JP6178088B2 - M2 macrophage differentiation inducer - Google Patents
M2 macrophage differentiation inducer Download PDFInfo
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- JP6178088B2 JP6178088B2 JP2013055275A JP2013055275A JP6178088B2 JP 6178088 B2 JP6178088 B2 JP 6178088B2 JP 2013055275 A JP2013055275 A JP 2013055275A JP 2013055275 A JP2013055275 A JP 2013055275A JP 6178088 B2 JP6178088 B2 JP 6178088B2
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- macrophages
- lactic acid
- differentiation
- rhamnosus
- balance
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Description
本発明は、乳酸菌を含むマクロファージの分化誘導剤、該乳酸菌又は該誘導剤を含む医薬組成物、栄養学的若しくは薬学的調製物、乳酸菌を含む食品、及び乳酸菌を用いたマクロファージの分化誘導方法、並びに該栄養学的若しくは薬学的調製物の製造方法に関する。 The present invention relates to a differentiation inducer for macrophages containing lactic acid bacteria, a pharmaceutical composition containing the lactic acid bacteria or the inducer, a nutritional or pharmaceutical preparation, a food containing lactic acid bacteria, and a method for inducing differentiation of macrophages using lactic acid bacteria, And a method for producing the nutritional or pharmaceutical preparation.
マクロファージは免疫系や炎症において重要な役割を果たす貪食細胞である。記号ではMφと表されうる。まず骨髄細胞から単球が分化し、これが血液中を循環して全身の組織に動員され、最終的にマクロファージに分化する。マクロファージにはM1とM2というサブセットがある。 Macrophages are phagocytic cells that play an important role in the immune system and inflammation. The symbol can be expressed as Mφ. First, monocytes differentiate from bone marrow cells, which circulate in the blood and are recruited to tissues throughout the body, and finally differentiate into macrophages. Macrophages have a subset of M1 and M2.
M1マクロファージは、LPSやIFN-γなどにより活性化され、高レベルのIL-12と低レベルのIL-10を分泌する。M1マクロファージは主にTh1依存性免疫反応(細胞性免疫)や炎症反応を担う。M2マクロファージはIL-4やIL-13により活性化され、高レベルのIL-10と低レベルのIL-12を産生する。またM2マクロファージはアルギナーゼ-1、キチナーゼ様タンパク質Ym1等の遺伝子を発現し、主として損傷の修復や炎症の抑制に関わる。このようにM1マクロファージとM2マクロファージは生体内で異なる役割を果たし、外的刺激による炎症の誘発そして損傷修復までの機構が正常に機能するにはM1マクロファージとM2マクロファージのバランスが重要となる。 M1 macrophages are activated by LPS and IFN-γ and secrete high levels of IL-12 and low levels of IL-10. M1 macrophages are mainly responsible for Th1-dependent immune responses (cellular immunity) and inflammatory responses. M2 macrophages are activated by IL-4 and IL-13 and produce high levels of IL-10 and low levels of IL-12. M2 macrophages express genes such as arginase-1 and chitinase-like protein Ym1, and are mainly involved in repairing damage and suppressing inflammation. Thus, M1 macrophages and M2 macrophages play different roles in vivo, and the balance between M1 macrophages and M2 macrophages is important for the normal functioning of inflammation-induced inflammation and damage repair by external stimuli.
したがってM1マクロファージとM2マクロファージのバランスの不均衡は種々の疾患又は障害の原因となりうる。脂肪組織内には、好酸球の産生するIL-4依存性のM2マクロファージが存在している。しかし食事により誘発された肥満モデルにおいて、脂肪組織マクロファージがM2極性からM1型の炎症促進性状態となり、インスリン抵抗性に寄与するとの報告がある(非特許文献1)。また、肥満の進行につれて、M2マクロファージがM1型にシフトし、肥満に伴う2型糖尿病の発症に関わりうるとの報告もある(非特許文献2)。
Thus, an imbalance in the balance between M1 macrophages and M2 macrophages can cause various diseases or disorders. IL-4 dependent M2 macrophages produced by eosinophils are present in the adipose tissue. However, in a diet-induced obesity model, there is a report that adipose tissue macrophages change from M2 polarity to M1 type pro-inflammatory state and contribute to insulin resistance (Non-patent Document 1). In addition, there is a report that M2 macrophages shift to M1 type as obesity progresses and can be involved in the development of
一方で特許文献1には、腸管バリア破綻を抑制する乳酸菌が記載されている。しかしながら、腸管バリア破綻の要因としては種々の要因があり、腸管バリア機能が破綻することとM1、M2マクロファージとの関連性は必ずしも明らかではない。特許文献1には、開示されている乳酸菌の脂質成分であるリポテイコ酸が腸管バリア機能破綻を予防する、との記載がある(段落[0008])。
On the other hand,
Habilらはプロバイオティクス細菌株がマクロファージのサイトカイン産生をモジュレートすることを報告した(非特許文献3)。非特許文献3の第285-286頁によると、加熱殺菌されたLactobacillus rhamnosus GG株(NCIMB 8824)による、既に分化したM1又はM2マクロファージのTNFα産生能、IL-6産生能、NFκB産生能に対する影響が検討された(第287-289頁Figures 1-3)。要約及び図の脚注にあるように、用いられているM1及びM2マクロファージは、CD14高発現及びCD14低発現THP-1-NFκBレポーター単球を、ホルボール12-ミリステート13-アセテートで、又は1,25-(OH)2ビタミンD3で分化させることにより作製されたものである。この文献で報告されているのは、あくまで分化したM1及びM2マクロファージのサイトカインプロファイルに対するプロバイオティクス調製物の影響である。
Habil et al. Reported that probiotic bacterial strains modulate macrophage cytokine production (Non-patent Document 3). According to pages 285-286 of Non-Patent
上記のようにマクロファージが分化する際、M1/M2バランスの不均衡により、種々の疾患が生じうる。M2マクロファージへの分化はIL-4やIL-13、ビタミンD3のフッ素化合物アナログなどにより誘導可能な場合があるものの、こうした物質は高価であったり、他の生理作用を有していたり、また食品への添加に適していないなどの問題がある。そこで種々の疾患の予防、治療のためM1/M2バランスの不均衡を是正すべく、M2マクロファージへの分化を誘導する、安全で簡便な方法が必要とされている。 When macrophages differentiate as described above, various diseases can occur due to imbalance in the M1 / M2 balance. Differentiation into M2 macrophages may be induced by IL-4, IL-13, or a fluorine compound analog of vitamin D3, but these substances are expensive, have other physiological effects, or are foods There is a problem such as being unsuitable for addition to the skin. Therefore, there is a need for a safe and simple method for inducing differentiation into M2 macrophages to correct the M1 / M2 balance imbalance for the prevention and treatment of various diseases.
本発明者らは、上記課題を解決するために鋭意検討した結果、驚くべきことにL. rhamnosus OLL2838(受託番号NITE P-313)がM2マクロファージ分化誘導作用を有することを見出し、本発明を完成させた。すなわち、本発明は次のとおりである。
[1] ラクトバチルス・ラムノーサス(Lactobacillus rhamnosus)を有効成分として含む、M2マクロファージ分化誘導剤。
[2] ラクトバチルス・ラムノーサス(L. rhamnosus)が、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)である、[1]に記載の分化誘導剤。
[3] [1]又は[2]に記載のM2マクロファージ分化誘導剤を含む、マクロファージのM1/M2バランスの不均衡に関連する疾患を治療又は予防するための医薬組成物。
[4] [1]又は[2]に記載のM2マクロファージ分化誘導剤を含む、マクロファージのM1/M2バランスの不均衡に関連する疾患を治療又は予防するための栄養学的若しくは薬学的調製物。
[5] ラクトバチルス・ラムノーサス(L. rhamnosus)を有効成分として含む、マクロファージのM1/M2バランスの不均衡に関連する疾患を治療又は予防するための医薬組成物。
[6] ラクトバチルス・ラムノーサス(L. rhamnosus)が、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)である、[5]に記載の医薬組成物。
[7] マクロファージのM1/M2バランスの不均衡に関連する疾患が、糖尿病及び動脈硬化からなる群より選択される疾患である、[3]、[5]又は[6]に記載の医薬組成物。
[8] マクロファージのM1/M2バランスの不均衡に関連する疾患が、糖尿病及び動脈硬化からなる群より選択される疾患である、[4]に記載の栄養学的若しくは薬学的調製物。
[9] ラクトバチルス・ラムノーサス(Lactobacillus rhamnosus)を有効成分として含む、マクロファージのM1/M2バランス調整剤又はマクロファージのM1/M2バランス調整用食品組成物。
[10] ラクトバチルス・ラムノーサス(L. rhamnosus)が、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)である、[9]に記載の調整剤又は食品組成物。
[11] ラクトバチルス・ラムノーサス(L. rhamnosus)を使用する工程を含む、in vitroでのM2マクロファージ分化誘導方法。
[12] ラクトバチルス・ラムノーサス(L. rhamnosus)が、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)である、[11]に記載の分化誘導方法。
[13] ラクトバチルス・ラムノーサス(L. rhamnosus)を配合する工程を含む、マクロファージのM1/M2バランスの不均衡に関連する疾患を治療又は予防するための栄養学的又は薬学的調製物の製造方法。
[14] マクロファージのM1/M2バランスの不均衡に関連する疾患が、糖尿病及び動脈硬化からなる群より選択される疾患である、[13]に記載の製造方法。
[15] ラクトバチルス・ラムノーサス(L. rhamnosus)が、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)である、[13]又は[14]に記載の製造方法。
As a result of diligent studies to solve the above-mentioned problems, the present inventors have surprisingly found that L. rhamnosus OLL2838 (Accession No. NITE P-313) has an M2 macrophage differentiation-inducing action and completed the present invention. I let you. That is, the present invention is as follows.
[1] An agent for inducing differentiation of M2 macrophages comprising Lactobacillus rhamnosus as an active ingredient.
[2] The differentiation-inducing agent according to [1], wherein Lactobacillus rhamnosus is L. rhamnosus specified by accession number NITE P-313.
[3] A pharmaceutical composition for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising the M2 macrophage differentiation inducer according to [1] or [2].
[4] A nutritional or pharmaceutical preparation for treating or preventing a disease associated with an imbalance in macrophage M1 / M2 balance, comprising the M2 macrophage differentiation inducer according to [1] or [2].
[5] A pharmaceutical composition for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising Lactobacillus rhamnosus as an active ingredient.
[6] The pharmaceutical composition according to [5], wherein Lactobacillus rhamnosus is L. rhamnosus identified by the accession number NITE P-313.
[7] The pharmaceutical composition according to [3], [5] or [6], wherein the disease associated with an imbalance in macrophage M1 / M2 balance is a disease selected from the group consisting of diabetes and arteriosclerosis. .
[8] The nutritional or pharmaceutical preparation according to [4], wherein the disease associated with an imbalance in macrophage M1 / M2 balance is a disease selected from the group consisting of diabetes and arteriosclerosis.
[9] A macrophage M1 / M2 balance regulator or a macrophage M1 / M2 balance food composition comprising Lactobacillus rhamnosus as an active ingredient.
[10] The adjusting agent or food composition according to [9], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.
[11] A method for inducing differentiation of M2 macrophages in vitro, comprising a step of using L. rhamnosus.
[12] The differentiation induction method according to [11], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.
[13] A method for producing a nutritional or pharmaceutical preparation for treating or preventing a disease associated with an imbalance in M1 / M2 balance of macrophages, comprising a step of blending L. rhamnosus .
[14] The production method according to [13], wherein the disease associated with an imbalance in M1 / M2 balance of macrophages is a disease selected from the group consisting of diabetes and arteriosclerosis.
[15] The production method according to [13] or [14], wherein Lactobacillus rhamnosus is L. rhamnosus specified by the accession number NITE P-313.
本発明に係る分化誘導剤を使用すると、M2マクロファージへの分化を誘導することができる。また、これによりM1/M2バランスの不均衡に関連する疾患や症状を軽減、緩和、治療又は予防することができる。 When the differentiation inducer according to the present invention is used, differentiation into M2 macrophages can be induced. This can also reduce, alleviate, treat or prevent diseases and symptoms associated with M1 / M2 imbalance.
本発明に係るM2マクロファージ分化誘導剤には乳酸菌(その菌体処理物も含まれる)を使用することができる。本明細書において乳酸菌とは、ブドウ糖を資化して乳酸を産生する菌をいい、生理学的性質としてはグラム陽性の球菌又は桿菌であり、運動性がなく、胞子形成能がなく、カタラーゼ陰性であるといった性質を有する。乳酸菌は食品発酵に使用されたり、哺乳動物の腸内細菌叢を構成するなど、極めて安全性の高い微生物と言える。本発明に用いることのできる乳酸菌としては、Lactobacillus属、Lactococcus属、Leuconostoc属、Pediococcus属、Streptococcus属、Weissella属、Tetragenococcus属、Oenococcus属、Enterococcus属、Vagococcus属、Carnobacterium属、Melissococcus属、Trichococcus属、Atopobium属のものが挙げられる。好ましくは本発明に用いることのできる乳酸菌としては、Lactobacillus属、とりわけLactobacillus rhamnosus種、より好ましくは受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(Lactobacillus rhamnosus)が挙げられるが、これに限定されない。また、M2マクロファージ分化誘導活性を保持するのであれば、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサスの変異株や形質転換株、受託番号NITE P-313で特定されるラクトバチルス・ラムノーサスに由来する株を使用することもできる。 As the M2 macrophage differentiation inducing agent according to the present invention, lactic acid bacteria (including treated cells thereof) can be used. In this specification, lactic acid bacteria refers to bacteria that assimilate glucose to produce lactic acid, and are physiologically gram-positive cocci or bacilli, have no motility, no spore-forming ability, and are catalase-negative. It has the following properties. Lactic acid bacteria can be said to be extremely safe microorganisms such as those used for food fermentation and constituting the intestinal bacterial flora of mammals. The lactic acid bacteria can be used in the present invention, Lactobacillus spp, Lactococcus spp, Leuconostoc spp, Pediococcus spp, Streptococcus spp, W e issella genus, Tetragenococcus genus, Oenococcus spp, Enterococcus spp, Vagococcus sp, Carnobacterium spp, Melissococcus genus, Trichococcus Genus, Atopobium genus. Preferably, as the lactic acid bacteria that can be used in the present invention, Lactobacillus genus, especially Lactobacillus rhamnosus species, more preferably Lactobacillus rhamnosus specified by the accession number NITE P-313 (Lactobacillus rhamnosus), but is not limited to this. In addition, if it retains M2 macrophage differentiation-inducing activity, a mutant or transformed strain of Lactobacillus rhamnosus specified by accession number NITE P-313, or Lactobacillus rhamnosus specified by accession number NITE P-313 Strains derived from can also be used.
受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(Lactobacillus rhamnosus)は腸管バリア機能の回復に寄与する株として、以前に単離されたものである(特許文献1参照)。以下にその寄託情報を記載する。
(1)寄託機関名:独立行政法人製品評価技術基盤機構 特許微生物寄託センター
(2)連絡先:郵便番号292-0818 千葉県木更津市かずさ鎌足2-5-8
電話番号0438-20-5580
(3)受託番号:NITE P-313
(4)識別のための表示:Lactobacillus rhamnosus OLL2838
(5)寄託日:平成19年2月14日。
Lactobacillus identified by accession number NITE P-313 (Lactobacillus) rhamnosus) was previously isolated as a strain that contributes to the recovery of intestinal barrier function (see Patent Document 1). The deposit information is described below.
(1) Depositary institution name: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: Postal code 292-0818 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture
Phone number 0438-20-5580
(3) Accession number: NITE P-313
(4) Display for identification: Lactobacillus rhamnosus OLL2838
(5) Date of deposit: February 14, 2007.
受託番号NITE P-313で特定されるラクトバチルス・ラムノーサス(L. rhamnosus)は、グラム陽性桿菌であり、BL寒天培地上で嫌気的に培養した際のコロニー形態は円形、白色、smooth型で円錐状に***する。生理学的特徴としては、ホモ乳酸発酵形式、15℃での発育性、ラムノース、リボース、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、メリチトース、マンニトール、ソルビトールに対する発酵性を有する。 Lactobacillus rhamnosus identified by accession number NITE P-313 is a Gram-positive bacilli, and the colony form when anaerobically cultured on BL agar is round, white, smooth and conical Raise into a shape. Physiological characteristics include homolactic fermentation, growth at 15 ° C., rhamnose, ribose, glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose, melittose, mannitol, sorbitol.
本発明に係るM2マクロファージ分化誘導剤及びこれを含む医薬組成物、栄養学的若しくは薬学的調製物又は食品組成物は、上記の乳酸菌を種々の形態で含みうる。例えば該乳酸菌は生菌又は死菌であってもよく、乳酸発酵物(乳酸菌飲料若しくは食品、酸乳、ヨーグルト等)また、菌体又は菌体断片、菌体処理物(破砕物、粉砕物、細胞溶解物等)であってもよい。このように、乳酸菌を含むM2マクロファージ分化誘導剤というとき、これは、上記のような生菌、死菌、発酵物、菌体、菌体断片、菌体処理物を包含するものとする。 The M2 macrophage differentiation-inducing agent and the pharmaceutical composition, nutritional or pharmaceutical preparation or food composition containing the same according to the present invention may contain the above-mentioned lactic acid bacteria in various forms. For example, the lactic acid bacteria may be live or dead bacteria, lactic acid fermented products (lactic acid bacteria beverages or foods, sour milk, yogurt, etc.), microbial cells or microbial cell fragments, microbial cell processed products (crushed material, crushed material, Cell lysate and the like). Thus, when referred to as an M2 macrophage differentiation inducer containing lactic acid bacteria, this includes live bacteria, dead bacteria, fermented products, bacterial cells, bacterial cell fragments, and processed bacterial cells as described above.
乳酸菌は、当技術分野で公知の適当な培地を用いて培養できる。培地の例としては、MRS培地(de Man J. C., Rogosa M. and Sharpe M. Elisabeth (1960) Appl. Bact. 23. 130-135)のような乳酸菌の選択培地や、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。菌体としては、このような培地を用いて培養した乳酸菌培養液から取得した菌体のみならず、培養終了後の乳酸菌培養液をそのまま、あるいは培養液を濃縮した物として使用することもできる。乳酸菌は生菌、死菌若しくはその混合物であってもよく、生菌体、湿潤菌体、乾燥菌(凍結乾燥、噴霧乾燥、真空乾燥、ドラム乾燥などによって得られうる)として用いられうる。死菌は加熱処理、加圧処理、加熱加圧処理、紫外線照射等により取得できる。本明細書では加熱処理を加熱殺菌と同義に用いることがある。生菌、死菌を問わず取得した菌体や培養液、濃縮物は、適当な媒体に再び懸濁させて懸濁物として使用してもよい。媒体としては、培養液、水、生理食塩水等が挙げられる。 Lactic acid bacteria can be cultured using an appropriate medium known in the art. Examples of media include lactic acid bacteria selection media such as MRS media (de Man JC, Rogosa M. and Sharpe M. Elisabeth (1960) Appl. Bact. 23. 130-135) and lactic acid bacteria culture media. Medium is used. That is, any medium can be used as long as it contains a nitrogen source, an inorganic substance 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 growth promoters. As the microbial cells, not only the microbial cells obtained from the lactic acid bacterium culture solution cultured using such a medium, but also the lactic acid bacterium culture solution after completion of the culture can be used as it is or as a concentrated product of the culture solution. The lactic acid bacterium may be a living bacterium, a dead bacterium, or a mixture thereof, and can be used as a alive cell, a wet cell, or a dry cell (which can be obtained by freeze drying, spray drying, vacuum drying, drum drying, or the like). Dead bacteria can be obtained by heat treatment, pressure treatment, heat pressure treatment, ultraviolet irradiation, or the like. In this specification, heat treatment may be used synonymously with heat sterilization. Cell bodies, culture solutions, and concentrates obtained regardless of whether they are live or dead, may be suspended again in an appropriate medium and used as a suspension. Examples of the medium include a culture solution, water, and physiological saline.
本発明において、有効成分である乳酸菌は、発酵物の形態であり得る。発酵物としては、乳酸菌飲料、乳酸菌食品、酸乳、発酵乳、ヨーグルト等が挙げられる。また、本発明では、有効成分である乳酸菌として乳酸菌処理物を用いることもできる。乳酸菌処理物としては、乳酸菌の菌体、乳酸菌含有物、発酵乳の濃縮物、ペースト化物、乾燥物など上記で述べた乳酸菌の菌体や菌体含有物を適当な装置を用いて破砕した破砕物、粉砕した粉砕物としたものが挙げられる。食品組成物は、乳酸発酵食品として製造された物以外にも、予め製造された各種食品に、本発明に係る乳酸菌、乳酸菌発酵物、菌体処理物、マクロファージM1/M2バランス調整剤、栄養学的若しくは薬学的調製物、又は医薬組成物を添加したものであってもよい。 In the present invention, the active ingredient lactic acid bacteria may be in the form of a fermented product. Examples of fermented products include lactic acid bacteria beverages, lactic acid bacteria foods, sour milk, fermented milk, and yogurt. Moreover, in this invention, a lactic acid bacteria processed material can also be used as lactic acid bacteria which are active ingredients. Lactic acid bacteria treated products include lactic acid bacteria, lactic acid bacteria-containing materials, fermented milk concentrates, pasted products, dried products, etc. And pulverized pulverized product. In addition to products manufactured as lactic acid fermented foods, the food composition can be applied to various foods manufactured in advance, lactic acid bacteria according to the present invention, fermented lactic acid bacteria, processed microbial cells, macrophage M1 / M2 balance regulator, nutrition Or a pharmaceutical preparation or a pharmaceutical composition may be added.
本発明において、M2マクロファージの分化を誘導する又は分化誘導を促進するとは、骨髄単核球由来の未分化マクロファージがさらにM1又はM2マクロファージへと分化する際、本発明に係る乳酸菌、M2マクロファージ分化誘導剤、又はこれを含む組成物を作用させない場合におけるM1/M2比と比較して、本発明に係る乳酸菌、M2マクロファージ分化誘導剤又はこれを含む組成物を作用させた場合のM1/M2比が小さくなること(すなわち系全体に占めるM2の割合が増えること)をいう。したがって、M2マクロファージへの分化を誘導する、との用語には、M1マクロファージへの分化が抑制されることも包含される。 In the present invention, to induce differentiation of M2 macrophages or to promote differentiation induction, when undifferentiated macrophages derived from bone marrow mononuclear cells further differentiate into M1 or M2 macrophages, lactic acid bacteria according to the present invention, induction of differentiation of M2 macrophages M1 / M2 ratio when the lactic acid bacteria according to the present invention, the M2 macrophage differentiation inducing agent or the composition containing the same is allowed to act is compared with the M1 / M2 ratio when the agent or the composition containing the same is not acted Smaller (that is, the proportion of M2 in the entire system increases). Therefore, the term “inducing differentiation into M2 macrophages” includes suppression of differentiation into M1 macrophages.
本発明に係る乳酸菌、M2マクロファージ分化誘導剤又はこれを含む組成物は、マクロファージのM1/M2バランスの不均衡に関連する疾患の治療又は予防に用いることができる。また、本発明に係る乳酸菌を含むM1/M2バランス調整剤又は食品組成物は、マクロファージのM1/M2バランスを調整するために使用できる。 The lactic acid bacterium, the M2 macrophage differentiation inducer or the composition containing the same according to the present invention can be used for treatment or prevention of a disease associated with an imbalance in macrophage M1 / M2 balance. Moreover, the M1 / M2 balance adjusting agent or food composition containing lactic acid bacteria according to the present invention can be used for adjusting the M1 / M2 balance of macrophages.
ここで、マクロファージのM1/M2バランスが取れているとは、生体における外部刺激に対する炎症応答から損傷修復に至るまでの一連の機構が正常に機能するようM1マクロファージとM2マクロファージとがバランスのとれた比で存在することをいう。マクロファージの正常なM1/M2バランスは、健常な個体又は健常な個体からなる集団におけるM1/M2存在比に基づいて決定でき、集団に基づいて決定する場合は、慣用の統計学的手法を用いることができる。M1/M2バランスが取れていない、正常な範囲から逸脱している、又は異常がある場合、本明細書ではこれをM1/M2バランスの不均衡、M1/M2バランスの崩れと呼ぶ。さらに、生体が外部刺激(炎症誘発性の刺激を含む)を受けた場合にM1/M2比は変化するが、健常者における変化範囲を超えてM1過剰/M2過小となる場合や、一時的にM1過剰/M2過小となった後に正常なバランスに戻らなかったり戻るのに長期間を要する場合も、「M1/M2バランスの不均衡」、「M1/M2バランスの崩れ」に包含されるものとする。したがって本明細書において、M1/M2バランスの不均衡とは、単に、M2と比較してM1マクロファージが過剰に存在する又はM1と比較してM2マクロファージが過小である場合をいうのみならず、生体が外部刺激(炎症誘発性の刺激を含む)を受けた場合に健常者において変動するM1/M2比からみて、被験体において変動するM1/M2比がM1過剰へと傾いている場合や、一時的なM1過剰への傾きから元のM1/M2比への回復を長時間に渡り示さない場合も含む。 Here, the M1 / M2 balance of macrophages means that M1 macrophages and M2 macrophages were balanced so that a series of mechanisms ranging from inflammatory responses to external stimuli to damage repair in the living body function normally. It exists in a ratio. Normal M1 / M2 balance of macrophages can be determined based on M1 / M2 abundance ratio in healthy individuals or populations of healthy individuals, using conventional statistical techniques when determining on a population basis Can do. When the M1 / M2 balance is not achieved, deviates from the normal range, or is abnormal, this is referred to as an M1 / M2 balance imbalance or an M1 / M2 balance collapse. Furthermore, the M1 / M2 ratio changes when the living body receives external stimuli (including pro-inflammatory stimuli), but if the M1 excess / M2 excess exceeds the change range in healthy subjects, or temporarily Even if it takes a long time to return to normal balance after M1 excess / M2 underestimation or it takes a long time to return, it is included in “M1 / M2 balance imbalance” and “M1 / M2 balance collapse”. To do. Therefore, in the present specification, the imbalance of the M1 / M2 balance not only refers to the case where M1 macrophages are excessively present compared to M2 or M2 macrophages are excessively small compared to M1, When the M1 / M2 ratio fluctuates in the subject when the subject receives external stimuli (including pro-inflammatory stimuli), the M1 / M2 ratio fluctuates in the subject tends to M1 excess, or temporarily This includes the case where the recovery to the original M1 / M2 ratio from the typical inclination to M1 excess is not shown for a long time.
本発明は特定の作用機序に限定されるものではないが、一実施形態において、本発明に係る乳酸菌、これを含む調整剤若しくは食品、M2マクロファージ分化誘導剤、又はこれを含む組成物は、M2マクロファージへの分化を誘導することにより、あるいはM1/M2バランスを調整することにより、M1/M2バランスの不均衡を是正し、又は崩れたM1/M2バランスを正常に回復させ、ひいてはマクロファージのM1/M2バランスの不均衡に関連する疾患を治療又は予防できる。本明細書においてM1/M2バランスの不均衡を是正する、崩れたM1/M2バランスを正常に回復させる、とは、M1/M2バランスの不均衡やバランスの崩れを正常なM1/M2バランスの取れている状態に戻すこと、又はそれを促進することをいう。また、M1/M2バランスを調整する、とはM1/M2バランスの不均衡を是正する、崩れたM1/M2バランスを正常に回復させるのみならず、M1/M2バランスの取れている状態を維持することも包含する。 Although the present invention is not limited to a specific mechanism of action, in one embodiment, the lactic acid bacteria according to the present invention, a regulator or food containing the same, an M2 macrophage differentiation inducing agent, or a composition containing the same, By inducing differentiation into M2 macrophages, or by adjusting the M1 / M2 balance, the M1 / M2 balance imbalance is corrected, or the disrupted M1 / M2 balance is restored to normal, and thus the M1 of the macrophages Can treat or prevent diseases associated with imbalance in the / M2 balance. In this specification, to correct the M1 / M2 balance imbalance and to restore the broken M1 / M2 balance to normal, the M1 / M2 balance imbalance and the balance imbalance are normal M1 / M2 balanced. It means returning to a state where it is in contact or promoting it. Adjusting the M1 / M2 balance not only corrects the M1 / M2 balance imbalance, restores the broken M1 / M2 balance to normal, but also maintains the M1 / M2 balance. It also includes.
マクロファージのM1/M2バランスの不均衡に関連する疾患としては、糖尿病、例えばインスリン抵抗性の増大による2型糖尿病、動脈硬化、例えば炎症に関連する動脈硬化、動脈の粥状硬化、アテローム性動脈硬化等が挙げられる。食事誘発肥満モデル動物において、脂肪組織マクロファージがM2極性からM1型の炎症促進性状態となり、インスリン抵抗性に寄与すると報告されている(非特許文献1)。また、肥満の進行につれて、M2マクロファージがM1型にシフトし、肥満に伴う2型糖尿病の発症に関わりうるとの報告もある(非特許文献2)。したがって当業者であれば、このような肥満モデルにおいてM2マクロファージへの分化を誘導することができれば、2型糖尿病を治療又は予防できる、と理解する。また、動脈の粥状硬化、アテローム性動脈硬化、は脂質異常と炎症の複合炎症であるが、IFN-γ等により活性化されたマクロファージが低比重リポタンパク(LDL)コレステロールを取り込んで泡沫細胞となり炎症を惹起することにより発症する。したがって当業者であれば、このような潜在的動脈硬化においてM2マクロファージへの分化を誘導することができれば、動脈硬化を予防できる、又はその進行を防止できると理解する。さらに、M1へのアンバランスが再発性脳脊髄炎を促進する、との報告(Mult Scler. 2011 Jan;17(1):2-15)や、M1極性化マクロファージが多発性硬化症の病理発症の一部であるとの報告(Int J Pharm. 2011 Sep 20;416(2):499-506)もある。当業者であれば、こうした報告から、M2マクロファージへの分化を誘導することやM1/M2バランスを回復することがこれらの疾患の治療予防につながり得ると理解する。このように本発明に係る乳酸菌、M2マクロファージ分化誘導剤又はこれを含む医薬組成物、栄養学的若しくは薬学的調製物又は食品組成物は、糖尿病予防、動脈硬化予防等に用いることができる。
Diseases associated with imbalance in macrophage M1 / M2 balance include diabetes, eg
本発明において、M2マクロファージ分化誘導剤は、本発明に係る乳酸菌の菌体(生菌、死菌を問わず、またそれらの混合物も包含する)を有効成分として含むように調製されたものであり、有効成分である菌体を単独で使用する場合のみならず、適宜必要な賦形剤等を添加し、栄養学的又は薬学的調製物、医薬組成物、食品組成物として使用することも含む。このような組成物の形態としては、錠剤や顆粒剤、カプセル剤、注射剤や液剤、ドライシロップ剤、散剤、シロップ剤などの各種剤形やサプリメントが例示されるがこれに限定されない。さらに、所望により、抗炎症剤、抗鎮痛剤、ビタミンなどの他の作用物質や、適当な賦形剤、結合剤、崩壊剤、滑沢剤、矯臭剤、溶解補助剤、懸濁化剤、コーティング剤、補助剤、保存料、香料、着色料等を加えることもできる。 In the present invention, the M2 macrophage differentiation inducing agent is prepared so as to contain as an active ingredient the lactic acid bacterium cells of the present invention (regardless of viable and dead bacteria, and also mixtures thereof). In addition to the use of the microbial cell as an active ingredient alone, it includes the addition of necessary excipients and the like as appropriate, and the use as nutritional or pharmaceutical preparations, pharmaceutical compositions, and food compositions. . Examples of such a composition include, but are not limited to, various dosage forms and supplements such as tablets and granules, capsules, injections and solutions, dry syrups, powders, and syrups. In addition, if desired, other active substances such as anti-inflammatory agents, anti-analgesic agents, vitamins, appropriate excipients, binders, disintegrants, lubricants, flavoring agents, solubilizing agents, suspending agents, Coating agents, adjuvants, preservatives, fragrances, coloring agents and the like can also be added.
本発明において、食品組成物とは、本発明に係る乳酸菌の菌体(生菌、死菌)、菌体成分、又はこれを含む分化誘導剤を添加した加工食品を意味する。これは、本発明に係る乳酸菌を用いて得られた食品、並びに通常の工程で得られた場合に比べて本発明の乳酸菌含量が同等若しくはそれ以上に高められた食品組成物を含む。食品組成物には、既存の食品、例えば、牛乳やヨーグルト、チーズ、発酵乳、豆腐、おかゆ、くず湯、お茶や果汁などからなる清涼飲料水、パン、ビスケット、クラッカー、ピッツァクラスト、調製粉乳、流動食、病者用食品、幼児用粉乳、授乳婦用粉乳等の食品(粉ミルクを含む)、栄養食品など、各種食用素材を原料にして製造された食品が含まれ、食品の製造時に上記乳酸菌又は気体成分を添加したもののみならず、栄養剤等のように、各種タンパク質(全脂粉乳、脱脂粉乳、部分脱脂粉乳、カゼイン、ホエー粉、ホエイタンパク質、ホエイタンパク質濃縮物、ホエイタンパク質分離物、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やレシチン、大豆タンパクなど植物性タンパク質など)、各種糖質(グルコース、フルクトース、ラムノース等の単糖類、ショ糖などの二糖類、キシリトールやグリセリンなどの多価アルコール、デキストリン、加工澱粉(デキストリン、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)、食物繊維などの多糖類など)、各種脂質(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や、大豆油、オアーム油、サフラワー油、コーン油、ナタネ油、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、不飽和脂肪酸など)、各種ビタミン(ビタミンA、カロテノイド類、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)や各種ミネラル(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)、有機酸(リンゴ酸、クエン酸、乳酸、酒石酸など)などの各種栄養素を任意の割合で混合し、そのまま、あるいはさらにそれらの混合物にゲル化剤を加え、粘度を調製した食品組成物も挙げられる。また、本発明に係る食品組成物には、いわゆる特定保健用食品も含まれる。これは糖尿病予防、動脈硬化予防、マクロファージのM1/M2バランスの不均衡に関連する疾患の予防又は改善等、本発明の作用に基づく効能を標榜可能とする食品や健康表示を具体的に表示することが公に許可された食品、栄養機能食品である。 In the present invention, the food composition means a processed food to which the lactic acid bacteria according to the present invention (live bacteria, dead bacteria), bacterial cell components, or a differentiation inducer containing the same is added. This includes foods obtained using the lactic acid bacteria according to the present invention, as well as food compositions in which the lactic acid bacteria content of the present invention is increased to the same or higher than when obtained in a normal process. The food composition includes existing foods such as milk, yogurt, cheese, fermented milk, tofu, porridge, kuzuyu, tea and fruit juice, bread, biscuits, crackers, pizza crust, formula milk, Includes foods made from various edible materials such as liquid foods, foods for the sick, infant milk powders, infant milk powders (including powdered milk), and nutritional foods. Or not only those with added gas components, but also various kinds of proteins (whole milk powder, skim milk powder, partially skimmed 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, egg protein, meat Animal and plant proteins such as proteins, plant proteins such as lecithin, soybean protein, etc., various carbohydrates (monosaccharides such as glucose, fructose, rhamnose, disaccharides such as sucrose, polyhydric alcohols such as xylitol and glycerin, dextrin, Processed starch (dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), polysaccharides such as dietary fiber), various lipids (lard, fish oil, etc., fractionated oils, hydrogenated oil, transesterification) Animal oils such as oil, soybean oil, oarm oil, safflower oil, corn oil, rapeseed oil, these fractionated oils, vegetable oils such as hydrogenated oil, transesterified oil, unsaturated fatty acids, etc.), various vitamins (Vitamin A, carotenoids, vitamin B group, vitamin C, vitamin D group, vitamin E, bi Min K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc.) and various minerals (calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium, etc.) Also, food compositions prepared by mixing various nutrients such as organic acids (malic acid, citric acid, lactic acid, tartaric acid, etc.) in any proportions, and adding a gelling agent to the mixture as it is or further adding them to adjust the viscosity It is done. The food composition according to the present invention includes so-called food for specified health use. This specifically displays foods and health indications that enable the indication of efficacy based on the action of the present invention, such as diabetes prevention, arteriosclerosis prevention, prevention or improvement of diseases related to imbalance in M1 / M2 balance of macrophages It is a food that is publicly permitted and a functional nutritional food.
当業者であれば、本発明に係る乳酸菌の摂取量(又は投与量)を適宜設定できる。摂取量は対象の年齢や性別、体重、症状あるいは使用目的によって異なるが、例えば成人のヒトにおける1回摂取量として、生菌であれば1×104〜1×1015個、好ましくは1×107〜1×1013個で用いることができるがこれに限定されない。死菌であれば、1×104〜1016個、好ましくは1×107〜1×1014個で使用できるがこれに限定されない。菌体濃度は使用態様にもよるが、例えば0.001〜100%(w/w)、好ましくは0.01〜100%(w/w)、さらに好ましくは0.1〜100%(w/w)とすることができる。また、乳酸菌発酵物や菌体処理物などとして用いる場合、好ましくは菌体に換算して上記範囲として使用する。 A person skilled in the art can appropriately set the intake (or dose) of lactic acid bacteria according to the present invention. The amount of intake varies depending on the age, sex, weight, symptom, or purpose of use of the subject. For example, as a single intake in an adult human, 1 × 10 4 to 1 × 10 15 cells, preferably 1 × Although it can be used by 10 < 7 > -1 * 10 < 13 > piece, it is not limited to this. As long as it is dead, it can be used at 1 × 10 4 to 10 16 , preferably 1 × 10 7 to 1 × 10 14 , but is not limited thereto. The cell concentration depends on the mode of use, but it may be, for example, 0.001 to 100% (w / w), preferably 0.01 to 100% (w / w), more preferably 0.1 to 100% (w / w). it can. Moreover, when using as a lactic-acid-bacteria fermented material, a microbial cell processed material, etc., Preferably it converts into a microbial cell and uses as said range.
乳酸菌の摂取量は、対象の年齢、性別、体重、症状、使用目的(治療、予防)によっても適宜増減され、医薬組成物又は食品組成物の種類や摂取量等によっても適宜調整されうる。また、摂取経路は経口投与、経管投与、経腸投与などが挙げられる。摂取又は投与対象はヒトに限らず、ペットや家畜、例えばイヌ、ネコ、サル、ウシ、ブタ、ウマなどの哺乳動物も含みうる。 The intake of lactic acid bacteria can be appropriately increased or decreased depending on the age, sex, weight, symptom, and intended use (treatment, prevention) of the subject, and can be adjusted as appropriate depending on the type and intake of the pharmaceutical composition or food composition. Examples of the route of intake include oral administration, tube administration, enteral administration, and the like. The subject of ingestion or administration is not limited to humans but may include pets and livestock, for example, mammals such as dogs, cats, monkeys, cows, pigs, horses.
本発明に係る医薬組成物、栄養学的若しくは薬学的調製物又は食品組成物は、マクロファージのM1/M2バランスの不均衡に関連する疾患を有する又はこれを発症しうる者にその治療、改善、軽減、緩和、予防のために使用できる。摂取は、医薬であれば食前や食後、食間が好ましく、食品組成物であれば食事の一品目として、あるいはその素材として食事の際に、また、サプリメント、栄養補助食品等として食事の間に摂取することができる。さらに、本発明に係る分化誘導剤はin vitroの実験系において細胞をM2マクロファージへと分化誘導させたい場合にも使用可能である。 The pharmaceutical composition, nutritional or pharmaceutical preparation or food composition according to the present invention has a disease associated with an imbalance in macrophage M1 / M2 balance or is capable of treating, improving, Can be used for mitigation, mitigation, prevention. Ingestion is preferably before meals, after meals, and between meals in the case of pharmaceuticals, and in the case of food compositions as one item of meal or as a material during meals, and during meals as supplements, dietary supplements, etc. can do. Furthermore, the differentiation inducer according to the present invention can also be used when it is desired to induce differentiation of cells into M2 macrophages in an in vitro experimental system.
すなわち本発明に係る乳酸菌を有効成分として含むM2マクロファージ分化誘導剤は、例えば研究用試薬としてin vitroで使用することができ、これにより未分化マクロファージのM2マクロファージへの分化を誘導することができる。このように本発明は、該分化誘導剤をin vitroにて未分化のマクロファージに接触させる工程を含む、M2マクロファージ分化誘導方法も提供する。一例として骨髄単核球を、マクロファージコロニー刺激因子(M-CSF)の存在下で培養し、骨髄由来M-CSF依存性マクロファージ(未分化のマクロファージ)を得る。未分化マクロファージとは、未だにM1又はM2マクロファージに分化していないマクロファージをいう。このとき、好ましくは樹状細胞は磁気細胞分離法等により系から除去しておく。この未分化マクロファージに、本発明に係る乳酸菌を添加し、場合によっては適当な因子(IL-4等)をさらに加え、M2マクロファージへの分化を誘導できる。分化誘導に用いる乳酸菌は、例えば未分化のマクロファージ2×105細胞に対し乳酸菌104〜108cfu、105〜107cfu、例えば2×106cfuとすることができる。M2マクロファージへの分化誘導は、ELISA、qPCR等の慣用法を用いて適当なマーカーを分析することにより確認できる。M2マクロファージのマーカーとしてはIL-10やCD206が挙げられる。逆にM1マクロファージへの分化誘導の減少又は抑制を確認することもでき、その場合、M1マクロファージのマーカーとしてはTNF-α、IL-12等が挙げられる。
That is, the M2 macrophage differentiation inducer containing the lactic acid bacterium according to the present invention as an active ingredient can be used, for example, in vitro as a research reagent, and thereby can induce differentiation of undifferentiated macrophages into M2 macrophages. Thus, the present invention also provides a method for inducing differentiation of M2 macrophages, comprising the step of bringing the differentiation inducer into contact with undifferentiated macrophages in vitro. As an example, bone marrow mononuclear cells are cultured in the presence of macrophage colony stimulating factor (M-CSF) to obtain bone marrow-derived M-CSF-dependent macrophages (undifferentiated macrophages). An undifferentiated macrophage refers to a macrophage that has not yet differentiated into M1 or M2 macrophages. At this time, the dendritic cells are preferably removed from the system by magnetic cell separation or the like. Lactic acid bacteria according to the present invention can be added to the undifferentiated macrophages, and in some cases, an appropriate factor (IL-4 or the like) can be further added to induce differentiation into M2 macrophages. The lactic acid bacteria used for differentiation induction can be, for example,
上記誘導方法を応用すれば、ある乳酸菌がM2マクロファージ分化誘導作用を有するか、簡便に確認することができる。すなわち、当業者であれば、慣用の方法に従って、未分化のマクロファージと試験する乳酸菌とを接触させ、M2マクロファージのマーカーを分析し、分化の有無又はその程度を確認することができる。例えば未分化のマクロファージ2×105細胞当たり、104〜108cfuの乳酸菌を接触させ、次いでIL-10やCD206をELISA、qPCR等により確認すればよい。なお、当業者であれば確認を96ウェルプレートを用いてハイスループットアッセイにより行うこともできる。 If the above induction method is applied, it can be easily confirmed whether a certain lactic acid bacterium has an M2 macrophage differentiation inducing action. That is, a person skilled in the art can contact an undifferentiated macrophage and a lactic acid bacterium to be tested according to a conventional method, analyze a marker of M2 macrophage, and confirm whether or not there is differentiation. For example, 10 4 to 10 8 cfu lactic acid bacteria may be contacted per 2 × 10 5 cells of undifferentiated macrophages, and then IL-10 and CD206 may be confirmed by ELISA, qPCR, or the like. A person skilled in the art can also confirm by a high-throughput assay using a 96-well plate.
以下の実施例は、例示のみを意図したものであり、本発明の技術的範囲を限定するものではない。 The following examples are intended for illustration only and are not intended to limit the technical scope of the present invention.
材料や試薬は特に断らない限り、市販されているか、又は当技術分野で慣用の手法、公知文献の手順に従って入手又は調製したものである。 Unless otherwise specified, the materials and reagents are commercially available, or obtained or prepared according to methods commonly used in the art and procedures in known literature.
[乳酸菌の調製]
本実施例では乳酸菌としてラクトバチルス・ラムノーサス(Lactobacillus rhamnosus)OLL2838(受託番号NITE P-313)を使用した。該乳酸菌を、MRS培地で18時間培養後、滅菌蒸留水で3回洗浄し、滅菌蒸留水を添加して1×108細胞/mLの菌体懸濁液を調製した。この菌体を75℃にて1時間加熱処理することにより殺菌した死菌を以下の実験に用いた。
[Preparation of lactic acid bacteria]
In this example, Lactobacillus rhamnosus (Lactobacillus) is used as the lactic acid bacterium. rhamnosus) OLL2838 (Accession Number NITE P-313) was used. The lactic acid bacteria were cultured in MRS medium for 18 hours, washed with sterilized distilled water three times, and sterilized distilled water was added to prepare a cell suspension of 1 × 10 8 cells / mL. The killed bacteria sterilized by heat-treating the cells at 75 ° C. for 1 hour were used in the following experiments.
[一般的実験方法]
ELISAについて簡単に説明すると、特異性の高い抗原抗体反応を利用し、酵素反応に基づく発色・発光をシグナルに用いることで、試料中の特定のタンパク質を検出・定量するものである。
[General experiment method]
Briefly describing ELISA, a specific protein in a sample is detected and quantified by using a highly specific antigen-antibody reaction and using color development / luminescence based on an enzyme reaction as a signal.
quantitative PCR (qPCR)について簡単に説明すると、定量PCRの一つで、ポリメラーゼ連鎖反応 (PCR) による増幅を経時的(リアルタイム)に測定することで、増幅率に基づいて鋳型となるDNAの定量を行うものである。 In brief, quantitative PCR (qPCR) is one of quantitative PCR. By measuring the polymerase chain reaction (PCR) amplification over time (real time), the template DNA can be quantified based on the amplification rate. Is what you do.
[マクロファージの分化誘導について]
Balb/cマウスの脛骨・大腿骨から採取された骨髄単核球(BMs)を取得し、マクロファージコロニー刺激因子(M-CSF)の存在下で6日間培養し、マクロファージに分化させた。具体的には採取されたBMsから非接着細胞を回収し、M-CSF(10ng/mLにて培養第1日及び第3日に添加)の存在下でフラスコで培養(1×106細胞/mL)して、骨髄由来M-CSF依存性マクロファージ(Mφ)に分化させた。
[Induction of differentiation of macrophages]
Bone marrow mononuclear cells (BMs) collected from tibia and femur bones of Balb / c mice were obtained, cultured in the presence of macrophage colony stimulating factor (M-CSF) for 6 days, and differentiated into macrophages. Specifically, non-adherent cells were collected from the collected BMs, and cultured in a flask (1 × 10 6 cells / ml) in the presence of M-CSF (added at 10 ng / mL on the first and third culture days). mL) and differentiated into bone marrow-derived M-CSF-dependent macrophages (Mφ).
このマクロファージについて、CD11c MicroBeadsを使用した磁気細胞分離法(MACS(登録商標))を用いてCD11c+の樹状細胞(DC)を除去し、2×105cellsという量のマクロファージに2×106細胞という量の上記L. rhamnosus加熱死菌体を添加し24時間培養した。次いで、これに10ng/mLのインターフェロン-γ(IFN-γ)又はインターロイキン-4(IL-4)を添加して、マクロファージをそれぞれM1マクロファージ又はM2マクロファージに分化させた。培養後の上清と細胞を回収し、qPCR及びELISAにより関連因子やマーカーであるIL-10、CD206、TNF-αを分析した。 For this macrophage, the CD11c + dendritic cells (DC) were removed using a magnetic cell separation method (MACS (registered trademark)) using CD11c MicroBeads, and 2 × 10 5 cells were subdivided into 2 × 10 5 cells. The above L. rhamnosus heat-killed cells were added and cultured for 24 hours. Subsequently, 10 ng / mL of interferon-γ (IFN-γ) or interleukin-4 (IL-4) was added thereto to differentiate the macrophages into M1 macrophages or M2 macrophages, respectively. The supernatant and cells after culture were collected, and related factors and markers such as IL-10, CD206, and TNF-α were analyzed by qPCR and ELISA.
[結果]
結果を図1〜4に示す。記号「HK-Lr」は加熱殺菌されたL. rhamnosusを表す。図1には、IL-10の産生量をELISAにより分析した結果を示す。図1中の「IL-4」は乳酸菌を添加せずインターロイキン-4のみを添加したものであるが、IL-10産生量は300pg/mLに満たない。これに対してIL-4及び加熱殺菌L. rhamnosus(HK-Lr)を添加した場合はIL-10産生量が1000pg/mLへと増大しており、M2マクロファージへの分化誘導が確認された。図1中の「none」はIL-4もIFN-γも添加していない場合であるが、これと比較してHK-Lrのみを添加した場合でもIL-10は有意に増大しており、M2マクロファージへの分化が誘導されている。また、IFN-γを添加した場合についても、HK-Lrの有無によりIL-10産生量に有意な差が見られ、同様にM2マクロファージへの分化誘導が観察された。
[result]
The results are shown in FIGS. The symbol “HK-Lr” represents heat-sterilized L. rhamnosus. FIG. 1 shows the results of analyzing the production amount of IL-10 by ELISA. “IL-4” in FIG. 1 is obtained by adding only interleukin-4 without adding lactic acid bacteria, but the amount of IL-10 produced is less than 300 pg / mL. In contrast, when IL-4 and heat sterilized L. rhamnosus (HK-Lr) were added, the IL-10 production amount increased to 1000 pg / mL, and differentiation induction into M2 macrophages was confirmed. “None” in FIG. 1 is the case where neither IL-4 nor IFN-γ is added, but IL-10 is significantly increased even when only HK-Lr is added, Differentiation into M2 macrophages is induced. In addition, when IFN-γ was added, a significant difference in IL-10 production was observed depending on the presence or absence of HK-Lr. Similarly, differentiation induction into M2 macrophages was observed.
図2には、IL-10/β-アクチンの量をqPCRにより分析した結果を示す。β-アクチンは、多くの組織や細胞中に共通して一定量発現する遺伝子であり、これと比較したIL-10発現量を分析することにより、正確なIL-10の発現量増大を解析することができる。IL-4単独の場合と比較して、IL-4+HK-Lrを添加した場合のIL-10/β-actin比が有意に増大しており、M2マクロファージへの分化が誘導されたことが確認された。 FIG. 2 shows the results of analyzing the amount of IL-10 / β-actin by qPCR. β-actin is a gene that is expressed in a certain amount in common in many tissues and cells. By analyzing the expression level of IL-10 in comparison with this, the exact increase in the expression level of IL-10 is analyzed. be able to. Compared to the case of IL-4 alone, the IL-10 / β-actin ratio was significantly increased when IL-4 + HK-Lr was added, confirming that differentiation into M2 macrophages was induced. It was.
図3には、M2マクロファージの表面抗原マーカーであるCD206のqPCR分析結果を示す。図中の縦軸の数値はCD206/β-アクチン比である。図3からも、IL-4及びHK-Lrを添加することにより、M2マクロファージへの分化が誘導されたことが確認された。 FIG. 3 shows the result of qPCR analysis of CD206, which is a surface antigen marker of M2 macrophages. The numerical value on the vertical axis in the figure is the CD206 / β-actin ratio. FIG. 3 also confirmed that differentiation into M2 macrophages was induced by adding IL-4 and HK-Lr.
図4には、M1マクロファージのマーカーであるTNF-αのqPCR分析結果を示す。図中の縦軸の数値はTNF-α/β-アクチン比である。IL-4添加した場合のTNF-α発現量は高くはないが、IL-4及びHK-Lrを添加した場合、TNF-αはほとんど発現せず、M1マクロファージへの分化はほとんど誘導されていないことが確認された。また、M1マクロファージへの分化誘導を促すIFN-γの存在下では一定量のTNF-αが発現したが、IFN-γの存在下で加熱殺菌したL. rhamnosus(HK-Lr)を添加したところTNF-αの発現量は有意に減少した。以上より、本発明に係るHK-LrはM1マクロファージへの分化誘導を抑制するか、又はM2マクロファージへの選択的分化を誘導することが確認された。 FIG. 4 shows the result of qPCR analysis of TNF-α, which is a marker for M1 macrophages. The numerical value on the vertical axis in the figure is the TNF-α / β-actin ratio. TNF-α expression level when IL-4 is added is not high, but when IL-4 and HK-Lr are added, TNF-α is hardly expressed and differentiation into M1 macrophages is hardly induced. It was confirmed. In addition, a certain amount of TNF-α was expressed in the presence of IFN-γ, which promotes differentiation into M1 macrophages, but L. rhamnosus (HK-Lr) that had been heat-sterilized in the presence of IFN-γ was added. The expression level of TNF-α was significantly reduced. From the above, it was confirmed that HK-Lr according to the present invention suppresses differentiation induction into M1 macrophages or induces selective differentiation into M2 macrophages.
本発明に係るM2マクロファージ分化誘導剤を用いることにより、骨髄単核球から得られた未分化のマクロファージを、選択的にM2マクロファージへと分化誘導することができる。また、本発明に係る分化誘導剤を含む組成物を用いることによりマクロファージのM1/M2バランスを調整することができ、ひいては糖尿病、動脈硬化といった疾患の治療又は予防に役立てることができる。 By using the M2 macrophage differentiation inducer according to the present invention, undifferentiated macrophages obtained from bone marrow mononuclear cells can be selectively induced to differentiate into M2 macrophages. Further, by using the composition containing the differentiation inducer according to the present invention, the M1 / M2 balance of macrophages can be adjusted, which can be used for the treatment or prevention of diseases such as diabetes and arteriosclerosis.
NITE P-313 NITE P-313
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