JP2001314172A5 - - Google Patents

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【００１６】核酸を添加した食餌を、通常のマウスに２週間自由に経口摂取させた後、ＩＥＬにおけるＴＣＲサブセット構成を、フローサイトメトリー（ＦＡＣＳ）で解析した。
【００１７】核酸摂取群におけるαβ-ＩＥＬの発現率（％）は、核酸非摂取群（対照群）のそれよりも有意（ｐ＜０．０５）に低くなり、逆にγδ-ＩＥＬの発現率は、対照群のそれよりも有意（ｐ＜０．０５）に高くなった（図１）。これを、核酸摂取群におけるαβ-ＩＥＬ/γδ-ＩＥＬの発現比、対照群におけるαβ-ＩＥＬ/γδ-ＩＥＬの発現比で比較すると、核酸摂取群の方が対照群よりも有意（ｐ＜０．０５）に低下した（図２）。これらの結果は、核酸を添加した食餌を経口摂取すると、ＩＥＬにおけるαβ-ＩＥＬとγδ-ＩＥＬの構成比が、γδ-ＩＥＬが高められる方法にシフトすることを示している。
【００１８】さらに、αβ-ＩＥＬおよびγδ-ＩＥＬのそれぞれにおけるＣＤ４とＣＤ８の発現比率をＦＡＣＳで個体別に解析した。
【００１９】αβ-ＩＥＬにおける核酸摂取群のＣＤ８αα⁺の発現率（％）が、対照群のそれよりも有意（ｐ＜０．０５）に低くなったのに対し、γδ-ＩＥＬにおける核酸摂取群のＣＤ８αα⁺の発現比率（％）は、対照群のそれよりも有意（ｐ＜０．０５）に高くなった（図３）。
【００２０】データは示さないが、核酸摂取群と対照群との間で、αβ-ＩＥＬにおけるＣＤ８αβ⁺の発現比率には差は認められなかったこと、ＣＤ４⁺、ＣＤ４⁺ＣＤ８⁺、の発現比率は、核酸摂取群と対照群で変わらなかったこと、γδ-ＩＥＬにおけるＣＤ４^-ＣＤ８^-は、核酸摂取群で僅かに増加したが、対照群と有意差はなかったことから、核酸摂取群において、αβ-ＩＥＬにおけるＣＤ８αα⁺の構成比率が低くなり、γδ-ＩＥＬにおけるＣＤ８αα⁺の構成比率が高くなることと、αβ-ＩＥＬとγδ-ＩＥＬの構成比率が、γδ-ＩＥＬの発現比率が高められる方向にシフトすることとは相関していることを示していると考えられる。したがって、核酸の経口摂取は、胸腺外でのγδ-ＩＥＬ発達・分化を促進すると考えられる。
【００２１】さらに、タンパク質抗原の経口摂取が、ＩＥＬにおけるＴＣＲサブセットの発現、およびＣＤ４とＣＤ８発現に及ぼす影響を、主要な食物アレルゲンである卵白アルブミン（ＯＶＡ）に特異的なＴ細胞抗原レセプター（ＴＣＲ）をもつトランスジェニックマウス（ＯＶＡ-ＴＣＲ Ｔｇ）で調べた。
【００２２】核酸を含む食餌とともにＯＶＡを含む水を自由に摂取したＯＶＡ-ＴＣＲ ＴｇマウスからＩＥＬを調製し、ＴＣＲサブセット構成、およびαβ-ＩＥＬとγδ-ＩＥＬにおけるそれぞれのＣＤ４とＣＤ８サブセット発現を、ＦＡＣＳで個体別に解析した。
【００２３】経口抗原存在下でも、αβ-ＩＥＬとγδ-ＩＥＬの構成比は、核酸の経口摂取により、γδ-ＩＥＬが高められる方向に有意（ｐ＜０.０５）にシフトした（図４）。ＣＤ８αα⁺の発現率（％）についても、γδ-ＩＥＬにおけるＣＤ８αα⁺の発現率は、対照群のそれよりも有意（ｐ＜０．０５）に高くなった（図５）。しかしながら、核酸摂取群、対照群ともにαβ-ＩＥＬの発現比率が、通常マウスの場合に比較して著明に増加した（図１と４参照）。
【００２４】一方、八村らは、ＯＶＡを経口摂取したＯＶＡＴＣＲ-Ｔｇマウスでは、ＯＶＡ特異的ＣＤ４⁺-ＩＥＬが増殖し、ＩＥＬ全体に占める割合が増加することを明らかにしている（八村敏志・他：日本農芸化学会誌, 73(5): 523-527, 1999）。
【００２５】２） 核酸の経口摂取が腸管上皮におけるサイトカイン産生に及ぼす影響核酸を添加した食餌とともにＯＶＡを含む水を自由に摂取したＯＶＡ-ＴＣＲＴｇマウスから調製したＩＥＬと、未感作のＢＡＬＢ/ｃマウスから調製しマイトマイシンＣ処理した脾細胞（抗原提示細胞）とを、ＯＶＡの存在下、あるいは非存在下で共培養すると、核酸摂取群において、ＯＶＡ特異的ＩＦＮ-γおよびＩＬ-２の産生増強が観察された（図６および７）。
【００２６】これは、核酸の経口摂取は、ＩＥＬ中の抗原特異的なＴｈ１タイプの細胞の増殖応答を誘導することを示していると考えられる。
【００２７】核酸の経口摂取はマウスの小腸上皮細胞のＩＬ-７産生を有意（ｐ＜０．０１）に増強する（図８）。
【００２８】また、核酸を添加した食餌とともにＯＶＡを含む水を自由に摂取したＯＶＡ-Ｔｇマウスの小腸上皮細胞のＩＬ-７産生およびＴＧＦ-β産生は、核酸摂取により有意（ｐ＜０．０１）に増強した（図９および１０）。
【００２９】３） 核酸の経口摂取が抗原特異的ＩｇＡ抗体産生に及ぼす影響【００３０】核酸の経口摂取は、腸管における抗原特異的ＩｇＡ抗体の産生を対照群に比較して有意（ｐ＜０．０５）に増強した（図１１）。
【００３１】ヌクレオチドをマウスに経口摂取させ、脾臓細胞や腹腔マクロファージのp70型のＩＬ-１２に与える影響を、生物学的測定法（Bioassay法）で検討した。また、in vitroでヌクレオチドとともにマクロファージを培養し、その上清中のＩＬ-１２活性をBioassay法で測定した。腹腔マクロファージのＩＬ-１２活性は、ＬＰＳ刺激下でヌクレオチド投与群の方がヌクレオチド非投与群に比べて有意に高くなった（図１２）。また、in vitroでヌクレオチドとともに腹腔マクロファージを培養した場合のＩＬ-１２活性は、ヌクレオチドを添加していない場合に比べて、有意差は見られないものの高くなる傾向が見られた（図１３）。
【００３２】脾臓リンパ細胞のＩＬ-１２活性は、ヌクレオチド投与群の方がヌクレオチド非投与群に比べて有意に高くなった（図１４）。
【００３３】以上のことから、核酸組成物の経口摂取は、マクロファージなどによるＩＬ-１２産生を促進し、ＩＬ-１２はナイーブＴ細胞からＴｈ１細胞への分化の促進、抹消血からＩＦＮ-γ産生を誘導することから、生体内でのＴｈ１／Ｔｈ２バランスをＴｈ１優位にシフトすることが期待される。その結果、Ｔｈ１／Ｔｈ２バランスが波綻し、Ｔｈ２優位に変異することに起因する疾患、例えば、アレルギー疾患や自己免疫疾患に対して、核酸組成物を含む飲食品を日常の食生活のなかで摂取することにより、その予防、改善あるいは治療が期待できる。
【００３４】また、ＩＬ-１２は、静止期のＴ細胞およびＮＫ細胞からのＩＦＮ-γの産生誘導、ＮＫ細胞活性の亢進、ＬＡＫ細胞活性の誘導、静止期Ｔ細胞のレクチン刺激による細胞増殖亢進などが知られており、核酸組成物を含む飲食品を日常の食生活のなかで摂取することにより、生体内での微生物および腫瘍細胞に対する排除能を高め、ＩＦＮ-γの産生増強をとおしてウイルスや腫瘍細胞に対する防御能を高めることが期待される。
【００３５】これらの結果から、核酸の経口摂取は、１）ＩＥＬにおけるＴ細胞のαβ-ＩＥＬサブセットとγδ-ＩＥＬサブセットの構成比がγδ-ＩＥＬ優位に高められ、それは、γδ-ＩＥＬにおけるＣＤ８αα⁺の比率の増加と、αβ-ＩＥＬにおけるＣＤ８αα⁺の比率の減少と相関しており、２）ＩＥＬにおいて、抗原特異的ＩＬ-２およびＩＦＮ-γの産生を促進し、３）腸管上皮細胞におけるＩＬ-７およびＴＧＦ-βの産生を促進し、４）抗原特異的ＩｇＡ抗体産生を誘導する、５）ＩＬ-１２産生を増強することが示された。
【００３６】一方、本発明者らは、ヌクレオチドを含む食餌を経口摂取したマウスは、１）パイエル板Ｔリンパ球のマイトジェン刺激に対する増殖応答能およびＩｇＡ産生能が、ストレス下（絶食）で促進されること、２）血清中のＩｇＥ産生が抑制されること、３）ＩｇＧ１／ＩｇＧ２aが低下すること、４）脾細胞のＩＦＮ-γの産生が促進され、ＩＬ-４産生が抑制されること、を示した（特開平9-323979）。さらにその後、本発明者らは、ヌクレオチドを含む食餌を経口摂取したマウスの血清中では、１）カゼイン特異的ＩｇＧ１は変化なかったが、ＩｇＧ２aは高まったこと、２）卵白アルブミン（ＯＶＡ）特異的ＩｇＧ１は変化なかったが、ＩｇＧ２aは高くなり、またＯＶＡ特異的ＩｇＥは低下したこと、３）血清中の全ＩｇＥが低くなったこと、を示した（特開平11-35468）。
【００３７】一方、γδ-ＩＥＬはＫＧＦを産生し小腸上皮細胞の成長、増殖を制御している（Boismenu, R. & Havran, W.L.: Science, 266: 1253, 1994; Komano, H. etal.: Proc. Natl. Acad. Sci., USA, 92: 6147, 1995）、分泌型ＩｇＡ産生を促進する（Fujihashi, K. et al.: J. Exp. Med., 183: 1929, 1996）、経口免疫寛容の誘導に必要である（Ke, Y. et al.: J. Immunol., 158: 3610-3618, 1997）、などから、ＩＥＬにおけるγδ-ＩＥＬの発現が高まる方向へのシフトは、腸管免疫系の賦活化に重要であると考えられる。
【００３８】また、Ｔｈ１細胞の産生するＩＦＮ-γは、上皮細胞の基底膜側に発現している分泌成分（serectory component: ＳＣ）産生に深く関与している（Taguchi,T. et al.: J. Immunol., 3736, 1991）。このＳＣとＩｇＡ形質細胞が産生する２量体のＩｇＡは結合して分泌型ＩｇＡを形成する。
【００３９】また、上皮細胞のＩＬ-７は、γδ-ＩＥＬのＩＬ-７レセプター（ＩＬ-７Ｒ）を介してγδ-ＩＥＬの分化・増殖に重要な役割を果たす（Fujihashi, K. et al.: Eur. J. Immunol., 27: 2133, 1997）。さらに、ＩＬ-２とＩＬ-７は胸腺由来のγδ-ＩＥＬやαβ-ＩＥＬに対して活性化因子として働いている（Okazaki,et al.: J. Immunol., 143: 2917, 1989）。
【００４０】また、ＴＧＦ-βは、免疫抑制性のサイトカインとして知られており、これを分泌するＴ細胞は経口免疫寛容における調節性Ｔ細胞として、免疫応答を抑制していることが示唆されている（八村敏志・他：日本農芸化学会誌, 73(5): 523-527, 1999）。また、ＴＧＦ-βは、ＩｇＡへのクラススイッチを行うことが知られており（Sonoda, E. et al.: J. Exp. Med., 170: 1415, 1989）、腸管におけるＩｇＡへの関与も示唆されている。
【００４１】ところで、ＣＤ４＋ヘルパーＴ細胞は、産生するサイトカインプロフィルとそれに基づくエフェクター機能によって、ＩＬ-２やＩＦＮ-γを産生するＴｈ１細胞と、ＩＬ-４やＩＬ-５、ＩＬ-１０を産生するＴｈ２細胞に分類される。このＴｈ１とＴｈ２のバランスの破綻は、がん、感染症、アレルギー、自己免疫疾患、動脈硬化など、さまざまな免疫病の発症に関与しているほか、ストレスと免疫、移植免疫、妊娠免疫にも重要な因子であることが知られている。核酸の経口摂取は、このＴｈ１とＴｈ２のバランスにおけるＴｈ２優位へのバランス破綻を、Ｔｈ１の発現を高めることにより、該バランスを改善することが期待される。
【００４２】結論として、有効量の核酸の経口摂取により、腸管免疫系に抗原特異的な分泌型ＩｇＡ抗体産生を促進して、有害な病原体などに対する防御能を高め、一方、抗原特異的な経口免疫寛容を効果的に誘導し、さらに、Ｔｈ１/Ｔｈ２細胞のバランスの破綻を改善し、結果、腸管免疫系、あるいは全身免疫系を賦活化することが期待される。
【００４３】本明細書における“核酸の有効量”は、例えば、ＩＥＬにおけるγδ-ＩＥＬの発現比率が高められること、腸管上皮におけるＩＦＮ-γ、ＩＬ-２、ＩＬ-７、およびＴＧＦ-βの産生が促進されること、抗原特異的ＩｇＡ抗体の産生が促進されること、血清中の抗原特異的ＩｇＥ抗体産生やｔｏｔａｌＩｇＥ抗体産生が抑制されること、脾細胞におけるＩＬ-４産生が抑制されること、ＩｇＧ１/ＩｇＧ２ａの比が低下すること、等を指標にして、公知のアッセイによりその有効量を決定し、さらに臨床症状等を勘案して、有効摂取量を決定することができる。
【００４４】経口的に摂取された核酸は、膵液中のリボヌクレアーゼ、デオキシリボヌクレアーゼによって加水分解を受け、ヌクレオチドになり、ホスファーターゼによってリン酸がはずれてヌクレオシドになり、さらにヌクレオシダーゼによって分解されて核酸塩基と五炭糖になる。胃腸からの吸収は、ヌクレオチド、ヌクレオシドとして行われる。
【００４５】ここでいう塩基は、アデニン、グアニン、ヒポキサンチン、キサンチン、シトシン、ウラシル、チミンのことである。ここでいうヌクレオシドは、ウリジン、アデノシン、グアノシン、シチジン、リポチミジン、デオキシアデノシン、デオキシグアノシン、デオキシウリジン、デオキシシチジン、チミジン、イノシン、キサントシンのことである。
【００４６】ここでいうヌクレオチドは、ヌクレオシドの糖部分にリン酸がエステル結合で結合している化合物のことで、結合するリン酸の位置はどこでもよく、結合するリン酸の数もいくつでもよい。また、例えば、１つのリン酸が 5'、3' 位の両方に結合する化合物もヌクレオチドに含める。この場合も結合するリン酸の数や位置はどこでもよい。
【００４７】ここでいう核酸は、ＲＮＡ、ＤＮＡなどのポリヌクレオチドや上記したヌクレオチドが結合したポリヌクレオチドのことである。本発明の核酸は、アデノシン-n'-一リン酸、シチジン-n'-一リン酸、グアノシン-n'-一リン酸、ウリジン-n'-一リン酸、チミジン-n'-一リン酸、およびイノシン-n'-一リン酸の１種または２種以上を含む核酸組成物を意味する。ここでn'は２'、３'、または５'を示す。
【００４８】本発明の核酸組成物は、特定の組み合わせの核酸を有効成分として含有するものであり、組み合わせを遊離形で示すと、次のとおりである。なお、核酸成分は、ＩＵＰＡＣ-ＩＵＢ規定、あるいは当該分野における慣用記号にしたがう。
【００４９】すなわち、アデノシンーリン酸（ＡＭＰ）／シチジンーリン酸（ＣＭＰ）／グアノシンーリン酸（ＧＭＰ）／ウリジンーリン酸（ＵＭＰ）／イノシン-n'-一リン酸（ＩＭＰ）の各組み合わせである。これらの組み合わせにおける各成分としては、公知のヌクレオシド、ヌクレオチド類、またはこれらの無毒性塩が含まれる。
【００５０】また、核酸成分の配合割合を、例えば核酸のモル比で表せば、モル比は、上記のアッセイ系で、核酸成分の種類、配合量等を決定し、さらに患者の病態等を勘案して決定することができる。これらの好ましい組み合わせの１例としては、モル比として、ＩＭＰ：ＡＭＰ：ＧＭＰ：ＣＭＰ：ＵＭＰ：ＡＭＰ＝１〜４：１〜４：１〜４：１〜４である。さらに好ましい一つの例として、ＧＭＰ：ＣＭＰ：ＵＭＰ：ＩＭＰ＝１：３〜４：１〜２：１〜３である。
【００５１】これらの核酸塩基のうちでの最適な核酸塩基の選択、あるいは組み合わせは、当業者であれば、上記アッセイ系で適切に決定することが可能である。したがって、そのようにして得られた核酸組成物も本発明に包含される。
【００５２】核酸はその由来に制限はなく、例えば、酵母、細菌、乳、魚介類、動物、植物由来が挙げられる。飲食品タイプの核酸組成物として使用する場合には、有効量の核酸成分（その処理物）をそのまま、使用したり、他の食品ないし食品成分と併用したりして適宜常法にしたがって使用できる。有効量の核酸成分を用いる本発明に係る組成物は、固体状（粉末、顆粒状その他）、ペースト状、液状ないし懸濁状のいずれでもよいが、甘味料、酸味料、ビタミン剤その他ドリンク剤製造に常用される各種成分を用いて、健康ドリンクに製剤化すると好適である。
【００５３】腸管免疫系の賦活化（抗原特異的ＩｇＡ抗体産生や免疫寛容）を目的として、核酸の有効量を、食品、健康食品、機能性食品、栄養補助食品、特定保健用食品、経腸栄養食品、あるいは経腸栄養剤等の医薬品製剤や医薬部外品製造のために配合することができる。それは周知・慣用技術である。
【００５４】プリン・ピリミジンの成人１日の必要量は、450〜700mgとされ、また、尿酸に蓄積を生じない最大安全量は、４g/日とされている（Rudolph, F.B. et al.: Nutrition, 6 : 45, 1990）。
【００５５】［試験例］ 以下、核酸の経口摂取が免疫系に及ぼす影響についての試験例を示すが、本発明はこれらの試験例に限定されるものではない。
【００５６】 統計処理データの統計的有意差は、Student's testによつて決定した。p＜０.０５における値を有意とみなした。
【００５７】 フローサイトメトリー一次抗体は、ビオチン化抗マウスＴＣＲβ(Ｈ５７-５９７)（PharMingen, SanDiego, CA, U.S.A）、ビオチン化抗マウスＣＤ４(Ｈ１２９.１９)（PharMingen）、およびビオチン化抗マウスＩＬ-２Ｒ抗体(ＣＤ２５)(７Ｄ４)(PharMingen)、を用いた。インキュベーションは全て遮光下に行った。細胞は、一次抗体、あるいはアイソタイプコントロール抗体とともに、氷上で３０分間インキュベートした。Ｈａｎｋ's平衡塩類溶液で細胞を洗浄後、Fluorescein isothiocyanate（ＦＩＴＣ）標識抗マウスＴＣＲδ抗体(ＧＬ３)(Cedar Lane Lab.)、ＦＩＴＣ標識抗マウスＣＤ４(Ｈ１２９.１９)（PharMingen）、phycoerythrin（ＰＥ）標識抗マウスＣＤ８α(５３-６.７)（GIBCO,Grand Island, NY, U.S.A）、ＦＩＴＣ標識抗マウスＣＤ８β(Ｙ８.７７)（Seikagaku Co., Tokyo, Japan)、ＰＥ標識抗マウスＴＣＲβ、(Ｈ８７-５９７)(PharMingen)、およびstreptavidin-Red 670(GIBCO)、とともに氷上で３０分間インキュベートした。細胞は、遠心して洗浄した。染色細胞は、３カラーによるフローサイトメトリー（Becton Dickinson FACSort）でＩＥＬsサブセットを解析した。解析には、Lysis IIソフトウエアを用いた。
【００５８】 サイトカインアッセイ（ＥＬＩＳＡ）アッセイバッファーとして３％ポリエチレングリコール６０００（Nacalai Tesque Inc., Japan）を含むリン酸緩衝液（ｐＨ７.４）を用いた（アッセイバッファー）。洗浄バッファーには、０.０５％Ｔｗｅｅｎ-２０を含むリン酸緩衝液（ｐＨ７.４）（ＰＢＳ-０．０５％Ｔｗｅｅｎ）を用いた。ＩＦＮ-γ、およびＩＬ-２は、ＥＬＩＳＡ法（enzyme-linked immunosorbentassay）で測定した。ラット抗マウスＩＦＮ-γ抗体（６Ａ２）(PharMingen)、ラット抗マウスＩＬ-２抗体（ＪＥＳ６-１Ａ１２）(PharMingen)と、ビオチン化ラット抗マウスＩＦＮ-γ抗体（ＸＭＧ１.２）(PharMingen)、ビオチン化ラット抗マウスＩＬ-２抗体（ＪＥＳ６-５Ｈ４)（PharMingen）を用いた。０.０５Ｍ-Ｔｒｉｓ緩衝液（ｐＨ８.９）で希釈したラット抗マウスＩＦＮ-γ抗体（１μg/ｍｌ）、あるいは０.１Ｍ-NaHCO₃で希釈したラット抗マウスＩＬ-２抗体（１μg/ｍｌ）を、マイクロタイタープレート（Nunc, Roskilde, Denmark）の各ウエルに分注した。４℃で一晩インキュベートし、抗体をウエルに吸着させた。ウエルを洗浄後、５％ＦＣＳを含むアッセイバッファーを加え、室温１時間ブロックした。ウエルを洗浄後、アッセイバッファーで希釈した培養上清を各ウエルに加え、２時間インキュベートした。ウエルを洗浄後、アッセイバッファーで希釈したビオチン化抗マウスＩＦＮ-γ抗体、またはビオチン化抗マウスＩＬ-２抗体を各ウエルに加え、２時間インキュベートした。洗浄後、アッセイバッファーで希釈したアルカリホスファターゼ標識ストレプトアビジン（Zymed Lab., South San Francisco, CA, U. S.A）を各ウエルに加え反応させた。ウエルを洗浄後、基質溶液［０.１Ｍジエタノールアミン緩衝液(ｐＨ９.８)に溶解した0.１％４ニトロフェニルホスフェイト（Tokyo Kasei Co.）］を各ウエルに加え、室温で１時間インキュベートした。５Ｎ-NaOHを加えて反応を停止し、４０５ｎｍにおける吸光値を測定した。ＩＬ-７およびＴＧＦ-βは、ＥＬＩＳＡ法で測定した。
【００５９】0.１Ｍ-NaHCO₃で希釈した抗マウスＩＬ-７抗体溶液（２μg/ｍｌ）(Genzyme)をマイクロタイタープレート（Nunc）の各ウエルに分注し、４℃一晩インキュベートし、抗体をウエルに吸着させた。ウエルを洗浄後、１０％ＯＶＡを含むアッセイバッファーをウエルに加え、室温１時間ブロックした。ウエルを洗浄後、0.１％Ｔｗｅｅｎ２０を含むアッセイバッファーで希釈した培養上清をウエルに加え、２時間インキュベートした。ウエルを洗浄後、0.１％Ｔｗｅｅｎ２０を含むアッセイバッファーで希釈してヒツジ抗マウスＩＬ-７抗体(１μg/ｍｌ）（R and D, Minneapolis, MN, USA）を各ウエルに加え２時間インキュベートした。ウエルを洗浄後、０.１％Ｔｗｅｅｎ２０を含むアッセイバッファーで希釈したビオチン化抗ヒツジＩｇＧ抗体を各ウエルに加え、１５分間反応させた。ウエルを洗浄後、アッセイバッファーで希釈したアルカリホスファターゼ標識ストレプトアビジンを各ウエルに加えた。ウエルを洗浄後、ＩＦＮ-γおよびＩＬ-２と同様に、基質溶液を各ウエルに加え、４０５ｎｍにおける吸光値を測定した。
【００６０】［試験例１］ＩＥＬのＴＣＲサブセット発現に及ぼす影響３週齢の雌性ＢＡＬＢ/ｃマウス［ＳＬＣ,Hamamatsu, Japan］は、ヌクレオチド非添加食餌［ＮＴ(−)］群（ｎ＝４）、およびヌクレオチド添加食餌［ＮＴ(＋)］群（ｎ＝４）、の２群にランダムに分けた。ＮＴ(−)、あるいはＮＴ(＋)を、それぞれ２週間自由摂取させた。ヌクレオチド［Yamasa Co.（Chosi, Japan）］組成を表２に示す。
【００６１】
【表２】

【００６２】また該ヌクレオチドを含みホエータンパク質（ＷＰＩ）［Davisco, International Inc.（Le Sueur, MN, U.S.A）］を基本とした食餌組成を表３に示す。
【００６３】
【表３】

【００６４】食餌の摂取後、小腸を摘出し、ＩＥＬを、Nannoらの方法（Nanno, M. et al.:J. Immunol., 153: 2014-2020, 1994）にしたがって調製した。マウスから小腸を摘出した。腸管腔内容物を洗い流した後、ポリエチレンチューブを貫通させ、一端を固定し、内腔が外側になるように反転した。反転した腸管を４等分し、５％ウシ胎仔血清（ＦＣＳ）を含むＨＢＳＳ４５ｍｌを入れた５０ｍｌコニカルチューブに移し、３７℃で４５分間、水平方向に１３５ｒｐｍ振とうした。振とう後、ガラスウールカラムで濾過して異物を取り除き、細胞を回収した。細胞は、３０％パーコール（Pharmacia, Uppsala, Sweden）に浮遊させ、１８００ｒｐｍで２５分間遠心した。パーコールを用いた密度勾配遠心（１８００ｒｐｍで２５分間遠心）により、４４％と７０％パーコールの境界に集積したＩＥＬを採取した。ＩＥＬの生存ＣＤ３⁺Ｔ細胞は、＞９０％であった。得られたＩＥＬのＴＣＲサブセット構成を、フローサイトメトリー（ＦＡＣＳ）で個体別に解析した。αβ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群における発現率、およびγδ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群における発現率を図１に示す。また、ＮＴ(−)群におけるαβ-ＩＥＬとγδ-ＩＥＬの発現比、およびＮＴ(＋)群におけるαβ-ＩＥＬとγδ-ＩＥＬの発現比を図２に示す。αβ-ＩＥＬの発現率（％）は、ＮＴ(＋)群がＮＴ(−)群のそれよりも有意（ｐ＜０．０５）に低くなり、γδ-ＩＥＬの発現率は、ＮＴ(＋)群がＮＴ(−)群のそれよりも有意（ｐ＜０．０５）に高くなった（図１）。ＮＴ(＋)群のαβ-ＩＥＬとγδ-ＩＥＬの発現比は、ＮＴ(−)群のαβ-ＩＥＬとγδ-ＩＥＬの発現比よりも有意（ｐ＜０．０５）に低下した（図２）。以上の結果、核酸の経口摂取は、αβ-ＩＥＬとγδ-ＩＥＬのバランスを、γδ-ＩＥＬ優位に誘導することが明らかとなった。
【００６５】［試験例２］ＩＥＬのＣＤ４、ＣＤ８サブセット発現に及ぼす影響αβ-ＩＥＬは、ＣＤ８αα⁺、ＣＤ８αβ⁺、ＣＤ４⁺、およびＣＤ４⁺ＣＤ８αα⁺の４つのサブセットに分類されるのに対し、γδ-ＩＥＬは、ほとんどがＣＤ８αα⁺で残りがＣＤ４^-ＣＤ８^-ある（表１参照）。試験例１の、αβ-ＩＥＬとγδ-ＩＥＬのＣＤ４とＣＤ８の発現率を、ＦＡＣＳで個体別に解析した（図３）。結果、αβ-ＩＥＬにおいては、ＮＴ(＋)群のＣＤ８αα⁺の発現比率がＮＴ(−)群のそれよりも有意に低下し、逆にγδ-ＩＥＬにおいては、ＮＴ(＋)群のＣＤ８αα⁺の発現比率がＮＴ(−)群のそれよりも有意に高くなった。一方、αβ-ＩＥＬにおけるＣＤ８αβ⁺の発現比率とγδ-ＩＥＬにおけるＣＤ８αβ⁺の発現比率には差は認められなかった（データは示さない）。また、ＣＤ４⁺、ＣＤ４⁺ＣＤ８⁺、およびＣＤ４^-ＣＤ８^-の発現比率は、ＮＴ(＋)群とＮＴ(−)群で変わらなかった。γδ-ＩＥＬＣＤ４^-ＣＤ８^-は、ＮＴ(＋)群で僅かに増加したが有意差はなかった（データは示さない）。
【００６６】これらの結果は、核酸の経口摂取による、γδ-ＩＥＬサブセットの優位な誘導が、γδ-ＩＥＬにおけるＣＤ８αα⁺の有意(ｐ＜０.０５)な上昇およびαβ-ＩＥＬにおけるＣＤ８αα⁺の有意（ｐ＜０.０５）な低下、と相関していることを示している。
【００６７】［試験例３］ＩＥＬのＴＣＲサブセット発現に及ぼす影響卵白アルブミン（ＯＶＡ）特異的ＴＣＲを発現するＴＣＲトランスジェニックマウス（ＯＶＡＴＣＲ-Ｔｇマウス）（ＯＶＡ２３-３；Sato, T. et al.: Eur.J. Immun., 24: 1512-1516, 1994）を用いた。
【００６８】３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスは、ＮＴ(−)群(ｎ=１０)、およびＮＴ(＋)群(ｎ=７)の２群に分けた。ＮＴ(−)、あるいはＮＴ(＋)を４週間自由摂取させた。この期間中、２％ＯＶＡを含む水を自由摂取させた。その後、ＯＶＡ-ＴＣＲ Ｔｇマウスは殺し、腸を摘出し、試験例１と同様にＩＥＬを調製した。
【００６９】ＩＥＬのＴＣＲサブセットの発現比率（％）、およびαβ-ＩＥＬとγδ-ＩＥＬのＣＤ４とＣＤ８サブセットの発現比率を、ＦＡＣＳで個体別に解析した。αβ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群における発現率、およびγδ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群における発現率を図４に示す。また、αβ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群におけるＣＤ８αα⁺サブセット発現比率、およびγδ-ＩＥＬのＮＴ(−)群とＮＴ(＋)群におけるＣＤ８αα⁺サブセット発現比率、を図５に示す。
【００７０】図５にＮＴ(−)群のαβ-ＩＥＬにおけるＣＤ８αα⁺サブセット発現比率、γδ-ＩＥＬにおけるそれらの発現比率をそれぞれ示す。抗原を摂取したＯＶＡ-ＴＣＲ Ｔｇマウスの場合、ＩＥＬにおけるαβ-ＩＥＬサブセットの発現比率が、通常のＢＡＬＢ／ｃマウスに比較して、図１と図４に示すように、ＮＴ(−)群においては約４８％から約８３％、ＮＴ(＋)群においては約４０％から約７７％と顕著に増加した。しかし、ＮＴ(＋)群におけるγδ-ＩＥＬの発現比率はＮＴ(−)群におけるそれに比較して有意に高く（図４）、これは、ＮＴ(＋)群のγδ-ＩＥＬにおけるＣＤ８αα⁺の発現比率が、ＮＴ(−)群におけるそれに比較して有意に高い（図５）ことと相関している。
【００７１】［試験例４］ＩＥＬにおける抗原特異的サイトカイン産生に及ぼす影響試験例３におけるＯＶＡ-ＴＣＲ Ｔｇマウスから得たＩＥＬは、１０％非働化ＦＣＳ、ペニシリン１００Ｕ/ｍｌ、ストレプトマイシン１００μg/ｍｌ、メルカプトエタノール５×１０^-5Ｍ、および２ｍＭ Ｌ-グルタミンを含むＲＰＭＩ１６４０培地（Nissui, Pharmaceutical Co., Tokyo）に、５.０×１０⁵細胞/１.０ｍｌ浮遊させ、９６ウエルプレートに、それぞれ、０.２ｍｌ/ウエルまいた（duplicate）。
【００７２】一方、抗原提示細胞として用いる脾細胞は、以下のように調製した。未感作のＢＡＬＢ/ｃマウスから脾臓を無菌的に摘出し、細かくきざんだ。細胞（１×１０⁸細胞）は、５０μg/ｍｌのマイトマイシンＣ（SIGMA, St. Louis, MO, U.S.A）を含む１ｍｌのＲＰＭＩ １６４０培地培養液中で、３７℃、４５分間インキュベートした。細胞は、0.８３％ＮＨ₄Ｃｌ溶液に浮遊させ、赤血球を溶解除去した。つぎに、細胞浮遊液は、ステンレススチールのメッシュに通し、均一の細胞浮遊液を得た。細胞は、ＲＰＭＩ-１６４０培地で２回洗浄した。マイトマイシンＣ処理した脾細胞（５×１０⁵細胞）とＴ細胞とを、５０μＭ濃度のＯＶＡ（Seikagaku Co.）の存在下、あるいは非存在下、５％ＣＯ₂/９５％ａｉｒの湿潤条件下で、３７℃２日間培養した。培養後、培養上清は、遠心して集め、−２５℃で貯蔵した。上清中のＩＦＮ-γ、およびＩＬ-２レベルをＥＬＩＳＡ法で測定した。ＩＦＮ-γについて図６に、ＩＬ-２について図７に示す。ＯＶＡ特異的ＩＦＮ-γの産生増強が、ＮＴ(−)群と比較して、有意な差はないがＮＴ(＋)群で観察された。ＮＴ(＋)群におけるＯＶＡ特異的ＩＬ-２産生は、ＮＴ(−)群よりも有意（ｐ＜０.０５）に高かった。
【００７３】［試験例５］腸管上皮細胞のＩＬ-７産生に及ぼす影響３週齢のＢＡＬＢ/ｃマウスにＮＴ(−)(ｎ＝８)とＮＴ(＋)(ｎ＝９)をそれぞれ２週間自由摂取させた。その後、小腸を摘出し小腸上皮細胞を調製した。小腸上皮細胞の培養は、Perreaultらの方法（Perreaul, N. and Beaulieu, J. F. :Exp. Cell. Res., 245: 34-42, 1998）にしたがった。小腸を縦に開き、ＰＢＳで洗浄し、４つのセグメントにカットした。これらのセグメントは、１０ｍｌのice-cold MatriSperse(Collaborative BiomedicalProducts, Becton Dickinson Lab.)を含む１５-ｍｌチューブに移した。小腸は、４℃で８〜１０時間攪拌なしでインキュベートした。つぎに、各チューブは、穏やかに振って上皮細胞を分離した。浮遊液は、ＰＢＳで２回洗浄（４℃、１２００ｒｐｍ、８分間）した。１０％ＦＣＳ、ペニシリン１００Ｕ/ｍｌ、ストレプトマイシン１００μｇ/ｍｌ、メルカプトエタノール５×１０^-5Ｍおよび２ｍｌ-グルタミンを添加した９６ウエルプレート中のＲＰＭＩ １６４０培地に浮遊させた。これらの上皮細胞は、５％ＣＯ₂インキュベーターに入れ、３７℃で１６時間培養した。培養上清のＩＬ-７レベルは、Sharmaらの方法（Sharma, S. et al.: Gene Therapy., 4: 1361-1370, 1997）にしたがい、ＥＬＩＳＡ法で測定した（図８）。ＮＴ(＋)群において、小腸上皮細胞のＩＬ-７産生が、ＮＴ(−)群のそれに比較して有意（ｐ＜０.０１）に増強していることが認められる。すなわち、核酸の経口摂取は小腸上皮細胞のＩＬ-７産生を増強することが明らかとなった。
【００７４】［試験例６］核酸の経口摂取が腸管上皮細胞のＩＬ-７およびＴＧＦ-β産生に及ぼす影響３週齢の雌性ＯＶＡ-ＴＣＲ ＴｇマウスにＮＴ(−)とＮＴ(＋)をそれぞれ４週間自由摂取させた。実験期間中、２％ＯＶＡを添加した水を自由に摂取させた。その後、小腸を摘出し、４℃で８時間マトリスパーゼ中に静置した。静置後、軽く振とうし、腸管を除き、マトリスパーゼ中の小腸上皮細胞を２回洗浄した。洗浄後、小腸上皮細胞を１０％ＦＣＳ含有ＲＰＭＩ-1640培地またはＦＣＳを含まないＲＰＭＩ-1640培地で１６時間培養した。培養後、上清中のＩＬ-７およびＴＧＦ-βをＥＬＩＳＡで測定した。その結果、小腸上皮細胞のＩＬ-７産生は、ＮＴ(+)食群の方がＮＴ(-)食群に比べて有意（ｐ＜０.０１）に高くなった（図９）。また、小腸上皮細胞のＴＧＦ-β産生も、ＮＴ(+)食群の方がＮＴ(-)食群に比べて有意（ｐ＜０.０５）有意に高くなった（図１０）。
【００７５】［試験例７］抗原特異的ＩｇＡ抗体産生に及ぼす影響３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウス にＮＴ(−)（ｎ＝１８）とＮＴ(＋)（ｎ＝１６）を自由摂取させた。実験期間中、２％ＯＶＡを添加した水を自由に摂取させた。６、７、８週齢で糞便を採取し、ＯＶＡ特異的ＩｇＡ抗体をＥＬＩＳＡ法で測定した。結果を図１１に示す。ＯＶＡ特異的ＩｇＡ抗体は、８週齢でＮＴ(＋)食群の方が有意に高くなった。したがって、核酸の経口摂取は、腸管免疫系の抗原特異的ＩｇＡの産生を増強することが明らかとなった。
【００７６】［試験例８］マクロファージの機能に及ぼす影響Whey Protein Isolate（ＷＰＩ）をタンパク源としたヌクレオチド無添加の食餌 (ＮＴ(-)食) および以下の割合で配合したヌクレオチドを０.４％添加した食餌 (ＮＴ(+)食) をそれぞれ１群６匹でＯＶＡ-ＴＣＲ Ｔｇマウスに３週齢から自由摂取させた。なお、このとき２％ＯＶＡ水溶液を自由摂取させた。チオグリコレートを腹腔投与し、投与後４日後（７週齢）に腹腔マクロファージを採取した。このマクロファージをＬＰＳ（0.1μg/mlおよび1μg/ml）刺激下で２日間培養し、その上清のＩＬ-１２活性をBioassay法で測定した。マウスＩＬ-１２のbioassayは、Skeenら (Skeen, M.J.ら, J Immunol 156:1196 - 1206, 1996) の方法で行った。すなわち、９６ウェルプレートに10μg/mlの抗ＩＬ-１２p40抗体をコーティングした。ウェルを洗浄し、様々な濃度のリコンビナントＩＬ-１２ (rＩＬ-１２) 、脾臓またはマクロファージの培養上清をウェルに加え、プレートを一晩インキュベートした。正常マウスの脾臓細胞を、CO2インキュベーター中で37℃２日間の培養後、培養上清を回収し、ＩＦＮ-γ濃度をELISAで測定した。既知のrＩＬ-１２濃度とＩＦＮ-γ濃度との検量線から上清中のＩＬ-１２濃度を外挿した。コーティングした抗体に、ＩＬ-１２p40とp70が結合するが、ＩＬ-１２p70だけがＩＦＮ-γ産生を誘導できる。その結果、ＬＰＳ濃度が0.1μg/mlでも1μg/mlいずれでも、ＩＬ-１２活性については、ＮＴ(+)食群の方が、ＮＴ(-)食群より有意に高くなった (図１２)。
【００７７】［試験例９］マクロファージの機能に及ぼす影響８週齢の雌性ＢＡＬＢ/cマウスにチオグリコレートを腹腔投与し、４日後（７週齢）、腹腔マクロファージを採取した。この腹腔マクロファージをヌクレオチド（０〜１０μg/ml）とともに１日間培養し、その上清中のＩＬ-１２活性をBioassay法で測定した。その結果、0.３２〜１０μg/mlのヌクレオチドを添加したときのＩＬ-１２活性は、ヌクレオチドを添加していない場合に比べて、有意差は見られないものの高くなる傾向が見られた(図１３)。
【００７８】［試験例１０］脾細胞におけるサイトカイン産生に及ぼす影響Whey Protein Isolateをタンパク源としたヌクレオチド無添加の食餌 (ＮＴ(-)食) および上記の割合で配合したヌクレオチドを０.４％添加した食餌 (ＮＴ(+)食) をそれぞれ3週齢のＯＶＡ-ＴＣＲ Ｔｇマウス（１群６匹）に、２％ＯＶＡ水溶液とともに自由摂取させ、７週齢で脾臓細胞を採取した。この脾臓細胞をＯＶＡ（100μg/ml）刺激下で2日間培養し、その上清のＩＬ-１２活性をBioassay法で測定した。その結果、脾臓細胞のＩＬ-１２活性については、ＮＴ(+)食群の方が、NT(-)食群より有意に高くなった (図１４)。
【００７９】
【実施例】 以下、実施例により本発明を具体的に説明するが、本発明は、これらの実施例に限定されるものではない。
【００８０】［実施例１］核酸関連物質として、下記の配合割合の核酸混合物を0.０１％の割合で市販の育児用調製粉乳（明治乳業 (株) 製）に配合した。
シチジン5' - 一リン酸 1.62g/kgグアノシン5' - 一リン酸 0.57g/kgイノシン5' - 一リン酸 1.10g/kgウリジン5'-一リン酸 0.71g/kg粉乳１gあたり、0.1mg、好ましくは0.01〜4mgを使用すればよい。また、一般に核酸関連物質の構成成分である塩基は、魚介類や肉などの食品に含まれているので安全である。したがって、上記範囲を越えて使用しても何ら差し支えはないし、予防ないし保健を目的とする場合は、上記範囲よりも少量使用してもよい。また、育児用粉乳以外の飲食品を調製する場合も、上記範囲を参考にしてヌクレオチド、ヌクレオシド、核酸 または塩基の使用量を定めればよい。
【００８１】［実施例２］アレルゲンとなるタンパク質を酵素分解した育児用調製粉乳（明治乳業 (株) 製; 調製粉乳A、調製粉乳B）に、ヌクレオチド、ヌクレオシド、核酸(RNA、DNA) またはその構成成分である塩基の混合物0.01%を配合した。この場合、調製粉乳Aは乳清タンパク質を酵素分解し、調製粉乳Bはカゼインを酵素分解したものである。しかし、酵素分解するタンパク質に制限はないし、分解に用いる酵素にも制限はない。製品の配合組成は下記表1の通りである。なお、ヌクレオチドの配合割合は実施例1の通りである。なお、本発明による育児用粉乳を製造するにあたり、核酸関連物質の構成成分である塩基は、上記の使用量を１例として使用することができるが、本発明においては、粉乳1gあたり、0.1mg、好ましくは0.01〜4mgを使用すればよい。また、上記範囲を越えて使用しても何ら差し支えはないし、予防ないし保健を目的とする場合は、上記範囲よりも少量使用してもよい。
【００８２】［実施例３］ビタミンC40gまたはビタミンCとクエン酸の等量混合物40g、グラニュー糖100g、コーンスターチと乳糖の等量混合物60gに、ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基を40g加えて十分に混合した。混合物を袋に詰め、1袋1.5gのスティック状栄養健康食品を100袋製造した。
【００８３】［実施例４］次の配合によりTCRγδ陽性Ｔ細胞とTCRαβ陽性Ｔ細胞の比率を高める製剤を製造した。(1)ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基50g、(2)ラクトース90g、(3)コーンスターチ29g、(4)ステアリン酸マグネシウム1g。先ず、(1)、(2)、(3) (但し17g) を混合し、(3) (但し7g) から調製したペーストとともに顆粒化した。得られた顆粒に (3) (但し5g) と (4) を加えてよく混合し、この混合物を圧縮錠剤機により圧縮して、1錠あたりヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基を10mg含有する錠剤100個を製造した。投与量は、患者の症状、年齢によっても異なるが、0.1〜1500mg/kg/dayで1日1〜4回投与する。本発明において用いるヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)、塩基は、本来食品由来のものであり、既述のように安全性にはほとんど問題はなく、したがって、上記容量を越えて、投与しても差し支えはない。また、健康の維持増進、保健栄養剤等としてこれを利用する場合は、上記容量より少ない量を長期間にわたって服用すればよい。また、既述のように本発明による錠剤は、経口投与以外の方法でも投与することができるが、静脈投与および筋肉投与の場合は0.01〜1200mg/kg/dayである。
【００８４】［実施例５］次の配合を用意した。(1)ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基 1g、(2)塩化ナトリウム 8g、(3)クロロブタノール 4g、(4)炭酸水素ナトリウム 1g。全成分を蒸留水1000mlに溶解し、これを500mlの点滴ビン2本に分注し、TCRγδ陽性Ｔ細胞とTCRαβ陽性Ｔ細胞の比率を高め、IgA抗体の産生を促進する製剤を製造した。
【００８５】［実施例６］次の配合を用意した。(1)ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基 0.5g、(2)殺菌乳 1l 。(1)を(2)に無菌的に混合し、ビン詰めした。本発明においては、殺菌乳 1l あたり、(1)を 0.01〜10gを混合すればよい。
【００８６】［実施例７］実施例1の配合割合のヌクレオチドを有効成分として用い、下記に示す組成にて皮膚外用剤を調製した。処方例の配合中、「適量」とは、全体で100％重量になる量を意味する。
【００８７】
処方例１ クリーム（１）
Ａ （重量％） モノステアリン酸 ポリエチレングリコール（40.E.O) ２.０ 自己乳化型モノステアリン酸グリセリン ５.０ ステアリン酸 ５.０ ベヘニルアルコール １.０ 流動パラフィン １０.０ トリオクタン酸グリセリル 1 ０.０ ビタミンE ０.１ ヌクレオチド １.０Ｂ グリセリン ５.０ エチルパラベン ０.１ 精製水 適量Ａに属する成分を加熱溶解する。別に、Ｂに属する成分を加熱溶解する。ＡにＢを添加して撹拌、乳化後、冷却してクリームを製造した。
【００８８】
処方例２ クリーム（２）
Ａ （重量％）
モノステアリン酸 ポリエチレングリコール（40.E.O） ２.０ 自己乳化型モノステアリン酸グリセリン ５.０ ステアリン酸 ５.０ ベヘニルアルコール １.０ 流動パラフィン １０.０ トリオクタン酸グリセリル １０.０ ビタミンE ０.２ ヌクレオチド ０.５Ｂ グリセリン ５.０ エチルパラベン ０.１ 精製水 適量Ａに属する成分を加熱溶解する。別に、Ｂに属する成分を加熱溶解する。ＡにＢを添加して撹拌、乳化後、冷却してクリームを製造した。
【００８９】
処方例３ 乳液Ａ （重量％）
モノステアリン酸 ポリオキシエチレンソルビタン（20.E.O） １.０ モノステアリン酸 ポリオキシエチレンソルビタン（60.E.O） ０.５ 親油型モノステアリン酸グリセリン １.０ ステアリン酸 ０.５ ベヘニルアルコール ０.５ アボガド油 ４.０ トリオクタン酸グリセリル ４.０ ヌクレオチド ５.０Ｂ １, ３ - ブチレングリコール ５.０ エチルパラベン ０.１ 精製水 適量Ａに属する成分を加熱溶解する。別に、Ｂに属する成分を加熱溶解する。ＡにＢを添加して撹拌、乳化後、冷却して乳液を製造した。
【００９０】
処方例４ 化粧水Ａ （重量％）
ポリオキシエチレン硬化ヒマシ油（60.E.O） ８.０ エタノール １５.０ ヌクレオチド ０.１ エチルパラベン ０.１ クエン酸 ０.１ クエン酸ナトリウム ０.３ １, ３ - ブチレングリコール ４.０ エデト酸二ナトリウム ０.０１ 精製水 適量上記の各成分を混合、均一に撹拌、溶解し、化粧水を製造した。
【００９１】
処方例５ 親水性軟膏Ａ （重量％）
ポリオキシエチレンセチルエーテル ２.０ グリセリンモノステアレート １０.０ 流動パラフィン １０.０ ワセリン ４.０ セタノール ５.０ ヌクレオチド ３.０Ｂ プロピレングリコール １０.０ メチルパラベン ０.１ 精製水 適量Ａに属する成分を加熱溶解する。別に、Ｂに属する成分を加熱溶解する。ＡにＢを添加して撹拌、乳化後、冷却して親水性軟膏を製造した。本発明においては、実施例1の配合割合のヌクレオチドだけでなく、ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基いずれを用いてもよい。また、上記範囲を越えて使用しても何ら差し支えはないし、上記範囲よりも少量使用してもよい。好ましくは、皮膚外用剤1gあたり、ヌクレオチド、ヌクレオシド、核酸 (RNA、DNA)またはその構成成分である塩基を0.1〜100mg使用すればよい。
【００９２】
【発明の効果】核酸組成物の経口摂取により、ＩＥＬにおけるαβ-ＩＥＬとγδ-ＩＥＬのサブセット構成比がγδ-ＩＥＬが高められる方向にシフトすることが明らかにされた。さらに、核酸組成物の経口摂取により、腸管上皮におけるサイトカイン（ＩＬ-２、ＩＬ-７、ＩＦＮ-γ、ＴＧＦ-βなど）の産生が亢進することが明らかにされた。さらに、腸管における抗原特異的分泌型ＩｇＡ抗体の産生が亢進することが明らかにされた。これらの結果、有効量の核酸の経口摂取により、腸管免疫系に抗原特異的な分泌型ＩｇＡ抗体産生を促進して、有害な病原体などに対する防御能を高め、一方、抗原特異的な経口免疫寛容を効果的に誘導し、さらに、Ｔｈ１/Ｔｈ２細胞のバランスの破綻を改善し、結果、腸管免疫系、あるいは全身免疫系を賦活化することが期待される。



【図面の簡単な説明】
【図１】 ３週齢の雌性ＢＡＬＢ/ｃマウスに、ヌクレオチド非添加食餌［ＮＴ(−)］、あるいはヌクレオチド添加食餌［ＮＴ(＋)］を、２週間自由摂取させた後の、ＮＴ(−)群におけるＴＣＲαβ⁺ＩＥＬとＴＣＲγδ⁺Ｔ細胞との構成比率（％）、およびＮＴ(＋)群におけるＴＣＲγδ⁺Ｔ細胞とＴＣＲγδ⁺Ｔ細胞との構成比率（％）、をそれぞれ示す（平均値±ＳＥ;ｐ＜０.０５）。
【図２】 同上の、ＮＴ(−)群におけるＩＥＬのＴＣＲαβ⁺Ｔ細胞とＴＣＲγδ⁺ＩＥＬとの比、およびＮＴ(＋)群におけるＴＣＲαβ⁺Ｔ細胞とＴＣＲγδ⁺Ｔ細胞との比、をそれぞれ示す（平均値±ＳＥ;^*ｐ＜０.０５）。
【図３】 同上のＩＥＬのＣＤ４分子およびＣＤ８分子の発現パターンのうち、ＮＴ(−)群におけるＴＣＲαβ⁺ＣＤ８αα⁺Ｔ細胞とＴＣＲγδ⁺ＣＤ８αα⁺ＩＥＬの構成比率（％）、およびＮＴ(＋)群におけるＴＣＲαβ⁺ＣＤ８αα⁺Ｔ細胞とＴＣＲγδ⁺ＣＤ８αα⁺Ｔ細胞との構成比率（％）、をそれぞれ示す（平均値±ＳＥ;^*ｐ＜０.０５）
【図４】 ３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスに、ＮＴ(−)、あるいはＮＴ(＋)を、２％のＯＶＡ添加した水とともに、４週間自由摂取させた後のＮＴ(−)群におけるＴＣＲαβ⁺Ｔ細胞とＴＣＲγδ⁺Ｔ細胞との構成比率（％）、およびＮＴ(＋)群におけるＴＣＲαβ⁺Ｔ細胞とＴＣＲγδ⁺Ｔ細胞との構成比率（％）、をそれぞれ示す（平均値±ＳＥ;ｐ＜０.０５）。
【図５】 同上のＩＥＬのＣＤ４分子、およびＣＤ８分子の発現パターンのうち、ＮＴ(−)群におけるＴＣＲαβ⁺ＣＤ８αα⁺Ｔ細胞とＴＣＲγδ⁺ＣＤ８αα⁺Ｔ細胞の構成比率（％）、およびＮＴ(＋)群におけるＴＣＲαβ⁺ＣＤ８αα⁺Ｔ細胞とＴＣＲγδ⁺ＣＤ８αα⁺Ｔ細胞の構成比率（％）、をそれぞれ示す（平均値±ＳＥ;^*ｐ＜０.０５）
【図６】 ３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスに、ＮＴ(−)、あるいはＮＴ(＋)を、２％のＯＶＡを添加した水とともに、４週間自由摂取させた後のＮＴ(−)群におけるＩＥＬのインターフェロン-γ（ＩＦＮ-γ）産生、あるいはＮＴ(＋)群におけるＩＦＮ-γ産生をそれぞれ示す（平均値±ＳＥ）。
【図７】 同上のＮＴ(−)群におけるＩＥＬのインターロイキン２（ＩＬ-２）産生、あるいはＮＴ(＋)群におけるＩＬ-２産生をそれぞれ示す。（平均値±ＳＥ;ｐ＜０.０５）。
【図８】 ３週齢の雌性ＢＡＬＢ/ｃマウスに、ＮＴ(−)、あるいはＮＴ(＋)を２週間自由摂取させた後のＮＴ(−)群における小腸上皮細胞のインターロイキン７（ＩＬ-７）産生、あるいはＮＴ(＋)群における小腸上皮細胞のインターロイキン７（ＩＬ-７）産生をそれぞれ示す。（平均値±ＳＥ;ｐ＜０.０１）。
【図９】 経口摂取されたヌクレオチドが、OVA-TCR Tgマウス (n=10) の小腸上皮細胞のＩＬ-７産生に与える影響（**; p <０.０１）を示す。(平均値±ＳＤ)
【図１０】 経口摂取されたヌクレオチドがＯＶＡ-ＴＣＲ Ｔｇマウス(n=4) のＩＥＬのＴＧＦ-β産生に与える影響（*; p <０.０５）を示す。(平均値±ＳＤ)
【図１１】 ３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスに、ＮＴ(−)、あるいはＮＴ(＋)を、２％のＯＶＡ添加した水とともに、自由摂取させ、６、７、および８週齢で、糞中のＯＶＡ特異的なＩｇＡ抗体量を測定した結果を示す（平均値±ＳＥ;p＜０.０５）
【図１２】 ３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスに、ＮＴ(−)、あるいはＮＴ(＋)を、２％のＯＶＡ添加した水とともに４週間自由摂取させ後（７週齢）の腹腔マクロファージのＩＬ-１２産生を示す（平均値±ＳＥ;p＜０.０５）。
【図１３】 ヌクレオチド存在下で培養した８週齢の雌性ＢＡＬＢ/ｃマウスの腹腔マクロファージのＩＬ-１２産生を示す（平均値±ＳＥ）。
【図１４】３週齢の雌性ＯＶＡ-ＴＣＲ Ｔｇマウスに、ＮＴ(−)、あるいはＮＴ(＋)を、２％のＯＶＡ添加した水とともに４週間自由摂取させ後（７週齢）の脾細胞のＩＬ-１２産生を示す（平均値±ＳＥ;p＜０.０５）。
[Document Name] Statement
[Title of the Invention] Immunostimulating composition
[Claims]
1. An immunostimulating food or drink comprising a nucleic acid composition as an active ingredient.
2. The food or drink according to claim 1, wherein the nucleic acid composition is a nucleic acid composition selected from the group consisting of a high molecular nucleic acid, a polynucleotide, a nucleotide, a nucleoside, and a nucleobase.
3. The food or drink according to claim 1, wherein the nucleic acid composition is a mixture of nucleotides and nucleosides.
4. The method according to claim 1, wherein the nucleotide is cytidylic acid, guanylic acid, adenyl. acid The food or drink according to claim 3, comprising at least one, or at least two of uridylic acid and inosinic acid.
5. The food or drink according to claim 3, wherein the nucleoside contains at least one, or two or more of cytidine, guanosine, adenosine, uridine, and inosine.
6. The immunostimulatory action shifts the subset composition ratio of αβ-IEL and γδ-IEL of intestinal interepithelial T lymphocytes (IEL) in the intestinal immune system in a direction to increase γδ-IEL. Food and drink as described in.
7. The food or drink according to claim 1, wherein the immunostimulatory effect is enhancement of production of IFN-γ, IL-2, IL-7, and TGF-β in intestinal epithelium.
8. The food or drink according to claim 1, wherein the immunostimulatory effect is an increase in IL-12 production in macrophages and splenocytes.
9. The food or drink according to any one of claims 6 to 8, wherein the immunostimulating effect is enhancement of cellular immunity.
10. The method according to claim 1, wherein the immunostimulatory action is an intestinal action, an antitumor action, an antimutant action, a blood pressure lowering action, an antiulcer action, and a lipid metabolism improving action. Food and drink.
11. The method according to claim 1, which is selected from the group consisting of foods, health foods, functional foods, dietary supplements, foods for specified health use, enteral nutritional foods, enteral nutritional supplements, and quasi-drugs. Food and drink described in Crab.
12. Use of the nucleic acid composition for producing the food or drink according to claim 11.

DETAILED DESCRIPTION OF THE INVENTION
[0001]
TECHNICAL FIELD The present invention relates to a food or drink having an immunostimulatory effect containing a nucleic acid composition as an active ingredient, and to the use of the nucleic acid composition for producing the food or drink.
[0002]
2. Description of the Related Art The intestinal tract is a digestive organ that digests and absorbs food to obtain necessary nutrients and energy, and suppresses an immune response to beneficial food components as much as possible, while preventing harmful pathogenic microorganisms and various toxins. On the other hand, it is also a first-class immune organ that rapidly elicits an immune defense response.
[0003] Intestinal immune organs are collectively referred to as gut-associated lymphoid tissue (GALT). GALT consists of Peyer's patches, mesenteric lymphocytes, lamina propria, and intestinal epithelial cells, working in concert. Among them, Peyer's patch and intestinal epithelium are considered to be particularly important as antigen entry routes.
In the intestinal immune system, a typical defense mechanism against harmful pathogenic microorganisms and toxins is a local immune response mainly based on IgA antibody production, and a typical mechanism for receiving beneficial foods is oral immunity. Tolerant.
[0005] IgA antibodies function to prevent invasion of pathogenic bacteria from the intestinal mucosa, neutralize toxins, and prevent invasion of allergens. These antigens are taken in from Peyer's patches, and B cells differentiate into IgA antibody-producing cells by the action of antigen-specific T cells, and the B cells migrate to the lamina propria and differentiate into IgA antibody secreting cells.
[0006] On the other hand, the intestinal epithelium, which is considered as another route of antigen entry, is composed of intestinal epithelial cells (IEC) and intestinal epithelium, which is a huge T cell population existing in a form embedded between them. It consists of lymphocytes (intraepithelial lymphocytes: IEL). IELs are mostly T cells. There are two types of molecules that T cells recognize antigens, that is, TCR molecules: αβ TCR composed of α and β chains and γδ TCR composed of γ and δ chains. IEL T cells differ greatly in properties from cells that make up Peyer's patches and lamina propria. Many of them are not dependent on the thymus and are presumed to have undergone unique selection of the intestinal tract, and many express γδ TCR (γδ-IEL) or CD8αα homodimer which hardly exist in the periphery. The antigens and functions currently recognized by the IEL have not been clarified. However, it is readily conceivable that the small intestinal epithelium at the forefront of the intestinal immune response plays an important role in the characteristic intestinal immune response such as induction of local IgA antibody production and oral tolerance. Therefore, with respect to GALT, active studies have been made on the interaction between the epithelial cells constituting the small intestinal epithelium and the IEL, and the interaction between the mucosal cells via the cytokine and its receptor.
[0007] For example, γδ-IEL produces keratinocyte growth factor (KGF) and controls the growth and proliferation of epithelial cells (Boismenu, R. and Havran, WL: Sience, 266: 1253, 1994), TCRδ ^-/- In mice, epithelial cell development is reduced (Komano, H .: et al .: Proc. Natl. Acad. Sci., 92: 6147-6151, 1995). Conversely, interleukin-7 secreted by epithelial cells. (IL-7) is deeply involved as a growth and activator of γδ-IEL (Fujihashi, K .: et al .: Proc. Natl. Acad. Sci., 93: 3613, 1996). 7 and IL-7 receptor-specific genes are deficient in the development and deficiency of these mucosal γδ-IELs. Mouse epithelial cells contain stem cell growth factor (SCF). This SCF is deeply involved in the proliferation and differentiation of γδ-IEL (Puddington, L. et al .: Immunity 1: 733, 1994), and γδ-IEL is synergistically produced by IL-2 and IL-7. Proliferating (Fujihashi, K. et al .: Proc. Naqtl. Acad. Sci., 93: 3613, 1996), TGF-β produced by epithelial cells as a class switch factor to IgA Provides a signal to prompt a gene conversion from μ chain of g gene into α-chain (Sonoda, E. et al .: J. Exp Med, 170:.. 1415, 1989), there are reports on.
[0008] Therefore, it is conceivable that the immune function of the intestinal immune system is enhanced by enhancing cytokine production in the small intestinal epithelium. An oral composition having such a cytokine production-enhancing action is considered to be useful for preventing or improving the function of the intestinal immune system or for activating immune activity. There have been reports on the effects of oral enteral nucleic acid administration or intravenous nucleic acid component administration on immune function (Masayoshi Usami et al. Chemistry and Biology. 36 (10): 620, 1998). There is no report on the effect of oral intake of nucleic acid components on cytokine production in intestinal epithelium.
[0009]
DISCLOSURE OF THE INVENTION The present invention provides a nucleic acid composition which can be expected to improve, prevent, or activate immune function of the intestinal immune system by enhancing cytokine production in the intestinal epithelium. Make it an issue. Another object of the present invention is to provide an oral composition containing an effective amount of the nucleic acid composition.
[0010]
Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, by oral ingestion of the nucleic acid composition, the subset composition ratio of αβ-IEL and γδ-IEL in IEL was It was found that the expression ratio of γδ-IEL was shifted in a direction to increase. In addition, it was found that oral ingestion of the nucleic acid composition promoted the production of IFN-γ, IL-2, IL-7, and TGF-β in the small intestinal epithelium. Furthermore, it has been found that oral ingestion of the nucleic acid composition induces production of an antigen-specific secretory IgA antibody.
That is, the present invention relates to (1) a food or drink having an immunostimulatory effect containing a nucleic acid composition as an active ingredient, and (2) a nucleic acid composition comprising a polymer nucleic acid, a polynucleotide, a nucleotide, a nucleoside, and a nucleobase. (1) the food or drink of (1), which is a nucleic acid composition selected from the group consisting of: (3) the food or drink of (1) or (2), wherein the nucleic acid composition is a mixture of nucleotides and nucleosides; Acid, guanylic acid, adenyl acid (3) a food or drink comprising at least one or two or more of uridylic acid and inosine acid; (5) the nucleoside is at least one or two or more of cytidine, guanosine, adenosine, uridine and inosine; And (6) the immunostimulatory effect increases the subset composition ratio of αβ-IEL and γδ-IEL of intestinal epithelial intercellular T lymphocytes (IEL) in the intestinal immune system, and increases γδ-IEL. (7) Food and drink of (1) wherein the immunostimulatory effect is enhanced production of IFN-γ, IL-2, IL-7, and TGF-β in intestinal epithelium; 8) The food or drink according to (1), wherein the immunostimulatory effect is enhanced production of IL-12 in macrophages and splenocytes, and (9) the food or drink according to any of (6) to (8), wherein the immunostimulatory effect is enhancement of cellular immunity. Goods, ( 0) The food or drink according to any of (1) or (6) to (8), wherein the immunostimulatory action is an intestinal action, an antitumor action, an antimutant action, a blood pressure lowering action, an anti-ulcer action, or a lipid metabolism improving action. (11) Any one of (1) to (11) selected from the group consisting of foods, health foods, functional foods, dietary supplements, foods for specified health uses, enteral nutritional foods, enteral nutritional agents, and quasi-drugs (12) Use of the nucleic acid composition for producing the food or drink of (11). Hereinafter, the present invention will be described in detail.
[0012]
DETAILED DESCRIPTION OF THE INVENTION T cells function by secreting cytokines. When a protein antigen is orally administered, a T cell response is induced, and a cytokine or an antibody-producing helper function to be analyzed is usually administered together with an adjuvant to enhance immune function. In this case, the effect of the adjuvant cannot be denied. The T cell response to oral administration of the protein antigen corresponds to the administration without adjuvant. Recently, it has become possible to analyze the weak T cell response when a protein antigen is orally administered without using an adjuvant by using a TCR-transgenic (TCR-Tg) mouse into which a TCR gene has been introduced. In normal mice, the frequency of antigen-specific T cells is extremely low and analysis is difficult, whereas in TCR-Tg mice, the frequency of T cells that recognize a single antigen is extremely high. Therefore, the present inventors have used TCR-Tg mice expressing a TCR recognizing ovalbumin (OVA) (OVA-TCR Tg mice) in addition to normal mice, and the oral ingestion of nucleic acids has a The effect on cell response was investigated.
Hereinafter, the present invention will be described with reference to Test Examples. However, the present invention is not limited to these Test Examples, and those skilled in the art will be able to further derive the present invention based on the Test Examples. It naturally includes the spread of technology.
1) Effect of oral ingestion of nucleic acid on T cell subset composition in IEL 80-90% of IEL present in the intestinal tract of mice is CD3 ⁺ T cells (this means that more than half of T cells distributed in other biological sites are CD4 ⁺ This is a striking feature considering that it is a T cell), and γδ-IEL is much more common and accounts for about half of peripheral blood lymphocytes. CD3 using anti-CD4 and anti-CD8 antibodies ⁺ When T cells were stained, 1) CD4 ^- CD8 ⁺ Group (CD8 ⁺ ), 2) CD4 ⁺ CD8 ⁺ Group, 3) CD4 ^- CD8 ^- Group, and 4) CD4 ⁺ CD8 ^- Group (CD4 ⁺ ) Can be classified into four subsets. Further, in these four subset groups, αβ-IEL is CD4 ⁺ Group, CD8 ⁺ Group, CD4 ⁺ CD8 ⁺ Γδ-IEL is present in the group ^- CD8 ⁺ Group, CD4 ^- CD8 ^- Found only in the group. The results are shown in Table 1 so that the whole picture of the T cell subset in the mouse IEL can be easily understood.
[0015]
[Table 1]

After a normal mouse was orally ingested with the diet to which the nucleic acid was added for two weeks, the composition of the TCR subset in the IEL was analyzed by flow cytometry (FACS).
The expression rate (%) of αβ-IEL in the nucleic acid ingestion group was significantly (p <0.05) lower than that in the non-nucleic acid ingestion group (control group), and conversely, the expression rate of γδ-IEL Was significantly (p <0.05) higher than that of the control group (FIG. 1). Comparing this with the expression ratio of αβ-IEL / γδ-IEL in the nucleic acid intake group and the expression ratio of αβ-IEL / γδ-IEL in the control group, the nucleic acid intake group is more significant than the control group (p <0). .05) (FIG. 2). These results indicate that the dietary ratio of αβ-IEL and γδ-IEL in the IEL is shifted to a method in which γδ-IEL is increased when the diet containing nucleic acid is orally ingested.
Further, the expression ratio of CD4 and CD8 in each of αβ-IEL and γδ-IEL was analyzed by FACS for each individual.
CD8αα of nucleic acid intake group in αβ-IEL ⁺ Expression rate (%) was significantly (p <0.05) lower than that of the control group, whereas the CD8αα of the nucleic acid intake group in γδ-IEL was ⁺ Was significantly (p <0.05) higher than that of the control group (FIG. 3).
Although data are not shown, CD8αβ in αβ-IEL was compared between the nucleic acid intake group and the control group. ⁺ No difference was observed in the expression ratio of CD4 ⁺ , CD4 ⁺ CD8 ⁺ , The expression ratio was the same in the nucleic acid ingestion group and the control group, and CD4 in γδ-IEL ^- CD8 ^- Was slightly increased in the nucleic acid ingestion group, but was not significantly different from the control group. Therefore, in the nucleic acid ingestion group, CD8αα in αβ-IEL was ⁺ Of CD8αα in γδ-IEL ⁺ It is considered that this indicates that there is a correlation between the increase in the composition ratio of αβ-IEL and γδ-IEL in the direction in which the expression ratio of γδ-IEL is increased. Therefore, it is considered that oral ingestion of nucleic acid promotes γδ-IEL development and differentiation outside the thymus.
In addition, the effect of oral intake of protein antigens on the expression of the TCR subset and on CD4 and CD8 expression in the IEL was demonstrated by the T cell antigen receptor (TCR) specific for the major food allergen, ovalbumin (OVA). ) Were examined in transgenic mice (OVA-TCR Tg).
OVA with a diet containing nucleic acids To IELs were prepared from OVA-TCR Tg mice that had free access to water containing them, and TCR subset composition and expression of CD4 and CD8 subsets in αβ-IEL and γδ-IEL were analyzed individually by FACS.
Even in the presence of an oral antigen, the composition ratio of αβ-IEL and γδ-IEL was significantly (p <0.05) shifted in the direction of increasing γδ-IEL by oral ingestion of nucleic acid (FIG. 4). . CD8αα ⁺ Expression rate (%) of CD8αα in γδ-IEL ⁺ Was significantly (p <0.05) higher than that of the control group (FIG. 5). However, the expression ratio of αβ-IEL in both the nucleic acid ingestion group and the control group was significantly increased as compared with the normal mouse (see FIGS. 1 and 4).
On the other hand, Hachimura et al. Reported that OVA-specific CD4 ⁺ -It has been clarified that IEL proliferates and its ratio in the whole IEL increases (Toshishi Yamura, et al .: Journal of the Japanese Society of Agricultural Chemistry, 73 (5): 523-527, 1999).
2) Influence of oral ingestion of nucleic acid on cytokine production in intestinal epithelium IEL prepared from OVA-TCRTg mice that freely ingested water containing OVA together with a diet supplemented with nucleic acid, and unsensitized BALB / c Prepared from mouse Might When co-cultured with mycin C-treated splenocytes (antigen presenting cells) in the presence or absence of OVA, enhanced production of OVA-specific IFN-γ and IL-2 was observed in the nucleic acid-ingested group. (FIGS. 6 and 7).
This is thought to indicate that oral ingestion of nucleic acids induces a proliferative response of antigen-specific Th1-type cells in the IEL.
Oral ingestion of nucleic acids significantly (p <0.01) enhances IL-7 production in mouse intestinal epithelial cells (FIG. 8).
In addition, IL-7 production and TGF-β production of small intestinal epithelial cells of OVA-Tg mice, which had freely consumed water containing OVA together with the diet to which the nucleic acid was added, were significantly (p <0.01) due to the nucleic acid ingestion. ) (FIGS. 9 and 10).
3) Influence of oral ingestion of nucleic acid on antigen-specific IgA antibody production Oral ingestion of nucleic acid significantly (p <0.sup.0) the production of antigen-specific IgA antibody in the intestinal tract as compared to the control group. 05) (FIG. 11).
Mice were orally ingested with nucleotides, and the effect of spleen cells and peritoneal macrophages on p70-type IL-12 was examined by a biological assay (Bioassay method). In addition, macrophages were cultured with nucleotides in vitro, and IL-12 activity in the supernatant was measured by the Bioassay method. The IL-12 activity of peritoneal macrophages under the LPS stimulation was significantly higher in the nucleotide administration group than in the nucleotide non-administration group (FIG. 12). In addition, when intraperitoneal macrophages were cultured with nucleotides in vitro, the IL-12 activity tended to be higher than in the case where nucleotides were not added, although there was no significant difference (FIG. 13).
The IL-12 activity of spleen lymphocytes was significantly higher in the nucleotide administration group than in the nucleotide non-administration group (FIG. 14).
As described above, oral ingestion of the nucleic acid composition promotes the production of IL-12 by macrophages and the like, and promotes the differentiation of naive T cells into Th1 cells and the production of IFN-γ from peripheral blood. Is expected to shift the Th1 / Th2 balance in vivo to Th1 dominance. As a result, the Th1 / Th2 balance is disrupted, and the mutation is predominantly Th2. To Prevention, improvement, or treatment of diseases caused by, for example, allergic diseases and autoimmune diseases can be expected by ingesting a food or drink containing the nucleic acid composition in a daily diet.
Further, IL-12 induces the production of IFN-γ from quiescent T cells and NK cells, enhances NK cell activity, induces LAK cell activity, and enhances cell proliferation of quiescent T cells by lectin stimulation. It is known that, by ingesting a food or drink containing a nucleic acid composition in a daily diet, the ability to eliminate microorganisms and tumor cells in vivo is enhanced, and the production of IFN-γ is enhanced. It is expected to enhance protection against viruses and tumor cells.
From these results, it was found that oral ingestion of nucleic acid 1) predominantly increased the composition ratio of αβ-IEL subset and γδ-IEL subset of T cells in IEL to γδ-IEL, indicating that CD8αα in γδ-IEL ⁺ Of CD8αα in αβ-IEL ⁺ 2) promotes the production of antigen-specific IL-2 and IFN-γ in the IEL, 3) promotes the production of IL-7 and TGF-β in intestinal epithelial cells, 4) induces antigen-specific IgA antibody production; and 5) enhances IL-12 production.
On the other hand, the present inventors have found that mice that orally ingested a diet containing nucleotides had the following effects: 1) the ability of Peyer's patch T lymphocytes to respond to mitogen stimulation of proliferation and IgA production under stress (fasting); 2) that serum IgE production is suppressed; 3) that IgG1 / IgG2a is reduced; 4) IFN-γ production of splenocytes is promoted, and IL-4 production is suppressed; (JP-A-9-323979). Further later, we found that in the sera of mice orally ingesting a diet containing nucleotides, 1) casein-specific IgG1 did not change, but IgG2a increased, 2) ovalbumin (OVA) -specific. IgG1 did not change, but IgG2a increased, and OVA-specific IgE decreased. 3) Total IgE in serum decreased (JP-A-11-35468).
On the other hand, γδ-IEL produces KGF and regulates growth and proliferation of small intestinal epithelial cells (Boismenu, R. & Havran, WL: Science, 266: 1253, 1994; Komano, H. et al .: Natl. Acad. Sci., USA, 92: 6147, 1995), promote secretory IgA production (Fujihashi, K. et al .: J. Exp. Med., 183: 1929, 1996), oral immunization Necessary for induction of tolerance (Ke, Y. et al .: J. Immunol., 158: 3610-3618, 1997), indicating that γδ-IEL expression increases in IEL of The shift is thought to be important for activation of the intestinal immune system.
IFN-γ produced by Th1 cells is deeply involved in the production of a secretory component (SC) expressed on the basement membrane side of epithelial cells (Taguchi, T. et al .: J. Immunol., 3736, 1991). The SC and the dimeric IgA produced by the IgA plasma cells combine to form secretory IgA.
Further, IL-7 in epithelial cells plays an important role in the differentiation and proliferation of γδ-IEL via the IL-7 receptor of γδ-IEL (IL-7R) (Fujihashi, K. et al. : Eur. J. Immunol., 27: 2133, 1997). Furthermore, IL-2 and IL-7 act as activators for thymus-derived γδ-IEL and αβ-IEL (Okazaki, et al .: J. Immunol., 143: 2917, 1989).
TGF-β is also known as an immunosuppressive cytokine, and it has been suggested that T cells secreting TGF-β suppress immune responses as regulatory T cells in oral tolerance. (Toshishi Yamura, et al .: Journal of the Japanese Society of Agricultural Chemistry, 73 (5): 523-527, 1999). In addition, TGF-β is known to perform class switching to IgA (Sonoda, E. et al .: J. Exp. Med., 170: 1415, 1989), and is also involved in IgA in the intestinal tract. Is suggested.
By the way, CD4 + helper T cells produce IL-2 and IFN-γ Th1 cells and IL-4, IL-5 and IL-10 by the cytokine profile produced and the effector function based thereon. It is classified into Th2 cells. This disruption in the balance between Th1 and Th2 has been implicated in the development of various immune diseases such as cancer, infectious diseases, allergies, autoimmune diseases, and atherosclerosis, as well as in stress and immunity, transplant immunity, and pregnancy immunity. It is known to be an important factor. Oral ingestion of nucleic acid is expected to improve the balance of Th1 and Th2 by increasing the expression of Th1 to break the balance toward Th2 dominance.
In conclusion, oral ingestion of an effective amount of nucleic acid promotes production of secretory IgA antibody specific to the intestinal tract immune system and enhances its ability to protect against harmful pathogens. It is expected to effectively induce immune tolerance and further improve the disruption of the Th1 / Th2 cell balance, thereby activating the intestinal immune system or the systemic immune system.
As used herein, the term “effective amount of nucleic acid” refers to, for example, that the expression ratio of γδ-IEL in IEL is increased, and that IFN-γ, IL-2, IL-7, and TGF-β are expressed in intestinal epithelium. Promotes production, promotes production of antigen-specific IgA antibody, suppresses antigen-specific IgE antibody production and total IgE antibody production in serum, suppresses IL-4 production in splenocytes Based on the fact that the ratio of IgG1 / IgG2a decreases, the effective amount can be determined by a known assay, and the effective intake amount can be determined in consideration of clinical symptoms and the like.
The orally ingested nucleic acid is hydrolyzed by ribonuclease and deoxyribonuclease in pancreatic juice to form nucleotides, phosphate is removed by phosphatase to form nucleosides, and further decomposed by nucleosidase to form nucleic acids. It becomes base and pentose. Gastrointestinal absorption is performed as nucleotides and nucleosides.
The base mentioned here is adenine, guanine, hypoxanthine, xanthine, cytosine, uracil and thymine. The nucleoside referred to here is uridine, adenosine, guanosine, cytidine, lipothymidine, deoxyadenosine, deoxyguanosine, deoxyuridine, deoxycytidine, thymidine, inosine, or xanthosine.
The nucleotide herein refers to a compound in which phosphoric acid is bonded to the sugar moiety of a nucleoside by an ester bond. The position of the phosphate to be bonded may be anywhere, and the number of bonded phosphates may be any number. Further, for example, a compound in which one phosphate binds to both the 5 ′ and 3 ′ positions is also included in the nucleotide. Also in this case, the number and position of the phosphate to be bound may be anywhere.
The nucleic acid referred to here is a polynucleotide such as RNA or DNA, or a polynucleotide to which the above-mentioned nucleotides are bound. The nucleic acid of the present invention comprises adenosine-n'-monophosphate, cytidine-n'-monophosphate, guanosine-n'-monophosphate, uridine-n'-monophosphate, and thymidine-n'-monophosphate. , And nucleic acid compositions comprising one or more of inosine-n'-monophosphate. Here, n ′ indicates 2 ′, 3 ′, or 5 ′.
The nucleic acid composition of the present invention contains a specific combination of nucleic acids as an active ingredient, and the combination is shown below in free form. The nucleic acid component complies with IUPAC-IUB regulations or conventional symbols in the art.
That is, each combination of adenosine-phosphate (AMP) / cytidine-phosphate (CMP) / guanosine-phosphate (GMP) / uridine-phosphate (UMP) / inosine-n'-monophosphate (IMP). Each component in these combinations includes known nucleosides, nucleotides, or non-toxic salts thereof.
If the compounding ratio of the nucleic acid component is expressed by, for example, the molar ratio of the nucleic acid, the molar ratio determines the type and the amount of the nucleic acid component in the above-mentioned assay system, and further takes into account the condition of the patient. Can be determined. One example of these preferable combinations is IMP: AMP: GMP: CMP: UMP: AMP = 1 to 4: 1 to 4: 1 to 4: 1 to 4 as a molar ratio. As a further preferred example, GMP: CMP: UMP: IMP = 1: 3 to 4: 1: 1-2: 1 to 3.
The selection or combination of the optimal nucleobases among these nucleobases can be appropriately determined by those skilled in the art using the above-described assay system. Therefore, the nucleic acid composition thus obtained is also included in the present invention.
The origin of the nucleic acid is not limited, and examples thereof include those derived from yeast, bacteria, milk, seafood, animals, and plants. When used as a food or drink type nucleic acid composition, an effective amount of the nucleic acid component (the processed product) can be used as it is, or used in combination with other foods or food components, and used in accordance with a conventional method as appropriate. . The composition according to the present invention using an effective amount of the nucleic acid component may be in the form of a solid (powder, granule or the like), a paste, a liquid or a suspension, but may be a sweetener, an acidulant, a vitamin and other drinks. It is preferable to formulate a health drink using various components commonly used in production.
For the purpose of activating the intestinal tract immune system (antigen-specific IgA antibody production and immune tolerance), an effective amount of nucleic acid is added to foods, health foods, functional foods, dietary supplements, specified health foods, enteral It can be formulated for the production of nutritional foods, pharmaceutical preparations such as enteral nutrition, and quasi-drugs. It is a well-known and conventional technique.
The required daily amount of purine pyrimidine for adults is 450-700 mg, and the maximum safe dose that does not cause accumulation of uric acid is 4 g / day (Rudolph, FB et al .: Nutrition , 6:45, 1990).
[Test Examples] Test examples of the effect of oral ingestion of nucleic acids on the immune system are shown below, but the present invention is not limited to these test examples.
[0056] Statistical processing Statistical significance of the data was determined by Student's test. Values at p <0.05 were considered significant.
[0057] Flow cytometry Primary antibodies include biotinylated anti-mouse TCRβ (H57-597) (PharMingen, San Diego, CA, USA), biotinylated anti-mouse CD4 (H129.19) (PharMingen), and biotinylated anti-mouse IL-2R antibody (CD25) (7D4) (PharMingen). All incubations were performed in the dark. Cells were incubated with primary antibody or isotype control antibody for 30 minutes on ice. After washing the cells with Hank's balanced salt solution, Fluorescein isothiocyanate (FITC) -labeled anti-mouse TCRδ antibody (GL3) (Cedar Lane Lab.), FITC-labeled anti-mouse CD4 (H129.19) (PharMingen), phycoerythrin (PE) -labeled anti-mouse Mouse CD8α (53-6.7) (GIBCO, Grand Island, NY, USA), FITC-labeled anti-mouse CD8β (Y8.77) (Seikagaku Co., Tokyo, Japan), PE-labeled anti-mouse TCRβ, (H87-597) ) (PharMingen), and streptavidin-Red 670 (GIBCO) for 30 minutes on ice. Cells were washed by centrifugation. The stained cells were analyzed for IELs subsets by flow cytometry with three colors (Becton Dickinson FACSort). Lysis II software was used for analysis.
[0058] Cytokine assay (ELISA) A phosphate buffer (pH 7.4) containing 3% polyethylene glycol 6000 (Nacalai Tesque Inc., Japan) was used as an assay buffer (assay buffer). As a washing buffer, a phosphate buffer (pH 7.4) containing 0.05% Tween-20 (PBS-0.05% Tween) was used. IFN-γ and IL-2 were measured by ELISA (enzyme-linked immunosorbent assay). Rat anti-mouse IFN-γ antibody (6A2) (PharMingen), rat anti-mouse IL-2 antibody (JES6-1A12) (PharMingen), biotinylated rat anti-mouse IFN-γ antibody (XMG1.2) (PharMingen), biotin Rat anti-mouse IL-2 antibody (JES6-5H4) (PharMingen) was used. Rat anti-mouse IFN-γ antibody (1 μg / ml) diluted with 0.05 M Tris buffer (pH 8.9) or 0.1 M NaHCO 3 _Three The rat anti-mouse IL-2 antibody (1 μg / ml) diluted in the above was dispensed into each well of a microtiter plate (Nunc, Roskilde, Denmark). After incubation at 4 ° C. overnight, the antibodies were adsorbed to the wells. After washing the wells, an assay buffer containing 5% FCS was added and blocked for 1 hour at room temperature. After washing the wells, the culture supernatant diluted with the assay buffer was added to each well and incubated for 2 hours. After washing the wells, a biotinylated anti-mouse IFN-γ antibody or a biotinylated anti-mouse IL-2 antibody diluted in assay buffer was added to each well and incubated for 2 hours. After washing, alkaline phosphatase-labeled streptavidin (Zymed Lab., South San Francisco, CA, USA) diluted with assay buffer was added to each well for reaction. After washing the wells, a substrate solution [0.1% 4-nitrophenyl phosphate (Tokyo Kasei Co.) dissolved in 0.1 M diethanolamine buffer (pH 9.8)] was added to each well and incubated at room temperature for 1 hour. . The reaction was stopped by adding 5N-NaOH, and the Absorbance value Was measured. IL-7 and TGF-β were measured by ELISA.
0.1 M NaHCO _Three The anti-mouse IL-7 antibody solution (2 μg / ml) (Genzyme) diluted in (1) was dispensed into each well of a microtiter plate (Nunc), incubated at 4 ° C. overnight, and the antibody was adsorbed to the well. After washing the wells, assay buffer containing 10% OVA was added to the wells and blocked for 1 hour at room temperature. After washing the wells, the culture supernatant diluted with assay buffer containing 0.1% Tween 20 was added to the wells and incubated for 2 hours. After washing the wells, the wells were diluted with assay buffer containing 0.1% Tween 20. hand Sheep anti-mouse IL-7 antibody (1 μg / ml) (R and D, Minneapolis, MN, USA) was added to each well and incubated for 2 hours. After washing the wells, a biotinylated anti-sheep IgG antibody diluted with an assay buffer containing 0.1% Tween 20 was added to each well and reacted for 15 minutes. After washing the wells, alkaline phosphatase-labeled streptavidin diluted with assay buffer was added to each well. After washing the wells, the substrate solution was added to each well, as in IFN-γ and IL-2, at 405 nm. Absorbance value Was measured.
[Test Example 1] Effect of IEL on TCR subset expression Three week old female BALB / c mice [SLC, Hamamatsu, Japan] were treated with a nucleotide-free diet [NT (-)] group (n = 4). , And the diet supplemented with nucleotides [NT (+)] (n = 4). NT (-) or NT (+) was freely taken for 2 weeks. The composition of the nucleotide [Yamasa Co. (Chosi, Japan)] is shown in Table 2.
[0061]
[Table 2]

Table 3 shows the dietary composition containing the nucleotides and based on whey protein (WPI) [Davisco, International Inc. (Le Sueur, MN, USA)].
[0063]
[Table 3]

After ingestion of the diet, the small intestine was removed and the IEL was prepared according to the method of Nanno et al. (Nanno, M. et al .: J. Immunol., 153: 2014-2020, 1994). The small intestine was removed from the mouse. After flushing the contents of the intestinal lumen, the polyethylene tube was pierced, one end was fixed, and the tube was inverted so that the lumen was on the outside. The inverted intestinal tract was divided into four equal parts, transferred to a 50 ml conical tube containing 45 ml of HBSS containing 5% fetal calf serum (FCS), and shaken horizontally at 135 ° C. for 45 minutes at 37 ° C. After shaking, foreign substances were removed by filtration through a glass wool column, and the cells were collected. Cells were suspended in 30% Percoll (Pharmacia, Uppsala, Sweden) and centrifuged at 1800 rpm for 25 minutes. IEL accumulated at the border between 44% and 70% Percoll was collected by density gradient centrifugation using Percoll (centrifugation at 1800 rpm for 25 minutes). IEL surviving CD3 ⁺ T cells were> 90%. The TCR subset composition of the obtained IEL was analyzed for each individual by flow cytometry (FACS). FIG. 1 shows the expression rates of αβ-IEL in the NT (−) group and NT (+) group, and the expression rates of γδ-IEL in the NT (−) group and NT (+) group. FIG. 2 shows the expression ratio of αβ-IEL and γδ-IEL in the NT (−) group, and the expression ratio of αβ-IEL and γδ-IEL in the NT (+) group. The expression rate (%) of αβ-IEL in the NT (+) group was significantly (p <0.05) lower than that in the NT (−) group, and the expression rate of γδ-IEL was NT (+). The group was significantly (p <0.05) higher than that of the NT (-) group (FIG. 1). The expression ratio of αβ-IEL and γδ-IEL in the NT (+) group was significantly (p <0.05) lower than the expression ratio of αβ-IEL and γδ-IEL in the NT (−) group (FIG. 2). ). As a result, it was revealed that oral ingestion of nucleic acid induces the balance between αβ-IEL and γδ-IEL to be superior to γδ-IEL.
[Test Example 2] Effect of IEL on CD4 and CD8 subset expression αβ-IEL was CD8αα ⁺ , CD8αβ ⁺ , CD4 ⁺ , And CD4 ⁺ CD8αα ⁺ Whereas γδ-IEL is mostly CD8αα ⁺ And the rest is CD4 ^- CD8 ^- (See Table 1). The expression rates of CD4 and CD8 of αβ-IEL and γδ-IEL in Test Example 1 were analyzed for each individual by FACS (FIG. 3). As a result, in αβ-IEL, CD8αα of NT (+) group ⁺ Is significantly lower than that in the NT (-) group, and conversely, in γδ-IEL, CD8αα in the NT (+) group ⁺ Was significantly higher than that of the NT (-) group. On the other hand, CD8αβ in αβ-IEL ⁺ Expression ratio and CD8αβ in γδ-IEL ⁺ No difference was observed in the expression ratio (data not shown). Also, CD4 ⁺ , CD4 ⁺ CD8 ⁺ , And CD4 ^- CD8 ^- Did not change between the NT (+) group and the NT (-) group. γδ-IELCD4 ^- CD8 ^- Increased slightly but not significantly in the NT (+) group (data not shown).
These results indicate that the predominant induction of the γδ-IEL subset by oral ingestion of nucleic acids indicates that CD8αα in γδ-IEL ⁺ (P <0.05) elevation of CD8αα in αβ-IEL ⁺ (P <0.05).
[Test Example 3] Effect of IEL on TCR subset expression TCR transgenic mice (OVATCR-Tg mice) expressing ovalbumin (OVA) -specific TCR (OVA23-3; Sato, T. et al .: Eur. J. Immun., 24: 1512-1516, 1994).
The 3-week-old female OVA-TCR Tg mice were divided into two groups: an NT (-) group (n = 10) and an NT (+) group (n = 7). They were allowed to freely take NT (-) or NT (+) for 4 weeks. During this period, water containing 2% OVA was freely available. Thereafter, the OVA-TCR Tg mouse was killed, the intestine was excised, and an IEL was prepared in the same manner as in Test Example 1.
The expression ratio (%) of the TCR subset of IEL and the expression ratio of CD4 and CD8 subsets of αβ-IEL and γδ-IEL were analyzed by FACS for each individual. FIG. 4 shows the expression rate of αβ-IEL in the NT (−) group and NT (+) group, and the expression rate of γδ-IEL in the NT (−) group and NT (+) group. CD8αα in the NT (−) group and the NT (+) group of αβ-IEL ⁺ Subset expression ratio and CD8αα in γδ-IEL NT (−) group and NT (+) group ⁺ The subset expression ratio is shown in FIG.
FIG. 5 shows CD8αα in αβ-IEL of the NT (−) group. ⁺ The subset expression ratio and their expression ratio in γδ-IEL are shown, respectively. Ingested antigen In the case of OVA-TCR Tg mice, the expression ratio of αβ-IEL subset in IEL is Normal BALB / c mouse As shown in FIGS. 1 and 4, in the NT (-) group, the increase was remarkably increased from about 48% to about 83%, and in the NT (+) group, from about 40% to about 77%. However, the expression ratio of γδ-IEL in the NT (+) group was significantly higher than that in the NT (−) group (FIG. 4), indicating that CD8αα in γδ-IEL in the NT (+) group. ⁺ Is significantly higher than that in the NT (-) group (FIG. 5).
[Test Example 4] Influence on antigen-specific cytokine production in IEL The IEL obtained from the OVA-TCR Tg mouse in Test Example 3 was 10% inactivated FCS, penicillin 100 U / ml, streptomycin 100 μg / ml, mercapto Ethanol 5 × 10 ^-Five M and 2 mM L-glutamine in RPMI 1640 medium (Nissui, Pharmaceutical Co., Tokyo) at 5.0 × 10 ^Five Cells / 1.0 ml were suspended and plated in 96-well plates at 0.2 ml / well, respectively.
On the other hand, spleen cells used as antigen presenting cells were prepared as follows. The spleen was aseptically removed from unsensitized BALB / c mice and minced. Cells (1 × 10 ⁸ The cells were incubated at 37 ° C. for 45 minutes in 1 ml of RPMI 1640 medium containing 50 μg / ml mitomycin C (SIGMA, St. Louis, MO, USA). Cells are 0.83% NH _Four The cells were suspended in a Cl solution to dissolve and remove erythrocytes. Next, the cell suspension was passed through a stainless steel mesh to obtain a uniform cell suspension. Cells were washed twice with RPMI-1640 medium. Mitomycin C treated splenocytes (5 × 10 ^Five Cells) and T cells in the presence or absence of 50 μM OVA (Seikagaku Co.) at 5% CO 2. _Two The cells were cultured at 37 ° C. for 2 days under a humid condition of / 95% air. After the culture, the culture supernatant was collected by centrifugation and stored at -25 ° C. IFN-γ and IL-2 levels in the supernatant were measured by ELISA. FIG. 6 shows IFN-γ and FIG. 7 shows IL-2. Enhanced production of OVA-specific IFN-γ was observed in the NT (+) group, though not significantly different, as compared to the NT (-) group. OVA-specific IL-2 production in the NT (+) group was significantly (p <0.05) higher than in the NT (-) group.
[Test Example 5] Influence of intestinal epithelial cells on IL-7 production Three-week-old BALB / c mice were treated with NT (-) (n = 8) and NT ( + ) (n = 9) were freely taken for 2 weeks. Thereafter, the small intestine was excised to prepare small intestinal epithelial cells. Culture of small intestinal epithelial cells followed the method of Perreault et al. (Perreaul, N. and Beaulieu, JF: Exp. Cell. Res., 245: 34-42, 1998). The small intestine was opened longitudinally, washed with PBS and cut into four segments. These segments were transferred to 15-ml tubes containing 10 ml of ice-cold MatriSperse (Collaborative Biomedical Products, Becton Dickinson Lab.). The small intestine was incubated at 4 ° C. for 8-10 hours without agitation. Next, each tube was shaken gently to separate epithelial cells. The suspension was washed twice with PBS (4 ° C., 1200 rpm, 8 minutes). 10% FCS, penicillin 100 U / ml, streptomycin 100 μg / ml, mercaptoethanol 5 × 10 ^-Five The cells were suspended in RPMI 1640 medium in a 96-well plate to which M and 2 ml-glutamine had been added. These epithelial cells have 5% CO _Two The cells were placed in an incubator and cultured at 37 ° C. for 16 hours. The IL-7 level of the culture supernatant was measured by ELISA according to the method of Sharma et al. (Sharma, S. et al .: Gene Therapy., 4: 1361-1370, 1997) (FIG. 8). It can be seen that in the NT (+) group, IL-7 production of small intestinal epithelial cells was significantly (p <0.01) enhanced as compared with that of the NT (-) group. That is, it was revealed that oral ingestion of nucleic acid enhances IL-7 production of small intestinal epithelial cells.
[Test Example 6] Influence of oral ingestion of nucleic acid on IL-7 and TGF-β production of intestinal epithelial cells NT (-) and NT (+) were administered to 3-week-old female OVA-TCR Tg mice, respectively. They were allowed free intake for 4 weeks. During the experimental period, water with 2% OVA was freely available. Thereafter, the small intestine was excised and allowed to stand in matricespase at 4 ° C. for 8 hours. After standing, the small intestinal epithelial cells in the matricesase were washed twice by removing the intestine by shaking lightly. After washing, small intestinal epithelial cells were cultured for 16 hours in RPMI-1640 medium containing 10% FCS or RPMI-1640 medium without FCS. After the culture, IL-7 and TGF-β in the supernatant were measured by ELISA. As a result, IL-7 production of small intestinal epithelial cells was significantly (p <0.01) higher in the NT (+) diet group than in the NT (−) diet group (FIG. 9). In addition, TGF-β production of small intestinal epithelial cells was significantly (p <0.05) significantly higher in the NT (+) diet group than in the NT (−) diet group (FIG. 10).
[Test Example 7] Effect on production of antigen-specific IgA antibody NT (-) (n = 18) and NT (+) (n = 16) were freely fed to 3-week-old female OVA-TCR Tg mice I let it. During the experimental period, water with 2% OVA was freely available. Feces were collected at 6, 7, and 8 weeks of age, and OVA-specific IgA antibodies were measured by ELISA. The results are shown in FIG. The OVA-specific IgA antibody was significantly higher in the NT (+) diet group at the age of 8 weeks. Therefore, it was revealed that oral ingestion of nucleic acid enhances production of antigen-specific IgA of the intestinal immune system.
Test Example 8 Effect on Macrophage Function Whey Protein Isolate (WPI) as a protein source was a nucleotide-free diet (NT (-) diet) and 0.4% of a nucleotide blended in the following ratio. OVA-TCR Tg mice were allowed to freely ingest the added diet (NT (+) diet) from 3 weeks old in 6 mice per group. At this time, a 2% OVA aqueous solution was freely taken. Thioglycolate was administered intraperitoneally, and peritoneal macrophages were collected 4 days after administration (7 weeks old). The macrophages were cultured under LPS (0.1 μg / ml and 1 μg / ml) stimulation for 2 days, and the IL-12 activity of the supernatant was measured by the Bioassay method. Bioassay of mouse IL-12 was performed according to the method of Skeen et al. (Skeen, MJ et al., J Immunol 156: 1196-1206, 1996). That is, a 96-well plate was coated with an anti-IL-12p40 antibody at 10 μg / ml. Wells were washed, various concentrations of recombinant IL-12 (rIL-12), spleen or macrophage culture supernatants were added to the wells and the plates were incubated overnight. After spleen cells of a normal mouse were cultured at 37 ° C. for 2 days in a CO 2 incubator, the culture supernatant was collected and the IFN-γ concentration was measured by ELISA. The IL-12 concentration in the supernatant was extrapolated from a calibration curve of known rIL-12 and IFN-γ concentrations. IL-12p40 and p70 bind to the coated antibody, but only IL-12p70 can induce IFN-γ production. As a result, at both LPS concentrations of 0.1 μg / ml and 1 μg / ml, the IL-12 activity was significantly higher in the NT (+) diet group than in the NT (−) diet group (FIG. 12). .
Test Example 9 Influence on Macrophage Function [0107] Thioglycolate was intraperitoneally administered to 8-week-old female BALB / c mice, and 4 days later (7-week old), peritoneal macrophages were collected. The peritoneal macrophages were cultured with nucleotides (0 to 10 μg / ml) for one day, and the IL-12 activity in the supernatant was measured by the Bioassay method. As a result, the IL-12 activity when 0.32 to 10 μg / ml nucleotides were added showed a tendency to increase although no significant difference was observed as compared to the case where no nucleotides were added (FIG. 13).
[Test Example 10] Influence on cytokine production in spleen cells A diet without nucleotides (NT (-) diet) using Whey Protein Isolate as a protein source and 0.4% of nucleotides mixed in the above ratio were added. The diet (NT (+) diet) was allowed to freely ingest 3 weeks old OVA-TCR Tg mice (6 mice per group) together with a 2% OVA aqueous solution, and spleen cells were collected at 7 weeks of age. The spleen cells were cultured under OVA (100 μg / ml) stimulation for 2 days, and the IL-12 activity of the supernatant was measured by the Bioassay method. As a result, the spleen cell IL-12 activity was significantly higher in the NT (+) diet group than in the NT (-) diet group (FIG. 14).
[0079]
EXAMPLES Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.
[Example 1] As a nucleic acid-related substance, a nucleic acid mixture having the following compounding ratio was compounded in a commercial infant formula (manufactured by Meiji Dairies Co., Ltd.) at a ratio of 0.01%.
Cytidine 5'-monophosphate 1.62 g / kg guanosine 5'-monophosphate 0.57 g / kg inosine 5'-monophosphate 1.10 g / kg uridine 5'-monophosphate 0.71 g / kg 0.1 g per 1 g of milk powder , Preferably 0.01-4 mg. In addition, bases, which are components of nucleic acid-related substances, are generally safe in foods such as fish and shellfish and meat. Therefore, there is no problem even if it is used beyond the above-mentioned range, and for the purpose of prevention or health, it may be used in a smaller amount than the above-mentioned range. When preparing foods and drinks other than infant milk powder, the amount of nucleotides, nucleosides, nucleic acids or bases may be determined with reference to the above range.
Example 2 A powdered infant formula (Meiji Dairy Co., Ltd .; powdered milk A, powdered milk B) obtained by enzymatically decomposing a protein to be an allergen contains nucleotides, nucleosides, nucleic acids (RNA, DNA) or a mixture thereof. 0.01% of a mixture of bases as constituent components was blended. In this case, the prepared milk powder A is obtained by enzymatically decomposing whey protein, and the prepared milk powder B is obtained by enzymatically decomposing casein. However, there is no restriction on the protein to be enzymatically decomposed, and no restriction on the enzyme used for the decomposition. The composition of the product is shown in Table 1 below. The mixing ratio of nucleotides is as in Example 1. In the production of the infant formula according to the present invention, the base as a component of the nucleic acid-related substance can be used as an example of the above-mentioned amount, but in the present invention, 0.1 mg per 1 g of the milk powder , Preferably 0.01-4 mg. Further, there is no problem even if it is used beyond the above range, and for the purpose of prevention or health, it may be used in a smaller amount than the above range.
Example 3 Nucleotides, nucleosides, nucleic acids (RNA, DNA) or their constituents were added to 40 g of vitamin C or 40 g of an equal mixture of vitamin C and citric acid, 100 g of granulated sugar, and 60 g of an equal mixture of corn starch and lactose. Was added and mixed well. The mixture was packed in a bag, and 100 bags of 1.5 g stick-shaped nutritional health food were prepared.
[Example 4] A preparation for increasing the ratio of TCRγδ-positive T cells to TCRαβ-positive T cells was prepared by the following formulation. (1) Nucleotide, nucleoside, nucleic acid (RNA, DNA) or its constituent 50 g base, (2) lactose 90 g, (3) corn starch 29 g, (4) magnesium stearate 1 g. First, (1), (2) and (3) (but 17 g) were mixed and granulated together with the paste prepared from (3) (but 7 g). (3) (However, 5 g) and (4) are added to the obtained granules and mixed well.The mixture is compressed by a compression tablet machine, and each tablet contains nucleotides, nucleosides, nucleic acids (RNA, DNA) or a component thereof. 100 tablets containing 10 mg of the base as a component were produced. The dosage varies depending on the condition and age of the patient, but is administered at a dose of 0.1 to 1500 mg / kg / day 1 to 4 times a day. The nucleotides, nucleosides, nucleic acids (RNA, DNA) and bases used in the present invention are originally derived from foods and have almost no safety problems as described above. There is no problem. In addition, in the case of using it as a health promotion or health supplement, etc., it is sufficient to take an amount smaller than the above-mentioned amount for a long period of time. Further, as described above, the tablet according to the present invention can be administered by a method other than oral administration, but in the case of intravenous administration and intramuscular administration, the dose is 0.01 to 1200 mg / kg / day.
Example 5 The following composition was prepared. (1) 1 g of nucleotides, nucleosides, nucleic acids (RNA, DNA) or bases thereof, (2) 8 g of sodium chloride, (3) 4 g of chlorobutanol, and (4) 1 g of sodium hydrogen carbonate. All the components were dissolved in 1000 ml of distilled water, and this was dispensed into two 500 ml drip bottles to increase the ratio of TCRγδ-positive T cells and TCRαβ-positive T cells, thereby producing a preparation that promotes the production of IgA antibodies.
Example 6 The following composition was prepared. (1) 0.5 g of nucleotides, nucleosides, nucleic acids (RNA, DNA) or bases thereof, and (2) 1 l of sterilized milk. (1) was aseptically mixed with (2) and bottled. In the present invention, 0.01 to 10 g of (1) may be mixed per liter of sterilized milk.
Example 7 A skin external preparation having the following composition was prepared using the nucleotides in the blending ratio of Example 1 as active ingredients. In the formulation of the formulation examples, “appropriate amount” means an amount that gives 100% by weight in total.
[0087]
Formulation Example 1 Cream (1)
A (% by weight) Polyethylene glycol monostearate (40.EO) 2.0 Self-emulsifying glyceryl monostearate 5.0 Stearic acid 5.0 Behenyl alcohol 1.0 Liquid paraffin 10.0 Glyceryl trioctanoate 1.0 Vitamin E 0.1 nucleotides 1.0B Glycerin 5.0 Ethylparaben 0.1 Purified water The components belonging to the appropriate amount A are dissolved by heating. Separately, the components belonging to B are dissolved by heating. B was added to A, stirred, emulsified and then cooled to produce a cream.
[0088]
Formulation Example 2 Cream (2)
A (% by weight)
Monostearic acid Polyethylene glycol (40.EO) 2.0 Self-emulsifying glyceryl monostearate 5.0 Stearic acid 5.0 Behenyl alcohol 1.0 Liquid paraffin 10.0 Glyceryl trioctanoate 10.0 Vitamin E 0.2 Nucleotide 0 .5B Glycerin 5.0 Ethylparaben 0.1 Purified water The components belonging to the appropriate amount A are dissolved by heating. Separately, the components belonging to B are dissolved by heating. B was added to A, stirred, emulsified and then cooled to produce a cream.
[0089]
Formulation Example 3 Emulsion A (% by weight)
Monostearic acid polyoxyethylene sorbitan (20.EO) 1.0 Monostearic acid polyoxyethylene sorbitan (60.EO) 0.5 Lipophilic glyceryl monostearate 1.0 Stearic acid 0.5 Behenyl alcohol 0.5 Avocado Oil 4.0 Glyceryl trioctanoate 4.0 Nucleotide 5.0B 1,3-butylene glycol 5.0 Ethylparaben 0.1 Purified water A component belonging to an appropriate amount A is heated and dissolved. Separately, the components belonging to B are dissolved by heating. B was added to A, stirred, emulsified, and cooled to produce an emulsion.
[0090]
Formulation Example 4 Lotion A (% by weight)
Polyoxyethylene hydrogenated castor oil (60.EO) 8.0 ethanol 15.0 nucleotides 0.1 ethylparaben 0.1 citric acid 0.1 sodium citrate 0.3 1,3-butylene glycol 4.0 edetate Sodium 0.01 Purified water An appropriate amount of each of the above components was mixed, uniformly stirred and dissolved to produce a lotion.
[0091]
Formulation Example 5 Hydrophilic ointment A (% by weight)
Polyoxyethylene cetyl ether 2.0 Glycerin monostearate 10.0 Liquid paraffin 10.0 Vaseline 4.0 Cetanol 5.0 Nucleotide 3.0B Propylene glycol 10.0 Methyl paraben 0.1 Purified water Heat the components belonging to A Dissolve. Separately, the components belonging to B are dissolved by heating. B was added to A, stirred, emulsified, and cooled to produce a hydrophilic ointment. In the present invention, not only the nucleotides in the mixing ratio of Example 1 but also any of nucleotides, nucleosides, nucleic acids (RNA, DNA) or bases that are components thereof may be used. Further, there is no problem even if it is used beyond the above range, and it may be used in a smaller amount than the above range. Preferably, 0.1 to 100 mg of nucleotides, nucleosides, nucleic acids (RNA, DNA) or bases that are components thereof are used per 1 g of the skin external preparation.
[0092]
EFFECT OF THE INVENTION It has been clarified that the oral composition of the nucleic acid composition shifts the ratio of the subset of αβ-IEL and γδ-IEL in the IEL in the direction of increasing γδ-IEL. Furthermore, it was revealed that oral ingestion of the nucleic acid composition enhances production of cytokines (such as IL-2, IL-7, IFN-γ, and TGF-β) in intestinal epithelium. Furthermore, it was revealed that production of an antigen-specific secretory IgA antibody in the intestinal tract was enhanced. As a result, oral ingestion of an effective amount of nucleic acid promotes production of secretory IgA antibody specific to the intestinal tract immune system, thereby enhancing the ability to protect against harmful pathogens and the like. Is expected to be effectively induced, and further, the disruption of the Th1 / Th2 cell balance is improved, and as a result, the intestinal immune system or the systemic immune system is activated.



[Brief description of the drawings]
FIG. 1 shows that three-week-old female BALB / c mice were allowed to freely take a diet without nucleotides [NT (−)] or a diet with nucleotides [NT (+)] for 2 weeks, and then to obtain NT (−). TCRαβ in the group ⁺ IEL and TCRγδ ⁺ Constituent ratio with T cells (%) and TCRγδ in NT (+) group ⁺ T cells and TCRγδ ⁺ The composition ratio (%) with T cells is shown (mean ± SE; p <0.05).
FIG. 2. ICR TCRαβ in NT (−) group ⁺ T cells and TCRγδ ⁺ Ratio to IEL, and TCRαβ in NT (+) group ⁺ T cells and TCRγδ ⁺ The ratio to T cells is shown (mean ± SE; ^* p <0.05).
FIG. 3 shows TCRαβ in the NT (−) group among the expression patterns of CD4 and CD8 ⁺ CD8αα ⁺ T cells and TCRγδ ⁺ CD8αα ⁺ IEL composition ratio (%) and TCRαβ in NT (+) group ⁺ CD8αα ⁺ T cells and TCRγδ ⁺ CD8αα ⁺ The percentage (%) relative to T cells is shown (mean ± SE; ^* p <0.05)
FIG. 4. NT (−) group after 3 weeks old female OVA-TCR Tg mice were allowed to freely ingest NT (−) or NT (+) with 2% OVA added water for 4 weeks. TCRαβ in ⁺ T cells and TCRγδ ⁺ Constituent ratio (%) with T cells, and TCRαβ in NT (+) group ⁺ T cells and TCRγδ ⁺ The composition ratio (%) with T cells is shown (mean ± SE; p <0.05).
FIG. 5 shows TCRαβ in NT (−) group among expression patterns of CD4 molecule and CD8 molecule of IEL in the above. ⁺ CD8αα ⁺ T cells and TCRγδ ⁺ CD8αα ⁺ T cell composition ratio (%) and TCRαβ in NT (+) group ⁺ CD8αα ⁺ T cells and TCRγδ ⁺ CD8αα ⁺ The percentage of T cells (%) is shown (mean ± SE; ^* p <0.05)
[FIG. 6] NT (−) after 3 weeks old female OVA-TCR Tg mice were allowed to freely ingest NT (−) or NT (+) together with water containing 2% OVA for 4 weeks. Group interferon-γ ( IFN -γ) production or in NT (+) group IFN Each shows -γ production (mean ± SE).
FIG. 7 shows IEL interleukin 2 (IL-2) production in the NT (-) group and IL-2 production in the NT (+) group, respectively. (Mean ± SE; p <0.05).
FIG. 8: Interleukin-7 (IL-) of small intestinal epithelial cells in the NT (−) group after 3 weeks of female BALB / c mice were allowed free intake of NT (−) or NT (+) for 2 weeks. 7) Production or interleukin 7 (IL-7) production of small intestinal epithelial cells in the NT (+) group, respectively. (Mean ± SE; p <0.01).
FIG. 9 shows the effect of nucleotides taken orally on IL-7 production of small intestinal epithelial cells of OVA-TCR Tg mice (n = 10) (**; p <0.01). (Average ± SD)
FIG. 10 shows the effect of nucleotides taken orally on TGF-β production of IEL in OVA-TCR Tg mice (n = 4) (*; p <0.05). (Average ± SD)
FIG. 11. Three week old female OVA-TCR Tg mice were allowed to freely ingest NT (−) or NT (+) together with water supplemented with 2% OVA, and at 6, 7 and 8 weeks of age. Shows the results of measuring the amount of OVA-specific IgA antibodies in feces (mean ± SE; p <0.05)
FIG. 12: Peritoneal macrophages after 3 weeks of female OVA-TCR Tg mice are allowed to freely ingest NT (−) or NT (+) together with 2% OVA-added water for 4 weeks (7 weeks of age) (Mean ± SE; p <0.05).
FIG. 13 8 cultured in the presence of nucleotides FIG. 9 shows IL-12 production of peritoneal macrophages of week-old female BALB / c mice (mean ± SE).
[FIG. 14] Splenocytes after 3 weeks of female OVA-TCR Tg mice were allowed to freely ingest NT (−) or NT (+) together with 2% OVA-added water for 4 weeks (7 weeks old) (Mean ± SE; p <0.05).