JP6619611B2 - Biomarker, method for assisting diagnosis of autoimmune hepatitis, and kit - Google Patents
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Description
本発明は、自己免疫性肝炎を診断するためのバイオマーカー、自己免疫性肝炎の診断補助方法、及び、自己免疫性肝炎を診断するためのキットに関する。 The present invention relates to a biomarker for diagnosing autoimmune hepatitis, a method for assisting diagnosis of autoimmune hepatitis, and a kit for diagnosing autoimmune hepatitis.
肝炎には、ウイルス性肝炎、アルコール性肝炎、自己免疫性肝炎（以下、「AIH：Autoimmune hepatitis」と略することがある。）が知られている。ウイルス性肝炎及びアルコール性肝炎については発症の原因物質が明らかとなっているが、AIHについてはまだ解明されていない。AIHは女性に多い疾患であり関節痛・全身倦怠感、食欲低下、悪心、黄疸、発熱等の症状を呈す。多くの症例では、副腎皮質ステロイド投与が極めて良く奏効し、投与によりAST、ALTは速やかに基準値内へと改善するが、治療開始が遅れた場合、有効性は低下する。 As hepatitis, viral hepatitis, alcoholic hepatitis, and autoimmune hepatitis (hereinafter sometimes abbreviated as “AIH: Autoimmune hepatitis”) are known. The causative agent of viral hepatitis and alcoholic hepatitis has been clarified, but AIH has not been elucidated yet. AIH is a disease common to women and presents symptoms such as joint pain, general malaise, decreased appetite, nausea, jaundice, and fever. In many cases, administration of corticosteroids has been very successful, and AST and ALT are quickly improved to the standard value by administration, but effectiveness is reduced if treatment is delayed.
AIHの診断は、特異性の高い診断マーカーが存在しないため、ウイルス性肝炎の有無を確認した後、血清トランスアミナーゼ・血清γグロブリン・自己抗体（抗核抗体、抗平滑筋抗体）の検査結果を総合的に判断してなされるものである。2013年版改訂厚生労働省「難治性の肝・胆道疾患に関する調査研究」班AIH診断指針でも、(1)他の原因による肝障害が否定される、(2)抗核抗体陽性あるいは抗平滑筋抗体陽性、(3)IgG高値（＞基準上限値1.1倍）、(4)組織学的にinterface hepatitisや形質細胞浸潤がみられる、(5)副腎皮質ステロイドが著効する、のうち上記項目で１を満たし、2〜5のうち3項目以上を認める場合をAIH典型例に該当するとしている。 Since there is no highly specific diagnostic marker for AIH diagnosis, after confirming the presence or absence of viral hepatitis, comprehensive test results of serum transaminase, serum γ globulin, and autoantibodies (antinuclear antibodies, anti-smooth muscle antibodies) Judgment is made by judgment. The 2013 edition revised Ministry of Health, Labor and Welfare “Survey and research on refractory liver and biliary tract diseases” group AIH diagnosis guidelines also (1) Liver disorders caused by other causes are denied, (2) Positive antinuclear antibody or antismooth muscle antibody , (3) high IgG level (> 1.1 times the upper limit of the standard), (4) histologically interface hepatitis and plasma cell infiltration, (5) corticosteroid is effective, 1 in the above item Satisfies and accepts 3 or more items from 2 to 5 as AIH typical cases.
特許文献１には、血清あるいは血漿中の所定の抗サイトケラチン抗体がAIH患者で有意に高値を示し、更に抗体の量が治療に伴って減少することが記載されている。 Patent Document 1 describes that a predetermined anti-cytokeratin antibody in serum or plasma shows a significantly high value in AIH patients, and that the amount of antibody decreases with treatment.
近年、血液試料中の肝特異的なmiRNAは既存の診断マーカーに比べて組織特異性が高いことから、高感度なマーカーとして注目されている。非特許文献１には、AIH患者では、血清内のmiR-941とmiR-1280の発現量が低下していることが記載されている。 In recent years, liver-specific miRNA in blood samples has been attracting attention as a highly sensitive marker because it has higher tissue specificity than existing diagnostic markers. Non-Patent Document 1 describes that in AIH patients, the expression levels of miR-941 and miR-1280 in serum are reduced.
しかし、上述の技術によってはAIHの迅速且つ的確な診断は十分であるとはいえない。 However, rapid and accurate diagnosis of AIH is not sufficient with the above-described techniques.
本発明はかかる問題点に鑑みてなされたものであって、迅速且つ的確なAIHの診断を可能とするバイオマーカー等を提供することを目的とする。 The present invention has been made in view of such problems, and an object of the present invention is to provide a biomarker or the like that enables rapid and accurate diagnosis of AIH.
本発明にかかるバイオマーカーは、自己免疫性肝炎を診断するためのバイオマーカーであって、miR-21-5pであることを特徴とする。 The biomarker according to the present invention is a biomarker for diagnosing autoimmune hepatitis, and is characterized by miR-21-5p.
本発明にかかる自己免疫性肝炎の診断補助方法は、生物学的試料中のmiR-21-5pの発現量を決定するステップを含むことを特徴とする。 The method for assisting diagnosis of autoimmune hepatitis according to the present invention comprises the step of determining the expression level of miR-21-5p in a biological sample.
本発明にかかるキットは、自己免疫性肝炎を診断するためのキットであって、生物学的試料におけるmiR-21-5pの発現量を検出又は定量する手段を含むことを特徴とする。 The kit according to the present invention is a kit for diagnosing autoimmune hepatitis, and includes a means for detecting or quantifying the expression level of miR-21-5p in a biological sample.
本発明によれば、AIHの迅速且つ的確な診断が可能となる。 According to the present invention, AIH can be diagnosed quickly and accurately.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
本実施形態にかかるバイオマーカーは、自己免疫性肝炎を診断するためのバイオマーカーであって、miR-21-5pである。 The biomarker according to this embodiment is a biomarker for diagnosing autoimmune hepatitis and is miR-21-5p.
miRNAは、タンパク質をコードしない22塩基程度の一本鎖RNAであり、標的mRNAの主に3’非翻訳領域に部分相補的に結合し、分解又は翻訳を抑制することにより、タンパク質の発現を負に制御することが知られている。miRNAは発生、分化、増殖、免疫、細胞死、恒常性等あらゆる生命現象に関与している。現在までにヒトでは約1500種類のmiRNAが同定されている。多くのmiRNAが様々な組織に広く分布しているが、いくつかのmiRNAは組織特異性の高いものが知られている。 miRNA is a single-stranded RNA of about 22 bases that does not encode protein, and binds to the 3 ′ untranslated region of the target mRNA partially in a complementary manner, thereby suppressing degradation or translation, thereby negatively expressing the protein. It is known to control. miRNAs are involved in all life phenomena such as development, differentiation, proliferation, immunity, cell death, and homeostasis. To date, approximately 1500 miRNAs have been identified in humans. Many miRNAs are widely distributed in various tissues, but some miRNAs are known to have high tissue specificity.
肝臓には多種類のmiRNAの発現が認められ、miR-941等のようにAIHのバイオマーカーも散見されるが、高感度なものは未だ見出されていない。本発明者はmiR-21-5pがAIHにおいて選択的に高発現することを見出し、かかる新知見に基づいて本件発明を完成させた。miR-21-5pは下記配列を有している。 Many types of miRNA are expressed in the liver, and AIH biomarkers such as miR-941 are also found in some cases, but no highly sensitive one has been found yet. The present inventor found that miR-21-5p is selectively highly expressed in AIH, and completed the present invention based on such new findings. miR-21-5p has the following sequence:
UAGCUUAUCAGACUGAUGUUGA・・・配列番号１
本明細書において「バイオマーカー」とは、自己免疫性肝炎の罹患の有無又は罹患の程度を診断するために、直接又は間接的に利用されるものである。
UAGCUUAUCAGACUGAUGUUGA ・ ・ ・ SEQ ID NO: 1
In the present specification, the “biomarker” is used directly or indirectly to diagnose the presence or absence of autoimmune hepatitis or the degree of the disease.
本実施形態においては、miR-21-5pの発現量の増加を指標として自己免疫性肝炎の診断がなされ、具体的には、(A)生物学的試料中に含まれるmiRNAを抽出する工程と、(B)生物学的試料中の本件miRNAの発現量を測定する工程と、(C)本件miRNAの発現量の増加を指標として自己免疫性肝炎を検出する工程と、を含む。 In the present embodiment, autoimmune hepatitis is diagnosed using an increase in the expression level of miR-21-5p as an index, and specifically, (A) a step of extracting miRNA contained in a biological sample; (B) measuring the expression level of the present miRNA in a biological sample, and (C) detecting autoimmune hepatitis using the increase in the expression level of the present miRNA as an index.
生物学的試料は、特に制限されることなく、被験者の血清、血液、唾液、腹水、尿、糞便、胃洗浄液、生検組織等が含まれ、例えば血清を好適に用いることができる。 The biological sample is not particularly limited, and includes serum, blood, saliva, ascites, urine, feces, gastric lavage fluid, biopsy tissue, and the like of the subject. For example, serum can be preferably used.
上記(A)工程におけるmiRNAを抽出する方法としては、被験者から得られる生物学的試料からmiRNAを含むRNAを抽出することのできる方法であれば特に制限されず、例えば、RNAzol RT試薬(Molecular Research Center社)でRNAを抽出後、miRCURY column(EXIQON社)にて精製することによって、miRNAを含むトータルRNAを抽出する方法が挙げられる。 The method for extracting miRNA in the step (A) is not particularly limited as long as it is a method capable of extracting RNA containing miRNA from a biological sample obtained from a subject.For example, RNAzol RT reagent (Molecular Research Center RNA), RNA is extracted, and then purified by miRCURY column (EXIQON) to extract total RNA containing miRNA.
上記(B)工程におけるmiRNAの発現量を測定する方法としては、特に限定されるものではないが、例えば定量PCR法（リアルタイムPCR法を含む）が挙げられる。この定量PCR法は、本件miRNAの配列を増幅し得るプライマーセットを用いる方法であり、本件miRNAの発現量を測定することが可能である限り特に制限されず、蛍光プローブ法（例えば、TaqMan（登録商標）プローブ法）、アガロース電気泳動法、SYBRグリーン法等の通常のPCR法を用いることができる。 The method for measuring the miRNA expression level in the step (B) is not particularly limited, and examples thereof include a quantitative PCR method (including a real-time PCR method). This quantitative PCR method is a method using a primer set capable of amplifying the sequence of the present miRNA, and is not particularly limited as long as the expression level of the present miRNA can be measured. The fluorescent probe method (for example, TaqMan (registered) (Trademark) probe method), agarose electrophoresis method, SYBR green method and other normal PCR methods can be used.
定量PCR法におけるプライマーセットとは、本件miRNAの配列を増幅し得るプライマー（ポリヌクレオチド）の組合せを意味する。上記プライマーとしては、本件miRNAの配列を増幅し得る限り特に限定されないが、フォワードプライマーとリバースプライマーとからなるプライマーセットを例示することができる。プライマーセットは、その配列情報に基づき当該技術分野において周知の方法を用いて化学合成等することにより得ることができる。 The primer set in the quantitative PCR method means a combination of primers (polynucleotides) that can amplify the sequence of the present miRNA. The primer is not particularly limited as long as the sequence of the present miRNA can be amplified, but a primer set composed of a forward primer and a reverse primer can be exemplified. The primer set can be obtained by chemical synthesis or the like using a method well known in the art based on the sequence information.
上記(C)工程において、上記のように測定された本件miRNAの発現量の増加は、具体的には、被験者の生物学的試料中の本件miRNAの発現量が、コントロールの生物学的試料中の本件miRNAの発現量（例えば、健常者の生物学的試料中の本件miRNAの発現量）に比して高い場合に、被験者がAIHであると診断することができる。 In the step (C), the increase in the expression level of the present miRNA measured as described above is, specifically, the expression level of the present miRNA in the biological sample of the subject is increased in the control biological sample. When the expression level of the present miRNA is higher than the expression level of the present miRNA (for example, the expression level of the present miRNA in a biological sample of a healthy person), the subject can be diagnosed as having AIH.
なお、自己免疫性肝炎が進行して肝組織の線維化が進むと肝硬変となるが、かかる場合にあっては本件miRNAの発現量は逆に低下する。これにより本件miRNAには、病態の経過が把握できるという利点がある。 In addition, when autoimmune hepatitis progresses and the fibrosis of the liver tissue progresses, cirrhosis occurs. In such a case, the expression level of the present miRNA decreases. As a result, the present miRNA has the advantage that the course of the disease can be grasped.
また、本実施形態による自己免疫性肝炎を診断するためのキットは、生物学的試料における本件miRNAの発現量を測定する手段を含むものであり、例えば、本件miRNAの配列を増幅し得る、フォワードプライマーとリバースプライマーとからなるプライマーセットを含む。 Further, the kit for diagnosing autoimmune hepatitis according to the present embodiment includes a means for measuring the expression level of the miRNA in a biological sample.For example, the forward miRNA sequence can be amplified. Includes a primer set consisting of a primer and a reverse primer.
無治療のAIH患者(n=5)血清と健常人(n=5)をmiRNAアレイでスクリーニングし、AIHで特異的に上昇しているmiRNAを同定した（図１）。 Untreated AIH patient (n = 5) sera and healthy individuals (n = 5) were screened with miRNA arrays to identify miRNAs that were specifically elevated with AIH (FIG. 1).
さらに、無治療AIH(n=5)、治療後寛解時(n=5)の血清を、同様にmiRNAアレイで解析し、AIHで特異的に上昇し、治療後、寛解時に正常レベルまで低下するmiRNAを絞り込んだ（図２）。これにより、miRNA-21-5pがAIHの急性期に特異的に血清中で増加しているmiRNAであることが判明した。 In addition, serum from untreated AIH (n = 5) and post-treatment remission (n = 5) was similarly analyzed with miRNA arrays, and increased specifically with AIH, and decreased to normal levels after treatment and remission. miRNA was narrowed down (FIG. 2). This revealed that miRNA-21-5p is a miRNA that is specifically increased in serum during the acute phase of AIH.
次にAIH患者(n=46)、慢性C型肝炎患者(n=40)、健常人(n=13)の血清を用い、確立したmiRNA-21-5p定量PCRで測定した。血清からのmiRNAはQIAGEN社のmiRNeasySerum/plasmaキットを用いて抽出した。miRNA発現レベルの測定において、miRNA抽出に起因する試料間の格差を補正するために、内部標準でmiRNAの発現レベルを補正した。単離手順の前にあらかじめcel-miR-39を血清に添加し内部標準とした。抽出したmiRNAはマイクロキャピラリー電気泳動により評価した(2100バイオアナライザー・アジレントテクノロジー)。 Next, sera from AIH patients (n = 46), chronic hepatitis C patients (n = 40), and healthy persons (n = 13) were used, and measured by established miRNA-21-5p quantitative PCR. MiRNA from serum was extracted using miRNeasySerum / plasma kit of QIAGEN. In measuring the miRNA expression level, the miRNA expression level was corrected with an internal standard in order to correct the disparity between samples due to miRNA extraction. Prior to the isolation procedure, cel-miR-39 was added to the serum in advance as an internal standard. The extracted miRNA was evaluated by microcapillary electrophoresis (2100 Bioanalyzer Agilent Technologies).
hsa-miR-21-5p、cel-miR-39はTaqman MicroRNA Assay (applied biosystems by Life Technoligies)を用いて逆転写し、リアルタイムPCR法にて発現量を分析した。発現量解析は、cel-miR-39を内部標準として、hsa-miR-21-5pを補正し比較Ct定量法(Comparative Ct Method,ΔΔCt法)を用いて計算し相対定量を行った。 hsa-miR-21-5p and cel-miR-39 were reverse transcribed using Taqman MicroRNA Assay (applied biosystems by Life Technologies), and the expression level was analyzed by real-time PCR. Expression level analysis was carried out using cel-miR-39 as an internal standard, hsa-miR-21-5p corrected and calculated using the comparative Ct quantification method (Comparative Ct Method, ΔΔCt method), and relative quantification was performed.
AIH患者においては、健常人及びCHC患者に比べ有意にmiRNA-21-5p発現量が増加していた（図３）。そしてAIH患者において、ステロイド治療（プレドニゾロン平均投与量40.3±11.6 mg/day 20-60mg/day)でmiRNA-21-5p発現量は低下することが判明した（図４）。 In AIH patients, miRNA-21-5p expression was significantly increased compared to healthy and CHC patients (FIG. 3). In AIH patients, it was found that miRNA-21-5p expression decreased with steroid treatment (prednisolone average dose 40.3 ± 11.6 mg / day 20-60 mg / day) (FIG. 4).
さらに、AIH患者の同一血清を用い肝炎の指標であるALT、ASTと、miRNA-21-5pとの相関を調べた所、強い相関を示すことが判明した（図５）。 Furthermore, when the correlation between ALT and AST, which are indicators of hepatitis, and miRNA-21-5p was examined using the same serum from AIH patients, it was found that the correlation was strong (FIG. 5).
また、肝硬変進展例では、血清miRNA-21-5pが有意に低下することが判明した（図６）。AIHの肝生検組織像との関連を検討した結果、miR-21-5pはAIH肝組織の線維化(staging)の進行と共に低下していた（図７）。なお、肝組織において門脈域より線維化が進展し小葉が改築され肝硬変へ進展する段階を線維化なし（F0）、門脈域の線維性拡大（F1）、線維性架橋形成（F2）、小葉のひずみを伴う線維性架橋形成（F3）までの４段階に区分され、さらに結節形成傾向が全体に認められる場合は肝硬変（F4）と分類される(Staging分類)。図７においてF4におけるmiRNA-21-5pの発現量は、健常者のmiRNA-21-5pの発現量とほぼ同程度であった。 It was also found that serum miRNA-21-5p was significantly decreased in cases of cirrhosis progression (FIG. 6). As a result of examining the relationship of AIH with liver biopsy histology, miR-21-5p decreased with the progression of AIH liver tissue fibrosis (FIG. 7). In the liver tissue, the stage where fibrosis progresses from the portal vein region and the lobule is remodeled and progresses to cirrhosis is not fibrotic (F0), the fibrosis of the portal vein region (F1), the formation of fibrous bridge (F2), It is divided into 4 stages up to fibrous bridge formation (F3) with lobular strain, and if the tendency of nodule formation is observed throughout, it is classified as cirrhosis (F4) (Staging classification). In FIG. 7, the expression level of miRNA-21-5p in F4 was almost the same as the expression level of miRNA-21-5p in healthy subjects.
また、miR-21-5pはAIH肝組織の肝炎の活動性(grading)の上昇と相関することが判明した（図８）。なお、肝組織において壊死・炎症所見はその程度により活動性なし（A0）、軽度活動性（A1）、中等度活動性（A2）、高度活動性（A3）の４段階に区分され、すなわち、活動性の評価はpiecemeal necrosis、小葉内の細胞浸潤と肝細胞の変性ならびに壊死（spotty necrosis、bridging necrosis等）で行われる(Grading分類)。 In addition, miR-21-5p was found to correlate with an increase in hepatic grading in AIH liver tissue (FIG. 8). In addition, necrosis / inflammation findings in liver tissue are classified into four stages according to the degree of inactivity (A0), mild activity (A1), moderate activity (A2), and high activity (A3). Evaluation of activity is performed by piecemeal necrosis, cell infiltration in leaflets, degeneration of hepatocytes and necrosis (spotty necrosis, bridging necrosis, etc.) (Grading classification).
また、同じ自己免疫性肝疾患である原発性胆汁性肝硬変症(PBC)との比較では、AIHのほうが有意に血清miR-21-5pが増加していることが判明した（図９）。なお、PBC患者のmiR-21-5pの発現量は、健常者のmiRNA-21-5pの発現量よりもやや高いものであった。 In comparison with primary biliary cirrhosis (PBC), which is the same autoimmune liver disease, it was found that AIH significantly increased serum miR-21-5p (FIG. 9). The expression level of miR-21-5p in PBC patients was slightly higher than the expression level of miRNA-21-5p in healthy subjects.
自己免疫性肝炎の診断に利用できる。 It can be used for diagnosis of autoimmune hepatitis.
配列番号１：マイクロRNA SEQ ID NO: 1 microRNA
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