JP5937015B2 - Delayed type hypersensitivity reducing agent - Google Patents
Delayed type hypersensitivity reducing agent Download PDFInfo
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- JP5937015B2 JP5937015B2 JP2012548826A JP2012548826A JP5937015B2 JP 5937015 B2 JP5937015 B2 JP 5937015B2 JP 2012548826 A JP2012548826 A JP 2012548826A JP 2012548826 A JP2012548826 A JP 2012548826A JP 5937015 B2 JP5937015 B2 JP 5937015B2
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- lactobacillus gasseri
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- delayed
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Description
本発明は、遅延型過敏症を軽減するための薬剤、およびこれを含有する遅延型過敏症軽減用の食品組成物等に関する。 The present invention relates to a drug for reducing delayed type hypersensitivity, a food composition for reducing delayed type hypersensitivity containing the same, and the like.
過敏性反応、即ちアレルギー疾患にはI型からIV型までの分類があり、それぞれでその発症メカニズムと症状は異なっている。I型アレルギーでは、抗原感作によって抗原特異的IgEの産生が誘導され、産生されたIgEが肥満細胞の表面に結合する。そこに体内に侵入してきた抗原がその特異的IgEによって捕捉されると、肥満細胞からヒスタミンなどのケミカルメディエーターが分泌される。これにより血管透過性の亢進や気管支収縮が起こり、くしゃみや鼻水などの即時型過敏性反応を呈する。
一方でIV型アレルギーは遅延型過敏性反応による疾患であり、抗原特異的なT細胞がマクロファージなどを活性化して炎症を引き起こし、皮膚などに腫脹を誘発する疾患である。There are different types of hypersensitivity reactions, ie allergic diseases, from type I to type IV, each of which has different onset mechanisms and symptoms. In type I allergy, antigen-specific IgE production is induced by antigen sensitization, and the produced IgE binds to the surface of mast cells. When the antigen that has entered the body is captured by its specific IgE, chemical mediators such as histamine are secreted from mast cells. This leads to increased vascular permeability and bronchoconstriction, and presents an immediate hypersensitivity reaction such as sneezing or runny nose.
On the other hand, type IV allergy is a disease caused by a delayed type hypersensitivity reaction, and antigen-specific T cells activate macrophages to cause inflammation and induce swelling in the skin.
これまでに、乳酸菌などの微生物による即時型過敏性反応への効果が記載された先行技術は多く存在している（特許文献１〜３）。例えば、特許文献３には、Lactobacillus属乳酸菌がアレルギー疾患に効果があることが記載されている。当該文献では、ヒト糞便から分離した、Lactobacillus gasseri OLL2809菌株が、即時型アレルギーに有用であることが記載されている。しかしながら、乳酸菌などの微生物と遅延型過敏性反応の関連を示唆する先行技術は少ない。例えば、特許文献４は、Lactobacillus plantarumのアトピー性皮膚炎緩和剤に関する発明である。当該文献には、アトピー性皮膚炎のアレルギー型は、即時型（I型）アレルギーと遅延型（IV型）アレルギーの側面を併せ持つことが背景技術に記載されている。しかし実施例は、即時型アレルギーモデル及びアトピー性動物モデルでの検証のみで、遅延型過敏性反応に対する効果は何ら実証されていない。さらに、特許文献５は、テトラジェノコッカス属、ペディオコッカス属もしくはロイコノストック属に属する乳酸菌のインターロイキン12産生促進剤に関する発明である。当該文献では、これら乳酸菌がインターロイキン12の産生を促進し、遅延型（IV型）過敏性反応を増強させ、細胞性免疫が増強したことが記載されている。同様に、非特許文献１にはLactobacillus casei Shirota株の生菌を経口投与することで、免疫記憶を亢進し、遅延型過敏性反応が増強したことが示されている。
この様に、乳酸菌などの微生物と遅延型過敏性反応との関連性については、インターロイキン12の産生促進などによる細胞性免疫の増強によって、遅延型過敏性反応が増強することついては報告されている。一方、乳酸菌などの微生物によって遅延型過敏症が軽減したり、遅延型過敏性反応を抑制するといった報告はなかった。So far, there are many prior arts that describe effects on immediate hypersensitivity reactions by microorganisms such as lactic acid bacteria (
As described above, it has been reported that the delayed hypersensitivity reaction is enhanced by the enhancement of cellular immunity by promoting the production of interleukin 12, etc., regarding the relationship between microorganisms such as lactic acid bacteria and delayed hypersensitivity reaction. . On the other hand, there has been no report that delayed type hypersensitivity is reduced or a delayed type hypersensitivity reaction is suppressed by microorganisms such as lactic acid bacteria.
本発明は、食経験があり長期間摂取における安全性が明確な、Lactobacillus gasseriの菌体画分を含む、遅延型過敏症の軽減用の薬剤を提供することを課題とする。また本発明は、これを含有する遅延型過敏症を軽減するための食品組成物を提供することを課題とする。 An object of the present invention is to provide a drug for alleviating delayed type hypersensitivity, including a bacterial fraction of Lactobacillus gasseri, which has a dietary experience and is clearly safe for long-term ingestion. Another object of the present invention is to provide a food composition for reducing delayed hypersensitivity containing the same.
本発明者らは、上記課題を解決するために、鋭意研究を行った。その結果、本発明者らは、乳酸菌の一種であるLactobacillus gasseriの菌体画分にCD4+T細胞に対する増殖抑制効果があることを明らかにした。また本発明者らは、Lactobacillus gasseri OLL2809菌株のRNA画分が、MyD88依存性シグナル伝達経路を介し、用量依存的にCD4+T細胞の増殖を抑制することを明らかにした。さらに本発明者らは、in vivoにおいてもLactobacillus gasseri OLL2809菌株が遅延型過敏性反応を抑制することを明らかにした。微生物の菌体画分がCD4+T細胞の増殖抑制に関与し、遅延型過敏性反応を抑制することを明らかにしたのは、本発明が初めてである。本発明はこのような知見に基づくものであり、以下の遅延型過敏症の軽減剤、および遅延型過敏症を軽減するための食品組成物などに関する。
〔１〕Lactobacillus gasseriの菌体画分を有効成分とする遅延型過敏症を軽減するための薬剤。
〔２〕前記菌体画分がRNA画分であることを特徴とする〔１〕記載の薬剤。
〔３〕前記Lactobacillus gasseriが、Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）であることを特徴とする〔１〕または〔２〕記載の薬剤。
〔４〕遅延型過敏症の軽減作用がCD4+ T細胞増殖の抑制作用を介するものである〔１〕〜〔３〕のいずれかに記載の薬剤。
〔５〕〔１〕〜〔４〕のいずれかに記載の薬剤の有効量を含有せしめた遅延型過敏症を軽減するための食品組成物または飲食品。
〔６〕乳児用調製粉乳、幼児用粉乳等食品、授乳婦用粉乳等食品、保健機能食品、病者用食品、または乳製品である〔５〕記載の食品組成物または飲食品。
〔７〕遅延型過敏症を軽減するための組成物の製造におけるLactobacillus gasseriの菌体画分の使用。
〔８〕前記菌体画分がRNA画分であることを特徴とする〔７〕記載の使用。
〔９〕前記Lactobacillus gasseriが、Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）であることを特徴とする〔７〕または〔８〕記載の使用。
〔１０〕遅延型過敏症の軽減作用がCD4+ T細胞増殖の抑制作用を介するものである〔７〕〜〔９〕のいずれかに記載の使用。
〔１１〕組成物が飲食品に配合されたものである、〔７〕〜〔１０〕のいずれかに記載の使用。
〔１２〕飲食品が、乳児用調製粉乳、幼児用粉乳等食品、授乳婦用粉乳等食品、保健機能食品、病者用食品、または乳製品である〔１１〕に記載の使用。
〔１３〕Lactobacillus gasseriの菌体画分の有効量を対象に投与する工程を含む、遅延型過敏症を軽減するための方法。
〔１４〕前記菌体画分がRNA画分であることを特徴とする〔１３〕記載の方法。
〔１５〕前記Lactobacillus gasseriが、Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）であることを特徴とする〔１３〕または〔１４〕記載の方法。
〔１６〕遅延型過敏症の軽減作用がCD4+ T細胞増殖の抑制作用を介するものである〔１３〕〜〔１５〕のいずれかに記載の方法。
〔１７〕遅延型過敏症の軽減に使用するためのLactobacillus gasseriの菌体画分。
〔１８〕前記菌体画分がRNA画分であることを特徴とする〔１７〕記載の菌体画分。
〔１９〕前記Lactobacillus gasseriが、Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）であることを特徴とする〔１７〕または〔１８〕記載の菌体画分。
〔２０〕遅延型過敏症の軽減作用がCD4+ T細胞増殖の抑制作用を介するものである〔１７〕〜〔１９〕のいずれかに記載の菌体画分。
〔２１〕飲食品に配合されたものである、〔１７〕〜〔２０〕のいずれかに記載の菌体画分。
〔２２〕飲食品が、乳児用調製粉乳、幼児用粉乳等食品、授乳婦用粉乳等食品、保健機能食品、病者用食品、または乳製品である〔２１〕に記載の菌体画分。In order to solve the above problems, the present inventors have conducted intensive research. As a result, the present inventors have clarified that the bacterial cell fraction of Lactobacillus gasseri, which is a kind of lactic acid bacteria, has a growth inhibitory effect on CD4 + T cells. Further, the present inventors have revealed that the RNA fraction of Lactobacillus gasseri OLL2809 strain suppresses the proliferation of CD4 + T cells in a dose-dependent manner through the MyD88-dependent signal transduction pathway. Furthermore, the present inventors have clarified that Lactobacillus gasseri OLL2809 strain suppresses delayed hypersensitivity reaction in vivo. The present invention is the first to show that the microbial cell fraction is involved in the suppression of proliferation of CD4 + T cells and suppresses the delayed hypersensitivity reaction. The present invention is based on such findings, and relates to the following delayed-type hypersensitivity alleviating agents, food compositions for reducing delayed-type hypersensitivity, and the like.
[1] A drug for reducing delayed hypersensitivity comprising a bacterial cell fraction of Lactobacillus gasseri as an active ingredient.
[2] The drug according to [1], wherein the bacterial cell fraction is an RNA fraction.
[3] The drug according to [1] or [2], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain (accession number: FERM BP-10542).
[4] The drug according to any one of [1] to [3], wherein the action for reducing delayed type hypersensitivity is mediated by an action for suppressing proliferation of CD4 + T cells.
[5] A food composition or food or drink for reducing delayed hypersensitivity containing an effective amount of the drug according to any one of [1] to [4].
[6] The food composition or food or drink according to [5], which is a food preparation such as infant formula, infant formula, food such as lactating milk, health functional food, sick food, or dairy product.
[7] Use of a bacterial cell fraction of Lactobacillus gasseri in the production of a composition for reducing delayed type hypersensitivity.
[8] The use according to [7], wherein the bacterial cell fraction is an RNA fraction.
[9] The use according to [7] or [8], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain (Accession Number: FERM BP-10542).
[10] The use according to any one of [7] to [9], wherein the delayed hypersensitivity alleviating action is mediated by a CD4 + T cell proliferation suppressing action.
[11] The use according to any one of [7] to [10], wherein the composition is formulated in a food or drink.
[12] The use according to [11], wherein the food or drink is a food product such as infant formula or infant formula, a food product such as infant formula, a health functional food, a sick food, or a dairy product.
[13] A method for reducing delayed hypersensitivity, comprising a step of administering an effective amount of a bacterial cell fraction of Lactobacillus gasseri to a subject.
[14] The method according to [13], wherein the bacterial cell fraction is an RNA fraction.
[15] The method according to [13] or [14], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain (Accession Number: FERM BP-10542).
[16] The method according to any one of [13] to [15], wherein the delayed hypersensitivity alleviating action is mediated by an inhibitory action on CD4 + T cell proliferation.
[17] A bacterial cell fraction of Lactobacillus gasseri for use in reducing delayed hypersensitivity.
[18] The bacterial cell fraction according to [17], wherein the bacterial cell fraction is an RNA fraction.
[19] The bacterial cell fraction according to [17] or [18], wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain (accession number: FERM BP-10542).
[20] The bacterial cell fraction according to any one of [17] to [19], wherein the effect of reducing delayed type hypersensitivity is mediated by the effect of suppressing the proliferation of CD4 + T cells.
[21] The bacterial cell fraction according to any one of [17] to [20], which is blended in a food or drink.
[22] The bacterial cell fraction according to [21], wherein the food or drink is a food product such as infant formula or infant formula, a food product such as infant formula, a health functional food, a sick food, or a dairy product.
本発明者らは後述する実施例において確認されたとおり、Lactobacillus gasseriの菌体画分が遅延型過敏症反応を抑制することを見出した。また、特に、Lactobacillus gasseri OLL2809菌株のRNA画分にその効果が高いことを見出した。Lactobacillus gasseriは食経験のある、安全性の高い乳酸菌である。従って、本発明の遅延型過敏症軽減剤、およびこれを含有する遅延型過敏症軽減用の食品組成物によって、遅延型過敏症を安全に軽減することが可能である。 The present inventors have found that the bacterial cell fraction of Lactobacillus gasseri suppresses the delayed hypersensitivity reaction, as confirmed in the examples described later. In particular, the present inventors have found that the effect is high in the RNA fraction of Lactobacillus gasseri OLL2809 strain. Lactobacillus gasseri is a highly safe lactic acid bacterium with dietary experience. Therefore, delayed hypersensitivity can be safely reduced by the delayed hypersensitivity reducing agent of the present invention and the food composition for reducing delayed hypersensitivity containing the same.
本発明は、Lactobacillus gasseriの菌体画分を含む遅延型過敏症を軽減するための薬剤を提供する。また本発明は、Lactobacillus gasseriの菌体画分を含む遅延型過敏性反応を抑制するための薬剤を提供する。さらに本発明は、Lactobacillus gasseriの菌体画分を含むCD4+T細胞の増殖抑制剤を提供する。本明細書においては特に断りのない限り、これら「軽減剤」、「遅延型過敏性反応の抑制剤」および「CD4+T細胞の増殖抑制剤」を「本発明の薬剤」と総称する。
また本発明は、Lactobacillus gasseriの菌体画分を含む遅延型過敏症軽減用組成物を提供する。また本発明は、Lactobacillus gasseriの菌体画分を含む遅延型過敏性反応の抑制用組成物を提供する。さらに本発明は、Lactobacillus gasseriの菌体画分を含むCD4+T細胞の増殖抑制用組成物を提供する。本明細書においては特に断りのない限り、これら「軽減用組成物」、「遅延型過敏性反応の抑制用組成物」および「CD4+T細胞の増殖抑制用組成物」を「本発明の組成物」と総称する。組成物としては医薬組成物、食品組成物が挙げられるがこれらに限定されない。The present invention provides a drug for reducing delayed type hypersensitivity containing a bacterial cell fraction of Lactobacillus gasseri. Moreover, this invention provides the chemical | medical agent for suppressing the delayed type hypersensitivity reaction containing the microbial cell fraction of Lactobacillus gasseri. The present invention further provides a growth inhibitor of CD4 + T cells containing a bacterial cell fraction of Lactobacillus gasseri. In the present specification, unless otherwise specified, these “reducing agents”, “inhibitors of delayed hypersensitivity reaction”, and “CD4 + T cell proliferation inhibitors” are collectively referred to as “agents of the present invention”.
The present invention also provides a delayed hypersensitivity reducing composition comprising a bacterial cell fraction of Lactobacillus gasseri. The present invention also provides a composition for suppressing delayed hypersensitivity reaction, which comprises a bacterial cell fraction of Lactobacillus gasseri. Furthermore, the present invention provides a composition for inhibiting the proliferation of CD4 + T cells, which contains a bacterial cell fraction of Lactobacillus gasseri. In the present specification, unless otherwise specified, these “reducing composition”, “composition for suppressing delayed hypersensitivity reaction”, and “composition for suppressing proliferation of CD4 + T cells” are referred to as “the composition of the present invention”. Collectively called "thing". Examples of the composition include, but are not limited to, a pharmaceutical composition and a food composition.
本明細書において「軽減」は、「改善」、「抑制」、「治療」と表現することもできる。従って本発明の「軽減」には、
・軽減
・改善
・抑制
・治療
が含まれる。In the present specification, “reduction” can also be expressed as “improvement”, “suppression”, and “treatment”. Therefore, “reduction” of the present invention includes
-Mitigation-Improvement-Suppression-Treatment included.
本発明の薬剤及び組成物は、その有効成分としてLactobacillus gasseriの菌体画分を含む。本発明の薬剤及び組成物は、医薬品または食品添加剤としても用いることができる。また、本発明の薬剤及び組成物は、Lactobacillus gasseriの菌体画分に加え、Lactobacillus gasseriの菌体画分以外の画分やLactobacillus gasseriそのもの（生菌）、Lactobacillus gasseriの死菌体を含んでもよい。あるいは生菌体を含まないことも可能である。つまり本発明の薬剤及び組成物は死菌体のみからなっていてもよい。
Lactobacillus gasseriの菌体画分は、菌体破砕液上清画分、菌体破砕液上清除タンパク質画分、RNA画分などがあげられるが、遅延型過敏性の軽減作用、遅延型過敏性反応の抑制作用、あるいはCD4+T細胞の増殖抑制作用を有する限りこれらに限定されない。本発明のLactobacillus gasseriの菌体画分として、好ましくは菌体破砕液上清画分、より好ましくはRNA画分が挙げられる。菌体破砕液上清画分、菌体破砕液上清除タンパク質画分、RNA画分は、実施例に記載の方法のほか、当業者に周知のRNA抽出方法や市販のRNA抽出キットを用いることによって調製することができる。
本発明のRNA画分は、RNAを含む限りRNA以外の核酸やタンパク質、細胞成分を含むものであってもよい。あるいはデオキシリボヌクレアーゼやプロテイナーゼなどの酵素処理を受けたものであってもよい。本発明のRNA画分の態様として、リボヌクレアーゼの作用を受けていないRNA抽出物を挙げることができるがこれに限定されない。The drug and composition of the present invention contain a bacterial cell fraction of Lactobacillus gasseri as its active ingredient. The agents and compositions of the present invention can also be used as pharmaceuticals or food additives. In addition to the bacterial cell fraction of Lactobacillus gasseri, the drug and composition of the present invention may contain a fraction other than the bacterial cell fraction of Lactobacillus gasseri, Lactobacillus gasseri itself (live bacteria), or dead bacterial cells of Lactobacillus gasseri. Good. Alternatively, it is possible not to include viable cells. That is, the chemical | medical agent and composition of this invention may consist only of dead microbial cells.
The cell fraction of Lactobacillus gasseri includes the cell suspension supernatant fraction, the cell suspension supernatant deproteinization fraction, the RNA fraction, etc., but the delayed hypersensitivity reducing action, delayed hypersensitivity reaction As long as it has an inhibitory action on CD4 + or proliferation inhibitory action on CD4 + T cells, it is not limited thereto. The bacterial cell fraction of Lactobacillus gasseri of the present invention is preferably a bacterial cell lysate supernatant fraction, more preferably an RNA fraction. For the cell lysate supernatant fraction, the cell lysate supernatant protein removal fraction, and the RNA fraction, in addition to the methods described in the Examples, use RNA extraction methods well known to those skilled in the art or commercially available RNA extraction kits. Can be prepared.
The RNA fraction of the present invention may contain nucleic acids other than RNA, proteins, and cell components as long as they contain RNA. Alternatively, it may be subjected to an enzyme treatment such as deoxyribonuclease or proteinase. Examples of the RNA fraction of the present invention include, but are not limited to, RNA extracts that are not subjected to the action of ribonuclease.
本発明の薬剤及び組成物は、その有効成分であるLactobacillus gasseriの菌体画分の他に、Lactobacillus gasseri以外の微生物を含んでいても良い。例えば、Lactobacillus属の乳酸菌であれば、Lactobacillus delbrueckii subsp. burgalicus、Lactobacillus delbrueckii subsp. lactis、Lactobacillus paracasei subsp. paracasei、Lactobacillus acidophilus、Lactobacillus helveticus、Lactobacillus helveticus subsp. jugurti、Lactobacillus acidophilus、Lactobacillus crispatus、Lactobacillus amylovorus、Lactobacillus gallinarum、Lactobacillus oris、Lactobacillus casei subsp. rhamnosus、Lactobacillus johnsonii、Lactobacillus fermentum、Lactobacillus brevisなどを挙げることができるが、これらに限定されない。本発明の薬剤及び組成物は、このようなLactobacillus属の乳酸菌や他の微生物を１種または２種以上を含んでもよい。本発明のLactobacillus gasseriとして、特に好ましくはLactobacillus gasseri OLL2809 (受託番号：FERM BP-10542)（本菌株は、独立行政法人産業技術総合研究所特許生物寄託センターに寄託されている）が挙げられるが、これに限定されない。 The drug and composition of the present invention may contain microorganisms other than Lactobacillus gasseri, in addition to the bacterial fraction of Lactobacillus gasseri which is an active ingredient. For example, if it is a lactic acid bacterium of the genus Lactobacillus, Lactobacillus delbrueckii subsp.burgalicus, Lactobacillus delbrueckii subsp.lactis, Lactobacillus paracasei subsp. Examples include, but are not limited to, gallinarum, Lactobacillus oris, Lactobacillus casei subsp. rhamnosus, Lactobacillus johnsonii, Lactobacillus fermentum, and Lactobacillus brevis. The drug and composition of the present invention may contain one or more of such Lactobacillus lactic acid bacteria and other microorganisms. As Lactobacillus gasseri of the present invention, Lactobacillus gasseri OLL2809 (Accession No .: FERM BP-10542) (this strain is deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary), It is not limited to this.
本発明者らは、ヒト成人の糞便より独自に分離したLacgtobacillus gasseriから、マウス由来CD4+ T細胞（ヘルパーT細胞）の増殖を阻害し、さらに、Lactobacillus gasseriの菌体画分が遅延型過敏性反応を抑制することを見出した。またさらに、ヒト成人の糞便より独自に分離したLacgtobacillus gasseriの中でも、Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）を選抜した。したがって本発明は、Lactobacillus gasseriの菌体画分を有効成分とする遅延型過敏症の軽減剤、またはこれらを含有する遅延型過敏症軽減用の食品組成物又は飲食品を提供する。The present inventors inhibit the growth of mouse-derived CD4 + T cells (helper T cells) from Lacgtobacillus gasseri uniquely isolated from human adult feces, and the bacterial fraction of Lactobacillus gasseri is delayed type hypersensitivity It was found to suppress the reaction. Furthermore, Lactobacillus gasseri OLL2809 strain (accession number: FERM BP-10542) was selected from among Lacgtobacillus gasseri uniquely isolated from human adult feces. Accordingly, the present invention provides a delayed-type hypersensitivity alleviating agent comprising a bacterial cell fraction of Lactobacillus gasseri as an active ingredient, or a food composition or food / drink for reducing delayed-type hypersensitivity containing these.
本発明者らは、Lactobacillus gasseri OLL2809菌株を独立行政法人産業技術総合研究所に寄託した。以下に、寄託を特定する内容を記載する。
（１）寄託機関名：独立行政法人産業技術総合研究所 特許生物寄託センター
（２）連絡先：日本国茨城県つくば市東１丁目１番地１ 中央第６
郵便番号 305-8566
電話番号 029-861-6029, 6079
（３）識別のための表示：Lactobacillus gasseri OLL2809（受託番号：FERM BP-10542）
（４）寄託日：平成１８年３月１日The present inventors deposited the Lactobacillus gasseri OLL2809 strain with the National Institute of Advanced Industrial Science and Technology. The contents specifying the deposit are described below.
(1) Depositary institution name: National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center (2) Contact information: 1st, 1st East, 1-chome Tsukuba, Ibaraki, Japan
Postal code 305-8566
Phone number 029-861-6029, 6079
(3) Indication for identification: Lactobacillus gasseri OLL2809 (Accession number: FERM BP-10542)
(4) Date of deposit: March 1, 2006
Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）は、グラム陽性桿菌であり、Lactobacilli MRS Agar（Difco）上でのコロニー形態は円形、淡黄色、扁平状である。生理学的特徴としては、ホモ乳酸発酵形式、45℃での発育性、グルコース、マンノース、フルクトース、ガラクトース、シュクロース、セロビオース、ラクトース、トレハロースに対する発酵性を有する。 Lactobacillus gasseri OLL2809 strain (Accession number: FERM BP-10542) is a Gram-positive gonococcus, and the colony form on Lactobacilli MRS Agar (Difco) is round, pale yellow, and flat. Physiological characteristics include homolactic fermentation, growth at 45 ° C., fermentability to glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose.
Lactobacillus gasseri OLL2809菌株（受託番号：FERM BP-10542）を培養するための培地としては、乳酸菌の培地に通常用いられる培地が使用される。すなわち主炭素源のほか窒素源、無機物その他の栄養素を程良く含有する培地ならば、いずれの培地も使用可能である。炭素源としては、ラクトース、グルコース、スクロース、フラクトース、澱粉加水分解物、廃糖蜜などが、使用菌の資化性に応じて使用できる。窒素源としては、カゼインの加水分解物、ホエータンパク質加水分解物、大豆タンパク質加水分解物等の有機窒素含有物が使用できる。ほかに、増殖促進剤として、肉エキス、魚肉エキス、酵母エキス等が用いられる。 As a medium for culturing Lactobacillus gasseri OLL2809 strain (Accession number: FERM BP-10542), a medium usually used for lactic acid bacteria is used. That is, any medium can be used as long as it contains a nitrogen source, inorganic substances and other nutrients in addition to the main carbon source. As the carbon source, lactose, glucose, sucrose, fructose, starch hydrolyzate, waste molasses and the like can be used according to the assimilation ability of the bacteria used. As the nitrogen source, organic nitrogen-containing substances such as casein hydrolyzate, whey protein hydrolyzate, and soy protein hydrolyzate can be used. In addition, meat extract, fish extract, yeast extract and the like are used as a growth promoter.
培養は嫌気条件下で行うことが望ましいが、通常用いられる液体静置培養などによる微好気条件下でもよい。嫌気培養には炭素ガス気層下で培養する方法などの公知の手法を適用することができるが、他の方法でもかまわない。培養温度は一般に30〜40℃が好ましいが、菌が生育する温度であれば他の温度条件でもよい。培養中の培地のpHは6.0〜7.0に維持することが好ましいが、菌が生育する温度であれば他のpH条件でもよい。また、バッチ培養条件下で培養することもできる。培養時間は通常10〜24時間が好ましいが、菌が生育することができる時間であれば、他の培養時間であってもよい。 The culture is desirably performed under anaerobic conditions, but may be performed under microaerobic conditions such as liquid stationary culture that is usually used. For anaerobic culture, known methods such as a method of culturing under a carbon gas gas layer can be applied, but other methods may be used. In general, the culture temperature is preferably 30 to 40 ° C. However, other temperature conditions may be used as long as the temperature allows the bacteria to grow. The pH of the medium during the culture is preferably maintained at 6.0 to 7.0, but may be other pH conditions as long as the temperature allows the bacteria to grow. It can also be cultured under batch culture conditions. The culture time is usually preferably 10 to 24 hours, but may be any other culture time as long as the bacteria can grow.
本発明に使用可能なLactobacillus gasseriも、グラム陽性桿菌である点や、その他の生理学的特徴について、Lactobacillus gasseri OLL2809と同様の特徴を示す。このようなLactobacillus gasseriは、当業者であればヒト糞便等から生理学的特徴に基づいて分離することができる。培養も、Lactobacillus gasseri OLL2809について上述したのと同様の方法によって可能である。 Lactobacillus gasseri that can be used in the present invention also has the same characteristics as Lactobacillus gasseri OLL2809 in that it is a Gram-positive rod and other physiological characteristics. Those skilled in the art can isolate such Lactobacillus gasseri from human feces and the like based on physiological characteristics. Culturing is also possible by the same method as described above for Lactobacillus gasseri OLL2809.
本発明の薬剤及び組成物は、Lactobacillus gasseriに何らかの処理を施した乳酸菌処理物としてのLactobacillus gasseriの菌体画分を含んでも良い。あるいは、Lactobacillus gasseriの菌体画分とLactobacillus gasseriの菌体画分を内包した乳酸菌そのものや、Lactobacillus gasseriを培養した培地を含んでもよい。例えばLactobacillus gasseriを乳酸菌懸濁液、乳酸菌培養物（菌体、培養上清液（培地成分を含む））、乳酸菌発酵物（乳酸菌飲料、酸乳、ヨーグルト等）等として使用することができる。 The chemical | medical agent and composition of this invention may also contain the microbial cell fraction of Lactobacillus gasseri as a lactic-acid-bacteria processed material which performed a certain process to Lactobacillus gasseri. Alternatively, a lactic acid bacterium itself encapsulating a bacterial cell fraction of Lactobacillus gasseri and a bacterial cell fraction of Lactobacillus gasseri, or a medium in which Lactobacillus gasseri is cultured may be included. For example, Lactobacillus gasseri can be used as a lactic acid bacterium suspension, lactic acid bacterium culture (bacteria, culture supernatant (including medium components)), lactic acid bacterium fermentation (lactic acid bacteria beverage, sour milk, yogurt, etc.) and the like.
本発明に用いられるLactobacillus gasseriに何らかの処理を施した乳酸菌処理物としては、例えばLactobacillus gasseriの菌体画分含有物、Lactobacillus gasseriの菌体画分を含有した発酵乳の濃縮物、Lactobacillus gasseriの菌体画分のペースト化物、Lactobacillus gasseriの菌体画分の乾燥物（噴霧乾燥物、凍結乾燥物、真空乾燥物、ドラム乾燥物から選ばれる少なくともひとつ）、Lactobacillus gasseriの菌体画分の液状物、Lactobacillus gasseriの菌体画分の希釈物、Lactobacillus gasseriの菌体画分の破砕物等が挙げられる。また、Lactobacillus gasseriの菌体画分を調製する際のLactobacillus gasseriとしては生菌体、湿潤菌、乾燥菌等が適宜使用可能である。あるいは、殺菌すなわち加熱殺菌処理、放射線殺菌処理、または破砕処理等を施した死菌体であってもよい。これらの処理工程は単独であっても複数を併用してもかまわない。このような乳酸菌処理物を、粉ミルクなど生物学的規格を有する医薬品および／または飲食品においても添加することも可能であり、医薬品および／または飲食品の形態などによらず様々な医薬品および／または飲食品に応用できる。 Lactobacillus gasseri used in the present invention as a lactic acid bacterium treated product, for example, Lactobacillus gasseri microbial fraction containing, Lactobacillus gasseri microbial concentrate, Lactobacillus gasseri bacterium Paste product of body fraction, dried product of bacterial cell fraction of Lactobacillus gasseri (at least one selected from spray-dried product, freeze-dried product, vacuum-dried product, and drum-dried product), liquid product of cell fraction of Lactobacillus gasseri , Dilution of the bacterial cell fraction of Lactobacillus gasseri, crushed material of the bacterial cell fraction of Lactobacillus gasseri, and the like. Further, as the Lactobacillus gasseri for preparing the bacterial cell fraction of Lactobacillus gasseri, live cells, wet bacteria, dry bacteria, and the like can be used as appropriate. Alternatively, it may be dead cells subjected to sterilization, that is, heat sterilization treatment, radiation sterilization treatment, or crushing treatment. These processing steps may be used alone or in combination. Such a processed product of lactic acid bacteria can also be added to pharmaceuticals and / or foods and drinks having biological standards such as powdered milk. Can be applied to food and drink.
本発明の薬剤及び組成物は、単独、または複数の混合物として経口投与したり、他の遅延型過敏症の軽減効果、予防効果、治療効果を有する化合物や微生物等と併用したりすることにより、ヒトおよび動物における遅延型過敏症の軽減に有効である。遅延型過敏症の種類は特に限定されないが、例えば、遅延型アトピー性皮膚炎、ツベルクリン反応、接触性皮膚炎、シェーグレン症候群、ギランバレー症候群、薬剤性肺炎等を挙げることができる。遅延型過敏症とは、これら遅延型過敏性反応を引き起こす疾患を包含し、遅延型過敏性反応に起因するアレルギー疾患と表現することもできる。 The drug and composition of the present invention can be orally administered alone or as a mixture, or can be used in combination with other delayed-type hypersensitivity reducing, preventing or therapeutic compounds or microorganisms. Effective in reducing delayed hypersensitivity in humans and animals. The type of delayed type hypersensitivity is not particularly limited, and examples thereof include delayed type atopic dermatitis, tuberculin reaction, contact dermatitis, Sjogren's syndrome, Guillain-Barre syndrome, and drug-induced pneumonia. Delayed type hypersensitivity includes diseases that cause these delayed type hypersensitivity reactions and can also be expressed as allergic diseases caused by delayed type hypersensitivity reactions.
遅延型過敏症の軽減効果、遅延型過敏性反応の抑制効果は、遅延型過敏症に特有の、当業者に周知の様々な症状の変化を観察することにより確認することができる。例えば身体の各組織や器官における腫脹や炎症、かゆみ、かぶれ等の変化を観察することにより、遅延型過敏症の改善効果、遅延型過敏性反応の抑制効果を確認することができるがこれらに限定されない。またCD4+T細胞の増殖抑制の効果も、例えば実施例に記載されている[3H]-チミジンの取り込み量を測定する方法、MTTアッセイなど当業者に周知の方法により確認することができる。The effect of reducing delayed type hypersensitivity and the effect of suppressing delayed type hypersensitivity reaction can be confirmed by observing various symptom changes known to those skilled in the art that are specific to delayed type hypersensitivity. For example, by observing changes in swelling, inflammation, itching, rash, etc. in each tissue or organ of the body, it is possible to confirm the improvement effect of delayed type hypersensitivity and the inhibitory effect of delayed type hypersensitivity reaction. Not. The effect of suppressing the proliferation of CD4 + T cells can also be confirmed by methods well known to those skilled in the art, such as a method for measuring the amount of [ 3 H] -thymidine incorporation described in the Examples and an MTT assay.
本発明の薬剤や組成物を医薬品または飲食品へ配合する配合量は、形態、剤型、症状、体重、用途などによって異なるため、特に限定されないが、あえて挙げるなら、0.001〜100 %(w/w)の含量で配合することができ、好ましくは0.01〜100%(w/w)、さらに好ましくは0.1〜100%(w/w)の含量で配合することができる。 The blending amount of the drug or composition of the present invention into a pharmaceutical product or food and drink is not particularly limited because it varies depending on the form, dosage form, symptom, body weight, use, etc., but if it is intentionally mentioned, 0.001 to 100% (w / It can be blended with a content of w), preferably 0.01 to 100% (w / w), more preferably 0.1 to 100% (w / w).
本発明の薬剤や組成物の医薬品または飲食品としての一日当たりの摂取量は年齢、症状、体重、用途などによって異なるため特に限定されないが、あえて挙げるなら0.1〜1000mg/kgを摂取することができ、好ましくは0.1〜100mg/kgを摂取することができる。
また本発明の薬剤は、経口投与又は非経口投与（筋肉内、皮下、静脈内、坐薬等）のいずれでも投与できる。The daily intake of the drug or composition of the present invention as a pharmaceutical product or food and drink is not particularly limited because it varies depending on age, symptoms, body weight, use, etc., but if it is mentioned, 0.1 to 1000 mg / kg can be ingested. Preferably, 0.1-100 mg / kg can be ingested.
The drug of the present invention can be administered either orally or parenterally (intramuscular, subcutaneous, intravenous, suppository, etc.).
本発明の薬剤や組成物は、医薬品または飲食品のいずれの形態でも利用することができる。例えば、医薬品として直接投与することにより、または特定保健用食品等の特別用途食品や栄養機能食品として直接摂取することにより、各種の遅延型過敏症を軽減することが期待される。また、各種飲食品（乳製品、牛乳、清涼飲料、発酵乳、ヨーグルト、チーズ、パン、ビスケット、クラッカー、ピッツァクラスト、調製粉乳（特に乳児用調製粉乳）、流動食、病者用食品、幼児用粉乳等食品、授乳婦用粉乳等食品、栄養食品、保健機能食品等）に本発明の薬剤や組成物、これらを構成する乳酸菌を添加し、これを摂取してもよい。Lactobacillus gasseriの菌体画分をそのまま使用し、あるいは、Lactobacillus gasseriの菌体画分を含む乳酸菌や、他の食品ないし食品成分と混合するなど、通常の飲食品組成物における常法にしたがって使用できる。また、その性状についても、通常用いられる飲食品の状態、例えば、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。
本発明の薬剤や組成物を含有する飲食品には、水、タンパク質、糖質、脂質、ビタミン類、ミネラル類、有機酸、有機塩基、果汁、フレーバー類等を主成分として使用することができる。タンパク質としては、例えば全脂粉乳、脱脂粉乳、部分脱脂粉乳、カゼイン、ホエー粉、ホエータンパク質、ホエータンパク質濃縮物、ホエータンパク質分離物、α―カゼイン、β―カゼイン、κ−カゼイン、β―ラクトグロブリン、α―ラクトアルブミン、ラクトフェリン、大豆タンパク質、鶏卵タンパク質、肉タンパク質等の動植物性タンパク質、これら加水分解物；バター、乳性ミネラル、クリーム、ホエー、非タンパク態窒素、シアル酸、リン脂質、乳糖等の各種乳由来成分などが挙げられる。糖質としては、糖類、加工澱粉（テキストリンのほか、可溶性澱粉、ブリティッシュスターチ、酸化澱粉、澱粉エステル、澱粉エーテル等）、食物繊維などが挙げられる。脂質としては、例えば、ラード、魚油等、これらの分別油、水素添加油、エステル交換油等の動物性油脂；パーム油、サフラワー油、コーン油、ナタネ油、ヤシ油、これらの分別油、水素添加油、エステル交換油等の植物性油脂などが挙げられる。ビタミン類としては、例えば、ビタミンA、カロチン類、ビタミンB群、ビタミンC、ビタミンD群、ビタミンE、ビタミンK群、ビタミンP、ビタミンQ、ナイアシン、ニコチン酸、パントテン酸、ビオチン、イノシトール、コリン、葉酸などが挙げられ、ミネラル類としては、例えば、カルシウム、カリウム、マグネシウム、ナトリウム、銅、鉄、マンガン、亜鉛、セレンなどが挙げられる。有機酸としては、例えば、リンゴ酸、クエン酸、乳酸、酒石酸などが挙げられる。これらの成分は、2種以上を組み合わせて使用することができ、合成品および／またはこれらを多く含む食品を用いてもよい。The drug or composition of the present invention can be used in any form of a pharmaceutical or a food or drink. For example, it is expected to reduce various types of delayed type hypersensitivity by directly administering as a pharmaceutical or by directly ingesting as a special-purpose food such as a food for specified health use or a nutritional functional food. In addition, various foods and drinks (dairy products, milk, soft drinks, fermented milk, yogurt, cheese, bread, biscuits, crackers, pizza crust, prepared milk powder (especially infant formula), liquid food, food for the sick, infants The drug or composition of the present invention and the lactic acid bacteria constituting them may be added to foods such as powdered milk, foods such as milk powder for nursing women, nutritional foods, health functional foods, etc. The bacterial cell fraction of Lactobacillus gasseri can be used as it is, or it can be used according to conventional methods in ordinary food and beverage compositions such as lactic acid bacteria containing the bacterial cell fraction of Lactobacillus gasseri and other foods or food ingredients. . Moreover, about the property, the state of the food / beverage products normally used, for example, any of solid (powder, granule, etc.), paste, liquid or suspension may be sufficient.
For foods and drinks containing the drug or composition of the present invention, water, proteins, carbohydrates, lipids, vitamins, minerals, organic acids, organic bases, fruit juices, flavors, and the like can be used as main components. . Examples of the protein include whole milk powder, skim milk powder, partially skim milk powder, casein, whey powder, whey protein, whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin , Α-lactalbumin, lactoferrin, soy protein, chicken egg protein, meat protein and other animal and plant proteins, hydrolysates thereof; butter, milk minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipid, lactose, etc. And various milk-derived components. Examples of the saccharide include saccharides, processed starch (in addition to text phosphorus, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber, and the like. Examples of the lipid include animal oils such as lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterified oil, etc .; palm oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils thereof, Examples include vegetable oils such as hydrogenated oils and transesterified oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline. Examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, and selenium. Examples of the organic acid include malic acid, citric acid, lactic acid, and tartaric acid. Two or more of these components can be used in combination, and a synthetic product and / or a food containing a large amount thereof may be used.
本発明の薬剤や組成物を医薬品として使用する場合には、種々の形態で投与することができる。その形態として、例えば、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、被覆錠剤、丸剤、液剤、懸濁剤、乳剤、注射剤、坐剤、浸剤、煎剤、チンキ剤等を挙げることができる。これらの各種製剤は、常法に従って主剤に対して必要に応じて賦形剤、結合剤、崩壊剤、滑沢剤、矯臭剤、溶解補助剤、懸濁剤、コーティング剤、充填剤、増量剤、保湿剤、界面活性剤などの医薬の製剤技術分野において通常使用し得る既知の補助剤を用いて製剤化することができる。また、この医薬製剤中に着色剤、保存剤、香料、風味剤、甘味剤等や他の医薬品を含有させてもよい。 When the drug or composition of the present invention is used as a pharmaceutical product, it can be administered in various forms. Examples of the form include tablets, capsules, granules, powders, syrups, coated tablets, pills, liquids, suspensions, emulsions, injections, suppositories, suppositories, decoction, tinctures and the like. it can. These various preparations are prepared in accordance with conventional methods as necessary with respect to the main agent, such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, suspension agents, coating agents, fillers, and bulking agents. In addition, it can be formulated using known adjuvants that can be usually used in the pharmaceutical formulation technical field such as humectants and surfactants. Moreover, you may contain a coloring agent, a preservative, a fragrance | flavor, a flavoring agent, a sweetening agent, etc. and other pharmaceuticals in this pharmaceutical formulation.
本発明の薬剤または組成物を構成するLactobacillus gasseriの菌体画分の量は、その目的、用途（薬剤、飲食品組成物）に応じて任意に定めることができる。本発明はこれに限定されないがその含量としては、全体量に対して通常、0.001〜100％（w/w）、特に0.01〜100％（w/w）が好ましい。 The amount of the bacterial cell fraction of Lactobacillus gasseri constituting the drug or composition of the present invention can be arbitrarily determined according to its purpose and use (drug, food and beverage composition). Although this invention is not limited to this, As the content, 0.001-100% (w / w) is preferable normally with respect to the whole quantity, Especially 0.01-100% (w / w) is preferable.
Lactobacillus gasseriの菌体画分を含む組成物や薬剤を飲食品や医薬品の製造に使用する場合、製造方法は当業者に周知の方法によって行うことができる。当業者であれば、Lactobacillus gasseriの菌体画分を他の成分と混合する工程、成形工程、殺菌工程、発酵工程、焼成工程、乾燥工程、冷却工程、造粒工程、包装工程等を適宜組み合わせ、所望の飲食品や医薬品を作ることが可能である。 When a composition or drug containing a bacterial cell fraction of Lactobacillus gasseri is used for the production of foods and drinks or pharmaceuticals, the production method can be carried out by methods well known to those skilled in the art. If it is a person skilled in the art, Lactobacillus gasseri's bacterial cell fraction is mixed with other ingredients, molding process, sterilization process, fermentation process, firing process, drying process, cooling process, granulation process, packaging process, etc. as appropriate It is possible to make desired foods and beverages and medicines.
またLactobacillus gasseriの菌体画分を各種乳製品の製造に使用する場合も、当業者に周知の方法で所望の乳製品を製造できる。ヨーグルトの場合の一例を示せば、Lactobacillus gasseriを用いてスターターを調製する工程、該スターターを前処理した牛乳に加えて培養する工程、冷却工程、フレーバーリング工程、充填工程等を経てヨーグルトを製造できる。チーズであれば、例えば、殺菌等の前処理をした牛乳に本発明の乳酸菌をスターターとして添加して乳酸発酵させる工程、レンネットを添加してチーズカードを生成する工程、カード切断工程、ホエー排出工程、加塩工程、熟成工程などを経て製造可能である。あるいは、前記各種乳製品の製造において、他の乳酸菌をスターターとして用い、製造工程中にLactobacillus gasseriまたはLactobacillus gasseriの菌体画分を添加してもよい。 Moreover, also when using the microbial cell fraction of Lactobacillus gasseri for manufacture of various dairy products, a desired dairy product can be manufactured by a method well-known to those skilled in the art. As an example of yogurt, yogurt can be manufactured through a process of preparing a starter using Lactobacillus gasseri, a process of adding the starter to pre-treated milk, a culture process, a cooling process, a flavoring process, a filling process, etc. . If it is cheese, for example, the step of adding lactic acid bacteria of the present invention as a starter to milk that has been pretreated such as sterilization, the step of lactic acid fermentation, the step of adding rennet to produce cheese curd, the card cutting step, whey discharge It can be manufactured through a process, a salting process, an aging process, and the like. Alternatively, in the production of the various dairy products, other lactic acid bacteria may be used as a starter, and Lactobacillus gasseri or a bacterial fraction of Lactobacillus gasseri may be added during the production process.
また本発明は、Lactobacillus gasseriの菌体画分を動物に経口投与する工程を含む、遅延型過敏症の軽減方法に関する。Lactobacillus gasseriの菌体画分が投与される対象としては哺乳動物が挙げられる。哺乳動物としてはヒト及びヒト以外の哺乳動物が挙げられ、好ましくはヒトやサルが挙げられ、より好ましくはヒトが挙げられる。
また本発明は、遅延型過敏症の軽減に使用するためのLactobacillus gasseriの菌体画分に関する。あるいは本発明は遅延型過敏症の軽減剤や軽減用組成物の製造におけるLactobacillus gasseriの菌体画分の使用に関する。また本発明は、遅延型過敏症の軽減のためのLactobacillus gasseriの菌体画分の使用に関する。また本発明は、Lactobacillus gasseriの菌体画分と薬学的に許容される担体を配合する工程を含む、遅延型過敏症の軽減剤や軽減用組成物の製造方法に関する。The present invention also relates to a method for reducing delayed hypersensitivity, comprising the step of orally administering a bacterial cell fraction of Lactobacillus gasseri to an animal. Mammals are examples of subjects to which the bacterial cell fraction of Lactobacillus gasseri is administered. Mammals include humans and mammals other than humans, preferably humans and monkeys, more preferably humans.
The present invention also relates to a bacterial fraction of Lactobacillus gasseri for use in reducing delayed hypersensitivity. Alternatively, the present invention relates to the use of a bacterial cell fraction of Lactobacillus gasseri in the manufacture of a delayed hypersensitivity alleviating agent or a reducing composition. The present invention also relates to the use of a bacterial fraction of Lactobacillus gasseri for the reduction of delayed type hypersensitivity. The present invention also relates to a method for producing a delayed-type hypersensitivity alleviating agent or a reducing composition comprising a step of blending a bacterial cell fraction of Lactobacillus gasseri and a pharmaceutically acceptable carrier.
あるいは本発明は、Lactobacillus gasseriの菌体画分を含む遅延型過敏性反応の抑制剤を提供する。また本発明は、Lactobacillus gasseriの菌体画分を動物に投与する工程を含む遅延型過敏性反応の抑制方法を提供する。さらに本発明は、遅延型過敏性反応の抑制に使用するためのLactobacillus gasseriの菌体画分に関する。あるいは本発明は、遅延型過敏性反応の抑制剤や抑制用組成物の製造におけるLactobacillus gasseriの菌体画分の使用に関する。あるいは本発明は、遅延型過敏性反応を抑制するためのLactobacillus gasseriの菌体画分の使用に関する。あるいは本発明は、Lactobacillus gasseriの菌体画分と薬学的に許容される担体を配合する工程を含む、遅延型過敏性反応の抑制剤や抑制用組成物の製造方法に関する。 Or this invention provides the inhibitor of the delayed type hypersensitivity reaction containing the microbial cell fraction of Lactobacillus gasseri. The present invention also provides a method for suppressing a delayed hypersensitivity reaction, comprising a step of administering a bacterial fraction of Lactobacillus gasseri to an animal. Furthermore, the present invention relates to a bacterial cell fraction of Lactobacillus gasseri for use in suppressing delayed hypersensitivity reactions. Alternatively, the present invention relates to the use of a bacterial cell fraction of Lactobacillus gasseri in the manufacture of a delayed hypersensitivity reaction inhibitor or a composition for suppression. Alternatively, the present invention relates to the use of a bacterial cell fraction of Lactobacillus gasseri for suppressing delayed hypersensitivity reactions. Or this invention relates to the manufacturing method of the inhibitor of delayed type hypersensitivity reaction, and the composition for suppression including the process of mix | blending the microbial cell fraction of Lactobacillus gasseri, and a pharmaceutically acceptable support | carrier.
さらに本発明は、Lactobacillus gasseriの菌体画分を含むCD4+T細胞の増殖抑制剤を提供する。また本発明は、Lactobacillus gasseriの菌体画分を動物に投与する工程を含むCD4+T細胞の増殖を抑制する方法を提供する。さらに本発明は、CD4+T細胞の増殖の抑制に使用するためのLactobacillus gasseriの菌体画分に関する。あるいは本発明は、CD4+T細胞の増殖抑制剤や増殖抑制用組成物の製造におけるLactobacillus gasseriの菌体画分の使用に関する。あるいは本発明は、CD4+T細胞の増殖を抑制するためのLactobacillus gasseriの菌体画分の使用に関する。あるいは本発明は、Lactobacillus gasseriの菌体画分と薬学的に許容される担体を配合する工程を含む、CD4+T細胞の増殖抑制剤や増殖抑制用組成物の製造方法に関する。
本発明の薬剤や組成物は、経口投与に好適な担体を配合することができる。本発明の薬剤や組成物は、遅延型過敏症の軽減を目的として、食品を兼ねて投与することができる。The present invention further provides a growth inhibitor of CD4 + T cells containing a bacterial cell fraction of Lactobacillus gasseri. The present invention also provides a method for inhibiting the proliferation of CD4 + T cells, comprising the step of administering a bacterial cell fraction of Lactobacillus gasseri to an animal. Furthermore, the present invention relates to a cell fraction of Lactobacillus gasseri for use in suppressing the proliferation of CD4 + T cells. Alternatively, the present invention relates to the use of a bacterial cell fraction of Lactobacillus gasseri in the production of a growth inhibitor or composition for inhibiting growth of CD4 + T cells. Alternatively, the present invention relates to the use of a bacterial fraction of Lactobacillus gasseri for suppressing the proliferation of CD4 + T cells. Alternatively, the present invention relates to a method for producing a CD4 + T cell growth inhibitor and a composition for inhibiting growth, which comprises a step of blending a bacterial cell fraction of Lactobacillus gasseri and a pharmaceutically acceptable carrier.
The drug and composition of the present invention can contain a carrier suitable for oral administration. The drug or composition of the present invention can be administered also as a food for the purpose of reducing delayed hypersensitivity.
以下に本発明を実施例に基づいてさらに説明するが、かかる実施例は本発明の例示であり、本発明を限定するものではない。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。The present invention will be further described below based on examples, but these examples are illustrative of the present invention and do not limit the present invention.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
脾臓CD4Spleen CD4 ++ T細胞の増殖応答の抑制効果 Suppressive effect of T cell proliferation response
１．材料と方法
〔動物〕
8〜10週齢の雌性BALB/cマウスは日本クレアから購入した。8〜10週齢の雌性のDO11.10 TCRトランスジェニックマウスは、卵白アルブミン（OVA）の323-339位に特異的な、I-Ad拘束性T細胞受容体(TCR)αβ鎖（BALB/cマウス由来）を発現するものを用いた。1. Materials and methods [animal]
8-10 week old female BALB / c mice were purchased from CLEA Japan. 8-10 week old female DO11.10 TCR transgenic mice are IA d- restricted T cell receptor (TCR) αβ chain (BALB / c mice, specific for ovalbumin (OVA) positions 323-339 The one expressing the origin) was used.
〔加熱処理細菌懸濁物およびその画分の調製〕
加熱処理菌体（乾燥菌体重量で3mg）は遠心分離によって集菌し、生理食塩水に5mg/mlの濃度で懸濁した。菌体懸濁液に0.5gのガラスビーズ（直径0.1mm; Sigma-Aldrich, St. Louis, MO）を加え、マルチビーズショッカー（安井機械）で破砕し、菌体破砕液を得た。破砕液を4℃、300×gで10分間遠心分離し、上清と沈殿画分に分画した。[Preparation of heat-treated bacterial suspension and its fractions]
Heat-treated cells (3 mg by dry cell weight) were collected by centrifugation and suspended in physiological saline at a concentration of 5 mg / ml. 0.5 g of glass beads (diameter 0.1 mm; Sigma-Aldrich, St. Louis, MO) was added to the cell suspension, and the cells were crushed with a multi-bead shocker (Yasui Kikai) to obtain a cell lysate. The disrupted solution was centrifuged at 4 ° C. and 300 × g for 10 minutes, and fractionated into a supernatant and a precipitate fraction.
乳酸菌株のうち、菌株名にMEPと記載された菌株は株式会社明治の保有菌株である。
また、MEP225101は、Lactobacillus gasseri種であり、MEP225102は、Lactobacillus casei種である。また、Lactobacillus gasseri OLL2809をLG2809と略して記載することがある。Among the lactic acid strains, the strain whose name is described as MEP is a strain possessed by Meiji Co., Ltd.
MEP225101 is a Lactobacillus gasseri species, and MEP225102 is a Lactobacillus casei species. In addition, Lactobacillus gasseri OLL2809 may be abbreviated as LG2809.
〔菌体破砕液上清の酵素処理〕
菌体破砕液の上清は以下の様に処理した。(1)37℃で4時間放置、(2)37℃で4時間放置し、その後96℃で10分間加熱処理、(3)100μg/mlのDNase I（Sigma-Aldrich）を加えて37℃で4時間放置し、その後96℃で10分間加熱処理してDNase Iを不活性化、(4)100μg/mlのプロテイナーゼK（Sigma-Aldrich）を加えて37℃で4時間放置し、その後96℃で10分間加熱処理して酵素を不活性化、(5) 100μg/mlのRNase A（Sigma-Aldrich）を加えて37℃で4時間放置し、その後Proteinase K処理で酵素を不活性化した。[Enzyme treatment of supernatant of disrupted bacterial cells]
The supernatant of the cell disruption solution was treated as follows. (1) Leave at 37 ° C for 4 hours, (2) Leave at 37 ° C for 4 hours, then heat treatment at 96 ° C for 10 minutes, (3) Add 100 µg / ml DNase I (Sigma-Aldrich) at 37 ° C Leave for 4 hours, then heat-treat at 96 ° C for 10 minutes to inactivate DNase I. (4) Add 100 µg / ml proteinase K (Sigma-Aldrich) and leave at 37 ° C for 4 hours, then 96 ° C (5) 100 μg / ml RNase A (Sigma-Aldrich) was added and left at 37 ° C. for 4 hours, followed by proteinase K treatment to inactivate the enzyme.
〔細菌からのRNAの抽出〕
細菌からのRNA抽出はRNeasy Mini Kit (Qiagen, Hilden Germany)を用いて行った。培養終了後の菌体（乾燥菌体重量で3mg）を遠心分離し、ペレットを生理食塩水に懸濁した。ペレットに0.5gのガラスビーズ（0.1mm）を加えてマルチビーズショッカーで破砕した。破砕液にBuffer RLTを加え、10秒間激しく混合した。遠心分離後、上清を新しいチューブに移し、等量の70%エタノールを加えてピペッティングした。破砕液をRNeasy spin columに移し、8000×gで15秒間遠心分離した。Buffer RW1をRNeasy spin columに加えて再度遠心分離し、洗浄した。Buffer RPEをRNeasy spin columに添加し、8000×gで2分間遠心分離し、洗浄した。30μlの水をRNeasy spin columに加え、8000×gで1分間遠心分離し、RNAを溶出した。抽出したRNAは使用時まで-80℃で保存した。[Extraction of RNA from bacteria]
RNA extraction from bacteria was performed using RNeasy Mini Kit (Qiagen, Hilden Germany). The cells after the culture (3 mg by dry cell weight) were centrifuged, and the pellets were suspended in physiological saline. 0.5 g glass beads (0.1 mm) were added to the pellets and crushed with a multi-bead shocker. Buffer RLT was added to the disrupted solution and mixed vigorously for 10 seconds. After centrifugation, the supernatant was transferred to a new tube and pipetted with an equal volume of 70% ethanol. The disrupted solution was transferred to RNeasy spin colum and centrifuged at 8000 × g for 15 seconds. Buffer RW1 was added to RNeasy spin colum and centrifuged again and washed. Buffer RPE was added to RNeasy spin colum, centrifuged at 8000 × g for 2 minutes and washed. 30 μl of water was added to RNeasy spin colum and centrifuged at 8000 × g for 1 minute to elute the RNA. The extracted RNA was stored at −80 ° C. until use.
〔マウス細胞株〕
マウス由来の細胞は10%のFCS（牛胎児血清、Gibco, Grand Island, NY）、2g/LのNaHCO3、100U/mlのペニシリン、100μg/mlのストレプトマイシン、50μM 2-メルカプトエタノール、300mg/L L-グルタミンを含むRPMI1640培地（日水製薬）で、37℃で5%のCO2インキュベーターで培養した。[Mouse cell line]
Mouse-derived cells are 10% FCS (fetal calf serum, Gibco, Grand Island, NY), 2 g / L NaHCO 3 , 100 U / ml penicillin, 100 μg / ml streptomycin, 50 μM 2-mercaptoethanol, 300 mg / L The cells were cultured in RPMI1640 medium (Nissui Pharmaceutical) containing L-glutamine at 37 ° C. in a 5% CO 2 incubator.
〔CD4+ T細胞増殖アッセイ〕
BALB/cマウスからの脾細胞の調製は既出の方法で行った。T細胞不含脾細胞は、脾細胞からMACS（磁気細胞分離装置）を用いて、Thy1.2マイクロビーズ（Miltenyi Biotec, Bergish Gladbach, Germany）によるネガティブセレクションを行って調製した。抗原提示細胞として調製したこれらの細胞は1×105cells/ウェルの濃度で、菌体懸濁液（3株）、LG2809菌体破砕液、LG2809由来RNA、マウス脾臓RNA（Ambion, Austin, TX）の存在下で、96ウェル平底プレートで、37℃で4時間、5%濃度のCO2インキュベーターで培養した。DO11.10マウス由来CD4+ T細胞は脾細胞からMACSを用いて、既出の方法によってCD4マイクロビーズ（Miltenyi Biotec）によるポジティブセレクションを行って調製した。分離したCD4+ T細胞（5×104cells/ウェル）およびOVA 323-339 (0.1, 0.3, 1μM)を、抗原提示細胞に各被験物質を加えた培養液に添加した。培養3日後、1ウェルに37kBqの[3H]-チミジンを添加し、さらに24時間培養した。March III harvester (Tomtec, Hamden, CT)を用いて細胞を回収し、細胞に取り込まれなかった[3H]-チミジンをTrilux1450 Microbeta counter (Wallac, Gaithersburg, MD)およびMicrobeta 270.004ソフトウェア(Wallac)を用いて解析した。
抗原提示細胞非存在下でのT細胞の活性化には、96ウェル平底プレートを10μg/mlの抗CD3εモノクローナル抗体（BD Biosciences, San Jose, CA）で、37℃で2時間コーティングした。BALB/cマウスまたはMyD88欠損マウス（BALB/cマウス由来）の脾細胞から単離したCD4+ T細胞を5×104cells/ウェルの濃度で、抗CD3εモノクローナル抗体でコーティングしたプレートに加え、2μg/mlの抗CD28モノクローナル抗体（BD Biosciences）と共に、LG2809またはRNA（LG2809由来またはマウス脾臓由来）の存在下で72時間培養した。また別の試験では、N-アセチルシステイン（NAC）を25mMの濃度で添加して培養した。細胞の増殖応答測定は上述の方法と同様に行った。[CD4 + T cell proliferation assay]
Preparation of splenocytes from BALB / c mice was performed by the method described above. T cell-free splenocytes were prepared from spleen cells using MACS (magnetic cell separator) and negative selection with Thy1.2 microbeads (Miltenyi Biotec, Bergish Gladbach, Germany). These cells were prepared as antigen-presenting cells at a concentration of 1 × 10 5 cells / well. Cell suspension (3 strains), LG2809 cell disruption solution, LG2809-derived RNA, mouse spleen RNA (Ambion, Austin, TX) ) In a 96-well flat-bottom plate at 37 ° C. for 4 hours in a 5% concentration CO 2 incubator. DO11.10 mouse-derived CD4 + T cells were prepared from spleen cells using MACS and positive selection using CD4 microbeads (Miltenyi Biotec) by the method described above. The separated CD4 + T cells (5 × 10 4 cells / well) and OVA 323-339 (0.1, 0.3, 1 μM) were added to a culture solution in which each test substance was added to antigen-presenting cells. After 3 days of culture, 37 kBq [ 3 H] -thymidine was added to 1 well, and the cells were further cultured for 24 hours. Cells were harvested using March III harvester (Tomtec, Hamden, CT), and [ 3 H] -thymidine that was not taken up by the cells using Trilux1450 Microbeta counter (Wallac, Gaithersburg, MD) and Microbeta 270.004 software (Wallac) And analyzed.
For activation of T cells in the absence of antigen presenting cells, 96-well flat bottom plates were coated with 10 μg / ml anti-CD3ε monoclonal antibody (BD Biosciences, San Jose, Calif.) At 37 ° C. for 2 hours. Add CD4 + T cells isolated from spleen cells of BALB / c mice or MyD88-deficient mice (derived from BALB / c mice) to a plate coated with anti-CD3ε monoclonal antibody at a concentration of 5 × 10 4 cells / well, 2 μg Cultured for 72 hours in the presence of LG2809 or RNA (from LG2809 or mouse spleen) with / ml anti-CD28 monoclonal antibody (BD Biosciences). In another test, N-acetylcysteine (NAC) was added at a concentration of 25 mM and cultured. Cell proliferation response was measured in the same manner as described above.
〔LG2809によるDO11.10由来脾臓CD4+ T細胞の増殖応答の抑制の検討方法〕
BALB/cマウス脾臓細胞からT細胞を除去した細胞(抗原提示細胞；APC)に、(図１B)加熱処理をしたLactobacillus属(LG2809、MEP225101、MEP225102(10μg dry weight/ml)) あるいは、(図１C)LG2809を濃度依存的に添加して、4時間培養した。DO11.10マウス由来脾臓CD4+ T細胞とOVAペプチド (1μM)を添加して96時間培養後の増殖応答を、[3H]-チミジン取り込み量を測定することで検討した（図１A）。各実験で対照は無添加区とした。グラフは平均値+標準偏差を示す。群間の有意差は、Tukey法を用いて多重検定した（a-c:異符号間で有意差あり、P<0.05）。
[Method for studying suppression of proliferation response of spleen CD4 + T cells derived from DO11.10 by LG2809]
BALB / c mouse spleen cells (antigen-presenting cells; APC) removed from T cells (FIG. 1B) heat-treated Lactobacillus genus (LG2809, MEP225101, MEP225102 (10 μg dry weight / ml) ) or (FIG. 1C) LG2809 was added in a concentration-dependent manner and cultured for 4 hours. The proliferation response after 96-hour culture after addition of DO11.10 mouse-derived splenic CD4 + T cells and OVA peptide (1 μM) was examined by measuring the amount of [ 3 H] -thymidine incorporation (FIG. 1A). In each experiment, the control was an additive-free group. The graph shows the average value + standard deviation. The significant difference between groups was subjected to a multiple test using the Tukey method (ac: significant difference between different signs, P <0.05).
〔増殖応答抑制に関与する菌体成分の探索方法〕
試験方法は上記のLG2809によるDO11.10由来脾臓CD4+ T細胞の増殖応答の抑制の検討方法（図1A）と同様に行った。加熱処理をしたLG2809 (heat-killed)、LG2809の破砕液 (homogenate),破砕液の上清 (sup)、破砕液の沈殿 (ppt) (0.1, 1, 10 および100μg dry weight LG2809/ml)をAPC に添加した（図2A）。LG2809の破砕上清 (100μg dry weight LG2809/ml)を、5パターンで処理をした。破砕上清は、DNase I (iii)、Proteinase K (iv)、そして RNase A (v)で処理した。DNase I およびProteinase K は96℃で10分間加熱することで不活化し、RNase A はProteinase K 処理で不活化した。破砕上清は37℃ で4時間放置 (i)、または 37℃で4 時間放置し、その後96℃で10分間加熱処理(ii)をすることで対照とした（図2B)。0.1、1および10μg/mlのLG2809 RNAを培養系に添加した（図2C)。1、10μg/mlのマウス脾臓からのRNAを培養系に添加した（図2D)。各実験で対照は無添加区とした。グラフは平均値+標準偏差を示す。群間の有意差は、Tukey法を用いて多重検定した（a-c:異符号間で有意差あり、P<0.05）。[Method of searching for bacterial cell components involved in growth response suppression]
The test method was the same as the above-described method for examining the suppression of proliferation response of DO11.10-derived splenic CD4 + T cells by LG2809 (FIG. 1A). Heat treated LG2809 (heat-killed), LG2809 homogenate, supernatant of supernatant (sup), precipitate of precipitate (ppt) (0.1, 1, 10 and 100μg dry weight LG2809 / ml) Added to APC (Figure 2A). LG2809 crushed supernatant (100 μg dry weight LG2809 / ml) was treated in 5 patterns. The disrupted supernatant was treated with DNase I (iii), Proteinase K (iv), and RNase A (v). DNase I and proteinase K were inactivated by heating at 96 ° C for 10 minutes, and RNase A was inactivated by proteinase K treatment. The disrupted supernatant was allowed to stand at 37 ° C. for 4 hours (i), or left at 37 ° C. for 4 hours, and then heat-treated at 96 ° C. for 10 minutes (ii) as a control (FIG. 2B). 0.1, 1 and 10 μg / ml LG2809 RNA was added to the culture system (FIG. 2C). 1, 10 μg / ml of mouse spleen RNA was added to the culture system (FIG. 2D). In each experiment, the control was an additive-free group. The graph shows the average value + standard deviation. The significant difference between groups was subjected to a multiple test using the Tukey method (ac: significant difference between different signs, P <0.05).
〔LG2809およびそのRNAによるCD4+ T細胞直接的な増殖応答の抑制の検討方法〕
あらかじめ、抗CD3εモノクローナル抗体（10μg/ml）をコーティングしたプレートに、BALB/cマウスから調製したCD4+ T細胞（1×105cells／ウェル）と、抗CD28モノクローナル抗体（2μg/ml）、加熱処理をしたLG2809（図3B)、または加熱処理をしたLG2809のRNA（図3C)、またはマウス脾臓由来のRNA（図3D）を、終濃度0.1〜10μg/mlになるように添加して96時間培養した。96時間培養の24時間前に、[3H]-チミジンをプレートに添加し、その取り込み量を測定することで検討した（図3A）。各実験で対照は無添加区とした。グラフは平均値+標準偏差を示す。群間の有意差は、Tukey法を用いて多重検定した（a-c:異符号間で有意差あり、P<0.05）。[Method of studying suppression of direct proliferation response of CD4 + T cells by LG2809 and its RNA]
A plate coated with anti-CD3ε monoclonal antibody (10 μg / ml) in advance, CD4 + T cells (1 × 10 5 cells / well) prepared from BALB / c mice, anti-CD28 monoclonal antibody (2 μg / ml), and heating LG2809 treated (Figure 3B), heat treated LG2809 RNA (Figure 3C), or mouse spleen-derived RNA (Figure 3D) added to a final concentration of 0.1-10 μg / ml for 96 hours Cultured. 24 hours before the 96-hour culture, [ 3 H] -thymidine was added to the plate and the amount taken up was measured (FIG. 3A). In each experiment, the control was an additive-free group. The graph shows the average value + standard deviation. The significant difference between groups was subjected to a multiple test using the Tukey method (ac: significant difference between different signs, P <0.05).
〔LG2809とそのRNAによる増殖応答の抑制効果におけるMyD88経路の関与の検討方法〕
あらかじめ、抗CD3εモノクローナル抗体（10μg/ml）をコーティングしたプレートに、（図4B）BALB/cマウス（Wild-type）、または（図4C）MyD88欠損BALB/cマウス（MyD88-/-）から調製したCD4+ T細胞（1×105cells／ウェル）と、抗CD28モノクローナル抗体（2μg/ml）、加熱処理をしたLG2809とそのRNAを、終濃度10μg/mlになるように添加し、96時間培養した。96時間培養の24時間前に、[3H]-チミジンをプレートに添加し、その取り込み量を測定することで検討した（図4A）。各実験で対照は無添加区とした。グラフは平均値+標準偏差を示す。群間の有意差は、Tukey法を用いて多重検定した（a,b:異符号間で有意差あり、P<0.05）。[Examination method of involvement of MyD88 pathway in inhibiting growth response by LG2809 and its RNA]
Prepared from BALB / c mice (Wild-type) or (Figure 4C) MyD88-deficient BALB / c mice (MyD88-/-) on a plate pre-coated with anti-CD3ε monoclonal antibody (10 μg / ml) CD4 + T cells (1 × 10 5 cells / well), anti-CD28 monoclonal antibody (2 μg / ml), heat-treated LG2809 and its RNA were added to a final concentration of 10 μg / ml for 96 hours. Cultured. 24 hours before the 96-hour culture, [ 3 H] -thymidine was added to the plate, and the amount taken up was measured (FIG. 4A). In each experiment, the control was an additive-free group. The graph shows the average value + standard deviation. The significant difference between groups was subjected to a multiple test using the Tukey method (a, b: significant difference between different signs, P <0.05).
〔LG2809による増殖応答の抑制効果における酸化ストレスの関与の検討方法〕
あらかじめ、抗CD3εモノクローナル抗体（10μg/ml）をコーティングしたプレートに、BALB/cマウス（Wild-type）から調製したCD4+ T細胞（1×105cells／ウェル）、N-アセチルシステイン（NAC、25mM）、抗CD28モノクローナル抗体（2μg/ml）、加熱処理をしたLG2809とそのRNA（終濃度10μg/ml)を添加し、96時間培養した。96時間培養の24時間前に、[3H]-チミジンをプレートに添加し、その取り込み量を測定することで検討した（図5A,B）。対照はLG2809またはLG2809由来RNAを加えない無添加区とした。グラフは平均値+標準偏差を示す。群間の有意差は、Studentのt検定を用いた（**：P<0.01）。[Examination method of involvement of oxidative stress in inhibitory effect of proliferation response by LG2809]
A plate coated with an anti-CD3ε monoclonal antibody (10 μg / ml) in advance, CD4 + T cells (1 × 10 5 cells / well) prepared from BALB / c mice (Wild-type), N-acetylcysteine (NAC, 25 mM), anti-CD28 monoclonal antibody (2 μg / ml), heat-treated LG2809 and its RNA (
〔LG2809のRNAによる遅延型過敏性（DTH）反応の測定方法〕
DO11.10マウスにフロイント完全アジュバント（CFA）に懸濁した100μgのOVAを、尾部から静脈注射して感作させた。14日後に、対照として50μlの生理食塩水を右足せきに注射し、5μgのOVAまたはOVAと被験物の混合物を含む50μlの生理食塩水を左足せきに注射した。試験群（各群ともn = 4）は以下の通りである。（1）OVAを含む生理食塩水を注射する群、(2)OVAおよびLG2809由来RNAを含む生理食塩水を注射する群、(3)OVA、LG2809由来RNAおよびNACを注射する群、(4)OVAおよびNACを注射する群。遅延型過敏反応は注射後24時間後に測定し、「左足せきの腫脹（肥厚）−右足せきの腫脹（肥厚）」から算出した（図6A、B、C）。グラフは平均値+標準偏差を示す。群間の有意差は、Tukey法を用いて多重検定した（a-c：異符号間で有意差あり、P<0.05）。[Measurement method of delayed hypersensitivity (DTH) reaction by LG2809 RNA]
DO11.10 mice were sensitized by intravenous injection of 100 μg OVA suspended in Freund's complete adjuvant (CFA) from the tail. After 14 days, 50 μl of saline was injected into the right foot as a control, and 50 μl of saline containing 5 μg of OVA or a mixture of OVA and test article was injected into the left foot. The test groups (n = 4 for each group) are as follows. (1) a group injecting physiological saline containing OVA, (2) a group injecting physiological saline containing OVA and LG2809-derived RNA, (3) a group injecting OVA, LG2809-derived RNA and NAC, (4) Group injected with OVA and NAC. Delayed type hypersensitivity reaction was measured 24 hours after injection, and was calculated from “swelling of left foot cough (thickening) −swelling of right foot cuff (thickening)” (FIGS. 6A, B, and C). The graph shows the average value + standard deviation. The significant difference between groups was subjected to a multiple test using the Tukey method (ac: significant difference between different signs, P <0.05).
〔統計解析〕
試験データは平均値+標準偏差で示した。統計学的有意差はTukey法による多重検定、またはStudentのt検定で解析した。P<0.05で有意と判断した。〔Statistical analysis〕
The test data is shown as mean value + standard deviation. Statistical significance was analyzed by Tukey's multiple test or Student's t test. P <0.05 was judged as significant.
２．結果
LG2809および2株（MEP225101、MEP225102）のLactobacillus属乳酸菌のCD4+ T細胞の活性化について検討するため、加熱処理をしたこれらの乳酸菌株の存在下で抗原刺激した際のDO11.10マウス由来CD4+ T細胞の増殖応答を測定した（図1A）。試験を行ったLactobacillus属乳酸菌株は全てCD4+ T細胞の増殖を抑制したが、中でもLG2809が最も強い抑制効果を示した（図1B）LG2809についてその抑制効果を検討したところ、用量依存的であることが確認された（図1C）。そこで、以降の試験ではLG2809を使用した。2. result
To examine the activation of CD4 + T cells of Lactobacillus genus lactic acid bacteria of LG2809 and 2 strains (MEP225101, MEP225102), CD4 + derived from DO11.10 mice when antigen-stimulated in the presence of these heat-treated lactic acid strains T cell proliferative response was measured (FIG. 1A). All tested Lactobacillus lactic acid strains suppressed the growth of CD4 + T cells, but LG2809 showed the strongest inhibitory effect (Fig. 1B). (Figure 1C). Therefore, LG2809 was used in subsequent tests.
LG2809のCD4+ T細胞増殖抑制効果の有効成分を同定するため、LG2809破砕物の各種画分の効果を検討した。その抑制効果はLG2809の破砕液上清に認められ、沈殿には認められなかった（図2A）。次に、LG2809破砕液上清画分の酵素処理の影響を検討した。その結果図2Bに示すとおり、対照と比較して未処置の菌体破砕液上清画分(i)では有意な抑制効果が認められた。未処置の菌体破砕液上清画分（i）と比較すると、加熱処理(ii)、DNase I処理(iii)、プロテイナーゼK処理(iv)では有意な影響は認められなかったが、RNase A処理（v）によってその抑制効果は消失した。さらに、LG2809から抽出したRNAは用量依存的にDO11.10マウス由来脾細胞CD4+ T細胞の増殖抑制効果を示した（図2C）。マウス脾臓由来のRNAは増殖抑制効果を示さなかった（図2D）。In order to identify the effective component of LG2809's CD4 + T cell proliferation inhibitory effect, the effect of various fractions of LG2809 crushed material was examined. The inhibitory effect was observed in the supernatant of LG2809 crushed liquid, but not in the precipitate (FIG. 2A). Next, the effect of enzyme treatment on the supernatant fraction of LG2809 crushing solution was examined. As a result, as shown in FIG. 2B, a significant inhibitory effect was observed in the untreated microbial cell supernatant supernatant fraction (i) compared to the control. Compared with the untreated bacterial cell supernatant supernatant fraction (i), heat treatment (ii), DNase I treatment (iii), and proteinase K treatment (iv) showed no significant effect, but RNase A The inhibitory effect disappeared by treatment (v). Furthermore, RNA extracted from LG2809 showed a growth-inhibitory effect on DO11.10 mouse-derived spleen CD4 + T cells in a dose-dependent manner (FIG. 2C). Mouse spleen-derived RNA did not show growth inhibitory effect (FIG. 2D).
LG2809およびそのRNAは、抗原刺激および抗原提示細胞によって活性化されたCD4+ T細胞の増殖抑制効果を示したので、これらがCD4+ T細胞に直接的に作用を及ぼしているのかを検討した。BALB/cマウスの脾細胞からCD4+ T細胞を単離し、抗CD3モノクローナル抗体および抗CD28モノクローナル抗体の刺激下で、LG2809（図3B)およびそのRNA（図3C)を添加して培養した。その結果、LG2809およびそのRNAは用量依存的にCD4+ T細胞の増殖を抑制したが、マウス由来のRNAでは効果は認められなかった（図3D）。これらの結果はLG2809およびそのRNAが、CD4+ T細胞に直接作用して増殖抑制応答を呈することを示すものであった。Since LG2809 and its RNA showed the effect of suppressing the proliferation of CD4 + T cells activated by antigen stimulation and antigen-presenting cells, it was examined whether these had a direct effect on CD4 + T cells. CD4 + T cells were isolated from spleen cells of BALB / c mice, and cultured under the stimulation of anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody by adding LG2809 (FIG. 3B) and its RNA (FIG. 3C). As a result, LG2809 and its RNA suppressed the proliferation of CD4 + T cells in a dose-dependent manner, but no effect was observed with mouse-derived RNA (FIG. 3D). These results indicated that LG2809 and its RNA acted directly on CD4 + T cells and exhibited a growth inhibitory response.
本効果にMyD88依存的なシグナル伝達経路が関与しているかを検討した結果を図4に示した。LG2809およびそのRNAはMyD88欠損マウス由来のCD4+ T細胞では増殖抑制効果を示さなかった。この結果は、LG2809およびそのRNAが、MyD88依存性シグナル伝達経路を介してCD4+ T細胞の増殖を抑制したことを示す。Fig. 4 shows the results of examining whether a MyD88-dependent signal transduction pathway is involved in this effect. LG2809 and its RNA did not show growth inhibitory effects on CD4 + T cells derived from MyD88-deficient mice. This result indicates that LG2809 and its RNA suppressed CD4 + T cell proliferation via MyD88-dependent signaling pathway.
LG2809およびそのRNAの抑制効果における活性酸素の関与を検討するために、酸化剤としてNACの作用を検討した。その結果、NACのみを添加した場合は対照と比較して試験に影響を与えなかったが、NACによりLG2809およびそのRNAの抑制効果は解除された（図5）。以上の結果から、LG2809およびそのRNAによるCD4+ T細胞増殖抑制効果は抗酸化剤により減弱した。つまり、酸素シグナルを必要とする事が明らかになった。In order to investigate the involvement of active oxygen in the inhibitory effect of LG2809 and its RNA, the action of NAC as an oxidizing agent was examined. As a result, the addition of NAC alone did not affect the test compared to the control, but NAC canceled the inhibitory effect of LG2809 and its RNA (FIG. 5). From the above results, the inhibitory effect of LG2809 and its RNA on CD4 + T cell proliferation was attenuated by antioxidants. In other words, it became clear that an oxygen signal was required.
LG2809由来RNAについて、抗原刺激したCD4+ T細胞の増殖抑制効果をin vivoの遅延型過敏反応で評価した（図6）。生理食塩水を注射したマウスの足せきでは腫脹が認められたが、LG2809由来RNAを投与したマウスでは腫脹の程度が低く、遅延型過敏応答が抑制されていることが確認された。さらに、NAC処置によってLG2809由来RNAによる遅延型過敏反応抑制効果が解除された。つまり、LG2809由来RNAは遅延型過敏反応を抑制した。この結果から、in vivoにおいてもLG2909由来RNAは活性酸素シグナルを介して遅延型過敏反応を抑制することが確認された。For LG2809-derived RNA, the effect of suppressing the proliferation of antigen-stimulated CD4 + T cells was evaluated by in vivo delayed hypersensitivity reaction (FIG. 6). Although swelling was observed in the footpads of mice injected with physiological saline, the degree of swelling was low in mice administered with RNA derived from LG2809, and it was confirmed that delayed hypersensitivity response was suppressed. Furthermore, the effect of suppressing delayed hypersensitivity reaction by LG2809-derived RNA was canceled by NAC treatment. That is, LG2809-derived RNA suppressed delayed hypersensitivity reaction. From this result, it was confirmed that RNA derived from LG2909 suppresses the delayed hypersensitivity reaction through an active oxygen signal even in vivo.
３．考察
本発明で、LG2809およびそのRNAがマウスCD4+ T細胞の増殖を直接的に抑制する効果を有することを明らかにした。また、この抑制効果はMyD88依存性シグナル伝達経路を介しており、抗酸化剤処理によって解除された。さらに、in vivoにおいてLG2809由来RNAが遅延型過敏反応を抑制する事も明らかにした。これらの結果は、RNAによるT細胞増殖抑制効果が、プロバイオティクスが炎症性疾患に対して有益な効果をもたらす機序になっていることを示すものである。
本発明で使用した3株のLactobacillus属乳酸菌の中で、LG2809が最も強い抑制効果を示した。本発明では、LG2809は、APC共培養実験モデルによりAPCと抗原の共刺激を誘導した場合だけでなく、抗CD3ε/CD28刺激で誘導した場合にもCD4+ T細胞の増殖を抑制した。また、LG2809およびそのRNAはアポトーシスを誘導しないことを確認している。従って、LG2809は樹状細胞に関与しないメカニズムで、またアポトーシスを誘導せずにCD4+ T細胞の増殖を抑制している。
本発明でLG2809のRNAがその関与成分であることを明らかにした。細菌のRNAがT細胞増殖抑制効果の関与成分であることを明らかにしたのは本発明が初めてである。RNAの増殖阻害活性の特異性を検討するため、ヒトおよびEscherichia coliから調製したRNAについてもその効果を検討した。興味深いことに、E. coli由来RNAはCD4+ T細胞増殖抑制効果を示したが、ヒト由来RNAは影響しなかった。RNAの調製方法による抑制効果の違いを検討するために、細菌からのRNA調製法によって抽出したBALB/cマウスの脾細胞RNAも評価したが、効果は認められなかった。これらの結果は細菌のRNAがCD4+ T細胞増殖抑制効果を有していることを示すものである。
ヒトの細胞には細菌のRNAに対して、TLR3とTLR7の膜結合型受容体および細胞質受容体であるNALP3（NACHT-LRR protein 3）の2種類の受容体がある。TLR3が二本鎖RNAを認識するのに対して、TLR7とNALP3は一本鎖RNAを認識する。これらの受容体の中で、TLR7のみがそのシグナル伝達経路にMyD88を要することが知られている。本発明では、LG2809およびそのRNAは、MyD88欠損マウス由来CD4+ T細胞には抑制効果を示さなかった。TLR7の発現はCD4+ T細胞でも認められ、TLR7のリガンドであるImiquimodもCD4+ T細胞増殖を強く抑制することも確認している。これらの知見はLG2809由来RNAはTLR7を介してその増殖抑制効果を示すものである。
遅延型過敏性反応は、細胞性免疫応答の典型的なin vivoの症状であり、その反応はT細胞の活性化と優先的な集積により特徴づけられる。遅延型過敏性反応はI型ヘルパーT細胞（Th1）細胞を介して起こることは一般的に知られている。LG2809由来RNAは、in vitroで脾細胞CD4+ T細胞の増殖を抑制したのと同様に、Th1細胞の増殖を阻害し、遅延型過敏性反応を抑制したといえる。抑制効果はin vivoでも認められているので、LG2809由来RNAは免疫抑制剤として炎症疾患への治療に有用と考えられる。さらに、LG2809由来RNAの抑制効果はNAC処理により消失したことから、LG2809由来RNAによるT細胞を介したin vivoでの免疫応答において、活性酸素種が影響を及ぼしたことが示される。
結論として、LG2809およびそのRNAはCD4+ T細胞の増殖を、MyD88依存性シグナル伝達経路を介して抑制し、その抑制効果は抗酸化剤により減弱した。これらの結果は、RNAによるT細胞増殖抑制効果は、プロバイオティクスによる炎症性疾患改善効果の機序であることを示すものである。LG2809およびそのRNAはCD4+ T細胞の過敏反応に起因する疾患をもつ患者の治療に有用であると考えられる。3. Discussion In the present invention, it has been clarified that LG2809 and its RNA have an effect of directly suppressing the proliferation of mouse CD4 + T cells. In addition, this inhibitory effect is mediated by MyD88-dependent signal transduction pathway and was canceled by antioxidant treatment. Furthermore, it was clarified that LG2809-derived RNA suppresses delayed hypersensitivity reaction in vivo. These results indicate that the T cell proliferation inhibitory effect of RNA is a mechanism by which probiotics have a beneficial effect on inflammatory diseases.
Among the three strains of Lactobacillus genus lactic acid bacteria used in the present invention, LG2809 showed the strongest inhibitory effect. In the present invention, LG2809 suppressed the proliferation of CD4 + T cells not only when APC and antigen costimulation was induced by the APC co-culture experimental model but also when induced by anti-CD3ε / CD28 stimulation. It has also been confirmed that LG2809 and its RNA do not induce apoptosis. Therefore, LG2809 suppresses the proliferation of CD4 + T cells by a mechanism not involved in dendritic cells and without inducing apoptosis.
In the present invention, it was clarified that the RNA of LG2809 is a component involved. This is the first time that the present invention has clarified that bacterial RNA is a component involved in the T cell growth inhibitory effect. In order to examine the specificity of RNA growth inhibitory activity, the effect of RNA prepared from human and Escherichia coli was also examined. Interestingly, E. coli-derived RNA showed a CD4 + T cell proliferation inhibitory effect, but human-derived RNA did not. In order to examine the difference in the inhibitory effect depending on the RNA preparation method, splenocyte RNA of BALB / c mice extracted by the RNA preparation method from bacteria was also evaluated, but no effect was observed. These results indicate that bacterial RNA has a CD4 + T cell proliferation inhibitory effect.
Human cells have two types of receptors for bacterial RNA: TLR3 and TLR7 membrane-bound receptors and the cytoplasmic receptor NALP3 (NACHT-LRR protein 3). TLR3 recognizes double-stranded RNA, whereas TLR7 and NALP3 recognize single-stranded RNA. Of these receptors, only TLR7 is known to require MyD88 in its signaling pathway. In the present invention, LG2809 and its RNA did not show a suppressive effect on CD4 + T cells derived from MyD88-deficient mice. TLR7 expression was also observed in CD4 + T cells, and Imiquimod, a ligand for TLR7, was also confirmed to strongly suppress CD4 + T cell proliferation. These findings indicate that LG2809-derived RNA exhibits its growth inhibitory effect via TLR7.
Delayed type hypersensitivity reactions are typical in vivo symptoms of cellular immune responses, which are characterized by T cell activation and preferential accumulation. It is generally known that delayed type hypersensitivity reactions occur via type I helper T cells (Th1) cells. LG2809-derived RNA inhibits the proliferation of spleen CD4 + T cells in vitro and inhibits the proliferation of Th1 cells and suppresses the delayed hypersensitivity reaction. Since the suppressive effect has been recognized in vivo, it is considered that LG2809-derived RNA is useful as an immunosuppressant for the treatment of inflammatory diseases. Furthermore, the inhibitory effect of LG2809-derived RNA disappeared by NAC treatment, indicating that reactive oxygen species had an effect on the immune response in vivo via T cells caused by LG2809-derived RNA.
In conclusion, LG2809 and its RNA inhibited CD4 + T cell proliferation via MyD88-dependent signaling pathway, and its inhibitory effect was attenuated by antioxidants. These results indicate that the T cell proliferation inhibitory effect by RNA is a mechanism of the inflammatory disease improving effect by probiotics. LG2809 and its RNA may be useful for the treatment of patients with diseases caused by CD4 + T cell hypersensitivity reactions.
本発明は食品産業や医薬産業において好ましく利用され得る。本発明の遅延型過敏症軽減剤によれば、食経験があり長期間摂取における安全性が明確なヒト腸内由来乳酸菌であるLactobacillus gasseriの菌体画分を用いて、遅延型過敏症の軽減効果を得ることができる。 The present invention can be preferably used in the food industry and the pharmaceutical industry. According to the delayed-type hypersensitivity reducing agent of the present invention, using a cell fraction of Lactobacillus gasseri, a human intestinal lactic acid bacterium that has a dietary experience and is safe for long-term ingestion, alleviates delayed-type hypersensitivity An effect can be obtained.
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