JP6947450B2 - Biomarkers, diagnostic compositions, and diagnostic kits - Google Patents
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Description
本発明は、特にバイオマーカー、診断用組成物、及び診断用キットに関する。 The present invention, biomarkers in particular, diagnostic compositions and diagnostic kits.
急性脳症は、小児期に多く発症する脳症である。我が国における急性脳症の定義は、Ｊａｐａｎ Ｃｏｍａ Ｓｃａｌｅ ２０以上（Ｇｌａｓｇｏｗ Ｃｏｍａ Ｓｃａｌｅ １０〜１１以下）の意識障害が急性に発症し、２４時間以上持続することである。急性脳症は、（１）ほとんどは、インフルエンザ、ＨＨＶ−６（突発性発疹）、ロタウィルス、ＲＳウィルス等の感染症の経過中に発症する、（２）多くは頭部ＣＴ、ＭＲＩで脳浮腫が描出される、（３）脳炎、髄膜炎等の他の疾患が否定される（髄液中の細胞数増加がない）という特徴がある。
急性脳症では、多くが死亡したり後遺症を生じたりするため、医学的、社会的に大きな問題を生じている。
Acute encephalopathy is an encephalopathy that often occurs in childhood. The definition of acute encephalopathy in Japan is that consciousness disorder of Japan Coma Scale 20 or more (Glasgow Coma Scale 10-11 or less) develops acutely and lasts for 24 hours or more. Acute encephalopathy (1) mostly develops during the course of infectious diseases such as influenza, HHV-6 (roseola), rotavirus, RS virus, etc. (3) Other diseases such as encephalitis and meningitis are denied (there is no increase in the number of cells in the spinal fluid).
Acute encephalopathy causes major medical and social problems because many people die or have sequelae.
ここで、急性脳症には、重篤な脳症である急性壊死性脳症（Ａｃｕｔｅ Ｎｅｃｒｏｔｉｚｉｎｇ Ｅｎｃｅｐａｈｌｏｐａｔｈｙ、以下「ＡＮＥ」と記載する。）、予後が比較的良好な脳梁膨大部脳症（Ｃｌｉｎｉｃａｌｌｙ ｍｉｌｄ ｅｎｃｅｐｈａｌｉｔｉｓ／ｅｎｃｅｐｈａｌｏｐａｔｈｙ ｗｉｔｈ ａ ｒｅｖｅｒｓｉｂｌｅ ｓｐｌｅｎｉａｌ ｌｅｓｉｏｎ、以下、「ＭＥＲＳ」と記載する。）、及び、けいれん重積型（二相性）急性脳症（Ａｃｕｔｅ ｅｎｃｅｐｈａｌｏｐａｔｈｙ ｗｉｔｈ ｂｉｐｈａｓｉｃ ｓｅｉｚｕｒｅｓ ａｎｄ ｌａｔｅ ｒｅｄｕｃｅｄ ｄｉｆｆｕｓｉｏｎ、以下「ＡＥＳＤ」と記載する。）と呼ばれる症候群が存在する。また、症候群として確立していないが、「一相性脳症」と考えられる、感染症による発熱後、けいれんを発症し、その後長時間（概ね２４時間以上）にわたり意識障害が遷延するが、時間経過とともに意識障害が改善する脳症が存在する。 Here, acute encephalopathy includes acute necrotizing encephalopathy (Acute Necrotizing Encepahropathy, hereinafter referred to as “ANE”), which is a serious encephalopathy, and callosal ampulla encephalopathy (Clinically mild encephalopathy) with a relatively good prognosis. with a reversible spiral encephalopathy (hereinafter referred to as "MERS"), and convulsive (biphasic) acute encephalopathy (Acute encephalopathy with biphasic encephalopathy) and "disephalopathy" and "disephalopathy". There is a syndrome called. In addition, although it has not been established as a syndrome, convulsions develop after fever due to an infectious disease, which is considered to be "monophasic encephalopathy," and then consciousness disorder persists for a long time (generally 24 hours or more), but with the passage of time. There is encephalopathy that improves disturbance of consciousness.
これらのうち一相性脳症及びＡＥＳＤでは、感染症により急激に発症して、けいれん及び意識障害を呈する１回目の発作が生じる。その後、一相性脳症では、自然軽快することが期待できるのに対し、ＡＥＳＤでは、１回目の発作からの数日後に、２回目のけいれん及び意識障害を呈する発作を起こすことが、臨床的な特徴である。すなわち、発作が１回だけ（一相性）の一相性脳症に対して、ＡＥＳＤ（二相性）では２回目の発作が起こる。そして、ＡＥＳＤは、罹病率が我が国で年間４００〜７００人と少ないものの、患者には、その後、麻痺、知的障害、てんかん等の厳しい神経学的後遺症が生じることが多いという問題があった。 Of these, monophasic encephalopathy and AESD develop first seizures that develop rapidly due to infection and present with convulsions and impaired consciousness. After that, monophasic encephalopathy can be expected to improve spontaneously, whereas AESD is clinically characterized by having a second seizure with convulsions and impaired consciousness several days after the first seizure. Is. That is, in contrast to monophasic encephalopathy with only one seizure (monophasic), AESD (biphasic) causes a second seizure. Although the morbidity rate of AESD is as low as 400 to 700 per year in Japan, there is a problem that patients often develop severe neurological sequelae such as paralysis, intellectual disability, and epilepsy.
ここで、従来の急性脳症の診断方法として、特許文献１を参照すると、（１）脳症が疑われる患者から採取した血液中のキノリン酸含有量及び／又はキヌレニン含有量を測定する、及び（２）前記キノリン酸含有量及び／又はキヌレニン含有量を、脳症非発症者又は脳症発症者の基準レベルと比較することで脳症を簡易かつ迅速に検出する技術が開示されている（以下、従来技術１とする。）。 Here, referring to Patent Document 1 as a conventional method for diagnosing acute encephalopathy, (1) the quinolinic acid content and / or the kynurenine content in blood collected from a patient suspected of having encephalopathy is measured, and (2). ) A technique for simply and quickly detecting encephalopathy by comparing the quinolinic acid content and / or the kynurenine content with a reference level of a person who does not develop encephalopathy or a person who develops encephalopathy is disclosed (hereinafter, prior art 1). ).
ここで、上述のように、病初期の１回目の発作では一相性脳症及びＡＥＳＤ共に、発熱、けいれん、意識障害を呈するものの、上述のように、予後が比較的良好な一相性脳症と異なり、ＡＥＳＤは、重篤な後遺症を残すことが多い。
しかしながら、ＡＥＳＤ用の診断用マーカーは知られておらず、従来技術１の診断方法でも、一相性脳症とＡＥＳＤとを区別することはできなかった。
このため、病初期にＡＥＳＤであることを検出することは難しかった。
Here, as described above, although both monophasic encephalopathy and AESD present with fever, convulsions, and impaired consciousness in the first seizure in the early stage of the disease, unlike monophasic encephalopathy having a relatively good prognosis as described above. AESD often leaves serious sequelae.
However, no diagnostic marker for AESD is known, and even with the diagnostic method of the prior art 1, it is not possible to distinguish between monophasic encephalopathy and AESD.
Therefore, it was difficult to detect AESD in the early stage of the disease.
本発明は、このような状況に鑑みてなされたものであり、上述の問題を解消することを目的とする。 The present invention has been made in view of such a situation, and an object of the present invention is to solve the above-mentioned problems.
本発明のバイオマーカーは、けいれん重積型（二相性）急性脳症の診断用のバイオマーカーであって、髄液中のＣｌｕｓｔｅｒｉｎのタンパク質マーカーであることを特徴とする。
本発明の診断用組成物は、けいれん重積型（二相性）急性脳症を診断するための診断用組成物であって、Ｃｌｕｓｔｅｒｉｎのタンパク質に特異的に結合する抗体を含むことを特徴とする。
本発明の診断用キットは、けいれん重積型（二相性）急性脳症を診断するための診断用キットであって、Ｃｌｕｓｔｅｒｉｎのタンパク質に特異的に結合する抗体を含むことを特徴とする。
The biomarker of the present invention is a biomarker for diagnosis of status epilepticus (biphasic) acute encephalopathy, and is characterized by being a protein marker of Crusterin in cerebrospinal fluid.
The diagnostic composition of the present invention is a diagnostic composition for diagnosing status epilepticus (biphasic) acute encephalopathy, and is characterized by containing an antibody that specifically binds to a protein of Crustin.
The diagnostic kit of the present invention is a diagnostic kit for diagnosing status epilepticus (biphasic) acute encephalopathy, and is characterized by containing an antibody that specifically binds to a protein of Crustin.
本発明によれば、Ｃｌｕｓｔｅｒｉｎのタンパク質の髄液中での発現量変化を指標とすることで、病初期にＡＥＳＤであることを検出するのに役立つバイオマーカーを提供することができる。
According to the present invention, by an index the expression level change in the cerebrospinal fluid of protein clusterin, it is possible to provide a biomarker which serves to detect that the AESD ill early.
＜実施の形態＞
本発明の発明者らは、一相性脳症と、二相性のＡＥＳＤとを病初期に判別するバイオマーカーを探索するため、鋭意実験を行った。その結果、髄液中に発現するタンパクのうち、いくつかのものの発現量が変化することを早期診断マーカーとして用いることで脳症を検出する方法を見いだし、本発明を完成するに至った。
<Embodiment>
The inventors of the present invention conducted diligent experiments in order to search for a biomarker that distinguishes monophasic encephalopathy from biphasic AESD in the early stage of the disease. As a result, we have found a method for detecting encephalopathy by using the change in the expression level of some of the proteins expressed in the cerebrospinal fluid as an early diagnostic marker, and have completed the present invention.
具体的には、本発明の発明者らは、ＡＥＳＤ患者３名と一相性脳症患者３名の病初期に採取したヒト髄液中で、プロテオーム解析を行った。髄液は１回目の発作から１０時間以内に集められた。この髄液について、ゲル電気泳動法（２Ｄ−ＤＩＧＥ）を使用し、発現が変化しているタンパク質を２Ｄ−ＤＩＧＥ法によって確認した。この結果として、ＡＥＳＤ患者からの髄液では、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｅｏｎ ａｎｔｉｇｅｎ ＣＤ１４及び免疫グロブリンの発現が上昇していた。これらは、免疫応答に関連するタンパク質であった。また、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎの発現が減少していた。これら発現が減少したタンパク質は、後述するようにアポトーシス、免疫学的応答、及び神経修復に関係していた。
患者の髄液中で、これらのタンパク質をバイオマーカーとして発現の定性、定量を行うことでＡＥＳＤの早期診断、早期治療が可能となることが期待される。
Specifically, the inventors of the present invention performed proteome analysis in human cerebrospinal fluid collected in the early stage of the disease in three AESD patients and three monophasic encephalopathy patients. Cerebrospinal fluid was collected within 10 hours of the first attack. For this cerebrospinal fluid, gel electrophoresis (2D-DIGE) was used, and proteins whose expression was changed were confirmed by the 2D-DIGE method. As a result, the expression of Monocyte differential antigen antibody CD14 and immunoglobulin was increased in the cerebrospinal fluid from AESD patients. These were proteins associated with the immune response. In addition, the expression of apolipoprotein E, gelsolin, Crustin, and α2-Macroglobulin was decreased. These reduced expression proteins were involved in apoptosis, immunological response, and nerve repair, as described below.
It is expected that early diagnosis and early treatment of AESD will be possible by qualitatively and quantifying the expression of these proteins in the cerebrospinal fluid of patients as biomarkers.
より具体的に説明すると、本発明の実施の形態に係る脳症の検出方法、バイオマーカー、診断用組成物、及び診断用キットの対象となる脳症は、けいれん重積型（二相性）急性脳症（ＡＥＳＤ）である。上述したように、ＡＥＳＤは、発熱に伴い、けいれん重積（１回目の発作）を起こした後に、一時的に意識状態が改善した後、２〜４日後に、再度けいれん発作や意識障害（２回目の発作）を起こす二相性の経過を示し、麻痺、知的障害やてんかん等の後遺症を残すことが多い疾患である。
本実施形態に係る脳症の検出方法の被験者は、急性脳症が疑われる患者であり、脳症非発症者、急性でない脳症発症者、及び脳症を引き起こす各種感染症の患者を含む。
More specifically, the encephalopathy targeted by the method for detecting encephalopathy, the biomarker, the composition for diagnosis, and the diagnostic kit according to the embodiment of the present invention is status epilepticus (biphasic) acute encephalopathy (). AESD). As mentioned above, AESD causes status epilepticus (first seizure) due to fever, and after a temporary improvement in consciousness, another seizure or impaired consciousness (2) 2 to 4 days later. It is a disease that shows a biphasic course that causes (second seizure) and often leaves aftereffects such as paralysis, intellectual disability, and epilepsy.
The subjects of the method for detecting encephalopathy according to the present embodiment are patients suspected of having acute encephalopathy, and include patients who do not develop encephalopathy, those who develop non-acute encephalopathy, and patients with various infectious diseases that cause encephalopathy.
また、本実施形態においては、上述のように、初回けいれん発症（１回目の発作）後、早期に髄液を採取し、この髄液中内で発現が変化しているタンパク質の発現量を、プロテオーム解析にて同定した。プロテオームは、特定の細胞若しくは生体試料が特定条件下に置かれた時に、その中に存在する全てのタンパク質を意味する。プロテオーム解析は、タンパク質の機能や各タンパク質が構築する機能ネットワークを明らかにする研究手法であり、こうして得られた知見は、疾病の早期診断や病態の解明、さらには創薬研究へと応用されている。 Further, in the present embodiment, as described above, the cerebrospinal fluid is collected at an early stage after the onset of the first seizure (first seizure), and the expression level of the protein whose expression is changed in the cerebrospinal fluid is determined. Identified by proteome analysis. Proteome means all proteins present in a particular cell or biological sample when placed under certain conditions. Proteome analysis is a research method that clarifies the functions of proteins and the functional networks constructed by each protein, and the findings obtained in this way are applied to early diagnosis of diseases, elucidation of pathological conditions, and drug discovery research. There is.
本発明の実施の形態に係るプロテオーム解析には、二次元電気泳動（Ｔｗｏ−ｄｉｍｅｎｓｉｏｎａｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ、２−ＤＥ）と質量分析（ｍａｓｓ ｓｐｅｃｔｒｏｍｅｔｒｙ、ＭＳ）を基礎としたプロテオーム解析を用いることが可能である。二次元電気泳動は、個々のタンパク質が持つ等電点の違いを利用した等電点電気泳動と、分子量の違いを利用したポリアクリルアミド電気泳動（ＳＤＳ−ＰＡＧＥ）とを組み合わせることによって、タンパク質を分離する方式である。これにより数百から数千種類のタンパク質を同時に分離し、タンパク質スポットとして可視化することができる。このタンパク質スポットは、酵素処理を行いペプチド断片とした後に、後述する質量分析にて、ペプチド断片の質量データ（ＭＳスペクトル）を計測する。ペプチドフィンガープリント法により、このＭＳスペクトラムを、既存のデータベースに登録されているタンパク質のＭＳスペクトラムと照合し、タンパク質の同定を行うことが可能である。また、タンデムマス質量分析（ＭＳ／ＭＳ）装置を用いることでより詳細な情報（ＭＳ／ＭＳスペクトル）を取得でき、高精度にタンパク質の同定が可能となる。 For the proteome analysis according to the embodiment of the present invention, it is possible to use a proteome analysis based on two-dimensional electrophoresis (Two-dimensional electrophoresis, 2-DE) and mass spectrometry (MS). Two-dimensional electrophoresis separates proteins by combining isoelectric focusing that utilizes the difference in isoelectric points of individual proteins and polyacrylamide gel electrophoresis (SDS-PAGE) that utilizes the difference in molecular weight. It is a method to do. This makes it possible to simultaneously separate hundreds to thousands of proteins and visualize them as protein spots. This protein spot is subjected to enzymatic treatment to obtain a peptide fragment, and then the mass data (MS spectrum) of the peptide fragment is measured by mass spectrometry described later. By the peptide fingerprint method, it is possible to identify the protein by collating this MS spectrum with the MS spectrum of the protein registered in the existing database. Further, by using a tandem mass spectrometry (MS / MS) apparatus, more detailed information (MS / MS spectrum) can be obtained, and proteins can be identified with high accuracy.
また、本発明の実施の形態に係るプロテオーム解析には、特に、蛍光標識二次元ディファレンスゲル電気泳動（２−Ｄｉｍｅｎｓｉｏｎａｌ Ｆｌｕｏｒｅｓｃｅｎｃｅ Ｄｉｆｆｅｒｅｎｃｅ Ｇｅｌ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ、２Ｄ−ＤＩＧＥ法）を用いることが好適である。２Ｄ−ＤＩＧＥ法では、異なる蛍光波長を持つ色素（ＣｙＤｙｅ：Ｃｙ２，Ｃｙ３，Ｃｙ５）を用い、泳動する前に各サンプルのタンパク質をあらかじめ標識（Ｃｙ３，Ｃｙ５）することで、同一のゲルで２群の発現量比較を行う。また、比較するサンプルを等量ずつ混合した内部標準（Ｃｙ２）を用いる。これにより、従来の二次元電気泳動法に比べて、２Ｄ−ＤＩＧＥ法では、ゲル間での泳動誤差が改善され、高感度で高精度に発現差異を解析可能である。さらに、２Ｄ−ＤＩＧＥではタンパク質を直接蛍光色素標識することで、比較的少量のタンパク質量でもスポットとして検出することが可能である。
本実施形態において、２Ｄ−ＤＩＧＥ法による急性脳症患者髄液を用いたプロテオーム解析により、初回けいれん後、早期に二相性の脳症を発症するリスクを判定するバイオマーカーを同定することが可能となった。
これにより、髄液内で変化している分子を測定することで、ＡＥＳＤの発症機序と病態を解明することが可能となる。
なお、本実施形態のプロテオーム解析として、その他の高性能な分析機器を駆使した解析法を用いてもよい。
Further, in the proteomics analysis according to the embodiment of the present invention, it is particularly preferable to use fluorescently labeled two-dimensional difference gel electrophoresis (2-Dimensional Fluorescence Difference Gel Electrophoresis, 2D-DIGE method). In the 2D-DIGE method, dyes having different fluorescence wavelengths (CyDye: Cy2, Cy3, Cy5) are used, and the proteins of each sample are labeled in advance (Cy3, Cy5) before electrophoresis, so that two groups of the same gel are used. The expression level of is compared. In addition, an internal standard (Cy2) in which equal amounts of samples to be compared are mixed is used. As a result, in the 2D-DIGE method, the migration error between gels is improved as compared with the conventional two-dimensional electrophoresis method, and the expression difference can be analyzed with high sensitivity and high accuracy. Furthermore, in 2D-DIGE, by directly labeling a protein with a fluorescent dye, even a relatively small amount of protein can be detected as a spot.
In the present embodiment, proteome analysis using cerebrospinal fluid of patients with acute encephalopathy by the 2D-DIGE method has made it possible to identify a biomarker for determining the risk of developing biphasic encephalopathy early after the initial convulsions. ..
This makes it possible to elucidate the pathogenic mechanism and pathophysiology of AESD by measuring the molecules that are changing in the cerebrospinal fluid.
As the proteome analysis of the present embodiment, an analysis method that makes full use of other high-performance analytical instruments may be used.
具体的には、本発明の実施の形態に係る脳症の検出方法は、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎからなる群の一種以上のタンパク質の髄液中での発現量変化を指標として、けいれん重積型（二相性）急性脳症であることを検出することを特徴とする。
また、本発明の実施の形態に係る脳症の検出方法は、指標として、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４については発現量が増加し、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎについては発現量が減少することに基づくことを特徴とする。
なお、本実施形態において、「一種以上」とは、各構成のうち一つ又は複数の組み合わせのいずれかであることを示す。すなわち、いずれか一つを用いてもよいし、これらのうち二つ〜全てについての任意の組み合わせを含む。
つまり、上述の脳症の検出方法の例においては、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎからなる群のタンパク質については、一つ又は複数のタンパク質の組み合わせのいずれかの変動を検出する。
Specifically, the method for detecting encephalopathy according to the embodiment of the present invention is the expression of one or more proteins in the cerebrospinal fluid of the group consisting of Monocyte differential antigen CD14, Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin. It is characterized by detecting convulsive-stacked (biphasic) acute encephalopathy using a change in amount as an index.
Further, in the method for detecting encephalopathy according to the embodiment of the present invention, as an index, the expression level of Monocite differentiation antigen CD14 increases, and the expression level of Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin decreases. It is characterized by being based on.
In the present embodiment, "one or more" means either one or a plurality of combinations of each configuration. That is, any one may be used, and any combination of two to all of these may be included.
That is, in the above-mentioned example of the method for detecting encephalopathy, for the proteins in the group consisting of Monocite differential antigen CD14, Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin, the variation of either one or a combination of multiple proteins. Is detected.
また、本発明の実施の形態に係るバイオマーカーは、けいれん重積型（二相性）急性脳症の診断用のバイオマーカーであって、髄液中のＭｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎからなる群の一種以上であることを特徴とする。 Further, the biomarker according to the embodiment of the present invention is a biomarker for diagnosing convulsive stacking type (biphasic) acute encephalopathy, and is a biomarker for diagnosing monocite differential antigen CD14 in cerebrospinal fluid, apolipoprotein E, Gelsolin, Crustin, It is characterized in that it is one or more of the group consisting of α2-Macroglobulin.
ここで、本発明の実施の形態に係るバイオマーカーとして、ＡＥＳＤで発現量が増加していたＭｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｅｏｎ ａｎｔｉｇｅｎ ＣＤ１４（以下、「ＣＤ１４」と記載する。）は、中枢神経系の感染及び傷害に対するマイクログリア応答に対応する重要なタンパクである。具体的に、ＣＤ１４は、ＴＬＲ（トール様受容体）４の補助受容体であり、ＬＰＳ（リポ多糖類）に対する応答を促進する。また、ＣＤ１４は、ＴＬＲ４及び関連する免疫受容体の機能を組み合わせて、ＴＬＲ及び非ＴＬＲの系により制御される。これにより、ＣＤ１４は、中枢神経系の感染及び無感染時のミクログリアのダメージ検出の機能を微調整し、ミクログリア活性化を補助する。 Here, as a biomarker according to the embodiment of the present invention, the Monocite differential antigen antigen CD14 (hereinafter referred to as “CD14”) whose expression level was increased in AESD is a microglia for infection and injury of the central nervous system. It is an important protein corresponding to the glial response. Specifically, CD14 is a co-receptor for TLRs (Toll-like receptors) 4 and promotes a response to LPS (lipopolysaccharides). CD14 is also regulated by TLR and non-TLR systems by combining the functions of TLR4 and related immune receptors. Thereby, the CD14 fine-tunes the function of detecting microglial damage in the case of infection and non-infection of the central nervous system, and assists the activation of microglia.
また、本発明の実施の形態に係るバイオマーカーとして、ＡＥＳＤで発現量が減少していたＡｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ（以下、「ＡｐｏＥ」と記載する。）は、アストロサイトやミクログリアでも産生、分泌されるタンパクである。また、ＡｐｏＥ受容体は、ニューロン、アストロサイト及びミクログリアの中で広く発現している。ＡｐｏＥは、髄液中のリポタンパクと結合し、神経細胞内に取り込まれる。すなわち、ＡｐｏＥは、神経細胞損傷後の修復期に必要なコレステロール等の脂質の神経細胞への輸送に関与している。また、コレステロール及び他の脂質はシナプス構造及び修復に重要な役割を果たす。具体的に、コレステロールは、薄膜とミエリン鞘の需要な原料であり、シナプスの完全性及び神経機能には必須の化合物である。
また、本発明の実施の形態に係るバイオマーカーとして、ＡＥＳＤで発現量が減少していたＧｅｌｓｏｌｉｎは、細胞分化や接着、アポトーシスに関係しているタンパクである。
また、本発明の実施の形態に係るバイオマーカーとして、ＡＥＳＤで発現量が減少していたＣｌｕｓｔｅｒｉｎ（以下、「ＣＬＵ」と記載する。）は、免疫グロブリンや補体等と複合体を形成する糖タンパク質である。ＣＬＵは、脳を含む多くの組織で広く発現され、マクロファージの誘引、補体攻撃抑制、アポトーシス阻害、膜の再形成等に関連する。また、ＣＬＵは、けいれん誘発性の神経細胞障害に対する保護に関連している。
Further, as a biomarker according to the embodiment of the present invention, apolipoprotein E (hereinafter referred to as "ApoE") whose expression level was reduced by AESD is a protein produced and secreted by astrocytes and microglia. be. ApoE receptors are also widely expressed in neurons, astrocytes and microglia. ApoE binds to lipoproteins in the cerebrospinal fluid and is taken up by nerve cells. That is, ApoE is involved in the transport of lipids such as cholesterol required for the repair phase after nerve cell damage to nerve cells. Cholesterol and other lipids also play important roles in synaptic structure and repair. Specifically, cholesterol is a sought-after source of thin films and myelin sheaths and is an essential compound for synaptic integrity and neural function.
Further, as a biomarker according to the embodiment of the present invention, gelsolin whose expression level was decreased by AESD is a protein involved in cell differentiation, adhesion, and apoptosis.
Further, as a biomarker according to the embodiment of the present invention, Crustin (hereinafter referred to as "CLU") whose expression level has been reduced by AESD is a sugar that forms a complex with immunoglobulin, complement and the like. It is a protein. CLU is widely expressed in many tissues including the brain and is associated with macrophage attraction, complement attack suppression, apoptosis inhibition, membrane remodeling and the like. CLU is also associated with protection against convulsive-induced neuronal damage.
これらをまとめると、従来、ＡＥＳＤの病理的な機構は、ニューロンの興奮毒性であると考えられていた。
これに対して、本実施形態によれば、ＡＥＳＤの免疫学的応答の関与が機構のうちの１つであることを示唆した。すなわち、脳症の初期で大量のステロイドを投与するステロイドパルス療法及び免疫抑制療法により、ＡＥＳＤの２回目の発作を抑える療法の開発が期待できる。
一方、本実施形態によれば、ＡＥＳＤでは、発現量減少したタンパク質はアポトーシス、免疫学的応答及び神経修復に関係していた。このため、ＡＥＳＤ患者においては、神経修復能力が他の脳症患者より低い可能性が示唆される。このため、各種の神経修復を促進する薬剤による治療により、ＡＥＳＤの後遺症を抑制することが期待できる。
なお、本実施形態のバイオマーカーは、患者がＡＥＳＤを発症していることを確認する発症マーカー、又は患者がＡＥＳＤを発症していないことを確認する健常マーカーのいずれとしても使用可能である。
Taken together, the pathological mechanism of AESD has traditionally been thought to be neuronal excitotoxicity.
In contrast, the present embodiment suggests that the involvement of the immunological response of AESD is one of the mechanisms. That is, the development of a therapy for suppressing the second attack of AESD can be expected by steroid pulse therapy and immunosuppressive therapy in which a large amount of steroid is administered in the early stage of encephalopathy.
On the other hand, according to the present embodiment, in AESD, the reduced expression protein was involved in apoptosis, immunological response and nerve repair. This suggests that nerve repair ability may be lower in AESD patients than in other encephalopathy patients. Therefore, it can be expected that the sequelae of AESD can be suppressed by treatment with various drugs that promote nerve repair.
The biomarker of the present embodiment can be used as either an onset marker for confirming that the patient has developed AESD or a healthy marker for confirming that the patient has not developed AESD.
また、本発明の実施の形態に係る診断用組成物は、けいれん重積型（二相性）急性脳症を診断するための診断用組成物であって、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎからなる群の一種以上のタンパク質に特異的に結合する抗体を含むことを特徴とする。
また、本発明の実施の形態に係る診断用キットは、けいれん重積型（二相性）急性脳症を診断するための診断用キットであって、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎからなる群の一種以上のタンパク質に特異的に結合する抗体を含むことを特徴とする。
In addition, the diagnostic composition according to the embodiment of the present invention is a diagnostic composition for diagnosing a spasmodic (biphasic) acute encephalopathy, and is a Monocite differential protein CD14, Apolipoprotein E, Gelsolin, Crustin. , And an antibody that specifically binds to one or more proteins in the group consisting of α2-Macroglobulin.
Further, the diagnostic kit according to the embodiment of the present invention is a diagnostic kit for diagnosing spasmodic (biphasic) acute encephalopathy, and is a diagnostic kit for diagnosing a convulsive stacking type (biphasic) acute encephalopathy, such as Monocite diffusion antibody CD14, Apolipoprotein E, Gelsolin, Crustin, and. It is characterized by containing an antibody that specifically binds to one or more proteins in the group consisting of α2-Macroglobulin.
ここで、従来、ＡＥＳＤにおいては、二相性の経過を見ないと診断が困難であるが、２回目のけいれん、意識障害の発症後に治療しても、予後の改善が見られないことが多かった。
これに対して本実施形態の診断用組成物、診断用キットにより、病初期の１回目のけいれん等の発作後に、髄液中のＭｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｅｏｎ ａｎｔｉｇｅｎ ＣＤ１４、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ、Ｇｅｌｓｏｌｉｎ、Ｃｌｕｓｔｅｒｉｎ、及びα２−Ｍａｃｒｏｇｌｏｂｕｌｉｎの一種以上の発現変化を診断し、早期治療を行うことで、予後の改善が得られる可能性がある。すなわち、髄液を取得して、本実施形態のバイオマーカーを計測することで、ＡＥＳＤの早期診断により適切な治療が行われ、後遺症が抑えられる可能性がある。
また、従来、ＡＥＳＤの治療は大量のステロイドを投与するステロイドパルス療法が適応であるものの、本実施形態の診断用組成物、診断用キットによりＡＥＳＤでなく「一相性脳症」であると診断された場合、自然軽快が期待される。このため、副作用の可能性のあるステロイドパルス療法をしなくてもよくなる。
Here, conventionally, in AESD, it is difficult to make a diagnosis without observing the biphasic course, but even if the treatment is performed after the onset of the second convulsion or disturbance of consciousness, the prognosis is often not improved. ..
On the other hand, according to the diagnostic composition and the diagnostic kit of the present embodiment, after the first seizure such as convulsions in the early stage of the disease, Monocite differentialatenantien CD14, Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin in the spinal fluid. Diagnosis of one or more changes in expression and early treatment may improve prognosis. That is, by acquiring cerebrospinal fluid and measuring the biomarker of the present embodiment, appropriate treatment may be performed by early diagnosis of AESD, and sequelae may be suppressed.
In addition, although steroid pulse therapy in which a large amount of steroid is administered is conventionally indicated for the treatment of AESD, it was diagnosed as "monophasic encephalopathy" instead of AESD by the diagnostic composition and diagnostic kit of the present embodiment. In that case, natural lightness is expected. This eliminates the need for steroid pulse therapy, which can have side effects.
ここで、本実施形態の診断用組成物及び診断用キットにおいて、バイオマーカーの各タンパク質の含有量は、公知の測定方法で測定可能である。この測定方法は、抗体を用いた方法及び質量分析法を用いることが可能である。 Here, in the diagnostic composition and the diagnostic kit of the present embodiment, the content of each protein of the biomarker can be measured by a known measuring method. As this measuring method, a method using an antibody and a mass spectrometry method can be used.
本実施形態のバイオマーカーの含有量を測定する際に抗体を用いる方法としては、例えば、一般的な、ウェスタンブロッティングやＥＬＩＳＡ（酵素免疫吸着測定法）を用いることが可能である。ウェスタンブロッティングは、髄液を必要に応じ緩衝液で希釈し、ドデシル硫酸ナトリウム（ＳＤＳ）を含むバッファーで溶解し、髄液をＳＤＳ−ＰＡＧＥで電気泳動して分子量に応じて分離する方式である。ゲル上の分離したタンパク質をニトロセルロース膜やＰＶＤＦ膜等に転写し、転写した膜をこれに一次抗体および二次抗体を添加し、二次抗体の識別標識を検出することにより、本実施形態のバイオマーカーのタンパク質を検出することが可能である。
また、ＥＬＩＳＡ法は、検出対象タンパク質について、特異的に結合する一次抗体と、この一次抗体に特異的に結合し、かつ標識（ラベル）する二次抗体により検出する方式である。検出は、例えば、それぞれの基質を加えた後、蛍光若しくは化学発光物質または酵素反応による可視光を計測する。
As a method of using an antibody when measuring the content of the biomarker of the present embodiment, for example, general Western blotting or ELISA (enzyme-linked immunosorbent assay) can be used. Western blotting is a method in which cerebrospinal fluid is diluted with a buffer solution as necessary, dissolved in a buffer containing sodium dodecyl sulfate (SDS), and the cerebrospinal fluid is electrophoresed on SDS-PAGE to separate it according to its molecular weight. By transferring the separated protein on the gel to a nitrocellulose membrane, PVDF membrane, etc., adding a primary antibody and a secondary antibody to the transferred membrane, and detecting the identification label of the secondary antibody, the present embodiment It is possible to detect biomarker proteins.
The ELISA method is a method in which a protein to be detected is detected by a primary antibody that specifically binds to the protein and a secondary antibody that specifically binds to and labels the primary antibody. For detection, for example, after adding each substrate, visible light due to fluorescence or chemiluminescent substance or enzymatic reaction is measured.
ここで、本実施形態のバイオマーカーのタンパク質を検出する抗体は、ポリクローナル抗体でも、モノクローナル抗体でも、ＶＨ領域やＶＬ領域のみ合成された抗体、ＶＨ領域や接着部位だけ合成された抗体様ペプチド等であってもよい。また、動物由来の抗体の場合には、例えば、ウサギ、ヤギ、マウス、ラット、ブタ、ヒツジ、イヌ、トリ、昆虫、その他の脊椎動物及び無脊椎動物等のものであってもよい。また、植物、酵母、古細菌、及び真正細菌、ｉｎ ｖｉｔｒｏの合成系等により合成された抗体を用いてもよい。また、抗体の作製は、本実施形態のバイオマーカーのタンパク質を用い、一般の方法で、動物を免疫して作成可能である。また、抗体としてモノクローナル抗体を用いる場合には、ハイブリドーマやファージディスプレイ法等により、一般的な方法で作成することが可能である。 Here, the antibody that detects the biomarker protein of the present embodiment is a polyclonal antibody, a monoclonal antibody, an antibody synthesized only in the VH region or the VL region, an antibody-like peptide synthesized only in the VH region or the adhesion site, or the like. There may be. In the case of animal-derived antibodies, for example, rabbits, goats, mice, rats, pigs, sheep, dogs, birds, insects, other vertebrates and invertebrates may be used. Further, an antibody synthesized by a plant, yeast, archaea, eubacteria, in vitro synthesis system, or the like may be used. Further, the antibody can be prepared by immunizing an animal by a general method using the protein of the biomarker of the present embodiment. When a monoclonal antibody is used as the antibody, it can be produced by a general method by a hybridoma, a phage display method, or the like.
また、本実施形態のバイオマーカーの含有量を測定する際に質量分析法を用いる場合には、高速液体クロマトグラフィー−質量分析計（ＬＣ−ＭＳ）法、ガスクロマトグラフィー−質量分析計（ＧＣ−ＭＳ）法、キャピラリー電気泳動−質量分析計（ＣＥ−ＭＳ）法、ＭＳ／ＭＳ分析、ＭＡＬＤＩ／ＴＯＦＭＳ分析、ＮＭＲ分析、酸アルカリ中和滴定、アミノ酸分析、酵素法、比色定量法、高速液体クロマトグラフィー、ガスクロマトグラフィー等の各種の手法を用いることが可能である。 When mass spectrometry is used when measuring the content of the biomarker of the present embodiment, high performance liquid chromatography-mass spectrometer (LC-MS) or gas chromatography-mass spectrometer (GC-) is used. MS) method, capillary electrophoresis-mass spectrometer (CE-MS) method, MS / MS analysis, MALDI / TOFMS analysis, NMR analysis, acid-alkali neutralization titration, amino acid analysis, enzyme method, colorimetric method, high performance liquid chromatography It is possible to use various methods such as chromatography and gas chromatography.
また、本実施形態のバイオマーカーの含有量は、上述の各タンパク質の濃度が上昇しているか、減少しているかについて、特定の閾値や統計的有意性を基に判定することが可能である。この判定により、ＡＥＳＤとして２回目の発作が生じるか否かの危険性を推定することも可能である。 In addition, the content of the biomarker of the present embodiment can be determined based on a specific threshold value or statistical significance as to whether the concentration of each of the above-mentioned proteins is increasing or decreasing. From this determination, it is also possible to estimate the risk of whether or not a second seizure will occur as AESD.
この本実施形態のバイオマーカーの特定の閾値は、例えば、ＬＣ−ＭＳ法を用いた測定で得られるグラフのピーク面積を標準化した値について、ＡＥＳＤと診断された患者が閾値以上に特定の割合で含まれ、一相性の患者が閾値未満に特定の割合で含まれ、カイ二乗検定値が最も有意となるような値を、各種統計分析プログラム等で算出可能である。また、本実施形態のバイオマーカーの各タンパク質の統計的有意差がある組み合わせについて算出し、この組み合わせについての閾値を設定してもよい。 The specific threshold value of the biomarker of this embodiment is, for example, a value obtained by standardizing the peak area of the graph obtained by measurement using the LC-MS method, at a specific ratio above the threshold value for patients diagnosed with AESD. It is possible to calculate a value in which the included and monophasic patients are included in a specific ratio below the threshold value and the chi-square test value is the most significant by various statistical analysis programs and the like. In addition, a combination of the biomarker proteins of the present embodiment having a statistically significant difference may be calculated and a threshold value may be set for this combination.
また、本実施形態の特定の閾値の設定により、バイオマーカーの感度及び特異度を算出してもよい。この場合の感度とはＡＥＳＤを正常に検出する割合であり、特異度は偽陽性の割合である。感度が低いと実際にＡＥＳＤである患者を正常と判定し辛くなり、得意度が低いとＡＥＳＤを誤判定することが多くなる。また、閾値、感度及び偽陽性率から、受診者動作特性（ＲＯＣ）曲線を求めてもよい。ＲＯＣ曲線の曲線下面積（ＲＯＣ−ＡＵＣ）は、バイオマーカーの性能を示す値である。このＲＯＣ−ＡＵＣのｐ値をｚ検定により算出してもよい。 In addition, the sensitivity and specificity of the biomarker may be calculated by setting a specific threshold value of the present embodiment. The sensitivity in this case is the rate at which AESD is normally detected, and the specificity is the rate of false positives. If the sensitivity is low, it becomes difficult to judge a patient who is actually AESD as normal, and if the degree of strength is low, the AESD is often misjudged. In addition, the receiver operating characteristic (ROC) curve may be obtained from the threshold value, sensitivity, and false positive rate. The area under the curve (ROC-AUC) of the ROC curve is a value indicating the performance of the biomarker. The p-value of this ROC-AUC may be calculated by the z-test.
なお、本実施形態のＡＥＳＤを発症する実験動物（モデル動物）により、ＡＥＳＤの治療に効果がある低分子の化合物等の薬剤をスクリーニングすることも可能である。すなわち、このモデル動物に治療薬の候補である化合物を投与し、投与前後で髄液を取得し、バイオマーカーの各タンパク質の発現量を測定し、比較する。そして、化合物の投与後に、バイオマーカーの各タンパク質の発現量が非ＡＥＳＤの患者の範囲に近づけば、投与した化合物は脳症の治療に効果的に用いられる可能性がある。
このように、本実施形態のバイオマーカーを用いることで、効果がある化合物を容易にスクリーニングすることも可能となる。
It is also possible to screen a drug such as a low-molecular-weight compound that is effective in treating AESD from the experimental animal (model animal) that develops AESD of the present embodiment. That is, a compound that is a candidate for a therapeutic drug is administered to this model animal, cerebrospinal fluid is obtained before and after administration, and the expression level of each protein of a biomarker is measured and compared. Then, if the expression level of each protein of the biomarker approaches the range of non-AESD patients after administration of the compound, the administered compound may be effectively used for the treatment of encephalopathy.
As described above, by using the biomarker of the present embodiment, it is possible to easily screen effective compounds.
次に図面に基づき本発明を実施例によりさらに説明するが、以下の具体例は本発明を限定するものではない。 Next, the present invention will be further described with reference to the drawings, but the following specific examples do not limit the present invention.
〔実験方法〕
（髄液サンプル）
対象は、患者の親権者のインフォームドコンセントを得たＡＥＳＤが３例（年齢：１１か月〜１歳３か月）の髄液（Ｓ１〜Ｓ３）であった。また、比較対象（ｃｏｎｔｒｏｌ）は、一相性脳症が３例（年齢：１１か月〜２歳０か月）の髄液（Ｃ１〜Ｃ３）であった。髄液は、初回けいれん後、１０時間以内（２時間〜１０時間）に採取した。髄液のタンパク濃度は採取直後に計測した。
各患者の臨床症状の特徴は、下記の表１の通りであった。
〔experimental method〕
(Cerebrospinal fluid sample)
The subjects were cerebrospinal fluid (S1 to S3) of 3 patients (age: 11 months to 1 year and 3 months) who gave informed consent to the parental authority of the patient. The control was the cerebrospinal fluid (C1 to C3) of 3 cases (age: 11 months to 2 years and 0 months) with monophasic encephalopathy. Cerebrospinal fluid was collected within 10 hours (2 to 10 hours) after the first convulsion. The protein concentration in the cerebrospinal fluid was measured immediately after collection.
The characteristics of clinical symptoms of each patient are as shown in Table 1 below.
（２Ｄ−ＤＩＧＥ用タンパク質サンプルの調整）
２Ｄ−ＤＩＧＥに適したタンパク質サンプルとするため、タンパク質３０μｇ分の髄液を２−Ｄ Ｃｌｅａｎ−ｕｐ Ｋｉｔ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）を用いて、塩等の夾雑物を除去した。タンパク質サンプルは６μｌのＬｙｓｉｓ Ｂｕｆｆｅｒ［３０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ ８．５），７Ｍ Ｕｒｅａ，２Ｍ Ｔｈｉｏｕｒｅａ，４％（ｗ／ｖ）ＣＨＡＰＳ］で再溶解し、タンパク濃度５μｇ／μｌとした。
(Preparation of protein sample for 2D-DIGE)
In order to prepare a protein sample suitable for 2D-DIGE, 30 μg of protein was used in 2-D Clean-up Kit (manufactured by GE Healthcare) to remove impurities such as salts. The protein sample was redissolved in 6 μl Lysis Buffer [30 mM Tris-HCl (pH 8.5), 7M Urea, 2M Thiourea, 4% (w / v) CHAPS] to a protein concentration of 5 μg / μl.
（タンパク質サンプルの蛍光標識）
蛍光試薬として、ＣｙＤｙｅ ＤＩＧＥ ｆｌｕｏｒ ｍｉｎｉｍａｌ ｄｙｅ −ｆｏｒ ２−Ｄ ｆｌｕｏｒｅｓｃｅｎｃｅ ｄｉｆｆｅｒｅｎｃｅ ｇｅｌ ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）を用い、標準的なプロトコルに従って、タンパク質サンプルの蛍光標識を行った。各１５μｇ（３μｌ）のタンパク質サンプルに対して、４００ｐｍｏｌのＣｙ３標識試薬乃至Ｃｙ５標識試薬を０．３μｌずつ混合し、暗所、氷上で３０分間静置して標識反応を行った。さらに内部標準として、全てのタンパク質サンプルから５μｇずつ集めた内部標準サンプルプールを作製し、内部標準サンプル３０μｇ（６μｌ）に対して、４００ｐｍｏｌのＣｙ２標準試薬を０．６μｌ混和し、同様に標識反応を行った。標識反応後のサンプルは、１０ｍＭリジンを蛍光標識試薬と等量添加し、氷上で１０分以上静置して反応を停止した。
同一ゲルに添加するタンパク質サンプルを、下記の表２のように混合した。
(Fluorescent labeling of protein samples)
As a fluorescent reagent, CyDye DIGE fluoro minimal die-for 2-D fluororescense difference gel electrophoresis (manufactured by GE Healthcare) was used, and the protein sample was fluorescently labeled according to a standard protocol. For each 15 μg (3 μl) of protein sample, 0.3 μl of 400 pmol of Cy3-labeled reagent to Cy5-labeled reagent was mixed, and the mixture was allowed to stand in the dark on ice for 30 minutes to carry out the labeling reaction. Furthermore, as an internal standard, an internal standard sample pool in which 5 μg was collected from all protein samples was prepared, and 0.6 μl of 400 pmol of Cy2 standard reagent was mixed with 30 μg (6 μl) of the internal standard sample, and the labeling reaction was carried out in the same manner. went. After the labeling reaction, 10 mM lysine was added in an equal amount to the fluorescent labeling reagent, and the sample was allowed to stand on ice for 10 minutes or more to stop the reaction.
The protein samples added to the same gel were mixed as shown in Table 2 below.
（２Ｄ−ＤＩＧＥ法）
標識反応後、混合した２Ｄ−ＤＩＧＥサンプルは、２ｘ ｓａｍｐｌｅ ｂｕｆｆｅｒ［７Ｍ Ｕｒｅａ，２Ｍ Ｔｈｉｏｕｒｅａ，４％（ｗ／ｖ）ＣＨＡＰＳ，１％（ｖ／ｖ）ＩＰＧ Ｂｕｆｆｅｒ ｐＨ ３−１１ ＮＬ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）， ２％（ｗ／ｖ）ＤＴＴ］を添加し、氷上で１０分間静置した。これに膨潤ｂｕｆｆｅｒ［７Ｍ Ｕｒｅａ，２Ｍ Ｔｈｉｏｕｒｅａ，４％（ｗ／ｖ）ＣＨＡＰＳ，０．５％（ｖ／ｖ）ＩＰＧ Ｂｕｆｆｅｒ ｐＨ ３−１１ ＮＬ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製），０．２％（ｗ／ｖ）ＤＴＴ，０．０００４％ＢＰＢ］を添加し、総タンパク質量１５０μｇに対して、総液体量が３５０μｌとなるように調整した。
このサンプルを固定化ｐＨ勾配（ＩＰＧ）ゲルであるＩｍｍｏｂｉｌｉｎｅ Ｄｒｙｓｔｒｉｐ １８ｃｍ ｐＨ ３−１１ ＮＬ（ＧＥ Ｈｅａｌｔｈｃａｒｅ社製）に添加し、等電点電気泳動を行った。等電点電気泳動には、Ｅｔｔａｎ ＩＰＧｐｈｏｒ Ｉｓｏｅｌｅｃｔｒｉｃ Ｆｏｃｕｓｉｎｇ Ｕｎｉｔ（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用い、サンプルを添加したＩＰＧゲルを２０℃、１２時間膨潤させた後、（１）５００Ｖ，１時間、（２）１０００Ｖ，１時間、（３）８０００Ｖ，８時間のプログラムで３７．５ｋＶｈｒに達するまで泳動した。
等電点電気泳動を行ったＩＰＧゲルは、ＳＤＳ化／還元処理Ｂｕｆｆｅｒ［５０ｍＭ Ｔｒｉｓ−ＨＣｌ，６Ｍ Ｕｒｅａ，３０％（ｖ／ｖ）Ｇｌｙｃｅｒｏｌ，１％（ｗ／ｖ）ＳＤＳ，０．２５％（ｗ／ｖ）ＤＴＴ］中で、１５分間、振盪した後、さらにＳＤＳ化／アルキル化処理Ｂｕｆｆｅｒ［５０ｍＭ Ｔｒｉｓ−Ｈｃｌ，６Ｍ Ｕｒｅａ，３０％（ｖ／ｖ）Ｇｌｙｃｅｒｏｌ，１％（ｗ／ｖ）ＳＤＳ，４．５％（ｗ／ｖ）Ｉｏｄｏａｃｅｔａｍｉｄｅ，０．００１％（ｖ／ｖ）ＢＰＢ］中で１５分間振盪し、ＳＤＳ化と還元アルキル化を行った。
２次元目のＳＤＳ−ポリアクリルアミドゲル電気泳動には、ＳＥ６００ Ｖｅｒｔｉｃａｌ Ｅｌｅｃｔｒｏｐｈｏｒｅｓｉｓ Ｓｙｓｔｅｍ（Ｈｏｅｆｅｒ社製）を用いた。無蛍光ガラスプレート（１８ｃｍ×１６ｃｍ）を用いて作製した１２％ＳＤＳ−ポリアクリルアミドゲル上に一次元展開したＩＰＧゲルを設置し、０．５％アガロースゲルで封入した。（１）８００Ｖ，２０ｍＡ，１５分間、（２）１０００Ｖ，６０ｍＡ，５時間のプログラムで、泳動の先端がゲルの底面に達するまで泳動を行った。
(2D-DIGE method)
After the labeling reaction, the mixed 2D-DIGE sample was 2x sample buffer [7M Urea, 2M Thiourea, 4% (w / v) CHAPS, 1% (v / v) IPG Buffer pH 3-11 NL (GE Healthcare). ), 2% (w / v) DTT] and allowed to stand on ice for 10 minutes. To this, swelling buffer [7M urea, 2M Thiourea, 4% (w / v) CHAPS, 0.5% (v / v) IPG Buffer pH 3-11 NL (manufactured by GE Healthcare), 0.2% (w /) v) DTT, 0.0004% BPB] was added to adjust the total liquid amount to 350 μl with respect to the total protein amount of 150 μg.
This sample was added to an immobilized pH gradient (IPG) gel, Immobiline Drytrip 18 cm pH 3-11 NL (manufactured by GE Healthcare), and isoelectric focusing was performed. For isoelectric focusing, Ettan IPGfor Isoelectric Focusing Unit (manufactured by Amersham Biosciences) was used, and the IPG gel to which the sample was added was swollen at 20 ° C. for 12 hours, and then (1) 500 V, 1 hour, (2). The program was run at 1000 V for 1 hour and (3) at 8000 V for 8 hours until it reached 37.5 kVhr.
IPG gels subjected to isoelectric point electrophoresis were subjected to SDS conversion / reduction treatment Buffer [50 mM Tris-HCl, 6M Urea, 30% (v / v) Glycerol, 1% (w / v) SDS, 0.25% ( After shaking for 15 minutes in w / v) DTT], further SDS / alkylation treatment Buffer [50 mM Tris-Hcl, 6M Urea, 30% (v / v) Glycerol, 1% (w / v) SDS , 4.5% (w / v) Iodoacetamide, 0.001% (v / v) BPB] for 15 minutes, and SDS formation and reduction alkylation were performed.
For the second-dimensional SDS-polyacrylamide gel electrophoresis, SE600 Vertical Electrophoresis System (manufactured by Hoefer) was used. A one-dimensionally developed IPG gel was placed on a 12% SDS-polyacrylamide gel prepared using a non-fluorescent glass plate (18 cm × 16 cm) and sealed with a 0.5% agarose gel. The electrophoresis was performed with a program of (1) 800 V, 20 mA, 15 minutes, and (2) 1000 V, 60 mA, 5 hours until the tip of the electrophoresis reached the bottom surface of the gel.
（画像解析及び発現差異解析）
２Ｄ−ＤＩＧＥゲルは、Ｔｙｐｈｏｏｎ ９４１０（Ａｍｅｒｓｈａｍ Ｂｉｏｓｃｉｅｎｃｅｓ社製）を用いて、Ｃｙ２、Ｃｙ３、Ｃｙ５の蛍光色素に対応する励起光／蛍光フィルター（Ｅｘｃｉｔａｔｉｏｎ／Ｅｍｉｓｓｉｏｎ Ｃｙ２＝４８８／５２２ｎｍ， Ｃｙ３＝５３２／５８０ ｎｍ，Ｃｙ５＝６３３／６７０ｎｍ）で蛍光標識タンパク質ゲル画像を取得した（Ｐｉｘｅｌ Ｓｉｚｅ＝１００μｍ）。
画像解析には、二次元電気泳動ゲル画像解析ソフトウェアであるＰｒｏｇｅｎｅｓｉｓ ＳａｍｅＳｐｏｔｓ（Ｎｏｎｌｉｎｅａｒ Ｄｙｎａｍｉｃｓ社製）を用い、ゲル画像の歪み補正とゲル間のスポットマッチングを行った。スポットマッチングとは、複数のゲルで同一位置に存在するスポットを検出することであり、対応するスポットがないものや適切なスポットの形態をなしていないものは解析から除外した。最終的にすべてのスポットについて、適切なマッチングができているか手動で確認した。そして、すべてのゲル画像に共通して存在するスポットを同定し、内部標準サンプルを介した定量値の標準化、Ａｎｏｖａによる有意差検定を行った。
(Image analysis and expression difference analysis)
The 2D-DIGE gel uses Typhon 9410 (manufactured by Amersham Biosciences) and is used as an excitation light / fluorescence filter (Excitation / Mission Cy2 = 488/522 nm, Cy3 = 532/580 nm) corresponding to the fluorescent dyes of Cy2, Cy3, and Cy5. , Cy5 = 633/670 nm), and a fluorescently labeled protein gel image was acquired (Pixel Size = 100 μm).
For image analysis, two-dimensional electrophoresis gel image analysis software, Progenesis SameSpots (manufactured by Nonlinear Dynamics) was used to correct the distortion of the gel image and perform spot matching between the gels. Spot matching is to detect spots existing at the same position in a plurality of gels, and those having no corresponding spots and those having no appropriate spot morphology were excluded from the analysis. In the end, I manually checked that all the spots were properly matched. Then, spots commonly present in all gel images were identified, quantitative values were standardized via an internal standard sample, and a significant difference test was performed by Anova.
（質量分析用サンプルの調整とゲル内消化）
２Ｄ−ＤＩＧＥ解析に使用したサンプルと同様に調整したタンパク質サンプル１５０μｇを用いて、上述の方法で二次元電気泳動を行い、質量分析用ゲルを作成した。質量分析用ゲルは、固定液［４０％（ｖ／ｖ）ｅｔｈａｎｏｌ、１０％（ｖ／ｖ）ａｃｅｔｉｃ ａｃｉｄ］中で１８時間振盪し、固定した後、Ｆｌａｍｉｎｇｏ Ｆｌｕｏｒｓｃｅｎｔ Ｇｅｌ Ｓｔａｉｎ（Ｂｉｏ−Ｒａｄ Ｌａｂｏｒａｔｏｒｉｅｓ社製）染色液（１ｘ）中で３時間、遮光、振盪して、染色した。次に染色後の質量分析用ゲルから、ＦｌｕｏｒｏＰｈｏｒｅｓｔａｒ ３０００（Ａｎａｔｅｃｈ社製）を用いて、タンパク質スポットの切り出しを行った。発現差異のあったタンパク質スポットを直径１．８ｍｍのゲルピッカーで切り出し、回収した。ゲル片は超純水とアセトニトリルで洗浄した後、減圧下で乾燥させた。次にゲル片に１０ｍＭ ＤＴＴ／１００ｍＭ ＮＨ4ＨＣＯ3溶液を加え、５６度で４５分間反応させた後、５５ｍＭ Ｉｏｄｏａｃｅｔａｍｉｄｅ／１００ｍＭ ＮＨ4ＨＣＯ3溶液を加え、室温で３０分間反応させ、還元アルキル化を行った。ゲル片を再度、超純水、アセトニトリル、１００ｍＭ ＮＨ4ＨＣＯ3で洗浄、乾燥させた後、トリプシン溶液（１２．５ｎｇ／μｌ トリプシン、５０ｍＭ ＮＨ4ＨＣＯ3）を添加し、氷上で４５分間静置した。続いて溶液を除去し、５０ｍＭ ＮＨ4ＨＣＯ3を加えて、３７℃で１６時間反応させた。トリプシンによってゲル内で消化されたペプチド断片は、２５ｍＭ ＮＨ4ＨＣＯ3、アセトニトリル、５％（ｖ／ｖ）ギ酸で回収し、減圧下で乾燥させた。
(Preparation of sample for mass spectrometry and in-gel digestion)
Using 150 μg of a protein sample prepared in the same manner as the sample used for 2D-DIGE analysis, two-dimensional electrophoresis was performed by the above method to prepare a gel for mass spectrometry. The gel for mass spectrometry is shaken in a fixing solution [40% (v / v) ethanol, 10% (v / v) acetic acid] for 18 hours, fixed, and then fixed, and then Flamingo Fluoroscent Gel Stain (manufactured by Bio-Rad Laboratories). ) Staining was carried out in a staining solution (1x) for 3 hours by shading and shaking. Next, protein spots were cut out from the stained gel for mass spectrometry using FluoroPhorestar 3000 (manufactured by Anatech). Protein spots having different expression differences were cut out with a gel picker having a diameter of 1.8 mm and collected. The gel pieces were washed with ultrapure water and acetonitrile, and then dried under reduced pressure. Next, 10 mM DTT / 100 mM NH 4 HCO 3 solution was added to the gel piece and reacted at 56 ° C. for 45 minutes, then 55 mM Iodoacetamide / 100 mM NH 4 HCO 3 solution was added and reacted at room temperature for 30 minutes for reduction alkylation. went. The gel pieces were washed again with ultrapure water, acetonitrile, and 100 mM NH 4 HCO 3 and dried, then a trypsin solution (12.5 ng / μl trypsin, 50 mM NH 4 HCO 3 ) was added, and the gel pieces were allowed to stand on ice for 45 minutes. bottom. Subsequently, the solution was removed, 50 mM NH 4 HCO 3 was added, and the mixture was reacted at 37 ° C. for 16 hours. Peptide fragments digested in gel with trypsin were recovered with 25 mM NH 4 HCO 3 , acetonitrile, 5% (v / v) formic acid and dried under reduced pressure.
（ＬＣ−ＭＳ／ＭＳとＭＳ／ＭＳ ｉｏｎ ｓｅａｒｃｈによるタンパク質同定）
ペプチド断片抽出液は、液体クロマトグラフ／タンデム質量分析装置として高速液体クロマトグラフ Ｐａｒａｄｉｇｍ ＭＳ４（Ｍｉｃｈｒｏｍ ＢｉｏＲｅｓｏｕｒｃｅｓ社製）及び質量分析装置 Ｆｉｎｎｉｇａｎ ＬＴＱ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を使用して、抽出液に含まれるペプチド断片の質量分析を行った。サンプルは、トラップカートリッジ（Ｐｅｐｔｉｄｅ Ｃａｐ Ｔｒａｐ， Ｍｉｃｈｒｏｍ ＢｉｏＲｅｓｏｕｒｃｅｓ社製）によりオンラインで脱塩・濃縮処理を行った後、ＬＣ部に導入した。ＬＣ部では、流速１５０μｌ／ｍｉｎで、固定相にＬ−ｃｏｌｕｍｎ Ｍｉｃｒｏ（０．１×５０ｍｍ、３μｍ、１２ｎｍ、化学物質評価研究機構社製）、移動相にＡ溶媒（２％アセトニトリル、０．１％ギ酸）とＢ溶媒（９０％アセトニトリル、０．１％ギ酸）を使用し、Ｂ溶媒の濃度を５％（０ｍｉｎ）から４５％（２０ｍｉｎ）まで直線勾配で上げ、ペプチド断片を連続的に溶出した。ペプチド断片は、ＭＳ部において、エレクトロスプレー法によるイオン化を行った後、イオントラップによって分離し、ＭＳスペクトル（質量範囲ｍ／ｚ ４５０−２０００）を取得した。さらに特定のｍ／ｚのイオン（プリカーサーイオン）を選択し、このイオンの衝突誘起解離によって生じるプロダクトイオンのＭＳ／ＭＳスペクトル（ＭＳ／ＭＳ解析）を取得した。得られたＭＳスペクトル及びＭＳ／ＭＳスペクトルについては、タンパク質同定解析ソフトＭＡＳＣＯＴＴＭ（Ｍａｔｒｉｘ Ｓｃｉｅｎｃｅ社製）に供し、ＭＳ／ＭＳ ｉｏｎ ｓｅａｒｃｈによるタンパク質同定を行った。なおタンパク質データベースとして、Ｓｗｉｓｓ−Ｐｒｏｔを使用した。
(Protein identification by LC-MS / MS and MS / MS ion search)
The peptide fragment extract is contained in the extract using a high performance liquid chromatograph Paradigm MS4 (manufactured by MicroBioResources) and a mass spectrometer Finnigan LTQ (manufactured by Thermo Fisher Scientific) as a liquid chromatograph / tandem mass spectrometer. Mass spectrometry of peptide fragments was performed. The sample was desalted and concentrated online by a trap cartridge (Peptide Cap Trap, manufactured by Microm BioResources), and then introduced into the LC section. In the LC section, at a flow velocity of 150 μl / min, the stationary phase was L-colum micro (0.1 × 50 mm, 3 μm, 12 nm, manufactured by Chemicals Evaluation and Research Institute), and the mobile phase was solvent A (2% acetonitrile, 0.1). % Formic acid) and B solvent (90% acetonitrile, 0.1% formic acid) were used, and the concentration of B solvent was increased from 5% (0 min) to 45% (20 min) with a linear gradient to continuously elute the peptide fragments. bottom. The peptide fragment was ionized by an electrospray method in the MS part and then separated by an ion trap to obtain an MS spectrum (mass range m / z 450-2000). Furthermore, a specific m / z ion (precursor ion) was selected, and the MS / MS spectrum (MS / MS analysis) of the product ion generated by the collision-induced dissociation of this ion was obtained. The obtained MS spectrum and MS / MS spectrum were subjected to protein identification analysis software MASCOTTM (manufactured by Matrix Science), and protein identification was performed by MS / MS ion search. Swiss-Prot was used as the protein database.
（候補タンパク質抽出）
同定タンパク質をＡＥＳＤで増加したタンパク質、減少したタンパク質に分け、Ｓｃａｆｆｏｌｄ（Ｐｒｏｔｅｏｍｅ Ｓｏｆｔｗａｒｅ， Ｉｎｃ．， ｈｔｔｐ：／／ｗｗｗ．ｐｒｏｔｅｏｍｅｓｏｆｔｗａｒｅ．ｃｏｍ）を用いて、ＧＯ（Ｇｅｎｅ Ｏｎｔｏｌｏｇｙ）解析を行いこれらがどのような機能、特徴を有するかを検討し、バイオマーカーの候補となるタンパク質を抽出した。
(Candidate protein extraction)
The identified proteins are divided into proteins increased and decreased by AESD, and GO (Gene Ontology) analysis is performed using Scaffold (Proteome Software, Inc., http: //www.proteomicsoffware.com). We examined whether they have functions and characteristics, and extracted proteins that are candidates for biomarkers.
〔結果〕
（２Ｄ−ＤＩＧＥを用いた網羅的なタンパク質発現解析と有意差を示すタンパク質スポットの同定）
図１に示すように、上記方法にてＡＥＳＤ症例の髄液、一相性脳症症例の髄液から抽出したタンパク質を２Ｄ−ＤＩＧＥによって分離し、Ｃｙ２、Ｃｙ３、Ｃｙ５の蛍光色素に対応する励起画像を取得した結果、それぞれのゲル画像においてタンパク質スポットを検出した。この検出スポットについて、スポットマッチングを行い、すべてのゲル画像において共通して存在するタンパク質スポット（マッチングスポット）を１１６３個同定した。
このマッチングスポットについて、発現差異解析を行ったところ、ＡＥＳＤと一相性脳症との間に１．３倍以上の発現量の有意差を認めたタンパク質スポットを２１個同定した。また、目視で２群に差があると思われたタンパク質スポットを２個、計２３個を同定した。
〔result〕
(Comprehensive protein expression analysis using 2D-DIGE and identification of protein spots showing significant differences)
As shown in FIG. 1, proteins extracted from the cerebrospinal fluid of AESD cases and the cerebrospinal fluid of monophasic encephalopathy cases by the above method are separated by 2D-DIGE, and excitation images corresponding to the fluorescent dyes of Cy2, Cy3, and Cy5 are obtained. As a result of acquisition, protein spots were detected in each gel image. Spot matching was performed on this detected spot, and 1163 protein spots (matching spots) commonly present in all gel images were identified.
When the expression difference analysis was performed on this matching spot, 21 protein spots in which a significant difference in expression level of 1.3 times or more was observed between AESD and monophasic encephalopathy were identified. In addition, two protein spots, which seemed to be visually different between the two groups, were identified, for a total of 23.
（ＡＥＳＤ髄液で発現差異を認めたタンパク質の同定）
発現差異を認めたタンパク質スポットについては、質量分析用ゲルからスポットの切り出しを行い、ＬＣ−ＭＳ／ＭＳとＭＳ／ＭＳ ｉｏｎ ｒｅｓｅａｒｃｈによるタンパク質同定を行った。発現差異を認めた２３個のタンパク質スポットのうち、１６個のスポットで解析が可能であった。同定したタンパク質の重複を除いた１１種類のタンパク質について、解析ソフトウェア「Ｓｃａｆｆｏｌｄ」を用いてＧＯ解析を行った。７種類はＡＥＳＤで発現が増加し、他の４種類のタンパク質はＡＥＳＤで発現が減少していた。
これらのタンパク質、発現増加の割合、ａｎｏｖａの算出結果等を、下記の表３に示す：
(Identification of proteins with different expression in AESD cerebrospinal fluid)
For protein spots with different expression, the spots were cut out from the gel for mass spectrometry, and the proteins were identified by LC-MS / MS and MS / MS ion research. Of the 23 protein spots with different expression differences, 16 spots could be analyzed. GO analysis was performed on 11 kinds of proteins excluding duplicates of the identified proteins using the analysis software "Scaffold". The expression of 7 kinds was increased by AESD, and the expression of the other 4 kinds of proteins was decreased by AESD.
Table 3 below shows these proteins, the rate of increase in expression, the calculation results of anova, and so on.
具体的には、ＡＥＳＤで発現が上昇したタンパク質は、Ｍｏｎｏｃｙｔｅ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ａｎｔｉｇｅｎ ＣＤ１４（図１の符号Ｅ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０８５７１）、Ｉｇ γ−１ ｃｈａｉｎ Ｃ ｒｅｇｉｏｎ（図１の符号Ａ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０１８５７．１）、Ｉｇ γ−２ ｃｈａｉｎ Ｃ ｒｅｇｉｏｎ（図１の符号Ｂ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０１８５９．２）、Ｉｇ κ ｃｈａｉｎ Ｖ−Ｉ ｒｅｇｉｏｎ（図１の符号Ｃ）、Ｉｇ κ ｃｈａｉｎ Ｖ−ＩＩＩ ｒｅｇｉｏｎ（図１の符号Ｃ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０１６２０．１）、Ｉｇ λ−２ ｃｈａｉｎ Ｃ ｒｅｇｉｏｎ（図１の符号Ｄ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０ＣＧ０５．１）、Ｉｇ ｈｅａｖｙ ｃｈａｉｎ Ｖ−ＩＩＩ ｒｅｇｉｏｎ（ＰＩＲ：Ｓ３４０１２）であった。
また、ＡＥＳＤで低下したタンパク質は、Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ（図１の符号ａ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０２６４９）、Ｇｅｌｓｏｌｉｎ（図１の符号ｂ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０６３９６）、Ｃｌｕｓｔｅｒｉｎ（図１の符号ｃ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ１０９０９）、α２−Ｍａｃｒｏｇｌｏｂｕｌｉｎ（図１の符号ｄ、ＵｎｉＰｒｏｔＫＢ／Ｓｗｉｓｓ−Ｐｒｏｔ：Ｐ０１０２３）であった。
Specifically, the proteins whose expression was increased by AESD are Monocite diffusion agent CD14 (reference numeral E in FIG. 1, UniProtKB / Swiss-Prot: P08571), Ig γ-1 chain Region (reference numeral A in FIG. 1, UniProtKB /). Swiss-Prot: P0185.7.1), Ig γ-2 chain Region (reference numeral B in FIG. 1, UniProtKB / Swiss-Prot: P018599.2), Ig κ chain VI region (reference numeral C in FIG. 1), Ig κ chain V-III region (reference numeral C in FIG. 1, UniProtKB / Swiss-Prot: P01620.1), Ig λ-2 chain Region (reference numeral D in FIG. 1, UniProtKB / Swiss-Prot: P0CG05.1), Ig It was a heavy chain V-III region (PIR: S34012).
The proteins lowered by AESD are Apolipoprotein E (reference numeral a in FIG. 1, UniProtKB / Swiss-Prot: P02649), Gelsolin (reference numeral b in FIG. 1, UniProtKB / Swiss-Prot: P06396), and Crustin (reference numeral in FIG. 1). c, UniProtKB / Swiss-Prot: P10909), α2-Macroglobulin (reference numeral d in FIG. 1, UniProtKB / Swiss-Prot: P01023).
以下に同定されたタンパク質の特徴を示す。
いずれのタンパク質も細胞外に局在する性質を有した。特にＡＥＳＤで上昇したタンパク質群は、炎症（免疫応答）に関連するタンパク質であった。一方、ＡＥＳＤで低下するタンパク質群として、ＧＳＮ（Ｇｅｌｓｏｌｉｎ）は繊毛形成に関与し、ＣＬＵ（Ｃｌｕｓｔｅｒｉｎ）は細胞外シャペロンとして働き、ストレス誘導性のタンパク質凝集を阻害する。ＡｐｏＥ（Ａｐｏｌｉｐｏｐｒｏｔｅｉｎ Ｅ）は抗酸化作用を示し、アミロイドβやｔａｕの重合を阻害する。Ａ２Ｍ（α２−Ｍａｃｒｏｇｌｏｂｕｌｉｎ）はプロテアーゼの阻害作用を有する。興味深いことにＡｐｏＥとＡ２Ｍは同じレセプターＬＲＰ−１（ＵｎｉＰｒｏｔ ＩＤ： Ｑ０７９５４）を共有している。ＬＲＰ−１は肝臓、肺臓以外に脳にも多く発現しており、神経機能にも関与している。
The characteristics of the identified proteins are shown below.
Both proteins had the property of being localized extracellularly. In particular, the protein group elevated by AESD was a protein related to inflammation (immune response). On the other hand, as a group of proteins that decrease in AESD, GSN (Gelsolin) is involved in cilia formation, and CLU (Crustin) acts as an extracellular chaperone and inhibits stress-induced protein aggregation. ApoE (Apolipoprotein E) exhibits antioxidant activity and inhibits the polymerization of amyloid β and tau. A2M (α2-Macroglobulin) has a protease inhibitory effect. Interestingly, ApoE and A2M share the same receptor LRP-1 (UniProt ID: Q07954). LRP-1 is abundantly expressed in the brain as well as in the liver and lungs, and is also involved in neural function.
なお、上記実施の形態の構成及び動作は例であって、本発明の趣旨を逸脱しない範囲で適宜変更して実行することができることは言うまでもない。 Needless to say, the configuration and operation of the above-described embodiment are examples, and can be appropriately modified and executed without departing from the spirit of the present invention.
本発明の脳症の検出方法、バイオマーカー、診断用組成物、及び診断用キットは、医師以外の検査技師等がＡＥＳＤの検出に用いることができ、産業上に利用することができる。 The encephalopathy detection method, biomarker, diagnostic composition, and diagnostic kit of the present invention can be used by a laboratory engineer other than a doctor for detecting AESD, and can be used industrially.
Claims (3)
髄液中のＣｌｕｓｔｅｒｉｎのタンパク質マーカーである
ことを特徴とするバイオマーカー。 A biomarker for the diagnosis of status epilepticus (biphasic) acute encephalopathy.
A biomarker characterized by being a protein marker of Crusterin in cerebrospinal fluid.
Ｃｌｕｓｔｅｒｉｎのタンパク質に特異的に結合する抗体を含む
ことを特徴とする診断用組成物。 A diagnostic composition for diagnosing status epilepticus (biphasic) acute encephalopathy.
A diagnostic composition comprising an antibody that specifically binds to a protein of Crustin.
Ｃｌｕｓｔｅｒｉｎのタンパク質に特異的に結合する抗体を含む
ことを特徴とする診断用キット。 A diagnostic kit for diagnosing status epilepticus (biphasic) acute encephalopathy.
A diagnostic kit comprising an antibody that specifically binds to a Crustin protein.
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