JP5757624B2 - Screening method for antiallergic factors - Google Patents
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本発明は、抗アレルギー因子のスクリーニング方法に関し、特に生体内の抗体生産細胞が生産するIgE抗体の生産を抑制する物質をスクリーニングする抗アレルギー因子のスクリーニング方法に関する。 The present invention relates to a screening method for antiallergic factors, and more particularly to a screening method for antiallergic factors that screens substances that suppress the production of IgE antibodies produced by antibody-producing cells in vivo.
近年、都市型疾患である花粉症やハウスダストによるアレルギー患者数は急速に増大している。特に花粉症は全国民の15〜20%が患者であるとされている。さらに将来的に、この数字は増えるとも言われており、深刻化している状況である。一方で、花粉症の症状は、鼻水や咳、目の痒みなど生活上に多大な影響を与える疾患であるにも関わらず、致命的ではないため、多くは本人の忍耐に依存し、また、病院でも様々な治療法が試されているものの、決め手となる治療法はなく、症状が激しい場合にステロイド系の抗炎症剤による緩和治療を行っている現状である。 In recent years, the number of allergic patients due to hay fever and house dust, which are urban diseases, has been rapidly increasing. In particular, hay fever is said to be 15 to 20% of the national population. Furthermore, it is said that this number will increase in the future, and it is getting worse. On the other hand, the symptoms of hay fever are diseases that have a great impact on life such as runny nose, cough, and itchy eyes, but they are not fatal, so many depend on their patience, Although various treatments have been tried in hospitals, there is no decisive treatment, and palliative treatment with steroidal anti-inflammatory drugs is being performed when symptoms are severe.
この強い炎症抑制作用をもつステロイド系の抗炎症剤は、ステロイドが生体内のホルモンの一部であるため体内では炎症抑制作用だけでなくホルモンとしての生理作用を有しているため、強い副作用も指摘されている。そのため、医師の厳密な管理の下で使用することが重要であり、また、患者はそうした副作用の影響を懸念する意見も多い。なお、機能性食品は医薬のように疾病治癒を目指すものではないが、日常の食生活に取り入れることで、手軽に疾病予防や疾病の症状緩和をもたらすため、患者の生活の質を改善することが可能になる手段として大きな期待が寄せられている。 This strong anti-inflammatory steroid-based anti-inflammatory agent has not only an anti-inflammatory action in the body but also a physiological action as a hormone because steroids are part of the hormone in the body, so there are also strong side effects. It has been pointed out. For this reason, it is important to use it under the strict supervision of a doctor, and many patients are concerned about the effects of such side effects. Functional foods do not aim to cure diseases like pharmaceuticals, but by incorporating them into daily eating habits, it is easy to prevent diseases and alleviate disease symptoms, so improve the quality of life for patients. There are great expectations as a means to make this possible.
ところで、花粉症やアトピー性皮膚炎などのアレルギー疾患は医学的にはI型アレルギー又は即時型アレルギーと呼ばれており、多くの研究者が発症メカニズムを研究している。今までに解明された知見によれば、以下のことが分かっている。まず、呼吸によって吸入されたアレルゲンである花粉は、体内の免疫反応としてIgE型の抗体と結合する。さらに、花粉とIgE抗体の結合物がマスト細胞(肥満細胞)と結合する。このことにより、このマスト細胞からヒスタミンやロイコトリエンという化学物質が放出され、これらの物質が鼻や目、気道の炎症を発生させる。つまり、花粉の体内侵入に対して、IgE型の抗体がアレルギーの引き金物質となっている。したがって、IgE型抗体の生体内における減少がアレルギー症状の抑制方法のひとつとなる。 By the way, allergic diseases such as hay fever and atopic dermatitis are medically called type I allergy or immediate allergy, and many researchers are studying the onset mechanism. According to the knowledge clarified so far, the following is known. First, pollen, which is an allergen inhaled by respiration, binds to an IgE-type antibody as an immune response in the body. Furthermore, the combined product of pollen and IgE antibody binds to mast cells (mast cells). As a result, chemical substances such as histamine and leukotriene are released from the mast cells, and these substances cause inflammation of the nose, eyes and airways. In other words, IgE type antibodies are the trigger for allergies against pollen invasion. Therefore, a decrease in IgE antibodies in vivo is one method for suppressing allergic symptoms.
また、本願発明者達は、ヒトのアレルギーの体内動態をモデル化したヒト末梢血リンパ球細胞による培養系を独自に構築し、食品成分中から抗アレルギー効果を示す成分の特定を行ってきた。この技術は、具体的には、アレルギーの引き金マーカー物質であるIgE抗体を生産するヒト血液細胞を培養するアレルギー発症モデル検出系の技術であり、血液細胞の培養液中に被検物質が添加されてIgE抗体生産量の減少が測定されて行われる。このような技術であれば、従来、マウス等の実験動物を大量に用いて検査する手法と異なり、10日前後の培養日数で数百種の検体を同時に検査できるため、極めて効率が高い。さらに、ヒト細胞を用いることから、探索した因子中の効果のある成分が見いだされた場合、生体でも効果を発揮する可能性が高く、探索に必要な時間的な短縮も可能である。また、探索した因子と細胞の相互作用を検討することで、因子の作用メカニズムを細胞レベルで解明できるため、食品機能の科学的な評価が可能であるなどの多くの優れた利点を有する(非特許文献1)。 In addition, the inventors of the present application have independently constructed a culture system using human peripheral blood lymphocyte cells that models the pharmacokinetics of human allergies, and have identified components exhibiting antiallergic effects from food components. Specifically, this technique is an allergy onset model detection system technique for culturing human blood cells that produce IgE antibody, which is an allergy trigger marker substance, and a test substance is added to the culture solution of blood cells. A decrease in IgE antibody production is measured and performed. Such a technique is extremely efficient because, unlike the conventional method of testing using a large amount of laboratory animals such as mice, hundreds of specimens can be tested simultaneously in about 10 days of culture. Furthermore, since a human cell is used, if an effective component in the searched factor is found, it is highly possible that the effect is exhibited in the living body, and the time required for searching can be shortened. In addition, by examining the interaction between the searched factor and the cell, it is possible to elucidate the action mechanism of the factor at the cellular level, and thus has many excellent advantages such as enabling scientific evaluation of food functions (non- Patent Document 1).
しかしながら、前述の細胞を用いるアレルギー発症モデル検出系は、生体から分離した免疫に関わる血液細胞を利用することから、検定の都度、健常人のドナー(提供者)から同意を得て血液細胞を提供してもらう必要があった。そのため、結果として検定に使用できる細胞数に限りがあるという問題があった。また、細胞の提供者による個人差が最終的な検定の結果に影響を与え、試験結果として得られたデータの再現が困難であるなどの問題があった。 However, since the allergic onset model detection system using cells described above uses blood cells related to immunity separated from the living body, blood cells are provided with the consent of healthy donors (providers) at each test. It was necessary to have you. As a result, there is a problem that the number of cells that can be used for the test is limited. In addition, there are problems such as individual differences among cell providers affecting the final test results, making it difficult to reproduce the data obtained as test results.
ゆえに、本発明は、利用できる細胞数も簡単に確保でき、かつ、個人差などの影響を受けない被検物質の抗アレルギー効果の再現性も期待できることを可能にする抗アレルギー因子のスクリーニング方法を提供することを目的とする。 Therefore, the present invention provides a screening method for an antiallergic factor that makes it possible to easily ensure the number of cells that can be used and to expect the reproducibility of the antiallergic effect of a test substance that is not affected by individual differences. The purpose is to provide.
本願発明者達は、血液由来の初代細胞を用いた抗アレルギースクリーニング系としてのアレルギー発症モデル検出系のメカニズムを詳細に検討し続けてきた。提供された血液細胞により、IgE抗体を生産するBリンパ球や免疫制御作用を有するTリンパ球などの混合細胞培養が実施された。この混合細胞培養において、Bリンパ球のIgE抗体の生産に伴って、Tリンパ球の細胞膜上にNOV遺伝子由来のタンパク質が発現していることが分かった。NOV遺伝子由来タンパク質は、細胞内で細胞活動の情報伝達物質であり、細胞の増殖信号および細胞の接着に関わる信号(シグナル伝達物質)に関与しているとされている。しかしながら、その働きは未だ研究中であり、報告はほとんどない。本願発明者達は、被検物質の抗アレルギー効果(抗アレルギー因子)に伴い、NOV由来タンパク質の発現量が変化することを見いだした。本願発明者達は、アレルギー発症モデル検出系の細胞群に、発症のない細胞群と比較してNOV遺伝子発現が5倍以上増大することを発見した。 The inventors of the present application have continued to examine in detail the mechanism of the allergy onset model detection system as an anti-allergy screening system using blood-derived primary cells. Using the provided blood cells, mixed cell culture of B lymphocytes producing IgE antibodies and T lymphocytes having an immunoregulatory effect was performed. In this mixed cell culture, it was found that a protein derived from the NOV gene was expressed on the cell membrane of T lymphocytes along with the production of IgE antibody of B lymphocytes. The NOV gene-derived protein is a cell activity information transmitter in the cell, and is considered to be involved in a cell proliferation signal and a signal related to cell adhesion (signal transmitter). However, its work is still under study and there are few reports. The inventors of the present application have found that the expression level of the NOV-derived protein changes with the antiallergic effect (antiallergic factor) of the test substance. The inventors of the present application have found that the expression of NOV gene is increased 5 times or more in the cell group of the allergy onset model detection system as compared with the cell group without the onset.
すなわち、本願発明者達は、Tリンパ球の細胞膜上のNOV由来のタンパク質の発現量が減少すると、それに伴いアレルギーの引き金物質であるIgE抗体の生産量が抑制されたことを見いだした。さらに、本願発明者達は、このNOV由来タンパク質が、抗アレルギー物質を被検物質にすると、被検物質に特異的に結合する性質を有することを併せて見いだした。言い換えれば、IgE抗体の生産量を抑制して、抗アレルギー効果を発揮する因子の探索には、IgE抗体量を調べる方法だけでなく、以下のスクリーニングを行えばよいことが判明した。 That is, the inventors of the present application have found that when the expression level of NOV-derived protein on the cell membrane of T lymphocytes decreases, the production amount of IgE antibody, which is an allergic trigger substance, is suppressed accordingly. Furthermore, the inventors of the present application have found that this NOV-derived protein has a property of specifically binding to a test substance when an antiallergic substance is used as the test substance. In other words, it has been found that not only the method of examining the amount of IgE antibody but also the following screening may be performed to search for a factor that exerts an antiallergic effect by suppressing the production amount of IgE antibody.
したがって、本発明の第一の観点は、生体内の抗体生産細胞が生産するIgE抗体の生産を抑制する物質をスクリーニングする抗アレルギー因子のスクリーニング方法であって、細胞株に発現するNOV由来タンパク質との関係により、IgE抗体の生産を抑制することによる抗アレルギー効果を発揮する被検物質をスクリーニングする判定ステップを含むものである。 Therefore, a first aspect of the present invention is a screening method for an antiallergic factor for screening a substance that suppresses the production of IgE antibodies produced by antibody-producing cells in vivo, comprising a NOV-derived protein expressed in a cell line and Therefore, the method includes a determination step of screening a test substance that exhibits an antiallergic effect by suppressing the production of IgE antibody.
具体的には、IgE抗体の産生を抑制する成分を探索するためのアッセイ方法であって、NOV由来タンパク質をプローブ(探索針)として、このNOV由来タンパク質と結合する被検物質が抗アレルギー物質であることを特徴とするスクリーニング手法である。抗アレルギー物質としてスクリーニングするものとして成功した具体例には、例えばイチゴ由来のグリセロアルデヒド3リン酸脱水素酵素というタンパク質が挙げられる。本願発明者達は、タンパク質性の抗アレルギー物質は検知可能だと判断している。そして、理論的には、NOVタンパク質と結合すればよいので、分子量が100以上あるような物質であれば、基本的にはスクリーニング可能と判断している。なお、本願発明者によって、タンパク質ではないが、現時点では、お茶の抗アレルギー物質と言われているカテキン類も、本スクリーニング系で調べることができることが分かっている。 Specifically, it is an assay method for searching for a component that suppresses the production of IgE antibody, and a test substance that binds to this NOV-derived protein is an antiallergic substance using the NOV-derived protein as a probe (search needle). It is a screening technique characterized by being. Specific examples of successful screening as antiallergic substances include, for example, a protein called glyceraldehyde 3-phosphate dehydrogenase derived from strawberries. The inventors of the present application have determined that protein antiallergic substances can be detected. Theoretically, since it only needs to bind to the NOV protein, it is basically determined that a substance having a molecular weight of 100 or more can be screened. In addition, although it is not protein by this inventor, it turns out that catechins currently said to be an antiallergic substance of tea can also be investigated by this screening system.
本発明の第二の観点は、抗アレルギー効果のある物質をスクリーニングする具体的な新規方法として、試薬であるNOVタンパク質と直接結合する能力の高い物質が該当物質であるとする方法である。すなわち、判定ステップにおいて、NOV由来タンパク質との結合親和性を比較して、高い被検物質をスクリーニングするものである。発明者らは、細胞株が、ヒトTリンパ球性白血病細胞株であり、NOV由来タンパク質が、遺伝子NOV由来のタンパク質である場合について、実験により具体的に示した。 The second aspect of the present invention is a method in which a substance having a high ability of directly binding to a reagent, NOV protein, is a relevant substance as a specific novel method for screening a substance having an antiallergic effect. That is, in the determination step, a high test substance is screened by comparing the binding affinity with the NOV-derived protein. The inventors specifically showed by experiments that the cell line is a human T lymphocytic leukemia cell line and the NOV-derived protein is a protein derived from the gene NOV.
本発明の第三の観点は、もう一つのスクリーニング新規方法として、NOVタンパク質が細胞膜上に発現しているTリンパ球を利用して、このTリンパ球の細胞培養液中に被検物質を添加した際、この細胞膜上のNOVタンパク質発現量が減少すれば、被検物質は抗アレルギー物質であるとする方法である。すなわち、判定ステップにおいて、前記細胞株の培養液中に前記被検物質を添加した場合に、前記細胞株が発現するNOV由来タンパク質の発現量を抑制する被検物質をスクリーニングするものである。発明者らは、細胞株が、ヒトT細胞性白血病細胞株Molt−4である場合について、実験により具体的に示した。Molt−4は無限増殖能を持つ細胞株であるため、継代培養することが可能になり、培養環境を一定に保つことができる。このことから、結果として、利用できる細胞数も簡単に安定して確保でき、かつ、個人差などの影響を受けない被検物質の抗アレルギー効果の再現性も期待できる。 The third aspect of the present invention is that, as another new screening method, a test substance is added to a cell culture solution of T lymphocytes using T lymphocytes in which NOV protein is expressed on the cell membrane. In this case, if the expression level of NOV protein on the cell membrane decreases, the test substance is an antiallergic substance. That is, in the determination step, when the test substance is added to the culture solution of the cell line, a test substance that suppresses the expression level of the NOV-derived protein expressed by the cell line is screened. The inventors specifically showed by experiments that the cell line was the human T-cell leukemia cell line Molt-4. Since Molt-4 is a cell line with infinite growth ability, it can be subcultured and the culture environment can be kept constant. As a result, the number of cells that can be used can be easily and stably secured, and reproducibility of the antiallergic effect of the test substance that is not affected by individual differences can be expected.
本願発明で使用しうる被検物質は、食品素材、食品の組成物、化学物質等である。これらの調製は、水溶性の物質の場合、水や生理食塩水、培養培地などを用いて行われ、非水溶性の場合、エタノールやメタノール、ジメチルスルホキシドなどの溶媒を用いて溶解させて行わればよい。 The test substances that can be used in the present invention are food materials, food compositions, chemical substances, and the like. In the case of water-soluble substances, these preparations are performed using water, physiological saline, culture medium, etc., and in the case of water-insoluble substances, they are dissolved by using a solvent such as ethanol, methanol, dimethyl sulfoxide or the like. That's fine.
本願の発明によれば、今までに無い抗アレルギー因子のスクリーニング方法が得られる。そして、スクリーニングに利用できる細胞数も簡単に確保でき、かつ、個人差などの影響を受けない被検物質の抗アレルギー効果の再現性も期待できることを可能にする抗アレルギー因子のスクリーニング方法が得られる。具体的には、細胞株による再現性の高い抗アレルギー因子の探索が可能になり、さらに、生化学的にNOVタンパク質と被検物質との結合能を調べることにより、極めて迅速且つ簡便に抗アレルギー物質をスクリーニングできることも可能になった。 According to the present invention, an unprecedented screening method for antiallergic factors can be obtained. In addition, it is possible to obtain a screening method for anti-allergic factors that makes it possible to easily secure the number of cells that can be used for screening and to expect the reproducibility of the anti-allergic effects of test substances that are not affected by individual differences. . Specifically, it becomes possible to search for a highly reproducible anti-allergic factor by cell lines, and furthermore, by examining the binding ability of NOV protein and a test substance biochemically, the anti-allergy can be extremely quickly and easily performed. It has become possible to screen substances.
本発明は、IgE生産抑制効果によって抗アレルギー能を有する物質のアッセイ方法に関する。より具体的には、本発明では、抗体生産細胞のIgE生産量の抑制に連動して作用するNOV由来タンパク質が被検物質と結合するため、その結合能を、例えば生体分子間相互作用装置などで検出する。このことにより、その結合親和性が高い物質をIgE産生抑制物質、ひいては、抗アレルギー物質として評価可能なアッセイ方法が実現できる。これは、NOV由来タンパク質をプローブとして生体分子間相互作用装置であるBIACOREセンサーチップCM5に固定化し、このチップに被検物質を作用させ、その結合を検出することで実現する。ここで、NOV由来タンパク質を細胞膜上に発現するヒトT細胞性白血病細胞株Molt−4がある。このヒトT細胞性白血病細胞株Molt−4の培養は、一般的な動物細胞培養方法で用いる牛胎児血清を増殖因子として10%程度含むRPMI1640培地などの基本合成培地を用いることにより可能である。なお、培養培地はこれに限らず、動物細胞培養培地であれば、市販の無血清培地なども利用できる。 The present invention relates to a method for assaying a substance having antiallergic activity due to an IgE production inhibitory effect. More specifically, in the present invention, a NOV-derived protein that works in conjunction with suppression of the IgE production amount of antibody-producing cells binds to a test substance. Detect with. This makes it possible to realize an assay method in which a substance having a high binding affinity can be evaluated as an IgE production inhibitor, and thus an antiallergic substance. This is realized by immobilizing the NOV-derived protein as a probe on a BIACORE sensor chip CM5, which is a biomolecular interaction device, allowing a test substance to act on this chip and detecting its binding. Here, there is a human T-cell leukemia cell line Molt-4 that expresses NOV-derived protein on the cell membrane. This human T cell leukemia cell line Molt-4 can be cultured by using a basic synthetic medium such as RPMI 1640 medium containing about 10% of fetal bovine serum used as a growth factor in a general animal cell culture method. The culture medium is not limited to this, and a commercially available serum-free medium can be used as long as it is an animal cell culture medium.
このMolt−4細胞の培養では、適切な細胞密度に調製した細胞懸濁液に、被検物質を培養液中に5%以内の終濃度になるよう添加することが行われる。数日間培養後、Molt−4細胞を回収して、細胞膜上のNOVタンパク質に結合する特異抗体を利用して蛍光色素でマーキングし、その蛍光量の増減を数値化して、蛍光量が減少したものを抗アレルギー物質とする。 In the cultivation of Molt-4 cells, a test substance is added to a cell suspension adjusted to an appropriate cell density so as to have a final concentration of 5% or less in the culture solution. After culturing for several days, Molt-4 cells are collected, marked with a fluorescent dye using a specific antibody that binds to the NOV protein on the cell membrane, and the increase or decrease in the amount of fluorescence is quantified, and the amount of fluorescence decreases Is an antiallergic substance.
なお、NOVタンパク質と被検試料との結合能の評価は、生体分子間相互作用装置を使用しているが、必ずしもこれに限らない。すなわち、タンパク質同士の結合の評価には、酵素抗体法やウェスタンブロッティング法などの生化学的評価法があり、これを利用することも可能である。 In addition, although evaluation of the binding ability of NOV protein and a test sample uses the biomolecule interaction apparatus, it does not necessarily restrict to this. That is, there is a biochemical evaluation method such as an enzyme antibody method or a Western blotting method for evaluating the binding between proteins, which can be used.
以下に本発明の実施例を例示するが、本発明は以下の実施例に限定されるものではない。 Examples of the present invention are illustrated below, but the present invention is not limited to the following examples.
[リンパ球の分離]
ヒト末梢リンパ球を取得するために、健常者の末梢血を密度勾配遠心法により血液分離した。健常人から末梢血をヘパリン入り真空管に採集し、あらかじめ15ml遠心管に分注しておいた4mlの血液分離剤(Ficoll;GE Healthcare)の上に液面を乱さないように5mlの末梢血を重層し、400×g、30minの条件で遠心した。そして、最上層の血漿の層を取得し、−24℃で保存した。次に、血漿層とFicoll層の間にあるリンパ球を回収後、基本合成培地ERDF(Kyokuto Seiyaku)で洗浄し、400×g、5minの条件で遠心、洗浄を3回繰り返した。取得したリンパ球は、−85℃で凍結保存し、解凍後はリンパ球をERDF培地で洗浄して使用した。
[Separation of lymphocytes]
In order to obtain human peripheral lymphocytes, the peripheral blood of healthy subjects was separated by density gradient centrifugation. Peripheral blood is collected from a healthy person in a vacuum tube containing heparin, and 5 ml of peripheral blood is placed on a 4 ml blood separation agent (Ficoll; GE Healthcare) previously dispensed into a 15 ml centrifuge tube so as not to disturb the liquid level. The layers were layered and centrifuged at 400 × g for 30 minutes. The uppermost plasma layer was obtained and stored at -24 ° C. Next, the lymphocytes between the plasma layer and the Ficoll layer were collected, washed with a basic synthetic medium ERDF (Kyokuto Seiyaku), and centrifuged and washed three times under conditions of 400 × g and 5 min. The obtained lymphocytes were stored frozen at −85 ° C., and after thawing, the lymphocytes were washed with ERDF medium and used.
[生体外アレルギー発症モデル細胞培養系]
5%ウシ胎児血清(FBS;Trace)及び10%ヒト血漿を含むERDF培地中に、生体外に取り出した2.5×106cells/mlのヒト末梢血リンパ球と免疫賦活剤としてムラミルジペプチド(MDP;SIGMA)を10mg/ml、インターロイキン−2、−4、−6(IL−2、−4、−6;R&D)を10ng/ml、さらにスギ花粉抗原(Cryj1;HAYASHIBARA)を100ng/mlの終濃度で加え、生体外アレルギー発症モデル培養系とした。添加した免疫賦活剤の主な働きは、MDPによる抗原への免疫応答を高めるアジュバンド活性、IL−2によるT細胞の増殖・分化の促進、IL−4によるIgEクラススイッチ誘導、IL−6によるB細胞の分化誘導である。これらの免疫賦活剤とCryj1を加えたヒト末梢血リンパ球を96穴プレートに各200ml分注し、37℃の5%CO2インキュベータにて10日間培養してIgE抗体産生を誘導した。なお、この培養系に含まれるヒト血漿とヒト末梢血リンパ球は同一個体のものを使用した。
[In vitro allergy development model cell culture system]
In an ERDF medium containing 5% fetal bovine serum (FBS; Trace) and 10% human plasma, 2.5 × 10 6 cells / ml human peripheral blood lymphocytes removed in vitro and muramyl dipeptide (MDP) as an immunostimulant SIGMA) at 10 mg / ml, interleukin-2, -4, -6 (IL-2, -4, -6; R & D) at 10 ng / ml, and cedar pollen antigen (Cryj1; HAYASHIBARA) at 100 ng / ml In addition to the final concentration, an in vitro allergy model culture system was obtained. The main functions of the added immunostimulant are adjuvant activity that enhances the immune response to the antigen by MDP, promotion of T cell proliferation and differentiation by IL-2, induction of IgE class switch by IL-4, and IL-6 This is induction of B cell differentiation. 200 ml of human peripheral blood lymphocytes added with these immunostimulants and Cryj1 were dispensed into 96-well plates, and cultured in a 5% CO 2 incubator at 37 ° C. for 10 days to induce IgE antibody production. The human plasma and human peripheral blood lymphocytes contained in this culture system were the same individuals.
[NOV由来タンパク質発現細胞とIgE抗体生産抑制効果との相関]
細胞の膜上に存在する分子を蛍光色素で標識することにより、定量的に解析可能なフローサイトメーターを用いて、細胞膜上のNOV由来タンパク質の発現確認を行なった。生体外アレルギー発症モデル細胞培養系でIgE抗体の生産を抑制する効果、すなわち、抗アレルギー効果を有する被検物質を添加して10日間培養した。Goat anti NOV IgG(R&D社,AF1640)とDonkey anti goat Ig’s FITC(Santa Cruz Biotechnology社,SC-3853)を用いてモデル培養系の細胞を蛍光抗体法による標識を行った。そして、細胞膜上のNOV分子の相対量を測定した。図1は、NOV由来タンパク質発現細胞の発現量とIgE抗体生産抑制効果との相関を示したグラフである。図1(A)の対照実験と比較して、図1(B)の抗アレルギー物質を添加した方が、グラフのピークが左にずれていることから、発現量が減少していることが分かる。図2は、図1に示した場合に用いた被検物質のIgE抗体生産量を示したグラフである。図2に示されているように、抗アレルギー効果を有する被検物質の入った培養系の細胞のみに、NOV由来タンパク質の発現が抑制されたことが示されている。
[Correlation between NOV-derived protein expressing cells and IgE antibody production inhibitory effect]
By labeling the molecules present on the cell membrane with a fluorescent dye, the expression of NOV-derived protein on the cell membrane was confirmed using a flow cytometer capable of quantitative analysis. The test substance which has the effect which suppresses the production of IgE antibody in an in vitro allergy onset model cell culture system, ie, an antiallergic effect, was added and cultured for 10 days. Cells of the model culture system were labeled by the fluorescent antibody method using Goat anti NOV IgG (R & D, AF1640) and Donkey anti goat Ig's FITC (Santa Cruz Biotechnology, SC-3853). Then, the relative amount of NOV molecules on the cell membrane was measured. FIG. 1 is a graph showing the correlation between the expression level of NOV-derived protein-expressing cells and the IgE antibody production inhibitory effect. Compared with the control experiment of FIG. 1 (A), the addition of the antiallergic substance of FIG. 1 (B) shows that the expression level is reduced because the peak of the graph is shifted to the left. . FIG. 2 is a graph showing the IgE antibody production amount of the test substance used in the case shown in FIG. As shown in FIG. 2, it is shown that the expression of the NOV-derived protein was suppressed only in the cells of the culture system containing the test substance having an antiallergic effect.
[NOV由来タンパク質を細胞膜上に発現している細胞の同定]
現在、NOV由来タンパク質を発現している細胞に関する報告がない。したがって、数種のヒト培養細胞株を用いて細胞内でのNOV遺伝子の発現を調べることで、どの細胞に発現しているかを同定した。使用した細胞株はヒトBリンパ球系細胞株SK729-1、ヒトTリンパ球系細胞株MOLT−4、ヒトBリンパ球系細胞株DND39を用いた。これらの細胞からRNAを抽出後、cDNAを合成し、リアルタイムPCRを行った。
[Identification of cells expressing NOV-derived protein on cell membrane]
There are currently no reports on cells expressing NOV-derived proteins. Therefore, by investigating the expression of the NOV gene in the cells using several types of cultured human cell lines, it was identified in which cells. The cell lines used were human B lymphocyte cell line SK729-1, human T lymphocyte cell line MOLT-4, and human B lymphocyte cell line DND39. After extracting RNA from these cells, cDNA was synthesized and real-time PCR was performed.
A)RNAの抽出
ISOGEN(日本ジーン社,311-02501)を用いて培養細胞1×107cellsを破砕し、これに0.2mlのクロロホルム(和光純薬社,038-02606)を加え、15秒間激しく震盪し室温で5分間静置させた後、12K×g、4℃で15分間遠心した。遠心後、水層を回収し、0.5mlのイソプロパノール(和光純薬社,168-21675)を加えて室温で10分間静置した。その後、12K×g、4℃で10分間遠心して上澄み液を取り除き、70%のエタノール(和光純薬社,057-00456)を加えた。続いて、12K×g、4℃で5分間再び遠心し、上澄み液を捨て、5分間風乾した。これにRNA濃度5μg/9μlになるようにddH2Oを加え、RNA試料とした。
A) RNA extraction
Cultivate cultured cells 1 × 10 7 cells using ISOGEN (Nippon Gene, 311-02501), add 0.2 ml of chloroform (Wako Pure Chemicals, 038-02606) to this, and shake vigorously for 15 seconds at room temperature. And allowed to stand for 5 minutes, and then centrifuged at 12K × g, 4 ° C. for 15 minutes. After centrifugation, the aqueous layer was collected, 0.5 ml of isopropanol (Wako Pure Chemicals, 168-21675) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Thereafter, the supernatant was removed by centrifugation at 12K × g and 4 ° C. for 10 minutes, and 70% ethanol (Wako Pure Chemical Industries, Ltd., 057-00456) was added. Subsequently, the mixture was centrifuged again at 12K × g and 4 ° C. for 5 minutes, and the supernatant was discarded and air-dried for 5 minutes. To this, ddH 2 O was added so that the RNA concentration was 5 μg / 9 μl to obtain an RNA sample.
B)cDNAの合成
単離したRNAからcDNAを合成するために、ThermoScriptTM RT-PCR System(Invitrogen,11146-016)を用いた。Random Hexamer 1μl、RNA9μl、10mM dNTP Mix 2μlをそれぞれ混和し、65℃で5分間インキュベートした。その後、5×cDNA Synthsis Buffer 4μl、0.1M DTT 1μl、RNaseOUTTM(40U/μl)1μl、DEPC-treated water 1μl、ThermoScriptTM RT(15units/μl)1μlをそれぞれに加え、混和した。その後、25℃で10分間、50℃で50分間、85℃で5分間反応させ、cDNAを合成した。その後、RNase H 1μlを加え、37℃で20分間インキュベートし、残存RNAを分解した。
B) Synthesis of cDNA The ThermoScript ™ RT-PCR System (Invitrogen, 11146-016) was used to synthesize cDNA from the isolated RNA. Random Hexamer (1 μl), RNA (9 μl), and 10 mM dNTP Mix (2 μl) were mixed and incubated at 65 ° C. for 5 minutes. Thereafter, 4 μl of 5 × cDNA Synthsis Buffer, 1 μl of 0.1M DTT, 1 μl of RNaseOUT ™ (40 U / μl), 1 μl of DEPC-treated water and 1 μl of ThermoScript ™ RT (15 units / μl) were added and mixed. Then, it was made to react at 25 degreeC for 10 minutes, 50 degreeC for 50 minutes, and 85 degreeC for 5 minutes, and synthesize | combined cDNA. Thereafter, 1 μl of RNase H was added and incubated at 37 ° C. for 20 minutes to decompose the remaining RNA.
C)リアルタイムPCR(NOV)
cDNAを鋳型として、cDNA 1μl、Forward Primer 1μl、Reverse Primer 1μl、iQTMSYBR(登録商標) Green Supermix(BIORAD社,170-8882)12.5μl、DEPC-treated water 9.5μlを混和し、NOV遺伝子のリアルタイムPCRを行なった。また、内部標準としてβ-actinを用いた。この際のPCR条件は、95℃で3分間酵素を活性化し、95℃で30秒、60℃で30秒、72℃で30秒を、40サイクルで実施した。用いたプライマーは下記の配列である。
C) Real-time PCR (NOV)
Using cDNA as a template, mix 1 μl of cDNA, 1 μl of Forward Primer, 1 μl of Reverse Primer, 12.5 μl of iQ ™ SYBR (registered trademark) Green Supermix (BIORAD, 170-8882), 9.5 μl of DEPC-treated water, and mix NOV gene Real-time PCR was performed. In addition, β-actin was used as an internal standard. The PCR conditions at this time were to activate the enzyme at 95 ° C. for 3 minutes, and carried out 40 cycles of 95 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds. The primers used have the following sequences.
NOV
Forward primer:5’-TGGTCCCTCATAGCCCTCAG-3’(配列番号:1)
Reverse primer:5’-CCCTGCTAATATGCTGTTTCTGC-3’(配列番号:2)
NOV
Forward primer: 5'-TGGTCCCTCATAGCCCTCAG-3 '(SEQ ID NO: 1)
Reverse primer: 5'-CCCTGCTAATATGCTGTTTCTGC-3 '(SEQ ID NO: 2)
β-actin
Forward primer:5’-GACTTCGAGCAAGAGATG-3’(配列番号:3)
Reverse primer:5’-GCCAGACAGCACTGTGTT-3’(配列番号:4)
β-actin
Forward primer: 5'-GACTTCGAGCAAGAGATG-3 '(SEQ ID NO: 3)
Reverse primer: 5'-GCCAGACAGCACTGTGTT-3 '(SEQ ID NO: 4)
図3は、ヒト培養細胞株を用いて細胞内でのNOV遺伝子の発現比較したグラフである。その結果、図3に示すように、MOLT−4細胞株にNOV遺伝子が多く発現していることが分かった。このことから、NOV由来のタンパク質は、ヒトTリンパ球系細胞株に発現していた。抗アレルギー効果の検出方法であるIgE抗体の生産抑制と本願発明で用いるTリンパ球細胞膜上のNOV由来タンパク質発現量の減少との間には相関関係があり、さらに、無限増殖能を有するMolt−4細胞株を用いて、NOV由来タンパク質発現量を検討することで、新規の抗アレルギー因子探索手法が確立できた。 FIG. 3 is a graph comparing the expression of NOV genes in cells using human cell lines. As a result, as shown in FIG. 3, it was found that many NOV genes were expressed in the MOLT-4 cell line. Thus, NOV-derived protein was expressed in human T lymphocyte cell lines. There is a correlation between the suppression of IgE antibody production, which is a method for detecting the antiallergic effect, and the decrease in the expression level of NOV-derived protein on the T lymphocyte cell membrane used in the present invention. By investigating the expression level of NOV-derived protein using 4 cell lines, a novel anti-allergic factor search method could be established.
[NOV由来タンパク質と抗アレルギー因子の結合能の評価]
NOV由来タンパク質と抗アレルギー因子との結合能を評価するため、分子同士の結合を動力学的に測定可能な生体分子間相互作用装置BIACOREを用いて測定した。すなわち、NOV由来タンパク質(R&D社,1640-NV)をBIACOREセンサーチップCM5(BIACORE社,BR-1000-12)に固定化し、抗アレルギー効果が認められる物質と抗アレルギー効果が認められない物質を120秒間流路に流して、NOV由来タンパク質との結合親和性をグラフ化した。図4は、NOV由来タンパク質と抗アレルギー因子との結合能の比較したグラフである。図4に示すように、抗アレルギー因子は、対照実験と比較して高い結合能を示すグラフの変化が記録されている。その結果、NOV由来タンパク質と抗アレルギー物質とは、結合親和性が高く、逆に、抗アレルギー物質でないものは、結合親和性は極めて低いことが分かった。抗アレルギー因子探索に、NOVタンパク質そのものを用いて、被検物質との結合能を測定することで新規に抗アレルギー因子を探索可能であることが分かった。
[Evaluation of binding ability of NOV-derived protein and antiallergic factor]
In order to evaluate the binding ability between the NOV-derived protein and the anti-allergic factor, the binding between molecules was measured using a biomolecular interaction device BIACORE capable of kinetically measuring. In other words, NOV-derived protein (R & D, 1640-NV) is immobilized on BIACORE sensor chip CM5 (BIACORE, BR-1000-12), and 120 substances that have anti-allergic effects and 120 substances that do not have anti-allergic effects are identified. Flow through the channel for 2 seconds and graphed the binding affinity with the NOV-derived protein. FIG. 4 is a graph comparing the binding ability of a NOV-derived protein and an antiallergic factor. As shown in FIG. 4, the anti-allergic factor has recorded a change in a graph showing a high binding ability compared to the control experiment. As a result, it was found that NOV-derived proteins and antiallergic substances have high binding affinity, and conversely, those that are not antiallergic substances have extremely low binding affinity. It was found that a novel anti-allergic factor can be searched by measuring the binding ability to the test substance using the NOV protein itself for the anti-allergic factor search.
Claims (2)
細胞株に発現するNOVタンパク質との結合親和性を用いて、前記IgE抗体の生産を抑制することによる抗アレルギー効果を発揮する被検物質をスクリーニングする判定ステップを含む抗アレルギー因子のスクリーニング方法。 A screening method for an antiallergic factor for screening a substance that suppresses production of IgE antibodies produced by antibody-producing cells in vivo,
With a binding affinity between NO V protein expressed in cell lines, anti-allergic factor of screening comprising determining step of screening a test substance to exert anti-allergic effects by inhibiting the production of the IgE antibody Method.
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