KR102118201B1 - Nanovesicles derived from Methylobacterium bacteria and Use thereof - Google Patents
Nanovesicles derived from Methylobacterium bacteria and Use thereof Download PDFInfo
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- KR102118201B1 KR102118201B1 KR1020190024889A KR20190024889A KR102118201B1 KR 102118201 B1 KR102118201 B1 KR 102118201B1 KR 1020190024889 A KR1020190024889 A KR 1020190024889A KR 20190024889 A KR20190024889 A KR 20190024889A KR 102118201 B1 KR102118201 B1 KR 102118201B1
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Abstract
본 발명은 메틸로박테리움 속 세균 유래 소포 및 이의 용도에 관한 것으로, 본 발명자들은 정상인에 비하여 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자의 임상샘플에 상기 소포가 유의하게 감소되어 있고, 상기 균주에서 분리한 소포를 투여하였을 때 대장균 유래 소포와 같은 병원성 소포에 의한 염증매개체 분비를 현저히 억제함을 실험적으로 확인하였는바, 본 발명에 따른 메틸로박테리움 속 세균 유래 소포는 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 진단방법, 및 상기 질환에 대한 예방, 개선 또는 치료용 조성물을 개발하기 위한 목적으로 유용하게 이용될 수 있을 것이다. The present invention relates to bacterial-derived vesicles of the genus Methylobacterium and uses thereof, and the present inventors have pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes mellitus, and Parkinson's. It has been experimentally confirmed that the vesicles are significantly reduced in the clinical sample of the ill patient, and when the vesicles isolated from the strain are administered, the inflammatory mediator secretion by the pathogenic vesicles such as E. coli-derived vesicles is significantly suppressed. According to the germ-derived vesicles of the genus Methylobacterium, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Parkinson's disease diagnosis method, and prevention and improvement of the disease Or it may be useful for the purpose of developing a therapeutic composition.
Description
본 발명은 메틸로박테리움 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 메틸로박테리움 속 세균에서 유래하는 나노소포를 이용한 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병 등의 진단방법, 및 상기 소포를 포함하는 상기 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to nanovesicles derived from bacteria of the genus Methylobacteria and uses thereof, more specifically, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction using nanovesicles derived from bacteria of the genus Methylobacteria. , Cardiomyopathy, angina pectoris, diabetes, or Parkinson's disease, and the like, and a composition for preventing, improving or treating the disease, including the vesicle.
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 있다.In the 21st century, while the importance of acute infectious diseases, which were previously recognized as infectious diseases, has become less important, chronic diseases accompanied by immune dysfunction caused by mismatch between humans and microbiomes determine the quality of life and human life. The disease pattern has changed to a major disease. As a refractory chronic disease in the 21st century, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric disease are major diseases that determine human lifespan and quality of life, and have become a major problem in public health.
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. It is known that the microorganisms that symbiotic with the human body reach 100 trillion times and are 10 times more than human cells, and the number of genes in the microorganism is more than 100 times that of the human gene. Microbiota (microbiota) refers to a microbial community (microbial community), including bacteria, archae, and eukarya.
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. Bacteria that live in our bodies and bacteria that exist in the surrounding environment secrete nanometer-sized vesicles to exchange information such as genes, low-molecular compounds, and proteins into other cells. The mucous membrane forms a physical barrier that cannot pass particles over 200 nanometers (nm) in size, and in the case of bacteria that symbiotic with the mucous membrane, the mucosa cannot pass through the mucous membrane, but the bacterial-derived vesicles are less than 100 nanometers in size. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacterial-derived vesicles are absorbed through epithelial cells of the mucous membrane to induce a local inflammatory response. In addition, vesicles that have passed through the epithelial cells are absorbed systemically through the lymphatic vessels, distributed to each organ, and distributed organs. It regulates immune and inflammatory responses. For example, vesicles derived from pathogenic Gram-negative bacteria such as E. coli ( Eshcherichia coli ) locally develop colitis and, when absorbed into blood vessels, promote systemic inflammatory and blood coagulation through vascular endothelial inflammatory responses. In addition, insulin is absorbed by muscle cells, etc., which act, and causes insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control 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 bacterial-derived vesicles produces a Th17 immune response characterized by the secretion of interleukin-17 cytokines, which secrete IL-6 upon exposure to bacterial-derived vesicles, This induces a Th17 immune response. Inflammation due to Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF-α) secreted by inflammatory cells such as macrophages in the process of inflammation plays an important role. In charge.
메틸로박테리움 속 세균은 토양 및 물에 존재하는 그람음성세균으로서 이 중에서 메틸로박테리움 젓갈리(Methylobacterium jeotgali)는 우리나라 젓갈에서 처음으로 동정된 호기성 세균이다. 그러나 아직까지 메틸로박테리움 속 세균이 세포밖으로 소포를 분비한다는 사실이 보고되지 않았고, 특히 암 또는 심혈관-뇌질환 등과 같은 난치성 질환의 진단 및 치료에 응용한 사례는 보고된 바가 없다.The bacteria in the genus Methylobacterium are Gram-negative bacteria present in soil and water. Among them, Methylobacterium jeotgali ) is the first aerobic bacteria identified in Korean salted fish. However, it has not been reported that bacteria in the genus Methylobacterium secrete extracellular vesicles, and there have been no reports of application to the diagnosis and treatment of incurable diseases such as cancer or cardiovascular-brain disease.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 메타게놈 분석을 통해 정상인에 비하여 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자 유래 샘플에서 메틸로박테리움 속 세균 유래 소포의 함량이 유의하게 감소되어 있음 확인하였다. 또한, 메틸로박테리움 속 세균에 속하는 메틸로박테리움 젓갈리 균에서 소포를 분리하여 대식세포에 처리하였을 때, 병원성 소포에 의한 IL-6 및 TNF-α 분비를 현저히 억제함을 확인한 바, 이에 기초하여 본 발명을 완성하였다. The present inventors, as a result of diligent research to solve the conventional problems as described above, through metagenome analysis, compared to normal people, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, And it was confirmed that the content of bacterial-derived vesicles in the genus Mycobacterium was significantly reduced in samples derived from Parkinson's disease patients. In addition, it was confirmed that when the vesicles were separated from the bacteria Methylobacterium belonging to the bacterium belonging to the genus Methylobacterium and processed into macrophages, IL-6 and TNF-α secretion by pathogenic vesicles were significantly suppressed. The present invention was completed on the basis of this.
이에, 본 발명은 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide an information providing method for diagnosis of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Parkinson's disease.
또한, 본 발명은 메틸로박테리움 속 세균 유래 소포를 유효성분으로 포함하는 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방, 개선 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다. In addition, the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, or Parkinson's disease prevention, improvement, including bacterial-derived vesicles from the genus Methylobacterium as an active ingredient Another object is to provide a composition for 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 problems, and other problems that are 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, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공 방법을 제공한다:In order to achieve the object of the present invention as described above, the present invention comprises the following steps, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Parkinson's disease Provides methods for providing information for diagnosis:
(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 a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 메틸로박테리움(Methylobacterium) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병으로 분류하는 단계.(c) Pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease when the content of extracellular vesicles derived from bacteria of the genus Methylobacterium is lower than that of normal persons through quantitative analysis of the PCR products Classified as an angina pectoris, diabetes, or Parkinson's disease.
또한, 본 발명은 하기의 단계를 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 진단 방법을 제공한다:In addition, the present invention provides a method for diagnosing pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, or Parkinson's disease, 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 a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 메틸로박테리움(Methylobacterium) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병으로 판정하는 단계.(c) Pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease when the content of extracellular vesicles derived from bacteria of the genus Methylobacterium is lower than that of normal persons through quantitative analysis of the PCR products , Anomalous angina, diabetes, or Parkinson's disease.
본 발명의 일 구현예로, 상기 (a) 단계에서의 샘플은 혈액 또는 소변일 수 있다. In one embodiment of the present invention, the sample in step (a) may be blood or urine.
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 primers of SEQ ID NO: 1 and SEQ ID NO: 2.
또한, 본 발명은 메틸로박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes mellitus, or prevention of Parkinson's disease, comprising vesicles derived from bacteria of the genus Methylobacteria as an active ingredient, or Provided is a pharmaceutical composition for treatment.
또한, 본 발명은 메틸로박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방 또는 개선용 식품 조성물을 제공한다. In addition, the present invention is a pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, or Parkinson's disease, comprising vesicles derived from bacteria of the genus Methylobacterium as an active ingredient, or Provided is a food composition for improvement.
또한, 본 발명은 메틸로박테리움 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방 또는 치료 방법을 제공한다. In addition, the present invention comprises the step of administering a pharmaceutical composition comprising a bacterial-derived vesicle from the genus Methylobacteria as an active ingredient, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy It provides a method for preventing or treating angina, diabetes, or Parkinson's disease.
또한, 본 발명은 메틸로박테리움 속 세균 유래 소포의, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방 또는 치료 용도를 제공한다. In addition, the present invention provides a preventive or therapeutic use of bacterial-derived vesicles of the genus Methylobacterium, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, or Parkinson's disease. .
본 발명의 일 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm인 것일 수 있다. In one embodiment of the present invention, the vesicle may have an average diameter of 10 to 200 nm.
본 발명의 다른 구현예로, 상기 소포는 메틸로박테리움 속 세균에서 자연적 또는 인공적으로 분비되는 것일 수 있다. In another embodiment of the present invention, the vesicle may be naturally or artificially secreted from bacteria of the genus Methylobacteria.
본 발명의 또 다른 구현예로, 상기 메틸로박테리움 속 세균 유래 소포는 메틸로박테리움 젓갈리 유래 소포일 수 있다. In another embodiment of the present invention, the bacterial-derived vesicles of the genus Methylobacterium may be vesicles derived from methylobacterium chopsticks.
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 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자의 혈액 또는 소변에 존재하는 메틸로박테리움 속 세균 유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. 또한, 메틸로박테리움 속 세균의 한 종인 메틸로박테리움 젓갈리를 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는바, 본 발명에 따른 메틸로박테리움 속 세균 유래 소포는 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병에 대한 진단방법, 및 상기 질환에 대한 예방, 개선 또는 치료용 조성물에 유용하게 이용될 수 있을 것으로 기대된다.The present inventors confirmed that in the case of intestinal bacteria, they 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 outside the body through the kidneys, liver, and lungs. Analysis of bacterial-derived vesicle metagenome present in pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, and methylobacterium present in the blood or urine of Parkinson's patients It was confirmed that the vesicles from the genus bacteria were significantly reduced compared to the normal person. In addition, it was observed that methylobacterium chopsticks, a species of bacteria of the genus Methylobacterium, were cultured in vitro to separate vesicles, and when administered to inflammatory cells in vitro, they significantly inhibited the secretion of inflammatory mediators by pathogenic vesicles. As described above, the bacterial-derived vesicles of the genus Methylobacterium according to the present invention include pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Parkinson's disease diagnostic method, and the above It is expected to be useful in compositions for preventing, improving or treating diseases.
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 그림이다.
도 2는 췌장암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 3은 담관암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 4는 유방암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 5는 난소암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 6은 방광암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 7은 림프종 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 8은 심근경색 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 9는 심근병증 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 10은 이형협심증 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 11은 당뇨병 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 12는 파킨슨병 환자 및 정상인 소변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 비교한 결과이다.
도 13은 메틸로박테리움 젓갈리 유래 소포의 항염증 및 면역조절 효과를 평가하기 위하여, 병원성 소포인 대장균 소포 (E. coli EV) 처리 전에 메틸로박테리움균 유래 소포를 전처리하여, 대장균 소포에 의한 염증매개체인 IL-6 및 TNF-α 분비에 미치는 영향을 평가한 결과이다.1A is a photograph of bacteria and bacterial-derived vesicles (EVs) administered orally to mice, and photographs of distribution patterns of bacteria and vesicles over time. FIG. 1B shows blood and
Figure 2 is a result of comparing the distribution of bacteria-derived vesicles in the genus Mycobacterium after performing metagenome analysis of bacterial-derived vesicles present in pancreatic cancer patients and normal blood.
3 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacterial-derived vesicles present in patients with bile duct cancer and normal blood.
4 is a result of comparing the distribution of bacterial-derived vesicles in the genus Methylbacterium after performing a metagenome analysis of bacterial-derived vesicles in breast cancer patients and normal blood.
5 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacterial-derived vesicles present in ovarian cancer patients and normal blood.
6 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacterial-derived vesicles present in patients with bladder cancer and normal blood.
7 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing metagenome analysis of bacterial-derived vesicles present in lymphoma patients and normal blood.
Figure 8 is a result of comparing the distribution of bacteria-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacteria in myocardial infarction patients and normal blood.
9 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacterial-derived vesicles present in patients with cardiomyopathy and normal blood.
10 is a result of comparing the distribution of bacterial-derived vesicles in the genus Mycobacterium after performing metagenome analysis of bacterial-derived vesicles present in patients with angina pectoris and normal blood.
11 is a result of comparing the distribution of bacterial-derived vesicles in the genus Methylbacterium after performing metagenome analysis of bacterial-derived vesicles present in diabetic patients and normal blood.
Figure 12 is a result of comparing the distribution of bacteria-derived vesicles in the genus Mycobacterium after performing a metagenome analysis of bacteria in Parkinson's disease patients and normal urine.
13 is to evaluate the anti-inflammatory and immunomodulatory effects of methylobacterium chopped-derived vesicles, pre-treatment with methylobacterium-derived vesicles before treatment with E. coli EV, a pathogenic vesicle, to E. coli vesicles It is the result of evaluating the effect on the secretion of inflammatory mediators IL-6 and TNF-α.
본 발명은 메틸로박테리움 속 세균 유래 소포 및 이의 용도에 관한 것이다. The present invention relates to bacterial-derived vesicles of the genus Methylobacteria and uses thereof.
본 발명자들은 메타게놈 분석을 통해 메틸로박테리움 속 세균 유래 소포가 정상인에 비하여 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자의 임상 샘플에 유의하게 감소되어 있음을 확인하여 질병을 진단할 수 있음을 확인하였다. 또한, 메틸로박테리움 속 세균에 속하는 메틸로박테리움 젓갈리로부터 소포를 분리하고 특성을 분석한 결과, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 등의 질환에 대한 예방, 개선 또는 치료용 조성물로 이용할 수 있음을 확인하였다.According to the present inventors, metagenome analysis shows that the bacterial-derived vesicles of the genus Methylobacterium have pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, and Parkinson's disease patients. It was confirmed that the disease can be diagnosed by confirming that the sample was significantly reduced. In addition, as a result of separating and analyzing vesicles from methylobacterium chopsticks belonging to bacteria in the genus Methylobacteria, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes mellitus And, it was confirmed that it can be used as a composition for preventing, improving or treating diseases such as Parkinson's disease.
이에, 본 발명은 하기의 단계를 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 진단을 위한 정보제공 방법을 제공한다:Accordingly, the present invention provides a method of providing information for the diagnosis of pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Parkinson's disease, including 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 a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 메틸로박테리움(Methylobacterium) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병으로 분류하는 단계.(c) Pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease when the content of extracellular vesicles derived from bacteria of the genus Methylobacterium is lower than that of normal persons through quantitative analysis of the PCR products Classified as an angina pectoris, diabetes, or Parkinson's disease.
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 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및/또는 파킨슨병 등의 발병 여부 및 질환의 수준 등을 판단하는 것이다.As used in the present invention, the term “diagnosis” means broadly determining the condition of a patient's illness across all aspects. The content of the judgment is disease name, etiology, disease type, severity, detailed aspects of the bed, presence of complications, and prognosis. Diagnosis in the present invention is to determine whether pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, cardiomyopathy, angina pectoris, diabetes, and/or Parkinson's disease, and the like, and the level of the disease.
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 메틸로박테리움 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.As used in the present invention, the term "nanovesicle (Nanovesicle)" or "vesicle (Vesicle)" refers to a structure made of nano-sized membranes secreted by various bacteria. Gram-negative bacteria-derived vesicles, or outer membrane vesicles (OMVs), contain not only endotoxins but also toxic proteins and bacterial DNA and RNA, and gram-positive bacteria-derived vesicles In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of the cell wall of bacteria. In the present invention, nanovesicles or vesicles are naturally secreted or artificially produced by bacteria of the genus Methylobacteria, 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.As used in the present invention, the term “metagenome” is also called “military genome” and refers to the sum of genomes including all viruses, bacteria, and fungi in isolated areas such as soil and animal intestines. It is used as a concept of genome to identify many microorganisms at once using a sequencer to analyze microorganisms that are not. In particular, metagenome does not refer to a genome or genome of one species, but a genome of all species of one environmental unit. This is a term that comes from the viewpoint that when defining a species in the course of biological development, it is functionally not only one existing species, but also various species interacting with each other to make a complete species. Technically, using rapid sequencing, it analyzes all DNA and RNA regardless of species, and is a target for techniques to identify all species in one environment and to identify interactions and metabolism.
본 발명에 있어서, 상기 샘플은 혈액 또는 소변일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the sample may be blood or urine, but is not limited thereto.
본 발명의 다른 양태로서, 본 발명은 메틸로박테리움 속 세균 유래 소포를 유효성분으로 포함하는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 또는 파킨슨병의 예방, 치료 또는 개선용 조성물을 제공한다. 상기 조성물은 식품 조성물 및 약학적 조성물을 포함하며, 본 발명에서 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명의 조성물은 구강분무제 또는 흡입제의 제형일 수 있다. As another aspect of the present invention, the present invention comprises a bacterial-derived vesicle from the genus Methylobacteria, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, or Provided is a composition for preventing, treating or improving Parkinson's disease. The composition includes a food composition and a pharmaceutical composition, and in the present invention, the food composition includes a health functional food composition. The composition of the present invention may be a formulation of an oral spray or inhalant.
본 발명에서 사용되는 용어, “예방”이란 본 발명에 따른 조성물의 투여에 의해 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및/또는 파킨슨병 등을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term “prevention” as used in the present invention means pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes mellitus, and/or Parkinson's disease by administration of the composition according to the present invention. It means any action that suppresses or delays the onset.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 조성물의 투여에 의해 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및/또는 파킨슨병 등에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used in the present invention, the term "treatment" means pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes mellitus, and/or Parkinson's disease by administration of the composition according to the present invention. It means all the actions that the symptoms of Korea improve or change beneficially.
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" refers to any action that at least reduces the severity of symptoms associated with the condition being treated.
상기 소포는 메틸로박테리움 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 are centrifuged, ultra-high-speed centrifugation, high-pressure treatment, extrusion, ultrasonic decomposition, cell lysis, homogenization, freeze-thaw, electroporation, mechanical decomposition, chemical treatment, filter, etc. Filtration, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for removing 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 present invention may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc. If necessary, it may further contain other conventional additives such as antioxidants, buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, and the like can be additionally added to prepare formulations for injection, pills, capsules, granules, or tablets, such as aqueous solutions, suspensions, and emulsions. Regarding suitable pharmaceutically acceptable carriers and formulations, the formulations described in Remington's literature can be used to formulate according to each component. The pharmaceutical composition of the present invention is not particularly limited in the formulation, but can be formulated as an injection, an inhalant, an external preparation for skin, or an oral intake.
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 may be administered orally or parenterally (eg, intravenously, subcutaneously, skin, nasal cavity, and airways) according to a desired method, and the dosage is the patient's condition and weight, disease Depending on the degree, drug type, route of administration and time, it can 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 ratio of rational benefit/risk applicable to medical treatment, and the effective dose level is the type of the patient's disease, the severity, the activity of the drug, the drug Sensitivity to treatment, time of administration, route of administration and rate of excretion, duration of treatment, factors including co-drugs and other factors well known in the medical field. The composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal 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 patient's age, sex, and body weight, and is generally administered in an amount of 0.001 to 150 mg per kg of body weight, preferably 0.01 to 100 mg daily or every other day Alternatively, it may be administered in 1 to 3 times a day. However, since the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., 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 health functional food composition. The food composition according to the present invention may be added as an active ingredient to the food or used with other food or food ingredients, it may be appropriately used according to a conventional method. The mixing amount of the active ingredient can be appropriately determined according to its purpose of use (for prevention or improvement). Generally, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less with respect to the raw materials, in the manufacture of a food or beverage. However, in the case of long-term intake for health and hygiene purposes or for 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 is an essential ingredient in the indicated proportions, and other ingredients than the above-mentioned active ingredient are not particularly limited and may contain various flavoring agents or natural carbohydrates, etc., as additional ingredients, as in conventional beverages. Examples of the natural carbohydrates described above include monosaccharides, such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, etc.; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract, for example rebaudioside A, glycyrrhizine, 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), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and It may contain salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonic acid used in carbonated beverages, and the like. These ingredients can be used independently or in combination. The proportions of these additives can 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 bacterial-derived vesicles are administered orally to the mouse to evaluate the absorption, distribution, and excretion of the bacterial and vesicles in the body. It was confirmed that it is absorbed within and is distributed systemically, and is excreted through the kidney, liver, etc. (see Example 1).
본 발명의 다른 실시예에서는, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자에 연령과 성별을 매칭한 정상인의 혈액 또는 소변에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 췌장암, 담관암, 유방암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증, 당뇨병, 및 파킨슨병 환자의 임상샘플에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3 내지 13 참조).In another embodiment of the present invention, pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes mellitus, and Parkinson's disease are separated from blood or urine of normal people matching age and gender Bacterial metagenome analysis was performed using one vesicle. As a result, bacterial-derived vesicles from the genus Mycobacterium are significant in clinical samples of patients with pancreatic cancer, bile duct cancer, breast cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris, diabetes, and Parkinson's disease, compared to normal samples. It was confirmed to be reduced (see Examples 3 to 13).
본 발명의 또 다른 실시예에서는, 메틸로박테리움 젓갈리 균주를 배양하여 이로부터 분비된 소포가 면역조절 및 항염증 효과를 나타내는지를 평가하였는데, 다양한 농도의 메틸로박테리움 젓갈리 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 메틸로박테리움 젓갈리 유래 소포가 효율적으로 억제함을 확인하였다(실시예 14 참조).In another embodiment of the present invention, the methylobacterium chopstick strain was cultured to evaluate whether the vesicles secreted therefrom show immunomodulatory and anti-inflammatory effects, and the vesicles derived from the various concentrations of methylobacterium chopstick were treated. After treatment on phagocytic cells, the inflammatory mediator vesicles were treated to evaluate the secretion of inflammatory mediators, and as a result, IL-6 and TNF-α secretion by E. coli-derived vesicles was effectively generated by methylobacterium chopped vesicles. Inhibition was confirmed (see Example 14).
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 embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예][Example]
실시예 1. 장내 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석Example 1 Analysis of intestinal bacteria and bacteria-derived vesicles in the body, absorption, distribution, and excretion patterns
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. To evaluate whether intestinal bacteria and bacterial-derived vesicles are systemically absorbed through the gastrointestinal tract, experiments were conducted in the following manner. Fluorescence-labeled intestinal bacteria and intestinal bacteria-derived vesicles were administered to the gastrointestinal tract at a dose of 50 μg to the gastrointestinal tract, respectively, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours. As a result of observing the whole mouse image, as shown in Fig. 1A, in the case of bacteria, it was not absorbed systemically, but in the case of bacterial-derived vesicles, it was absorbed systemically 5 minutes after administration, and fluorescence in the bladder 3 hours after administration. It was observed that the vesicle was excreted by the urinary system. In addition, it was found that the vesicles remained in the body until 12 hours of administration.
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.After evaluating intestinal bacteria and intestinal bacteria-derived vesicles systemically, infiltrating into various organs, fluorescently labeled 50 μg of bacteria and bacterial-derived vesicles were administered as described above, followed by 12 hours of administration Later, blood, heart, lung, liver, kidney, spleen, fat, and muscle were collected. As a result of fluorescence observation in the collected tissues, as shown in FIG. 1B, it was found that bacterial-derived vesicles were distributed in blood, heart, lung, liver, spleen, fat, muscle, and kidney, but bacteria were not absorbed.
실시예 2. 임상샘플에서 세균 유래 소포 메타게놈 분석Example 2. Metagenome analysis of bacterial-derived vesicles in clinical samples
혈액, 소변 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 시켰다. 다시 한 번 0.22㎛ filter를 사용하여 박테리아 및 이물질을 제거한 후, Type 90ti 로터로 150,000 x g, 4℃에서 3시간동안 초고속원심분리방법을 사용하여 상등액을 버리고 덩어리진 pellet을 생리식염수(PBS)로 녹였다. Clinical samples such as blood and urine were first placed in a 10 ml tube, and the suspension was settled by centrifugation (3,500 x g, 10 min, 4°C), and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign matter using a 0.22㎛ filter, transferred to a centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes, discarding substances smaller than 50 kD and concentrating to 10 ml. Once again, the bacteria and foreign matter were removed using a 0.22㎛ filter, and the supernatant was discarded using a ultra-high-speed centrifugation method at 150,000 xg, 4℃ for 3 hours using a Type 90ti rotor, and the agglomerated pellet was dissolved in physiological saline (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 모았다. 그리고 Nanodrop을 이용하여 DNA 양을 정량하였다. 이후, 상기 추출된 DNA에 세균 유래 DNA가 존재하는지 확인하기 위하여 하기 표 1에 나타낸 16s rDNA primer로 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 allow the DNA inside to come out of the lipid, and then cooled on ice for 5 minutes. Then, to remove the remaining suspended solids, centrifugation was performed at 10,000 x g at 4°C for 30 minutes, and only the supernatant was collected. And the amount of DNA was quantified using Nanodrop. Subsequently, in order to confirm that the bacterial-derived DNA was present in the extracted DNA, PCR was performed with the 16s rDNA primer shown in Table 1 below to confirm that the bacterial-derived gene was present in the extracted gene.
16S rDNA
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 quality score파일로 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, genus는 94%, family는 90%, order는 85%, class는 80%, phylum은 75% 시퀀스 유사도를 기준으로 클러스터링을 하고 각 OTU의 phylum, class, order, family, 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).The DNA extracted by the above method is amplified using the 16S rDNA primer described above, followed by sequencing (Illumina MiSeq sequencer), the results are output to a Standard Flowgram Format (SFF) file, and GS FLX software (v2.9) is used. To convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file, and then check the readability of the read, and remove the part with a window (20 bps) average base call accuracy of less than 99% (Phred score <20). Did. For Operational Taxonomy Unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Bacteria having a sequence similarity of 97% or higher at the genus level using clustering, classification of phylum, class, order, family, and genus levels of each OTU, and 16B RNA sequence database (108,453 sequences) of BLASTN and GreenGenes Was profiled (QIIME).
실시예 3. 췌장암환자의 혈액 내 세균 유래 소포 메타게놈 분석Example 3. Metagenome analysis of bacterial-derived vesicles in the blood of pancreatic cancer patients
실시예 2의 방법으로 췌장암환자 176명의 혈액과, 나이와 성별을 매칭한 정상인 271명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 췌장암환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 2 및 도 2 참조).In the method of Example 2, the blood of 176 patients with pancreatic cancer and 271 normal people with matching age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 2 and FIG. 2).
실시예 4. 담관암환자 혈액 세균 유래 소포 메타게놈 분석Example 4. Analysis of vesicle metagenome from blood bacteria in patients with bile duct cancer
실시예 2의 방법으로 담관암환자 79명의 혈액과, 나이와 성별을 매칭한 정상인 259명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 담관암환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 3 및 도 3 참조).In the method of Example 2, the blood of 79 patients with bile duct cancer and the blood of 259 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of bile duct cancer patients compared to normal blood (see Table 3 and FIG. 3).
실시예 5. 유방암환자 혈액 세균 유래 소포 메타게놈 분석Example 5. Analysis of vesicle metagenome from blood bacteria in breast cancer patients
실시예 2의 방법으로 유방암환자 96명의 혈액과, 나이와 성별을 매칭한 정상인 192명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 유방암환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 4 및 도 4 참조).In the method of Example 2, the blood of 96 breast cancer patients and the blood of 192 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of breast cancer patients compared to normal blood (see Table 4 and FIG. 4).
실시예 6. 난소암환자 혈액 세균 유래 소포 메타게놈 분석Example 6 Analysis of vesicle metagenome from blood bacteria in patients with ovarian cancer
실시예 2의 방법으로 난소암환자 137명의 혈액과, 나이와 성별을 매칭한 정상인 139명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 난소암환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 5 및 도 5 참조).In the method of Example 2, the blood of 137 patients with ovarian cancer and the blood of 139 normal people who matched age and gender were extracted from the vesicles present in the blood, followed by metagenome analysis, and then methylobacterium. Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of ovarian cancer patients compared to normal blood (see Table 5 and FIG. 5).
실시예 7. 방광암환자 혈액 세균 유래 소포 메타게놈 분석Example 7. Analysis of vesicle metagenome from blood bacteria in bladder cancer patients
실시예 2의 방법으로 방광암환자 91명의 혈액과, 나이와 성별을 매칭한 정상인 176명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 방광암환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 6 및 도 6 참조).In the method of Example 2, the blood of 91 patients with bladder cancer and the blood of 176 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 6 and FIG. 6).
실시예 8. 림프종환자 혈액 세균 유래 소포 메타게놈 분석Example 8. Analysis of bacterial vesicle metagenome from lymphoma patients
실시예 2의 방법으로 림프종환자 63명의 혈액과, 나이와 성별을 매칭한 정상인 53명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 림프종환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 7 및 도 7 참조).In the method of Example 2, the blood of 63 lymphoma patients and the blood of 53 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of lymphoma patients compared to normal blood (see Table 7 and FIG. 7).
실시예 9. 심근경색환자 혈액 세균 유래 소포 메타게놈 분석Example 9. Analysis of bacterial vesicle metagenome from myocardial infarction patient
실시예 2의 방법으로 심근경색 환자 57명의 혈액과, 나이와 성별을 매칭한 정상인 163명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심근경색환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 8 및 도 8 참조).In the method of Example 2, blood from 57 patients with myocardial infarction and blood from 163 normal people who matched age and gender were extracted from the vesicles present in the blood, followed by metagenome analysis, and then methylobacterium. Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 8 and FIG. 8).
실시예 10. 심근병증환자 혈액 세균 유래 소포 메타게놈 분석Example 10. Analysis of bacterial vesicle metagenome from cardiomyopathy patients
실시예 2의 방법으로 심근병증 환자 72명의 혈액과, 나이와 성별을 매칭한 정상인 163명의 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심근병증환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 9 및 도 9 참조).The method of Example 2 extracts genes from the blood of 72 patients with cardiomyopathy, and vesicles present in the blood of 163 normal humans with matching age and gender, and then performs a metagenome analysis, followed by analysis of bacterial-derived vesicles in the genus Mycobacterium. Distribution was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of cardiomyopathy patients compared to normal blood (see Table 9 and FIG. 9).
실시예 11. 이형협심증환자 혈액 세균 유래 소포 메타게놈 분석Example 11. Analysis of vesicle metagenome derived from blood bacteria in angina pectoris patients
실시예 2의 방법으로 이형협심증 환자 80명의 혈액과, 나이와 성별을 매칭한 정상인 80명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 이형협심증환자의 혈액에 메틸로박테리움 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 pectoris and the blood of 80 normal people with matching age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed, followed by methylobacterium. Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of patients with angina pectoris compared to normal blood (see Table 10 and FIG. 10).
실시예 12. 당뇨병환자 혈액 세균 유래 소포 메타게놈 분석Example 12. Diabetic patient blood bacteria-derived vesicle metagenome analysis
실시예 2의 방법으로 당뇨병환자 61명의 혈액과, 나이와 성별을 매칭한 정상인 122명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 당뇨병환자의 혈액에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 11 및 도 11 참조).In the method of Example 2, the blood of 61 diabetic patients and the blood of 122 normal people who matched age and gender were extracted from the vesicles present in the blood, and then a metagenome analysis was performed. The distribution of bacterial-derived vesicles was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in the blood of diabetic patients compared to normal blood (see Table 11 and FIG. 11).
실시예 13. 파킨슨병환자 소변 세균 유래 소포 메타게놈 분석Example 13. Metagenome analysis of urine bacteria derived from Parkinson's disease
실시예 2의 방법으로 파킨슨병환자 39명의 소변과, 나이와 성별을 매칭한 정상인 76명의 소변을 대상으로, 소변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 메틸로박테리움 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 소변에 비하여 파킨슨병환자의 소변에 메틸로박테리움 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 12 및 도 12 참조).In the method of Example 2, the urine of 39 patients with Parkinson's disease and the normal urine of 76 who matched age and gender were extracted from the vesicles present in the urine, and then a metagenome analysis was performed, followed by methylobacterium. Distribution of vesicles from genus bacteria was evaluated. As a result, it was confirmed that the bacterial-derived vesicles were significantly reduced in urine of Parkinson's disease patients compared to normal urine (see Table 12 and FIG. 12).
실시예 14. 메틸로박테리움 젓갈리 유래 소포의 항염증 효과Example 14. Anti-inflammatory effect of vesicles derived from methylobacterium chopped
상기 실시예의 결과를 바탕으로, 메틸로박테리움 속 세균에 속하는 메틸로박테리움 젓갈리 균주를 배양한 후 이의 소포를 분리하였다. 메틸로박테리움 젓갈리 균주를 37℃ 호기성 챔버에서 흡광도(OD600)가 1.0 내지 1.5가 될 때까지 BHI(brain heart infusion) 배지에서 배양한 후 sub-culture 하였다. 이후 균주가 포함되어 있지 않은 배지 상등액을 회수하여 10,000 g, 4 ℃에서 15분 동안 원심분리하고 0.45 μm 필터에 거른 후 거른 상등액을 100 kDa hollow 필터 맴브레인으로 QuixStand benchtop system(GE Healthcare, UK)을 이용하여 ultrafiltration을 통해 200 ㎖ 부피로 농축하였다. 이후 농축시킨 상등액을 다시 한번 0.22 μm 필터로 필터링 하고, 걸러진 상등액을 150,000 g, 4 ℃에서 3시간 동안 초원심분리한 후 펠렛을 DPBS로 현탁하였다. 다음으로 10 %, 40 %, and 50 % 옵티프렙 용액(Axis-Shield PoC AS, Norway)을 이용해 밀도구배 원심분리를 수행하였고, 저밀도 용액 제조를 위해 옵티프렙 용액을 HEPES-buffered saline (20 mM HEPES, 150 mM NaCl, pH 7.4)에 희석하여 이용하였다. 200,000 g, 4 ℃ 조건으로 2시간 동안 원심분리를 수행한 후 윗층에서부터 1 ㎖의 동일한 볼륨으로 분획된 각 용액을 150,000 g, 4 ℃ 조건으로 3시간 동안 추가로 초원심분리를 실시하였다. 이후 BCA assay를 이용해 단백질을 정량하였고, 얻어진 소포에 대하여 실험을 실시하였다. Based on the results of the above examples, after culturing the methylobacterium chopstick strain belonging to the bacteria of the genus Methylobacterium, its vesicles were separated. The Methyl bacterium chopped strain was cultured in a brain heart infusion (BHI) medium until the absorbance (OD 600 ) was 1.0 to 1.5 in a 37°C aerobic chamber, and then sub-cultured. Subsequently, the culture medium supernatant that does not contain the strain was collected, centrifuged at 10,000 g, 4° C. for 15 minutes, filtered through a 0.45 μm filter, and the filtered supernatant was used as a 100 kDa hollow filter membrane using the QuixStand benchtop system (GE Healthcare, UK). And concentrated to 200 ml volume through ultrafiltration. Thereafter, the concentrated supernatant was filtered once again with a 0.22 μm filter, and the filtered supernatant was ultracentrifuged at 150,000 g and 4° C. for 3 hours, and then the pellet was suspended with DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solution (Axis-Shield PoC AS, Norway), and the Optiprep solution was HEPES-buffered saline (20 mM HEPES) , 150 mM NaCl, pH 7.4). After centrifugation was performed for 2 hours under conditions of 200,000 g and 4°C, each solution fractionated into the same volume of 1 ml from the upper layer was further ultracentrifuged for 3 hours under conditions of 150,000 g and 4°C. Subsequently, the protein was quantified using the BCA assay, and the obtained vesicle was tested.
메틸로박테리움 젓갈리 유래 소포가 염증세포에서 염증매개체 분비에 대한 영향을 알아보기 위해, 마우스 대식세포주인 Raw 264.7 세포에 메틸로박테리움 젓갈리 유래 소포를 다양한 농도(0.1, 1, 10 ㎍/㎖)로 처리한 후, 염증질환 병원성 소포인 대장균 유래 소포 (E. coli EV)를 처리하여 염증매개체 (IL-6, TNF-α 등)의 분비량을 측정하였다. 보다 구체적으로, Raw 264.7 세포를 1 x 105 개씩 24-well 세포 배양 플레이트에 분주한 후, 24시간 동안 DMEM 완전배지에서 배양시켰다. 이후, 배양 상층액을 1.5 ml 튜브에 모아 3000 g에서 5분간 원심분리하여 상층액을 모아 4 ℃에 보관해두었다가 ELISA 분석을 진행하였다. In order to investigate the effect of methylobacterium chopstick-derived vesicles on inflammatory mediator secretion from inflammatory cells, various concentrations (0.1, 1, 10 ㎍/) of methylobacterium chopstick-derived vesicles were added to mouse macrophages, Raw 264.7 cells. ㎖), followed by treatment with inflammatory disease pathogenic vesicles from E. coli ( E. coli EV) to measure the secretion of inflammatory mediators (IL-6, TNF-α, etc.). More specifically, after dispensing 1×10 5 Raw 264.7 cells into 24-well cell culture plates, the cells were cultured in DMEM complete medium for 24 hours. Thereafter, the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, and the supernatant was collected and stored at 4°C for ELISA analysis.
그 결과, 도 13에 나타난 바와 같이 메틸로박테리움 젓갈리 유래 소포를 전 처리 한 경우, 대장균 유래 소포에 의한 IL-6 및 TNF-α 분비가 현저히 억제됨을 확인하였다. 이는, 대장균 유래 소포와 같은 병원성 소포에 의해 유도되는 염증의 발생을 메틸로박테리움 젓갈리 유래 소포가 효율적으로 억제할 수 있음을 의미한다.As a result, as shown in Figure 13, when the pre-treatment of vesicles derived from Methyl bacterium chopped, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles is significantly inhibited. This means that vesicles derived from Methyl bacterium chopstick can effectively suppress the occurrence of inflammation caused 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 above description of the present invention is for illustration only, and a person having ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily modified to 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 illustrative in all respects and not restrictive.
<110> MD Healthcare Inc. <120> Nanovesicles derived from Methylobacterium bacteria and Use thereof <130> MP19-052 <150> KR 10-2018-0026036 <151> 2018-03-05 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 16S_V3_F <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 16S_V4_R <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55 <110> MD Healthcare Inc. <120> Nanovesicles derived from Methylobacterium bacteria and Use thereof <130> MP19-052 <150> KR 10-2018-0026036 <151> 2018-03-05 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> 16S_V3_F <400> 1 tcgtcggcag cgtcagatgt gtataagaga cagcctacgg gnggcwgcag 50 <210> 2 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> 16S_V4_R <400> 2 gtctcgtggg ctcggagatg tgtataagag acaggactac hvgggtatct aatcc 55
Claims (10)
(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
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 메틸로박테리움(Methylobacterium) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 하나 이상의 질환을 가지고 있을 것으로 예측하는 단계를 포함하는, 질환 발병 예측을 위한 정보제공방법으로서,
상기 (a) 단계에서의 샘플은 혈액 또는 소변이고,
상기 (a) 단계에서의 샘플이 혈액일 경우, 상기 질환은 췌장암, 담관암, 난소암, 방광암, 림프종, 심근경색, 심근병증, 이형협심증 및 당뇨병으로 이루어진 군으로부터 선택된 하나 이상의 질환이며,
상기 (a) 단계에서의 샘플이 소변일 경우, 상기 질환은 파킨슨병인 것을 특징으로 하는, 질환 발병 예측을 위한 정보제공방법.
(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on a gene sequence present in 16S rDNA, and then obtaining each PCR product; And
(c) through the quantitative analysis of the PCR product, predicting the onset of disease, comprising predicting that if the content of extracellular vesicles derived from bacteria in the genus Methylobacterium is low compared to a normal person, one or more diseases will occur. As a method for providing information,
The sample in step (a) is blood or urine,
When the sample in step (a) is blood, the disease is at least one disease selected from the group consisting of pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, lymphoma, myocardial infarction, myocardial disease, angina pectoris and diabetes,
When the sample in the step (a) is urine, characterized in that the disease is Parkinson's disease, information providing method for predicting disease development.
Methylobacterium jeotgali ( Methylobacterium jeotgali ) pharmaceutical composition for anti-inflammatory comprising a vesicle derived from.
상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 약학적 조성물.
According to claim 3,
The vesicle is characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
상기 소포는 메틸로박테리움 젓갈리(Methylobacterium jeotgali)에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 약학적 조성물.According to claim 3,
The vesicle is characterized in that it is naturally or artificially secreted from Methylobacterium jeotgali ( Methylobacterium jeotgali ), pharmaceutical composition.
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