KR101657051B1 - Marker composition for diagnosis of chronic obstructive pulmonary disease - Google Patents
Marker composition for diagnosis of chronic obstructive pulmonary disease Download PDFInfo
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Abstract
본 발명은 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 제제를 포함하는, 만성폐쇄성폐질환 (chronic obstructive pulmonary disease, COPD) 진단용 조성물, 상기 조성물을 포함하는 만성폐쇄성폐질환 진단 키트 및 상기 조성물을 이용한 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법에 관한 것이다. 본 발명의 마커 유전자인 SKIL은 만성폐쇄성폐질환 환자에서 정상인에 비해 현저하게 발현이 증가하기 때문에, 만성폐쇄성폐질환의 발병 유무를 정확하고 빠르게 진단하는데 유용하게 이용될 수 있다. The present invention relates to a method for measuring the expression level of mRNA of mRNA of SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD), a kit for diagnosing chronic obstructive pulmonary disease including the composition, and a method for providing information for diagnosis of chronic obstructive pulmonary disease using the composition . SKIL, which is a marker gene of the present invention, is remarkably increased in patients with chronic obstructive pulmonary disease as compared with normal individuals, so SKIL can be used to accurately and quickly diagnose the onset of COPD.
Description
본 발명은 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 제제를 포함하는, 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD) 진단용 조성물, 상기 조성물을 포함하는 만성폐쇄성폐질환 진단 키트, 상기 마커를 이용한 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법에 관한 것이다.
The present invention relates to a method for measuring the expression level of mRNA of mRNA of SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) A composition for the diagnosis of chronic obstructive pulmonary disease (COPD), a kit for diagnosing chronic obstructive pulmonary disease comprising the composition, and a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker .
만성폐쇄성폐질환(Chronic Obstructive Pulmonary Disease, COPD)은 유해한 입자나 가스의 흡입에 의해 폐에 비정상적인 염증 반응이 일어나면서 이로 인해 점차 기류 제한이 진행되어 폐 기능이 저하되고 호흡곤란을 유발하게 되는 호흡기 질환이다. 폐기종, 만성 기관지염 등이 이에 속한다.Chronic Obstructive Pulmonary Disease (COPD) is a chronic obstructive pulmonary disease (COPD) that is caused by abnormal inflammation of the lungs caused by inhalation of harmful particles or gases, to be. Emphysema, and chronic bronchitis.
만성폐쇄성폐질환은 전세계적으로 5대 사망원인이며 2020 년에는 4대 사망원인에 이를 것으로 추정된다. 우리나라에서도 유병률이 7.9%에 이르며, 1년간 만성폐쇄성폐질환으로 인한 국민건강보험 지출이 600억 원에 이르러 보건학적으로나 경제적으로 중요한 질환이다. 만성폐쇄성폐질환은 지속적으로 진행하는 병으로 다른 만성 질환처럼 조기 진단과 예방이 중요할 것으로 보인다. 대표적인 만성 질환인 고혈압과 당뇨의 경우 혈압 및 혈당과 같은 명확한 진단기준이 있고, 관리를 할 경우 합병증과 사망률을 낮춘다는 연구결과들이 있어 조기 진단에 대한 활발한 노력이 있다. 그러나 만성폐쇄성폐질환에 대해서는 조기 진단에 대해서 논쟁이 많고, 우리나라에서는 특별한 지침도 마련되어 있지 않다. It is estimated that chronic obstructive pulmonary disease is the fifth leading cause of death in the world and the fourth leading cause of death in 2020. In Korea, the prevalence rate is 7.9%, and the national health insurance expenditure due to chronic obstructive pulmonary disease for one year reaches 60 billion won, which is a health and economically important disease. Chronic obstructive pulmonary disease (COPD) is an ongoing disease that requires early diagnosis and prevention as other chronic diseases. There are studies that have clear diagnostic criteria such as hypertension and diabetes mellitus, which are typical chronic diseases, and lowered complications and mortality when they are administered, and there is active effort for early diagnosis. However, there is much debate about the early diagnosis of chronic obstructive pulmonary disease, and no specific guidelines are available in Korea.
이렇듯 만성폐쇄성폐질환은 예방 가능하고 치료 가능한 질병이며, 적절한 진단과 그에 따른 치료가 매우 중요하나, 만성폐쇄성폐질환을 진단하기 위한 바이오 마커에 대해서는 연구가 미미한 실정이다. Thus, chronic obstructive pulmonary disease is a preventable and treatable disease, and proper diagnosis and treatment are very important. However, biomarkers for diagnosing chronic obstructive pulmonary disease are rarely studied.
또한, SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) 유전자에 의해 코딩되는 단백질은 TGFβ를 통해 세포 성장 및 분화를 조절하는 SMAD 경로의 구성 요소로 알려져 있으나, SKIL 유전자와 만성폐쇄성폐질환과의 관계에 대해서는 밝혀진 바가 거의 없다.In addition, proteins encoded by SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) It is known as a component of the SMAD pathway that regulates differentiation, but the relationship between SKIL gene and chronic obstructive pulmonary disease has not been elucidated.
이에 본 발명자들은 만성폐쇄성폐질환(Chronic Obstructive Pulmonary Disease, COPD)을 진단하기 위한 마커를 개발하고자 연구를 계속한 결과, 만성폐쇄성폐질환 환자에서 정상인에 비해 현저하게 발현이 증가하는 유전자인 SKIL을 확인함으로써, 본 발명을 완성하였다.
Accordingly, the present inventors have continued to develop a marker for diagnosing chronic obstructive pulmonary disease (COPD). As a result, the present inventors have found that SKIL, a gene whose expression is remarkably increased in patients with chronic obstructive pulmonary disease Thereby completing the present invention.
본 발명의 목적은 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN)을 포함하는 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD) 진단용 바이오마커 조성물을 제공하는 것이다.It is an object of the present invention to provide a method of treating chronic obstructive pulmonary disease (COPD) including SKIL (SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, pulmonary disease (COPD)) diagnostic biomarker composition.
본 발명의 목적은 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 제제를 포함하는, 만성폐쇄성폐질환 진단용 조성물을 제공하는 것이다. It is an object of the present invention to provide a composition for diagnosing chronic obstructive pulmonary disease, which comprises an agent for measuring an expression level of mRNA of SKIL gene or a protein thereof.
본 발명의 또 다른 목적은 상기 조성물을 포함하는 만성폐쇄성폐질환 진단 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.
본 발명의 또 다른 목적은 상기 마커를 이용하여 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법을 제공하는 것이다.
It is still another object of the present invention to provide a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker.
상기 과제를 해결하기 위하여, 본 발명은 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN)을 포함하는 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD) 진단용 바이오마커 조성물을 제공한다.In order to solve the above-mentioned problems, the present invention provides a method for preventing chronic obstructive lung disease including SKIL (Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN) A biomarker composition for the diagnosis of chronic obstructive pulmonary disease (COPD) is provided.
또한, 본 발명은 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 제제를 포함하는, 만성폐쇄성폐질환 진단용 조성물을 제공한다. The present invention also provides a composition for the diagnosis of chronic obstructive pulmonary disease, which comprises an agent for measuring an expression level of mRNA of SKIL gene or a protein thereof.
또한, 본 발명은 상기 조성물을 포함하는 만성폐쇄성폐질환 진단 키트를 제공한다. The present invention also provides a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.
또한, 본 발명은 상기 마커를 이용하여 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법을 제공한다.
In addition, the present invention provides a method for providing information for diagnosis of chronic obstructive pulmonary disease using the marker.
본 발명의 마커 유전자 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN)은 만성폐쇄성폐질환 환자에서 정상인에 비해 현저하게 발현이 증가하기 때문에, 만성폐쇄성폐질환의 발병 유무를 정확하고 빠르게 진단하는데 유용하게 이용될 수 있다.
SKIL-like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Protein, SnoN) Since the expression is remarkably increased, it can be usefully used for accurately and quickly diagnosing the onset of COPD.
도 1은 정상인의 폐조직, 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD)의 일종인 만성 폐쇄성 기관지염(Chronic obstructive bronchitis) 및 폐기종(Emphysema) 환자의 폐조직, 및 만성폐쇄성폐질환(COPD)이 아닌 폐질환의 일종인 기관지확장증(Bronchietasis) 환자의 폐조직에서의 SKIL 단백질의 발현 정도를 나타낸 도이다.
도 2는 정상인 및 만성폐쇄성폐질환 환자의 혈장 내 SKIL 단백질의 발현 정도를 나타낸 도이다.FIG. 1 is a schematic view of a lung tissue of a normal person, chronic obstructive bronchitis and a lung obstructive bronchitis, which is a kind of chronic obstructive pulmonary disease (COPD), and chronic obstructive pulmonary disease (COPD) FIG. 2 is a graph showing the expression level of SKIL protein in the lung tissue of a bronchial asthma patient, which is a kind of non-lung disease.
2 is a graph showing the expression level of SKIL protein in plasma of a normal human and chronic obstructive pulmonary disease patients.
이하 본 발명에 대하여 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명은 SKIL(SKI like oncogene, Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, Ski-Related Protein, SnoN)을 포함하는 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD) 진단용 바이오마커 조성물을 제공한다.
The present invention relates to a method of treating a chronic obstructive pulmonary disease (SKN) including SKIL (Ski-Like Protein, SNO, Ski-Related Oncogene SnoN, SnoA, Ski-Related Oncogene, SnoI, , COPD) diagnostic biomarker composition.
본 발명에서 "진단"은 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명의 목적상, 진단은 만성폐쇄성폐질환의 발병 여부 또는 발병 가능성 여부를 확인하는 것이다. In the present invention, "diagnosis" means identifying the presence or characteristic of a pathological condition. For the purpose of the present invention, the diagnosis is to ascertain whether or not the onset of chronic obstructive pulmonary disease is feasible.
본 발명의 만성폐쇄성폐질환의 종류에는 만성폐쇄성기관지염(Chronic obstructive bronchitis), 만성세기관지염(Chronic bronchiolitis) 및 폐기종(Emphysema) 등이 포함된다. The types of chronic obstructive pulmonary disease of the present invention include chronic obstructive bronchitis, chronic bronchiolitis, and emphysema.
본 발명에서 "마커"란 만성폐쇄성폐질환을 진단할 수 있는 물질로, 만성폐쇄성폐질환과 관련된 폴리펩타이드, 핵산 (예: mRNA 등), 지질, 당지질, 당단백질, 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자 등을 포함한다. 본 발명의 목적상, 마커는 SKIL로 만성폐쇄성폐질환 환자에서 현저하게 발현이 증가하는 유전자이다.
In the present invention, the term "marker" refers to a substance capable of diagnosing chronic obstructive pulmonary disease, including a polypeptide associated with chronic obstructive pulmonary disease, a nucleic acid (e.g., mRNA), a lipid, a glycolipid, a glycoprotein, a sugar (monosaccharide, And the like), and the like. For the purposes of the present invention, the marker is SKIL, a gene whose expression is markedly increased in patients with chronic obstructive pulmonary disease.
또한, 본 발명은 SKIL(SKI like oncogene, Gene ID: 6498) 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 제제를 포함하는, 만성폐쇄성폐질환(chronic obstructive pulmonary disease, COPD) 진단용 조성물을 제공한다.The present invention also provides a composition for diagnosing chronic obstructive pulmonary disease (COPD) comprising an agent for measuring an expression level of mRNA of SKIL (SKI like oncogene, Gene ID: 6498) gene or a protein thereof .
본 발명에서 “mRNA 발현 수준 측정”이란 만성폐쇄성폐질환을 진단하기 위하여 생물학적 시료에서 만성폐쇄성폐질환 관련 유전자 SKIL의 mRNA 존재 여부와 발현 정도를 확인하는 과정으로, mRNA의 양을 측정하여 이루어진다. 이를 위한 분석 방법으로는 예를 들어, 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Realtime RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting), DNA 칩 등이 있으나, 이에 제한되는 것은 아니다. 본 발명에서 유전자의 mRNA 수준을 측정하는 제제는 바람직하게는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브이다. In the present invention, " measurement of mRNA expression level " is a process of confirming the presence and expression level of mRNA of SKIL, a chronic obstructive pulmonary disease-related gene, in a biological sample in order to diagnose chronic obstructive pulmonary disease. For example, RT-PCR, competitive RT-PCR, real-time RT-PCR, and RNase protection assay (RPA). RNase protection assay, Northern blotting, DNA chip, and the like. The agent for measuring the mRNA level of a gene in the present invention is preferably an antisense oligonucleotide, a primer pair or a probe.
본 발명에서 "안티센스"는 안티센스 올리고머가 왓슨-크릭 염기쌍 형성에 의해 RNA 내의 표적 서열과 혼성화되어, 표적서열 내에서, 전형적으로 mRNA와 RNA:올리고머 헤테로이중체의 형성을 허용하는, 뉴클레오티드염기의 서열 및 서브 유닛간 백본을 갖는 올리고머를 지칭한다. 올리고머는 표적 서열에 대한 정확한 서열 상보성 또는 근사 상보성을 가질 수 있다. 이 안티센스 올리고머는 mRNA의 번역을 차단 또는 저해하고 mRNA의 스플라이스 변이체를 생산하는 mRNA의 프로세싱 과정을 변화시킬 수 있다.In the present invention, "antisense" means that the antisense oligomer is hybridized with the target sequence in the RNA by Watson-Crick base pairing to form the sequence of the nucleotide base, typically within the target sequence, And an oligomer having a backbone between subunits. Oligomers may have an exact sequence complement or approximate complementarity to the target sequence. This antisense oligomer can alter the processing of mRNA that blocks or inhibits translation of mRNA and produces splice variants of mRNA.
본 발명에서 "프라이머"는 짧은 자유 3말단 수산화기를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 중합효소 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. "Primer" in the present invention means a short nucleic acid sequence capable of forming a base pair with a complementary template with a short free 3-terminal hydroxyl group and functioning as a starting point for template strand copying. Primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.
본 발명에서 "프로브"란 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하며 표지(Labelling)되어 있어서 특정 mRNA의 존재 유무를 확인 할 수 있다. 프로브는 올리고 뉴클레오티드 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있다. 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다.In the present invention, "probe" means a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides that can specifically bind to mRNA, and is labeled to detect the presence or absence of a specific mRNA Can be confirmed. The probe can be produced in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe. Selection of suitable probes and hybridization conditions can be modified based on what is known in the art.
본 발명의 만성폐쇄성폐질환 관련 유전자인 SKIL의 핵산 서열이 유전자 은행에 등록되어 있으므로 당업자는 상기 서열을 바탕으로 이들 유전자의 특정 영역을 특이적으로 증폭하는 안티센스 올리고뉴클레오티드, 프라이머 쌍 또는 프로브를 디자인할 수 있다.Since the nucleic acid sequence of SKIL which is a gene related to chronic obstructive pulmonary disease of the present invention is registered in the gene bank, a person skilled in the art will design an antisense oligonucleotide, a primer pair or a probe that specifically amplifies a specific region of these genes based on the sequence .
본 발명의 안티센스 올리고뉴클레오티드, 프라이머 또는 프로브는 포스포르 아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산 서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오티드 하나 이상의 동족체로의 치환, 및 뉴클레오티드 간의 변형, 예를 들면, 하전되지 않은 연결체(예: 메틸 포스포네이트, 포스포트리에스테르, 포스포로아미 데이트, 카바메이트 등) 또는 하전된 연결체(예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다.The antisense oligonucleotides, primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution with one or more of the natural nucleotide analogs, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, Amidates, carbamates, etc.) or charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.).
본 발명에서 “단백질 발현 수준 측정”이란 만성폐쇄성폐질환을 진단하기 위하여 생물학적 시료에서 만성폐쇄성폐질환 관련 유전자인 SKIL로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 단백질의 양을 측정하여 이루어진다. 이를 위한 분석 방법으로는 웨스턴 블랏, ELISA (enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip) 등이 있으나 이로 제한되는 것은 아니다. 본 발명에서 단백질 발현 수준을 측정하는 제제는 바람직하게는 항체이다. In the present invention, " measurement of protein expression level " is a process of confirming the presence and expression level of protein expressed from SKIL, a gene related to chronic obstructive pulmonary disease, in a biological sample in order to diagnose COPD. . Examples of the assay methods include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis But are not limited to, tissue immuno staining, immunoprecipitation assays, complement fixation assays, fluorescence activated cell sorters (FACS), protein chips, and the like. The agent for measuring the protein expression level in the present invention is preferably an antibody.
본 발명에서, “항체”란 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 마커 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다.In the present invention, " antibody " means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies.
본 발명의 마커는 만성폐쇄성폐질환 환자에서 정상인에 비해 현저하게 발현이 증가하기 때문에, 만성폐쇄성폐질환의 발병 유무를 정확하고 빠르게 진단하는데 유용하게 이용될 수 있다.
Since the marker of the present invention is remarkably increased in patients with chronic obstructive pulmonary disease as compared with normal subjects, the marker can be used to accurately and quickly diagnose the incidence of COPD.
또한, 본 발명은 상기 조성물을 포함하는 만성폐쇄성폐질환 진단 키트를 제공한다. The present invention also provides a kit for the diagnosis of chronic obstructive pulmonary disease comprising the above composition.
본 발명의 키트는 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 확인함으로써 만성폐쇄성폐질환의 진단을 할 수 있다. 본 발명의 키트에는 만성폐쇄성폐질환 진단을 위한 프라이머, 프로브, 항체 등 뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액, 또는 장치가 더 포함될 수 있으며, 바람직하게는 RT-PCR 키트, 마이크로어레이 칩 키트, DNA 칩 키트, 단백질 칩 키트의 형태일 수 있으나 이에 제한되지 않는다. The kit of the present invention can diagnose chronic obstructive pulmonary disease by confirming the expression level of the SKIL gene mRNA or its protein. The kit of the present invention may further include one or more other component compositions, solutions, or devices suitable for the analysis method as well as primers, probes, and antibodies for diagnosing COPD, preferably RT-PCR A kit, a microarray chip kit, a DNA chip kit, and a protein chip kit.
구체적인 일례로서, 본 발명에서 상기 마커 유전자들의 mRNA 발현 수준을 측정하기 위한 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일수 있다. RT-PCR 키트는 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합 효소 및 역전사효소와 같은 효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다.As a specific example, in the present invention, the kit for measuring the mRNA expression level of the marker genes may be a kit containing essential elements necessary for performing RT-PCR. RT-PCR kits contain enzymes such as test tubes or other appropriate containers, reaction buffers, deoxynucleotides (dNTPs), Taq-polymerase and reverse transcriptase, DNase, RNase inhibitors, DEPC Water (DEPC-water), sterile water, and the like.
또한, 본 발명의 키트는 마이크로어레이를 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. 마이크로어레이 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판을 포함하고 기판은 정량 대조구 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있으며, 본 발명의 마커를 이용하여 당업계에서 통상적으로 사용되는 제조 방법에 의하여 용이하게 제조될 수 있다. 마이크로어레이를 제작하기 위해서, 상기 탐색된 마커를 탐침 DNA 분자로 이용하여 DNA 칩의 기판상에 고정화시키기 위해 파이조일렉트릭(piezoelectric) 방식을 이용한 마이크로피펫팅(micropipetting)법 또는 핀(pin) 형태의 스폿터(spotter)를 이용한 방법 등을 사용하는 것이 바람직하나 이에 제한되지 않는다. 상기 마이크로어레이 칩의 기판은 아미노-실란(amino-silane), 폴리-L-라이신(poly-Llysine) 및 알데히드(aldehyde)로 이루어진 군에서 선택되는 활성기가 코팅된 것이 바람직하나, 이에 제한되지 않는다. 또한, 상기 기판은 슬라이드 글래스, 플라스틱, 금속, 실리콘, 나일론 막 및 니트로셀룰로스 막(nitrocellulose membrane)으로 이루어진 군에서 선택되는 것이 바람직하나 이에 제한되지 않는다.In addition, the kit of the present invention may be a kit including essential elements necessary for performing the microarray. The microarray kit may include a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe and the substrate may include a cDNA corresponding to a quantitative control gene or a fragment thereof, Can be easily produced by a production method commonly used in the art. In order to fabricate a microarray, a micropipetting method using a piezo electric method or a micropipetting method using a pin-shaped method to immobilize the detected marker on a substrate of a DNA chip using the probe as a probe DNA molecule A method using a spotter or the like is preferably used, but the present invention is not limited thereto. The substrate of the microarray chip is preferably coated with an activator selected from the group consisting of amino-silane, poly-L-lysine and aldehyde, but is not limited thereto. In addition, the substrate is preferably selected from the group consisting of slide glass, plastic, metal, silicon, nylon film, and nitrocellulose membrane, but is not limited thereto.
또한, 본 발명의 키트는 DNA 칩을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. DNA 칩 키트는 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드(oligonucleotide)가 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한 기판은 대조군 유전자 또는 그의 단편에 해당하는 cDNA 또는 올리고뉴클레오티드를 포함할 수 있다.
In addition, the kit of the present invention may be a kit containing essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate on which a cDNA or an oligonucleotide corresponding to a gene or a fragment thereof is attached, and reagents, preparations, enzymes, and the like for producing a fluorescent-labeled probe. The substrate may also comprise a cDNA or oligonucleotide corresponding to a control gene or fragment thereof.
또한, 본 발명은In addition,
(a) 생물학적 시료로부터 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하는 단계; 및 (a) measuring the level of expression of an SKIL gene mRNA or protein thereof from a biological sample; And
(b) 상기 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 정상 대조구 시료에서 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준과 비교하는 단계;를 포함하는 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법을 제공한다. (b) comparing the expression level of mRNA of SKIL gene or its protein with the level of mRNA of SKIL gene or protein thereof in a normal control sample, and do.
본 발명에서 “생물학적 시료”는 개체로부터 분리된 전혈, 혈청, 혈장, 타액, 뇨, 객담, 림프액, 세포 등을 포함하며, 바람직하게는 폐조직이나, 이에 제한되지 않는다. In the present invention, the term "biological sample" includes whole blood, serum, plasma, saliva, urine, sputum, lymph fluid, cells and the like isolated from an individual, preferably lung tissue.
본 발명에서 mRNA 발현 수준을 측정하는 방법은 역전사 중합효소반응(RT-PCR), 경쟁적 역전사 중합효소반응(Competitive RT-PCR), 실시간 역전사 중합효소반응(Realtime RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블랏팅(Northern blotting), DNA 칩 등이 있으나 이에 제한되지 않는다.Methods for measuring mRNA expression levels in the present invention include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA RNase protection assay, Northern blotting, DNA chip, and the like.
본 발명에서 단백질의 발현 수준을 측정하는 방법은 웨스턴 블랏, ELISA (enzyme linked immunosorbent assay), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip) 등이 있으나 이에 제한되지 않는다.Methods for measuring the expression level of a protein in the present invention include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip. However, there are limitations on the use of immunoassays, It does not.
본 발명의 마커 유전자 SKIL은 만성폐쇄성폐질환 환자에서 정상인에 비해 현저하게 발현이 증가하기 때문에, 정상 대조구 시료와의 비교를 통해, 만성폐쇄성폐질환의 발병 유무를 정확하고 빠르게 진단할 수 있다.
Since the marker gene SKIL of the present invention is remarkably increased in patients with chronic obstructive pulmonary disease as compared with that of normal individuals, the presence or absence of COPD can be accurately and quickly diagnosed through comparison with a normal control sample.
이하, 본 발명을 실시예에 의거하여 보다 구체적으로 설명한다. 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.
실시예 1. RNA 시퀀싱을 이용한 만성폐쇄성폐질환 (COPD) 환자의 폐조직 내 SKIL 유전자 발현 정도 측정Example 1. Measurement of SKIL gene expression level in lung tissue of patients with chronic obstructive pulmonary disease (COPD) using RNA sequencing
1-1. mRNA 샘플의 준비1-1. Preparation of mRNA samples
아산병원 (서울, 대한민국)의 만성폐쇄성폐질환(COPD) 환자 100명 및 정상인 (대조구) 100명의 폐 조직으로부터 종래 공지된 방법에 따라 mRNA를 수득하였다.
MRNA was obtained from lung tissues of 100 patients with chronic obstructive pulmonary disease (COPD) and 100 normal controls (control) of Asan Hospital (Seoul, Korea) according to conventionally known methods.
1-2. RNA 시퀀싱1-2. RNA sequencing
상기 1-1.에서 수득한 샘플로부터 종래 공지된 방법에 따라 RNA 시퀀싱 (QIAGEN, RNA-Seq Experiment)을 수행한 후, 분석을 통하여 통계적으로 유의하게 정상인의 폐조직에 비하여 차별발현하는 유전자를 확인하였다.RNA sequencing (QIAGEN, RNA-Seq Experiment) was performed according to a conventionally known method from the sample obtained in 1-1. Then, the gene that expresses differentially compared to the lung tissue of the normal person was identified statistically significantly Respectively.
만성폐쇄성질화 환자의 폐 조직에서는 대조군인 정상인의 폐 조직에 비해 SKIL 유전자가 약 163% 발현이 증가하였음을 확인하였고, 이 결과는 통계적으로 유의하다(p=8.95E-11). 이상의 결과를 통해 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 측정하여, 만성폐쇄성폐질환을 진단할 수 있음을 알 수 있다.
In the lung tissue of patients with chronic obstructive nitrification, the expression of SKIL gene was increased by about 163% compared to the lung tissue of the normal control group. This result was statistically significant (p = 8.95E-11). These results indicate that SKIL gene mRNA or its protein expression levels can be measured to diagnose chronic obstructive pulmonary disease.
실시예 2. 면역조직화학법을 이용한 만성폐쇄성폐질환 (COPD) 환자의 폐조직 내 SKIL 단백질 발현 여부 확인Example 2 Confirmation of Expression of SKIL Protein in Pulmonary Tissue of Patients with COPD by Immunohistochemistry
만성폐쇄성폐질환(COPD) 환자의 폐조직에서 특이적으로 SKIL 단백질의 발현이 증가하는지를 확인하기 위하여 정상인의 폐조직, 만성폐쇄성폐질환(COPD)의 일종인 만성 폐쇄성 기관지염(Chronic obstructive bronchitis) 및 폐기종(Emphysema) 환자의 폐조직, 만성폐쇄성폐질환(COPD)이 아닌 폐질환의 일종인 기관지확장증(Bronchietasis) 환자의 폐조직에 대하여 면역조직화학법을 수행하였다.In order to confirm whether the expression of SKIL protein increases specifically in the lung tissue of patients with COPD, it is necessary to evaluate the expression of SKIL protein in lung tissues of normal persons, chronic obstructive bronchitis (COPD) We performed immunohistochemistry on the lung tissue of patients with bronchial asthma, a type of pulmonary disease that is not a chronic obstructive pulmonary disease (COPD).
상기 면역조직화학법을 수행하기 위하여 시판중인 조직 어레이 슬라이드(tissue array slide, Pantomics, LUD481)을 구입한 후, 파라핀 조직 슬라이드에 SKIL 유전자에 대한 항체(1:50, abcam)를 이용하여 면역조직화학법을 수행하였다. 그 결과를 도 1에 나타내었다.To perform the immunohistochemistry, a commercially available tissue array slide (Pantomics, LUD481) was purchased. Immunohistochemistry was performed on the paraffin tissue slides using an antibody against the SKIL gene (1:50, abcam) The law was carried out. The results are shown in Fig.
도 1에 나타낸 바와 같이 만성폐쇄성폐질환(COPD)의 일종인 만성 폐쇄성 기관지염(Chronic obstructive bronchitis) 및 폐기종(Emphysema) 환자의 폐조직에서는 정상인의 폐조직에 비하여 SKIL 유전자의 발현이 증가하였으나, 만성폐쇄성폐질환(COPD)이 아닌 폐질환의 일종인 기관지확장증(Bronchietasis) 환자의 폐조직에서는 정상인의 폐조직에 비하여 SKIL 유전자의 발현이 증가하지 않았음을 확인하였다. 이상의 결과를 통해 상기 유전자의 단백질 발현 수준을 측정하여, 만성폐쇄성폐질환을 진단할 수 있음을 알 수 있다.
As shown in FIG. 1, the expression of SKIL gene was increased in the lung tissues of patients with chronic obstructive bronchitis and Emphysema, which is a kind of chronic obstructive pulmonary disease (COPD), compared with normal lung tissues, The expression of SKIL gene was not increased in pulmonary tissue of bronchial asthma, which is a kind of pulmonary disease other than pulmonary disease (COPD). The above results indicate that the protein expression level of the gene can be measured to diagnose chronic obstructive pulmonary disease.
실시예 3. ELISA 방법을 이용한 만성폐쇄성폐질환 (COPD) 환자의 혈장 내 SKIL 단백질양 측정Example 3 Measurement of SKIL Protein Level in Plasma of Patients with COPD Using ELISA
만성폐쇄성폐질환(COPD) 환자의 혈장에서 특이적으로 SKIL 단백질의 발현이 증가하는지를 확인하기 위하여 정상인의 혈장 및 만성폐쇄성폐질환(COPD) 환자의 혈장에 대하여 ELISA(enzyme linked immunosorbent assay) 방법을 수행하였다.In order to confirm that the expression of SKIL protein increases specifically in the plasma of patients with COPD, an enzyme linked immunosorbent assay (ELISA) is performed on the plasma of normal human patients with COPD and patients with chronic obstructive pulmonary disease (COPD) Respectively.
상기 ELISA 방법을 수행하기 위하여 SKIL에 대한 ELISA 키트(Cloud-clone corp., SEM234Hu)를 구입하였다. 상기 ELISA 키트를 이용하여 아산병원 (서울, 대한민국)의 정상인 20명 및 만성폐쇄성폐질환 (COPD) 환자 20명으로부터 수득한 혈장에 대하여 혈장 내 SKIL 단백질의 양을 측정하였다. 이 때 혈장은 상기 ELISA 키트에서 제공하는 버퍼로 8분의 1의 농도로 희석하여 사용하였다. 그 결과를 도 2에 나타내었다.To perform the ELISA method, an ELISA kit (Cloud-clone corp., SEM234Hu) for SKIL was purchased. Using the above ELISA kit, the amount of SKIL protein in plasma was measured for plasma obtained from 20 healthy persons and 20 COPD patients from Asan Medical Center (Seoul, Korea). Plasma was diluted to 1/8 concentration with the buffer provided in the ELISA kit. The results are shown in Fig.
도 2에 나타낸 바와 같이, 만성폐쇄성폐질환(COPD) 환자의 혈장에서 정상인의 혈장에 비해 혈장 내 SKIL 단백질의 양이 유의하게 증가되었음을 확인하였다. 이상의 결과를 통해 상기 유전자의 혈액 내 단백질 발현 수준을 측정하여, 만성폐쇄성폐질환을 진단할 수 있음을 알 수 있다.As shown in FIG. 2, it was confirmed that the amount of SKIL protein in the plasma was significantly increased in the plasma of patients with COPD compared with the plasma of normal persons. The above results indicate that the protein expression level in the blood of the gene can be measured to diagnose chronic obstructive pulmonary disease.
Claims (11)
(b) 상기 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준을 정상 대조구 시료에서 SKIL 유전자의 mRNA 또는 이의 단백질의 발현 수준과 비교하는 단계;를 포함하는 만성폐쇄성폐질환의 진단을 위한 정보 제공 방법. (a) measuring the level of expression of an SKIL gene mRNA or protein thereof from a biological sample; And
(b) comparing the expression level of the mRNA of the SKIL gene or a protein thereof with the level of mRNA of the SKIL gene or a protein thereof in a normal control sample.
The method of claim 8, wherein the level of expression of the protein in step (a) is measured by Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), and Protein Chip Assay are used for immunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, wherein the method is one or more selected from the group consisting of a protein chip, and a method for diagnosing chronic obstructive pulmonary disease.
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| KR101944665B1 (en) * | 2017-02-24 | 2019-02-01 | 주식회사 엠디헬스케어 | Method for diagnosis of chronic obstructive airway disease using analysis of bacteria metagenome |
| WO2018155950A1 (en) * | 2017-02-24 | 2018-08-30 | 주식회사 엠디헬스케어 | Method for diagnosing diabetes through bacterial metagenome analysis |
| WO2018155967A1 (en) * | 2017-02-24 | 2018-08-30 | 주식회사 엠디헬스케어 | Method for diagnosing chronic obstructive airway disease through bacterial metagenome analysis |
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