KR102230479B1 - Nanovesicles derived from Turicibacter bacteria and Use thereof - Google Patents
Nanovesicles derived from Turicibacter bacteria and Use thereof Download PDFInfo
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- KR102230479B1 KR102230479B1 KR1020190021663A KR20190021663A KR102230479B1 KR 102230479 B1 KR102230479 B1 KR 102230479B1 KR 1020190021663 A KR1020190021663 A KR 1020190021663A KR 20190021663 A KR20190021663 A KR 20190021663A KR 102230479 B1 KR102230479 B1 KR 102230479B1
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Abstract
본 발명은 투리시박터 속 세균 유래 소포 및 이의 용도에 관한 것으로, 본 발명자들은 정상인에 비하여 대장암 및 폐암 환자의 임상샘플에서 상기 소포가 유의하게 감소되어 있었고, 상기 균주에서 분리한 소포를 투여하였을 때 대장균 유래 소포와 같은 병원성 소포에 의한 염증매개체 분비를 현저히 억제함을 실험적으로 확인하였는바, 본 발명에 따른 투리시박터 속 세균 유래 소포는 대장암 또는 폐암의 진단방법, 및 상기 질환에 대한 예방 또는 치료용 조성물을 개발하기 위한 목적으로 유용하게 이용될 수 있을 것이다. The present invention relates to vesicles derived from bacteria of the genus Turicibacter and their uses, and the present inventors have significantly reduced the vesicles in clinical samples of colon cancer and lung cancer patients compared to normal people, and administered vesicles isolated from the strain. It was experimentally confirmed that the secretion of inflammatory mediators by pathogenic vesicles such as E. coli-derived vesicles was significantly suppressed. Or it may be usefully used for the purpose of developing a therapeutic composition.
Description
본 발명은 투리시박터 속 세균 유래 나노소포 및 이의 용도에 관한 것으로, 보다 구체적으로 투리시박터 속 세균에서 유래하는 나노소포를 이용한 대장암 또는 폐암 등의 진단방법, 및 상기 소포를 포함하는 상기 질환의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a method for diagnosing colon cancer or lung cancer, such as colon cancer or lung cancer using nanovesicles derived from bacteria of the genus Turicibacter and its use, and more specifically, to a method for diagnosing colon cancer or lung cancer using nanovesicles derived from bacteria of the genus Turicibacter, and the disease including the vesicles It relates to a composition for the prevention, improvement or treatment of.
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. 특히 암 발생에는 원인인자에 의한 면역기능 이상과 이와 동반된 만성염증의 중요성이 부각되고 있다.As the 21st century enters, the importance of acute infectious diseases, previously recognized as infectious diseases, has become less important, while chronic diseases accompanied by immune dysfunction caused by the incongruity between humans and microbiomes determine the quality of life and human longevity. The disease pattern has changed as a major disease. As a refractory chronic disease in the 21st century, cancer, cardiovascular disease, chronic lung disease, metabolic disease, and neuro-psychiatric diseases are major diseases that determine human lifespan and quality of life, and are becoming a major problem for public health. In particular, the importance of immune dysfunction caused by causative factors and chronic inflammation associated with cancer is being emphasized.
한편, 인체에 공생하는 미생물은 100조에 이르러 인간 세포보다 10배 많으며, 미생물의 유전자수는 인간 유전자수의 100배가 넘는 것으로 알려지고 있다. 미생물총(microbiota 혹은 microbiome)은 주어진 거주지에 존재하는 진정세균(bacteria), 고세균(archaea), 진핵생물(eukarya)을 포함한 미생물 군집(microbial community)을 말한다. 우리 몸에 공생하는 세균 및 주변 환경에 존재하는 세균은 다른 세포로의 유전자, 저분자화합물, 단백질 등의 정보를 교환하기 위하여 나노미터 크기의 소포(vesicle)를 분비한다. 점막은 200 나노미터(nm) 크기 이상의 입자는 통과할 수 없는 물리적인 방어막을 형성하여 점막에 공생하는 세균인 경우에는 점막을 통과하지 못하지만, 세균 유래 소포는 크기가 100 나노미터 크기 이하라서 비교적 자유롭게 점막을 통하여 상피세포를 통과하여 우리 몸에 흡수된다. 국소적으로 분비된 세균 유래 소포는 점막의 상피세포를 통해 흡수되어 국소 염증반응을 유도할 뿐만 아니라, 상피세포를 통과한 소포는 림프관을 통해 전신적으로 흡수되어 각 장기로 분포하고, 분포된 장기에서 면역 및 염증반응을 조절한다. 예를 들어, 대장균(Eshcherichia coli)와 같은 병원성 그람음성세균에서 유래하는 소포는 국소적으로 대장염을 일으키고, 혈관으로 흡수된 경우에 혈관 내피세포 염증반응을 통해 전신적인 염증반응 및 혈액응고를 촉진시키고, 또한 인슐린이 작용하는 근육세포 등에 흡수되어선 인슐린저항성과 당뇨병을 유발한다. 반면, 유익한 세균에서 유래하는 소포는 병원성 소포에 의한 면역기능 및 대사기능 이상을 조절하여 질병을 조절할 수 있다. On the other hand, it is known that the number of microorganisms living symbiotically in the human body reaches 100 trillion, which is 10 times more than that of human cells, and the number of genes in microorganisms is more than 100 times the number of human genes. The microbiota (or microbiome) refers to a microbial community, including bacteria, archaea, and eukarya, present in a given habitat. Bacteria living in our body and bacteria existing in the surrounding environment secrete nanometer-sized vesicles to exchange information such as genes, small molecule compounds, and proteins to other cells. The mucous membrane forms a physical barrier through which particles larger than 200 nanometers (nm) cannot pass, and bacteria that coexist in the mucous membrane cannot pass through the mucous membrane. It passes through the 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 mucous membrane to induce a local inflammatory reaction, and vesicles that have passed 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, through vascular endothelial inflammatory reactions, systemic inflammatory reactions and blood coagulation are promoted. , In addition, insulin is absorbed into muscle cells that act, causing insulin resistance and diabetes. On the other hand, vesicles derived from beneficial bacteria can control disease by regulating immune and metabolic function 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 against factors such as vesicles derived from bacteria is a Th17 immune response characterized by the secretion of interleukin (IL)-17 cytokines, which is secreted by IL-6 when exposed to bacterial vesicles. This induces a Th17 immune response. Inflammation caused by Th17 immune response is characterized by neutrophil infiltration, and tumor necrosis factor-alpha (TNF-α) secreted by inflammatory cells such as macrophages plays an important role in the process of inflammation. In charge.
투리시박터 속 세균은 동물의 장에서 많이 발견되는 그람양성세균이다. 그러나 상기 세균의 임상적인 중요성은 알려지지 않았고, 특히 투리시박터 속 세균이 세포밖으로 소포를 분비한다는 사실이 보고되지 않았을 뿐만 아니라 암과 같은 난치성 질환의 진단 및 치료에 응용한 사례는 보고된 바가 없다.The bacteria of the genus Turicibacter are Gram-positive bacteria that are often found in the intestines of animals. However, the clinical importance of the bacterium is not known, and in particular, the fact that the bacteria of the genus Turicibacter secretes vesicles out of the cells has not been reported, and no cases have been reported that have been applied to the diagnosis and treatment of intractable diseases such as cancer.
본 발명자들은 상기와 같은 종래의 문제점을 해결하기 위해 예의 연구한 결과, 메타게놈 분석을 통해 정상인에 비하여 대장암 및 폐암 환자 유래 샘플에서 투리시박터 속 세균 유래 소포의 함량이 유의하게 감소되어 있음 확인하였다. 또한, 투리시박터 속 세균에 속하는 투리시박터 상구이니스 균에서 소포를 분리하여 대식세포에 처리하였을 때, 병원성 소포에 의한 IL-6 및 TNF-α 분비를 현저히 억제함을 확인한 바, 이에 기초하여 본 발명을 완성하였다.As a result of intensive research to solve the conventional problems as described above, the present inventors confirmed that the content of vesicles derived from bacteria in Turicibacter in samples derived from colon cancer and lung cancer patients was significantly reduced compared to normal subjects through metagenomic analysis. I did. In addition, it was confirmed that when vesicles were isolated from the bacteria of the genus Turicibacter, and treated with macrophages, the secretion of IL-6 and TNF-α by pathogenic vesicles was remarkably suppressed. The present invention has been completed.
이에, 본 발명은 대장암 또는 폐암의 진단을 위한 정보제공방법을 제공하는 것을 목적으로 한다.Accordingly, an object of the present invention is to provide a method of providing information for diagnosis of colon cancer or lung cancer.
또한, 본 발명은 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는 대장암 또는 폐암의 예방, 개선 또는 치료용 조성물을 제공하는 것을 다른 목적으로 한다.In addition, another object of the present invention is to provide a composition for preventing, improving or treating colon cancer or lung cancer comprising vesicles derived from bacteria of the genus Turicibacter as an active ingredient.
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 problems mentioned above, 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, 대장암 또는 폐암의 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 diagnosis of colon cancer or lung cancer, 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) using a primer pair prepared based on the gene sequence present in 16S rDNA with respect to the extracted DNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 투리시박터 (Turicibacter) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암 또는 폐암으로 분류하는 단계.(c) Classifying as colorectal cancer or lung cancer when the content of extracellular vesicles derived from bacteria of the genus Turicibacter is lower than that of a normal person through quantitative analysis of the PCR product.
또한 본 발명은 하기의 단계를 포함하는, 대장암 또는 폐암의 진단 방법을 제공한다:In addition, the present invention provides a method for diagnosing colon cancer or lung cancer, 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) using a primer pair prepared based on the gene sequence present in 16S rDNA with respect to the extracted DNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 투리시박터 (Turicibacter) 속 세균 유래 세포밖 소포의 함량이 낮을 경우 대장암 또는 폐암으로 판정하는 단계.(c) determining a colon cancer or lung cancer when the content of extracellular vesicles derived from bacteria of the genus Turicibacter is lower than that of a normal person through quantitative analysis of the PCR product.
본 발명의 일 구현예로, 상기 (a) 단계에서의 샘플은 혈액 또는 대변 일 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 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는, 대장암 또는 폐암의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating colon cancer or lung cancer, comprising as an active ingredient vesicles derived from bacteria of the genus Turicibacter.
또한, 본 발명은 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는, 대장암 또는 폐암의 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving colon cancer or lung cancer, comprising vesicles derived from bacteria of the genus Turicibacter as an active ingredient.
또한, 본 발명은 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는, 대장암 또는 폐암의 예방 또는 치료용 흡입제 조성물을 제공한다.In addition, the present invention provides an inhalant composition for preventing or treating colon cancer or lung cancer, comprising vesicles derived from bacteria of the genus Turicibacter as an active ingredient.
또한, 본 발명은 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는 약학적 조성물을 개체에 투여하는 단계를 포함하는, 대장암 또는 폐암의 예방 또는 치료 방법을 제공한다. In addition, the present invention provides a method for preventing or treating colon cancer or lung cancer, comprising administering to an individual a pharmaceutical composition containing vesicles derived from bacteria of the genus Turicibacter as an active ingredient.
또한, 본 발명은 투리시박터 속 세균 유래 소포의, 대장암 또는 폐암의 예방 또는 치료 용도를 제공한다. In addition, the present invention provides a use of vesicles derived from bacteria of the genus Turicibacter, for the prevention or treatment of colon cancer or lung cancer.
본 발명의 일 구현예로, 상기 소포는 평균 직경이 10 내지 200 nm 일 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 투리시박터 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 the bacteria of the genus Turicibacter.
본 발명의 다른 구현예로, 상기 투리시박터 속 세균 유래 소포는 투리시박터 상구이니스 유래 소포일 수 있다.In another embodiment of the present invention, the vesicles derived from bacteria of the genus Turis bacterium may be vesicles derived from Turis bacterium sangguinis.
본 발명자들은 장내 세균인 경우에는 체내에 흡수되지 않지만, 세균 유래 소포인 경우에는 상피세포를 통해 체내에 흡수되어, 전신적으로 분포하고, 신장, 간, 폐를 통해 체외로 배설됨을 확인하였고, 환자 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 통해 대장암 및 폐암 환자의 혈액 또는 대변에 존재하는 투리시박터 속 세균 유래 소포가 정상인에 비하여 유의하게 감소되어 있음을 확인하였다. 또한, 투리시박터 속 세균의 한 종인 투리시박터 상구이니스를 체외에서 배양하여 소포를 분리하여, 체외에서 염증세포에 투여하였을 때, 병원성 소포에 의한 염증매개체 분비를 유의하게 억제함을 관찰하였는 바, 본 발명에 따른 투리시박터 속 세균 유래 소포는 대장암 또는 폐암에 대한 진단방법, 및 상기 질환에 대한 예방, 개선 또는 치료용 조성물로 유용하게 이용될 수 있을 것으로 기대된다.The present inventors confirmed that intestinal bacteria are not absorbed into the body, but bacterial-derived vesicles are absorbed into the body through epithelial cells, distributed systemically, and excreted outside the body through the kidneys, liver, and lungs. Through metagenomic analysis of vesicles derived from bacteria present in the colon cancer and lung cancer patients, it was confirmed that vesicles derived from bacteria of the genus Turicibacter, present in the blood or feces, were significantly reduced compared to those of normal subjects. In addition, it was observed that when vesicles were separated by culturing Turicibacter Sangguinis, a species of bacteria of the genus Turicibacter in vitro, and administered to inflammatory cells in vitro, it was observed that the secretion of inflammatory mediators by pathogenic vesicles was significantly suppressed. , It is expected that the vesicle derived from bacteria of the genus Turicibacter according to the present invention can be usefully used as a diagnostic method for colon cancer or lung cancer, and a composition for preventing, improving or treating the disease.
도 1a는 마우스에 세균과 세균 유래 소포 (EV)를 구강으로 투여한 후, 시간별로 세균과 소포의 분포양상을 촬영한 사진이고, 도 1b는 구강으로 투여한 후 12시간째에, 혈액, 신장, 간, 및 여러 장기를 적출하여, 세균과 소포의 체내 분포양상을 평가한 결과이다.
도 2는 대장암 환자 및 정상인 대변에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 투리시박터 속 세균 유래 소포의 분포를 비교한 결과이다.
도 3은 폐암 환자 및 정상인 혈액에 존재하는 세균 유래 소포 메타게놈 분석을 실시한 후, 투리시박터 속 세균 유래 소포의 분포를 비교한 결과이다.
도 4는 투리시박터 상구이니스 유래 소포의 항염증 및 면역조절 효과를 평가하기 위하여, 병원성 소포인 대장균 소포 (E. coli EV) 처리 전에 투리시박터 상구이니스 유래 소포를 전처리하여, 대장균 소포에 의한 염증매개체인 IL-6 및 TNF-α 분비에 미치는 영향을 평가한 결과이다.Figure 1a is a photograph of the distribution pattern of bacteria and vesicles by time after oral administration of bacteria and vesicles derived from bacteria (EV) to a mouse, and Figure 1b is a photograph taken 12 hours after oral administration, blood, kidney , Liver, and various organs were excised, and the distribution pattern of bacteria and vesicles in the body was evaluated.
2 is a result of comparing the distribution of vesicles derived from bacteria in the genus Turicibacter after performing metagenomic analysis of vesicles derived from bacteria present in the stool of colon cancer patients and normal humans.
3 is a result of comparing the distribution of vesicles derived from bacteria in the genus Turicibacter after performing metagenome analysis of vesicles derived from bacteria present in blood of lung cancer patients and normal people.
Figure 4 is to evaluate the anti-inflammatory and immunomodulatory effects of vesicles derived from Turicibacter sangguinis, pretreatment of vesicles derived from Turicibacter sangguinis before treatment with Escherichia coli vesicle (E. coli EV), which is a pathogenic vesicle, by E. coli vesicles. This is the result of evaluating the effect on the secretion of IL-6 and TNF-α, which are inflammatory mediators.
본 발명은 투리시박터 속 세균 유래 소포 및 이의 용도에 관한 것이다. The present invention relates to vesicles derived from bacteria of the genus Turicibacter and to uses thereof.
본 발명자들은 메타게놈 분석을 통해 투리시박터 속 세균 유래 소포가 정상인에 비하여 대장암 및 폐암 환자의 임상 샘플에 유의하게 감소되어 있음을 확인하여 질병을 진단할 수 있음을 확인하였다. 또한, 투리시박터 속 세균에 속하는 투리시박터 상구이니스(Turicibacter sanguinis)로 부터 소포를 분리하고 특성을 분석한 결과, 대장암 및 폐암에 대한 예방, 개선 또는 치료용 조성물로 이용할 수 있음을 확인하였다.The present inventors confirmed that the disease can be diagnosed by confirming that the vesicles derived from bacteria in Turicibacter genus are significantly reduced in clinical samples of colon cancer and lung cancer patients compared to normal people through metagenomic analysis. In addition, as a result of separating and analyzing vesicles from Turicibacter sanguinis belonging to the bacteria of the genus Turicibacter, it was confirmed that it can be used as a composition for preventing, improving or treating colon cancer and lung cancer. .
이에, 본 발명은 하기의 단계를 포함하는, 대장암 또는 폐암의 진단을 위한 정보제공 방법을 제공한다:Accordingly, the present invention provides a method for providing information for diagnosis of colon cancer or lung cancer, 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) using a primer pair prepared based on the gene sequence present in 16S rDNA with respect to the extracted DNA, and then obtaining each PCR product; And
(c) 상기 PCR 산물의 정량분석을 통하여 정상인에 비하여 투리시박터 (Turicibacter) 속 세균 유래 소포의 함량이 낮을 경우 대장암 또는 폐암으로 분류하는 단계.(c) Classifying as colorectal cancer or lung cancer when the content of vesicles derived from bacteria of the genus Turicibacter is lower than that of a normal person through quantitative analysis of the PCR product.
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 및/또는 폐암 등의 발병 여부 및 질환의 수준 등을 판단하는 것이다. The term "diagnosis" used in the present invention means to judge the condition of a patient's disease in all aspects in a broad sense. The contents of the judgment are the name of the disease, etiology, disease type, severity, detailed mode of the bed, the presence or absence of complications, and the prognosis. In the present invention, the diagnosis is to determine the onset of colon cancer and/or lung cancer 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 투리시박터 속 세균에서 자연적으로 분비되거나 또는 인공적으로 생산하는 것으로, 구형의 형태이며, 10 내지 200 nm의 평균 직경을 가지고 있다.The term "nanovesicle" or "vesicle" used in the present invention refers to a structure made of a nano-sized membrane secreted from various bacteria. Vesicles derived from gram-negative bacteria or outer membrane vesicles (OMVs) contain toxic proteins and bacterial DNA and RNA as well as endotoxin (lipopolysaccharide), and vesicles derived from gram-positive bacteria In addition to proteins and nucleic acids, it has 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 Turicibacter, 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 is also referred to as “military genome”, and refers to the sum of genomes including all viruses, bacteria, fungi, etc. in isolated areas such as soil and animal intestines. It is used as a concept of the genome to describe the identification of many microorganisms at once by using a sequencer to analyze microbes that do not. In particular, metagenome does not refer to the genome or genome of one species, but refers to a kind of mixed genome as the genome of all species in one environmental unit. This is a term that came from the point of view that not only one existing species functionally but also various species interact with each other to create a complete species when defining a species in the course of the development of biology in an ohmic way. Technically, it is the subject of a technique that uses rapid sequencing to analyze all DNA and RNA regardless of species, to identify all species within one environment, and to identify interactions and metabolisms.
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 투리시박터 속 세균 유래 소포를 유효성분으로 포함하는, 대장암 또는 폐암의 예방, 치료 또는 개선용 조성물을 제공한다. 상기 조성물은 식품 조성물, 흡입제 조성물 및 약학적 조성물을 포함하며, 본 발명에서 식품 조성물은 건강기능식품 조성물을 포함한다. 본 발명의 조성물은 구강분무제 제형일 수 있다.As another aspect of the present invention, the present invention provides a composition for preventing, treating or improving colon cancer or lung cancer, comprising as an active ingredient vesicles derived from bacteria of the genus Turicibacter. The composition includes a food composition, an inhalant composition, and a pharmaceutical composition, and the food composition in the present invention includes a health functional food composition. The composition of the present invention may be an oral spray formulation.
본 발명에서 사용되는 용어, “예방”이란 본 발명에 따른 조성물의 투여에 의해 대장암 및/또는 폐암 등을 억제시키거나 발병을 지연시키는 모든 행위를 의미한다.The term "prevention" used in the present invention refers to any action that suppresses or delays the onset of colon cancer and/or lung cancer by administration of the composition according to the present invention.
본 발명에서 사용되는 용어, “치료”란 본 발명에 따른 조성물의 투여에 의해 대장암 또는 폐암 등에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. The term "treatment" used in the present invention refers to any action in which symptoms for colon cancer or lung cancer are improved or advantageously changed by administration of the composition according to the present invention.
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 a parameter related to the condition being treated, for example, symptoms.
상기 소포는 투리시박터 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 obtained by centrifugation, ultra-high-speed centrifugation, high-pressure treatment, extrusion, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical decomposition, chemical treatment, and filter. Separation can be performed using one or more methods selected from the group consisting of filtration, gel filtration chromatography, pre-flow electrophoresis, and capillary electrophoresis. In addition, a process such as washing to remove impurities and concentration of the obtained vesicle may be further included.
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 contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in formulation, and includes, but is limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome, and the like. It is not, and other conventional additives such as antioxidants and buffers may be further included as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Regarding suitable pharmaceutically acceptable carriers and formulations, it can be preferably formulated according to each component using a method disclosed in Remington's literature. The pharmaceutical composition of the present invention is not particularly limited in its formulation, but may be formulated as an injection, an inhalant, an external preparation for the skin, or an oral ingestion.
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 according to a desired method (for example, intravenous, subcutaneous, skin, nasal, airway), and the dosage is the patient's condition, weight, and disease. It depends on the degree of the drug, the form of the drug, the route of administration and the time, but 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, a pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type of disease, severity, drug activity, drug Sensitivity, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, 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 administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with a conventional therapeutic agent, and may be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and this can be easily determined by a person 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 per 1 kg of body weight is administered daily or every other day. Alternatively, it can be administered in 1 to 3 times a day. However, since it may increase or decrease depending on the route of administration, the severity of obesity, sex, weight, age, etc., the dosage amount does not limit the scope of the present invention in any way.
본 발명의 흡입제 조성물은 유효성분을 흡입제에 그대로 첨가하거나 다른 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 치료용)에 따라 적합하게 결정될 수 있다.The inhalant composition of the present invention may be added as it is to the inhalant or used together with other ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or treatment).
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 to the food as it is, or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, in the manufacture of food or beverage, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than 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 not particularly limited in other ingredients other than containing the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as an additional ingredient, such as a normal beverage. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, 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 a person 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 and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives can also be appropriately selected by a person 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, vesicles derived from bacteria and bacteria are administered orally to mice to evaluate the absorption, distribution, and excretion patterns of bacteria and vesicles in the body. In the case of bacteria, vesicles are not absorbed through the intestinal tract, but vesicles are administered for 5 minutes. It was confirmed that it was absorbed within, distributed systemically, and excreted through the kidneys and liver (see Example 1).
본 발명의 다른 실시예에서는, 대장암 및 폐암 환자에 연령과 성별을 매칭한 정상인의 혈액 또는 대변에서 분리한 소포를 이용하여 세균 메타게놈 분석을 실시하였다. 그 결과, 정상인 샘플에 비하여, 대장암 및 폐암 환자의 임상샘플에 투리시박터 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다(실시예 3 및 4 참조).In another embodiment of the present invention, bacterial metagenomic analysis was performed using vesicles isolated from blood or feces of a normal person whose age and sex were matched to colon cancer and lung cancer patients. As a result, it was confirmed that the vesicles derived from bacteria in Turicibacter genus were significantly reduced in the clinical samples of colon cancer and lung cancer patients compared to the normal sample (see Examples 3 and 4).
본 발명의 또 다른 실시예에서는, 투리시박터 상구이니스 균주를 배양하여 이로부터 분비된 소포가 면역조절 및 항염증 효과를 나타내는지를 평가하였는데, 다양한 농도의 투리시박터 상구이니스 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 투리시박터 상구이니스 유래 소포가 효율적으로 억제함을 확인하였다(실시예 5 참조). In another embodiment of the present invention, vesicles secreted therefrom were evaluated by culturing a strain of Turicibacter sangguinis to evaluate whether they exhibit immunomodulatory and anti-inflammatory effects, and vesicles derived from Turicibacter sangguinis at various concentrations were applied to macrophages. After treatment, vesicles derived from Escherichia coli, which is a causative agent of inflammatory disease, were treated to evaluate the secretion of inflammatory mediators. As a result, it was found that the vesicles derived from Turicibacter sangguinis efficiently inhibited the secretion of IL-6 and TNF-α by the vesicles derived from E. coli. Confirmed (see Example 5).
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, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of 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 absorption, distribution, and excretion of intestinal bacteria and vesicles derived from bacteria in the body
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 intestinal bacteria and vesicles derived from bacteria are systemically absorbed through the gastrointestinal tract, an experiment was performed as follows. Intestinal bacteria and vesicles derived from intestinal bacteria were administered to the gastrointestinal tract at a dose of 50 μg, respectively, and fluorescence was measured after 0 minutes, 5 minutes, 3 hours, 6 hours, and 12 hours. As a result of observing the entire mouse image, as shown in Fig. 1A, in the case of bacteria, it was not systemically absorbed, but in the case of bacterial-derived vesicles, it was systemically absorbed 5 minutes after administration, and fluorescence in the
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 systemic absorption of intestinal bacteria and vesicles derived from intestinal bacteria, in order to evaluate the infiltrate into various organs, 50 μg of bacteria and vesicles derived from bacteria, labeled with fluorescence, 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 observing fluorescence in the collected tissues, as shown in FIG. 1B, it was found that bacteria-derived vesicles were distributed in blood, heart, lung, liver, spleen, fat, muscle, and kidney, but the bacteria were not absorbed.
실시예 2. 임상샘플에서 세균 유래 소포 메타게놈 분석Example 2. Analysis of bacterial-derived vesicle metagenomics in clinical samples
혈액, 대변과 같은 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㎛ 필터(filter)를 사용하여 박테리아 및 이물질을 제거한 후, Type 90ti 로터로 150,000 x g, 4℃에서 3시간동안 초고속원심분리방법을 사용하여 상등액을 버리고 덩어리진 펠렛(pellet)을 생리식염수(PBS)로 녹였다. Clinical samples such as blood and feces 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 bacteria and foreign substances using a 0.22 μm filter, it was transferred to a centrifugal filter 50 kD and centrifuged at 1500 x g for 15 minutes at 4° C., discarding substances smaller than 50 kD, and concentrating to 10 ml. Once again, after removing bacteria and foreign substances by using a 0.22㎛ filter, discard the supernatant using a high-speed centrifugation method at 150,000 xg at 4℃ for 3 hours with a Type 90ti rotor and physiologically circulate the lumped pellets. It was dissolved in saline solution (PBS).
상기 방법으로 분리한 소포 100㎕를 100℃에서 끓여서 내부의 DNA를 지질 밖으로 나오게 하고 그 후 얼음에 5분 동안 식힌 다음 남은 부유물을 제거하기 위하여 10,000 x g, 4℃에서 30분간 원심분리하고 상등액만을 모으고 Nanodrop을 이용하여 DNA 양을 정량하였다. 이후, 상기 추출된 DNA에 세균 유래 DNA가 존재하는지 확인하기 위하여 하기 표 1에 나타낸 16s rDNA 프라이머(primer)로 PCR을 수행하여 상기 추출된 유전자에 세균 유래 유전자가 존재하는 것을 확인하였다.Boil 100 µl of the vesicles separated by the above method at 100°C to allow the DNA inside to come out of the lipids, cool on ice for 5 minutes, and then centrifuge at 10,000 xg for 30 minutes at 4°C to remove the remaining suspended matter, and collect only the supernatant. The amount of DNA was quantified using Nanodrop. Thereafter, in order to confirm the presence of bacterial-derived DNA in the extracted DNA, PCR was performed with the 16s rDNA primer shown in Table 1 below, and it was confirmed that the bacterial-derived gene was present 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, 속(genus)은 94%, 과(family)는 90%, 목(order)은 85%, 강(class)은 80%, 문(phylum)은 75% 시퀀스 유사도를 기준으로 클러스터링을 하고 각 OTU의 문(phylum), 강(class), 목(order), 과(family), 속(genus) 레벨의 분류를 수행하고, BLASTN와 GreenGenes의 16S RNA 시퀀스 데이터베이스 (108,453 시퀀스)를 이용하여 속 수준에서 97% 이상의 시퀀스 유사도 갖는 세균을 프로파일링 하였다 (QIIME).The DNA extracted by the above method was amplified using the above 16S rDNA primer, followed by sequencing (Illumina MiSeq sequencer), outputting the result as a Standard Flowgram Format (SFF) file, and using GS FLX software (v2.9). Then, the SFF file was converted into a sequence file (.fasta) and a nucleotide quality score file, and then the credit rating of the lead was checked, and the portion with an average base call accuracy of less than 99% (Phred score <20) in the window (20 bps) was removed. . For Operational Taxonomy Unit (OTU) analysis, clustering is performed according to sequence similarity using UCLUST and USEARCH, and the genus is 94%, the family is 90%, the order is 85%, and the strong ( Class) is 80%, phylum is clustered based on sequence similarity of 75%, and each OTU's phylum, class, order, family, and genus level Classification was performed, and bacteria having a sequence similarity of 97% or more at the genus level were profiled using BLASTN and GreenGenes' 16S RNA sequence database (108,453 sequences) (QIIME).
실시예 3. 대장암환자의 대변 내 세균 유래 소포 메타게놈 분석Example 3. Bacterial-derived vesicle metagenomic analysis in stool of colon cancer patient
실시예 2의 방법으로 대장암환자 29명의 대변과, 정상인 358명의 대변을 대상으로, 대변 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 투리시박터 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 대변에 비하여 대장암환자의 대변에 투리시박터 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 2 및 도 2 참조).In the stool of 29 patients with colorectal cancer and the stool of 358 normal people by the method of Example 2, the gene was extracted from the vesicles present in the stool and metagenomic analysis was performed, and then the distribution of the vesicles derived from bacteria in the genus Turicibacter was analyzed. Evaluated. As a result, it was confirmed that vesicles derived from bacteria of the genus Turicibacter were significantly reduced in the stool of a colon cancer patient compared to the stool of a normal person (see Table 2 and FIG. 2).
실시예Example 4. 폐암환자 혈액 세균 유래 소포 4. Blood Bacteria-derived Vesicles of Lung Cancer Patients 메타게놈Metagenome 분석 analysis
실시예 2의 방법으로 폐암환자 318명의 혈액과, 나이와 성별을 매칭한 정상인 234명의 혈액을 대상으로, 혈액 내에 존재하는 소포에서 유전자를 추출하여 메타게놈 분석을 수행한 후, 투리시박터 속 세균 유래 소포의 분포를 평가하였다. 그 결과, 정상인 혈액에 비하여 폐암환자의 혈액에 투리시박터 속 세균 유래 소포가 유의하게 감소되어 있음을 확인하였다 (표 3 및 도 3 참조).The blood of 318 lung cancer patients with the method of Example 2 and the blood of 234 normal people whose age and sex were matched were subjected to metagenomic analysis by extracting genes from vesicles present in the blood, and then bacteria in Turicibacter. The distribution of the derived vesicles was evaluated. As a result, it was confirmed that the blood of lung cancer patients was significantly reduced in vesicles derived from bacteria of the genus Turicibacter compared to normal blood (see Table 3 and FIG. 3).
실시예 5. 투리시박터 상구이니스 유래 소포의 항염증 효과Example 5. Anti-inflammatory effect of vesicles derived from Turicibacter Sangguinis
상기 실시예의 결과를 바탕으로, 투리시박터 속 세균에 속하는 투리시박터 상구이니스 균주를 배양한 후 이의 소포를 분리하였다. 투리시박터 상구이니스 균주를 37℃ 혐기성 챔버에서 흡광도(OD600)가 1.0 내지 1.5가 될 때까지 BHI(brain heart infusion) 배지에서 배양한 후 서브컬쳐(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 한외여과(ultrafiltration)을 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 %, 및 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 a strain of Turicibacter sangguinis belonging to the bacteria of the genus Turicibacter, the vesicles thereof were isolated. Turisibacter Sangguinis strain was cultured in BHI (brain heart infusion) medium until the absorbance (OD 600 ) became 1.0 to 1.5 in an anaerobic chamber at 37° C. and then sub-cultured. Afterwards, the medium supernatant containing no strain was collected, centrifuged at 10,000 g, 4 ℃ 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). Then, it was concentrated to a volume of 200 ml through ultrafiltration. Afterwards, 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 the pellet was suspended in DPBS. Next, density gradient centrifugation was performed using 10%, 40%, and 50% Optiprep solutions (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 subjected to ultracentrifugation at 150,000 g and 4° C. for 3 hours. Subsequently, the protein was quantified using BCA assay, and an experiment was performed on the obtained vesicles.
투리시박터 상구이니스 유래 소포가 염증세포에서 염증매개체 분비에 대한 영향을 알아보기 위해, 마우스 대식세포주인 Raw 264.7 세포에 투리시박터 상구이니스 유래 소포를 다양한 농도(0.1, 1, 10 ㎍/㎖)로 처리한 후, 염증질환 병원성 소포인 대장균 유래 소포 (E. coli EV 1.0 ㎍/㎖)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(Dulbeco’s Modified Eagle’s Medium) 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 vesicles derived from Turicibacter sangguinis on the secretion of inflammatory mediators in inflammatory cells, vesicles derived from Turicibacter sangguinis were added in various concentrations (0.1, 1, 10 ㎍/㎖) in Raw 264.7 cells, a mouse macrophage cell line. After treatment with inflammatory disease pathogenic vesicles, E. coli-derived vesicles ( E. coli EV 1.0 μg/ml) was treated to measure the amount of secretion of inflammatory mediators (IL-6, TNF-α, etc.). More specifically, 1 x 10 5 of Raw 264.7 cells were dispensed into a 24-well cell culture plate, and then cultured in DMEM (Dulbeco's Modified Eagle's Medium) 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 4에 나타난 바와 같이 투리시박터 상구이니스 유래 소포를 전 처리 한 경우, 대장균 유래 소포에 의한 IL-6 및 TNF-α 분비가 현저히 억제됨을 확인하였다. 상기 결과는, 대장균 유래 소포와 같은 병원성 소포에 의해 유도되는 염증의 발생을 투리시박터 상구이니스 유래 소포가 효율적으로 억제할 수 있음을 의미한다.As a result, as shown in Figure 4, when pre-treatment of the vesicles derived from Turicibacter sangguinis, it was confirmed that IL-6 and TNF-α secretion by E. coli-derived vesicles were significantly suppressed. The above results mean that the vesicles derived from Turicibacter sangguinis can effectively inhibit 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 above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified 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 and non-limiting in all respects.
<110> MD Healthcare Inc. <120> Nanovesicles derived from Turicibacter bacteria and Use thereof <130> MP19-014 <150> KR 10-2018-0023212 <151> 2018-02-26 <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 Turicibacter bacteria and Use thereof <130> MP19-014 <150> KR 10-2018-0023212 <151> 2018-02-26 <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 (11)
Turicibacter sanguinis (Turicibacter sanguinis), comprising as an active ingredient derived vesicles, anti-inflammatory pharmaceutical composition.
상기 소포는 평균 직경이 10 내지 200 nm인 것을 특징으로 하는, 약학적 조성물.
The method of claim 3,
The vesicle is characterized in that the average diameter of 10 to 200 nm, pharmaceutical composition.
상기 소포는 투리시박터 상구이니스에서 자연적 또는 인공적으로 분비되는 것을 특징으로 하는, 약학적 조성물.
The method of claim 3,
The vesicle is characterized in that the natural or artificial secretion from Turicibacter sangguinis, pharmaceutical composition.
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