JP2617758B2 - Antibody production enhancer - Google Patents
Antibody production enhancerInfo
- Publication number
- JP2617758B2 JP2617758B2 JP63069267A JP6926788A JP2617758B2 JP 2617758 B2 JP2617758 B2 JP 2617758B2 JP 63069267 A JP63069267 A JP 63069267A JP 6926788 A JP6926788 A JP 6926788A JP 2617758 B2 JP2617758 B2 JP 2617758B2
- Authority
- JP
- Japan
- Prior art keywords
- cells
- breve
- cell
- antibody production
- peyer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、抗体産生増強型免疫賦活剤に関するもので
ある。TECHNICAL FIELD The present invention relates to an antibody production-enhancing immunostimulant.
腸管は、消化・吸収器官としての機能のみならず、多
数のリンパ組織を持ち、生体の局所防御的役割をも担っ
ている。特に、そのリンパ球から腸管内に分泌される抗
体(特にIgA抗体)は、有害菌の定着、病原菌の体内へ
の侵入およびアレルゲンの体内吸収を阻止するので、そ
の重要性が注目されている。The intestinal tract not only functions as a digestive / absorbing organ, but also has a large number of lymphoid tissues and plays a local protective role in the living body. In particular, antibodies secreted into the intestinal tract from the lymphocytes (especially IgA antibodies) are attracting attention because they prevent the colonization of harmful bacteria, the invasion of pathogenic bacteria into the body, and the absorption of allergens into the body.
ところで、人間の腸管に常在する嫌気性グラム陽性桿
菌・ビフィドバクテリウム菌は、E.coli感染を阻止する
作用や抗生剤誘発下痢症の改善作用を示し、宿主にとっ
て有用な菌とされている。そして、その免疫賦活作用に
ついては、たとえば静脈投与されたビフィドバクテリウ
ム・インファンティス生菌が脾臓による抗体産生を増強
することが報告されている(厚井芳則他:日本細菌学雑
誌,32巻,220頁)。しかしながら、脾臓は全身性免疫組
織であるから、ビフィドバクテリウム・インファンティ
スその他のビフィドバクテリウム菌が経口摂取されたと
き上述のような腸管内分泌抗体の産生にどのような影響
を及ぼすかは、上記事実からは予想できない。By the way, anaerobic Gram-positive bacilli and Bifidobacterium resident in the human intestinal tract show an action of inhibiting E. coli infection and an action of ameliorating antibiotic-induced diarrhea, and are regarded as useful bacteria for the host. I have. Regarding its immunostimulatory effect, it has been reported that, for example, live Bifidobacterium infantis bacteria administered intravenously enhance antibody production by the spleen (Atsui Yoshinori et al .: Japanese Bacteriological Magazine, 32). Vol., P. 220). However, since the spleen is a systemic immune system, how does Bifidobacterium infantis and other Bifidobacterium bacteria orally affect the production of intestinal endocrine antibodies as described above? Cannot be predicted from the above facts.
本発明者らは、消化管粘膜免疫系の主要な組織であり
分泌抗体産生細胞の前駆細胞を有するパイエル板の細胞
を賦活し、腸管内分泌抗体産生を増加させることのでき
る免疫賦活剤の開発を目的として鋭意研究を行なったと
ころ、ビフィドバクテリウム・ブレーベに、パイエル板
細胞の抗体産生を増強させ、骨髄由来細胞(以下、B細
胞という)の増殖を促進する作用があり、腸管感染やア
レルギーの防御に先立つ可能性を見いだし、本発明を完
成するに至った。The present inventors have developed an immunostimulant capable of activating cells of the Peyer's patch, which is a major tissue of the gastrointestinal mucosal immune system and having precursor cells for secretory antibody-producing cells, and increasing intestinal endocrine antibody production. As a result of extensive research, Bifidobacterium breve has the effect of enhancing antibody production of Peyer's patch cells and promoting the proliferation of bone marrow-derived cells (hereinafter referred to as B cells). Have found a possibility prior to the defense of the present invention, and have completed the present invention.
すなわち本発明は、ビフィドバクテリウム・ブレーベ
の菌体がパイエル板細胞の増殖を促進するとともにパイ
エル板細胞による抗体産生を増強し、ひいては免疫力を
強化する作用を利用する免疫賦活剤を提供するものであ
る。That is, the present invention provides an immunostimulant that utilizes the action of the cells of Bifidobacterium breve to promote the growth of Peyer's patch cells, enhance antibody production by Peyer's patch cells, and thus enhance immunity. It is a thing.
本発明で用いるビフィドバクテリウム・ブレーベは、
特定の菌株である必要はなく、ザ・アメリカン・タイプ
カルチャー・コレクションのカタログに記載されている
ような公知菌株その他任意のものを用いることができ
る。Bifidobacterium breve used in the present invention,
The strain does not need to be a specific strain, and a known strain such as described in a catalog of the American Type Culture Collection or any other strain can be used.
本発明の免疫賦活剤に用いるビフィドバクテリウム・
ブレーベの菌体は、凍結乾燥菌体および死菌体(たとえ
ば熱処理やホルマリン処理によって得られた死菌体)の
いずれであってもよく、原料菌体取得のための培養法お
よび菌体分離法にも、制限はない。Bifidobacterium used in the immunostimulant of the present invention
The breve cells may be either freeze-dried cells or dead cells (for example, dead cells obtained by heat treatment or formalin treatment). But there is no limit.
菌体は、そのまま本発明の免疫賦活剤として提供して
もよいが、常法により、粉末剤、ドリンク剤、錠剤等に
製剤化することもできる。The microbial cells may be provided as they are as the immunostimulant of the present invention, but they can also be formulated into powders, drinks, tablets and the like by a conventional method.
次に、本発明の免疫賦活剤の上記作用について、実験
例を示して説明する。なお、実験に用いたビフィドバク
テリウム・ブレーベ(以下、B・ブレーベと略記)の菌
体の調製法および実験法は、次のとおりである。Next, the above effects of the immunopotentiator of the present invention will be described with reference to experimental examples. The method for preparing the cells of Bifidobacterium breve (hereinafter, abbreviated as B. breve) used in the experiment and the experimental method are as follows.
(1)菌体調製法 B・ブレーベのスターターをVLL培地(組成後記)に
接種し、嫌気条件下、37℃で18時間培養した。遠心分離
して得られた菌体は、生理食塩水で洗浄し、再度遠心分
離して集菌した。得られた湿菌体を凍結乾燥して、乾燥
菌体(菌数1×1011/g)を得た。(1) Bacterial cell preparation method A B. breve starter was inoculated into a VLL medium (described later) and cultured at 37 ° C. for 18 hours under anaerobic conditions. The cells obtained by centrifugation were washed with physiological saline, centrifuged again and collected. The obtained wet cells were freeze-dried to obtain dried cells (cell count 1 × 10 11 / g).
(2)パイエル板細胞の分離: パイエル板をマウスの小腸から無菌的に取り出し、Di
spase溶液(1.5mg Dispase/ml Joklik−modified MEM)
に入れ、30〜40分間、37℃で攪拌し、溶液中に出てきた
single cellを回収した。この操作を4〜5回繰り返
し、遠心洗浄することにより、パイエル板細胞を得た。(2) Separation of Peyer's patch cells: Aspirate the Peyer's patches from the small intestine of the mouse and
spase solution (1.5mg Dispase / ml Joklik-modified MEM)
And stir at 37 ° C for 30-40 minutes, then come out into solution
I collected a single cell. This operation was repeated 4 to 5 times and centrifugally washed to obtain Peyer's patch cells.
(3)抗LPS(リポポリサッカライド)抗体産生量の測
定: 96穴平底プレートを用い、1穴あたり5×105個のパ
イエル板細胞と1×107個のB・ブレーベ菌体を入れ、
5%牛胎児血清を含むRPMI1640培地(組成後記)で、5
%CO2/Airの雰囲気下、37℃で7日間培養し、その上清
の抗LPS抗体量をELISA法で測定した。(3) Measurement of anti-LPS (lipopolysaccharide) antibody production amount: Using a 96-well flat bottom plate, 5 × 10 5 Peyer's patch cells and 1 × 10 7 B. breve cells were placed per well.
In RPMI1640 medium containing 5% fetal calf serum (composition described later),
The cells were cultured at 37 ° C. for 7 days in an atmosphere of% CO 2 / Air, and the amount of the anti-LPS antibody in the supernatant was measured by ELISA.
(4)抗羊赤血球プラーク形成細胞の測定: 上記(3)の場合と同様にパイエル板細胞とB・プレ
ーベ菌体とを平底プレートに入れ、更に1×105個の羊
赤血球を添加し、5%CO2/Airの雰囲気下、37℃で4日
間培養後、Cuningham−Szenberg法を用いて測定した。(4) Measurement of anti-sheep erythrocyte plaque-forming cells: As in the case of (3) above, the Peyer's patch cells and B. prebe cells were placed in a flat bottom plate, and 1 × 10 5 sheep red blood cells were added, After culturing at 37 ° C. for 4 days in an atmosphere of 5% CO 2 / Air, measurement was performed using the Cuningham-Szenberg method.
(5)B細胞増殖促進作用: 上記(3)と同様、パイエル板細胞あるいは各分画細
胞にB・ブレーベ菌体を加え、72時間培養した後、0.5
μci3H−TdRを添加し、18時間後、3H−TdRの細胞への取
り込みをシンチレーションカウンターで測定した。(5) B cell proliferation promoting action: Similar to the above (3), B. breve cells were added to Peyer's patch cells or each fraction cell, and cultured for 72 hours.
μci 3 H-TdR was added, and 18 hours later, uptake of 3 H-TdR into cells was measured with a scintillation counter.
(6)B細胞富画分の分画(Panning法): 直径10cmのシャーレに50μg抗マウスIgs/ml RPMI164
0を10ml注ぎ、室温に2.5時間静置し、コートする。RPMI
1640培地で3回洗浄後、非特異的吸着を防ぐため、5ml
の5%牛胎児血清を含むRPMI1640培地を注ぎ、1時間室
温に放置し、抗体コートシャーレを作成する。これに5
×107パイエル板細胞/10ml 5%FBSを含むPRMI1640培地
を入れ、室温で1時間付着させ、その後、2回静かに洗
浄して、非付着細胞を除く。次に5%牛胎児血清を含む
PRMI1640培地10mlを注ぎ、氷上に15分間静置して付着細
胞を浮遊させ、遠心分離してこれを回収する。(6) Fractionation of B cell-rich fraction (Panning method): 50 μg anti-mouse Igs / ml RPMI164 was added to a dish having a diameter of 10 cm.
Pour 10 ml of 0, leave at room temperature for 2.5 hours and coat. RPMI
After washing 3 times with 1640 medium, 5 ml to prevent non-specific adsorption
RPMI1640 medium containing 5% fetal bovine serum is poured and left at room temperature for 1 hour to prepare an antibody-coated dish. 5 to this
× 10 7 Peyer's patch cells / 10 ml Add PRMI1640 medium containing 5% FBS and allow to adhere for 1 hour at room temperature, then gently wash twice to remove non-adherent cells. Next contains 5% fetal calf serum
Pour 10 ml of PRMI1640 medium, leave on ice for 15 minutes to suspend adherent cells, and collect by centrifugation.
(7)B細胞画分の分画: 全細胞数5×107のB細胞富画分を直径10cmのプラス
チックシャーレに注ぎ、5%CO2/Airの雰囲気下、37℃
で1時間培養し、付着マクロファージ系細胞を除去し、
非付着細胞を回収する。(7) Fractionation of B cell fraction: B cell rich fraction of total cell number 5 × 10 7 was poured into a plastic petri dish having a diameter of 10 cm, and 37 ° C. in an atmosphere of 5% CO 2 / Air.
For 1 hour to remove adherent macrophage cells,
Collect non-adherent cells.
(8)VLL培地: 塩溶液(I) 75 ml/ 0.3%K2HPO4 75 ml/ トリプチカーゼ 10 g/ 酵母エキス 5 g/ 牛肉エキス 2 g/ 0.1%レサズリン 1 ml/ 8%Na2CO3 50 ml/ 3%L−システイン塩酸塩 10 ml/ 0.1%ヘミン 7 ml/ ラクターゼ 5 g/ pH 6.8 塩溶液(I):KH2PO40.3%,NaCl0.6% (NH4)2SO40.3
%,CaCl20.03% MgSO40.03% (9)RPMI1640培地: PRMI1640(Boehringer Mannhaim) 10.85g/ ペニシリン 10 万単位/ ストレプトマイシン 100 mg/ HEPES(pH7.2) 15 mg/ pH6.8 実験例1 B・ブレーベの抗体産生増強作用を調べた。(8) VLL medium: Salt solution (I) 75 ml / 0.3% K 2 HPO 4 75 ml / Trypticase 10 g / Yeast extract 5 g / Beef extract 2 g / 0.1% Resazurin 1 ml / 8% Na 2 CO 3 50 ml / 3% L-cysteine hydrochloride 10 ml / 0.1% hemin 7 ml / lactase 5 g / pH 6.8 Salt solution (I): KH 2 PO 4 0.3%, NaCl 0.6% (NH 4 ) 2 SO 4 0.3
%, CaCl 2 0.03% MgSO 4 0.03% (9) RPMI1640 medium: PRMI1640 (Boehringer Mannhaim) 10.85 g / penicillin 100,000 units / streptomycin 100 mg / HEPES (pH 7.2) 15 mg / pH 6.8 Experimental Example 1 The breve was investigated for its antibody production enhancing action.
抗LPS抗体産生増強作用:図1に示したように、抗LPS
抗体はB・ブレーベを添加することにより増加した。な
お、パイエル板細胞は生体内で種々のLPSで感作されて
いるので、菌体添加のみ(LPS無添加)でパイエル板細
胞による抗LPS抗体産生が増強されたと結論することが
できる。Anti-LPS antibody production enhancing effect: As shown in FIG.
Antibodies were increased by adding B. breve. In addition, since Peyer's patch cells are sensitized in vivo with various LPS, it can be concluded that the anti-LPS antibody production by Peyer's patch cells was enhanced only by adding bacterial cells (without adding LPS).
抗羊赤血球プラーク形成細胞増強作用:図2に示した
ように、生体にとって未感作の抗原である羊赤血球の場
合であっても、B・ブレーベは当該抗原とともに加える
と抗体産生量を増加させた。Anti-sheep erythrocyte plaque-forming cell enhancing effect: As shown in Fig. 2, even in the case of sheep erythrocytes, which are unsensitized antigens to the living body, B. breve increases the amount of antibody production when added together with the antigen. Was.
実験例2 B・ブレーベのB細胞増殖促進作用について調べた。Experimental Example 2 B. breve was examined for its B cell proliferation promoting action.
図3に示したように、B・ブレーベ菌体を添加すると
細胞増殖は亢進した。そして、増殖細胞は、表1に示し
たように、B細胞を豊富に含む画分の細胞であった。ま
た、表2に示したように、B・ブレーベは胸腺由来細胞
(T細胞)欠損マウスであるヌードマウスのパイエル板
細胞の増殖も促進した。また、表3に示したように、B
・ブレーベは、B細胞を豊富に含む画分からマクロファ
ージ系細胞を除去したB細胞画分の増殖は促進しなかっ
たが、図4に示したように、このB細胞画分にB・ブレ
ーベ添加マクロファージ系細胞の培養上清を加えること
により、再び細胞の増殖をひき起こすことができた。As shown in FIG. 3, addition of B. breve cells accelerated cell growth. Then, as shown in Table 1, the proliferating cells were cells in a fraction rich in B cells. In addition, as shown in Table 2, B. breve also promoted the proliferation of Peyer's patch cells in nude mice, which are thymus-derived cell (T cell) -deficient mice. In addition, as shown in Table 3, B
-Breve did not promote the proliferation of the B cell fraction obtained by removing macrophage cells from the B cell-rich fraction, but as shown in FIG. By adding the culture supernatant of the lineage cells, it was possible to cause cell proliferation again.
以上のことから、B・ブレーベによるB細胞の増殖促
進は、B・ブレーベ刺激マクロファージ系細胞から放出
される因子を介していることが明らかになった。From the above, it was revealed that the promotion of B cell proliferation by B. breve was mediated by factors released from B. breve-stimulated macrophage cells.
表2 パイエル板細胞の増殖に及ぼすB・ブレーベの影響分裂誘導物質 活性化指数 B・ブレーベ 2.0 LPS 4.3 ConA 1.0 表3 B・ブレーベの細胞増殖促進作用に及ぼすプラスチック
付着細胞の影響 分裂誘導物質 活性化指数 無処理 6.8 B細胞富画分 6.9 B細胞画分 1.3 B細胞不含画分 1.5 〔発明の効果〕 本発明の免疫賦活剤は、ビフィドバクテリウム・ブレ
ーベが消化管粘膜免疫系の主要組織であるパイエル板細
胞の増殖を促進するとともに該細胞による抗体産生を増
強する作用を利用するものであり、経口投与によってパ
イエル板細胞による抗体産生を増強し、ひいては感染症
を防ぎ腸管からのアレルギー源吸収を防ぐのに有効であ
る。 Table 2 Peyer's patches affect mitotic inducer activation of plastic adherent cells on affected mitotic inducer activation index B · breve 2.0 LPS 4.3 ConA 1.0 Table 3 cell growth promoting action of B · breve of B · breve on proliferation of cells Untreated index 6.8 B cell rich fraction 6.9 B cell fraction 1.3 B cell free fraction 1.5 [Effect of the Invention] The immunostimulant of the present invention is obtained by using Bifidobacterium breve as the main tissue of the gastrointestinal mucosal immune system. Which utilizes the action of promoting the growth of Peyer's patch cells and enhancing the production of antibodies by the cells, and enhancing the antibody production by Peyer's patch cells by oral administration, thereby preventing infectious diseases and thereby allergic sources from the intestinal tract. It is effective to prevent absorption.
また、上述のように本発明による免疫賦活剤は経口投
与が可能であり、また乳製品の製造に広く使用されてい
るビフィドバクテリウム・ブレーベを有効成分とするも
のであるから、安全性の点でも心配がなく、きわめて使
用し易いものである。さらに、微生物菌体を有効成分と
するものではあるが、それは死菌体で差し支えないか
ら、製剤化や保存、使用も容易である。In addition, as described above, the immunostimulant of the present invention can be administered orally, and contains Bifidobacterium breve, which is widely used in the production of dairy products, as an active ingredient. It is very easy to use without any concerns. Furthermore, although microbial cells are used as an active ingredient, they can be dead cells, so that preparation, storage and use are easy.
したがって、本発明の免疫賦活剤は、腸管内感染やア
レルギーの予防薬として使用するほか、栄養剤や、いわ
ゆる健康食品等の配合成分として広く利用することも可
能である。Therefore, the immunostimulant of the present invention can be used as a preventive agent for intestinal infection and allergy, and can also be widely used as a nutritional component and a compounding component for so-called health foods.
図1〜図4は実験例の結果を示すグラフである。 図中、+は添加、−は非添加を意味する。 1 to 4 are graphs showing the results of the experimental example. In the figure, + means addition, and-means no addition.
Claims (1)
有効成分として含有することを特徴とする抗体産生増強
型免疫賦活剤。1. An immunostimulating agent for enhancing antibody production, comprising a cell of Bifidobacterium breve as an active ingredient.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63069267A JP2617758B2 (en) | 1988-03-25 | 1988-03-25 | Antibody production enhancer |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP63069267A JP2617758B2 (en) | 1988-03-25 | 1988-03-25 | Antibody production enhancer |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH01242532A JPH01242532A (en) | 1989-09-27 |
JP2617758B2 true JP2617758B2 (en) | 1997-06-04 |
Family
ID=13397742
Family Applications (1)
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JP63069267A Expired - Fee Related JP2617758B2 (en) | 1988-03-25 | 1988-03-25 | Antibody production enhancer |
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JP (1) | JP2617758B2 (en) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH07106142B2 (en) * | 1989-04-21 | 1995-11-15 | 株式会社ヤクルト本社 | Bifidobacterium longum or Bifidobacterium breve capable of inducing IgA |
DE69219768T2 (en) * | 1992-07-06 | 1997-08-28 | Societe Des Produits Nestle S.A., Vevey | Milk bacteria |
JP2506314B2 (en) * | 1993-10-27 | 1996-06-12 | 全国農業協同組合連合会 | Prophylactic / therapeutic agent and feed for sepsis in poultry and pigs |
AU727940B2 (en) * | 1997-08-21 | 2001-01-04 | New Zealand Dairy Board | Immunity enhancing lactic acid bacteria |
ATE350010T1 (en) | 2001-02-23 | 2007-01-15 | Richter Chem Lab | COMPOSITION FOR TOPICAL USE |
JP2006298783A (en) * | 2005-04-18 | 2006-11-02 | Nisshin Sugar Mfg Co Ltd | Immunostimulating composition |
WO2008153377A1 (en) * | 2007-06-15 | 2008-12-18 | N.V. Nutricia | Nutrition with non-viable bifidobacterium and non-digestible oligosaccharide |
EP2065048A1 (en) * | 2007-11-30 | 2009-06-03 | Institut Pasteur | Use of a L. casei strain, for the preparation of a composition for inhibiting mast cell activation |
ATE546149T1 (en) * | 2009-05-11 | 2012-03-15 | Nestec Sa | PREVENTION AND TREATMENT OF ALLERGIC DIARRHEA |
ITMI20091034A1 (en) * | 2009-06-11 | 2010-12-12 | Parmalat Spa | PROBIOTIC SPECIES OF BIFIDOBACTERIUM BREVE |
JP5409439B2 (en) * | 2010-02-26 | 2014-02-05 | チュンヤン ペーパー カンパニー リミテッド | Composition for enhancing immune function, comprising Kouzo extract |
JP5936392B2 (en) | 2012-03-02 | 2016-06-22 | 松谷化学工業株式会社 | IgA production promoter |
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JPS5592322A (en) * | 1978-12-07 | 1980-07-12 | Meiji Seika Kaisha Ltd | Antitumor immunity activator and its preparation |
JPS5658491A (en) * | 1979-06-18 | 1981-05-21 | Meiji Seika Kaisha Ltd | Immunoactivating substance ies-1638 having antitumor activity, and its preparation |
JPS58203913A (en) * | 1982-05-25 | 1983-11-28 | Meiji Milk Prod Co Ltd | High polymer polysaccharide substance mps-82 and its use |
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1988
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