JP2021129513A - Mirna biomarkers for diagnosis of diffuse alveolar damage type drug-induced interstitial pneumonia - Google Patents
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Abstract
Description
本発明は、びまん性肺胞障害型薬剤性間質性肺炎の発症や病勢の診断を補助するための方法に関する。 The present invention relates to a method for assisting in the diagnosis of the onset and pathology of diffuse alveolar disorder type drug-induced interstitial pneumonia.
薬剤性間質性肺炎は医薬品による肺の間質の炎症性有害作用の総称である。間質性肺炎は、薬剤性やじん肺、膠原病などの明確な原因に伴うものと、原因が不明な特発性とに分けられ、さらに様々な病型に分類される。それぞれ治療法や予後が異なるため、これらを鑑別することは臨床上重要である。 Drug-induced interstitial pneumonia is a general term for the inflammatory adverse effects of drugs on the lung interstitium. Interstitial pneumonia is divided into those associated with clear causes such as drug-induced pneumoconiosis and collagen disease, and idiopathic diseases of unknown cause, and further classified into various types. Differentiation of these is clinically important because each has different treatments and prognosis.
薬剤性間質性肺炎は、特発性や既知の肺疾患との類似性に基づいて、病型の診断が行われる（非特許文献1）。間質性肺炎の病型の中で、特に重篤な病型は、びまん性肺胞傷害（diffuse alveolar damage、DAD）である。DADは、急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例に見られる典型的な病型であり(非特許文献２)、特発性においても薬剤性においても、DADは治療反応性に乏しく予後が悪い（非特許文献１、２）。そのため、DADに対しては、早期の検出と診断が特に重要とされる。また、DAD以外の病型としては、器質化肺炎（organizing pneumonia、OP）、非特異性間質性肺炎（nonspecific interstitial pneumonia、NSIP）などが挙げられるが、どのパターンにも当てはまらない例や複数のパターンが混在する例など、画像検査や病理検査では分類が困難な例も存在し、画像パターン分類には限界がある（非特許文献3） The type of drug-induced interstitial pneumonia is diagnosed based on idiopathic or similarities with known lung diseases (Non-Patent Document 1). Among the types of interstitial pneumonia, the most serious type is diffuse alveolar damage (DAD). DAD is a typical type of disease found in acute exacerbation cases of acute respiratory distress syndrome and idiopathic interstitial pneumonia (Non-Patent Document 2), and DAD is responsive to treatment, both idiopathic and drug-induced. Poor and poor prognosis (Non-Patent Documents 1 and 2). Therefore, early detection and diagnosis are of particular importance for DAD. Disease types other than DAD include organizing pneumonia (OP) and nonspecific interstitial pneumonia (NSIP), but there are cases and multiple cases that do not fit into any pattern. There are cases where it is difficult to classify by image inspection or pathological examination, such as cases where patterns are mixed, and there is a limit to image pattern classification (Non-Patent Document 3).
薬剤性間質性肺炎の診断には、自己免疫性疾患による肺炎や医薬品に起因しない間質性肺炎の可能性を排除する必要があり、確定診断に時間を要することも多く、画像診断や病理診断だけでは経済的にも体力的にも患者に大きな負担を強いるため、バイオマーカーの利用が有用とされる。 For the diagnosis of drug-induced interstitial pneumonia, it is necessary to eliminate the possibility of pneumonia due to autoimmune diseases and interstitial pneumonia not caused by drugs, and it often takes time to make a definitive diagnosis. The use of biomarkers is useful because diagnosis alone imposes a heavy burden on patients both economically and physically.
間質性肺炎のバイオマーカーとしては、現在、シアル化糖鎖抗原KL-6（KL-6）、肺サーファクタントプロテインA（SP-A）、肺サーファクタントプロテインD（SP-D）が、診断の補助に利用されており、KL-6はDADに対する陽性率が高いことが知られている（非特許文献4, 5）。これらは、薬剤性肺障害を反映する指標であるが、喫煙や間質性肺炎以外の疾患でも増加することもあり、肺障害の重症度とは相関しない。KL-6は、薬剤投与前値を基準に経過を追うことが推奨されているものの、既存の間質性肺炎の増悪や日和見感染症でも上昇する。すなわち、これらのマーカーは、DADに特異的ではなく、病型を鑑別するマーカーとしての有用性は低い。また、KL-6、SP-D、およびSP-Aは病状の改善後も長期間高値を示す（非特許文献4, 5）という問題があることから、新たなバイオマーカーが必要とされている。これまでに、UBE2T、HK1、PMSE1、USO1、IFI16、GLTPなどのタンパク質や（特許文献1）、自己抗体（特許文献2）などがマーカー候補として提案、特許申請されているものの、薬剤性微小機能性RNAの一つであるmicroRNA(miRNA)が薬剤性間質性肺炎のバイオマーカーのなりうることを示す報告はこれまでになされていない。
Currently, sialylated sugar chain antigen KL-6 (KL-6), pulmonary surfactant protein A (SP-A), and pulmonary surfactant protein D (SP-D) assist in diagnosis as biomarkers of interstitial pneumonia. KL-6 is known to have a high positive rate for DAD (
本発明は、DAD型薬剤性間質性肺炎の診断、病勢、およびその病型を診断、予測するためのmiRNAバイオマーカーの開発を行うことを目的とする。 An object of the present invention is to develop a miRNA biomarker for diagnosing, pathologically, and diagnosing and predicting DAD-type drug-induced interstitial pneumonia.
752種のmiRNAに対するLNA-enhanced Probe/Primersを用いたmiRCURY LNA microRNA PCR法にて、薬剤性間質性肺炎患者の血清試料を対象にmiRNAの網羅的発現解析を行った。本研究では種々の薬剤性間質性肺炎病型のうち、治療応答性かつ臨床予後が悪いびまん性肺胞傷害（DAD）に焦点を当て、miRCURY LNA microRNA PCRで算出される補正サイクル値（ΔCq値）から、健常成人や回復期群に対して当該疾患の急性期で大きく変動するmiRNAを、Fold change値と効果量（Hedge’s g値）を指標に探索した。その結果、測定した752種のmiRNAの中から、DADの急性期に有意な発現低下を示すmiRNAを3種類（has-miR-150-5p, has-miR-10a-5p, has-miR-340-5p）同定した。 Comprehensive expression analysis of miRNA was performed on serum samples of drug-induced interstitial pneumonia patients by miRCURY LNA microRNA PCR method using LNA-enhanced Probe / Primers for 752 types of miRNA. This study focuses on diffuse alveolar injury (DAD), which is treatment-responsive and has a poor clinical prognosis, among various drug-induced interstitial pneumonia types, and the corrected cycle value (ΔCq) calculated by miRCURY LNA microRNA PCR. Values) were used to search for miRNAs that fluctuate significantly in the acute phase of the disease in healthy adults and convalescent groups, using the Fold change value and the amount of effect (Hedge's g value) as indicators. As a result, out of the 752 types of miRNAs measured, 3 types of miRNAs (has-miR-150-5p, has-miR-10a-5p, has-miR-340) showing a significant decrease in the expression of DAD in the acute phase -5p) Identified.
DADで発現変動が認められた3種のmiRNAについて、ROC曲線（Receiver Operating Characteristic curve) 解析の結果、DAD患者と健常成人を高い精度（area under the ROC curve: AUROC 0 .85以上）で鑑別した。また、これらmiRNAの病勢診断における診断能についても、既存マーカーであるKL-6 (AUROC 0.74)より高く, SP-D (0.90)と同程度であり、これら３種のmiRNAを組み合わせた診断モデルは健常成人並びにDAD回復期症例に対し、既存マーカーより高い診断能(AUROC 0.98以上)を示した。 As a result of ROC curve (Receiver Operating Characteristic curve) analysis, DAD patients and healthy adults were differentiated with high accuracy (area under the ROC curve: AUROC 0.85 or higher) for the three miRNAs whose expression was observed to fluctuate in DAD. .. In addition, the diagnostic ability of these miRNAs in pathological diagnosis is higher than the existing marker KL-6 (AUROC 0.74) and comparable to SP-D (0.90), and a diagnostic model combining these three types of miRNAs is available. It showed higher diagnostic ability (AUROC 0.98 or higher) than existing markers in healthy adults and DAD convalescent cases.
DADで発現変動が認められた3種のmiRNAについて、Taqman Advanced miRNA Assay法を用いて、その診断能について検証したところ、いずれのmiRNAも健常成人に対して有意な診断能を示した。また、miRCURY LNA microRNA PCR法で得られた結果と同様に、3種のmiRNAを組み合わせることにより、既存マーカーであるKL-6やSP-Dと比べて高いAUROC (0.96)でDADの急性期と回復期症例を識別することができた。さらに、SP-Dと上記3種のmiRNAsを組み合わせることで、感度・特異度100％(AUROC 1.0)でDADの病勢診断を行うことができることが認められた。加えて、3種miRNAsの組み合わせによる診断モデルはDADとその他薬剤性間質性肺炎の病型であるOP(AUROC 0.93)及びNSIP(AUROC 0.88)を、既存マーカーより高い精度で識別可能であった。 When the diagnostic ability of the three miRNAs whose expression was observed to fluctuate in DAD was verified using the Taqman Advanced miRNA Assay method, all miRNAs showed significant diagnostic ability in healthy adults. In addition, similar to the results obtained by the miRCURY LNA microRNA PCR method, by combining three types of miRNA, AUROC (0.96), which is higher than the existing markers KL-6 and SP-D, is associated with the acute phase of DAD. Convalescent cases could be identified. Furthermore, it was confirmed that by combining SP-D and the above three types of miRNAs, it is possible to diagnose the pathology of DAD with 100% sensitivity and specificity (AUROC 1.0). In addition, the diagnostic model using the combination of three miRNAs was able to identify DAD and other types of drug-induced interstitial pneumonia, OP (AUROC 0.93) and NSIP (AUROC 0.88), with higher accuracy than existing markers. ..
has-miR-150-5p, has-miR-10a-5p, has-miR-340-5pの組み合わせ診断モデルを用いてDADと非薬剤性間質性肺炎の肺疾患との診断能を評価したところ、細菌感染症以外（AUROC 0.77）の肺疾患すべてに対して高い診断能が認められた（AUROC > 0.87）。また、本miRNAモデルにSP-Dを加えることで、細菌感染症を含むすべての対照肺疾患に対して高い診断能が認められた（AUROC > 0.89）。以上より、has-miR-150-5p, has-miR-10a-5p, has-miR-340-5p、あるいはそれらの組み合わせによるmiRNA診断モデルが、DAD型薬剤性間質性肺炎の特異的診断に有用なバイオマーカーとなりうることが示された。 The diagnostic ability of DAD and non-drug interstitial pneumonia with lung disease was evaluated using a combined diagnostic model of has-miR-150-5p, has-miR-10a-5p, and has-miR-340-5p. , High diagnostic ability was observed for all lung diseases other than bacterial infection (AUROC 0.77) (AUROC> 0.87). In addition, by adding SP-D to this miRNA model, high diagnostic ability was observed for all control lung diseases including bacterial infections (AUROC> 0.89). Based on the above, the miRNA diagnostic model using has-miR-150-5p, has-miR-10a-5p, has-miR-340-5p, or a combination thereof can be used for specific diagnosis of DAD-type drug-induced interstitial pneumonia. It has been shown that it can be a useful biomarker.
本発明は、これらの知見に基づいて、完成されたものであり、本発明の要旨は以下の通りである。
（１）hsa-miR-10a-5p、hsa-miR-340-5及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することを含む、DADの検査方法。
（２）発現を測定するmiRNAが、hsa-miR-10a-5p、hsa-miR-340-5及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAであり、測定値がDADの病勢診断を補助する(１)記載の方法。
（３）発現を測定するmiRNAが、hsa-miR-10a-5p、hsa-miR-340-5及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAであり、測定値がDADの特異的診断を補助する(１)記載の方法。
（４）発現を測定するmiRNAが、hsa-miR-10a-5p、hsa-miR-340-5、hsa-miR-150-5pの組み合わせである(２)記載の方法。
（５）発現を測定するmiRNAが、hsa-miR-10a-5p、hsa-miR-340-5、hsa-miR-150-5pの組み合わせである(３)記載の方法。
（６）hsa-miR-10a-5p、hsa-miR-340-5、hsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することができる試薬を含む、DADの検査のためのキット。
The present invention has been completed based on these findings, and the gist of the present invention is as follows.
(1) For at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5 and hsa-miR-150-5p, the gene expression in the sample derived from the subject is measured. How to test for DAD, including that.
(2) The miRNA whose expression is to be measured is at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5 and hsa-miR-150-5p, and the measured value. Assists in the diagnosis of DAD disease (1).
(3) The miRNA whose expression is to be measured is at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5 and hsa-miR-150-5p, and the measured value. Assists in the specific diagnosis of DAD (1).
(4) The method according to (2), wherein the miRNA whose expression is measured is a combination of hsa-miR-10a-5p, hsa-miR-340-5, and hsa-miR-150-5p.
(5) The method according to (3), wherein the miRNA whose expression is measured is a combination of hsa-miR-10a-5p, hsa-miR-340-5, and hsa-miR-150-5p.
(6) For at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5, and hsa-miR-150-5p, the gene expression in the sample derived from the subject is measured. A kit for testing DAD that contains reagents that can be used.
本発明により見いだされたmiRNAの発現量を、単独、あるいは複数項目を測定することにより、薬剤性間質性肺炎疑いの患者がびまん性肺胞傷害なのか否か、また患者の病勢や間質性肺炎の活動性について、高い精度で診断することができる。
By measuring the expression level of miRNA found by the present invention alone or by measuring multiple items, whether or not the patient suspected of having drug-induced interstitial pneumonia has diffuse alveolar injury, and the patient's pathology and interstitium. The activity of pneumonia can be diagnosed with high accuracy.
以下、本発明の実施の形態を詳細に説明する。
本発明は、hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における遺伝子発現を測定することを含む、びまん性肺胞障害の検査方法を提供する。
Hereinafter, embodiments of the present invention will be described in detail.
The present invention measures gene expression in a sample derived from a subject for at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p. Provided are methods of testing for diffuse alveolar disorders, including doing so.
びまん性肺胞傷害は、急性で高度の肺胞傷害により出現する肺病変で、CTでは広範な浸潤影やスリガラス様陰影に、牽引性気管支拡張や蜂巣肺などの構造改変所見が認められるのが特徴で、びまん性肺胞傷害を呈する病態としては、急性呼吸窮迫症候群（ARDS）や急性間質性肺炎（AIP）、特発性肺線維症（IPF）をはじめとする慢性間質性肺炎の急性増悪などが挙げられる。本発明の方法において、びまん性肺胞傷害は特発性であっても良いが薬剤性であることがより好ましい。本発明の方法は、びまん性肺胞障害の診断、例えば、びまん性肺胞障害の病勢診断や特異的診断を補助することができる。 Diffuse alveolar injury is a lung lesion that develops due to acute and severe alveolar injury, and CT shows extensive infiltrative and suriglass-like shadows with structural alterations such as traction bronchiectal dilatation and honeycombing. Characteristically, the pathological conditions that present with diffuse alveolar injury include acute chronic interstitial pneumonia such as acute respiratory distress syndrome (ARDS), acute interstitial pneumonia (AIP), and idiopathic pulmonary fibrosis (IPF). Exacerbations can be mentioned. In the method of the present invention, diffuse alveolar injury may be idiopathic, but more preferably drug-induced. The method of the present invention can assist in the diagnosis of diffuse alveolar disorder, for example, pathological diagnosis or specific diagnosis of diffuse alveolar disorder.
hsa-miR-10a-5p（miRBase ID: MIMAT0000253）は、精巣上体、消化管、腎臓、膵臓、肺等に発現することが報告されているmiRNAである。本遺伝子は、膵臓がん細胞において抗がん剤の治療抵抗性獲得に寄与することが報告されている（J Exp Clin Cancer Res. 2018 Apr 3;37(1):76.）。加えて、本遺伝子は胃がん細胞において、がんの転移を促進することが明らかとなっている(Oncol Lett. 2017 Mar; 13(3): 1131-1136.)。一方で、脂肪組織中のマクロファージにより産生される本遺伝子は、脂肪組織における炎症の抑制並びに脂肪組織のリモデリングにも関与する(Mol Metab. 2019, 29: 86-98.)。hsa-miR-10a-5pの塩基配列を配列番号１に示す。
hsa-miR-10a-5p (miRBase ID: MIMAT0000253) is a miRNA that has been reported to be expressed in the epididymis, gastrointestinal tract, kidneys, pancreas, lungs, etc. This gene has been reported to contribute to the acquisition of treatment resistance of anticancer drugs in pancreatic cancer cells (J Exp Clin Cancer Res. 2018
hsa-miR-340-5p（miRBase ID: MIMAT0004692）は、中枢神経系、硬膜や筋肉に高く発現するmiRNAであり、肺における発現も確認されている。本miRNAの機能については、これまでに炎症性サイトカインの産生抑制をおこなうことが知られている(Genes Genomics. 2019, 41:713-721.)。また、本遺伝子については、肺がん細胞の成長や転移を抑制することも報告されている(Cell Mol Biol Lett. 2019, 24: 34. Gene. 2019, 683:47-53.)。hsa-miR-340-5pの塩基配列を配列番号２に示す。 hsa-miR-340-5p (miRBase ID: MIMAT0004692) is a miRNA highly expressed in the central nervous system, dura mater and muscles, and its expression in the lungs has also been confirmed. The function of this miRNA has been known to suppress the production of inflammatory cytokines (Genes Genomics. 2019, 41: 713-721.). It has also been reported that this gene suppresses the growth and metastasis of lung cancer cells (Cell Mol Biol Lett. 2019, 24: 34. Gene. 2019, 683: 47-53.). The nucleotide sequence of hsa-miR-340-5p is shown in SEQ ID NO: 2.
hsa-miR-150-5p（miRBase ID: MIMAT0000451）は、肺、膵臓、リンパ節、胸膜等様々な部位に発現するmiRNAであり、がん抑制遺伝子として機能することが種々のがん細胞で報告されている(Mol Ther Nucleic Acids. 2019, 7;16:675-685. Biochem Biophys Res Commun. 2019, 12; 515:85-91. Auris Nasus Larynx. 2018, 45:854-865)。一方で、本分子は線維芽細胞において、炎症性サイトカインの産生や繊維化促進にも関与することが報告されている(Clin Rheumatol. 2019, doi: 10.1007/s10067-019-04894-7.. J Cell Physiol. 2019, doi: 10.1002/jcp.29386.)。 hsa-miR-150-5pの塩基配列を配列番号３に示す。 hsa-miR-150-5p (miRBase ID: MIMAT0000451) is a miRNA expressed in various sites such as lung, pancreas, lymph node, and pleura, and has been reported to function as a tumor suppressor gene in various cancer cells. (Mol Ther Nucleic Acids. 2019, 7; 16: 675-685. Biochem Biophys Res Commun. 2019, 12; 515: 85-91. Auris Nasus Larynx. 2018, 45: 854-865). On the other hand, it has been reported that this molecule is also involved in the production of inflammatory cytokines and promotion of fibrosis in fibroblasts (Clin Rheumatol. 2019, doi: 10.1007 / s10067-019-04894-7 .. J Cell Physiol. 2019, doi: 10.1002 / jcp.29386.). The nucleotide sequence of hsa-miR-150-5p is shown in SEQ ID NO: 3.
本発明において、被験者は、びまん性肺胞傷害の発症が疑われる哺乳動物、医薬品による間質性肺炎の発症が疑われる哺乳動物などであるが、発症の危険性が考えられるすべての哺乳動物を対象としてもよい。哺乳動物は、典型的にはヒトである。被験者由来の試料としては、被験者から得た細胞、組織、体液など、具体的には、被験者の血液（例えば、全血、血清、血漿、血漿交換外液など）や気管支肺胞洗浄液などを例示することができる。通常の血液検査（臨床検査）で得られる全血、血清あるいは血漿を血液サンプルとして使用するとよい。 In the present invention, the subjects are mammals suspected of developing diffuse alveolar injury, mammals suspected of developing interstitial pneumonia due to pharmaceuticals, etc. It may be a target. Mammals are typically humans. Examples of the sample derived from the subject include cells, tissues, and body fluids obtained from the subject, specifically, the subject's blood (for example, whole blood, serum, plasma, plasma exchange external fluid, etc.), bronchoalveolar lavage fluid, and the like. can do. Whole blood, serum or plasma obtained by a normal blood test (clinical test) may be used as a blood sample.
本発明の方法において、被験者由来の試料における発現の測定は、試料中の上記miRNAの存在量を測定すればよい。測定する手段としては、特に限定されることなく公知の方法を用いればよく、ノーザンブロット法、RT-PCR法、リアルタイムPRC法、miRNAマイクロアレイ法、Small RNA Sequencingなどを公知の方法として挙げることができる。 In the method of the present invention, the expression in a sample derived from a subject may be measured by measuring the abundance of the above miRNA in the sample. As the measuring means, a known method may be used without particular limitation, and examples of the known method include Northern blotting, RT-PCR, real-time PRC, miRNA microarray, and Small RNA Sequencing. ..
上記miRNAの発現を測定するためには、上記miRNAと特異的にハイブリダイズできる核酸プローブを用いるとよい（ノーザンブロット法で測定する場合）。あるいはまた、上記miRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを用いてもよい（RT-PCR法で測定する場合）。さらに、上述の核酸プローブとプライマーセットの組み合わせを用いてもよい（リアルタイムPCR法で測定する場合）。核酸プローブ及び核酸プライマーは、上記miRNAの遺伝子情報（上述）に基づいて設計することができる。核酸プローブは、通常、約１５〜１５００塩基のものが適当である。核酸プローブは、放射性元素、蛍光色素、酵素などで標識するとよい。核酸プライマーは、通常、約１５〜３０塩基のものが適当である。核酸プライマーを放射性元素、蛍光色素、酵素などで標識してもよい。 In order to measure the expression of the above miRNA, it is preferable to use a nucleic acid probe capable of specifically hybridizing with the above miRNA (when measuring by Northern blotting). Alternatively, at least one pair of nucleic acid primers capable of specifically amplifying the cDNA synthesized using the above miRNA as a template may be used (when measured by the RT-PCR method). Furthermore, the combination of the above-mentioned nucleic acid probe and primer set may be used (when measuring by the real-time PCR method). Nucleic acid probes and nucleic acid primers can be designed based on the above-mentioned genetic information of miRNA (described above). A nucleic acid probe of about 15 to 1500 bases is usually suitable. The nucleic acid probe may be labeled with a radioactive element, a fluorescent dye, an enzyme or the like. As the nucleic acid primer, one having about 15 to 30 bases is usually suitable. Nucleic acid primers may be labeled with radioactive elements, fluorescent dyes, enzymes and the like.
発現を測定するmiRNAは１種類でもよいし、複数種類であってもよい。複数のmiRNA発現データを参照することにより、より正確な評価が可能となりうる。複数のmiRNA発現を同時に検出するためには、マルチプレックスリアルタイムPCR（複数種の蛍光プローブを使用）、ＤＮＡアレイ（プローブを基板に固定）（Nat Rev Drug Discov. 2002, 1:951-960）、small RNA Sequencing等の検出法を用いてもよい。 The expression of miRNA may be one type or a plurality of types. More accurate evaluation may be possible by referring to multiple miRNA expression data. For simultaneous detection of multiple miRNA expressions, multiplex real-time PCR (using multiple fluorescent probes), DNA array (probeing on substrate) (Nat Rev Drug Discov. 2002, 1: 951-960), A detection method such as small RNA Sequencing may be used.
hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における発現を測定し、その発現レベルが低い場合（例えばΔCq値が高い）に、びまん性肺胞傷害を発症している可能性が高いと判定し、前記レベルが高い場合（例えばΔCq値が低い）に、びまん性肺胞傷害を発症している可能性が低いと判定することができる。 For at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p, the expression in the sample derived from the subject was measured and the expression level thereof. If the value is low (for example, the ΔCq value is high), it is determined that there is a high possibility of developing diffuse alveolar injury, and if the level is high (for example, the ΔCq value is low), the diffuse alveolar injury is detected. It can be determined that the possibility of developing the disease is low.
よって、本発明の方法は、びまん性肺胞傷害の診断（びまん性肺胞傷害の発症の有無の判定）を補助することができる。 Therefore, the method of the present invention can assist in the diagnosis of diffuse alveolar injury (determination of the presence or absence of the onset of diffuse alveolar injury).
本発明の一つの例として、びまん性肺胞傷害の診断は、以下のような基準で行うことができる。被験者から採取した血清における上記miRNAの少なくとも１種の発現を測定し、発現解析から得られるΔCq値、またはmiRNA組み合わせ診断モデルにΔCq値を代入して得られる診断スコアに対する予め設定されたカットオフ値や基準値よりも低い値（診断スコアの場合は基準値より高値）が得られた場合、被験者はびまん性肺胞傷害を発症していると評価する。この予め設定するカットオフ値は、当業者が適宜設定することができる。例えば、びまん性肺胞傷害を発症していない健常成人の定量値の95％信頼区間を基準値としたり、ROC曲線からカットオフ値を設定したりすることができる。あるいは、過去の測定値と比較して、上記miRNAの少なくとも一つが下降の傾向を辿った場合、びまん性肺胞傷害の発症の可能性を疑う。 As an example of the present invention, the diagnosis of diffuse alveolar injury can be made according to the following criteria. A preset cutoff value for the ΔCq value obtained by measuring the expression of at least one of the above miRNAs in the serum collected from the subject and substituting the ΔCq value into the miRNA combination diagnostic model. If a value lower than the reference value (or higher than the reference value in the case of a diagnostic score) is obtained, the subject is evaluated as having diffuse alveolar injury. This preset cutoff value can be appropriately set by those skilled in the art. For example, the 95% confidence interval of the quantitative value of a healthy adult who has not developed diffuse alveolar injury can be used as a reference value, or the cutoff value can be set from the ROC curve. Alternatively, if at least one of the above miRNAs follows a downward trend compared to past measurements, the possibility of developing diffuse alveolar injury is suspected.
本発明の方法は、びまん性肺胞傷害の病勢診断に利用できる。本明細書において、「病勢診断」とは、びまん性肺胞傷害と診断された被験者の、病態の程度や変化（重症度、治療効果等）の判断を意味する。hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における発現レベルが低い場合に、びまん性肺胞傷害の急性期にある可能性が高いと判定することができる。 The method of the present invention can be used for pathological diagnosis of diffuse alveolar injury. As used herein, the term "disease diagnosis" means the judgment of the degree and change (severity, therapeutic effect, etc.) of the pathological condition of a subject diagnosed with diffuse alveolar injury. Diffuse when at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p has low expression levels in subject-derived samples. It can be determined that there is a high possibility that the patient is in the acute phase of sexual alveolar injury.
また、本発明の方法は、びまん性肺胞傷害の特異的診断に利用できる。本明細書において、「特異的診断」とは、被験者が、びまん性肺胞傷害パターンの間質性肺炎なのか否かを判定することを指す。びまん性肺胞傷害は、間質性肺炎の病型の中でも特に重篤な病型であるので、びまん性肺胞傷害の特異的診断に利用できることは、臨床上の意義が大きい。hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における発現レベルが低い場合に、病型がびまん性肺胞傷害である可能性が高いと判定することができる。 In addition, the method of the present invention can be used for specific diagnosis of diffuse alveolar injury. As used herein, "specific diagnosis" refers to determining whether a subject has interstitial pneumonia with a diffuse alveolar injury pattern. Since diffuse alveolar injury is a particularly serious form of interstitial pneumonia, it is of great clinical significance that it can be used for specific diagnosis of diffuse alveolar injury. Disease when at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p has low expression levels in subject-derived samples. It can be determined that the type is likely to be diffuse alveolar injury.
本発明の方法をびまん性肺胞傷害の特異的診断に利用する場合には、発現を測定するmiRNAが、hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5p、又はそれらの組み合わせであるとよい。組み合わせとしては、hsa-miR-10a-5pとhsa-miR-340-5pの組み合わせ、hsa-miR-10a-5pとhsa-miR-150-5pの組み合わせ、hsa-miR-340-5pとhsa-miR-150-5pの組み合わせ又はhsa-miR-10a-5pとhsa-miR-340-5pとhsa-miR-150-5pの組み合わせを例示することができ、hsa-miR-10a-5pとhsa-miR-340-5pとhsa-miR-150-5pの組み合わせが好ましい。さらに、既存の間質性肺炎マーカーであるKL-6またはSP-Dを組み合わせてもよい。KL-6 (Krebs von den Lungen-6)は、II型肺胞上皮細胞が産生するO−グリコシド結合型糖タンパク質MUC1に存在する糖鎖抗原である。これまでに血清KL-6の測定は、特発性間質性肺炎、過敏性間質性肺炎、薬剤性間質性肺炎を含む、間質性肺炎全般に特異性の高い臨床検査として用いられてきた。SP-D（UniProtKB ID: P35247）は、II型肺胞上皮細胞により産生および分泌される肺サーファクタントタンパク質の一種であり、生体中では肺特異的に発現し、細菌やウイルスに対する生体防御機能に寄与することが知られている。肺で産生されたSP-Dは血液中にも存在することが知られており、血清SP-Dは特発性間質性肺炎、膠原病肺、および過敏性肺炎などの肺疾患で高値を示すことが報告されている。本分子はこれまでに臨床検査において、間質性肺炎の臨床的診断や病勢診断のためのバイオマーカーとして活用されてきた。血清中におけるKL-6並びにSP-Dの発現は、酵素免疫測定法や化学発光酵素免疫測定法等の免疫血清学的検査法により、測定することができる。 When the method of the present invention is used for the specific diagnosis of diffuse alveolar injury, the miRNAs whose expression is measured are hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-. 5p or a combination thereof is preferable. The combinations are hsa-miR-10a-5p and hsa-miR-340-5p, hsa-miR-10a-5p and hsa-miR-150-5p, hsa-miR-340-5p and hsa- The combination of miR-150-5p or the combination of hsa-miR-10a-5p and hsa-miR-340-5p and hsa-miR-150-5p can be exemplified, and the combination of hsa-miR-10a-5p and hsa- A combination of miR-340-5p and hsa-miR-150-5p is preferred. In addition, the existing interstitial pneumonia markers KL-6 or SP-D may be combined. KL-6 (Krebs von den Lungen-6) is a sugar chain antigen present in the O-glycosidic glycoprotein MUC1 produced by type II alveolar epithelial cells. So far, measurement of serum KL-6 has been used as a highly specific clinical test for interstitial pneumonia in general, including idiopathic interstitial pneumonia, hypersensitive interstitial pneumonia, and drug-induced interstitial pneumonia. rice field. SP-D (UniProtKB ID: P35247) is a type of pulmonary surfactant protein produced and secreted by type II alveolar epithelial cells. It is expressed specifically in the lung in the living body and contributes to the biological defense function against bacteria and viruses. It is known to do. SP-D produced in the lungs is also known to be present in the blood, and serum SP-D is elevated in lung diseases such as idiopathic interstitial pneumonitis, collagen disease lung, and hypersensitivity pneumonitis. Has been reported. This molecule has been used as a biomarker for clinical diagnosis and disease diagnosis of interstitial pneumonia in clinical examinations. The expression of KL-6 and SP-D in serum can be measured by immunoserologic tests such as enzyme immunoassay and chemiluminescent enzyme immunoassay.
さらに、hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、びまん性肺胞傷害の急性期にある可能性が高いと判定された被験者由来の試料における発現を１回又は異なる時期に複数回測定し、発現レベルがカットオフ値もしくは基準値に近いレベルにまで上昇した場合に、治療によりびまん性肺胞傷害から回復したと判定し、前記レベルが低いあるいは上昇しない場合に、治療によりびまん性肺胞傷害から回復していない、あるいは、回復が不十分であると判定することができる。本発明の方法は、びまん性肺胞傷害の病勢診断の他、予後の検査、治療効果の確認にも利用できる。 In addition, at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p may be in the acute phase of diffuse alveolar injury. Diffuse alveolar mellitus by treatment when the expression level in a sample derived from a subject judged to be highly sex is measured once or multiple times at different times and the expression level rises to a level close to the cutoff value or the reference value. If it is determined that the patient has recovered from the injury and the level is low or does not increase, it can be determined that the treatment has not recovered from the diffuse alveolar injury or the recovery is insufficient. The method of the present invention can be used not only for diagnosing the pathology of diffuse alveolar injury, but also for prognosis examination and confirmation of therapeutic effect.
被験者がびまん性肺胞傷害を発症している可能性が高いと判断された場合には、薬剤性が疑われる場合は、被疑薬はすみやかに中止するとよい。次いで、喀痰や血清の感染症検査、画像検査、気管支肺胞洗浄検査や病理検査などの各種検査を組み合わせて行い、びまん性肺胞傷害の診断を確定させるとよい。びまん性肺胞傷害の診断が下された時は、すみやかに副腎皮質ステロイドの投与を開始するとよい。日本呼吸器学会の治療指針では、メチルプレドニゾロン500〜1000 mg/日を3日間投与するパルス療法を行い、プレドニゾロン換算で0.5〜1.0 mg/kg/日で継続し、漸減することが推奨されている。漸減の速度に一定の基準はなく、治療反応性をみながら減らしていく。ステロイド治療に抵抗性、あるいは難治性の肺障害では、免疫抑制薬（シクロスポリン、タクロリムスなど）や好中球エラスターゼ阻害薬（シベレスタット）の投与、ポリミキシンB固定化線維カラム（PMX）療法などを組み合わせた集学的治療を行う。これらの治療は薬剤性肺障害に対するエビデンスに乏しく保険適用もないため、実際にはIPFの急性増悪やARDSに準じて用いられる。呼吸不全に対しては、高流量酸素投与、非侵襲的陽圧換気療法、あるいは気管内挿管下での人工呼吸管理など、重症度に応じた対処を行う。 If it is determined that the subject is likely to have diffuse alveolar injury and drug-induced, the suspected drug should be discontinued immediately. Next, it is advisable to perform various tests such as sputum and serum infection tests, imaging tests, bronchoalveolar lavage tests, and pathological tests in combination to confirm the diagnosis of diffuse alveolar injury. When a diagnosis of diffuse alveolar injury is made, administration of corticosteroids should be started immediately. The treatment guidelines of the Japanese Respiratory Society recommend that pulse therapy in which methylprednisolone 500 to 1000 mg / day is administered for 3 days be continued at 0.5 to 1.0 mg / kg / day in terms of prednisolone and gradually decreased. .. There is no fixed standard for the rate of tapering, and the rate of tapering is reduced while observing treatment responsiveness. For pulmonary disorders that are resistant to steroid treatment or refractory, immunosuppressive drugs (cyclosporine, tacrolimus, etc.), neutrophil elastase inhibitor (sivelestat), and polymixin B-immobilized fiber column (PMX) therapy are combined. Perform multidisciplinary treatment. Since these treatments have little evidence for drug-induced lung injury and are not covered by insurance, they are actually used according to acute exacerbations of IPF and ARDS. Respiratory failure should be treated according to severity, such as high-flow oxygen administration, non-invasive positive pressure ventilation therapy, or mechanical ventilation management under endotracheal intubation.
本発明は、hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における発現を測定すること、その測定値に基づき、被験者がびまん性肺胞傷害を発症している可能性が高いと評価された場合には、その被験者に治療を施すことを含む、びまん性肺胞傷害の治療方法も包含する。 The present invention measures the expression of at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p in a sample derived from a subject. A method for treating diffuse alveolar injury, including treating the subject if it is evaluated that the subject is likely to have diffuse alveolar injury based on the measured values. Also includes.
本発明は、hsa-miR-10a-5p、hsa-miR-340-5p及びhsa-miR-150-5pからなる群より選択される少なくとも１種のmiRNAについて、被験者由来の試料における発現を測定することができる試薬を含む、びまん性肺胞傷害の検査のためのキットも提供する。 The present invention measures the expression of at least one miRNA selected from the group consisting of hsa-miR-10a-5p, hsa-miR-340-5p and hsa-miR-150-5p in a sample derived from a subject. Kits for testing for diffuse alveolar injury, including reagents that can be provided, are also provided.
一つの例として、本発明のキットは、上記miRNAと特異的にハイブリダイズできる核酸プローブを試薬として含む。核酸プローブは基板に固定されていてもよい。キットには、さらに、生体試料を採取するための器具、血液凝固剤、被験者由来の試料からRNAを抽出するための試薬類、RNAを検出するための試薬類、取扱説明書などが含まれてもよい。取扱説明書には、キットの使用方法の他、びまん性肺胞傷害、あるいは急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。 As an example, the kit of the present invention contains as a reagent a nucleic acid probe capable of specifically hybridizing with the above miRNA. The nucleic acid probe may be fixed to the substrate. The kit also includes equipment for collecting biological samples, blood coagulants, reagents for extracting RNA from samples derived from subjects, reagents for detecting RNA, instruction manuals, etc. May be good. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for cases of diffuse alveolar injury or acute exacerbation of acute respiratory distress syndrome or idiopathic interstitial pneumonia. ..
さらに別の一例として、本発明のキットは上記miRNAを鋳型として合成されるcDNAを特異的に増幅できる少なくとも１対の核酸プライマーを試薬として含む。キットには、さらに、被験者由来の試料を採取するための器具、血液凝固剤、被験者由来の試料からRNAを抽出するための試薬類、抽出RNAを逆転写するための試薬、標的cDNAを検出するためのPCR反応試薬、取扱説明書などが含まれるとよい。取扱説明書には、キットの使用方法の他、びまん性肺胞傷害、急性呼吸窮迫症候群や特発性間質性肺炎の急性増悪症例の評価及び／又は鑑別基準なども記載しておくとよい。 As yet another example, the kit of the present invention contains at least one pair of nucleic acid primers capable of specifically amplifying cDNA synthesized using the above miRNA as a reagent as a reagent. The kit also detects instruments for collecting subject-derived samples, blood coagulants, reagents for extracting RNA from subject-derived samples, reagents for reverse transcribing the extracted RNA, and target cDNA. It is preferable to include PCR reaction reagents for this purpose, instruction manuals, and the like. In addition to how to use the kit, the instruction manual should also describe the evaluation and / or differentiation criteria for acute exacerbation cases of diffuse alveolar injury, acute respiratory distress syndrome and idiopathic interstitial pneumonia.
本発明のキットには、この他に、標的miRNA発現量の補正用に使用する外部スパイクコントロールのmiRNAの標準品、陽性コントロールmiRNA、バッファー、反応停止液、洗浄液、反応容器などを含めてもよい。
本発明のキットは、さらに、KL-6やSP-Dを特異的に認識できる抗体あるいは核酸アプタマーを含んでもよい。抗体及びアプタマーはマイクロタイタープレートや磁気ビーズ、セルロース膜や基板に固定されていてもよい。あるいは、SP-DのmRNAと特異的にハイブリダイズできる核酸プローブを含んでもよい。核酸プローブは基板に固定されていてもよい。
In addition, the kit of the present invention may include a standard product of an external spike control miRNA used for correcting the target miRNA expression level, a positive control miRNA, a buffer, a reaction terminator, a washing solution, a reaction vessel, and the like. ..
The kit of the present invention may further contain an antibody or nucleic acid aptamer capable of specifically recognizing KL-6 or SP-D. Antibodies and aptamers may be immobilized on microtiter plates, magnetic beads, cellulose membranes or substrates. Alternatively, a nucleic acid probe capable of specifically hybridizing with SP-D mRNA may be included. The nucleic acid probe may be fixed to the substrate.
本発明のキットは、疾病を診断するための医薬品として用いることができる。
The kit of the present invention can be used as a pharmaceutical product for diagnosing a disease.
以下、実施例により本発明を更に詳細に説明する。
〔実施例１〕
（１）検体
解析に用いたヒト間質性肺炎試料については、4箇所の拠点病院（信州大学、日本医科大学、千葉大学、広島大学）、国立医薬品食品衛生研究所、木原財団、アステラス製薬、及び第一三共において、各研究倫理委員会の承認を得て収集・解析した。
Hereinafter, the present invention will be described in more detail with reference to Examples.
[Example 1]
(1) Regarding human interstitial pneumonia samples used for sample analysis, four base hospitals (Shinshu University, Japan Medical University, Chiba University, Hiroshima University), National Institute of Pharmaceutical and Food Sanitation, Kihara Foundation, Astellas Pharma, And Daiichi Sankyo collected and analyzed with the approval of each research ethics committee.
医薬品による間質性肺炎の発症が疑われた患者に対して薬剤性間質性肺炎の急性期（最悪期付近）および回復期に採血を上記の拠点病院にて行った。入院時は早朝空腹時に、外来時は随時、採血した。各拠点病院において、患者の同意の下、血清採取用の7 mLの血液凝固促進剤入り採血管を用いて採血を行い、室温で30分放置後、3,000 rpm×10分（15℃〜20℃）遠心分離を行った。採取した血清は-80℃にて凍結保存した。 For patients suspected of developing interstitial pneumonia due to drugs, blood was collected at the above-mentioned base hospital during the acute phase (near the worst phase) and convalescent phase of drug-induced interstitial pneumonia. Blood was collected early in the morning on an empty stomach at the time of admission and at any time during the outpatient department. At each base hospital, with the consent of the patient, blood was collected using a 7 mL blood collection tube containing a blood coagulation promoter for serum collection, left at room temperature for 30 minutes, and then 3,000 rpm x 10 minutes (15 ° C to 20 ° C). ) Centrifugation was performed. The collected serum was cryopreserved at -80 ° C.
miRCURY LNA microRNA PCRを用いたバイオマーカー候補miRNAsのスクリーニング解析には、画像診断においてびまん性肺胞傷害（DAD）パターンを呈する薬剤性間質性肺炎症例の急性期に採取された血清検体9例、並びに回復期に採取された血清検体3例を供した。一方、器質化肺炎（OP）パターンの症例については、急性期16例、回復期13例、非特異性間質性肺炎（NSIP）パターンの症例は、急性期18例、回復期16例を用いた。健常成人検体としては、ノイエス株式会社にて収集されたボランティア健常成人（72例）の血清検体を用いた。
Taqman Advanced miRNA Assay法を用いたバイオマーカー候補miRNAsの検証試験では、薬剤性間質性肺炎の急性期と診断された血清検体を42例 (DAD型8例、OP型19例、NSIP型15例) ならびにその回復期検体22例 (DAD型2例、OP型9例、NSIP型11例) を供した。また、対照肺疾患として、同系薬剤投与間質性肺炎非発症例(20例)、肺がん(46例)、細菌感染症(14例)、肺非結核性抗酸菌症(17例)、特発性間質性肺炎(22例)、膠原病肺(20例)、慢性閉塞性肺疾患(14例)及び気管支喘息(12例)、並びに健康成人(30例)を測定に供した。
Screening analysis of biomarker candidate miRNAs using miRCURY LNA microRNA PCR included 9 serum samples collected during the acute phase of drug-induced interstitial lung disease showing a diffuse alveolar injury (DAD) pattern in diagnostic imaging. In addition, 3 serum samples collected during the recovery period were provided. On the other hand, 16 cases of acute phase and 13 cases of convalescent phase are used for cases of organizing pneumonia (OP) pattern, and 18 cases of acute phase and 16 cases of convalescent phase are used for cases of nonspecific interstitial pneumonia (NSIP) pattern. board. As a healthy adult sample, a serum sample of volunteer healthy adults (72 cases) collected by Neues Co., Ltd. was used.
In a validation study of biomarker candidate miRNAs using the Taqman Advanced miRNA Assay method, 42 serum samples (8 DAD, 19 OP, 15 NSIP) were diagnosed with the acute phase of drug-induced interstitial pneumonia. ) And 22 convalescent specimens (2 DAD type, 9 OP type, 11 NSIP type) were provided. In addition, as control lung diseases, interstitial pneumonia non-onset cases (20 cases), lung cancer (46 cases), bacterial infections (14 cases), pulmonary non-tuberculous mycobacteriosis (17 cases), idiopathic drug administration Interstitial pneumonia (22 cases), collagen disease lung (20 cases), chronic obstructive pulmonary disease (14 cases) and bronchial asthma (12 cases), and healthy adults (30 cases) were used for measurement.
（２）RNA抽出及びcDNA合成
血清検体からのtotal RNA抽出は、miRNeasy（登録商標） Mini Kit （Qiagen）を用いて行った。miRCURY LNA microRNA PCR測定用のcDNAの合成には、Universal cDNA Synthesis Kit II（Qiagen）を使用した。一方、Taqman Advanced miRNA Assay測定用のcDNAの合成には、TaqmanTM Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific)を用いた。なお、Taqman Advanced miRNA Assayの測定用試料調製では、外因性スパイクコントロールとして、cel-miR-54-3p (0.2 fmol)を各血清試料に添加した後に、total RNA抽出及びcDNA合成を行った。
(2) RNA extraction and cDNA synthesis Total RNA extraction from serum samples was performed using miRNeasy® Mini Kit (Qiagen). Universal cDNA Synthesis Kit II (Qiagen) was used to synthesize cDNA for miRCURY LNA microRNA PCR measurement. On the other hand, the Taqman TM Advanced miRNA cDNA synthesis kit (Thermo Fisher Scientific) was used for the synthesis of cDNA for Taqman Advanced miRNA Assay measurement. In the sample preparation for measurement of Taqman Advanced miRNA Assay, total RNA extraction and cDNA synthesis were performed after adding cel-miR-54-3p (0.2 fmol) to each serum sample as an exogenous spike control.
（３）測定手法並びに補正手法
miRNAの網羅的発現解析には、QUIAGEN社のmiRCURY LNA microRNA PCR システムを用いた。本システムは、Locked Nucleic Acid (RNA修飾核酸)プライマーにより、752種のmiRNAを特異的に検出する測定法である。測定データの解析には、GenExソフトウェア（Multid）を用いて、[1]Interplate calibration(IPC)、[2]Cut off（Cq>37を除外）、[3]Call rate（10％未満を除外）、[4]Missing data（各プローブの最大Cq値＋１で非検出検体を補完）、[5]Normfinder Normalizationという手順により、下記の式で定義されるΔCqを算出した。ΔCq値は標的miRNAの内標遺伝子（リファレンスプローブ）に対する相対発現量を表しており、本値が大きいほど発現量が相対的に低いことを意味する。
The miRCURY LNA microRNA PCR system from QUIAGEN was used for comprehensive expression analysis of miRNA. This system is a measurement method that specifically detects 752 types of miRNA using Locked Nucleic Acid (RNA-modified nucleic acid) primers. Use GenEx software (Multid) to analyze measurement data: [1] Interplate calibration (IPC), [2] Cut off (excluding Cq> 37), [3] Call rate (excluding less than 10%) , [4] Missing data (maximum Cq value of each probe + 1 complements undetected specimens), [5] Normfinder Normalization, and ΔCq defined by the following formula was calculated. The ΔCq value represents the relative expression level of the target miRNA with respect to the internal standard gene (reference probe), and the larger this value is, the lower the expression level is.
一方、選定したバイオマーカー候補miRNA (hsa-miR-10a-5p; MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG（配列番号１）, hsa-miR-150-5p; MIMAT0000451, UCUCCCAACCCUUGUACCAGUG（配列番号３）, hsa-miR-340-5p; MIMAT0004692, UUAUAAAGCAAUGAGACUGAUU（配列番号２）)の検証試験では、各標的miRNAに特異的なプローブ並びにプライマーを含むTaqman Advanced miRNA Assay（Thermo Fisher Scientific社, hsa-miR-10a-5p; 479241_mir, hsa-miR-150-5p; 477918_mir, hsa-miR-340-5p; 478042_mir, cel-miR-54-3p; 478410_mir）を用いた。本測定における各miRNAの発現量(ΔCq値)は、Cq(標的miRNA)からCq(外部スパイクコントロール;cel-miR-54-3p)を差し引くことにより求めた。なお、miRCURY LNA microRNA PCR システムの解析と同様、算出されたΔCq値が大きいほど、標的miRNAの発現量が相対的に低いことを表す。 On the other hand, selected biomarker candidate miRNAs (hsa-miR-10a-5p; MIMAT0000253, UACCCUGUAGAUCCGAAUUUGUG (SEQ ID NO: 1), hsa-miR-150-5p; MIMAT0000451, UCUCCCAACCCUUGUACCAGUG (SEQ ID NO: 3), hsa-miR-340-5p In the validation test of MIMAT0004692, UUAUAAAGCAAUGAGACUGAUU (SEQ ID NO: 2), the Taqman Advanced miRNA Assay (Thermo Fisher Scientific, hsa-miR-10a-5p; 479241_mir, hsa-miR) containing probes and primers specific for each target miRNA. -150-5p; 477918_mir, hsa-miR-340-5p; 478042_mir, cel-miR-54-3p; 478410_mir) was used. The expression level (ΔCq value) of each miRNA in this measurement was determined by subtracting Cq (external spike control; cel-miR-54-3p) from Cq (target miRNA). As in the analysis of the miRCURY LNA microRNA PCR system, the larger the calculated ΔCq value, the lower the expression level of the target miRNA.
（４）DAD型薬剤性間質性肺炎バイオマーカー候補miRNAの選定
miRCURY LNA microRNA PCR測定データにおける各miRNAのΔCq値を用いて、DAD(9例)と回復期(32例)で、効果量Hedge’s gの値が1.0以上かつFold change値が2倍以上の変動を示すmiRNAのうち、最も発現変動が大きい3種のmiRNA（hsa-miR-340−5p, hsa-miR-10a-5p, has-miR-150-5p）を抽出した。抽出したmiRNAについて、各薬剤性間質性肺炎の病型と健常成人（図1Ａ）または回復期（図1Ｂ）の間の発現量を比較したところ、いずれのmiRNAもDADの急性期において最も顕著な発現変動を示すことが明らかとなった(図1)。
(4) Selection of DAD-type drug-induced interstitial pneumonia biomarker candidate miRNA
Using the ΔCq value of each miRNA in the miRCURY LNA microRNA PCR measurement data, the effect size Hedge's g value is 1.0 or more and the Fold change value is more than doubled in DAD (9 cases) and recovery period (32 cases). Among the miRNAs shown, the three miRNAs (hsa-miR-340-5p, hsa-miR-10a-5p, has-miR-150-5p) with the largest expression fluctuations were extracted. Comparing the expression levels of the extracted miRNAs between the type of drug-induced interstitial pneumonia and healthy adults (Fig. 1A) or convalescent stage (Fig. 1B), all miRNAs were most prominent in the acute phase of DAD. It was clarified that the expression fluctuated (Fig. 1).
（５）びまん性肺胞傷害に特異的なバイオマーカー候補miRNAの診断能
表1では、スクリーニングデータセットを用いて各病型の薬剤性間質性肺炎の急性期と正常成人並びに回復期の間における、3種類のDADマーカー候補miRNA並びに既存間質性肺炎マーカー(KL-6, SP-D)のROC曲線下面積値(AUROC)を示している。いずれのmiRNAも健常成人と比べた際に、DADの急性期においてAUROC値が0.85以上の高い診断能を示した(表1)。一方、これらmiRNAの回復期例に対する病勢診断能についてROC解析したところ、健常成人と同様に、いずれのmiRNAもDADの急性期において最も高い診断能を示した(表1)。また、本解析で算出されたDADと回復期との間における各miRNA単体の診断能は、KL6(0.74)とSP-D（0.90）と同程度(AUROC 079-0.87)であった。これらの結果は、上記3種miRNAがDADの検出及び病勢診断に有用なバイオマーカーとなることを示唆している。
(5) Diagnostic ability of biomarker candidate miRNA specific for diffuse alveolar injury Table 1 shows the diagnostic ability of miRNA for each type of drug-induced interstitial pneumonia between the acute phase and normal adult and convalescent phase using the screening dataset. The area values (AUROC) under the ROC curve of the three types of DAD marker candidate miRNA and the existing interstitial pneumonia markers (KL-6, SP-D) are shown in. All miRNAs showed high diagnostic ability with an AUROC value of 0.85 or higher in the acute phase of DAD when compared with healthy adults (Table 1). On the other hand, ROC analysis of the disease diagnostic ability of these miRNAs in convalescent cases showed that all miRNAs showed the highest diagnostic ability in the acute phase of DAD, as in healthy adults (Table 1). In addition, the diagnostic ability of each miRNA alone between DAD and convalescent phase calculated in this analysis was similar to that of KL6 (0.74) and SP-D (0.90) (AUROC 079-0.87). These results suggest that the above three miRNAs are useful biomarkers for DAD detection and disease diagnosis.
複数のバイオマーカー候補miRNAを組み合わせることで、診断精度の向上が期待される。そこで次に、DADの急性期および回復期の間における各miRNAのΔCq値を用いて多重ロジスティック回帰によりDAD病勢診断のための診断モデル[診断スコア＝2.13554372×ΔCq(hsa-miR-10a-5p)+2.73022292×ΔCq(hsa-miR-340-5p)+0.75374455 ΔCq(hsa-miR-150-5p)-28.655941]を作製した。作製した診断モデルにより求められる診断スコアを用いて、各病型の急性期と健常成人のAUROCを解析したところ、DADにおいてAUROC 0.98以上の極めて高い診断能が認められた(表1)。また、本モデルによるOP及びNSIP病勢診断能がAUROC0.7以下であったことは、スクリーニングにて同定されたバイオマーカー候補miRNAの組み合わせにより、DADの病勢を特異的に、かつ高い確度で診断できることを示している。 Combining multiple biomarker candidate miRNAs is expected to improve diagnostic accuracy. Therefore, next, a diagnostic model for diagnosing DAD disease by multiple logistic regression using the ΔCq value of each miRNA during the acute phase and convalescent phase of DAD [diagnosis score = 2.13554372 × ΔCq (hsa-miR-10a-5p)) +2.73022292 × ΔCq (hsa-miR-340-5p) +0.75374455 ΔCq (hsa-miR-150-5p) -28.655941] was prepared. When the AUROC of each type of disease in the acute phase and healthy adults was analyzed using the diagnostic score obtained from the prepared diagnostic model, an extremely high diagnostic ability of AUROC 0.98 or higher was observed in DAD (Table 1). In addition, the OP and NSIP disease diagnostic ability of this model was AUROC 0.7 or less, which means that the disease of DAD can be diagnosed specifically and with high accuracy by the combination of biomarker candidate miRNAs identified by screening. Is shown.
（６）びまん性肺胞傷害マーカー候補miRNAのバイオマーカー性能の検証
薬剤性間質性肺炎に対するmiCURRY LNA microRNA PCRを用いた上記の試験結果が、測定手法に依存せず普遍的であることを示すため、別群の薬剤性間質性肺炎ならびに対照肺疾患試料中におけるhsa-miR-340−5p, hsa-miR-10a-5p及び has-miR-150-5pの発現量(ΔCq値)をTaqman Advanced miRNA Assayにより測定した。まず各病型の薬剤性間質性肺炎症例と健常成人をROC曲線により解析したところ、いずれのmiRNAについてもOPやNSIPと比べ、DADの急性期において有意な診断能が認められた（表2）。次に、病勢診断能について解析したところ、DAD症例においてhsa-miR-10a-5p及びhsa-miR-150-5pは既存間質性肺炎マーカーであるSP-Dと同定度の診断能を示した(表3)。次に、Taqman assayの測定データに基づき、DADの急性期および回復期間における各miRNAのΔCq値を用いて多重ロジスティック回帰によりDAD病勢診断のための診断モデル[診断スコア＝4.21381729×ΔCq(hsa-miR-10a-5p)-4.6569967×ΔCq(hsa-miR-340-5p)+1.58717808×ΔCq(hsa-miR-150-5p)-28.451547]を作製した。本モデルから得られる診断スコアをもとに、DADと健常成人ならびに回復期との比較をしたところ、それぞれAUROC値0.97（vs. 健常成人、表2）及び0.96（vs. 回復期、表3）で、互いを識別できることが示された。さらに、3種miRNAとSP-Dの組み合わせによる診断モデルでは、AUROC 1.0 (感度・特異度100％)でDADの病勢を診断することが可能であった（表3）。一方、病勢診断において、3種miRNAにKL-6を追加した診断モデルではAUROCの上昇は認められなかった。
(6) Verification of biomarker performance of candidate miRNAs for diffuse alveolar injury The above test results using miCURRY LNA microRNA PCR for drug-induced interstitial pneumonia are shown to be universal regardless of measurement method. Therefore, the expression levels (ΔCq values) of hsa-miR-340-5p, hsa-miR-10a-5p and has-miR-150-5p in different groups of drug-induced interstitial pneumonia and control lung disease samples were measured by Taqman. Measured by Advanced miRNA Assay. First, when drug-induced interstitial pneumonia cases and healthy adults of each disease type were analyzed by ROC curve, significant diagnostic ability was observed in the acute phase of DAD compared with OP and NSIP for all miRNAs (Table 2). ). Next, when the disease diagnostic ability was analyzed, hsa-miR-10a-5p and hsa-miR-150-5p showed the diagnostic ability of SP-D, which is an existing interstitial pneumonia marker, and the degree of identification in DAD cases. (Table 3). Next, based on the measurement data of the Taqman assay, a diagnostic model for diagnosing DAD disease by multiple logistic regression using the ΔCq value of each miRNA in the acute phase and recovery period of DAD [diagnosis score = 4.21381729 × ΔCq (hsa-miR) -10a-5p) -4.6569967 × ΔCq (hsa-miR-340-5p) +1.58717808 × ΔCq (hsa-miR-150-5p) -28.451547] was prepared. A comparison of DAD with healthy adults and convalescents based on the diagnostic scores obtained from this model revealed that AUROC values were 0.97 (vs. healthy adults, Table 2) and 0.96 (vs. Convalescent, Table 3), respectively. It was shown that they can be distinguished from each other. Furthermore, in a diagnostic model using a combination of three miRNAs and SP-D, it was possible to diagnose the pathology of DAD with AUROC 1.0 (sensitivity /
（７）miRNA組み合わせDAD診断モデルによる薬剤性間質性肺炎の病型診断の有用性
図2Aは、3種のバイオマーカー候補miRNAを組み合わせたDAD診断モデル、並びに既存マーカーの各病型の薬剤性間質性肺炎の急性期における定量値を比較した結果である。3種miRNAを組み合わせたDAD診断モデルでは、OPおよびNSIPと比べ、DADの急性期において有意に高い診断スコアが観察された（図2A）。しかしながら、各薬剤性間質性肺炎の病型間でKL-6及びSP-Dの定量値に差異は認められなかった（図2A）。
(7) Usefulness of drug-induced interstitial pneumonia disease type diagnosis using miRNA-combined DAD diagnostic model Figure 2A shows a DAD diagnostic model that combines three types of biomarker candidate miRNAs, as well as the drug-induced disease of each type of existing marker. This is the result of comparing the quantitative values in the acute phase of interstitial pneumonia. In the DAD diagnostic model combining the three miRNAs, a significantly higher diagnostic score was observed in the acute phase of DAD compared to OP and NSIP (Fig. 2A). However, there was no difference in the quantitative values of KL-6 and SP-D among the types of drug-induced interstitial pneumonia (Fig. 2A).
DAD診断モデルから得られる診断スコアを用いて、DADのOP及びNSIPに対する診断能（AUROC）を解析したところ、OPで0.93、NSIPで0.88と既存マーカーより高値を示した（図2B）。このことから、本診断モデルは、薬剤性間質性肺炎患者からDADを特異的に検出することにも応用可能であることが示された。 When the diagnostic ability (AUROC) of DAD for OP and NSIP was analyzed using the diagnostic score obtained from the DAD diagnostic model, the OP was 0.93 and the NSIP was 0.88, which were higher than the existing markers (Fig. 2B). From this, it was shown that this diagnostic model can also be applied to the specific detection of DAD in patients with drug-induced interstitial pneumonia.
（８）びまん性肺胞傷害マーカー候補miRNAによる対照肺疾患との鑑別
DAD及び対照肺疾患症例における、DADマーカー候補miRNA単体の血清中発現量、及びそれらを元にDAD診断モデルから算出された診断スコアをプロットし、統計的に比較した（図3）。その結果、hsa-miR-340-5pついて、DADと細菌性感染症患者との間で有意なレベル差が認められた（図3）。また、miR-10a-5pについては、同系薬剤投与間質性肺炎非発症例、肺がん、膠原病肺、及び気管支喘息との間で統計学上有意なレベル差が認められた（図3）。さらに、miR-150-5pについては、同系薬剤投与間質性肺炎非発症例、肺非結核性抗酸菌症、特発性間質性肺炎、膠原病肺、気管支喘息との間で有意なレベル差が認められた（図3）。一方、DAD症例におけるDAD診断モデルの診断スコアは細菌感染症以外のすべての対照肺疾患と比べ有意に高かった（図3）。
(8) Differentiation from control lung disease by diffuse alveolar injury marker candidate miRNA
In DAD and control lung disease cases, the serum expression level of the DAD marker candidate miRNA alone and the diagnostic score calculated from the DAD diagnostic model based on them were plotted and statistically compared (Fig. 3). As a result, a significant level difference was observed between DAD and patients with bacterial infections for hsa-miR-340-5p (Fig. 3). Regarding miR-10a-5p, a statistically significant level difference was observed between non-onset cases of interstitial pneumonia administered with similar drugs, lung cancer, collagen disease lung, and bronchial asthma (Fig. 3). Furthermore, for miR-150-5p, significant levels were observed among patients with non-symptomatic interstitial pneumonia, nontuberculous mycobacteriosis of the lung, idiopathic interstitial pneumonia, collagen disease lung, and bronchial asthma. A difference was observed (Fig. 3). On the other hand, the diagnostic score of the DAD diagnostic model in DAD cases was significantly higher than that of all control lung diseases other than bacterial infection (Fig. 3).
次に、DADとそれ以外の肺疾患との診断能の評価を行った（表4）。既存間質性肺炎マーカーであるSP-Dは、ほとんどの対照肺疾患と良好な診断能を示すものの、DADと特発性間質性肺炎(AUROC 0.55)及び膠原病肺(AUROC 0.74)の識別が不良であることが明らかとなった。既存マーカーであるKL-6についても同様の傾向が確認できたが、各種肺疾患とDADの診断能はSP-Dより劣っていた。一方で、今回同定されたmiRNAのうち、DADとのAUROC値が0.8を超えた肺疾患は、hsa-miR-340-5pが1種、hsa-miR-10a-5pが4種、hsa-miR-150-5pが4種であった（表4）。特に、hsa-miR-10a-5p及びhsa-miR-150-5pは既存マーカーが識別不良となる膠原病肺に対し、高い診断能を示した（表4）。また、hsa-miR-150-5pは特発性間質性肺炎に対しても、有意な診断能(0.78)を示した。さらに、これらmiRNAを組み合わせたDAD診断モデルは、細菌感染症以外すべての対照肺疾患で、0.85を超える高い診断能を示した（表4）。 Next, the diagnostic ability of DAD and other lung diseases was evaluated (Table 4). SP-D, an existing interstitial pneumonia marker, shows good diagnostic ability with most control lung diseases, but distinguishes DAD from idiopathic interstitial pneumonia (AUROC 0.55) and collagen disease lung (AUROC 0.74). It turned out to be defective. A similar tendency was confirmed for the existing marker KL-6, but the diagnostic ability of various lung diseases and DAD was inferior to that of SP-D. On the other hand, among the miRNAs identified this time, the lung diseases whose AUROC value with DAD exceeded 0.8 were hsa-miR-340-5p (1 type), hsa-miR-10a-5p (4 types), and hsa-miR. There were 4 types of -150-5p (Table 4). In particular, hsa-miR-10a-5p and hsa-miR-150-5p showed high diagnostic ability for collagen disease lungs in which existing markers were poorly identified (Table 4). Hsa-miR-150-5p also showed significant diagnostic ability (0.78) for idiopathic interstitial pneumonia. Furthermore, the DAD diagnostic model combining these miRNAs showed a high diagnostic ability of over 0.85 in all control lung diseases except bacterial infections (Table 4).
3種miRNAと既存マーカーとの組み合わせによる対照肺疾患との鑑別度の向上にむけて、本DAD診断モデルにKL-6またはSP-Dを加えた際の診断能について解析を行った。その結果、KL-6と3種miRNAの組み合わせでは、3種miRNAによるDAD診断モデルの診断能を大きく向上させることはできなかったものの、KL-6単独では診断困難であった特発性間質性肺炎や膠原病肺との鑑別において高い診断能を示した（表4）。一方、本ＤＡＤ診断モデルにSP-Dを加えた場合は、DADとすべての対照肺疾患を極めて良好に鑑別可能であることが明らかとなった（表4）。以上の結果より、本研究により同定したmiRNAsは、単体あるいは組み合わせることで、DADを特異的に検出することが可能で、かつ既存マーカー単体では識別困難である肺疾患との鑑別を補助できる新たなDADバイオマーカー分子である。 In order to improve the degree of differentiation from control lung disease by combining the three miRNAs with existing markers, we analyzed the diagnostic ability when KL-6 or SP-D was added to this DAD diagnostic model. As a result, the combination of KL-6 and 3 miRNAs could not significantly improve the diagnostic ability of the DAD diagnostic model using 3 miRNAs, but idiopathic interstitial disease, which was difficult to diagnose with KL-6 alone. It showed high diagnostic ability in differentiation from pneumonia and collagen disease lung (Table 4). On the other hand, when SP-D was added to this DAD diagnostic model, it became clear that DAD and all control lung diseases could be distinguished very well (Table 4). Based on the above results, the miRNAs identified in this study can be used alone or in combination to specifically detect DAD, and can assist in differentiation from lung diseases, which are difficult to identify with existing markers alone. It is a DAD biomarker molecule.
表１．スクリーニングデータセットにおけるびまん性肺胞障害バイオマーカー候補miRNAの診断能
miRCURY LNA microRNA PCRにて同定された3種のDADマーカー候補miRNA並びに既存の間質性肺炎マーカー（KL-6, SP-D）について、正常成人や回復期例との診断を比較するためのROC曲線解析を行い、算出されたAUROC値を示した。薬剤性間質性肺炎の病型で、A-DADはびまん性肺胞障害の急性期症例、A-OPは器質化肺炎の急性期症例、A-NSIPは非特異性間質性肺炎の急性期症例を表している。
ROC for comparing the diagnosis of three DAD marker candidate miRNAs identified by miRCURY LNA microRNA PCR and existing interstitial pneumonia markers (KL-6, SP-D) with normal adults and convalescent cases. Curve analysis was performed and the calculated AUROC value was shown. Drug-induced interstitial pneumonia, A-DAD is an acute case of diffuse alveolar disorder, A-OP is an acute case of organized pneumonia, and A-NSIP is an acute case of nonspecific interstitial pneumonia. Represents a stage case.
表２．検証試験データセットにおける健常成人と薬剤性間質性肺炎患者の診断能
Taqman Advanced miRNA assay測定を行った3種のDADマーカー候補miRNAおよびその組み合わせによるDAD診断モデルについて、正常成人との診断を比較するためのROC曲線解析を行い、算出されたAUROC値を示した。薬剤性間質性肺炎の病型で、A-DADはびまん性肺胞障害の急性期症例、A-OPは器質化肺炎の急性期症例、A-NSIPは非特異性間質性肺炎の急性期症例を表している。
ROC curve analysis was performed to compare the diagnosis with normal adults for the three DAD marker candidate miRNAs measured by Taqman Advanced miRNA assay and the DAD diagnostic model based on the combination, and the calculated AUROC value was shown. Drug-induced interstitial pneumonia, A-DAD is an acute case of diffuse alveolar disorder, A-OP is an acute case of organized pneumonia, and A-NSIP is an acute case of nonspecific interstitial pneumonia. Represents a stage case.
表３．検証試験データセットにおけるびまん性肺胞障害バイオマーカー候補miRNAの病勢診断能
Taqman Advanced miRNA assay測定を行った3種のDADマーカー候補miRNA、既存の間質性肺炎マーカー（KL-6, SP-D）、それら組み合わせによるDAD診断モデルの病勢診断能についてROC曲線解析を行い、算出されたAUROC値を示した。薬剤性間質性肺炎の病型で、A-DADはびまん性肺胞障害の急性期症例、A-OPは器質化肺炎の急性期症例、A-NSIPは非特異性間質性肺炎の急性期症例を表している。
ROC curve analysis was performed on the pathological diagnostic ability of the DAD diagnostic model using the three DAD marker candidate miRNAs measured by Taqman Advanced miRNA assay, the existing interstitial pneumonia markers (KL-6, SP-D), and their combination. The calculated AUROC value is shown. Drug-induced interstitial pneumonia, A-DAD is an acute case of diffuse alveolar disorder, A-OP is an acute case of organized pneumonia, and A-NSIP is an acute case of nonspecific interstitial pneumonia. Represents a stage case.
表４．対照肺疾患に対するびまん性肺胞障害マーカー候補miRNAの診断能の評価
DADマーカー候補miRNA、SP-D、KL-6並びにそれらの組み合わせによるDAD診断モデルを用いて、DADと対照肺疾患の診断能についてROC曲線解析を行い、算出されたAUROC値を記載した。
ROC curve analysis was performed for the diagnostic ability of DAD and control lung disease using the DAD marker candidates miRNA, SP-D, KL-6, and a DAD diagnostic model based on the combination thereof, and the calculated AUROC value was described.
本発明は、体外診断薬、臨床検査などに利用できる。 The present invention can be used for in vitro diagnostic agents, clinical tests, and the like.
＜配列番号１＞
hsa-miR-10a-5pの塩基配列を示す。
＜配列番号２＞
hsa-miR-340-5pの塩基配列を示す。
＜配列番号３＞
hsa-miR-150-5pの塩基配列を示す。
＜配列番号４＞
hsa-let-7d-3pの塩基配列を示す。
＜配列番号５＞
hsa-miR-21-5pの塩基配列を示す。
＜配列番号６＞
hsa-miR-30d-5pの塩基配列を示す。
＜配列番号７＞
hsa-miR-30e-5pの塩基配列を示す。
＜配列番号８＞
hsa-652-3pの塩基配列を示す。
<SEQ ID NO: 1>
The base sequence of hsa-miR-10a-5p is shown.
<SEQ ID NO: 2>
The base sequence of hsa-miR-340-5p is shown.
<SEQ ID NO: 3>
The base sequence of hsa-miR-150-5p is shown.
<SEQ ID NO: 4>
The base sequence of hsa-let-7d-3p is shown.
<SEQ ID NO: 5>
The base sequence of hsa-miR-21-5p is shown.
<SEQ ID NO: 6>
The base sequence of hsa-miR-30d-5p is shown.
<SEQ ID NO: 7>
The base sequence of hsa-miR-30e-5p is shown.
<SEQ ID NO: 8>
The base sequence of hsa-652-3p is shown.
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| JP2013502931A (en) * | 2009-08-28 | 2013-01-31 | アスラジェン， インコーポレイテッド | MiRNA biomarkers for lung disease |
| JP2015504307A (en) * | 2011-11-22 | 2015-02-12 | インターミューン， インコーポレイテッド | Methods for diagnosing and treating idiopathic pulmonary fibrosis |
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| JP2013502931A (en) * | 2009-08-28 | 2013-01-31 | アスラジェン， インコーポレイテッド | MiRNA biomarkers for lung disease |
| JP2015504307A (en) * | 2011-11-22 | 2015-02-12 | インターミューン， インコーポレイテッド | Methods for diagnosing and treating idiopathic pulmonary fibrosis |
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| CN114525331A (en) * | 2021-11-23 | 2022-05-24 | 中山大学 | Detection product for rapidly identifying severe pneumonia |
| CN114525331B (en) * | 2021-11-23 | 2024-04-26 | 中山大学 | Detection product for rapidly identifying severe pneumonia |
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