WO2018124744A1 - Method for diagnosing liver disease via bacterial metagenomic analysis - Google Patents

Method for diagnosing liver disease via bacterial metagenomic analysis Download PDF

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WO2018124744A1
WO2018124744A1 PCT/KR2017/015578 KR2017015578W WO2018124744A1 WO 2018124744 A1 WO2018124744 A1 WO 2018124744A1 KR 2017015578 W KR2017015578 W KR 2017015578W WO 2018124744 A1 WO2018124744 A1 WO 2018124744A1
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derived
extracellular vesicles
bacteria
liver
decrease
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김윤근
박태성
윤정환
임상섭
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주식회사 엠디헬스케어
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  • Liver cancer is one of the three major cancers in the liver, competing with stomach cancer and lung cancer for the second highest cancer mortality rate, commonly referred to as Hepatocellular carcinoma (HCC). It affects people in their 40s and 60s, and four times more men than women. Liver cancer is more likely to occur if you have an underlying liver disease. If you have cirrhosis of any cause, it is a risk factor for liver cancer. Liver cirrhosis occurs in 75-85% of liver cancer patients, and 10-30% of cirrhosis patients develop liver cancer. Viral hepatitis is also an important risk factor. Among them, hepatitis B is involved in 60-70% of liver cancer patients, and hepatitis C is involved in about 10% of liver cancer patients.
  • HCC Hepatocellular carcinoma
  • the present inventors In order to diagnose liver disease based on the causative factors of cirrhosis and liver cancer, the present inventors extracted genes from extracellular vesicles derived from bacteria from blood, a sample derived from a subject, and performed a metagenome analysis on them. Identified bacterial extracellular vesicles that can act as a causative agent of liver disease, and the like, the present invention was completed based on this.
  • the present invention provides a method for providing information for diagnosing liver disease, comprising the following steps.
  • the present invention provides a method for diagnosing liver disease, comprising the following steps.
  • compared to the normal sample derived from the step (c) by comparing the increase or decrease in the content of one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales It may be to diagnose liver cancer.
  • the content of at least one family bacteria-derived extracellular vesicles selected from the group consisting of Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae in comparison to the sample derived from the normal in step (c) It may be to diagnose liver cancer by comparing the increase and decrease.
  • liver cancer may be diagnosed by comparing the increase and decrease of the content of Trabulsiella genus bacteria-derived extracellular vesicles in comparison with the sample derived from the cirrhosis transducer in step (c).
  • the amount of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, and Succiniclasticum is increased in comparison with the sample derived from the normal person in the step (c). It may be to compare the diagnosis of liver cirrhosis.
  • the sample may be blood, and the blood may be whole blood, serum, plasma, or blood monocytes.
  • Extracellular vesicles secreted by the bacteria present in the environment can be absorbed directly into the body and have a direct impact on the development of cancer, liver disease is difficult to diagnose early due to difficult early diagnosis before symptoms appear, so the human-derived according to the present invention Metagenome analysis of bacterial or bacterial-derived extracellular vesicles using a sample predicts the risk of developing liver disease in advance, allowing early diagnosis and prediction of risk groups of liver disease, and delaying the onset or preventing the onset through proper management. In addition, early diagnosis is possible even after the onset of the disease, thereby reducing the incidence of liver disease and increasing the therapeutic effect. In addition, metagenome analysis predicts causative factors in patients diagnosed with hepatic disease, thereby avoiding exposure to causative factors, and improving the progression of liver disease or preventing recurrence.
  • Figure 1 is for evaluating the distribution of bacteria-derived extracellular vesicles in the body
  • Figure 1a after the administration of oral intestinal bacteria (Bacteria) and bacteria-derived vesicles (EV) in the mouth hourly (0, 5min, 3h, 6h, and 12h) is a photograph taken of their distribution
  • Figure 1b is 12 hours after the oral administration of intestinal bacteria (Bacteria) and bacteria-derived extracellular vesicles (EV) to the blood and various organs (heart, Lung, liver, kidney, spleen, adipose tissue, and muscles), and the photographs of the distribution of the bacterial and extracellular vesicles.
  • FIG. 3 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 4 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 5 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles in the liver cancer patients and liver cirrhosis blood.
  • EVs bacteria-derived vesicles
  • FIG. 6 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacterial-derived vesicles from liver cirrhosis cancer patients and normal blood, and performing a metagenome analysis.
  • EVs bacteria-derived vesicles
  • FIG. 7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles from liver cirrhosis cancer patients and normal blood.
  • EVs bacteria-derived vesicles
  • the present invention relates to a method for diagnosing liver disease through bacterial metagenomic analysis, and the present inventors extracted a gene from bacterial extracellular vesicles using a sample derived from a subject and performed a metagenome analysis on the liver disease. Bacterial-derived extracellular vesicles that could act as causative factors were identified.
  • the present invention comprises the steps of (a) extracting DNA from the extracellular vesicles isolated from the subject sample;
  • an information providing method for diagnosing liver disease comprising comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
  • the term "diagnostic liver cancer” means to determine whether there is a possibility of developing liver cancer, relatively high chance of developing liver cancer, or whether liver cancer has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing liver cancer for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of liver cancer and selecting the most appropriate treatment regimen.
  • the term "diagnosing cirrhosis” means to determine whether there is a possibility of developing cirrhosis, a relatively high chance of developing cirrhosis, or whether cirrhosis has already occurred.
  • the method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing cirrhosis for any particular patient.
  • the methods of the present invention can be used clinically to determine treatment by early diagnosis of cirrhosis and selecting the most appropriate treatment regimen.
  • metagenome used in the present invention, also referred to as “metagenome”, refers to the total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, It is mainly used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms which are not cultured.
  • metagenome does not refer to one species of genome or genome, but refers 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.
  • 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.
  • metagenome analysis was preferably performed using bacterial-derived extracellular vesicles isolated from serum.
  • the subject sample may be blood, and the blood may preferably be whole blood, serum, plasma, or blood monocytes, but is not limited thereto.
  • metagenome analysis was performed on extracellular vesicles derived from bacteria in the blood of normal people, liver cirrhosis patients, and liver cancer patients, and were classified into phylum, class, order, and family. Analyzes at the,, and genus levels were respectively identified to identify bacterial-derived vesicles that could actually cause the development of liver cancer and cirrhosis.
  • liver cancer patients as a result of analyzing the bacteria-derived metagenome at the genus level, there is a significant difference between liver cancer patients and normal vesicles derived from Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella (See Example 4).
  • metagenome analysis of extracellular vesicles derived from bacteria in the blood of liver cirrhosis and liver cancer patients was carried out, including phylum, class, order, family, Analyzes at the genus and genus levels, respectively, identified bacterial vesicles that can actually cause liver cancer in cirrhosis transducers.
  • liver-derived liver cancer patients As a result of analyzing the bacteria-derived metagenome at the genus level, there was a significant difference between the liver-derived liver cancer patients and liver cirrhosis vesicles derived from Trabulsiella genus (see Example 5).
  • the metagenome analysis of extracellular vesicles derived from bacteria in the blood of normal people and liver cirrhosis was performed, including phylum, class, order, family, and Analyzes at the genus level identified bacterial vesicles that could actually cause liver cancer in asthma patients.
  • the present invention through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from blood by metagenomic analysis to identify bacteria-derived vesicles significantly changed in liver cancer patients compared to normal people and liver cirrhosis Meta-genomic analysis confirmed that liver cancer can be diagnosed by analyzing the increase and decrease of the content of bacterial-derived vesicles at each level.
  • the present invention through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from the blood by performing a genome analysis of bacteria-derived vesicles with significantly changed content in liver cirrhosis compared to normal people , Metagenomic analysis confirmed that liver cirrhosis can be diagnosed by analyzing the increase and decrease of the content of the bacteria-derived vesicles at each level.
  • Example 1 Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
  • the fluorescently labeled 50 ⁇ g of bacteria and bacteria-derived vesicles were administered in the same manner as above 12 hours.
  • Blood, Heart, Lung, Liver, Kidney, Spleen, Adipose tissue, and Muscle were extracted from mice.
  • the intestinal bacteria (Bacteria) were not absorbed into each organ, whereas the intestinal bacteria-derived extracellular vesicles (EV) were detected in the tissues, as shown in FIG. And distribution in liver, kidney, spleen, adipose tissue, and muscle.
  • the blood was first placed in a 10 ml tube and centrifuged (3,500 ⁇ g, 10 min, 4 ° C.) to settle the suspended solids to recover only the supernatant and then transferred to a new 10 ml tube. After removing the bacteria and foreign substances from the recovered supernatant using a 0.22 ⁇ m filter, transfer to centripreigugal filters (50 kD) and centrifuged at 1500 xg, 4 °C for 15 minutes to discard the material smaller than 50 kD and 10 ml Concentrated until.
  • centripreigugal filters 50 kD
  • PCR was performed using the 16S rDNA primer shown in Table 1 to amplify the gene and perform sequencing (Illumina MiSeq sequencer). Output the result as a Standard Flowgram Format (SFF) file, convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and window (20 bps) The part with the average base call accuracy of less than 99% (Phred score ⁇ 20) was removed.
  • SFF Standard Flowgram Format
  • the Operational Taxonomy Unit performed UCLUST and USEARCH for clustering according to sequence similarity. Specifically, the clustering is based on 94% genus, 90% family, 85% order, 80% class, and 75% sequence similarity. OTU's door, river, neck, family and genus level classifications were performed, and bacteria with greater than 97% sequence similarity were analyzed using BLASTN and GreenGenes' 16S DNA sequence database (108,453 sequences) (QIIME).
  • metagenome sequencing was performed after separating vesicles from the blood of 86 liver cancer patients and 331 normal humans with age and gender matching.
  • the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • the diagnostic performance of liver cancer is improved when developing a diagnostic model with one or more biomarkers in bacteria of Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella. Significantly (see Table 4 and FIG. 4).
  • Example 5 Separated from liver cirrhosis and liver cancer patients blood Germ-derived parcel Metagenome Analysis-based Liver Cancer Diagnosis Model
  • Example 3 By the method of Example 3, vesicles were isolated from blood of 84 liver cancer patients and 86 cirrhosis transducers, and metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
  • Cirrhosis Liver cancer t-test Training set Test set Taxon Mean SD Mean SD p-value Ratio AUC sensitivity specificity AUC sensitivity specificity g__Trabulsiella 0.0197 0.0325 0.0436 0.0436 0.0001 2.22 0.73 0.83 0.47 0.55 0.62 0.42
  • liver cancer and cirrhosis By predicting the risk of liver cancer and cirrhosis in advance through metagenomic analysis on the genes present in bacteria-derived extracellular parcel vesicles using a human-derived sample according to the present invention, early diagnosis and prediction of risk groups of liver disease are made through appropriate management. It can delay the onset or prevent the onset, and early diagnosis even after the occurrence of cirrhosis or liver cancer can reduce the incidence of liver disease and increase the therapeutic effect.

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Abstract

The present invention relates to a method for diagnosing a liver disease, such as liver cancer and cirrhosis, via bacterial metagenomic analysis and, more particularly, to a method for diagnosing liver cancer and cirrhosis by performing bacterial metagenomic analysis using a subject-derived sample, and by analyzing an increase or decrease in the content of a specific bacterium-derived extracellular vesicle. An extracellular vesicle secreted from a bacterium present in the environment can be absorbed into the body and directly affect the occurrence of inflammation and cancer, and a liver disease, such as liver cancer and cirrhosis, is difficult to diagnose early on before any symptom appears, which makes efficient treatment difficult. As such, through the metagenomic analysis on a gene present in a bacterium-derived extracellular vesicle using a human body-derived sample according to the present invention, the risk of the onset of liver cancer and cirrhosis can be predicted in advance, enabling early diagnosis and prediction of a liver disease risk group and delay of the time of the onset or prevention of the onset through proper care, and early diagnosis is still possible even after the onset of cirrhosis or liver cancer, which can lower the incidence rate of the liver disease and enhance the treatment effect.

Description

세균 메타게놈 분석을 통한 간질환 진단 방법 Diagnosis of liver disease through bacterial metagenome analysis
본 발명은 세균 메타게놈 분석을 통해 간암 및 간경변 등의 간질환을 진단하는 방법에 관한 것으로서, 보다 구체적으로는 피검체 유래 샘플을 이용해 세균 메타게놈 분석을 수행하여 특정 세균 유래 세포밖 소포의 함량 증감을 분석함으로써 간질환을 진단하는 방법에 관한 것이다.The present invention relates to a method for diagnosing liver diseases such as liver cancer and cirrhosis through bacterial metagenome analysis, and more specifically, to increase or decrease the content of specific bacterial-derived extracellular vesicles by performing bacterial metagenome analysis using a sample derived from a subject. The present invention relates to a method for diagnosing liver disease.
간암(liver cancer)은 간에서 발생하는, 암 사망률 2위를 놓고 위암, 폐암과 다투는 3대 암 중 하나로서 흔히 간세포암 (Hepatocellular carcinoma, HCC)를 말한다. 40~60대에 호발하며, 남자가 여자보다 4배 정도 많이 발생한다. 기저 간질환이 있는 경우 간암이 발생하기 쉬운데, 원인 불문하고 간경변이 있으면 간암의 위험인자가 된다. 간암 환자의 75~85%에서 간경변이 있으며, 간경변환자의 10~30%가 간암이 발생한다고 한다. 또한 바이러스성 간염이 있는 경우도 중요한 위험 인자인데, 그중에서도 B형간염이 간암환자의 60~70%에서, C형간염이 간암환자의 10%정도가 관련된다고 한다. 우리나라에서 B형간염이 있는 사람의 간암발생률은 정상인의 6배라고 하며, 일본, 미국, 유럽에서는 C형간염이 간암의 가장 중요한 위험인자라고 알려져 있다. 또한 술이 중요한 위험인자인데, 우리나라 간암의 10~20%가 알코올성 간 질환에서 발생한다. 그 외 대사질환, 예를 들어 혈색소증, 윌슨병 등을 가지고 있는 경우에도 간암 발생이 증가할 수 있으며, 경구피임약 (에스트로겐이 함유된 제품)도 간암의 발생 위험을 높인다. 당뇨, 비만, 조영제 사용 등도 간암 발생과 연관이 있는 것으로 알려져 있다. 대표적인 간세포암의 종양표지자는 알파태아단백 (Alphafetoprotein, AFP)으로서 혈액검사로 측정한다. 다른 종양표지자도 마찬가지지만 민감도 50~80%, 특이도 60~90%로 그다지 높지 않기 때문에 다른 검사 결과와 같이 해석해야 하는 한계가 있다. 최근에 나온 표지자로 PIVKA-II라고 하는 단백질도 있는데, 민감도 60-90%, 특이도 90%로 알파태아단백보다 좋다. Liver cancer is one of the three major cancers in the liver, competing with stomach cancer and lung cancer for the second highest cancer mortality rate, commonly referred to as Hepatocellular carcinoma (HCC). It affects people in their 40s and 60s, and four times more men than women. Liver cancer is more likely to occur if you have an underlying liver disease. If you have cirrhosis of any cause, it is a risk factor for liver cancer. Liver cirrhosis occurs in 75-85% of liver cancer patients, and 10-30% of cirrhosis patients develop liver cancer. Viral hepatitis is also an important risk factor. Among them, hepatitis B is involved in 60-70% of liver cancer patients, and hepatitis C is involved in about 10% of liver cancer patients. In Korea, the incidence of liver cancer in people with hepatitis B is six times that of normal people. Hepatitis C is known to be the most important risk factor for liver cancer in Japan, the United States, and Europe. Alcohol is also an important risk factor, and 10-20% of Korean liver cancer occurs in alcoholic liver disease. Liver cancers may also increase with metabolic diseases such as hemochromatosis and Wilson's disease, and oral contraceptives (products containing estrogen) also increase the risk of developing liver cancer. Diabetes, obesity and the use of contrast agents are also known to be associated with liver cancer. Tumor markers of representative hepatocellular carcinoma are alphafetoprotein (AFP) and are measured by blood tests. The same is true for other tumor markers, but its sensitivity is 50-80% and specificity 60-90%. A recent marker is a protein called PIVKA-II, which is 60-90% sensitive and 90% specific, better than alpha-fetoprotein.
간부전 (肝不全, hepatic failure)은 간이 정상적인 생리 작용으로서의 단백질 합성과 대사 기능을 수행할 수 없는 상태를 말하고, 간기능부전 (肝機能不全) 또는 간기능상실(肝機能喪失)이라고도 한다. 간부전에는 두 가지 형태가 알려져 있는데, 급성 간부전은 간 문제로 인한 첫 증상 (황달 등)을 보인 후 4주 내로 간성 뇌증 (혼란, 인사불성, 혼수상태)이 진행되고, 단백질 (알부민 등) 혈액 응고 단백질의 생산이 줄어든다. 만성 간부전은 과도한 음주, B형 간염 혹은 C형 간염 바이러스, 자가면역, 유전적 원인, 철, 구리의 과잉섭취 등 대사적 원인 등에 의해 발생하는데, 간경변 (肝硬便, 영어: liver Cirrhosis)을 수반한다. 간경변은 간의 조직 세포에 문제가 생겨 간이 굳고 오므라드는 것이 특징이고, 간경화, 간섬유증으로도 부른다. 간경변은 광범위한 간세포 파괴의 결과로 섬유조직의 증식과 재생성 결절 형성의 형태학적 특징을 보이며, 2차적으로 간내혈관의 변형 및 간기능의 저하가 초래되는 질병이다. 간의 섬유화는 서서히 진행되어 상당한 경과가 있더라도 간기능 부전이 일어나기 전까지 자각증상이 없다. 따라서 조기에 간경변의 발생을 진단하는 것은 매우 어렵다. Liver failure (은 不全, hepatic failure) refers to a condition in which the liver can not perform protein synthesis and metabolic function as a normal physiological function, also referred to as liver failure (能 機能 不全) or liver failure (肝 機能 喪失). There are two known forms of hepatic insufficiency. Acute hepatic insufficiency develops hepatic encephalopathy (confusion, non-incompatibility, coma) within 4 weeks after the first symptoms of liver problems (such as jaundice), and blood clotting of proteins (albumin, etc.). The production of protein is reduced. Chronic liver failure is caused by excessive drinking, hepatitis B or hepatitis C virus, autoimmunity, genetic causes, and metabolic causes such as excessive intake of iron and copper, which are accompanied by cirrhosis (肝 硬 便, English: liver Cirrhosis). do. Cirrhosis is characterized by problems with liver tissue cells, causing the liver to harden and retract, also called cirrhosis and liver fibrosis. Hepatic cirrhosis is a disease that results in extensive hepatocellular destruction, showing morphological characteristics of fibrous tissue proliferation and regenerative nodule formation. Secondly, hepatic vascular deformation and deterioration of liver function are caused. Fibrosis of the liver progresses slowly and there is no subjective symptoms until significant progress is made. Therefore, it is very difficult to diagnose the occurrence of cirrhosis early.
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 세균(bacteria), 고세균(archaea), 진핵생물(eukarya)을 포함한 미생물 군집(microbial community)을 말하고, 장내 미생물총은 사람의 생리현상에 중요한 역할을 하며, 인체 세포와 상호작용을 통해 인간의 건강과 질병에 큰 영향을 미치는 것으로 알려져 있다. 우리 몸에 공생하는 세균은 다른 세포로의 유전자, 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 대개 100 나노미터 크기 이하라서 비교적 자유롭게 점막을 통화하여 우리 몸에 흡수된다.On the other hand, the symbiosis of the human body reaches 100 trillion times 10 times more than human cells, the number of genes of the microorganism is known to be more than 100 times the number of human genes. Microbiota (microbiota or microbiome) refers to a microbial community including bacteria, archaea and eukarya that exist in a given settlement.Intestinal microbiota is an important role in human physiology. It is known to have a great effect on human health and disease through interaction with human cells. The symbiotic bacteria secrete nanometer-sized vesicles to exchange information about genes and proteins in other cells. The mucous membrane forms a physical protective film that particles larger than 200 nanometers (nm) in size can't pass through, so that the symbiotic bacteria cannot pass through the mucosa, but bacterial-derived vesicles are usually less than 100 nanometers in size. It freely speaks to the mucous membrane and is absorbed by our body.
환경 유전체학이라고도 불리는 메타게놈학은 환경에서 채취한 샘플에서 얻은 메타게놈 자료에 대한 분석학이라고 할 수 있다(국내 공개특허 제2011-0073049호). 최근 16s 리보솜 RNA(16s rRNA) 염기서열을 기반으로 한 방법으로 인간의 미생물총의 세균 구성을 목록화하는 것이 가능해졌으며, 16s 리보솜 RNA의 유전자인 16s rDNA 염기서열을 차세대 염기서열분석 (next generation sequencing, NGS) platform을 이용하여 분석한다. 그러나 간암 발병에 있어서, 혈액 등의 인체 유래물에서 세균 유래 소포에 존재하는 메타게놈 분석을 통해 간암의 원인인자를 동정하고 간암 발병 위험도를 진단하는 방법에 대해서는 보고된 바가 없다.Metagenomics, also called environmental genomics, can be said to be an analysis of metagenomic data obtained from samples taken from the environment (Korean Patent Publication No. 2011-0073049). Recently, it has become possible to list the bacterial composition of the human microflora by a method based on 16s ribosomal RNA (16s rRNA) sequencing. Next generation sequencing of 16s rDNA sequencing gene of 16s ribosomal RNA is performed. , NGS) platform to analyze. However, in the development of liver cancer, there has been no report on a method for identifying the causative factors of liver cancer and diagnosing the risk of liver cancer through metagenomic analysis of bacteria-derived vesicles in human derivatives such as blood.
본 발명자들은 간경변 및 간암의 원인인자를 기반으로 간질환을 진단하기 위하여, 피검체 유래 샘플인 혈액에서 세균 유래 세포밖 소포로부터 유전자를 추출하고 이에 대하여 메타게놈 분석을 수행하였으며, 그 결과 간암 및 간경변 등의 간질환의 원인인자로 작용할 수 있는 세균 유래 세포밖 소포를 동정하였는바, 이에 기초하여 본 발명을 완성하였다.In order to diagnose liver disease based on the causative factors of cirrhosis and liver cancer, the present inventors extracted genes from extracellular vesicles derived from bacteria from blood, a sample derived from a subject, and performed a metagenome analysis on them. Identified bacterial extracellular vesicles that can act as a causative agent of liver disease, and the like, the present invention was completed based on this.
이에, 본 발명은 세균 유래 세포밖 소포에 대한 메타게놈 분석을 통해 간암을 진단하기 위한 정보제공방법을 제공하는 것을 목적으로 한다. 또한, 본 발명은 세균 유래 세포밖 소포에 대한 메타게놈 분석을 통해 간경변환자에서 간암을 진단하기 위한 정보제공방법을 제공하는 것을 목적으로 한다. 또한, 본 발명은 세균 유래 세포밖 소포에 대한 메타게놈 분석을 통해 간경변을 진단하기 위한 정보제공방법을 제공하는 것을 목적으로 한다. Accordingly, an object of the present invention is to provide a method for providing information for diagnosing liver cancer through metagenome analysis of extracellular vesicles derived from bacteria. In addition, an object of the present invention is to provide a method for providing information for diagnosing liver cancer in a liver cirrhosis through a metagenomic analysis of bacterial extracellular vesicles. In addition, an object of the present invention is to provide a method for providing information for diagnosing liver cirrhosis through a metagenomic analysis of bacterial extracellular vesicles.
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, 간질환 진단을 위한 정보제공방법을 제공한다. In order to achieve the object of the present invention as described above, the present invention provides a method for providing information for diagnosing liver disease, comprising the following steps.
(a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from a subject sample;
(b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
(c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계.Comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
또한, 본 발명은 하기의 단계를 포함하는, 간질환 진단방법을 제공한다.In addition, the present invention provides a method for diagnosing liver disease, comprising the following steps.
(a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from a subject sample;
(b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
(c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계.Comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
또한, 본 발명은 하기의 단계를 포함하는, 간질환의 발병 위험도 예측방법을 제공한다. In addition, the present invention provides a method for predicting the risk of developing liver disease, comprising the following steps.
(a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from a subject sample;
(b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
(c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계.Comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
본 발명의 일구현예로, 상기 (c) 단계에서 정상인 유래 샘플과 비교하여 Lactobacillales, Enterobacteriales, 및 Bacillales로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간암을 진단하는 것일 수 있다. In one embodiment of the present invention, compared to the normal sample derived from the step (c) by comparing the increase or decrease in the content of one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales It may be to diagnose liver cancer.
본 발명의 다른 구현예로, 상기 (c) 단계에서 정상인 유래 샘플과 비교하여 Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, 및 Staphylococcaceae로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간암을 진단하는 것일 수 있다.In another embodiment of the present invention, the content of at least one family bacteria-derived extracellular vesicles selected from the group consisting of Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae in comparison to the sample derived from the normal in step (c) It may be to diagnose liver cancer by comparing the increase and decrease.
본 발명의 다른 구현예로, 상기 (c) 단계에서 정상인 유래 샘플과 비교하여 Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간암을 진단하는 것일 수 있다.In another embodiment, the step (c) of the at least one genus bacteria-derived extracellular vesicles selected from the group consisting of Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella compared to the sample derived from normal It may be to diagnose liver cancer by comparing the increase and decrease of the content.
본 발명의 다른 구현예로, 상기 (c) 단계에서 간경변환자 유래 샘플과 비교하여 Trabulsiella 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간암을 진단하는 것일 수 있다. In another embodiment of the present invention, liver cancer may be diagnosed by comparing the increase and decrease of the content of Trabulsiella genus bacteria-derived extracellular vesicles in comparison with the sample derived from the cirrhosis transducer in step (c).
본 발명의 다른 구현예로, 상기 (c) 단계에서 정상인 유래 샘플과 비교하여 Streptococcaceae, Moraxellaceae, Staphylococcaceae로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간경변을 진단하는 것일 수 있다.In another embodiment of the present invention, cirrhosis is compared by increasing or decreasing the content of one or more family bacterial-derived extracellular vesicles selected from the group consisting of Streptococcaceae, Moraxellaceae, and Staphylococcaceae in comparison to the sample derived from the normal in step (c). It may be to diagnose.
본 발명의 다른 구현예로, 상기 (c) 단계에서 정상인 유래 샘플과 비교하여 Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, Succiniclasticum로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하여 간경변을 진단하는 것일 수 있다.In another embodiment of the present invention, the amount of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, and Succiniclasticum is increased in comparison with the sample derived from the normal person in the step (c). It may be to compare the diagnosis of liver cirrhosis.
본 발명의 또 다른 구현예로, 상기 샘플은 혈액일 수 있으며, 상기 혈액은 전혈, 혈청, 혈장, 또는 혈액 단핵구일 수 있다. In another embodiment of the invention, the sample may be blood, and the blood may be whole blood, serum, plasma, or blood monocytes.
환경에 존재하는 세균에서 분비되는 세포밖 소포는 체내에 흡수되어 암 발생에 직접적인 영향을 미칠 수 있으며, 간질환은 증상이 나타나기 전 조기진단이 어려워 효율적인 치료가 어려운 실정이므로, 본 발명에 따른 인체 유래 샘플을 이용한 세균 또는 세균 유래 세포밖　소포의 메타게놈 분석을 통해 간질환 발병의 위험도를 미리 예측함으로써 간질환의 위험군을 조기에 진단 및 예측하여 적절한 관리를 통해 발병 시기를 늦추거나 발병을 예방할 수 있으며, 발병 후에도 조기진단 할 수 있어 간질환의 발병률을 낮추고 치료효과를 높일 수 있다. 또한, 간질환으로 진단받은 환자에서 메타게놈 분석을 통해 원인인자를 예측하여, 원인인자에 대한 노출을 피함으로써 간질환의 경과를 좋게 하거나, 재발을 막을 수 있다. Extracellular vesicles secreted by the bacteria present in the environment can be absorbed directly into the body and have a direct impact on the development of cancer, liver disease is difficult to diagnose early due to difficult early diagnosis before symptoms appear, so the human-derived according to the present invention Metagenome analysis of bacterial or bacterial-derived extracellular vesicles using a sample predicts the risk of developing liver disease in advance, allowing early diagnosis and prediction of risk groups of liver disease, and delaying the onset or preventing the onset through proper management. In addition, early diagnosis is possible even after the onset of the disease, thereby reducing the incidence of liver disease and increasing the therapeutic effect. In addition, metagenome analysis predicts causative factors in patients diagnosed with hepatic disease, thereby avoiding exposure to causative factors, and improving the progression of liver disease or preventing recurrence.
도 1은 체내에서 세균 유래 세포밖 소포의 분포양상을 평가하기 위한 것으로, 도 1a는 마우스에 장내 세균(Bacteria) 및 세균 유래 소포(EV)를 구강으로 투여한 후 시간별(0, 5min, 3h, 6h, 및 12h)로 이들의 분포양상을 촬영한 사진이고, 도 1b는 마우스에 장내 세균(Bacteria) 및 세균 유래 세포밖 소포(EV)를 구강으로 투여하고 12시간 후 혈액 및 다양한 장기(심장, 폐, 간, 신장, 비장, 지방조직, 및 근육)를 적출하여 상기 세균 및 세포밖 소포의 분포양상을 촬영한 사진이다. Figure 1 is for evaluating the distribution of bacteria-derived extracellular vesicles in the body, Figure 1a after the administration of oral intestinal bacteria (Bacteria) and bacteria-derived vesicles (EV) in the mouth hourly (0, 5min, 3h, 6h, and 12h) is a photograph taken of their distribution, Figure 1b is 12 hours after the oral administration of intestinal bacteria (Bacteria) and bacteria-derived extracellular vesicles (EV) to the blood and various organs (heart, Lung, liver, kidney, spleen, adipose tissue, and muscles), and the photographs of the distribution of the bacterial and extracellular vesicles.
도 2는 간암환자 및 정상인 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 목(order) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.FIG. 2 shows the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the neck level after separation of bacteria-derived vesicles from liver cancer patients and normal blood.
도 3은 간암환자 및 정상인 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 과(family) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.3 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
도 4는 간암환자 및 정상인 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 속(genus) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.4 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles from liver cancer patients and normal blood, and performing a metagenome analysis.
도 5는 간암환자 및 간경변환자 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 속(genus) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.5 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level by separating the bacteria-derived vesicles in the liver cancer patients and liver cirrhosis blood.
도 6은 간경변암환자 및 정상인 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 과(family) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.6 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the family level by separating bacterial-derived vesicles from liver cirrhosis cancer patients and normal blood, and performing a metagenome analysis.
도 7은 간경변암환자 및 정상인 혈액에서 세균 유래 소포를 분리한 후, 메타게놈 분석을 수행하여 속(genus) 수준에서 진단적 성능이 유의한 세균 유래 소포(EVs)의 분포를 나타낸 결과이다.7 is a result showing the distribution of bacteria-derived vesicles (EVs) with significant diagnostic performance at the genus level after separating the bacteria-derived vesicles from liver cirrhosis cancer patients and normal blood.
본 발명은 세균 메타게놈 분석을 통해 간질환을 진단하는 방법에 관한 것으로서, 본 발명자들은 피검체 유래 샘플을 이용해 세균 유래 세포밖 소포로부터 유전자를 추출하고 이에 대하여 메타게놈 분석을 수행하였으며, 간질환의 원인인자로 작용할 수 있는 세균 유래 세포밖 소포를 동정하였다. The present invention relates to a method for diagnosing liver disease through bacterial metagenomic analysis, and the present inventors extracted a gene from bacterial extracellular vesicles using a sample derived from a subject and performed a metagenome analysis on the liver disease. Bacterial-derived extracellular vesicles that could act as causative factors were identified.
이에, 본 발명은 (a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;Thus, the present invention comprises the steps of (a) extracting DNA from the extracellular vesicles isolated from the subject sample;
(b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
(c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 포함하는 간질환 진단을 위한 정보제공방법을 제공한다. Provides an information providing method for diagnosing liver disease, comprising comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the sequencing of the PCR product.
본 발명에서 사용되는 용어, "간암 진단" 이란 환자에 대하여 간암이 발병할 가능성이 있는지, 간암이 발병할 가능성이 상대적으로 높은지, 또는 간암이 이미 발병하였는지 여부를 판별하는 것을 의미한다. 본 발명의 방법은 임의의 특정 환자에 대한 간암 발병 위험도가 높은 환자로써 특별하고 적절한 관리를 통하여 발병 시기를 늦추거나 발병하지 않도록 하는데 사용할 수 있다. 또한, 본 발명의 방법은 간암을 조기에 진단하여 가장 적절한 치료방식을 선택함으로써 치료를 결정하기 위해 임상적으로 사용될 수 있다.As used herein, the term "diagnostic liver cancer" means to determine whether there is a possibility of developing liver cancer, relatively high chance of developing liver cancer, or whether liver cancer has already occurred. The method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing liver cancer for any particular patient. In addition, the methods of the present invention can be used clinically to determine treatment by early diagnosis of liver cancer and selecting the most appropriate treatment regimen.
본 발명에서 사용되는 용어, "간경변 진단" 이란 환자에 대하여 간경변이 발병할 가능성이 있는지, 간경변이 발병할 가능성이 상대적으로 높은지, 또는 간경변이 이미 발병하였는지 여부를 판별하는 것을 의미한다. 본 발명의 방법은 임의의 특정 환자에 대한 간경변 발병 위험도가 높은 환자로써 특별하고 적절한 관리를 통하여 발병 시기를 늦추거나 발병하지 않도록 하는데 사용할 수 있다. 또한, 본 발명의 방법은 간경변을 조기에 진단하여 가장 적절한 치료방식을 선택함으로써 치료를 결정하기 위해 임상적으로 사용될 수 있다.As used herein, the term "diagnosing cirrhosis" means to determine whether there is a possibility of developing cirrhosis, a relatively high chance of developing cirrhosis, or whether cirrhosis has already occurred. The method of the present invention can be used to prevent or delay the onset of the disease through special and appropriate management as a patient at high risk of developing cirrhosis for any particular patient. In addition, the methods of the present invention can be used clinically to determine treatment by early diagnosis of cirrhosis and selecting the most appropriate treatment regimen.
본 발명에서 사용되는 용어, "메타게놈(metagenome)"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 또는 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. 본 발명에서는 바람직하게 혈청에서 분리한 세균 유래 세포밖　소포를 이용하여 메타게놈 분석을 실시하였다.The term "metagenome" used in the present invention, also referred to as "metagenome", refers to the total of the genome including all viruses, bacteria, fungi, etc. in an isolated area such as soil, animal intestine, It is mainly used as a concept of genome explaining the identification of many microorganisms at once using sequencer to analyze microorganisms which are not cultured. In particular, metagenome does not refer to one species of genome or genome, but refers 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. In the present invention, metagenome analysis was preferably performed using bacterial-derived extracellular vesicles isolated from serum.
본 발명에 있어서, 상기 피검체 샘플은 혈액일 수 있고, 상기 혈액은 바람직하게 전혈, 혈청, 혈장, 또는 혈액 단핵구일 수 있으나, 이것으로 제한되는 것은 아니다.In the present invention, the subject sample may be blood, and the blood may preferably be whole blood, serum, plasma, or blood monocytes, but is not limited thereto.
본 발명의 실시예에서는 정상인, 간경변환자, 및 간암환자의 혈액 내 세균 유래 세포밖 소포에 대한 메타게놈 분석을 실시하였으며, 문(phylum), 강(class), 목(order), 과(family), 및 속(genus) 수준에서 각각 분석하여 실제로 간암 및 간경변 발생의 원인으로 작용할 수 있는 세균 유래 소포를 동정하였다.In an embodiment of the present invention, metagenome analysis was performed on extracellular vesicles derived from bacteria in the blood of normal people, liver cirrhosis patients, and liver cancer patients, and were classified into phylum, class, order, and family. Analyzes at the,, and genus levels were respectively identified to identify bacterial-derived vesicles that could actually cause the development of liver cancer and cirrhosis.
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 목 수준에서 분석한 결과, Lactobacillales, Enterobacteriales, 및 Bacillales 목 세균 유래 소포가 간암환자와 정상인 사이에 유의한 차이가 있었다(실시예 4 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacterial-derived metagenome at the neck level, there was a significant difference between Lactobacillales, Enterobacteriales, and Bacillales neck bacteria-derived vesicles between liver cancer patients and normal people (see Example 4). ).
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 과 수준에서 분석한 결과, Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, 및 Staphylococcaceae 과 세균 유래 소포가 간암환자와 정상인 사이에 유의한 차이가 있었다(실시예 4 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacteria-derived metagenome at the excessive level, there was a significant difference between Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae and bacterial-derived vesicles between normal and liver cancer patients ( See Example 4).
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 속 수준에서 분석한 결과, Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, 및 Trabulsiella 속 세균 유래 소포가 간암환자와 정상인 사이에 유의한 차이가 있었다(실시예 4 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacteria-derived metagenome at the genus level, there is a significant difference between liver cancer patients and normal vesicles derived from Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella (See Example 4).
본 발명의 또 다른 실시예에서는 간경변환자와 간암환자의 혈액 내 세균 유래 세포밖 소포에 대한 메타게놈 분석을 실시하였으며, 문(phylum), 강(class), 목(order), 과(family), 및 속(genus) 수준에서 각각 분석하여 실제로 간경변환자에서 간암 발생의 원인으로 작용할 수 있는 세균 유래 소포를 동정하였다.In another embodiment of the present invention, metagenome analysis of extracellular vesicles derived from bacteria in the blood of liver cirrhosis and liver cancer patients was carried out, including phylum, class, order, family, Analyzes at the genus and genus levels, respectively, identified bacterial vesicles that can actually cause liver cancer in cirrhosis transducers.
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 속 수준에서 분석한 결과, Trabulsiella 속 세균 유래 소포가 간암환자와 간경변환자 사이에 유의한 차이가 있었다(실시예 5 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacteria-derived metagenome at the genus level, there was a significant difference between the liver-derived liver cancer patients and liver cirrhosis vesicles derived from Trabulsiella genus (see Example 5).
본 발명의 또 다른 실시예에서는 정상인과 간경변환자의 혈액 내 세균 유래 세포밖 소포에 대한 메타게놈 분석을 실시하였으며, 문(phylum), 강(class), 목(order), 과(family), 및 속(genus) 수준에서 각각 분석하여 실제로 천식환자에서 간암 발생의 원인으로 작용할 수 있는 세균 유래 소포를 동정하였다.In another embodiment of the present invention, the metagenome analysis of extracellular vesicles derived from bacteria in the blood of normal people and liver cirrhosis was performed, including phylum, class, order, family, and Analyzes at the genus level identified bacterial vesicles that could actually cause liver cancer in asthma patients.
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 과 수준에서 분석한 결과, Streptococcaceae, Moraxellaceae, 및 Staphylococcaceae 과 세균 유래 소포가 간경변환자와 정상인 사이에 유의한 차이가 있었다(실시예 6 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacteria-derived metagenome at the excessive level, there was a significant difference between Streptococcaceae, Moraxellaceae, and Staphylococcaceae and bacteria-derived vesicles between liver cirrhosis and normal people (see Example 6). ).
보다 구체적으로 본 발명의 일실시예에서는, 세균 유래 메타게놈을 속 수준에서 분석한 결과, Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, 및 Succiniclasticum 속 세균 유래 소포가 간경변환자와 정상인 사이에 유의한 차이가 있었다(실시예 6 참조).More specifically, in one embodiment of the present invention, as a result of analyzing the bacteria-derived metagenome at the genus level, there was a significant difference between the cirrhosis and normal people of the bacteria-derived vesicles of Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, and Succiniclasticum. See Example 6.
본 발명은 상기와 같은 실시예 결과를 통해, 혈액으로부터 분리한 세균 유래 세포밖 소포에 대하여 메타게놈 분석을 실시함으로써 정상인 및 간경변환자와 비교하여 간암환자에서 함량이 유의하게 변화한 세균 유래 소포들을 동정하였으며, 메타게놈 분석을 통해 상기 각 수준에서 세균 유래 소포들의 함량 증감을 분석함으로써 간암을 진단할 수 있음을 확인하였다.The present invention, through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from blood by metagenomic analysis to identify bacteria-derived vesicles significantly changed in liver cancer patients compared to normal people and liver cirrhosis Meta-genomic analysis confirmed that liver cancer can be diagnosed by analyzing the increase and decrease of the content of bacterial-derived vesicles at each level.
또한, 본 발명은 상기와 같은 실시예 결과를 통해, 혈액으로부터 분리한 세균 유래 세포밖 소포에 대하여 메타게놈 분석을 실시함으로써 정상인과 비교하여 간경변환자에서 함량이 유의하게 변화한 세균 유래 소포들을 동정하였으며, 메타게놈 분석을 통해 상기 각 수준에서 세균 유래 소포들의 함량 증감을 분석함으로써 간경변을 진단할 수 있음을 확인하였다.In addition, the present invention, through the results of the above embodiment, by identifying the bacteria-derived extracellular vesicles isolated from the blood by performing a genome analysis of bacteria-derived vesicles with significantly changed content in liver cirrhosis compared to normal people , Metagenomic analysis confirmed that liver cirrhosis can be diagnosed by analyzing the increase and decrease of the content of the bacteria-derived vesicles at each level.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[[ 실시예Example ]]
실시예Example 1. 장내 세균 및 세균 유래 소포의 체내 흡수, 분포, 및 배설 양상 분석 1. Analysis of absorption, distribution, and excretion of intestinal bacteria and bacterial-derived vesicles
장내 세균과 세균 유래 소포가 위장관을 통해 전신적으로 흡수되는 지를 평가하기 위하여 다음과 같은 방법으로 실험을 수행하였다. 마우스의 위장에 형광으로 표지한 장내세균과 장내 세균 유래 소포를 각각 50 μg의 용량으로 위장관으로 투여하고 0분, 5분, 3시간, 6시간, 12시간 후에 형광을 측정하였다. 마우스 전체 이미지를 관찰한 결과, 도 1a에 나타낸 바와 같이, 상기 세균(Bacteria)인 경우에는 전신적으로 흡수되지 않았지만, 세균 유래 소포(EV)인 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 entire image of the mouse, as shown in FIG. 1A, the bacteria (Bacteria) were not absorbed systemically, but in the case of bacteria-derived vesicles (EV), they were absorbed systemically 5 minutes after administration and administered. After 3 hours, the bladder was strongly observed, 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.
장내세균과 장내 세균유래 소포가 전신적으로 흡수된 후, 여러 장기로 침윤된 양상을 평가하기 위하여, 형광으로 표지한 50 μg의 세균과 세균유래 소포를 상기의 방법과 같이 투여한 다음 12시간째에 마우스로부터 혈액(Blood), 심장(Heart), 폐(Lung), 간(Liver), 신장(Kidney), 비장(Spleen), 지방조직(Adipose tissue), 및 근육(Muscle)을 적출하였다. 상기 적출한 조직들에서 형광을 관찰한 결과, 도1b에 나타낸 바와 같이, 상기 장내 세균(Bacteria)은 각 장기에 흡수되지 않은 반면, 상기 장내 세균 유래 세포밖 소포(EV)는 혈액, 심장, 폐, 간, 신장, 비장, 지방조직, 및 근육에 분포하는 것을 확인하였다.After the systemic absorption of enterobacteriaceae and enteric bacteria-derived vesicles systemically, in order to assess the invasion of various organs, the fluorescently labeled 50 μg of bacteria and bacteria-derived vesicles were administered in the same manner as above 12 hours. Blood, Heart, Lung, Liver, Kidney, Spleen, Adipose tissue, and Muscle were extracted from mice. As shown in FIG. 1B, the intestinal bacteria (Bacteria) were not absorbed into each organ, whereas the intestinal bacteria-derived extracellular vesicles (EV) were detected in the tissues, as shown in FIG. And distribution in liver, kidney, spleen, adipose tissue, and muscle.
실시예Example 2. 혈액으로부터 소포 분리 및 DNA 추출 2. Isolation of Vesicles and DNA Extraction from Blood
혈액으로부터 소포를 분리하고 DNA를 추출하기 위해, 먼저 10 ㎖ 튜브에 혈액을 넣고 원심분리(3,500 x g, 10min, 4℃)를 실시하여 부유물을 가라앉혀 상등액만을 회수한 후 새로운 10 ㎖ 튜브에 옮겼다. 0.22 ㎛ 필터를 사용하여 상기 회수한 상등액으로부터 세균 및 이물질을 제거한 후, 센트리프랩튜브(centripreigugal filters 50 kD)에 옮기고 1500 x g, 4℃에서 15분간 원심분리하여 50 kD 보다 작은 물질은 버리고 10 ㎖까지 농축 시켰다. 다시 한 번 0.22 ㎛ 필터를 사용하여 박테리아 및 이물질을 제거한 후, Type 90ti 로터로 150,000 x g, 4℃에서 3시간 동안 초고속원심분리방법을 사용하여 상등액을 버리고 덩어리진 pellet을 생리식염수(PBS)로 녹여 소포를 수득하였다. To separate the vesicles from the blood and extract the DNA, the blood was first placed in a 10 ml tube and centrifuged (3,500 × g, 10 min, 4 ° C.) to settle the suspended solids to recover only the supernatant and then transferred to a new 10 ml tube. After removing the bacteria and foreign substances from the recovered supernatant using a 0.22 ㎛ filter, transfer to centripreigugal filters (50 kD) and centrifuged at 1500 xg, 4 ℃ for 15 minutes to discard the material smaller than 50 kD and 10 ml Concentrated until. Once again, remove the bacteria and foreign substances using a 0.22 ㎛ filter, discard the supernatant using ultra-fast centrifugation for 3 hours at 150,000 xg, 4 ℃ with a Type 90ti rotor and dissolve the agglomerated pellet in physiological saline (PBS) Vesicles were obtained.
상기 방법에 따라 혈액으로부터 분리한 소포 100 ㎕를 100℃에서 끓여서 내부의 DNA를 지질 밖으로 나오게 한 후 얼음에 5분 동안 식혔다. 다음으로 남은 부유물을 제거하기 위하여 10,000 x g, 4℃에서 30분간 원심분리하고 상등액 만을 모은 후 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.According to the above method, 100 μl of the vesicles isolated from blood were boiled at 100 ° C. to let the DNA inside the lipid out and then cooled on ice for 5 minutes. Next, in order to remove the remaining suspended matter, centrifugation at 10,000 x g, 4 ℃ for 30 minutes, and collected only the supernatant and quantified the DNA amount 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.
primerprimer 서열order 서열번호SEQ ID NO:
16S rDNA16S rDNA 16S_V3_F16S_V3_F 5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3'5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3 ' 1One
16S_V4_R16S_V4_R 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-35'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3 22
실시예Example 3. 혈액 내 소포에서 추출한 DNA를 이용한  3. Using DNA extracted from vesicles in blood 메타게놈Metagenome 분석 analysis
상기 실시예 2의 방법으로 유전자를 추출한 후, 상기 표1에 나타낸 16S rDNA 프라이머를 사용하여 PCR을 실시하여 유전자를 증폭시키고 시퀀싱(Illumina MiSeq sequencer)을 수행하였다. 결과를 Standard Flowgram Format(SFF) 파일로 출력하고 GS FLX software(v2.9)를 이용하여 SFF 파일을 sequence 파일(.fasta)과 nucleotide quality score 파일로 변환한 다음 리드의 신용도 평가를 확인하고, window(20 bps) 평균 base call accuracy가 99% 미만(Phred score <20)인 부분을 제거하였다. 질이 낮은 부분을 제거한 후, 리드의 길이가 300 bps 이상인 것만 이용하였으며(Sickle version 1.33), 결과 분석을 위해 Operational Taxonomy Unit(OTU)은 UCLUST와 USEARCH를 이용하여 시퀀스 유사도에 따라 클러스터링을 수행하였다. 구체적으로 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, 강, 목, 과, 속 레벨의 분류를 수행하고, BLASTN와 GreenGenes의 16S DNA 시퀀스 데이터베이스(108,453 시퀀스)를 이용하여 97% 이상의 시퀀스 유사도 갖는 박테리아를 분석하였다(QIIME).After the gene was extracted by the method of Example 2, PCR was performed using the 16S rDNA primer shown in Table 1 to amplify the gene and perform sequencing (Illumina MiSeq sequencer). Output the result as a Standard Flowgram Format (SFF) file, convert the SFF file into a sequence file (.fasta) and a nucleotide quality score file using GS FLX software (v2.9), check the credit rating of the lead, and window (20 bps) The part with the average base call accuracy of less than 99% (Phred score <20) was removed. After removing the low quality part, only the lead length was 300 bps or more (Sickle version 1.33), and the Operational Taxonomy Unit (OTU) performed UCLUST and USEARCH for clustering according to sequence similarity. Specifically, the clustering is based on 94% genus, 90% family, 85% order, 80% class, and 75% sequence similarity. OTU's door, river, neck, family and genus level classifications were performed, and bacteria with greater than 97% sequence similarity were analyzed using BLASTN and GreenGenes' 16S DNA sequence database (108,453 sequences) (QIIME).
실시예Example 4. 정상인과 간암환자 혈액에서 분리한  4. Separated from normal and liver cancer patients blood 세균유래Germ-derived 소포  parcel 메타게놈Metagenome 분석 기반 간암 진단모형 Analysis-based Liver Cancer Diagnosis Model
상기 실시예 3의 방법으로, 간암환자 86명과 나이와 성별을 매칭한 정상인 331명의 혈액에서 소포를 분리한 후 메타게놈 시퀀싱을 수행하였다. 진단모형 개발은 먼저 t-test에서 두 군 사이의 p값이 0.05 이하이고, 두 군 사이에 2배 이상 차이가 나는 균주를 선정하고 난 후, logistic regression analysis 방법으로 진단적 성능 지표인 AUC(area under curve), 민감도, 및 특이도를 산출하였다.By the method of Example 3, metagenome sequencing was performed after separating vesicles from the blood of 86 liver cancer patients and 331 normal humans with age and gender matching. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
혈액 내 세균유래 소포를 목(order) 수준에서 분석한 결과, Lactobacillales, Enterobacteriales, 및 Bacillales 목 세균에서 하나 이상의 바이오마커로 진단모형을 개발하였을 때, 간암에 대한 진단적 성능이 유의하게 나타났다 (표 2 및 도 2 참조).Analysis of bacterial vesicles in the blood at the order level showed significant diagnostic performance for liver cancer when developing a diagnostic model with one or more biomarkers in Lactobacillales, Enterobacteriales, and Bacillales throat bacteria (Table 2). And FIG. 2).
　 정상대조군Normal control 간암Liver cancer t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
o__Lactobacillaleso__Lactobacillales 0.09720.0972 0.06300.0630 0.03450.0345 0.03450.0345 0.00000.0000 0.360.36 0.870.87 0.920.92 0.420.42 0.890.89 0.950.95 0.450.45
o__Enterobacterialeso__Enterobacteriales 0.07220.0722 0.05150.0515 0.17470.1747 0.17470.1747 0.00000.0000 2.422.42 0.800.80 0.980.98 0.440.44 0.740.74 0.990.99 0.350.35
o__Bacillaleso__Bacillales 0.03600.0360 0.02950.0295 0.11820.1182 0.11820.1182 0.00000.0000 3.283.28 0.870.87 0.960.96 0.530.53 0.870.87 0.960.96 0.500.50
혈액 내 세균유래 소포를 과(family) 수준에서 분석한 결과, Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, 및 Staphylococcaceae 과 세균에서 하나 이상의 바이오마커로 진단모형을 개발하였을 때, 간암에 대한 진단적 성능이 유의하게 나타났다 (표 3 및 도 3 참조).Family-level analysis of bacterial vesicles in the blood showed that diagnostic performance for liver cancer was significantly improved when the diagnostic model was developed with one or more biomarkers in Streptococcaceae, Pasteurellaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae. (See Table 3 and FIG. 3).
　 정상대조군Normal control 간암Liver cancer t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
f__Streptococcaceaef__Streptococcaceae 0.05730.0573 0.05520.0552 0.01280.0128 0.01280.0128 0.00000.0000 0.220.22 0.860.86 0.940.94 0.480.48 0.880.88 0.940.94 0.550.55
f__Pasteurellaceaef__Pasteurellaceae 0.00410.0041 0.00820.0082 0.00180.0018 0.00180.0018 0.00100.0010 0.430.43 0.680.68 1.001.00 0.020.02 0.640.64 1.001.00 0.050.05
f__Enterobacteriaceaef__Enterobacteriaceae 0.07220.0722 0.05150.0515 0.17470.1747 0.17470.1747 0.00000.0000 2.422.42 0.800.80 0.980.98 0.440.44 0.740.74 0.990.99 0.350.35
f__Moraxellaceaef__Moraxellaceae 0.03950.0395 0.04750.0475 0.10610.1061 0.10610.1061 0.00000.0000 2.692.69 0.790.79 0.960.96 0.180.18 0.870.87 0.990.99 0.400.40
f__Staphylococcaceaef__Staphylococcaceae 0.01980.0198 0.02430.0243 0.10540.1054 0.10540.1054 0.00000.0000 5.315.31 0.900.90 0.960.96 0.550.55 0.860.86 0.950.95 0.600.60
혈액 내 세균유래 소포를 속(genus) 수준에서 분석한 결과, Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, 및 Trabulsiella 속 세균에서 하나 이상의 바이오마커로 진단모형을 개발하였을 때, 간암에 대한 진단적 성능이 유의하게 나타났다 (표 4 및 도 4 참조).As a result of analyzing genotype-derived vesicles in the blood at the genus level, the diagnostic performance of liver cancer is improved when developing a diagnostic model with one or more biomarkers in bacteria of Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, and Trabulsiella. Significantly (see Table 4 and FIG. 4).
　 정상대조군Normal control 간암Liver cancer t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
g__Streptococcusg__Streptococcus 0.05680.0568 0.05530.0553 0.01240.0124 0.01240.0124 0.00000.0000 0.220.22 0.860.86 0.940.94 0.480.48 0.880.88 0.940.94 0.500.50
g__Blautiag__Blautia 0.00950.0095 0.01340.0134 0.00430.0043 0.00430.0043 0.00000.0000 0.450.45 0.680.68 0.990.99 0.020.02 0.690.69 1.001.00 0.050.05
g__Klebsiellag__Klebsiella 0.02210.0221 0.02940.0294 0.05460.0546 0.05460.0546 0.00000.0000 2.482.48 0.740.74 0.960.96 0.210.21 0.720.72 0.970.97 0.150.15
g__Acinetobacterg__Acinetobacter 0.01790.0179 0.02430.0243 0.07250.0725 0.07250.0725 0.00000.0000 4.054.05 0.800.80 0.960.96 0.300.30 0.820.82 0.980.98 0.400.40
g__Staphylococcusg__Staphylococcus 0.01980.0198 0.02430.0243 0.10530.1053 0.10530.1053 0.00000.0000 5.325.32 0.900.90 0.960.96 0.550.55 0.860.86 0.950.95 0.600.60
g__Trabulsiellag__Trabulsiella 0.00740.0074 0.01470.0147 0.04360.0436 0.04360.0436 0.00000.0000 5.905.90 0.820.82 0.960.96 0.500.50 0.780.78 0.970.97 0.450.45
실시예Example 5. 간경변환자와 간암환자 혈액에서 분리한  5. Separated from liver cirrhosis and liver cancer patients blood 세균유래Germ-derived 소포  parcel 메타게놈Metagenome 분석 기반 간암 진단모형 Analysis-based Liver Cancer Diagnosis Model
상기 실시예 3의 방법으로, 간암환자 84명과 간경변환자 86명의 혈액에서 소포를 분리한 후 메타게놈 시퀀싱을 수행하였다. 진단모형 개발은 먼저 t-test에서 두 군 사이의 p값이 0.05 이하이고, 두 군 사이에 2배 이상 차이가 나는 균주를 선정하고 난 후, logistic regression analysis 방법으로 진단적 성능 지표인 AUC(area under curve), 민감도, 및 특이도를 산출하였다.By the method of Example 3, vesicles were isolated from blood of 84 liver cancer patients and 86 cirrhosis transducers, and metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
혈액 내 세균유래 소포를 속(genus) 수준에서 분석한 결과, Trabulsiella속 세균을 바이오마커로 진단모형을 개발하였을 때, 간암에 대한 진단적 성능이 유의하게 나타났다 (표 5 및 도 5 참조).As a result of analyzing the bacteria-derived vesicles in the blood at the genus level, when the diagnostic model was developed as a biomarker of the genus Trabulsiella, the diagnostic performance for liver cancer was significant (see Table 5 and FIG. 5).
　 간경변Cirrhosis 간암Liver cancer t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
g__Trabulsiellag__Trabulsiella 0.01970.0197 0.03250.0325 0.04360.0436 0.04360.0436 0.00010.0001 2.222.22 0.730.73 0.830.83 0.470.47 0.550.55 0.620.62 0.420.42
실시예Example 6. 정상인과 간경변 혈액에서 분리한  6. Separated from normal and cirrhosis blood 세균유래Germ-derived 소포  parcel 메타게놈Metagenome 분석 기반 간경변 진단모형 Analysis-based Liver Cirrhosis Diagnosis Model
상기 실시예 3의 방법으로, 간암환자 84명과 정상인 331명의 혈액에서 소포를 분리한 후 메타게놈 시퀀싱을 수행하였다. 진단모형 개발은 먼저 t-test에서 두 군 사이의 p값이 0.05 이하이고, 두 군 사이에 2배 이상 차이가 나는 균주를 선정하고 난 후, logistic regression analysis 방법으로 진단적 성능 지표인 AUC(area under curve), 민감도, 및 특이도를 산출하였다.In the method of Example 3, vesicles were isolated from blood of 84 liver cancer patients and 331 normal humans, and then metagenome sequencing was performed. In the development of the diagnostic model, the strains whose p-value between the two groups is 0.05 or less and more than two times different between the two groups are selected in the t-test. under curve), sensitivity, and specificity.
혈액 내 세균유래 소포를 과(family) 수준에서 분석한 결과, Streptococcaceae, Moraxellaceae, 및 Staphylococcaceae 과 세균에서 하나 이상의 바이오마커로 진단모형을 개발하였을 때, 간경변에 대한 진단적 성능이 유의하게 나타났다 (표 6 및 도 6 참조).The analysis of blood-derived vesicles at the family level showed significant diagnostic performance for liver cirrhosis when developing a diagnostic model with one or more biomarkers in Streptococcaceae, Moraxellaceae, and Staphylococcaceae and bacteria (Table 6). And FIG. 6).
　 정상대조군Normal control 간경변Cirrhosis t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
f__Streptococcaceaef__Streptococcaceae 0.05730.0573 0.05520.0552 0.01830.0183 0.01830.0183 0.00000.0000 0.320.32 0.800.80 0.970.97 0.220.22 0.860.86 0.960.96 0.250.25
f__Moraxellaceaef__Moraxellaceae 0.03950.0395 0.04750.0475 0.08210.0821 0.08210.0821 0.00000.0000 2.082.08 0.710.71 0.980.98 0.080.08 0.750.75 0.970.97 0.130.13
f__Staphylococcaceaef__Staphylococcaceae 0.01980.0198 0.02430.0243 0.05740.0574 0.05740.0574 0.00000.0000 2.892.89 0.740.74 0.970.97 0.230.23 0.680.68 0.970.97 0.210.21
혈액 내 세균유래 소포를 속(genus) 수준에서 분석한 결과, Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, 및 Succiniclasticum 속 세균에서 하나 이상의 바이오마커로 진단모형을 개발하였을 때, 간경변에 대한 진단적 성능이 유의하게 나타났다 (표 7 및 도 7 참조).As a result of analyzing genotype-derived vesicles in the blood at genus level, the diagnostic performance of liver cirrhosis was significantly increased when developing a diagnostic model with one or more biomarkers in Streptococcus, Acinetobacter, Trabulsiella, Staphylococcus, and Succiniclasticum bacteria. (See Table 7 and FIG. 7).
　 정상대조군Normal control 간경변Cirrhosis t-testt-test Training SetTraining set Test SetTest set
TaxonTaxon MeanMean SDSD MeanMean SDSD p-valuep-value RatioRatio AUCAUC sensitivitysensitivity specificityspecificity AUCAUC sensitivitysensitivity specificityspecificity
g__Streptococcusg__Streptococcus 0.05680.0568 0.05530.0553 0.01770.0177 0.01770.0177 0.00000.0000 0.310.31 0.840.84 0.960.96 0.290.29 0.790.79 0.960.96 0.230.23
g__Acinetobacterg__Acinetobacter 0.01790.0179 0.02430.0243 0.04190.0419 0.04190.0419 0.00000.0000 2.342.34 0.700.70 0.980.98 0.100.10 0.720.72 1.001.00 0.040.04
g__Trabulsiellag__Trabulsiella 0.00740.0074 0.01470.0147 0.01970.0197 0.01970.0197 0.00110.0011 2.662.66 0.690.69 0.990.99 0.030.03 0.700.70 1.001.00 0.120.12
g__Staphylococcusg__Staphylococcus 0.01980.0198 0.02430.0243 0.05740.0574 0.05740.0574 0.00000.0000 2.892.89 0.710.71 0.980.98 0.210.21 0.760.76 1.001.00 0.230.23
g__Succiniclasticumg__Succiniclasticum 0.00000.0000 0.00020.0002 0.00360.0036 0.00360.0036 0.00820.0082 241.16241.16 0.670.67 1.001.00 0.090.09 0.660.66 0.990.99 0.100.10
상기 진술한 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 description of the present invention set forth above is for illustrative purposes, and one of ordinary skill in the art may understand that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 인체 유래 샘플을 이용한 세균 유래 세포밖　소포에 존재하는 유전자에 대하여 메타게놈 분석을 통해 간암 및 간경변 발병의 위험도를 미리 예측함으로써 간질환의 위험군을 조기에 진단 및 예측하여 적절한 관리를 통해 발병 시기를 늦추거나 발병을 예방할 수 있으며, 간경변 혹은 간암 발병 후에도 조기진단 할 수 있어 간질환의 발병률을 낮추고 치료효과를 높일 수 있다. By predicting the risk of liver cancer and cirrhosis in advance through metagenomic analysis on the genes present in bacteria-derived extracellular parcel vesicles using a human-derived sample according to the present invention, early diagnosis and prediction of risk groups of liver disease are made through appropriate management. It can delay the onset or prevent the onset, and early diagnosis even after the occurrence of cirrhosis or liver cancer can reduce the incidence of liver disease and increase the therapeutic effect.

Claims (14)

  1. (a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from a subject sample;
    (b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
    (c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
    상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
    상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 포함하는, 간질환 진단을 위한 정보제공방법.Comparing the increase and decrease of the content of the bacterial-derived extracellular vesicles in a sample derived from normal people and liver cirrhosis through the sequencing of the PCR product, information providing method for diagnosing liver disease.
  2. 제1항에 있어서, The method of claim 1,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간암을 진단하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.In step (c), the liver product is diagnosed by comparing the increase or decrease in the content of bacterial-derived extracellular vesicles in a sample derived from a normal person and liver cancer patient by sequencing PCR products, information for diagnosing liver disease. How to Provide.
  3. 제2항에 있어서, The method of claim 2,
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum Comparing the increase or decrease of the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of, Information providing method for diagnosing liver disease.
  4. 제1항에 있어서, The method of claim 1,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간암을 진단하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.In the step (c), liver cancer is diagnosed by comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the liver cirrhosis and liver cancer patient-derived sample by sequencing the PCR product, for diagnosing liver disease How to Provide Information.
  5. 제4항에 있어서, The method of claim 4, wherein
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum Comparing the increase or decrease of the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of, Information providing method for diagnosing liver disease.
  6. 제1항에 있어서, The method of claim 1,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간경변을 진단하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.In step (c), liver cirrhosis is characterized by comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in a sample derived from a normal person and a cirrhosis transducer by sequencing the PCR product, information for diagnosing liver disease. How to Provide.
  7. 제6항에 있어서, The method of claim 6,
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단을 위한 정보제공방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum Comparing the increase or decrease of the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of, Information providing method for diagnosing liver disease.
  8. (a) 피검체 샘플에서 분리한 세포밖 소포로부터 DNA를 추출하는 단계;(a) extracting DNA from extracellular vesicles isolated from a subject sample;
    (b) 상기 추출한 DNA에 대하여 서열번호 1 및 서열번호 2의 프라이머 쌍을 이용하여 PCR을 수행하는 단계; 및(b) performing PCR using the primer pairs of SEQ ID NO: 1 and SEQ ID NO: 2 on the extracted DNA; And
    (c) 상기 PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는(c) comparing the increase and decrease in the content of bacterial-derived extracellular vesicles in the sample derived from normal and liver cancer patients by sequencing the PCR product; or
    상기 PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계; 또는Comparing the increase or decrease in the content of bacterial extracellular vesicles from the liver cirrhosis and liver cancer patient-derived samples by sequencing the PCR product; or
    상기 PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 포함하는, 간질환 진단방법.Comprising the step of comparing the increase and decrease of the content of the bacteria-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through the PCR product sequencing.
  9. 제8항에 있어서, The method of claim 8,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 정상인과 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간암을 진단하는 것을 특징으로 하는, 간질환 진단방법.In the step (c), liver cancer diagnosis method, characterized in that the liver cancer through the step of comparing the increase and decrease of the content of the bacterial-derived extracellular vesicles in the sample derived from normal people and liver cancer patients by sequencing the PCR product.
  10. 제9항에 있어서, The method of claim 9,
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum A method for diagnosing liver disease, characterized by comparing the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of:
  11. 제8항에 있어서, The method of claim 8,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 간경변환자와 간암환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간암을 진단하는 것을 특징으로 하는, 간질환 진단방법.In the step (c), liver cancer diagnosis method, characterized in that the liver cancer through the step of comparing the increase and decrease of the content of bacterial-derived extracellular vesicles in the sample derived from liver cirrhosis and liver cancer patients by sequencing the PCR product.
  12. 제11항에 있어서, The method of claim 11,
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum A method for diagnosing liver disease, characterized by comparing the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of:
  13. 제8항에 있어서, The method of claim 8,
    상기 (c)단계에서, PCR 산물의 서열분석을 통하여 정상인과 간경변환자 유래 샘플에서 세균 유래 세포밖 소포의 함량 증감을 비교하는 단계를 통하여 간경변을 진단하는 것을 특징으로 하는, 간질환 진단방법.In step (c), the liver disease diagnosis method, characterized in that the liver cirrhosis is diagnosed by comparing the increase and decrease of the content of bacteria-derived extracellular vesicles in the sample derived from normal people and liver cirrhosis through PCR product sequencing.
  14. 제13항에 있어서, The method of claim 13,
    상기 (c)단계에서, 락토바실레아레스(Lactobacillales, 엔테로박테리아레스(Enterobacteriales, 및 간균목(Bacillales)으로 이루어진 군으로부터 선택되는 1종 이상의 목(order) 세균 유래 세포밖 소포,In the step (c), one or more order bacteria-derived extracellular vesicles selected from the group consisting of Lactobacillales, Enterobacteriales, and Bacillales,
    스트렙토코카시에(Streptococcaceae), 파스테우렐라시에(Pasteurellaceae), 엔테로박테리아시에(Enterobacteriaceae), 모락셀라과(Moraxellaceae), 및 포도상구균과(Staphylococcaceae)로 이루어진 군으로부터 선택되는 1종 이상의 과(family) 세균 유래 세포밖 소포, 또는At least one family selected from the group consisting of Streptococcaceae, Pasteurllaceae, Enterobacteriaceae, Moraxellaceae, and Staphylococcaceae ) Extracellular vesicles derived from bacteria, or
    연쇄상구균(Streptococcus), 블라우티아(Blautia), 클렙시엘라(Klebsiella), 아시네토박터(Acinetobacter), 포도상구균속(Staphylococcus), 트라불시엘라(Trabulsiella), 및 숙시니클라스티쿰속(Succiniclasticum)로 이루어진 군으로부터 선택되는 1종 이상의 속(genus) 세균 유래 세포밖 소포의 함량 증감을 비교하는 것을 특징으로 하는, 간질환 진단방법.Streptococcus, Blautia, Klebsiella, Acinetobacter, Staphylococcus, Trabulsiella, and Succiniclasticum A method for diagnosing liver disease, characterized by comparing the increase or decrease in the content of one or more genus bacteria-derived extracellular vesicles selected from the group consisting of:
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