JP2020187145A - Biomarker determination method, biomarker, composition for diagnosis, and kit for diagnosis - Google Patents
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Abstract
Description
本発明は、特にバイオマーカー判定方法、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 particularly relates to a biomarker determination method , a biomarker, a diagnostic composition, and a diagnostic kit.
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, and RS virus, and (2) most of them are cerebral edema by head CT and MRI. (3) Other diseases such as encephalitis and meningitis are denied (there is no increase in the number of cells in the cerebrospinal 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 (hereinafter referred to as “ANE”), which is a serious encephalopathy, and clinically mild encephalopathy / encephalopathy, which has a relatively good prognosis. with a reversible spiral encephalopathy (hereinafter referred to as "MERS"), and acute encephalopathy with biphasic encephalopathy with "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, both monophasic encephalopathy and AESD present with fever, convulsions, and impaired consciousness in the first seizure in the early stage of the disease, but 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 the diagnostic method of the prior art 1 could not 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判定方法は、バイオマーカー判定方法であって、α2−Macroglobulinのタンパク質の髄液中での発現量変化を指標として、バイオマーカーの含有量を判定することを特徴とする。
本発明のバイオマーカー判定方法は、前記指標は、発現量が減少することに基づくことを特徴とする。
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α2−Macroglobulin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、α2−Macroglobulin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、α2−Macroglobulin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 determination method of the present invention is a biomarker determination method, and is characterized in that the content of a biomarker is determined using a change in the expression level of a protein of α 2-Macroglobulin in the cerebrospinal fluid as an index. ..
Biomarkers determination method of the present invention, the index is characterized in that it is based on reducing the outgoing current amount.
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 α 2-Macroglobulin in cerebrospinal fluid.
The diagnostic composition of the present invention is a diagnostic composition for diagnosing status epilepticus (biphasic) acute encephalopathy, and contains an antibody that specifically binds to a protein of α 2-Macroglobulin. It is a feature.
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 α 2-Macroglobulin. To do.
本発明によれば、α2−Macroglobulin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判定方法を提供することができる。 According to the present invention, a biomarker determination method useful for detecting AESD in the early stage of a disease is provided by using a change in the expression level of a protein of α 2-Macroglobulin in the cerebrospinal fluid as an index. Can be done.
<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 changes in the expression levels of some of the proteins expressed in cerebrospinal fluid as early diagnostic markers, 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 proteomics 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 early 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 proteomics analysis according to the embodiment of the present invention, it is possible to use a proteomics 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 differences in the isoelectric points of individual proteins and polyacrylamide gel electrophoresis (SDS-PAGE) that utilizes the differences 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 enzyme 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 fluorescence-labeled two-dimensional difference gel electrophoresis (2-Dimensional Fluorescense 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, proteomics analysis using cerebrospinal fluid of patients with acute encephalopathy by the 2D-DIGE method has made it possible to identify biomarkers that determine 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 Monocite diffusion antigen CD14, Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin. It is characterized by detecting status epilepticus (biphasic) acute encephalopathy using a change in amount as an index.
In addition, in the method for detecting encephalopathy according to the embodiment of the present invention, as an index, the expression level of Monocite diffusion antigen CD14 is increased, and the expression level of Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin is decreased. It is characterized by being based on.
In addition, in this embodiment, "one or more" means either one or a combination of a plurality 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 diffusion 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, 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. is there. In addition, ApoE receptors are 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 in 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 capacity may be lower in AESD patients than in other encephalopathy patients. Therefore, treatment with various drugs that promote nerve repair can be expected to suppress the sequelae of AESD.
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 convulsive stacking type (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 convulsive stacking type (biphasic) acute encephalopathy, and is a diagnostic kit for diagnosing convulsive stacking type (biphasic) acute encephalopathy, such as Monocite differential protein 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 in many cases, the prognosis is not improved even if the treatment is performed after the onset of the second convulsion or disturbance of consciousness. ..
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, Monocyte defertiention antigen CD14, Apolipoprotein E, Gelsolin, Crustin, and α2-Macroglobulin in the cerebrospinal fluid. Diagnosis of one or more changes in the expression of the disease and early treatment may improve the 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 needed, 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 in which only the VH region or the VL region is synthesized, an antibody-like peptide in which only the VH region or the adhesion site is synthesized, 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-speed 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-speed liquid 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 biomarker content 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 the 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 seizure. 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 dye-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 a dark place 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 the same amount as 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, an 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)
Two-dimensional electrophoresis was performed by the above method using 150 μg of a protein sample prepared in the same manner as the sample used for 2D-DIGE analysis to prepare a gel for mass spectrometry. The gel for mass spectrometry is shaken in a fixative [40% (v / v) ethanol, 10% (v / v) acetic acid] for 18 hours, 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 are 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 ) is added, and the gel pieces are allowed to stand on ice for 45 minutes. did. The solution was then removed, 50 mM NH 4 HCO 3 was added and reacted at 37 ° C. for 16 hours. Peptide fragments digested in gel by 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 Michrom BioResources) 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-column Micro (0.1 × 50 mm, 3 μm, 12 nm, manufactured by Chemical Substances Evaluation and Research Organization), and the mobile phase was solvent A (2% acetonitrile, 0.1). % Formic acid) and B solvent (90% acetonitrile, 0.1% formic acid) were used to increase the concentration of B solvent from 5% (0 min) to 45% (20 min) on a linear gradient to continuously elute peptide fragments. did. The peptide fragment was ionized by an electrospray method in the MS section 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 increased proteins and decreased proteins by AESD, and GO (Gene Onlogy) 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 the protein, the rate of increase in expression, the calculation result of anova, etc.
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 antigen CD14 (reference numeral E in FIG. 1, UniProtKB / Swiss-Prot: P08571), Ig γ-1 chain Cregion (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 V-I region (reference numeral C in FIG. 1), Ig κ chain V-III region (reference C in FIG. 1, UniProtKB / Swiss-Prot: P01620.1), Ig λ-2 chain Region (reference D in FIG. 1, UniProtKB / Swiss-Prot: P0CG05.1), Ig It was a havey 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 ciliary 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 laboratory technicians other than doctors to detect AESD, and can be used industrially.
Claims (5)
α2−Macroglobulinのタンパク質の髄液中での発現量変化を指標として、バイオマーカーの含有量を判定する
ことを特徴とするバイオマーカー判定方法。 It is a biomarker judgment method
A method for determining a biomarker, which comprises determining the content of a biomarker using a change in the expression level of a protein of α 2-Macroglobulin in the cerebrospinal fluid as an index.
ことを特徴とする請求項1に記載のバイオマーカー判定方法。 The index is, the biomarker determination method according to claim 1, characterized in that based on a decrease in the outgoing current amount.
髄液中のα2−Macroglobulin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 α 2-Macroglobulin in cerebrospinal fluid.
α2−Macroglobulin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 α 2-Macroglobulin.
α2−Macroglobulin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 protein of α 2-Macroglobulin.
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