WO2011126105A1 - 免疫系の異常疾患または関節系の異常疾患への罹患またはこれらの病勢を判定するための検査方法 - Google Patents
免疫系の異常疾患または関節系の異常疾患への罹患またはこれらの病勢を判定するための検査方法 Download PDFInfo
- Publication number
- WO2011126105A1 WO2011126105A1 PCT/JP2011/058875 JP2011058875W WO2011126105A1 WO 2011126105 A1 WO2011126105 A1 WO 2011126105A1 JP 2011058875 W JP2011058875 W JP 2011058875W WO 2011126105 A1 WO2011126105 A1 WO 2011126105A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- joint
- mirna
- abnormal
- expression level
- derived
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/04—Endocrine or metabolic disorders
- G01N2800/046—Thyroid disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/06—Gastro-intestinal diseases
- G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/20—Dermatological disorders
- G01N2800/202—Dermatitis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/26—Infectious diseases, e.g. generalised sepsis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/285—Demyelinating diseases; Multipel sclerosis
Definitions
- the present invention measures the expression level of miRNA in a blood sample or a blood sample derived from a joint, and tests for determining whether or not the immune system abnormal disease and / or abnormal disease of the joint system or the disease state of these diseases Regarding the method.
- MicroRNA is a small RNA that is not translated into a protein having a length of about 22 bases (Non-patent Document 1). miRNA regulates transcription by binding to 3'UTR of messenger RNA, and is related in various ways such as cell metabolism, organogenesis and malignant tumor. It has long been thought that RNA is not present in serum or plasma from which cellular components have been removed because RNase is present in blood and is immediately degraded even when RNA is added. However, recently, miRNA-141 has been shown as a specific marker for bladder cancer in serum and plasma (Non-patent Document 2). On the other hand, it is generally known that miRNA in cells and miRNA released from cells do not match (Non-patent Document 3). For this reason, since the fluctuation
- An object of the present invention is to provide a method (in vitro diagnostic method) for detecting the morbidity of abnormal diseases of the immune system and / or abnormal diseases of the joint system, and furthermore, the collection and management of the sample is simple.
- an object of the present invention is to provide a test method capable of testing a disease with high accuracy.
- the inventors of the present invention have (1) abnormal diseases of the immune system and / or abnormal diseases of the joint system using a specific miRNA in a blood-derived liquid sample as a marker. It is found that the presence or absence can be examined, and (2) the presence of specific miRNA groups in joint-derived liquid samples is found, and abnormal diseases of the immune system and / or joints are detected using these miRNA groups as markers. It has been found that abnormal diseases of the system can be discriminated, and (3) it has been found that the disease state of abnormal diseases of the immune system and / or abnormal diseases of the joint system can be examined by a specific miRNA group in a blood-derived liquid sample.
- the ratio of the concentration in the joint-derived fluid sample divided by the concentration in the blood-derived fluid sample is used to determine whether the immune system abnormal disease and / or joint system
- the present invention has been completed by finding that the disease state of an abnormal disease can be examined.
- the present invention encompasses the following.
- a test method for determining a disease state of an abnormal disease of the immune system and / or an abnormal disease of the joint system including a comparison step of comparing the expression level between different collection times.
- the “liquid sample” means a liquid in a state where cell components are removed from a collected material.
- the present invention even a sample from which cell components have been removed can be examined with high accuracy, so that it is possible to omit the temperature and time management conventionally required for maintaining cell activity.
- joint-related tissues such as synovial tissue and bone tissue can be examined without taking them, the patient's pain and possible complications can be reduced.
- the miRNA concentrations of SEQ ID NOs: 1 to 5 are shown in synovial fluid collected from patients with rheumatoid arthritis (RA) and osteoarthritis patients (OA).
- the number of samples is 30 for each patient.
- the ROC curve and area under ROC curve (AUC) for the determination of the rheumatoid arthritis patient using the miRNA of sequence number 2 in plasma, or the determination of the osteoarthritis patient are shown. There are 30 samples in each group. 2 shows the correlation between TJC / DAS28 expression level in plasma of miRNAs of SEQ ID NOs: 1 to 5 in specimens collected from patients with rheumatoid arthritis.
- the correlation between the synovial fluid / plasma miRNA ratio and TJC is shown for miRNAs of SEQ ID NOs: 1 to 5 in specimens collected from patients with rheumatoid arthritis. An approximate straight line is shown for a P value of 0.1 or less.
- detection of abnormal diseases of the immune system and / or abnormal diseases of the joint system is not limited to the following, but for example, determination of morbidity of the diseases, Discrimination, determination of disease state, and the like are included.
- the expression level of at least one miRNA selected from miRNAs having the sequences represented by SEQ ID NOs: 1 to 5 shown in Table 1 below in blood or joint-derived liquid samples is used as an index. Abnormal diseases and / or abnormal diseases of the joint system are detected.
- blood-derived liquid samples include serum, plasma, culture supernatant of blood-derived cells (supernatant obtained by culturing blood-derived cells in a culture solution), and particularly samples from plasma. It is preferable from the viewpoint of simplicity. These liquid samples can be collected by a known or conventional method.
- joint-derived liquid samples include joint fluid supernatants, culture supernatants of various tissue cells related to joints (for example, articular cartilage tissue, synovial tissue, bone tissue, ligament tissue, and cells derived therefrom).
- a supernatant obtained by culturing related cells in a culture solution), and a joint fluid supernatant is particularly preferable from the viewpoint of simplicity.
- These liquid samples can be collected by a known or conventional method.
- Abnormal diseases of the immune system that can be examined include, for example, rheumatoid arthritis, juvenile rheumatoid arthritis, rheumatic fever, Bazedeau disease, Hashimoto's disease, systemic lupus erythematosus, periarteritis nodosa, polyarteritis nodosa, mixed connective tissue Disease, polymyositis, dermatomyositis, sarcoidosis, scleroderma, psoriatic arthritis, Behcet's disease, seronegative arthritis, ankylosing spondylitis, ulcerative colitis, Crohn's disease, atopic dermatitis, polymyalgia rheumatica Autoimmune hepatitis, relapsing polychondritis, multiple sclerosis, viral infection, sepsis, Sjogren's syndrome, antiphospholipid antibody syndrome, adult still disease, amyloidosis and the like.
- Rheumatoid arthritis, juvenile rheumatoid arthritis, and rheumatic fever are preferable, and among these, rheumatoid arthritis can be examined with high accuracy.
- rheumatoid arthritis and juvenile rheumatoid arthritis belong to joint system abnormal diseases because they involve joint destruction.
- Other abnormal joint diseases that can be examined include osteoarthritis, cartilage damage, gout, pseudogout, joint infection (suppurative arthritis), bone system diseases, fractures, intra-articular ligament damage, half-moon Examples include plate damage, sprains, osteonecrosis, osteoporosis, Charcot arthritis, osteochondral tumors, and soft tissue tumors.
- Preferred examples include osteoarthritis, cartilage damage, bone fracture, intra-articular ligament damage, meniscus damage, sprain, and osteonecrosis. Among these, it is possible to examine with high accuracy, particularly in osteoarthritis.
- the method of the present invention is useful for all mammals, and examples of mammals include humans, apes, rodents, dogs, cats, and rabbits, with human beings being particularly desirable.
- RNA is first extracted from a blood-derived liquid sample or a joint-derived liquid sample obtained from a patient.
- RNA extraction methods include guanidine-cesium chloride ultracentrifugation, acidic phenol method, and spin column method.
- miRNA in RNA obtained from a blood-derived liquid sample or a joint-derived liquid sample is detected.
- the detection method is not particularly limited as long as it can measure the expression level of miRNA, and examples thereof include real-time RT-PCR method and microarray method. The details of these methods are described, for example, in the microRNA experiment protocol written by Kazuyoshi Kawafu.
- the miRNA expression level analysis kit of the present invention includes at least one primer or probe capable of analyzing the expression level of miRNA selected from the group consisting of SEQ ID NOs: 1 to 5.
- a buffer solution, an enzyme, etc. can be suitably included according to the measurement system.
- a test method for determining the disease state of an abnormal disease of the immune system and / or an abnormal disease of the joint system including a measurement step of comparing and a comparison step of comparing the expression level in different collection time periods.
- the expression level of at least one miRNA shown in SEQ ID NOs: 1 to 5 in the liquid sample is measured over time, and different collections are obtained.
- a significant correlation is found with the disease state, and for example, it can be determined that the disease state progresses as the expression level of the miRNA decreases.
- “disease” means the momentum of progression of the disease, and indicates that the disease tends to get worse as the expression level of the miRNA decreases with time.
- the sampling interval over time can be appropriately set in determining the patient's disease state.
- the sampling interval is, for example, 7 days to 1 year, preferably 30 days to It may be 180 days apart.
- the disease state of abnormal diseases of the immune system and / or abnormal diseases of the joint system using at least one miRNA (preferably SEQ ID NOs: 1 to 3) shown in SEQ ID NOs: 1 to 5 is used.
- This test method prepares a blood-derived fluid sample and a joint-derived fluid sample collected over time, and measures the expression level of at least one miRNA shown in SEQ ID NOs: 1 to 5 from each sample. And the ratio (Ea / Eb) of the miRNA expression level (Ea) obtained from the joint-derived liquid sample and the miRNA expression level (Eb) obtained from the blood-derived liquid sample. And comparing with time period.
- each liquid sample for obtaining the expression level ratio (Ea / Eb) is collected from the same patient, for example, within 7 days, preferably within 1 day.
- the ratio (Ea / Eb) was high. It can be judged that the disease progresses as the time goes.
- the miRNA expression level ratio (Eb / Ea) is preferably the ratio of the miRNA concentration in synovial fluid to the plasma concentration.
- the expression level of the miRNA shown in SEQ ID NO: 2 is measured to determine abnormal diseases of the immune system (for example, rheumatoid arthritis) and / or Or the test method which determines the morbidity of the abnormal disease (for example, osteoarthritis) of a joint system is included.
- the threshold of the patient is determined according to a statistical method described later. A significant correlation is shown for the presence or absence of abnormal diseases of the immune system and / or abnormal diseases of the joint system.
- the immune system abnormality is about 84% sensitivity and about 80% specificity. It can be determined that the condition has a disease (eg, rheumatoid arthritis) and / or an abnormal disease of the joint system (eg, osteoarthritis).
- This threshold value is merely an example, and may be appropriately changed by statistical processing.
- an immune system abnormal disease is measured by measuring the expression level of at least one miRNA selected from the group consisting of SEQ ID NOs: 1, 3, 4, and 5 in a joint-derived liquid sample. It includes a test method for determining (for example, rheumatoid arthritis) and / or abnormal diseases of the joint system (for example, osteoarthritis). According to this test method, the expression level of the miRNA present in the joint-derived liquid sample collected from the subject is measured, and the threshold is determined according to a statistical method described later, so that the subject is immune system A significant correlation is shown as to whether you are suffering from an abnormal disease and / or an abnormal disease of the joint system.
- the miRNAs of SEQ ID NOs: 1, 3, 4, and 5 have a sensitivity of 75% specificity 78% and sensitivity 73% specificity at thresholds of 40 pmol / L, 11 pmol / L, 0.04 pmol / L, and 2.5 pmol / L, respectively. It can be determined that osteoarthritis is 72%, sensitivity 77% specificity 70%, sensitivity 56% specificity 80%.
- the sensitivity is 75% specificity.
- Rheumatoid arthritis can be determined with 78%, sensitivity 73%, specificity 72%, sensitivity 77%, specificity 70%, sensitivity 56%, specificity 80%. Note that these threshold values are examples, and may be appropriately changed by statistical processing.
- Peripheral blood and synovial fluid were obtained from patients with rheumatoid arthritis and osteoarthritis, and 30 samples of peripheral blood were obtained from healthy individuals, with the approval of the ethics committee of the graduate School of Medicine of Kyoto University. Peripheral blood was collected in a test tube containing EDTA-2K.
- Real-time RT-PCR After synthesizing cDNA with NCode VILO miRNA cDNA Synthesis Kit (Invitrogen), real-time RT-PCR was performed using ABI Prism 7300 Sequence Detection System (Applied Biosystems) and EXPRESS SYBR GreenER qPCR SuperMix (Invitrogen).
- the primers used are as follows: hsa-miR-16, 5'-TAG-CAG-CAC-GTA-AAT-ATT-GGC-G-3 '(SEQ ID NO: 6) hsa-miR-132, 5'-TAA-CAG-TCT-ACA-GCC-ATG-GTC-G-3 '(SEQ ID NO: 7) hsa-miR-146a, 5'-TGA-GAA-CTG-AAT-TCC-ATG-GGT-T-3 '(SEQ ID NO: 8) hsa-miR-155, 5'-TTA-ATG-CTA-ATC-GTG-ATA-GGG-GTA-3 '(SEQ ID NO: 9) hsa-miR-223, 5'-TGT-CAG-TTT-GTC-AAA-TAC-CCC-A-3 '(SEQ ID NO: 10) cel-miR-39, 5'-CGT-
- a PCR product inserted into a pTAC-1 vector (Biodynamic Laboratory, Inc.) was prepared in advance, and a standard curve was prepared with serial dilutions. Based on cel-miR-39 added to each sample at a known concentration, the absolute concentration of miRNA in each sample was calculated.
- the concentrations of miR-16, miR-146a, miR-155, and miR-223 in synovial fluid were p ⁇ 0.05, and rheumatoid arthritis patients were higher than osteoarthritis patients. If the positive values of rheumatoid arthritis are 40 pmol / L, 11 pmol / L, 0.04 pmol / L, 2.5 pmol / L or more, respectively, sensitivity is 75% specificity 78%, sensitivity 73% specificity 72%, sensitivity 77% specificity Rheumatoid arthritis can be judged with 70% sensitivity, 56% sensitivity and 80% specificity. As shown in FIG.
- miR-16, miR-132, miR-146a, miR-155, miR-223 plasma concentrations were correlated with TJC (number of painful joints).
- TJC number of painful joints.
- the joint fluid / plasma concentration ratio of miR-16, miR-132, and miR-146a was correlated with TJC (the number of painful joints).
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
血液中にはRNaseが存在し、RNAを付加しても即座に分解されるため、細胞成分を除去した血清または血漿中にRNAは存在しないと長年考えられていた。しかし近年、血清や血漿中で、miRNA-141が膀胱癌に特異的なマーカーとして示された(非特許文献2)。
一方で、一般に細胞中のmiRNAと細胞から放出されるmiRNAとは一致しないことが知られている(非特許文献3)。このため、細胞内や組織内で増加または低下しているmiRNAの血清または血漿中での変動量は不確実であるため、予測性に欠ける。
(2)経時的に採取された血液由来の液体サンプルまたは関節由来の液体サンプルを準備する準備工程と、前記サンプルから配列番号1~5に示される少なくとも一つのmiRNAの発現量を測定する測定工程と、異なる採取時期の間で前記発現量を比較する比較工程とを含む免疫系の異常疾患及び/または関節系の異常疾患の病勢の判定を行う検査方法。
(3)経時的に採取された血液由来の液体サンプルおよび関節由来の液体サンプルを準備する準備工程と、それぞれのサンプルから配列番号1~5に示される少なくとも一つのmiRNAの発現量を測定する測定工程と、関節由来サンプルから得られた前記miRNAの発現量(Ea)と血液由来サンプルから得られた前記miRNAの発現量(Eb)との比(Ea/Eb)を異なる採取時期の間で比較する工程とを含む、免疫系の異常疾患及び/または関節系の異常疾患の病勢の判定を行う検査方法。
(4)血液由来の液体サンプル中、配列番号2に示されるmiRNAの発現量を測定する、免疫系の異常疾患及び/または関節系の異常疾患の罹患の判定を行う検査方法。
(5)関節由来の液体サンプル中、配列番号1、3、4および5からなる群から選択された少なくとも一つのmiRNAの発現量を測定する、免疫系の異常疾患及び/または関節系の異常疾患の判別を行う検査方法。
(6)血液由来の液体サンプルが血漿であり、かつ、関節由来の液体サンプルが、関節液である、(1)~(5)のいずれかの方法。
(7)配列番号1~5からなる群から選択されるmiRNAの発現量を分析することのできるプライマーまたはプローブの、免疫系の異常疾患及び/または関節系の異常疾患の検出用キットの製造への使用。
なお、本発明において、「液体サンプル」とは、採取物より細胞成分を取り除いた状態の液体を意味する。
また、滑膜組織や骨組織などの関節関連組織を採取せずに検査できるため、患者の苦痛および合併症の可能性を軽減することができる。
本発明方法における、免疫系の異常疾患及び/または関節系の異常疾患の検出(in vitro診断)には、以下に限定されるものではないが、例えば、当該疾患の罹患の判定、当該疾患の判別、当該疾患の病勢の判定等が含まれる。
本発明では、下記表1に示す配列番号1~5で表される配列からなるmiRNAから選択した少なくとも1つのmiRNAの、血液または関節由来の液体サンプル中での発現量を指標として、免疫系の異常疾患及び/または関節系の異常疾患を検出する。
また、上記以外の検査可能な関節系異常疾患としては、例えば、変形性関節症、軟骨損傷、痛風、偽痛風、関節感染(化膿性関節炎)、骨系統疾患、骨折、関節内靭帯損傷、半月板損傷、捻挫、骨壊死、骨粗鬆症、シャルコー関節炎、骨軟骨腫瘍、軟部腫瘍などを挙げることができる。好ましくは変形性関節症、軟骨損傷、骨折、関節内靭帯損傷、半月板損傷、捻挫、骨壊死が挙げられ、これらのうち、特に変形性関節症において高い精度で検査することが可能である。
次いで血液由来液体サンプルまたは関節由来液体サンプルから得られたRNA中のmiRNAを検出する。検出方法は、miRNAの発現量を測定できるものである限り特に限定されないが、例えば、リアルタイムRT-PCR法、マイクロアレイ法などが挙げられる。これらの方法は、例えば、microRNA実験プロトコール 著 河府 和義 羊土社にその詳細が記載されている。
前記配列番号1~5からなる群から選択されるmiRNAの発現量を分析することができるプライマーまたはプローブは、当業者であれば、これらの塩基配列に基づいて容易に設計し合成して使用することができる。
本発明のmiRNAの発現量の分析キットとしては、前記配列番号1~5からなる群から選択されるmiRNAの発現量を分析することができるプライマーまたはプローブを少なくとも一つ以上含む。その他、測定系に合わせて、適宜、緩衝液や酵素などを含むことができる。
本発明では、第一の態様として、経時的に採取された血液または関節由来の液体サンプルを準備する準備工程と、前記サンプルから配列番号1~5に示される少なくとも一つのmiRNAの発現量を測定する測定工程と、異なる採取時期間で前記発現量を比較する比較工程とを含む、免疫系の異常疾患及び/または関節系の異常疾患の病勢の判定を行う検査方法が含まれる。
第二の態様に係る検査方法においては、好ましくはmiRNAの発現量の比(Eb/Ea)は、前記miRNAの関節液中濃度と血漿中濃度との比であるのが好ましい。
この検査方法によれば、患者から採取した配列番号2のmiRNAの発現量と、健常者から採取した前記miRNAの発現量とを比較すると、後述する統計手法に従って閾値を決定することにより、患者の免疫系の異常疾患及び/または関節系の異常疾患の罹患の有無について有意な相関が示される。
より詳細には、例えば、被検者について、血漿中配列番号2のmiRNA濃度が閾値67pmol/L以下であった場合に、約84%の感度と約80%の特異度をもって、免疫系の異常疾患(例えば関節リウマチ)及び/または関節系の異常疾患(例えば変形性関節症)を有する状態であると判定することができる。なお、この閾値は例示であり、統計処理により適宜変動してもよい。
この検査方法によれば、被検者から採取した関節由来の液体サンプル中に存在する前記miRNAの発現量を測定し、後述する統計手法に従って閾値を決定することにより、被検者が免疫系の異常疾患及び/または関節系の異常疾患のいずれに罹患しているのかについて、有意な相関が示される。
例えば、関節リウマチに罹患している場合は、変形性関節症より前記miRNAの発現量が高くなる。従って、配列番号1、3、4および5のmiRNAは、それぞれ閾値が40pmol/L、11pmol/L、0.04pmol/L、2.5pmol/L以下で感度75%特異度78%、感度73%特異度72%、感度77%特異度70%、感度56%特異度80%で変形性関節症であると判別することができる。あるいは、関節リウマチの陽性値を、配列番号1、3、4および5のmiRNAに関して、それぞれ40pmol/L、11pmol/L、0.04pmol/L、2.5pmol/L以上とすると、それぞれ感度75%特異度78%、感度73%特異度72%、感度77%特異度70%、感度56%特異度80%で関節リウマチと判断することができる。なお、これらの閾値は例示であり、統計処理により適宜変動してもよい。
(実験対象集団)
関節リウマチ患者および変形性関節症患者より末梢血および関節液を、健常人より末梢血をそれぞれ30検体ずつ、京都大学大学院医学研究科医の倫理委員会の承認のもと取得した。末梢血はEDTA-2Kを含む試験管に採取した。
細胞成分を取り除くため、末梢血および関節液は400gで7分間遠心分離し上清を解析まで-20℃で凍結保存した。
100μLの血漿または関節液上清に、酸性フェノール法を用いた試薬であるIsogen LS(日本ジーン社)を750μL加えてRNaseの活性を失活させた後、25fmolの合成したcel-miR-39を外部標準として加え懸濁液を得た。ついで、この懸濁液に更に200μLのクロロホルムを加えた後、12,000gで15分間遠心分離して300μLの液相を採取した。この液相よりHigh Pure miRNA Isolation Kit(ロッシュ社)を用いて全RNAを抽出した。
NCode VILO miRNA cDNA Synthesis Kit(Invitrogen社)にてcDNAを合成した後に、リアルタイムRT-PCRをABI Prism 7300 Sequence Detection System(Applied Biosystems社)、EXPRESS SYBR GreenER qPCR SuperMix(Invitrogen社)を用いて実施した。
使用したプライマーは以下の通りである:
hsa-miR-16, 5’-TAG-CAG-CAC-GTA-AAT-ATT-GGC-G-3’(配列番号6)
hsa-miR-132, 5’-TAA-CAG-TCT-ACA-GCC-ATG-GTC-G-3’(配列番号7)
hsa-miR-146a, 5’-TGA-GAA-CTG-AAT-TCC-ATG-GGT-T-3’(配列番号8)
hsa-miR-155, 5’-TTA-ATG-CTA-ATC-GTG-ATA-GGG-GTA-3’(配列番号9)
hsa-miR-223, 5’-TGT-CAG-TTT-GTC-AAA-TAC-CCC-A-3’(配列番号10)
cel-miR-39, 5’-CGT-CAC-CGG-GTG-TAA-ATC-AGC-TTG-3’(配列番号11)
リバースプライマーはNCode VILO miRNA cDNA Synthesis Kitの添付品を用いた。
各miRNAの発現レベルを定量するために、PCR産物をpTAC-1 vector((株)バイオダイナミック研究所)に挿入したものを予め作成し、連続希釈物で標準曲線を作成した。各サンプルに既知の濃度で添加してあるcel-miR-39をもとに各サンプルのmiRNAの絶対濃度を計算した。
(1)関節リウマチ患者と変形性関節症患者の関節液中miRNAの濃度を両側t検定を用いて検定した。
(2)関節リウマチ患者と変形性関節症患者の血漿中miRNA濃度の両疾患間の違いを両側t検定を用いて検定した。
(3)関節リウマチ患者において、関節リウマチの病勢を示すTJC(疼痛関節数)、DAS28スコアに関し、(a)血漿中miRNA濃度、(b)関節液中miRNA濃度、(c)関節液中miRNA濃度を血漿中miRNA濃度で除した値(Ea/Eb)の相関について、それぞれピアソンの積率相関係数を用いて統計的に解析した。
図1に示すように、関節液中miR-16、miR-146a、miR-155およびmiR-223の濃度はp≦0.05で関節リウマチ患者が変形性関節症患者より高かった。関節リウマチの陽性値をそれぞれ40pmol/L、11pmol/L、0.04pmol/L、2.5pmol/L以上とすると、それぞれ感度75%特異度78%、感度73%特異度72%、感度77%特異度70%、感度56%特異度80%で関節リウマチと判断することができる。
図2に示すように、正常人由来の血漿中miR-132は関節リウマチ患者または変形性関節症患者より有意に高かった。また、陽性値を67pmol/L以下とした場合に、関節リウマチおよび変形性関節症それぞれに対して約84%の感度と約80%の特異度をもって、疾患を有する状態であると判定することができる。また、ROC曲線のAUCはそれぞれ0.90、0.91と高値であることからも優れた検査であることが示される。
図3に示すように、miR-16血漿中濃度は現在最もよく使われている関節リウマチの疾患活動性評価基準であるDAS28と相関した。miR-16、miR-132、miR-146a、miR-155、miR-223血漿中濃度はTJC(疼痛関節数)と相関が認められた。
図4に示すようにmiR-16、miR-132、miR-146aの関節液/血漿濃度比は、TJC(疼痛関節数)と相関が認められた。
以上、本発明を特定の態様に沿って説明したが、当業者に自明の変形や改良は本発明の範囲に含まれる。
Claims (7)
- 血液または関節由来の液体サンプル中に含まれる配列番号1~5からなる群から選択される少なくとも1つのmiRNAを指標とする、免疫系の異常疾患及び/または関節系の異常疾患の検出方法。
- 経時的に採取された血液由来の液体サンプルまたは関節由来の液体サンプルを準備する準備工程と、
前記サンプルから配列番号1~5に示される少なくとも一つのmiRNAの発現量を測定する測定工程と、
異なる採取時期の間で前記発現量を比較する比較工程とを含む、
免疫系の異常疾患及び/または関節系の異常疾患の病勢の判定を行う検査方法。 - 経時的に採取された血液由来の液体サンプルおよび関節由来の液体サンプルを準備する準備工程と、
それぞれのサンプルから配列番号1~5に示される少なくとも一つのmiRNAの発現量を測定する測定工程と、
関節由来サンプルから得られた前記miRNAの発現量(Ea)と血液由来サンプルから得られた前記miRNAの発現量(Eb)との比(Ea/Eb)を異なる採取時期の間で比較する工程とを含む、免疫系の異常疾患及び/または関節系の異常疾患の病勢の判定を行う検査方法。 - 血液由来の液体サンプル中、配列番号2に示されるmiRNAの発現量を測定する、免疫系の異常疾患及び/または関節系の異常疾患の罹患の判定を行う検査方法。
- 関節由来の液体サンプル中、配列番号1、3、4および5からなる群から選択された少なくとも一つのmiRNAの発現量を測定する、免疫系の異常疾患及び/または関節系の異常疾患の判別を行う検査方法。
- 血液由来の液体サンプルが血漿であり、かつ、関節由来の液体サンプルが、関節液である、請求項1~5のいずれか一項に記載の方法。
- 配列番号1~5からなる群から選択されるmiRNAの発現量を分析することのできるプライマーまたはプローブの、免疫系の異常疾患及び/または関節系の異常疾患の検出用キットの製造への使用。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2012509711A JPWO2011126105A1 (ja) | 2010-04-08 | 2011-04-08 | 免疫系の異常疾患または関節系の異常疾患への罹患またはこれらの病勢を判定するための検査方法 |
EP11766005.0A EP2557179A4 (en) | 2010-04-08 | 2011-04-08 | ANALYSIS METHOD FOR DETERMINING IMMUNE SYSTEM FAILURE, MANIFESTATION OF JOINT SYSTEM FAILURE, OR PROGRESSION THEREOF |
US13/639,278 US20130022985A1 (en) | 2010-04-08 | 2011-04-08 | Examination method to determine contraction or activity of diseases related immune system or joint system |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2010-101467 | 2010-04-08 | ||
JP2010101467 | 2010-04-08 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2011126105A1 true WO2011126105A1 (ja) | 2011-10-13 |
Family
ID=44763039
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/058875 WO2011126105A1 (ja) | 2010-04-08 | 2011-04-08 | 免疫系の異常疾患または関節系の異常疾患への罹患またはこれらの病勢を判定するための検査方法 |
Country Status (4)
Country | Link |
---|---|
US (1) | US20130022985A1 (ja) |
EP (1) | EP2557179A4 (ja) |
JP (1) | JPWO2011126105A1 (ja) |
WO (1) | WO2011126105A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014034685A1 (ja) | 2012-08-27 | 2014-03-06 | 国立大学法人京都大学 | マイクロrnaによる関節リウマチの診断 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20170183734A1 (en) * | 2014-05-22 | 2017-06-29 | Servicio Andaluz De Salud | Circulating mirnas as biomarkers of therapy effectiveness in rheumatoid arthritis patients treated with anti-tnf alpha |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009100342A2 (en) * | 2008-02-08 | 2009-08-13 | Medimmune, Llc | Disease markers and uses thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2386637B1 (en) * | 2001-09-28 | 2018-05-16 | Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. | Microrna molecules |
-
2011
- 2011-04-08 JP JP2012509711A patent/JPWO2011126105A1/ja active Pending
- 2011-04-08 WO PCT/JP2011/058875 patent/WO2011126105A1/ja active Application Filing
- 2011-04-08 EP EP11766005.0A patent/EP2557179A4/en not_active Withdrawn
- 2011-04-08 US US13/639,278 patent/US20130022985A1/en not_active Abandoned
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009100342A2 (en) * | 2008-02-08 | 2009-08-13 | Medimmune, Llc | Disease markers and uses thereof |
Non-Patent Citations (13)
Title |
---|
AMBROS V: "microRNAs: tiny regulators with great potential", CELL, vol. 107, 2001, pages 823 - 826 |
FULCI V. ET AL.: "miR-223 is overexpressed in T-lymphocytes of patients affected by rheumatoid arthritis", HUM. IMMUNOL., vol. 71, no. 2, 1 February 2010 (2010-02-01), pages 206 - 211, XP026871884 * |
HANSON E.K. ET AL.: "Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs", ANAL. BIOCHEM., vol. 387, no. 2, 15 April 2009 (2009-04-15), pages 303 - 314, XP026007607 * |
HENEGHAN H.M. ET AL.: "MicroRNAs as Novel Biomarkers for Breast Cancer", J. ONCOL., vol. 2010, 2009, XP008161993 * |
MITCHELL P.S. ET AL.: "Circulating microRNAs as stable blood-based markers for cancer detection", PROC. NATL. ACAD. SCI. USA., vol. 105, no. 30, 29 July 2008 (2008-07-29), pages 10513 - 10518, XP002518102 * |
MITCHELL PS ET AL.: "Circulating microRNAs as stable blood-based markers for cancer detection", PROC NATL ACAD SCI USA, vol. 105, 2008, pages 10513 - 10518 |
MURATA K. ET AL.: "Plasma and synovial fluid microRNAs as potential biomarkers of rheumatoid arthritis and osteoarthritis", ARTHRITIS RES. THER., vol. 12, no. 3, 2010, pages R86, XP021085214 * |
NAKASA T. ET AL.: "Expression of microRNA-146 in rheumatoid arthritis synovial tissue", ARTHRITIS RHEUM., vol. 58, no. 5, May 2008 (2008-05-01), pages 1284 - 1292, XP055024560 * |
PAULEY K.M. ET AL.: "Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients", ARTHRITIS RES. THER., vol. 10, no. 4, 2008, pages R101, XP021046804 * |
See also references of EP2557179A4 |
STANCZYK J. ET AL.: "Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis", ARTHRITIS RHEUM., vol. 58, no. 4, April 2008 (2008-04-01), pages 1001 - 1009, XP002559944 * |
VALADI H ET AL.: "Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells", NAT CELL BIOL, vol. 9, 2007, pages 654 - 659 |
ZHU W. ET AL.: "Circulating microRNAs in breast cancer and healthy subjects", BMC RES. NOTES, vol. 2, no. 89, 19 May 2009 (2009-05-19), XP021060778 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2014034685A1 (ja) | 2012-08-27 | 2014-03-06 | 国立大学法人京都大学 | マイクロrnaによる関節リウマチの診断 |
Also Published As
Publication number | Publication date |
---|---|
EP2557179A4 (en) | 2013-09-25 |
JPWO2011126105A1 (ja) | 2013-07-11 |
US20130022985A1 (en) | 2013-01-24 |
EP2557179A1 (en) | 2013-02-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20230313300A1 (en) | Biomarkers of traumatic brain injury | |
EP3283643B1 (en) | Means for diagnosing, predicting or monitoring pneumocystis pneumonia | |
Halimi et al. | Clinical translation of human microRNA 21 as a potential biomarker for exposure to ionizing radiation | |
WO2017054325A1 (zh) | 乳腺癌联合诊断标志物及检测试剂盒 | |
Wang et al. | Plasma microRNA-586 is a new biomarker for acute graft-versus-host disease | |
KR20200002241A (ko) | 비만 진단을 위한 마이크로RNA-26b 또는 마이크로RNA-4449 바이오마커 및 이의 용도 | |
Narita et al. | Expression of microRNAs in plasma and in extracellular vesicles derived from plasma for dogs with glioma and dogs with other brain diseases | |
CN104694623A (zh) | 一种用于肺癌诊断的血浆miRNA标志物及应用 | |
TWI571514B (zh) | 評估罹患大腸直腸癌風險的方法 | |
WO2011126105A1 (ja) | 免疫系の異常疾患または関節系の異常疾患への罹患またはこれらの病勢を判定するための検査方法 | |
WO2014034685A1 (ja) | マイクロrnaによる関節リウマチの診断 | |
CN110305953B (zh) | 检测miRNA表达量的系统在制备区分结核性脑膜炎和病毒性脑膜炎产品中的应用 | |
ES2688737A1 (es) | Método para diagnosticar placa ateroesclerótica inestable | |
Zhong et al. | WT1 expression in circulating RNA as a minimal residual disease marker for AML patients after stem-cell transplantation | |
CN112534068A (zh) | 使用液体活检多种癌基因生物标志物的乳腺癌早期诊断及治疗后监测的方法 | |
CN115851958A (zh) | 检测胰腺癌相关基因甲基化的引物、探针、试剂盒及方法 | |
CN112522391B (zh) | hsa_circ_0008961作为痛风诊断标志物的应用 | |
CN114196747A (zh) | 一种与代谢相关脂肪性肝病发生发展关联的生物标志物 | |
Nateghi et al. | Circulating miR-193b-3p and miR-376a-3p involved in Iranian patients with multiple sclerosis | |
CN112662774A (zh) | 一种肝癌循环肿瘤细胞标志物及其应用 | |
WO2020127499A2 (en) | Mirnas as biomarkers for parkinson's syndrome | |
Salim et al. | Investigation on staging of breast cancer using miR-21 as a biomarker in the serum | |
Xu et al. | The Plasma Exosomal MicroRNA let-7c-5p as a Potential Circulating Biomarker for Mycobacterium Infection in HIV Population | |
Hadi et al. | Altered Expression of Circulating miR-377 and miR-98 in Relapsing-remitting Multiple Sclerosis | |
CN105969895A (zh) | 一种使用逆转录pcr检测hla-b*5801基因表达的检测方法及试剂盒 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 11766005 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2012509711 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13639278 Country of ref document: US |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2011766005 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2011766005 Country of ref document: EP |