KR20190106731A - Nanovesicles derived from Prevotella bacteria and Use thereof - Google Patents
Nanovesicles derived from Prevotella bacteria and Use thereof Download PDFInfo
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- KR20190106731A KR20190106731A KR1020190025020A KR20190025020A KR20190106731A KR 20190106731 A KR20190106731 A KR 20190106731A KR 1020190025020 A KR1020190025020 A KR 1020190025020A KR 20190025020 A KR20190025020 A KR 20190025020A KR 20190106731 A KR20190106731 A KR 20190106731A
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- vesicles
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- bacteria
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- prevotella
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Abstract
Description
본 발명은 프레보텔라 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 프레보텔라 속 세균에서 유래하는 나노소포를 이용한 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병 등의 진단방법, 및 상기 소포를 포함하는 상기 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다. The present invention relates to nano-vesicles derived from the bacteria of the genus Prebotella, and more specifically, to colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation using nano-vesicles derived from the genus Prebotella bacteria Or a diagnostic method for diabetes and the like, and a composition for preventing, ameliorating 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, acute infectious diseases, which were previously recognized as infectious diseases, have become less important, while chronic diseases with immune dysfunctions caused by incompatibility between humans and microbiomes determine the quality of life and human life. As a major disease, the disease pattern has changed. As the refractory chronic disease of the 21st century, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric disease are major diseases that determine human life and quality of life, which is a major problem in public health.
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. The microorganisms symbiotic to the human body reaches 100 trillion times more than human cells, and the number of genes of microorganisms is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) is a microbial community including microbes, archaea and eukarya that exist in a given settlement.
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. The symbiotic bacteria in the body and the bacteria present in the environment secrete nanometer-sized vesicles to exchange information such as genes, low molecular weight compounds, and proteins with other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through. If the bacteria are symbiotic bacteria, the mucosa cannot pass through the mucous membrane, but the bacteria-derived vesicles are 100 nanometers in size or less. It passes through epithelial cells through the mucous membrane and is absorbed by our body. Locally secreted bacterial-derived vesicles are absorbed through the epithelial cells of the mucosa to induce local inflammatory responses, as well as vesicles passing through the epithelial cells are systemically absorbed through the lymphatic vessels and distributed to each organ. Regulates immune and inflammatory responses For example, vesicles derived from pathogenic Gram-negative bacteria, such as Eshcherichia coli , locally cause colitis and, when absorbed into blood vessels, promote systemic inflammatory and blood coagulation through vascular endothelial inflammatory responses. In addition, when insulin is absorbed into muscle cells, it causes insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control the disease by controlling immune and metabolic abnormalities caused by pathogenic vesicles.
세균에서 유래하는 소포 등의 인자에 대한 면역반응은 인터루킨(Interleukin, 이하 IL)-17 사이토카인 분비를 특징으로 하는 Th17 면역반응이 발생하는데, 이는 세균 유래 소포에 노출 시 IL-6가 분비되고, 이는 Th17 면역반응을 유도한다. Th17 면역반응에 의한 염증은 호중구 침윤을 특징으로 하고, 염증이 발생하는 과정에서 호중구, 대식세포 등과 같은 염증세포에서 분비되는 종양괴사인자-알파(tumor necrosis factor-alpha, 이하 TNF-α)가 중요한 역할을 담당한다. The immune response to factors such as vesicles derived from bacteria causes a Th17 immune response characterized by the secretion of Interleukin (IL) -17 cytokines, which secrete IL-6 upon exposure to bacterial vesicles, This induces a Th17 immune response. Inflammation by the Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF-α), which is secreted from inflammatory cells such as neutrophils and macrophages, is important during inflammation. Play a role.
프레보텔라 속 세균은 구강 및 여성 질에 공생하는 그람음성세균으로서, 구강 및 호흡기 감염의 주요 원인균으로 알려져 있다. 아프리카와 유럽 어린이 대변에 대한 메타게놈 분석 결과, 아프리카 어린이 대변의 53%가 프레보텔라 속 세균이었던 반면, 유럽 어린이 대변에선 관찰되지 않았고, 이러한 연구결과로부터 프로포텔라 속 세균의 분포가 고섬유, 고탄수화물 식이와 밀접한 관련이 있음을 알 수 있다. 프로보텔라 속 세균 중에서 프레보텔라 인터메디아(Prevotella intermedia) 균은 스테로이드호르몬을 성장인자로 사용하기 때문에, 임신 중의 여성의 질에 상기 균의 분포가 증가한다고 알려져 있다. 그러나 아직까지 프레보텔라 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 of the genus Prebotella are Gram-negative bacteria symbiotic to the oral cavity and female vagina, and are known as the main cause of oral and respiratory infections. A metagenome analysis of feces in African and European children found that 53% of African feces were bacterial in the prebotella, but not in the feces of European children. It can be seen that it is closely related to the high carbohydrate diet. Among the bacteria of the genus Probotella, Prevotella intermedia ) bacteria are known to increase the distribution of these bacteria in the vagina of pregnant women because steroid hormones are used as growth factors. However, it has not been reported that the bacteria of the genus Prebotella secrete extracellular vesicles, and in particular, there have been no cases of application to the diagnosis and treatment of intractable diseases such as cancer or cardiovascular-brain diseases.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 메타게놈 분석을 통해 정상인에 비하여 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 당뇨병 환자 유래 샘플에서 프레보텔라 속 세균 유래 소포의 함량이 유의하게 감소되어 있음 확인하였다. 또한, 프레보텔라 속 세균에 속하는 프레보텔라 인터메디아 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 earnestly researched to solve the above-mentioned conventional problems. As a result of meta-genome analysis, the present inventors have compared colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and diabetes-derived samples. It was confirmed that the content of the vesicles derived from the genus Prebotella was significantly reduced. In addition, when the vesicles were isolated from the prebotella intermedibacteria belonging to the genus Prebotella, and treated with macrophages, it was confirmed that significantly inhibiting the secretion of IL-6 and TNF-α by pathogenic vesicles. The present invention has been completed.
이에, 본 발명은 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide a method for providing information for the diagnosis of colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes.
또한, 본 발명은 프레보텔라 속 세균 유래 소포를 유효성분으로 포함하는 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 예방, 개선 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다.The present invention also provides a composition for the prevention, improvement or treatment of colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes mellitus comprising a vesicle-derived vesicles of the genus Prebotella as an active ingredient. For other purposes.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 하기의 단계를 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 진단을 위한 정보제공 방법을 제공한다:In order to achieve the object of the present invention as described above, the present invention provides a method for providing information for the diagnosis of colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes mellitus to provide:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 프레보텔라(Prevotella)속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병으로 분류하는 단계.(c) colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, if the content of extracellular vesicles derived from Prevotella sp. Classify as diabetes.
또한, 본 발명은 하기의 단계를 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 진단 방법을 제공한다:The present invention also provides a method for diagnosing colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes, comprising the following steps:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 프레보텔라(Prevotella)속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병으로 판정하는 단계.(c) colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, if the content of extracellular vesicles derived from Prevotella sp. Determining diabetes.
본 발명의 일 구현예로, 상기 (a) 단계에서의 샘플은 혈액 또는 대변인 것일 수 있다. In one embodiment of the present invention, the sample in step (a) may be blood or feces.
본 발명의 다른 구현예로, 상기 (b) 단계에서의 프라이머쌍은 서열번호 1 및 서열번호 2의 프라이머 일 수 있다. In another embodiment of the present invention, the primer pair in step (b) may be a primer of SEQ ID NO: 1 and SEQ ID NO: 2.
또한, 본 발명은 프레보텔라 속 세균 유래 소포를 유효성분으로 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes mellitus, including the vesicles derived from the genus Prebotella as an active ingredient. .
또한, 본 발명은 프레보텔라 속 세균 유래 소포를 유효성분으로 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 예방 또는 개선용 식품 조성물을 제공한다. The present invention also provides a food composition for the prevention or improvement of colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes mellitus, including the vesicles derived from the genus Prebotella as an active ingredient.
또한, 본 발명은 프레보텔라 속 세균 유래 소포의, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 예방 또는 치료 용도를 제공한다. The present invention also provides a prophylactic bacteria-derived vesicle, colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes.
또한, 본 발명은 프레보텔라 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 예방 또는 치료 방법을 제공한다. In addition, the present invention comprises administering to the subject a pharmaceutical composition comprising a vesicle derived from the genus Prebotella as an active ingredient, colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or Provided is a method for preventing or treating diabetes.
본 발명의 일 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm인 것일 수 있다.In one embodiment of the present invention, the vesicles may have an average diameter of 10 to 200 nm.
본 발명의 다른 구현예로, 상기 소포는 프레보텔라 속 세균에서 자연적 또는 인공적으로 분비되는 것일 수 있다.In another embodiment of the present invention, the vesicles may be secreted naturally or artificially from bacteria of the genus Prebotella.
본 발명의 또 다른 구현예로, 상기 프레보텔라 속 세균 유래 소포는 프레보텔라 인터메디아 유래 소포일 수 있다.In another embodiment of the present invention, the vesicles derived from the genus Prebotella may be vesicles derived from Prebotella intermedia.
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 혈액 또는 대변에 존재하는 세균 유래 소포 메타게놈 분석을 통해 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 당뇨병 환자의 혈액 또는 대변에 존재하는 프레보텔라 속 세균 유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. 또한, 프레보텔라 속 세균의 한 종인 프레보텔라 인터메디아를 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 프레보텔라 속 세균 유래 소포는 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병에 대한 진단방법, 및 상기 질환에 대한 예방 개선 또는 치료용 조성물에 유용하게 이용될 수 있을 것으로 기대된다.The present inventors confirmed that intestinal bacteria are not absorbed into the body, but in the case of bacterial-derived vesicles, they are absorbed into the body through epithelial cells, distributed systemically, and excreted in vitro through the kidneys, liver, and lungs. Bacterial-derived vesicles in the stool are analyzed by metagenomic analysis of colonic, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and prevotelella vesicles in the blood or stool of diabetic patients. It was confirmed that significantly reduced compared to. In addition, the prevesella intermedia, a species of the genus Prebotella, was cultured in vitro to separate vesicles, and when administered to inflammatory cells in vitro, it was observed that the secretion of inflammatory mediators caused by pathogenic vesicles was significantly inhibited. , Bacterial-derived vesicles according to the present invention is colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or a diagnostic method for diabetes, and preventive improvement or treatment composition for the disease It is expected to be useful.
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 대장암 환자 및 정상인 대변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 3은 췌장암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 4는 담관암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 5는 난소암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 6은 방광암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 7은 심근경색 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 8은 심방세동 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 9는 당뇨병 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 프레보텔라 속 세균 유래 소포의 분포를 비교한 결과이다.
도 10은 프레보텔라 인터메디아 유래 소포의 항염증 및 면역조절 효과를 평가하기 위하여, 병원성 소포인 대장균 소포 (E. coli EV) 처리 전에 프레보텔라 인터메디아(Prevotella intermedia) 균 유래 소포를 전처리하여, 대장균 소포에 의한 염증매개체인 IL-6 및 TNF-α 분비에 미치는 영향을 평가한 결과이다.Figure 1a is a picture of the distribution of bacteria and vesicles by time after oral administration of bacteria and bacteria-derived vesicles (EV) to the mouse, Figure 1b is 12 hours after oral administration, blood, kidney Figure shows the distribution of bacteria, vesicles and vesicles in the body, liver and various organs.
Figure 2 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after performing a bacterial-derived vesicle metagenome analysis present in colorectal cancer patients and normal feces.
Figure 3 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after the analysis of bacteria-derived vesicles metagenome present in pancreatic cancer patients and normal blood.
Figure 4 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after the analysis of bacteria-derived vesicles metagenome present in patients with bile duct cancer.
Figure 5 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after performing a bacterial-derived vesicle metagenome analysis present in ovarian cancer patients and normal blood.
Figure 6 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after performing a bacterial-derived vesicle metagenome analysis present in bladder cancer patients and normal blood.
Figure 7 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after the analysis of bacteria-derived vesicles metagenome present in patients with myocardial infarction and normal blood.
8 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after analyzing the bacteria-derived vesicles metagenome present in atrial fibrillation patients and normal blood.
Figure 9 is a result of comparing the distribution of bacteria-derived vesicles of the genus Prebotella after the analysis of bacteria-derived vesicles metagenome present in diabetic patients and normal blood.
10 is a frame beams inter telra media to evaluate the anti-inflammatory and immunomodulatory effect of the resulting package, before the package pathogenic E. coli vesicles (E. coli EV) correction processing pre telra Inter Media (Prevotella intermedia )) The results were evaluated by pretreatment of vesicles derived from E. coli vesicles to secrete IL-6 and TNF-α, which are mediators of inflammatory mediators.
본 발명은 프레보텔라 속 세균 유래 소포 및 이의 용도에 관한 것이다. The present invention relates to vesicles derived from the genus Prebotella and their use.
본 발명자들은 메타게놈 분석을 통해 프레보텔라 속 세균 유래 소포가 정상인에 비하여 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 당뇨병 환자의 임상 샘플에 유의하게 감소되어 있음을 확인하여 질병을 진단할 수 있음을 확인하였다. 또한, 프레보텔라 속 세균에 속하는 프레보텔라 인터메디아로 부터 소포를 분리하고 특성을 분석한 결과, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병 등의 질환에 대한 예방, 개선 또는 치료용 조성물로 이용할 수 있음을 확인하였다.The inventors found that metagenome analysis showed that vesicles derived from the genus Prebotella were significantly reduced in clinical samples of colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and diabetic patients. Confirmation confirmed that the disease can be diagnosed. In addition, the vesicles were isolated and characterized from the prebotella intermedia, which belongs to the bacteria of the genus Prebotella, and analyzed for diseases such as colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes mellitus. It was confirmed that it can be used as a composition for the prevention, improvement or treatment.
이에, 본 발명은 하기의 단계를 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 진단을 위한 정보제공 방법을 제공한다:Accordingly, the present invention provides a method for providing information for diagnosing colon cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes, comprising the following steps:
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) 상기 추출한 DNA에 대하여 16S rDNA에 존재하는 유전자 서열에 기초하여 제작한 프라이머 쌍을 이용하여 PCR(Polymerase Chain Reaction)을 수행한 후, 각각의 PCR 산물을 수득하는 단계; 및(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 프레보텔라(Prevotella)속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병으로 분류하는 단계.(c) colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, if the content of extracellular vesicles derived from Prevotella sp. Classify as diabetes.
본 발명에서 사용되는 용어, “진단”이란 넓은 의미로는 환자의 병의 실태를 모든 면에 걸쳐서 판단하는 것을 의미한다. 판단의 내용은 병명, 병인, 병형, 경중, 병상의 상세한 양태, 합병증의 유무, 및 예후 등이다. 본 발명에서 진단은 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및/또는 당뇨병 등의 발병 여부 및 질환의 수준 등을 판단하는 것이다. As used herein, the term "diagnosis" in the broad sense means to determine the actual condition of the patient's disease in all aspects. The content of the judgment is the name of the disease, the etiology, the type of disease, the seriousness, the detailed mode of the condition, the presence or absence of complications, and the prognosis. Diagnosis in the present invention is to determine the incidence and level of disease, such as colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and / or diabetes.
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 프레보텔라 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.As used herein, the term "nanovesicle" or "vesicle" refers to a structure of nanoscale membranes secreted by various bacteria. Gram-negative bacteria-derived vesicles or outer membrane vesicles (OMVs) contain toxic proteins, bacterial DNA and RNA as well as lipopolysaccharides, and gram-positive bacteria-derived vesicles. In addition to proteins and nucleic acids, it also contains peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls. In the present invention, the nano-vesicles or vesicles are naturally secreted or artificially produced by the bacteria of the genus Prebotella, and have a spherical shape and have an average diameter of 10 to 200 nm.
본 발명에서 사용되는 용어, “메타게놈”이란 “군유전체”라고도 하며, 흙, 동물의 장 등 고립된 지역 내의 모든 바이러스, 세균, 곰팡이 등을 포함하는 유전체의 총합을 의미하는 것으로, 주로 배양이 되지 않는 미생물을 분석하기 위해서 서열분석기를 사용하여 한꺼번에 많은 미생물을 동정하는 것을 설명하는 유전체의 개념으로 쓰인다. 특히, 메타게놈은 한 종의 게놈, 유전체를 말하는 것이 아니라, 한 환경단위의 모든 종의 유전체로서 일종의 혼합유전체를 말한다. 이는 오믹스적으로 생물학이 발전하는 과정에서 한 종을 정의할 때 기능적으로 기존의 한 종뿐만 아니라, 다양한 종이 서로 상호작용하여 완전한 종을 만든다는 관점에서 나온 용어이다. 기술적으로는 빠른 서열분석법을 이용해서, 종에 관계없이 모든 DNA, RNA를 분석하여, 한 환경 내에서의 모든 종을 동정하고, 상호작용, 대사작용을 규명하는 기법의 대상이다.The term "metagenome" used in the present invention, also referred to as "gunoelectric", refers to the sum total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, mainly culture It is used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms that are not. In particular, the metagenome does not refer to one genome or genome, but to a kind of mixed dielectric as the genome of all species of one environmental unit. This is a term from the point of view of defining a species in the course of the evolution of biology in terms of functional species as well as various species that interact with each other to create a complete species. Technically, rapid sequencing is used to analyze all DNA and RNA, regardless of species, to identify all species in one environment, and to identify interactions and metabolism.
본 발명에 있어서, 상기 샘플은 혈액 또는 대변일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the sample may be blood or feces, but is not limited thereto.
본 발명의 다른 양태로서, 본 발명은 프레보텔라 속 세균 유래 소포를 유효성분으로 포함하는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병의 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. In another aspect of the present invention, the present invention comprises a vesicle from the genus Prebotella as an active ingredient, prevention, treatment or improvement of colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes It provides a composition for. The composition comprises a food composition and a pharmaceutical composition, in the present invention the food composition comprises a nutraceutical composition. The composition of the present invention may be a formulation of an oral nebulizer or inhalant.
본 발명에서 사용되는 용어, “예방”이란 본 발명에 따른 조성물의 투여에 의해 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및/또는 당뇨병 등을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.As used herein, the term "prevention" refers to inhibiting or delaying the onset of colon cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and / or diabetes by administration of a composition according to the present invention. It means all the acts to make.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 조성물의 투여에 의해 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및/또는 당뇨병 등에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. As used herein, the term "treatment" is improved or advantageously improved by the administration of the composition according to the present invention for colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and / or diabetes It means all the acts.
본 발명에서 사용되는 용어, “개선”이란 치료되는 상태와 관련된 파라미터, 예를 들면 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. As used herein, the term “improvement” means any action that at least reduces the parameters associated with the condition being treated, for example, the extent of symptoms.
상기 소포는 프레보텔라 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-fast centrifugation, autoclaving, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical degradation, chemical treatment, filter The separation can be carried out using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, it may further include a process for washing to remove impurities, concentration of the obtained vesicles and the like.
본 발명에 따른 약학적 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 상기 약학적으로 허용 가능한 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 사이클로덱스트린, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 리포좀 등을 포함하지만 이에 한정되지 않으며, 필요에 따라 항산화제, 완충액 등 다른 통상의 첨가제를 더 포함할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제, 윤활제 등을 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립, 또는 정제로 제제화할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제화에 관해서는 레밍턴의 문헌에 개시되어 있는 방법을 이용하여 각 성분에 따라 바람직하게 제제화할 수 있다. 본 발명의 약학적 조성물은 제형에 특별한 제한은 없으나 주사제, 흡입제, 피부 외용제, 또는 경구 섭취제 등으로 제제화할 수 있다. The pharmaceutical composition according to the invention may comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are conventionally used in the preparation, and include, but are not limited to, saline solution, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. If necessary, other conventional additives such as antioxidants and buffers may be further included. In addition, diluents, dispersants, surfactants, binders, lubricants and the like may be additionally added to formulate injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions and the like. Suitable pharmaceutically acceptable carriers and formulations can be preferably formulated according to the individual components using methods disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in formulation, but may be formulated as an injection, inhalant, external preparation for skin, oral ingestion, and the like.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여(예를 들어, 정맥 내, 피하, 피부, 비강, 기도에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, skin, nasal, airways) according to the desired method, and the dosage is determined by the condition and weight of the patient, disease Depending on the degree, drug form, route of administration, and time, it may be appropriately selected by those skilled in the art.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, 약학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/ 위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 따른 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the pharmaceutically effective amount means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment, and the effective dose level refers to the type of disease, the severity, the activity of the drug and the drug. Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 약학적 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.001 내지 150 mg, 바람직하게는 0.01 내지 100 mg을 매일 또는 격일 투여하거나 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary depending on the age, sex and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg daily or every other day, per kg of body weight Or divided into 1 to 3 times a day. However, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.
본 발명의 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명에 따른식품 조성물은 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에 본 발명의 조성물은 원료에 대하여 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.The food composition of the present invention includes a nutraceutical composition. The food composition according to the present invention may be used as it is, or may be used in combination with other foods or food ingredients, or may be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined according to the purpose of use (prevention or improvement). Generally, in the preparation of food or beverages the compositions of the invention are added in amounts of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of prolonged intake for health and hygiene purposes or health control purposes, the amount may be below the above range.
본 발명의 식품 조성물은 지시된 비율로 필수 성분으로서 상기 유효성분을 함유하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물, 예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The food composition of the present invention, in addition to containing the active ingredient as an essential ingredient in the indicated ratio, there are no particular restrictions on other ingredients, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The proportion of the natural carbohydrate can be appropriately determined by the choice of those skilled in the art.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다. In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. These components can be used independently or in combination. The proportion of such additives may also be appropriately selected by those skilled in the art.
본 발명의 일 실시예에서는 세균 및 세균 유래 소포를 마우스 경구로 투여하여 세균 및 소포의 체내 흡수, 분포, 및 배설 양상을 관찰한 바, 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 bacteria-derived vesicles orally administered to observe the body absorption, distribution, and excretion of the bacteria and vesicles in the body, in the case of bacteria is not absorbed through the intestinal membrane, the vesicles are administered 5 It was confirmed that it was absorbed within minutes and distributed systemically and excreted through the kidney, liver, and the like (see Example 1).
본 발명의 다른 실시예에서는, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 당뇨병 환자에 연령과 성별을 매칭한 정상인의 혈액 또는 대변에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 및 당뇨병 환자의 임상샘플에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3 내지 10 참조).In another embodiment of the present invention, bacterial metabolism using colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and vesicles isolated from blood or stool of a normal person whose age and sex were matched to a diabetic patient. Genomic analysis was performed. As a result, it was confirmed that the vesicle-derived bacteria of prebotella were significantly reduced in clinical samples of colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and diabetic patients compared to normal samples ( See examples 3 to 10).
본 발명의 또 다른 실시예에서는, 프레보텔라 인터메디아 균주를 배양하여 이로부터 분비된 소포가 면역조절 및 항염증 효과를 나타내는지를 평가하였는데, 다양한 농도의 프레보텔라 인터메디아 유래 소포를 대식세포에 처리한 후, 염증질환 원인인자인 대장균 유래 소포를 처리하여 염증매개체 분비를 평가한 결과, 대장균 유래 소포에 의한 TNF-α 분비를 프레보텔라 인터메디아 유래 소포가 효율적으로 억제함을 확인하였다(실시예 11 참조). In another embodiment of the present invention, the prebotella intermedia strains were cultured to evaluate whether the secreted vesicles exhibit immunomodulatory and anti-inflammatory effects. After treatment, the E. coli-derived vesicles, which are the causative agent of inflammatory diseases, were treated to evaluate the secretion of inflammatory mediators. As a result, it was confirmed that prebotella intermedia-derived vesicles effectively suppressed TNF-α secretion by E. coli-derived vesicles. See example 11.)
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1. 장내 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석Example 1 Analysis of Uptake, Distribution, and Excretion of Intestinal Bacteria and Bacterial-Derived Vesicles
장내 세균과 세균 유래 소포가 위장관을 통해 전신적으로 흡수되는 지를 평가하기 위하여 다음과 같은 방법으로 실험을 수행하였다. 마우스의 위장에 형광으로 표지한 장내세균과 장내 세균 유래 소포를 각각 50 μg의 용량으로 위장관으로 투여하고 0분, 5분, 3시간, 6시간, 12시간 후에 형광을 측정하였다. 마우스 전체 이미지를 관찰한 결과, 도 1a에 나타낸 바와 같이, 세균인 경우에는 전신적으로 흡수되지 않았지만, 세균 유래 소포인 경우에는, 투여 후 5분에 전신적으로 흡수되었고, 투여 3시간 후에는 방광에 형광이 진하게 관찰되어, 소포가 비뇨기계로 배설됨을 알 수 있었다. 또한, 소포는 투여 12시간까지 체내에 존재함을 알 수 있었다. In order to evaluate whether the intestinal bacteria and bacteria-derived vesicles are absorbed systemically through the gastrointestinal tract, experiments were performed as follows. Fluorescently labeled enterobacteriaceae and enteric bacteria-derived vesicles were administered to the gastrointestinal tract at doses of 50 μg, respectively, and the fluorescence was measured after 0, 5, 3, 6 and 12 hours. As a result of observing the whole image of the mouse, as shown in FIG. 1A, the bacteria were not absorbed systemically, but in the case of bacterial-derived vesicles, they were absorbed systemically 5 minutes after administration, and the bladder fluoresced 3 hours after administration. This was observed strongly, indicating that the vesicles were excreted by the urinary system. In addition, the vesicles were found to exist in the body until 12 hours of administration.
또한, 장내세균과 장내 세균 유래 소포가 전신적으로 흡수된 후, 여러 장기로 침윤된 양상을 평가하기 위하여, 형광으로 표지한 50 μg의 세균과 세균 유래 소포를 상기의 방법과 같이 투여한 후, 투여 12시간 후에 혈액, 심장, 폐, 간, 신장, 비장, 지방, 근육을 채취하였다. 채취한 조직에서 형광을 관찰한 결과, 도 1b에 나타낸 바와 같이, 세균 유래 소포가 혈액, 심장, 폐, 간, 비장, 지방, 근육, 신장에 분포하였으나, 세균은 흡수되지 않음을 알 수 있었다.In addition, after the intestinal bacteria and enteric bacteria-derived vesicles are absorbed systemically, 50 μg of fluorescently labeled bacteria and bacteria-derived vesicles are administered in the same manner as described above in order to evaluate the infiltration into various organs. After 12 hours, blood, heart, lungs, liver, kidneys, spleen, fat and muscle were collected. As a result of fluorescence observed in the collected tissue, as shown in Figure 1b, the bacteria-derived vesicles were distributed in the blood, heart, lung, liver, spleen, fat, muscle, kidney, it can be seen that the bacteria are not absorbed.
실시예 2. 임상샘플에서 세균 유래 소포 메타게놈 분석Example 2 Bacterial-derived Vesicular Metagenome Analysis in Clinical Samples
혈액, 대변 등의 임상샘플을 먼저 10 ml 튜브에 넣고 원심분리법(3,500 x g, 10min, 4℃)으로 부유물을 가라앉히고 상등액만을 새로운 10 ml 튜브에 옮겼다. 0.22㎛ 필터를 사용하여 세균 및 이물질을 제거한 후, 센트리프랩튜브 (centrifugal filters 50 kD)에 옮겨서 1500 x g, 4℃에서 15분간 원심분리하여 50 kD 보다 작은 물질은 버리며 10 ml 까지 농축 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 of blood, feces, etc. were first placed in 10 ml tubes and the suspension was allowed to settle by centrifugation (3,500 x g, 10 min, 4 ° C.) and only the supernatant was transferred to a new 10 ml tube. After removing the bacteria and foreign substances using a 0.22㎛ filter, and transferred to centrifugal filters (centrifugal filters 50 kD) and centrifuged at 1500 x g, 4 ℃ for 15 minutes to discard the material smaller than 50 kD concentrated to 10 ml. Once again, the bacteria and foreign substances were removed using a 0.22㎛ filter, and the supernatant was discarded using ultra-centrifugation for 3 hours at 150,000 xg and 4 ℃ using a Type 90ti rotor, and the lumped 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 let the internal DNA come out of the lipid and then cooled on ice for 5 minutes. And centrifuged at 10,000 x g, 4 ℃ 30 minutes to remove the remaining suspended solids and collected only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, PCR was performed with the 16s rDNA primer shown in Table 1 to confirm whether the bacteria-derived DNA exists in the extracted DNA, and it was confirmed that the bacteria-derived gene exists in the extracted gene.
상기 방법으로 추출한 DNA를 상기의 16S rDNA 프라이머를 사용하여 증폭을 한 다음 시퀀싱을 수행하고 (Illumina MiSeq sequencer), 결과를 Standard Flowgram Format (SFF) 파일로 출력하고 GS FLX software (v2.9)를 이용하여 SFF 파일을 sequence 파일 (.fasta)과 nucleotide qualityscore파일로 변환한 다음 리드의 신용도 평가를 확인하고, window (20 bps) 평균 base call accuracy가 99% 미만 (Phred score <20)인 부분을 제거하였다. Operational Taxonomy Unit (OTU) 분석을 위해서는 UCLUST와 USEARCH를 이용하여 시퀀스 유사도에 따라 클러스터링을 수행하고, 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).DNA extracted by the above method was amplified using the above 16S rDNA primers, followed by sequencing (Illumina MiSeq sequencer), and the results were outputted in a Standard Flowgram Format (SFF) file, using GS FLX software (v2.9). After converting the SFF file into a sequence file (.fasta) and a nucleotide qualityscore file, the credit rating of the lead was confirmed, and the window (20 bps) average base call accuracy was less than 99% (Phred score <20). . For operational taxonomy unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, genus 94%, family 90%, order 85%, class 80%, phylum 75% sequence similarity Clustering is based on the phylum, class, order, family, and genus levels of each OTU, and BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) is used to identify bacteria with greater than 97% sequence similarity at the genus level. Was profiled (QIIME).
실시예 3. 대장암환자의 대변 내 세균 유래 소포 메타게놈 분석Example 3 Analysis of Bacterial-Derived Vesicle Metagenome in Feces of Colon Cancer Patients
실시예 2의 방법으로 대장암환자 29명의 대변과, 나이와 성별을 매칭한 정상인 358명의 대변을 대상으로, 대변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 대변에 비하여 대장암환자의 대변에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 2 및 도 2 참조).In the method of Example 2, the genes were extracted from vesicles in the stool, and the stool of 29 colon cancer patients and 358 feces of normal age and sex were analyzed. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles of the genus Prebotella were significantly reduced in the stool of colorectal cancer patients compared to the normal stool (see Table 2 and Figure 2).
실시예 4. 췌장암환자의 혈액 내 세균 유래 소포 메타게놈 분석Example 4 Analysis of Bacterial-Derived Vesicular Metagenome in 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, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 췌장암환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 3 및 도 3 참조).In the method of Example 2, 176 bloods of pancreatic cancer patients and 271 bloods of normal people whose age and sex were matched were extracted from vesicles present in the blood and subjected to metagenome analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Prebotella were significantly reduced in the blood of pancreatic cancer patients compared to normal blood (see Table 3 and FIG. 3).
실시예 5. 담관암환자 혈액 세균 유래 소포 메타게놈 분석Example 5 Analysis of Vesicular Metagenome Derived from Bacterial Cancer Patients
실시예 2의 방법으로 담관암환자 79명의 혈액과, 나이와 성별을 매칭한 정상인 259명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 담관암환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 4 및 도 4 참조).The blood of 79 patients with cholangiocarcinoma patients and 259 normal blood patients whose ages and genders were matched by the method of Example 2 were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Prebotella was significantly reduced in the blood of bile duct cancer patients compared to normal blood (see Table 4 and FIG. 4).
실시예 6. 난소암환자 혈액 세균 유래 소포 메타게놈 분석Example 6 Analysis of Blood Bacteria Derived from Ovarian Cancer Patients
실시예 2의 방법으로 난소암환자 137명의 혈액과, 나이와 성별을 매칭한 정상인 139명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 난소암환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 5 및 도 5 참조).In the method of Example 2, 137 blood of ovarian cancer patients and 139 normal blood of age and sex matched were extracted from vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Prebotella 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 Blood Bacteria Derived from 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, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 방광암환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 6 및 도 6 참조).In the method of Example 2, the genes were extracted from the vesicles in the bladder cancer patients and 176 normal blood patients whose ages and genders were matched. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Prebotella were significantly reduced in the blood of bladder cancer patients compared to normal blood (see Table 6 and FIG. 6).
실시예 8. 심근경색환자 혈액 세균 유래 소포 메타게놈 분석Example 8. Vesicular Metagenome Analysis of Blood Bacteria from Myocardial Infarction Patients
실시예 2의 방법으로 심근경색 환자 57명의 혈액과, 나이와 성별을 매칭한 정상인 163명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심근경색환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 7 및 도 7 참조).The blood of 57 patients with myocardial infarction and 163 normal blood patients whose age and sex were matched were extracted from the vesicles in the blood and subjected to metagenomic analysis. The distribution of bacterial derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Prebotella were significantly reduced in the blood of myocardial infarction patients compared to normal blood (see Table 7 and FIG. 7).
실시예 9. 심방세동환자 혈액 세균 유래 소포 메타게놈 분석Example 9. Vesicular Metagenome Analysis of Blood Bacteria Derived from Atrial Fibrillation Patients
실시예 2의 방법으로 심방세동 환자 34명의 혈액과, 나이와 성별을 매칭한 정상인 62명의 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 심방세동환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 8 및 도 8 참조).Gene extraction from vesicles in the blood of 34 patients with atrial fibrillation and bleeding in 62 blood of normal and matched age and sex by the method of Example 2, followed by metagenome analysis, distribution of vesicles derived from bacteria of the genus Prebotella Was evaluated. As a result, it was confirmed that the vesicle-derived bacteria of the genus Prebotella were significantly reduced in blood of atrial fibrillation patients compared to normal blood (see Table 8 and FIG. 8).
실시예 10. 당뇨병환자 혈액 세균 유래 소포 메타게놈 분석Example 10 Analysis of Vesicular Metagenome from Blood Bacteria from Diabetic Patients
실시예 2의 방법으로 당뇨병환자 61명의 혈액과, 나이와 성별을 매칭한 정상인 122명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 프레보텔라 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 당뇨병환자의 혈액에 프레보텔라 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 9 및 도 9 참조).In the method of Example 2, the blood of 61 diabetic patients and 122 normal blood of age and sex matched were extracted from the vesicles in the blood and subjected to a metagenome analysis, followed by bacteria of the genus Prebotella. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the vesicles derived from the genus Prebotella were significantly reduced in the blood of diabetic patients compared to the normal blood (see Table 9 and FIG. 9).
실시예Example 11. 11. 프레보텔라Prevotela 인터메디아Intermedia 유래 소포의 항염증 효과 Anti-inflammatory Effects of Derived Vesicles
상기 실시예의 결과를 바탕으로, 프레보텔라 속 세균에 속하는 프레보텔라 인터메디아 균주를 장에서 분리한 후 이를 체외에서 배양하여 이의 소포를 분리하였다. 프레보텔라 인터메디아 균주를 37℃ 혐기성 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 example, the prebotella intermedia strains belonging to the genus Prebotella were isolated in the intestine and then cultured in vitro to separate the vesicles thereof. Prebotella intermedia strains were incubated in BHI (brain heart infusion) medium until absorbance (OD 600 ) was 1.0 to 1.5 in an anaerobic chamber at 37 ° C. and then sub-cultured. Thereafter, the culture supernatant containing no strain was recovered, centrifuged at 10,000 g, 4 ° C. for 15 minutes, filtered through a 0.45 μm filter, and the filtered supernatant was used as a 100 kDa hollow filter membrane using a QuixStand benchtop system (GE Healthcare, UK). Concentrated to 200 ml volume by ultrafiltration. Then, the concentrated supernatant was once again filtered with a 0.22 μm filter, and the filtered supernatant was ultracentrifuged at 150,000 g, 4 ° C. for 3 hours, and the pellet was suspended in DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solution (Axis-Shield PoC AS, Norway) .Optiprep solution was prepared using HEPES-buffered saline (20 mM HEPES) to prepare low density solution. , 150 mM NaCl, pH 7.4) was used. After centrifugation at 200,000 g, 4 ° C for 2 hours, each solution fractionated with the same volume of 1 ml from the upper layer was further subjected to ultracentrifugation for 15 hours at 150,000 g, 4 ° C. Thereafter, the protein was quantified by BCA assay, and the obtained vesicles were tested.
프레보텔라 인터메디아 유래 소포가 염증세포에서 염증매개체 분비에 대한 영향을 알아보기 위해, 마우스 대식세포주인 Raw 264.7 세포에 프레보텔라 인터메디아 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. To investigate the effect of prebotella intermediated vesicles on inflammatory mediator secretion in inflammatory cells, prebotella intermediated vesicles were injected into various concentrations (0.1, 1, 10 μg / ml) in raw 264.7 cells. E. coli ( E. coli) vesicles EV) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF-α, etc.). More specifically, 1 × 10 5 Raw 264.7 cells were dispensed into 24-well cell culture plates, and then cultured in DMEM complete medium for 24 hours. Then, the culture supernatant was collected in a 1.5 ml tube, centrifuged at 3000 g for 5 minutes, the supernatant was collected and stored at 4 ° C., followed by ELISA analysis.
그 결과, 도 10에 나타난 바와 같이 프레보텔라 인터메디아 유래 소포를 전 처리 한 경우, 대장균 유래 소포에 의한 TNF-α 분비가 현저히 억제됨을 확인하였다. 이는, 대장균 유래 소포와 같은 병원성 소포에 의해 유도되는 염증의 발생을 프레보텔라 인터메디아 유래 소포가 효율적으로 억제할 수 있음을 의미한다.As a result, as shown in Fig. 10, when pre-vesicles derived from Prebotella intermedia, it was confirmed that the secretion of TNF-α by E. coli-derived vesicles is significantly suppressed. This means that the prebotella intermedia-derived vesicles can effectively suppress the occurrence of inflammation induced by pathogenic vesicles such as E. coli-derived vesicles.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
<110> MD Healthcare Inc. <120> Nanovesicles derived from Prevotella bacteria and Use thereof <130> MP19-059 <150> KR 10-20180026145 <151> 2018-03-06 <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 Prevotella bacteria and Use <130> MP19-059 <150> KR 10-20180026145 <151> 2018-03-06 <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)
(a) 정상인 및 피검자 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;
(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 프레보텔라(Prevotella) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암, 췌장암, 담관암, 난소암, 방광암, 심근경색, 심방세동, 또는 당뇨병으로 분류하는 단계.
A method of providing information for diagnosing colorectal cancer, pancreatic cancer, bile duct cancer, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, or diabetes, comprising the following steps:
(a) extracting DNA from extracellular vesicles isolated from normal and subject samples;
(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; And
(c) colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, if the content of extracellular vesicles derived from bacteria of Prevotella is lower than that of normal people through quantitative analysis of the PCR products Classify as diabetes.
상기 (a) 단계에서의 샘플은 혈액 또는 대변인 것을 특징으로 하는, 정보제공 방법.
The method of claim 1,
And the sample in step (a) is blood or feces.
Pharmaceutical for preventing or treating at least one disease selected from the group consisting of colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and diabetes mellitus, comprising vesicles derived from bacteria of the genus Prevotella Composition.
상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 약학적 조성물.
The method of claim 3,
The vesicles are characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
상기 소포는 프레보텔라(Prevotella) 속 세균에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 약학적 조성물.
The method of claim 3,
The vesicles are characterized in that the natural or artificial secretion from the bacteria of the genus Prevotella , pharmaceutical composition.
상기 프레보텔라(Prevotella) 속 세균 유래 소포는 프레보텔라 인터메디아(Prevotella intermedia) 유래 소포인 것을 특징으로 하는, 약학적 조성물.
The method of claim 3,
The frame beam telra (Prevotella) derived from bacteria belonging to the genus parcel frame beams inter telra media (Prevotella intermedia), pharmaceutical composition, characterized in that the resulting package.
Food for preventing or ameliorating at least one disease selected from the group consisting of colorectal cancer, pancreatic cancer, cholangiocarcinoma, ovarian cancer, bladder cancer, myocardial infarction, atrial fibrillation, and diabetes mellitus, comprising vesicles derived from bacteria of the genus Prevotella Composition.
상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 식품 조성물.
The method of claim 7, wherein
The vesicles are characterized in that the average diameter of 10 to 200 nm, food composition.
상기 소포는 프레보텔라(Prevotella) 속 세균에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 식품 조성물.
The method of claim 7, wherein
The vesicles are characterized in that they are secreted naturally or artificially from bacteria of the genus Prevotella , food composition.
상기 프레보텔라(Prevotella) 속 세균 유래 소포는 프레보텔라 인터메디아(Prevotella intermedia) 유래 소포인 것을 특징으로 하는, 식품 조성물.The method of claim 7, wherein
The frame beam telra (Prevotella) derived from bacteria belonging to the genus parcel frame beams inter telra media (Prevotella intermedia), a food composition, characterized in that the resulting package.
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