp. Was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of patients with atopic dermatitis as compared with the blood of normal humans (see FIG. 7).

[実施例9.鬱病患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で鬱病患者72人と年齢と性別をマッチングした正常ヒト80人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて鬱病患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図8参照)。
[Example 9. Metagenomic analysis of bacterial vesicles in the blood of depressed patients]
In the blood of 72 depressed patients and 80 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of depressed patients as compared with the blood of normal humans (see FIG. 8).

[実施例10.乳癌患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で乳癌患者102人と年齢と性別をマッチングした正常ヒト100人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて乳癌患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図9参照)。
[Example 10. Metagenomic analysis of bacterial vesicles in the blood of breast cancer patients]
In the blood of 102 breast cancer patients and 100 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynebacterium was performed. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of breast cancer patients as compared with the blood of normal humans (see FIG. 9).

[実施例11.認知症患者の血液で細菌由来の小胞のメタゲノム分析]
前記実施例3の方法で認知症患者73人と年齢と性別をマッチングした正常ヒト70人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて認知症患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した(図10参照)。
[Example 11. Metagenomic analysis of bacterial vesicles in the blood of dementia patients]
In the blood of 73 dementia patients and 70 normal humans whose age and sex were matched by the method of Example 3, genes were extracted from vesicles existing in the blood and metagenome analysis was performed, and then corynea. The distribution of vesicles derived from Corynebacterium was evaluated. As a result, it was confirmed that vesicles derived from Corynebacterium spp. Are significantly reduced in the blood of dementia patients as compared with the blood of normal humans (see FIG. 10).

[実施例12.鼻ポリープ患者及び正常対照群の鼻粘膜のメタゲノム分析を通じたコリネバクテリウム属細菌由来の小胞の浸潤様相]
前記実施例3の方法で鼻ポリープ患者(非アレルギー性患者43人及びアレルギー性患者45人)及び正常対照群39人の鼻粘膜で細菌由来の小胞の浸潤様相を評価するためにメタゲノム分析を行った。その結果、アレルギー性鼻ポリープ患者と非アレルギー性鼻ポリープ患者の鼻腔組織では、正常対照群に比べてコリネバクテリウム属細菌由来の小胞が有意に減少しており、鼻ポリープ患者でアレルギー性の有無とコリネバクテリウム属細菌由来の小胞の浸潤程度には差がなかった(図11参照)。
[Example 12. Infiltration aspect of vesicles derived from Corynebacterium spp. Through metagenome analysis of nasal mucosa in patients with nasal polyps and normal controls]
Metagenome analysis was performed by the method of Example 3 to evaluate the infiltration aspect of bacterial vesicles in the nasal mucosa of nasal polyp patients (43 non-allergic patients and 45 allergic patients) and 39 normal control groups. went. As a result, in the nasal tissues of patients with allergic nasal polyps and patients with non-allergic nasal polyps, vesicles derived from Corinebacterium spp. There was no difference between the presence or absence and the degree of infiltration of vesicles derived from Polynebacterium spp. (See FIG. 11).

[実施例13.コリネバクテリウム・グルタミクム培養液から小胞分離]
コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)菌株を培養した後、その小胞を分離して特性を分析した。まず、コリネバクテリウム・グルタミクム菌株を37℃の培養器で吸光度(OD 600)が1.0~1.5になるまでMRS(de Man-Rogosa and Sharpe)培地で培養した後、LB(Luria-Bertani)培地に継代培養(sub-culture)した。その後、菌株が含まれている培養液を回収して10,000×g、4℃で20分間遠心分離して菌体を除去し、0.22μmフィルタで濾過した。
[Example 13. Vesicle separation from Corynebacterium glutamicum culture medium]
After culturing a strain of Corynebacterium glutamicum, the vesicles were isolated and analyzed for their characteristics. First, the Corynebacterium glutamicum strain was cultured in an MRS (de Man-Logosa and Sharpe) medium in an incubator at 37 ° C. until the absorbance (OD 600) reached 1.0 to 1.5, and then LB (Luria-). Subculture (sub-culture) was carried out in Bertani) medium. Then, the culture broth containing the strain was collected, centrifuged at 10,000 × g at 4 ° C. for 20 minutes to remove the cells, and filtered through a 0.22 μm filter.

前記濾過した上澄液を100kDa Pellicon 2 Cassetteフィルタメンブレイン(Merck Millipore、US)でMasterFlex ポンプシステム(Cole-Parmer、US)を用いて精密濾過(microfiltration)を通じて50ml以下の体積で濃縮し、濃縮させた上澄液をもう一度0.22μmフィルタで濾過した。その後、BCA(Bicinchoninic acid)assayを用いてタンパク質を定量し、得られた小胞に対して下記実験を実施した。 The filtered supernatant is concentrated by microfiltration in a 100 kDa Cellicon 2 Cassette filter membrane (Merck Millipore, US) using a MasterFlex pump system (Cole-Parmer, US) to a volume of 50 ml or less. The supernatant was once again filtered through a 0.22 μm filter. Then, the protein was quantified using BCA (Bicinchoninic acid) assay, and the following experiment was carried out on the obtained vesicles.

[実施例14.コリネバクテリウム・グルタミクム由来の小胞の炎症誘発効果]
炎症細胞でコリネバクテリウム・グルタミクム由来の小胞(Corynebacterium glutamicum EV)の炎症メディエーター(IL-6、TNF-α)の分泌に対する影響を確認するために、マウスマクロファージ株であるRaw 264.7細胞にコリネバクテリウム・グルタミクム由来の小胞を多様な濃度(0.1、1、10μg/ml)で処理した後、細胞死滅とELISAを進行した。
[Example 14. Anti-inflammatory effect of vesicles derived from Corynebacterium glutamicum]
In order to confirm the effect of Corynebacterium glutamicum-derived vesicles (Corynebacterium glutamicum EV) on the secretion of inflammatory mediators (IL-6, TNF-α) in inflammatory cells, a mouse macrophage strain, Raw 264.7 cells, was used. After treatment of vesicles derived from Corynebacterium glutamicum at various concentrations (0.1, 1, 10 μg / ml), cell killing and ELISA proceeded.

より具体的に、48-well細胞培養プレート中に5×10個ずつ分注したRaw264.7細胞にDMEM(Dulbecco’s Minimum Essential Medium)無血清培地で希釈した多様な濃度のコリネバクテリウム・グルタミクム由来の小胞を処理して12時間の間培養した。その後、細胞培養液を1.5mlチューブに集めて3000×gで5分間遠心分離し、上澄液を集めて-80℃で保管した後、ELISAを進めた。 More specifically, various concentrations of corynebacterium diluted in DMEM (Dulvecco's Minimum Essential Medium) serum-free medium in Raw264.7 cells dispensed in 5 × 10 4 cells in a 48-well cell culture plate. Vesicles derived from Glutamicum were treated and cultured for 12 hours. Then, the cell culture solution was collected in a 1.5 ml tube and centrifuged at 3000 × g for 5 minutes, and the supernatant was collected and stored at −80 ° C., and then ELISA was proceeded.

ELISAを行うために、捕捉抗体をPBSに希釈させて96 wellのポリスチレンプレートに作用濃度に合わせて50μlずつ分注した後、4℃で一晩反応させた。その後、PBST(0.05% tween-20が入っているPBS)溶液100μlで3回ずつ洗った後、RD(1% BSAが入っているPBS)溶液100μlを分注して常温で1時間の間ブロッキングした。サンプル及びスタンダードを濃度に合わせて50μlずつ分注して常温で2時間の間反応させた後、PBST 100μlで3回洗った後、検出抗体をRDに希釈させて作用濃度に合わせて50μlずつ分注して常温で2時間の間反応させた。PBST 100μlで3回洗った後、Streptavidin-HRP(R&D system、USA)をRDに1/40で希釈させて50μlずつ分注して常温で20分間反応させた。最後に、PBST 100μlで3回洗った後、TMB(3,3’,5,5’-Tetramethylbenzidine)基質(SurModics、USA)50μlを分注して5分~20分後に発色が進行されたとき、1M硫酸溶液を50μlずつ分注して反応を止め、SpectraMax M3 microplate reader(Molecular Devices、USA)を用いて450nmで吸光度を測定した。 To perform ELISA, the capture antibody was diluted with PBS and dispensed into a 96-well polystyrene plate in 50 μl increments according to the concentration of action, followed by overnight reaction at 4 ° C. Then, wash 3 times with 100 μl of PBST (PBS containing 0.05% Tween-20) solution, and then dispense 100 μl of RD (PBS containing 1% BSA) solution for 1 hour at room temperature. Blocked for a while. After dispensing 50 μl of the sample and standard according to the concentration and reacting at room temperature for 2 hours, washing with 100 μl of PBST three times, diluting the detected antibody with RD and dividing by 50 μl according to the action concentration. It was poured and reacted at room temperature for 2 hours. After washing 3 times with 100 μl of PBST, Streptavidin-HRP (R & D system, USA) was diluted 1/40 with RD, dispensed in 50 μl portions, and reacted at room temperature for 20 minutes. Finally, after washing 3 times with 100 μl of PBST, 50 μl of TMB (3,3', 5,5'-Tetramethylbenzidine) substrate (SurModics, USA) was dispensed and color development progressed 5 to 20 minutes later. The reaction was stopped by dispensing 50 μl of a 1 M sulfuric acid solution at a time, and the absorbance was measured at 450 nm using a SpectraMax M3 microplate reader (Molecular Devices, USA).

その結果、図12に示したように、コリネバクテリウム・グルタミクム由来の小胞(CGT101)の処理による細胞死滅は観察されなかった(図12参照)。また、図13a及び図13bに示したように、炎症細胞での炎症メディエーターの分泌様相を評価した結果、陽性対照群である大腸菌由来の小胞(E.coli EV 1μg/ml)の処理時に比べてコリネバクテリウム・グルタミクム由来の小胞(CGT101)の処理時、IL-6(図13a)及びTNF-α(図13b)の分泌がより一層減少していることを確認した。 As a result, as shown in FIG. 12, no cell death was observed by the treatment of the vesicle (CGT101) derived from Corynebacterium glutamicum (see FIG. 12). In addition, as shown in FIGS. 13a and 13b, as a result of evaluating the secretory aspect of the inflammatory mediator in the inflammatory cells, compared with the treatment of vesicles derived from Escherichia coli (E. coli EV 1 μg / ml), which is a positive control group. It was confirmed that the secretion of IL-6 (FIG. 13a) and TNF-α (FIG. 13b) was further reduced during the treatment of vesicles (CGT101) derived from Escherichia coli glutamicum.

[実施例15.コリネバクテリウム・グルタミクム由来の小胞の抗炎症効果]
前記実施例14の結果を土台に、コリネバクテリウム・グルタミクム由来の小胞の抗炎症効果を評価するために、多様な濃度(0.1、1、10μg/ml)のコリネバクテリウム・グルタミクム由来の小胞(CGT101)をマウスマクロファージ株に12時間前処理した後、病原性原因因子である大腸菌由来の小胞1μg/mlを処理し、12時間後に炎症性サイトカインの分泌をELISAで測定した。
[Example 15. Anti-inflammatory effect of vesicles derived from Corynebacterium glutamicum]
Based on the results of Example 14, various concentrations (0.1, 1, 10 μg / ml) derived from Corynebacterium glutamicum were used to evaluate the anti-inflammatory effect of the vesicles derived from Corynebacterium glutamicum. (CGT101) was pretreated with a mouse macrophage strain for 12 hours, followed by treatment with 1 μg / ml of vesicles derived from Escherichia coli, which is a pathogenic causative factor, and 12 hours later, the secretion of inflammatory cytokines was measured by ELISA.

その結果、コリネバクテリウム・グルタミクム由来の小胞を前処理するとき、大腸菌由来の小胞の刺激による炎症細胞に分泌されるTNF-αの量が顕著に抑制されることを確認した(図14参照)。これは、病原性小胞である大腸菌由来の小胞のような炎症誘発因子により誘導される炎症反応をコリネバクテリウム・グルタミクム由来の小胞が効率的に抑制できることを意味する。 As a result, it was confirmed that when pretreatment of vesicles derived from Corynebacterium glutamicum, the amount of TNF-α secreted into inflammatory cells stimulated by vesicles derived from Escherichia coli was significantly suppressed (FIG. 14). reference). This means that vesicles derived from Corynebacterium glutamicum can efficiently suppress inflammatory reactions induced by pro-inflammatory factors such as vesicles derived from Escherichia coli, which are pathogenic vesicles.

上述した本発明の説明は例示のためのもので、本発明が属する技術分野において通常の知識を有した者は、本発明の技術的思想や必須的な特徴を変更しなくても他の具体的な形態に容易に変形が可能であることが理解できる。したがって、以上で記述した実施例は、全ての面で例示的なものであり、限定的ではないことで理解すべきである。 The above description of the present invention is for illustration purposes only, and a person who has ordinary knowledge in the technical field to which the present invention belongs may use other specific materials without changing the technical idea or essential features of the present invention. It can be understood that it can be easily transformed into a typical form. Therefore, it should be understood that the examples described above are exemplary in all respects and are not limiting.

本発明によるコリネバクテリウム属細菌由来の小胞は、粘膜の防御膜を通過して粘膜の上皮細胞に吸収されて全身的に分布し、腎臓、肝臓、肺を通じて体外に排泄されることが確認され、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者の血液又は鼻粘膜で有意に減少していることが確認され、病原性小胞によるTNF-αなどの炎症メディエーターの分泌を有意に抑制できるところ、炎症性疾患の予防又は治療用組成物に有用に用いられ得るという点で、産業的利用価値が大きいと予想される。 It was confirmed that the vesicles derived from the genus Corinebacterium according to the present invention pass through the protective membrane of the mucosa, are absorbed by the epithelial cells of the mucosa, are distributed systemically, and are excreted from the body through the kidneys, liver, and lungs. It was confirmed that there was a significant decrease in the blood or nasal mucosa of patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, and TNF-α due to pathogenic vesicles. Since it can significantly suppress the secretion of inflammatory mediators such as inflammatory mediators, it is expected to have great industrial utility value in that it can be usefully used in compositions for the prevention or treatment of inflammatory diseases.

Claims (17)

下記のステップを含む、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断のための情報提供方法:
(a)正常ヒト及び被検者のサンプルから分離した小胞からDNAを抽出するステップ;
(b)前記抽出したDNAに対して16S rDNAに存在する遺伝子配列に基づいて製作したプライマー対を用いてPCR(Polymerase Chain Reaction)を行った後、それぞれのPCR産物を収得するステップ;及び
(c)前記PCR産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム(Corynebacterium)属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された1つ以上の疾患であると判定するステップ。
Information method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, including the following steps:
(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corynebacterium spp. Is lower than that of normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia. And a step of determining one or more diseases selected from the group consisting of nasal polyps.
前記ステップ(a)でのサンプルが、血液、小便、大便、唾液又は鼻粘膜であることを特徴とする、請求項1に記載の情報提供方法。 The information providing method according to claim 1, wherein the sample in step (a) is blood, urine, stool, saliva, or nasal mucosa. コリネバクテリウム(Corynebacterium)属細菌由来の小胞を有効成分として含むことを特徴とする、炎症疾患の予防又は治療用薬学的組成物。 A pharmaceutical composition for the prevention or treatment of inflammatory diseases, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. 前記コリネバクテリウム属細菌由来の小胞が、コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)から分泌されることを特徴とする、請求項3に記載の炎症疾患の予防又は治療用薬学的組成物。 The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles derived from the genus Corynebacterium are secreted from Corynebacterium glutamicum. 前記小胞が、平均直径が10~200nmであることを特徴とする、請求項3に記載の炎症疾患の予防又は治療用薬学的組成物。 The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles have an average diameter of 10 to 200 nm. 前記小胞が、コリネバクテリウム属細菌から自然的又は人工的に分泌されることを特徴とする、請求項3に記載の炎症疾患の予防又は治療用薬学的組成物。 The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 3, wherein the vesicles are naturally or artificially secreted from a bacterium belonging to the genus Corynebacterium. 前記炎症疾患が、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であることを特徴とする、請求項3に記載の炎症疾患の予防又は治療用薬学的組成物。 3. The inflammatory disease is one or more selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A pharmaceutical composition for the prevention or treatment of inflammatory diseases according to. コリネバクテリウム(Corynebacterium)属細菌由来の小胞を有効成分として含むことを特徴とする、炎症疾患の予防又は改善用食品組成物。 A food composition for preventing or ameliorating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. 前記コリネバクテリウム属細菌由来の小胞が、コリネバクテリウム・グルタミクム(Corynebacterium glutamicum)から分泌されることを特徴とする、請求項8に記載の炎症疾患の予防又は改善用食品組成物。 The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles derived from the genus Corynebacterium are secreted from Corynebacterium glutamicum. 前記小胞が、平均直径が10~200nmであることを特徴とする、請求項8に記載の炎症疾患の予防又は改善用食品組成物。 The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles have an average diameter of 10 to 200 nm. 前記小胞が、コリネバクテリウム属細菌から自然的又は人工的に分泌されることを特徴とする、請求項8に記載の炎症疾患の予防又は改善用食品組成物。 The food composition for preventing or ameliorating an inflammatory disease according to claim 8, wherein the vesicles are naturally or artificially secreted from a bacterium belonging to the genus Corynebacterium. 前記炎症疾患が、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であることを特徴とする、請求項8に記載の炎症疾患の予防又は改善用食品組成物。 8. Claim 8 characterized in that the inflammatory disease is one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. A food composition for preventing or ameliorating an inflammatory disease according to. コリネバクテリウム(Corynebacterium)属細菌由来の小胞を有効成分として含むことを特徴とする、炎症疾患の予防又は改善用化粧料組成物。 A cosmetic composition for preventing or ameliorating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. コリネバクテリウム(Corynebacterium)属細菌由来の小胞を有効成分として含むことを特徴とする、炎症疾患の予防又は治療用吸入剤組成物。 An inhaler composition for preventing or treating an inflammatory disease, which comprises a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. 下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法:
(a)正常ヒト及び被検者のサンプルから分離した小胞からDNAを抽出するステップ;
(b)前記抽出したDNAに対して16S rDNAに存在する遺伝子配列に基づいて製作したプライマー対を用いてPCR(Polymerase Chain Reaction)を行った後、それぞれのPCR産物を収得するステップ;及び
(c)前記PCR産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム(Corynebacterium)属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。
Diagnostic method of one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps including the following steps:
(A) Step of extracting DNA from vesicles isolated from normal human and subject samples;
(B) After performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, the steps of obtaining each PCR product; and (c). ) When the content of vesicles derived from Corynebacterium spp. Is lower than that of normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia. And a step of determining one or more diseases selected from the group consisting of nasal polyps.
コリネバクテリウム(Corynebacterium)属細菌由来の小胞を有効成分として含む組成物を個体に投与するステップを含むことを特徴とする、炎症疾患の予防又は治療方法。 A method for preventing or treating an inflammatory disease, which comprises a step of administering to an individual a composition containing a vesicle derived from a bacterium belonging to the genus Corynebacterium as an active ingredient. コリネバクテリウム(Corynebacterium)属細菌由来の小胞の炎症疾患に用いられる薬剤を生産するための用途。 Uses for producing drugs used for inflammatory diseases of vesicles derived from Corynebacterium spp.
JP2021532852A 2018-12-10 2019-11-20 Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof Active JP7186470B2 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
KR10-2018-0158622 2018-12-10
KR20180158622 2018-12-10
KR10-2019-0132137 2019-10-23
KR1020190132137A KR102223406B1 (en) 2018-12-10 2019-10-23 Nanovesicles derived from Corynebacterium bacteria and Use thereof
PCT/KR2019/015901 WO2020122449A1 (en) 2018-12-10 2019-11-20 Nano-vesicle derived from corynebacterium sp. bacteria and use thereof

Publications (3)

Publication Number Publication Date
JP2022512469A true JP2022512469A (en) 2022-02-04
JPWO2020122449A5 JPWO2020122449A5 (en) 2022-04-20
JP7186470B2 JP7186470B2 (en) 2022-12-09

Family

ID=71075621

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2021532852A Active JP7186470B2 (en) 2018-12-10 2019-11-20 Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof

Country Status (2)

Country Link
JP (1) JP7186470B2 (en)
WO (1) WO2020122449A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013503858A (en) * 2009-09-04 2013-02-04 イオン メディックス インコーポレイテッド Gram-positive bacteria-derived extracellular vesicles and uses thereof
JP2013507354A (en) * 2009-10-08 2013-03-04 イオン メディックス インコーポレイテッド Composition comprising indoor air-derived extracellular vesicle and use thereof

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9201072B2 (en) * 2009-09-01 2015-12-01 Aeon Medix Inc. Gut flora-derived extracellular vesicles, and method for searching for a disease model, vaccine, and candidate drug and for diagnosis using the same
KR20110082480A (en) * 2010-01-11 2011-07-19 포항공과대학교 산학협력단 Composition comprising extracellular vesicles derived from mammals, and use thereof
KR101940426B1 (en) * 2016-12-28 2019-01-18 주식회사 엠디헬스케어 Method for diagnosis of colon tumor using analysis of bacteria metagenome
KR101944665B1 (en) * 2017-02-24 2019-02-01 주식회사 엠디헬스케어 Method for diagnosis of chronic obstructive airway disease using analysis of bacteria metagenome
KR102597602B1 (en) 2018-05-18 2023-11-02 삼성전자주식회사 thin film formation apparatus and thin film formation apparatus using the same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013503858A (en) * 2009-09-04 2013-02-04 イオン メディックス インコーポレイテッド Gram-positive bacteria-derived extracellular vesicles and uses thereof
JP2013507354A (en) * 2009-10-08 2013-03-04 イオン メディックス インコーポレイテッド Composition comprising indoor air-derived extracellular vesicle and use thereof

Also Published As

Publication number Publication date
WO2020122449A1 (en) 2020-06-18
JP7186470B2 (en) 2022-12-09

Similar Documents

Publication Publication Date Title
JP7300762B2 (en) Nanovesicles derived from bacteria belonging to the genus Lactobacillus and uses thereof
JP7132661B2 (en) Nanovesicles derived from bacteria of the genus Sphingomonas and uses thereof
JP6959665B2 (en) Nanovesicles derived from Proteus bacteria and their uses
KR20180018354A (en) Nanovesicles derived from Bacillus bacteria and Use thereof
JP7433665B2 (en) Nanovesicles derived from Micrococcus bacteria and their uses
JP2022046611A (en) Nanovesicles derived from bacteria of lactococcus genus and use thereof
JP7291424B2 (en) Deinococcus-derived nanovesicles and uses thereof
JP7186470B2 (en) Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof
KR102223406B1 (en) Nanovesicles derived from Corynebacterium bacteria and Use thereof
KR102124794B1 (en) Nanovesicles derived from Geobacillus bacteria and Use thereof
EP3910074A1 (en) Nanovesicles derived from bacteria of genus deinococcus, and use thereof
JP2022517962A (en) Nanovesicles derived from Rhodococcus bacteria and their uses
CN113286897A (en) Nanocysomes derived from Rhodococcus bacteria and uses thereof

Legal Events

Date Code Title Description
A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20210708

A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20210708

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20220412

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20220712

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20221011

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20221108

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20221121

R150 Certificate of patent or registration of utility model

Ref document number: 7186470

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150