JP2019128317A - Marker and kit for diagnosing multiple sclerosis - Google Patents
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Abstract
Description
本発明は、多発性硬化症を検出又は発症予測するための診断マーカーに関する。また、本発明は、多発性硬化症を検出又は発症予測するための診断キットに関する。 The present invention relates to diagnostic markers for detecting or predicting the onset of multiple sclerosis. The present invention also relates to a diagnostic kit for detecting or predicting the onset of multiple sclerosis.
多発性硬化症（multiple sclerosis; MS）とは中枢性脱髄疾患の一つで、神経のミエリン鞘が破壊され脳、脊髄、視神経などに病変が起こり、多様な神経症状が再発と寛解を繰り返す疾患で、日本では特定疾患に認定されている指定難病である。病名は、神経を包む組織（ミエリン鞘）が破壊されて生じる硬化が多数の領域で発生することに由来している。 Multiple sclerosis (MS) is a central demyelinating disease in which the myelin sheath of nerves is destroyed, resulting in lesions in the brain, spinal cord, optic nerve, etc., and various neurological symptoms recur and recur. It is a designated intractable disease that is recognized as a specific disease in Japan. The name of the disease is derived from the fact that the sclerosis caused by destruction of the tissue (myelin sheath) that wraps the nerves occurs in many areas.
多発性硬化症の判定には、MRI検査や、脳脊髄液採取により蛋白質の総量を測定することが行われる（非特許文献１）。しかし、MRI検査では、病巣の一部しか発見できない場合があり、また脳脊髄液検査でも、疼痛や神経損傷の可能性など患者に負担がかかるという問題がある。 For determination of multiple sclerosis, it is carried out to measure the total amount of protein by MRI examination or cerebrospinal fluid collection (Non-patent Document 1). However, in MRI, only a part of the lesion may be found, and in cerebrospinal fluid examination, there is a problem that the patient is burdened such as pain and possibility of nerve damage.
近年、多発性硬化症等の中枢神経系の炎症性疾患の患者において、脳の炎症部位でTSPO（Translocator protein 18kDa）の発現がある程度上昇していること（非特許文献２および３）が複数報告されている。TSPOは、MBR（ミトコンドリア型ベンゾジアゼピン受容体）、またはPBR（末梢性ベンゾジアゼピン受容体）とも呼ばれるタンパク質であり、IBS（過敏性腸症候群）に代表されるストレス性疾患の薬理標的として注目されている。しかしながらこの検出方法は多発性硬化症を明確に検出できるものではなく、また多発性硬化症の発症を予測できるものでもない。 In recent years, in patients with central nervous system inflammatory diseases such as multiple sclerosis, TSPO (Translocator protein 18 kDa) expression has increased to some extent at the inflammatory sites in the brain (Non-Patent Documents 2 and 3). It is done. TSPO is a protein also called MBR (mitochondrial benzodiazepine receptor) or PBR (peripheral benzodiazepine receptor), and has attracted attention as a pharmacological target of stress disorder represented by IBS (irritable bowel syndrome). However, this detection method cannot clearly detect multiple sclerosis and cannot predict the onset of multiple sclerosis.
多発性硬化症の患者には、寛解期と再発期とが交互に現れる。寛解期とは、比較的健康に過ごせる時期であり、再発期とは、症状が急に出現し悪化する時期である。多発性硬化症の治療法としては、再発期には副腎皮質ステロイドの大量投与等の炎症を和らげる治療を行い、寛解期には抗痙攣剤や筋弛緩剤の投与を行なう方法が適用される。しかしこれらの薬剤では、多発性硬化症の進行を食い止める事はできない。 In patients with multiple sclerosis, remission and relapse appear alternately. The remission phase is the time when you can stay relatively healthy, and the relapsing phase is the time when symptoms suddenly appear and get worse. As a method for treating multiple sclerosis, a method of treating inflammation such as a large dose of corticosteroids in the relapsing period is performed, and a method of administering an anticonvulsant or a muscle relaxant is applied in the remission period. However, these agents can not stop the progression of multiple sclerosis.
多発性硬化症の効果的な治療法としてインターフェロン療法がある（非特許文献４）。インターフェロン療法は多発性硬化症の長期的な予後を改善する治療薬の中心的な薬剤として、インターフェロンβ1a(IFNβ-1a)とβ1b(IFNβ-1b)が国際的に利用されている。しかしながら、インターフェロン療法の効果には個人差が認められ、神経細胞の障害を抑制する効果は少なくとも１年程度の長期間にわたる投与の後でないと判定する事ができない。インターフェロン療法では自己注射を必要とする事、注射部位の発赤、頭痛、関節痛等の副作用が認められる場合もある。 There is interferon therapy as an effective treatment for multiple sclerosis (Non-patent Document 4). Interferon therapy is used internationally for interferon β1a (IFNβ-1a) and β1b (IFNβ-1b) as the central drugs for improving the long-term prognosis of multiple sclerosis. However, individual differences in the effects of interferon therapy are recognized, and it can not be determined that the effect of suppressing nerve cell damage can not be determined after administration over a long period of at least one year. Interferon therapy may require self-injection and may have side effects such as redness at the injection site, headache, and arthralgia.
本発明はかかる問題点に鑑みてなされたものであって、多発性硬化症を的確に検出でき、且つ、多発性硬化症の発症を予測できる診断マーカーを提供することを目的とする。 The present invention has been made in view of such problems, and it is an object of the present invention to provide a diagnostic marker which can accurately detect multiple sclerosis and can predict the onset of multiple sclerosis.
本発明にかかる多発性硬化症の検出又は発症予測のための診断マーカーは、S100A9タンパク質であることを特徴とする。 The diagnostic marker for detecting or predicting the occurrence of multiple sclerosis according to the present invention is the S100A9 protein.
本発明にかかる多発性硬化症の検出又は発症予測のための診断キットは、検体から単離された生物学的試料から、多発性硬化症を検出又は発症予測するための診断キットであって、S100A9タンパク質に対する特異的結合物質又はS100A9を増幅するプライマーセットを備えることを特徴とする。 A diagnostic kit for detecting or predicting the onset of multiple sclerosis according to the present invention is a diagnostic kit for detecting or predicting the onset of multiple sclerosis from a biological sample isolated from a specimen, It is characterized by comprising a specific binding substance for S100A9 protein or a primer set for amplifying S100A9.
本発明によれば、多発性硬化症を的確に検出でき、また多発性硬化症の発症を予測できる。特に、本発明によれば多発性硬化症の発症を予測できるため発症を遅延させる、治療計画を立てるなどの対策が可能となり、本発明により得られる社会的利益は大きい。 According to the present invention, multiple sclerosis can be accurately detected, and the onset of multiple sclerosis can be predicted. In particular, according to the present invention, since the onset of multiple sclerosis can be predicted, it is possible to take measures such as delaying the onset and making a treatment plan, and the social benefits obtained by the present invention are great.
以下、添付の図面を参照して本発明の実施形態について具体的に説明するが、当該実施形態は本発明の原理の理解を容易にするためのものであり、本発明の範囲は、下記の実施形態に限られるものではなく、当業者が以下の実施形態の構成を適宜置換した他の実施形態も、本発明の範囲に含まれる。 Hereinafter, embodiments of the present invention will be specifically described with reference to the accompanying drawings. However, the embodiments are for facilitating understanding of the principle of the present invention, and the scope of the present invention is as follows. The present invention is not limited to the embodiments, and other embodiments in which those skilled in the art appropriately replace the configurations of the following embodiments are also included in the scope of the present invention.
本発明にかかる多発性硬化症の検出又は発症予測のための診断マーカーは、S100A9タンパク質である。 A diagnostic marker for detecting or predicting the onset of multiple sclerosis according to the present invention is the S100A9 protein.
S100A9タンパク質としては、配列番号１、２または３に記載のアミノ酸配列を含むタンパク質が例示できるが、これらに限定されるものではない。本発明のS100A9タンパク質には、配列番号１、２または３に記載のアミノ酸配列を含むタンパク質と機能的に同等なタンパク質も含まれる。そのようなタンパク質としては、例えば、配列番号１、２または３に記載のアミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、挿入、および／または付加したアミノ酸配列からなり、配列番号１、２または３に記載のアミノ酸配列を含むタンパク質と機能的に同等な単離されたタンパク質が挙げられる。 Examples of the S100A9 protein include, but are not limited to, proteins containing the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3. The S100A9 protein of the present invention includes proteins functionally equivalent to the protein comprising the amino acid sequence set forth in SEQ ID NO: 1, 2, or 3. As such a protein, for example, it consists of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence described in SEQ ID NO: 1, 2 or 3; Alternatively, an isolated protein functionally equivalent to a protein containing the amino acid sequence described in 3 is mentioned.
本発明にかかる多発性硬化症の検出又は発症予測のための診断キットは、検体から単離された生物学的試料から、多発性硬化症を検出又は発症予測するための診断キットであって、S100A9タンパク質に対する特異的結合物質又はS100A9を増幅するプライマーセットを備える。 A diagnostic kit for detecting or predicting the onset of multiple sclerosis according to the present invention is a diagnostic kit for detecting or predicting the onset of multiple sclerosis from a biological sample isolated from a specimen, A specific binding substance for S100A9 protein or a primer set for amplifying S100A9 is provided.
特異的結合物質としては、例えば、抗体、抗体断片、アプタマー等が挙げられる。抗体は、例えば、マウス等の動物にS100A9タンパク質又はその部分ペプチドを抗原として免疫することにより作製することができる。あるいは、ファージライブラリー等の抗体ライブラリーのスクリーニング等により作製することができる。抗体断片としては、Fv、Fab、scFv等が挙げられる。上記の抗体又は抗体断片は、ポリクローナルであってもよく、モノクローナルであってもよい。 Specific binding substances include, for example, antibodies, antibody fragments, aptamers and the like. The antibody can be produced, for example, by immunizing an animal such as a mouse with the S100A9 protein or a partial peptide thereof as an antigen. Alternatively, it can be prepared by screening an antibody library such as a phage library. Examples of antibody fragments include Fv, Fab, scFv and the like. The above antibody or antibody fragment may be polyclonal or monoclonal.
アプタマーとは、標識物質に対する特異的結合能を有する物質である。アプタマーとしては、核酸アプタマー、ペプチドアプタマー等が挙げられる。S100A9タンパク質に特異的結合能を有する核酸アプタマーは、例えば、systematic evolution of ligand by exponential enrichment(SELEX)法等により選別することができる。 An aptamer is a substance having a specific binding ability to a labeling substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers having specific binding ability to S100A9 protein can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method.
特異的結合物質は、S100A9タンパク質に特異的に結合することができれば特に制限されず、市販のものであってもよい。また、特異的結合物質は、担体上に固定されてプロテインチップ等を構成していてもよい。 The specific binding substance is not particularly limited as long as it can specifically bind to the S100A9 protein, and may be commercially available. The specific binding substance may be fixed on a carrier to constitute a protein chip or the like.
S100A9を増幅するプライマーセットとしては、診断の対象とする動物種に由来するS100A9のcDNAを増幅することができるものであれば特に限定されない。例えば、S100A9のcDNAを増幅するプライマーとしては、配列番号４に示す塩基配列からなるフォワードプライマー及び配列番号５に示す塩基配列からなるリバースプライマーのセット等が挙げられる。 The primer set for amplifying S100A9 is not particularly limited as long as it can amplify S100A9 cDNA derived from the animal species to be diagnosed. For example, as a primer for amplifying S100A9 cDNA, a forward primer consisting of the nucleotide sequence shown in SEQ ID NO: 4 and a set of reverse primers consisting of the nucleotide sequence shown in SEQ ID NO: 5 can be mentioned.
本実施形態の診断キットを用いた診断は、例えば次のようにして行うことができる。 Diagnosis using the diagnosis kit of the present embodiment can be performed, for example, as follows.
診断キットがS100A9タンパク質に対する特異的結合物質を用いるものである場合、まず、患者から採取された生検試料及び対照となる正常組織を試料として、上記の特異的結合物質を用いた組織染色、ELISA、ウエスタンブロッティング、フローサイトメトリー等を行うことにより、生検試料及び正常組織中のS100A9タンパク質の発現量を測定する。 When the diagnostic kit uses a specific binding substance for S100A9 protein, first, a biopsy sample collected from a patient and a normal tissue as a control are used as a sample, tissue staining using the above specific binding substance, ELISA By performing Western blotting, flow cytometry, etc., the expression level of S100A9 protein in a biopsy sample and normal tissue is measured.
続いて、生検試料中のS100A9タンパク質の発現量が、正常組織中のS100A9タンパク質の発現量と比較して高い場合には、多発性硬化症を発症していると診断できるか、又は、発症の可能性が高いと診断できる。 Subsequently, when the expression level of S100A9 protein in the biopsy sample is higher than the expression level of S100A9 protein in normal tissue, it can be diagnosed as having developed multiple sclerosis or Can be diagnosed as having a high probability.
または、正常組織中のS100A9タンパク質の発現量を予め測定して基準値を設定しておき、生検試料中のS100A9タンパク質の発現量が上記基準値と比較して高い場合には、多発性硬化症を発症していると診断できるか、又は、発症の可能性が高いと診断できる。 Alternatively, if the expression level of S100A9 protein in normal tissue is measured in advance and a reference value is set, and the expression level of S100A9 protein in the biopsy sample is higher than the reference value, multiple sclerosis Can be diagnosed as having developed symptoms or can be diagnosed as having a high possibility of developing.
診断キットがS100A9遺伝子を増幅するプライマーセットを用いるものである場合、まず、患者から採取された生検試料及び対照となる正常組織を試料として、定量的RT-PCR等を行うことにより、生検試料及び正常組織中のS100A9のmRNAの発現量を測定する。 When the diagnostic kit uses a primer set that amplifies the S100A9 gene, a biopsy sample is first obtained by performing quantitative RT-PCR etc. using a biopsy sample collected from a patient and a normal tissue as a control as a sample. The expression level of S100A9 mRNA in the sample and normal tissue is measured.
続いて、生検試料中のS100A9のmRNAの発現量が、正常組織中のS100A9のｍＲＮＡの発現量と比較して高い場合には、多発性硬化症を発症していると診断できるか、又は、発症の可能性が高いと診断できる。 Subsequently, if the expression level of S100A9 mRNA in the biopsy sample is high compared to the expression level of S100A9 mRNA in normal tissue, it can be diagnosed as having multiple sclerosis, or It can be diagnosed that there is a high possibility of onset.
あるいは、正常組織中のS100A9のmRNAの発現量を予め測定して基準値を設定しておき、生検試料中のS100A9のmRNAの発現量が前記基準値と比較して高い場合には、多発性硬化症を発症していると診断できるか、又は、発症の可能性が高いと診断できる。 Alternatively, if the expression level of S100A9 mRNA in normal tissue is measured in advance and a reference value is set, and the expression level of S100A9 mRNA in the biopsy sample is higher than the reference value, It can be diagnosed as having developed sclerosis, or can be diagnosed as having a high possibility of onset.
１．EAE誘導
SJL/Jマウス(雌性，10週齢)に、Proteolipid proteinの139-151番目のアミノ酸配列(配列番号６：HCLGKWLGHPDKF,His Cys Leu GlyLys Trp Leu Gly His Pro Asp Lys Phe)に該当するペプチド(PLP139-151)を、完全フロイントアジュバントと共に免疫し、EAEを誘導した。なお、実験的アレルギー性脳脊髄炎(EAE：Experimental autoimmune encephalomyelitis)は、中枢神経組織由来の蛋白質抗原やペプチドを免疫することによって誘導される自己免疫モデルである。多発性硬化症(MS)と多くの病態を共有することから、MSの病態研究、治療法開発において使用されている。定法に従い、臨床症状を6段階で毎日評価した。
1. EAE guidance
In SJL / J mice (female, 10 weeks old), a peptide (PLP 139 ) corresponding to amino acid sequence 139-151 of Proteolipid protein (SEQ ID NO: 6: HCLGKWLGHPDKF, His Cys Leu GlyLys Trp Leu Gly His Pro Asp Lys Phe) -151 ) was immunized with complete Freund's adjuvant to induce EAE. Experimental allergic encephalomyelitis (EAE) is an autoimmune model induced by immunizing protein antigens and peptides derived from central nervous tissues. Since it shares many pathologies with multiple sclerosis (MS), it is used in pathological research and development of therapeutic methods for MS. Clinical symptoms were evaluated daily in six stages according to a standard method.
ペプチド免疫後、9日目(pre-EAE)、12日目(acute-EAE)、21日目(chronic-EAE)にマウスを解剖し、中枢神経系(Central nervous system; CNS)試料を採取した。図１は、controlマウスとacute-EAEマウスとにおいて、小脳白質、延髄、海馬采と視床の間の軟膜領域におけるヘマトキシリン・エオシン(HE)染色図(a,b)と、抗S100A9抗体を用いた免疫組織化学(Immunohistochemistry; IHC)による解析図(c,d)と、イメージング質量分析(Imaging mass spectrometry; IMS)による解析図(e,f)とである。図１において、a,b,c,dのスケールバーは5 mmであり、e,fのスケールバーは1 mmである。 After peptide immunization, mice were dissected on day 9 (pre-EAE), day 12 (acute-EAE), day 21 (chronic-EAE), and central nervous system (CNS) samples were collected. . FIG. 1 shows the immunity using hematoxylin and eosin (HE) staining (a, b) and anti-S100A9 antibody in the cerebellar white matter, medulla oblongata, and the buffy coat region between the hippocampal fistula and thalamus in control mice and acute-EAE mice. It is the analysis figure (c, d) by histochemistry (Immunohistochemistry; IHC), and the analysis figure (e, f) by imaging mass spectrometry (IMS). In FIG. 1, the scale bars of a, b, c, d are 5 mm and the scale bars of e, f are 1 mm.
２．イメージング質量分析(Imaging mass spectrometry; IMS)
acute-EAEマウス脳から、クリオスタットで10 μm厚の新鮮凍結切片を作製した。組織切片を、エタノール、カルノア液で洗浄後、マトリックスとしてシナピン酸を噴霧した。質量分析装置は、UltrafleXtreme(Bruker Daltonics)を用いた。UltrafleXtremeは、最高性能のMALDI-TOF&TOF/TOFシステムであり、TOF/TOFモードで2 kHzの解析が可能である。データ解析は、fleximaging(Bruker Daltonics)ソフトウェアを用いて、EAEマウス組織に特異的なm/z 値を検索し、描出した。
2. Imaging mass spectrometry (IMS)
Fresh frozen sections of 10 μm thickness were prepared from acute-EAE mouse brain on a cryostat. The tissue sections were washed with ethanol and Carnoy's solution and sprayed with sinapinic acid as a matrix. The mass spectrometer used UltrafleXtreme (Bruker Daltonics). UltrafleXtreme is the highest performance MALDI-TOF & TOF / TOF system, capable of 2 kHz analysis in TOF / TOF mode. Data analysis was performed using fleximaging (Bruker Daltonics) software to retrieve and depict m / z values specific to EAE mouse tissues.
IMSにより、EAEマウス脳に特異的に検出されるm/z 12,971を見出した(図1f黒矢印)。この分子群は健常マウス脳には見られない(図1e)。m/z 12,917を調べたところ、S100A9タンパク質であることが判明した(Caldwell et al, Wound Repair Reagen 16(3):442-449 (2008))。S100A9タンパク質は、EAE病巣の進行と深く関わっており、多発性硬化症のバイオマーカーであることが判明した。 IMSs found m / z 12,971 specifically detected in EAE mouse brain (FIG. 1f, black arrow). This group of molecules is not found in the healthy mouse brain (FIG. 1e). The m / z 12,917 was examined and found to be the S100A9 protein (Caldwell et al, Wound Repair Reagen 16 (3): 442-449 (2008)). The S100A9 protein is closely associated with the progression of EAE foci and has been found to be a biomarker for multiple sclerosis.
３．免疫組織化学(Immunohistochemistry; IHC)
新鮮凍結切片を4%パラホルムアルデヒド含有リン酸緩衝生理食塩水にて固定を行った。0.3%過酸化水素含有メタノールで内在性Peroxidaseを不活性化後、10%ヤギ血清でブロッキングを行った。一次抗体として、抗S100A9抗体、二次抗体として、ビオチン標識抗ラットIgG抗体を用いた。アビジン-ビオチン複合体を形成させ、3,3’-diaminobenzidine(DAB)法により検出を行った。
3. Immunohistochemistry (IHC)
Fresh frozen sections were fixed with phosphate buffered saline containing 4% paraformaldehyde. After inactivating endogenous peroxidase with methanol containing 0.3% hydrogen peroxide, blocking was performed with 10% goat serum. Anti-S100A9 antibody was used as a primary antibody, and biotin-labeled anti-rat IgG antibody was used as a secondary antibody. The avidin-biotin complex was formed and detection was performed by the 3,3'-diaminobenzidine (DAB) method.
ヘマトキシリン・エオシン(HE)染色により、acute-EAEマウス脳の小脳白質、延髄、海馬采と視床の間の軟膜領域において異所性細胞集塊(浸潤細胞群)を認めた(図.b矢印)。この細胞集塊は健常マウス脳には見られない(図1a)。抗S100A9抗体を用いたIHCによる検証の結果、S100A9タンパク質の陽性反応はm/z 12,971と一致した(図.d,f実線矢柱)。本所見は、浸潤細胞群と共局在した。さらに、小脳分子層においても、m/z 12,971及びS100A9タンパク質陽性領域を認めた(図d,f点線矢柱)。 Hematoxylin and eosin (HE) staining revealed ectopic cell clumps (infiltrating cell groups) in the cerebellar white matter, medulla oblongata, and buffy coat area between hippocampal fistula and thalamus of acute-EAE mouse brain (Fig. B arrow). This cell cluster is not found in the healthy mouse brain (FIG. 1a). As a result of IHC verification using an anti-S100A9 antibody, the positive reaction of S100A9 protein was consistent with m / z 12,971 (Fig. D, f solid arrows). This finding colocalized with the infiltrating cell population. Furthermore, m / z 12,971 and S100A9 protein positive regions were also observed in the cerebellar molecular layer (Fig. D, f dotted arrowheads).
４．発症前から慢性期までの解析
図２は、controlマウスとacute-EAEマウスとにおいて脳切片のIMS法による解析結果を示す図であり、そのうち(a)はcontrolであり、(b)はpre-EAEマウス脳であり、(c)はacute-EAEマウス脳であり、(d)はchronic-EAEマウス脳である。UltrafleXtreme(Bruker Daltonics)を用いた質量分析による結果、IMSにより、EAEマウス脳に特異的に検出されるm/z 12,971は、acute-EAEマウス脳のみならず、chronic-EAEマウス脳においても、更には、pre-EAEマウス脳においても認められた。
4. Analysis from pre-onset to chronic phase Figure 2 shows the results of analysis of brain sections by IMS method in control mice and acute-EAE mice, wherein (a) is control and (b) is pre- EAE mouse brain, (c) is an acute-EAE mouse brain, and (d) is a chronic-EAE mouse brain. As a result of mass spectrometry using UltrafleXtreme (Bruker Daltonics), m / z 12,971 specifically detected in EAE mouse brain by IMS is further detected not only in acute-EAE mouse brain but also in chronic-EAE mouse brain Was also observed in the pre-EAE mouse brain.
図３は、controlマウスとacute-EAEマウスとにおいて、小脳白質、延髄、海馬采と視床の間の軟膜領域におけるHE染色図であり、そのうち(a)はcontrolであり、(b)はpre-EAEマウス脳であり、(c)はacute-EAEマウス脳であり、(d)はchronic-EAEマウス脳である。免疫組織化学分析による結果、EAEマウス脳に特異的に検出されるS100A9タンパク質は、acute-EAEマウス脳のみならず、chronic-EAEマウス脳においても、更には、pre-EAEマウス脳においても認められた。 FIG. 3 is a HE staining diagram of cerebellar white matter, medulla oblongata, and pia mater region between hippocampal fistula and thalamus in control mice and acute-EAE mice, wherein (a) is control and (b) is pre-EAE. (C) is an acute-EAE mouse brain, (d) is a chronic-EAE mouse brain. As a result of immunohistochemical analysis, S100A9 protein specifically detected in EAE mouse brain is found not only in acute-EAE mouse brain but also in chronic-EAE mouse brain and also in pre-EAE mouse brain The
これらの結果から、S100A9タンパク質は、多発性硬化症の発症前においても認められ、発症予測のための診断マーカーとしても利用できることが判明した。 From these results, it was found that the S100A9 protein was recognized even before the onset of multiple sclerosis and can also be used as a diagnostic marker for predicting the onset.
５．S100A9タンパク質の特異的阻害剤タスキニモド投与による治療効果
SJL/Jマウス(雌性，10週齢)にペプチド(PLP139-151)を完全フロイントアジュバントと共に免疫し、EAEを誘導したEAEマウスに、ペプチド免疫後、S100A9タンパク質の特異的阻害剤としてタスキニモドを2日に1回、経口投与した。Controlにはタスキニモドを投与しなかった。
5. Therapeutic effect of S100A9 protein specific inhibitor Taskinimod
After immunizing SJL / J mice (female, 10 weeks old) with peptide (PLP 139-151 ) with complete Freund's adjuvant and inducing EAE in EAE mice induced with EAE, tasquinimod 2 as a specific inhibitor of S100A9 protein after peptide immunization It was orally administered once a day. Taskinimod was not administered to Control.
EAEの臨床スコアは、０：正常、１：尾の完全下垂、２：歩行困難、３：後肢の完全脱力、４：体幹部の麻痺、５：前肢麻痺を含む後肢の完全脱力、６：死亡にて評価した。臨床症状は、ペプチド免疫後９〜14日程度から明らかとなり、その後数日で最大スコアに達する。 The clinical score of EAE is 0: normal, 1: full fall of tail, 2: walking difficulty, 3: full weakness of hindlimb, 4: paralysis of trunk, 5: complete weakness of hindlimb including paralysis of forelimbs, 6: death It evaluated by. Clinical symptoms become apparent from about 9 to 14 days after peptide immunization, and reach a maximum score several days thereafter.
図４は、controlマウスとS100A9タンパク質の特異的阻害剤投与マウスとにおいて、EAEの臨床スコアを示す解析図である。図４から、S100A9タンパク質の特異的阻害剤は、多発性硬化症の発症を遅らせることが認められる。また、S100A9タンパク質の特異的阻害剤は、多発性硬化症の治療に十分な効果があることが認められる。なお、図４において、経過日数０日〜８日は臨床症状が現れておらず、図面の煩雑さを裂けるために、day21(非投与群)のみ記載し、その他の記号は省略されている。また経過日数９日目ではday9(非投与群)、day9(投与群)及びday12(非投与群)の記号が省略されているが、これは臨床スコアが０に近いものであったからである。 FIG. 4 is an analysis diagram showing clinical scores of EAE in control mice and mice administered with a specific inhibitor of S100A9 protein. FIG. 4 shows that a specific inhibitor of S100A9 protein delays the onset of multiple sclerosis. Moreover, it is recognized that the specific inhibitor of S100A9 protein has a sufficient effect for the treatment of multiple sclerosis. In FIG. 4, clinical symptoms do not appear on the lapsed days 0 to 8, and only day 21 (non-administration group) is shown and other symbols are omitted in order to break up the complexity of the drawing. On the 9th day, the symbols day9 (non-administered group), day9 (administered group), and day12 (non-administered group) are omitted because the clinical score was close to zero.
多発性硬化症の診断に利用できる。 It can be used to diagnose multiple sclerosis.
配列番号４，５：プライマー
配列番号６：ペプチド
SEQ ID NO: 4, 5: Primer SEQ ID NO: 6: Peptide
Claims (3)
S100A9タンパク質に対する特異的結合物質又はS100A9を増幅するプライマーセットを備える、多発性硬化症の検出又は発症予測のための診断キット。 A diagnostic kit for detecting or predicting the onset of multiple sclerosis from a biological sample isolated from a sample, comprising:
A diagnostic kit for detecting or predicting multiple sclerosis, comprising a specific binding substance for S100A9 protein or a primer set for amplifying S100A9.
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