JP5714267B2 - Stem cell undifferentiation maintenance agent and proliferation promoter - Google Patents
Stem cell undifferentiation maintenance agent and proliferation promoter Download PDFInfo
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- JP5714267B2 JP5714267B2 JP2010189136A JP2010189136A JP5714267B2 JP 5714267 B2 JP5714267 B2 JP 5714267B2 JP 2010189136 A JP2010189136 A JP 2010189136A JP 2010189136 A JP2010189136 A JP 2010189136A JP 5714267 B2 JP5714267 B2 JP 5714267B2
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Description
本発明は、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を有効成分として含有することを特徴とする、幹細胞に対して特異的な未分化維持及び／又は増殖促進剤に関する。また、本発明は、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を含有する培養液を用いることで、移植時に生着率が極めて高い幹細胞を調製する方法及び／又は調製された幹細胞に関する。 The present invention comprises a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof as an active ingredient, and is specific for stem cells. The present invention relates to a differentiation maintenance and / or growth promoter. Further, the present invention uses a culture solution containing a compound containing one or more selected from bryonolic acid or a salt thereof, a bryonolic acid dicarboxylic acid ester or a salt thereof, and has a very high survival rate at the time of transplantation. The present invention relates to a method for preparing a stem cell and / or a prepared stem cell.
脊椎動物、特に哺乳動物の組織は、傷害もしくは疾患、又は加齢などに伴い細胞・臓器の損傷が起こった場合、再生系が働き、細胞・臓器の損傷を回復しようとする。この作用に、当該組織に備わる幹細胞が大きな役割を果たしている。幹細胞は、あらゆる細胞に分化する多能性を有しており、この性質により損傷部の細胞・組織を再生することで回復に導くと考えられている。このような幹細胞を応用した、次世代の医療である再生医療に期待が集まっている。 In the case of vertebrate, particularly mammalian tissue, when cell or organ damage occurs due to injury or disease, or aging, the regeneration system works to try to recover the damage of the cell or organ. Stem cells provided in the tissue play a major role in this effect. Stem cells have the pluripotency to differentiate into all kinds of cells, and it is considered that this property leads to recovery by regenerating the cells and tissues in the damaged part. Expectations are gathered for regenerative medicine, which is the next generation of medicine using such stem cells.
哺乳動物における幹細胞研究で最も進んでいる組織は骨髄である。骨髄には生体の造血幹細胞が存在しており、すべての血液細胞の再生の源であることが明らかにされた。さらに骨髄には、造血幹細胞とは別に、その他の組織（例えば、骨、軟骨、筋肉、脂肪組織など）へ分化可能な幹細胞が存在していることが報告されている（非特許文献１参照）。
さらに、近年、骨髄以外にも、肝臓、膵臓、脂肪組織など、あらゆる臓器・組織に幹細胞が存在することが明らかにされ、各臓器・組織の再生および恒常性維持を司っていることがわかってきた（非特許文献２〜５参照）。このような各組織に存在する幹細胞は可塑性に優れており、今まで自己複製が不可能であった臓器や組織の根本的な再生にも利用できる可能性がある。また、近年では、胚性幹細胞（ＥＳ細胞）や遺伝子導入により人工的に作製された幹細胞（人工多能性幹細胞：ｉＰＳ細胞）などの技術も進歩しており、様々な幹細胞が再生医療に活用できると期待されている（特許文献１〜２参照）。
The most advanced tissue in stem cell research in mammals is the bone marrow. Bone marrow contains living hematopoietic stem cells, which have been shown to be the source of all blood cell regeneration. Furthermore, it has been reported that in the bone marrow, there are stem cells that can be differentiated into other tissues (for example, bone, cartilage, muscle, adipose tissue, etc.) in addition to hematopoietic stem cells (see Non-Patent Document 1). .
Furthermore, in recent years, it has been clarified that stem cells exist in all organs / tissues such as liver, pancreas, and adipose tissue in addition to bone marrow, and it is known that they are responsible for regeneration and homeostasis of each organ / tissue. (See Non-Patent Documents 2 to 5). Such stem cells present in each tissue are excellent in plasticity and may be used for fundamental regeneration of organs and tissues that have been impossible to self-replicate until now. In recent years, technologies such as embryonic stem cells (ES cells) and stem cells artificially produced by gene transfer (artificial pluripotent stem cells: iPS cells) have also advanced, and various stem cells are used in regenerative medicine. It is expected to be possible (see Patent Documents 1 and 2).
このような中、これら幹細胞の能力（多能性）を細胞移植治療や組織工学（再生医療や再生美容）の分野において上手く活用するために、治療に用いる幹細胞を調製するための増殖技術や培養技術などの開発が進められている（非特許文献６〜７参照）。 Under such circumstances, proliferation techniques and cultures for preparing stem cells used for treatment in order to make effective use of the ability (pluripotency) of these stem cells in the field of cell transplantation treatment and tissue engineering (regenerative medicine and regenerative beauty). Development of technology and the like is underway (see Non-Patent Documents 6 to 7).
特に、幹細胞を生体外で増殖させる場合、幹細胞の能力である多能性を維持した状態、すなわち、未分化な状態を維持させたまま増殖させることが極めて重要である。また、この未分化な状態を保ちつつ、治療に必要な数になるまで幹細胞を増殖させる必要がある。もし、この増殖時に幹細胞の未分化状態が維持できず分化が進んでしまった場合、最終的に調製された幹細胞の能力（多能性）は失われていることになり、目的の治療効果（臓器、組織の再生など）は発揮されにくい。 In particular, when proliferating stem cells in vitro, it is extremely important to proliferate while maintaining the pluripotency that is the ability of stem cells, that is, maintaining an undifferentiated state. Moreover, it is necessary to proliferate stem cells until the number necessary for treatment is maintained while maintaining this undifferentiated state. If the undifferentiated state of the stem cells cannot be maintained during this proliferation and the differentiation has progressed, the ability (pluripotency) of the finally prepared stem cells has been lost, and the desired therapeutic effect ( Regeneration of organs and tissues is difficult to exert.
また、幹細胞は様々な細胞へ分化する能力を備えていることから、幹細胞の増殖時に分化が進行した場合、幹細胞以外の細胞も混入し増殖してしまう可能性がある。この場合、最終的に調製された幹細胞には、幹細胞以外の細胞が含まれることになり、目的の治療効果（臓器、組織の再生など）は発揮されにくい。 In addition, since stem cells have the ability to differentiate into various cells, when differentiation progresses during the proliferation of stem cells, cells other than stem cells may also be mixed and proliferated. In this case, the finally prepared stem cells contain cells other than the stem cells, and the intended therapeutic effect (eg, regeneration of organs and tissues) is difficult to be exhibited.
以上より、今後、再生医療に幹細胞を用いることを考えた場合、幹細胞の未分化状態を維持させたまま移植に必要な数まで増殖させ、また、移植時には組織への生着率が高い幹細胞を調製する技術が必要である。これには、幹細胞の未分化状態を維持するとともに、幹細胞のみを特異的に増殖させ、かつ移植時に組織への生着が高い幹細胞を調製する技術が必要である。 Based on the above, when considering the use of stem cells for regenerative medicine in the future, the stem cells can be proliferated to the number necessary for transplantation while maintaining the undifferentiated state of the stem cells. A technique to prepare is needed. This requires a technique for preparing stem cells that maintain the undifferentiated state of the stem cells, specifically proliferate only the stem cells, and have high engraftment in the tissue at the time of transplantation.
現在までに、幹細胞の未分化状態を維持させたまま増殖させる技術について、幾つか報告はあるが、未だ発展途上である。例えば、胚性幹細胞（ＥＳ細胞）や造血幹細胞は、支持細胞（ストローマ細胞、もしくはフィーダー細胞）と共培養することで未分化を維持することができる（非特許文献８〜１０、特許文献３参照）。しかし、最近になってフィーダー細胞由来の内在性ウイルスによる異種動物間の感染例が報告されており（非特許文献１１参照）、医療用途を目的とした幹細胞の培養には適していない。 To date, there have been some reports on techniques for growing stem cells while maintaining the undifferentiated state of stem cells, but they are still under development. For example, embryonic stem cells (ES cells) and hematopoietic stem cells can be maintained undifferentiated by co-culture with supporting cells (stromal cells or feeder cells) (see Non-Patent Documents 8 to 10 and Patent Document 3). ). However, recently, an example of infection between different animals caused by endogenous virus derived from feeder cells has been reported (see Non-Patent Document 11), and is not suitable for culturing stem cells for medical purposes.
その他の方法に、サイトカインを複雑に組合せることによって幹細胞の未分化状態を維持させる方法がある。例えば、マウスＥＳ細胞は、ＬＩＦ（白血病抑制因子（Ｌｅｕｋｅｍｉａ Ｉｎｈｉｂｉｔｏｒｙ Ｆａｃｔｏｒ））を培地に添加することによって、未分化性が維持される（非特許文献１２、特許文献４参照）。その他、トロンボポイエチン（ＴＰＯ）、インターロイキン６（ＩＬ−６）、ＦＬＴ−３リガンド、および幹細胞因子（ＳＣＦ）の存在下で、未分化性を維持させることが胚性幹細胞、体性幹細胞などで報告されている（特許文献５、非特許文献１３参照）。 As another method, there is a method of maintaining an undifferentiated state of a stem cell by complexly combining cytokines. For example, mouse ES cells are maintained in an undifferentiated state by adding LIF (Leukemia Inhibitory Factor) to the medium (see Non-patent Document 12 and Patent Document 4). In addition, maintaining undifferentiation in the presence of thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF) is an embryonic stem cell, somatic stem cell, etc. (See Patent Document 5 and Non-Patent Document 13).
さらに近年では、人工的に幹細胞を調製する技術やそれを増殖させるための技術として塩基性線維芽細胞増殖因子（ｂＦＧＦ）などを添加する方法などが発明されている（特許文献６参照）。また、幹細胞を純化する技術として、特殊な培養液（ｂＦＧＦ含有）を用いることで幹細胞の未分化状態を維持させつつ増殖させる方法が発明されている（特許文献７参照）。いずれもｂＦＧＦなどの増殖因子やサイトカインを添加することで、幹細胞の未分化状態を維持させつつ増殖を促進する技術が開発されている。しかし、このような技術で使用されている増殖因子やサイトカインなどは、極めて高価であり、製造メーカーやロット間での品質や効能の違いなどの問題があり使用は難しい。加えて、これまで汎用されているＬＩＦなどの未分化維持に効果を示す成分は、極めて特定の細胞系統に限定的であり、特に霊長類のＥＳ細胞や体性幹細胞においては、ＬＩＦの添加のみでは未分化状態を維持することができないことが明らかにされている（非特許文献９参照）。 Furthermore, in recent years, a method of adding a basic fibroblast growth factor (bFGF) or the like has been invented as a technique for artificially preparing stem cells or a technique for growing them (see Patent Document 6). In addition, as a technique for purifying stem cells, a method of proliferating while maintaining the undifferentiated state of stem cells by using a special culture solution (containing bFGF) has been invented (see Patent Document 7). In either case, a technique has been developed that promotes proliferation while maintaining the undifferentiated state of stem cells by adding growth factors such as bFGF and cytokines. However, the growth factors and cytokines used in such a technique are extremely expensive, and are difficult to use due to problems such as differences in quality and efficacy between manufacturers and lots. In addition, components that are effective for maintaining undifferentiation such as LIF, which have been widely used so far, are limited to specific cell lines, and in particular, in primate ES cells and somatic stem cells, only the addition of LIF Is not able to maintain an undifferentiated state (see Non-Patent Document 9).
このように、これまでの技術では、特に幹細胞の未分化状態を維持する効果が不十分であり、幹細胞の増殖は可能ではあるが、同時に幹細胞から分化した細胞や初期段階に混入した幹細胞以外の細胞も増殖してしまい、最終的に調製された幹細胞には幹細胞以外の細胞が混在する状態になってしまう。 As described above, in the conventional technology, in particular, the effect of maintaining the undifferentiated state of the stem cells is insufficient, and the proliferation of the stem cells is possible, but at the same time, other than the cells differentiated from the stem cells or the stem cells mixed in the initial stage. The cells also proliferate, and the finally prepared stem cells are mixed with cells other than the stem cells.
すなわち、現在までに報告されている幹細胞の未分化状態の維持及び増殖技術は、いずれも移植用の幹細胞を調製し、実際に移植した場合に満足いく効果を望めるものは少なく、開発途上の技術であると考えられている。また、これまでの技術は、煩雑な操作を必要としたり、幹細胞の未分化状態の維持効果が低いことから、最終的には幹細胞以外の細胞の増殖を招き、これを移植する際には、幹細胞以外の細胞を移植することになるため、幹細胞の組織への生着率が極めて低下する。故に、今後の再生医療において、治療に用いる幹細胞を調製する場合、幹細胞の未分化状態を維持させる技術と、かつこの未分化状態を維持させたまま、幹細胞のみを必要な数まで増殖させる技術の開発が望まれている。これには、幹細胞に対して選択的に未分化維持と増殖促進効果を示す成分や培養技術の開発が必要である。また、これらの技術により調製された幹細胞を、生体組織に効率よく生着させる必要がある。つまり、安全かつ簡便で効率的に、幹細胞特異的に未分化状態を維持させたまま増殖させ、その他の細胞に対しては増殖効果を示さず、かつ移植時の生体組織への生着率の高い幹細胞の調製技術が求められていた。 In other words, none of the techniques for maintaining and proliferating the undifferentiated state of stem cells that have been reported so far can produce satisfactory effects when preparing stem cells for transplantation and actually transplanting them. It is considered to be. In addition, the conventional techniques require complicated operations or have a low effect of maintaining the undifferentiated state of the stem cells, so that eventually the cells other than the stem cells proliferate, and when transplanted, Since cells other than stem cells are transplanted, the engraftment ratio of stem cells to the tissue is extremely reduced. Therefore, when preparing stem cells for treatment in future regenerative medicine, a technique for maintaining the undifferentiated state of the stem cells and a technique for proliferating only the stem cells to the required number while maintaining the undifferentiated state. Development is desired. For this purpose, it is necessary to develop components and culture techniques that selectively exhibit undifferentiation maintenance and proliferation promoting effects on stem cells. Further, it is necessary to efficiently engraft stem cells prepared by these techniques in living tissues. In other words, it is safe, convenient, and efficient, proliferating while maintaining an undifferentiated state specific to stem cells, showing no proliferative effect on other cells, and the rate of engraftment in living tissue at the time of transplantation High stem cell preparation technology has been demanded.
かかる状況に鑑み、本発明は、上記のような従来技術における問題点を解決し、哺乳動物の幹細胞に対して未分化状態を維持させたまま、幹細胞特異的な増殖促進効果を示す幹細胞の未分化維持及び／又は増殖促進剤を開発し、かつ、移植時に生体組織への生着率の高い幹細胞とその調製技術を提供することにある。 In view of such circumstances, the present invention solves the problems in the prior art as described above, and the stem cells that exhibit stem cell-specific proliferation-promoting effects while maintaining an undifferentiated state with respect to mammalian stem cells have not been obtained. An object of the present invention is to develop a differentiation maintenance and / or growth promoter and to provide a stem cell having a high engraftment rate in a living tissue at the time of transplantation and a preparation technique thereof.
本発明者らは、上記課題の解決に向けて鋭意検討を行った結果、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物に、幹細胞に対して特異的な未分化維持効果と増殖促進効果を見出し、さらに、当該化合物を含有した培養液を用いて調製した移植用の幹細胞を、実際に移植した際に組織への生着率が極めて向上することを明らかにし、本発明を完成するに至った。 As a result of intensive studies aimed at solving the above-mentioned problems, the present inventors have developed a compound containing one or more selected from bryonolic acid or a salt thereof, a bryonolic acid dicarboxylic acid ester or a salt thereof, into a stem cell. On the other hand, a specific undifferentiation maintenance effect and a proliferation promoting effect were found, and furthermore, the transplantation stem cells prepared using a culture solution containing the compound had an extremely high survival rate in tissues when transplanted. It has been clarified that this has been improved, and the present invention has been completed.
即ち本発明は、以下のとおりである。
（１）下記一般式（Ａ）で示されるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を含有することを特徴とする、幹細胞の未分化維持剤。
（化１）一般式（Ａ）
（式中、Ｒ１はＨ又はジカルボン酸エステル残基（その塩を含む）であり、Ｒ２は、カルボキシル基又はその塩である）
（２）一般式（Ａ）で示されるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を含有することを特徴とする、幹細胞の増殖促進剤。
（３）一般式（Ａ）で示されるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を含有する培地を用いて培養することを特徴とする、幹細胞の調製方法。
（４）（３）に記載の幹細胞の調製方法を用いて調製した幹細胞。
（５）一般式（Ａ）で示されるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を含有することを特徴とする、幹細胞用途の培地。
That is, the present invention is as follows.
(1) A non-stem cell comprising a compound containing one or more selected from the group consisting of bryonolic acid represented by the following general formula (A) or a salt thereof, a bryonolic acid dicarboxylic acid ester or a salt thereof Differentiation maintenance agent.
(Formula 1) General formula (A)
(Wherein R 1 is H or a dicarboxylic acid ester residue (including a salt thereof), and R 2 is a carboxyl group or a salt thereof)
(2) Proliferation promotion of stem cells characterized by containing a compound containing one or more selected from bryonolic acid represented by the general formula (A) or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof Agent.
(3) It is characterized by culturing using a medium containing a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof represented by the general formula (A). A method for preparing stem cells.
(4) Stem cells prepared using the stem cell preparation method according to (3).
(5) A medium for use in stem cells, comprising a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof represented by the general formula (A) .
本発明に用いるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物は、上記一般式（Ａ）で示される。 The compound containing 1 type (s) or 2 or more types chosen from the bryonolic acid or its salt used for this invention, a bryonolic acid dicarboxylic acid ester, or its salt is shown by the said general formula (A).
一般式（Ａ）で示される化合物は、ブリオノール酸から誘導することができる。ブリオノール酸とは、下記一般式（Ｂ）で示される酸性五環性トリテルペノイドであり、ウリ科植物の培養細胞を低濃度のオーキシンを含む培地中で暗所にて培養することによって生産することができる（特許文献８、非特許文献１４参照）。 The compound represented by the general formula (A) can be derived from brionolic acid. Bryonolic acid is an acidic pentacyclic triterpenoid represented by the following general formula (B), which can be produced by culturing cultured cells of Cucurbitaceae plants in a dark place in a medium containing a low concentration of auxin. Yes (see Patent Document 8 and Non-Patent Document 14).
（化２）一般式（Ｂ）
(Formula 2) General formula (B)
例えば、ウリ科植物（ヘチマ、メロンなど）のカルスを常法により誘導し、その懸濁培養細胞を作製する。この培養細胞を１０−６〜１０−８Ｍの２，４−ジクロロフェノキシ酢酸を含む液体培地に接種し、暗所で１〜３週間、回転振とう培養する。得られた培養細胞を乾燥した後、エタノールで抽出し、濃縮乾固する。得られた抽出物を結晶化させるとブリオノール酸が得られる。また、ウリ科植物、例えば、メロン、カボチャやカラスウリなどの根から同様にして抽出し、シリカゲルカラムで分画後、再結晶によりブリオノール酸を得ることができる。 For example, calli of cucurbitaceae plants (such as loofah and melon) are induced by a conventional method to produce suspension culture cells thereof. The cultured cells are inoculated into a liquid medium containing 10 −6 to 10 −8 M 2,4-dichlorophenoxyacetic acid, and cultured in a dark place for 1 to 3 weeks with rotary shaking. The obtained cultured cells are dried, extracted with ethanol, and concentrated to dryness. When the resulting extract is crystallized, bryonolic acid is obtained. Further, bryonolic acid can be obtained by extraction from roots of cucurbitaceae plants, for example, melon, pumpkin and crow cucumber in the same manner, fractionation with a silica gel column, and recrystallization.
ジカルボン酸エステル残基に関して、ジカルボン酸としては、分子内にカルボキシル基を２個有するものであれば特に限定されず、シュウ酸、マロン酸、コハク酸、グルタル酸、アジピン酸、ピメリン酸、スベリン酸、アゼライン酸、セバシン酸などの脂肪族飽和ジカルボン酸、マレイン酸、フマル酸などの脂肪族不飽和ジカルボン酸、フタル酸、イソフタル酸、テレフタル酸などの芳香族ジカルボン酸などが例示される。上記ジカルボン酸エステルは、常法によりブリオノール酸を無水コハク酸や無水グルタル酸などと反応させることにより、容易に調製することができる。 Regarding the dicarboxylic acid ester residue, the dicarboxylic acid is not particularly limited as long as it has two carboxyl groups in the molecule. Oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid And aliphatic saturated dicarboxylic acids such as azelaic acid and sebacic acid, aliphatic unsaturated dicarboxylic acids such as maleic acid and fumaric acid, and aromatic dicarboxylic acids such as phthalic acid, isophthalic acid and terephthalic acid. The dicarboxylic acid ester can be easily prepared by reacting bryonolic acid with succinic anhydride, glutaric anhydride or the like by a conventional method.
ブリオノール酸又はブリオノール酸ジカルボン酸エステルは、塩を形成していてもよく、塩としては、カルボキシル基が、塩を形成しており、生理学的に許容されれば特に制限されるものではない。好適にはナトリウム、カリウム塩等のアルカリ金属塩、カルシウム、マグネシウム塩等のアルカリ土類金属塩、亜鉛、アルミニウム、アンモニウム等の第四級アミン塩、及びリジン，アルギニン塩等のアミノ酸塩などが例示される。上記塩類は、ブリオノール酸又はブリオノール酸ジカルボン酸エステルから常法により容易に誘導することができる。 The bryonolic acid or the bryonolic acid dicarboxylic acid ester may form a salt, and the salt is not particularly limited as long as the carboxyl group forms a salt and is physiologically acceptable. Preferred examples include alkali metal salts such as sodium and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, quaternary amine salts such as zinc, aluminum and ammonium, and amino acid salts such as lysine and arginine salts. Is done. The salts can be easily derived from bryonolic acid or bryonolic acid dicarboxylic acid ester by a conventional method.
ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物は、本発明の未分化維持剤、増殖促進剤、培地などに対して、乾燥物として０．００００１〜１０重量％であることが好ましく、０．０００１〜１重量％含まれる濃度で使用することが最も好ましい。０．００００１重量％未満であると本発明の効果が十分に発揮されにくい場合がある。 A compound containing one or more selected from bryonolic acid or a salt thereof, brionolic acid dicarboxylic acid ester or a salt thereof is 0 as a dry product with respect to the undifferentiation maintaining agent, growth promoter, medium, etc. of the present invention. It is preferably 0.0001 to 10% by weight, and most preferably used at a concentration of 0.0001 to 1% by weight. If it is less than 0.00001% by weight, the effects of the present invention may not be sufficiently exerted.
また、本発明の幹細胞に対して特異的な未分化維持効果や増殖促進効果を示す化合物や、これにより調製された幹細胞、またその調製方法などは、細胞培養用添加剤、研究用試薬、医療用試薬、細胞移植剤をはじめとし、医薬品、医薬部外品、化粧品、食品等への配合や応用が可能である。 In addition, compounds exhibiting a specific undifferentiation maintenance effect and proliferation promoting effect on the stem cells of the present invention, stem cells prepared thereby, and methods for preparing the same are used for cell culture additives, research reagents, medical treatments, etc. It can be used in pharmaceuticals, quasi drugs, cosmetics, foods, etc.
本発明の幹細胞としては、本発明の目的に沿うものであれば、胚性の幹細胞（ＥＳ細胞）、もしくは、骨髄、血液、皮膚、脂肪組織、脳、肝臓、膵臓、腎臓、筋肉やその他の組織に存在する、体性の幹細胞、さらには遺伝子導入などにより人工的に調製された幹細胞、また、当該細胞の初代培養細胞、継代培養細胞、凍結細胞いずれであってもよい。好ましくは、骨髄、血液、皮膚、脂肪組織由来の幹細胞に対してより効果を発揮する。また、哺乳動物における、幹細胞の分化の方向性、および、分化の過程等について同等の特性を持っていれば、全ての哺乳動物に応用が可能である。例えば、ヒト、サル、マウス、ラット、モルモット、ウサギ、ネコ、イヌ、ウマ、ウシ、ヒツジ、ヤギ、ブタ等の哺乳動物の幹細胞に対して効果を発揮することができる。 The stem cells of the present invention are embryonic stem cells (ES cells) or bone marrow, blood, skin, adipose tissue, brain, liver, pancreas, kidneys, muscles, and the like as long as they meet the purpose of the present invention. Somatic stem cells present in tissues, stem cells artificially prepared by gene transfer or the like, and primary culture cells, subculture cells, or frozen cells of the cells may be used. Preferably, it is more effective against bone marrow, blood, skin, and adipose tissue-derived stem cells. Moreover, if it has the same characteristic about the direction of differentiation of a stem cell in a mammal, the process of differentiation, etc., it can apply to all mammals. For example, the effect can be exerted on stem cells of mammals such as humans, monkeys, mice, rats, guinea pigs, rabbits, cats, dogs, horses, cows, sheep, goats and pigs.
本発明の幹細胞を培養する培地、又は同時に用いる添加剤としては、例えば以下のものを使用できるが、限定されるものではない。 As the medium for culturing the stem cells of the present invention, or the additive used at the same time, for example, the following can be used, but is not limited.
具体的には、細胞の生存に必要な成分（無機塩、炭水化物、ホルモン、必須アミノ酸、非必須アミノ酸、ビタミン、脂肪酸）を含む基本培地、例えば、Ｄｕｌｂｅｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ（Ｄ−ＭＥＭ）、Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（ＭＥＭ）、ＲＰＭＩ１６４０，Ｂａｓａｌ Ｍｅｄｉｕｍ Ｅａｇｌｅ（ＢＭＥ）、Ｄｕｌｂｅｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ：Ｎｕｔｒｉｅｎｔ Ｍｉｘｔｕｒｅ Ｆ−１２（Ｄ−ＭＥＭ／Ｆ−１２）、Ｇｌａｓｇｏｗ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ（Ｇｌａｓｇｏｗ ＭＥＭ）、ハンクス液（Ｈａｎｋ’ｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ）に増殖因子として塩基性線維芽細胞増殖因子（ｂＦＧＦ）、白血球遊走阻止因子（ＬＩＦ）の少なくともいずれか１種を添加した培地が用いられ、好ましくは、これら増殖因子の全てが含有されたものである。また、必要に応じて、上皮細胞増殖因子（ＥＧＦ）、腫瘍壊死因子（ＴＮＦ）、ビタミン類、インターロイキン類、インスリン、トランスフェリン、ヘパリン、ヘパラン硫酸、コラーゲン、フィブロネクチン、プロゲステロン、セレナイト、Ｂ２７−サプリメント、Ｎ２−サプリメント、ＩＴＳ−サプリメント、抗生物質などが含有されてもよい。 Specifically, a basic medium containing components necessary for cell survival (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, fatty acids), such as Dulbecco's Modified Eagle Medium (D-MEM), Minum Essential Medium (MEM), RPMI 1640, Basal Medium Eagle (BME), Dulbecco's Modified Eagle Medium (H), Numeric Medium F-12 (D-MEM / F-12) Hank's balanced salt solution) is a basic fibroblast growth factor (growth factor) FGF), medium supplemented with at least one kind of leukocyte migration inhibitory factor (LIF) is used, preferably those that all of these growth factors is contained. If necessary, epidermal growth factor (EGF), tumor necrosis factor (TNF), vitamins, interleukins, insulin, transferrin, heparin, heparan sulfate, collagen, fibronectin, progesterone, selenite, B27-supplement, N2-supplements, ITS-supplements, antibiotics, and the like may be included.
また、上記以外には、１〜２０％の含有率で血清が含まれることが好ましい。しかし、血清はロットの違いにより成分が異なり、その効果にバラツキがあるため、ロットチェックを行った後に使用することが好ましい。 In addition to the above, it is preferable that serum is contained at a content of 1 to 20%. However, since serum has different components depending on the lot and there are variations in the effects, it is preferable to use the serum after a lot check.
市販品としては、インビトロジェン製の間葉系幹細胞基礎培地や、三光純薬製の間葉系幹細胞基礎培地、ＴＯＹＯＢＯ社製のＭＦ培地、Ｓｉｇｍａ社製のハンクス液（Ｈａｎｋ’ｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ）などを用いることができる。 Commercially available products include Mesenchymal stem cell basal medium manufactured by Invitrogen, Mesenchymal stem cell basal medium manufactured by Sanko Junyaku, MF medium manufactured by TOYOBO, Hank's balanced salt solution manufactured by Sigma, etc. Can be used.
幹細胞の培養に用いられる培養器は、幹細胞の培養が可能なものであれば特に限定されないが、例えば、フラスコ、シャーレ、ディッシュ、プレート、チャンバースライド、チューブ、トレイ、培養バック、ローラーボトルなどが挙げられる。 The incubator used for stem cell culture is not particularly limited as long as stem cells can be cultured. Examples thereof include flasks, petri dishes, dishes, plates, chamber slides, tubes, trays, culture bags, and roller bottles. It is done.
培養器は、細胞非接着性であっても接着性であってもよく、目的に応じて適宜選択される。細胞接着性の培養器は、細胞との接着性を向上させる目的で、細胞外マトリックス等による細胞支持用基質などで処理したものを用いてもよい。細胞外基質としては、例えば、コラーゲン、ゼラチン、ポリ−Ｌ−リジン、ポリ−Ｄ−リジン、ラミニン、フィブロネクチンなどが挙げられる。 The incubator may be non-cell-adhesive or adhesive, and is appropriately selected according to the purpose. As the cell-adhesive incubator, for the purpose of improving the adhesion with cells, a cell-treated culture vessel treated with a cell support substrate using an extracellular matrix or the like may be used. Examples of the extracellular matrix include collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin and the like.
培養条件としては、適宜設定できる。例えば、培養温度は、特に限定されるものではないが約３０〜４０℃、好ましくは約３７℃が良い。ＣＯ２ガス濃度は、例えば約１〜１０％、好ましくは約２〜５％が良い。 Culture conditions can be set as appropriate. For example, the culture temperature is not particularly limited, but is about 30 to 40 ° C, preferably about 37 ° C. The CO 2 gas concentration is, for example, about 1 to 10%, preferably about 2 to 5%.
本発明のブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物は、幹細胞に対して極めて高い未分化状態の維持効果を示した。さらに、増殖促進効果に関しては、幹細胞以外の細胞に対しては増殖促進効果を示さず、幹細胞のみに対する極めて特異的な増殖促進効果を見出した。また、このブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を用いて調製した幹細胞を生体組織へ移植した場合、従来の方法に比べて組織への生着率が著しく向上した。以上より、本発明は、組織の再生治療、再生美容に役立つものであり、組織の再生の分野において大きく貢献できるものである。 The compound containing one or two or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof of the present invention showed an extremely high undifferentiated state maintaining effect on stem cells. Furthermore, with regard to the growth promoting effect, the present inventors have found a very specific growth promoting effect only on stem cells without showing a growth promoting effect on cells other than stem cells. In addition, when stem cells prepared by using a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof are transplanted into a living tissue, the tissue is compared with a conventional method. The engraftment rate to the markedly improved. As described above, the present invention is useful for tissue regeneration treatment and regeneration beauty, and can greatly contribute to the field of tissue regeneration.
以下、次に本発明を詳細に説明するため、具体的かつ詳細な実施例を挙げるが、本発明はこれらに何ら限定されるものではない。 Hereinafter, in order to describe the present invention in detail, specific and detailed examples will be given, but the present invention is not limited thereto.
まず、実施例として本発明に用いるブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物の製造例および幹細胞の未分化状態の維持効果を示す実験例を挙げる。 First, as an example, a production example of a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof used in the present invention, and an experiment showing an effect of maintaining an undifferentiated state of a stem cell Give an example.
以下に、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物の製造例を示す。 Below, the manufacture example of the compound containing the 1 type (s) or 2 or more types chosen from a bryonolic acid or its salt, a bryonolic acid dicarboxylic acid ester, or its salt is shown.
製造例１ ブリオノール酸
ヘチマの子葉を４．５×１０−６Ｍの２，４−ジクロロフェノキシ酢酸（２，４−Ｄ）及び２．３×１０−６Ｍ のカイネチンを含むムラシゲとスクーグの寒天培地に置床し、常法によりカルスを誘導した。一ヶ月後、このカルスを同液体培地に入れて９０ｒｐｍで回転振とう培養し、その懸濁培養細胞を調製した。この培養細胞を１０−７Ｍの２，４−Ｄを含むムラシゲとスクーグの液体培地に接種し、暗所で２週間、回転振とう培養した。得られた培養細胞を凍結乾燥した後、エタノールにて抽出し、抽出液を濃縮乾固した。得られた抽出物をエタノールに再溶解させ、続いて再結晶させブリオノール酸の針状結晶を得た。ブリオノール酸含量は、細胞の乾燥重量に対して１．２％であった。
Production Example 1 Bryonolic acid Agar of Murashige and Skoog containing cotyledons of loofah containing 4.5 × 10 −6 M 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.3 × 10 −6 M kinetin It was placed on the medium and calli were induced by a conventional method. One month later, this callus was placed in the same liquid medium and cultured with shaking at 90 rpm to prepare suspension culture cells. The cultured cells were inoculated into Murashige and Skoog liquid medium containing 10 −7 M 2,4-D, and cultured with shaking in the dark for 2 weeks. The obtained cultured cells were lyophilized and extracted with ethanol, and the extract was concentrated to dryness. The obtained extract was redissolved in ethanol and subsequently recrystallized to obtain needle crystals of bryonolic acid. The bryonolic acid content was 1.2% based on the dry weight of the cells.
製造例２ サクシニルブリオノール酸
ブリオノール酸１０ｇ、無水コハク酸６４ｇをピリジン５５０ｍＬに溶解し、１００℃にて８時間反応させた。放冷後、１Ｎ塩酸６．５Ｌ中に反応液を加え、３℃にて放置し、析出した結晶をろ別し、精製水にて洗浄した。
ろ別した固形分をジエチルエーテル３Ｌに溶解し、精製水にて洗浄後、ジエチルエーテルを留去し、乾燥して固形分１３ｇを得た。 固形分１３ｇをエタノール１００ｍＬに加熱溶解し再結晶することにより、白色固形分としてサクシニルブリオノール酸１２ｇを得た。サクシニルブリオノール酸を一般式（Ｃ）に示す。
Production Example 2 Succinylbryonolic acid 10 g of bryonolic acid and 64 g of succinic anhydride were dissolved in 550 mL of pyridine and reacted at 100 ° C. for 8 hours. After allowing to cool, the reaction solution was added to 6.5 L of 1N hydrochloric acid and allowed to stand at 3 ° C., and the precipitated crystals were separated by filtration and washed with purified water.
The solid content separated by filtration was dissolved in 3 L of diethyl ether and washed with purified water, and then diethyl ether was distilled off and dried to obtain 13 g of a solid content. 13 g of solid content was dissolved by heating in 100 mL of ethanol and recrystallized to obtain 12 g of succinyl bryonolic acid as a white solid content. Succinyl bryonolic acid is shown in general formula (C).
（化３）一般式（Ｃ）
(Formula 3) General formula (C)
製造例３ サクシニルブリオノール酸二カリウム
サクシニルブリオノール酸１２ｇを脱水テトラヒドロフラン（ＴＨＦ）３００ｍｌに溶解し、１Ｎ水酸化カリウム水溶液４５ｍＬを添加した。追加でＴＨＦを徐々に加えて沈殿が生成し始めたら約１時間熟成し、総液量１２００ｍＬまでＴＨＦを加え、３℃、１日後、ろ別し、黄色固形分１５ｇを得た。
黄色固形分１５ｇを１００ｍＬエタノールに加熱溶解し、ＴＨＦを徐々に加え白濁し始めたらろ過をし、ろ液を３℃で１日放置した。固形分をろ別し、エタノール１００ｍＬに加熱溶解し、３℃、１日後、ろ過乾燥し、白色〜微黄色固形分のサクシニルブリオノール酸二カリウム１１ｇを得た。サクシニルブリオノール酸二カリウムを一般式（Ｄ）に示す。
Production Example 3 Dipotassium succinylbrionolate 12 g of succinylbrionolate was dissolved in 300 ml of dehydrated tetrahydrofuran (THF), and 45 mL of 1N aqueous potassium hydroxide solution was added. Additional THF was gradually added and when a precipitate started to form, the mixture was aged for about 1 hour, THF was added to a total liquid volume of 1200 mL, and the mixture was filtered at 3 ° C. for 1 day to obtain 15 g of a yellow solid.
15 g of a yellow solid was dissolved in 100 mL of ethanol by heating, THF was gradually added, and when it became white turbid, it was filtered, and the filtrate was left at 3 ° C. for 1 day. The solid content was separated by filtration, dissolved by heating in 100 mL of ethanol, and filtered and dried at 3 ° C. for 1 day to obtain 11 g of dipotassium succinylbrionolate having a white to slightly yellow solid content. Dipotassium succinylbrionolate is shown in general formula (D).
（化４）一般式（Ｄ）
(Formula 4) General formula (D)
以下に、実施例１で示した製造例１〜３のブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を用いた、幹細胞の未分化状態維持効果及び細胞増殖促進効果の実験例とその結果を示す。 Below, the undifferentiated state of the stem cell using the compound containing 1 type (s) or 2 or more types chosen from the bryonolic acid of the manufacture examples 1-3 shown in Example 1, or its salt, bryonolic acid dicarboxylic acid ester, or its salt Experimental examples and results of the maintenance effect and the cell growth promoting effect are shown.
実験例１ 幹細胞に対する未分化状態維持効果の評価
Ｄｕｌｂｅｃｃｏ’ｓ Ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ Ｍｅｄｉｕｍ培養液（Ｇｉｂｃｏ社製）に、ウシ胎児血清（ＦＢＳ、１５％、Ｓｉｇｍａ社製）、ヌクレオシド液（１００倍希釈、大日本製薬社製）、非必須アミノ酸液（１００倍希釈、大日本製薬社製）、β２−メルカプトエタノール液（１００倍希釈、大日本製薬社製）、Ｌ−グルタミン液（１００倍希釈、大日本製薬社製）、ペニシリン（１００ｕｎｉｔ／ｍＬ、Ｓｉｇｍａ社製）とストレプトマイシン（１００μｇ／ｍＬ、Ｓｉｇｍａ社製）を加えて調製した培地を用いて、マウス胚性幹細胞（マウスＥＳ細胞：コスモバイオ社製）を、６ｃｍディッシュに１×１０５個播種し、各試料（製造例１〜３）を最終濃度が０．００１％になるように添加し、３日間培養を続けた。次に細胞をＰＢＳ（−）にて３回洗浄した後、ラバーポリスマンにて集め、血球計数板にて細胞数をカウントした後、ＣｅｌＬｙｔｉｃ（Ｓｉｇｍａ社製）にてタンパク質を抽出し、未分化状態の測定を豊岡らの報告に従って行った（文献：豊岡やよい，Ｍｏｌｅｃｕｌａｒ Ｍｅｄｉｃｉｎｅ臨時増刊号 再生医学，２００３，１０６−１１５）。すなわち、幹細胞の未分化状態を示しているオクタマーバインディングプロテイン３／４タンパク質（Ｏｃｔ３／４タンパク質）の発現量を指標に、培養開始時に播種した幹細胞（１×１０５個）が発現していたＯｃｔ３／４タンパク質の量を１００％未分化状態とし、各試料（製造例１〜３）を添加して３日間培養した後のＯｃｔ３／４タンパク質の量をウエスタンブロッティング法にて定量解析し、培養開始時と３日間培養後のＯｃｔ３／４タンパク質の量を比較することで、未分化状態の維持効果について評価した。なお、これまでに幹細胞の未分化維持効果を示す物質として報告されている塩基性線維芽細胞増殖因子（ｂＦＧＦ）（特表２０１０−５０００４７号公報）を陽性対照として用いて同様な評価を行った。
Experimental Example 1 Evaluation of effect of maintaining undifferentiated state on stem cells In Dulbecco's Modified Eagle Medium culture solution (Gibco), fetal bovine serum (FBS, 15%, Sigma), nucleoside solution (100-fold dilution, Dainippon) Manufactured by Pharmaceutical Co., Ltd.), non-essential amino acid solution (100-fold dilution, manufactured by Dainippon Pharmaceutical), β2-mercaptoethanol solution (100-fold diluted, manufactured by Dainippon Pharmaceutical), L-glutamine solution (100-fold diluted, Dainippon Pharmaceutical) Mouse embryonic stem cells (mouse ES cells: manufactured by Cosmo Bio) using a medium prepared by adding penicillin (100 units / mL, manufactured by Sigma) and streptomycin (100 μg / mL, manufactured by Sigma). 1 × 10 5 seeded in a 6 cm dish, and each sample (Production Examples 1 to 3) has a final concentration of 0.001%. The culture was continued for 3 days. Next, the cells were washed 3 times with PBS (−), collected with a rubber policeman, counted with a hemocytometer, and then extracted with CelLytic (Sigma), undifferentiated state. Was measured according to the report of Toyooka et al. (Reference: Yayoi Toyooka, Extraordinary issue of Molecular Medicine, Regenerative Medicine, 2003, 106-115). That is, stem cells (1 × 10 5 ) seeded at the start of culture were expressed using the expression level of octamer binding protein 3/4 protein (Oct3 / 4 protein) indicating the undifferentiated state of stem cells as an index. After the amount of Oct3 / 4 protein is 100% undifferentiated, each sample (Production Examples 1 to 3) is added and cultured for 3 days, the amount of Oct3 / 4 protein is quantitatively analyzed by Western blotting and cultured. The effect of maintaining the undifferentiated state was evaluated by comparing the amount of Oct3 / 4 protein at the start and after culturing for 3 days. In addition, the same evaluation was performed using the basic fibroblast growth factor (bFGF) (Japanese translations of PCT publication 2010-500047 gazette) currently reported as a substance which shows the undifferentiation maintenance effect of a stem cell as a positive control until now. .
具体的な評価方法としては、ウエスタンブロッティング法にて培養開始時の幹細胞の単一細胞数（１×１０５個）に発現しているＯｃｔ３／４タンパク質量（Ａとする）と化合物を添加して３日間培養し、増殖させた後の単一細胞数（１×１０５個）で発現しているＯｃｔ３／４タンパク質量（Ｂとする）を求め、次の式より培養３日後の未分化状態（％）を算出した。培養３日後の未分化状態の算出式＝Ｂ／Ａ×１００（％）。 As a specific evaluation method, an amount of Oct3 / 4 protein (referred to as A) expressed in a single number of stem cells (1 × 10 5 ) at the start of culture by Western blotting and a compound were added. The amount of Oct3 / 4 protein (referred to as B) expressed in the number of single cells (1 × 10 5 cells) after culturing for 3 days and proliferating was determined, and undifferentiated 3 days after culturing using the following formula: The state (%) was calculated. Calculation formula of undifferentiated state after 3 days of culture = B / A × 100 (%).
これらの試験結果を表１に示した。その結果、陽性対照物質（ｂＦＧＦ）と比較して、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）全てにおいて顕著な幹細胞の未分化状態維持効果が認められた。以上より、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物の極めて優れた幹細胞の未分化状態維持効果を明らかにした。なお、本実験例で用いた幹細胞以外にも、体性の幹細胞や遺伝子導入により人工的に調製した幹細胞についても同様な試験を行ったところ、顕著な幹細胞の未分化状態維持効果を認めた。 The test results are shown in Table 1. As a result, as compared with the positive control substance (bFGF), all of the compounds (Production Examples 1 to 3) containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof The effect of maintaining the undifferentiated state of stem cells was observed. As mentioned above, the very outstanding stem cell undifferentiated state maintenance effect of the compound containing 1 type, or 2 or more types chosen from a bryonolic acid or its salt, a bryonolic acid dicarboxylic acid ester, or its salt was clarified. In addition to the stem cells used in this experimental example, similar tests were performed on somatic stem cells and stem cells artificially prepared by gene transfer, and a remarkable effect of maintaining the undifferentiated state of stem cells was observed.
実験例２ 幹細胞に対する特異的な細胞増殖促進効果の評価
生体組織は、大きく外胚葉、中胚葉、内胚葉の組織に分類され、これら組織を構成する細胞は、約２００種類程度存在すると考えられていることから、その中でも代表として、外胚葉組織の細胞である角化細胞（ＤＳファーマバイオメディカル社製）、中胚葉組織の細胞である線維芽細胞（ＤＳファーマバイオメディカル社製）、内胚葉組織の細胞である肝臓細胞（ＨＥＰＧ２）とヒト体性幹細胞を用いて、これら細胞に対するブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）の細胞特異的な増殖促進効果について評価を行った。すなわち、これら細胞の中で、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）が特異的に幹細胞のみに対して増殖促進効果を示すかについて評価した。具体的には、これら細胞を、それぞれを６ｃｍディッシュに１×１０６個播種し、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）を最終濃度が０．００１％になるように添加し、３日間培養を続けた。次に細胞をＰＢＳ（−）にて３回洗浄した後、それぞれの細胞をラバーポリスマンにて集め、細胞数をカウントした。
それぞれの細胞に対して化合物未添加時の総細胞数をコントロール（１００％）とした場合の、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）添加時のそれぞれの細胞数の増減（％）を算出し、各細胞に対する細胞増殖促進効果の評価を行った。なお、これまでに幹細胞の増殖促進効果を示す物質として報告されている塩基性線維芽細胞増殖因子（ｂＦＧＦ）（特許文献７参照）を陽性対照として用いて同様な評価を行った。
Experimental Example 2 Evaluation of Specific Cell Proliferation Promoting Effect on Stem Cells Biological tissues are broadly classified into ectoderm, mesoderm, and endoderm tissues, and it is considered that there are about 200 types of cells constituting these tissues. Among them, keratinocytes (DS Pharma Biomedical), which are cells of ectoderm tissue, fibroblasts (DS Pharma Biomedical), which are cells of mesoderm tissue, and endoderm tissue are representative of them. A compound containing 1 or 2 or more types selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof, using liver cells (HEPG2) and human somatic stem cells, which are cells of 1-3) The cell-specific proliferation promoting effect of 1) was evaluated. That is, among these cells, a compound (Production Examples 1 to 3) containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof specifically for only stem cells. It was evaluated whether it showed a growth promoting effect. Specifically, each of these cells is seeded at 1 × 10 6 cells in a 6 cm dish, and a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof (production) Examples 1 to 3) were added to a final concentration of 0.001%, and the culture was continued for 3 days. Next, the cells were washed three times with PBS (−), and then each cell was collected with a rubber policeman, and the number of cells was counted.
1 type or 2 or more types chosen from bryonolic acid or its salt, bryonolic acid dicarboxylic acid ester, or its salt when the total cell number at the time of a compound non-addition with respect to each cell is made into control (100%) is included. The increase / decrease (%) in the number of cells at the time of adding the compound (Production Examples 1 to 3) was calculated, and the effect of promoting cell proliferation on each cell was evaluated. In addition, the same evaluation was performed using the basic fibroblast growth factor (bFGF) (refer patent document 7) reported as a substance which shows the proliferation promotion effect of a stem cell until now as a positive control.
これらの試験結果を表２に示した。その結果、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）は、幹細胞のみに対して顕著な細胞増殖促進効果を示した。この効果は、線維芽細胞、角化細胞や肝臓細胞に対しては見られず、幹細胞に対して特異的なものであった。また、これまで幹細胞増殖促進物質として使用されているｂＦＧＦは、幹細胞以外に角化細胞、線維芽細胞や肝臓細胞に対しても有意な増殖効果を示した。以上より、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物は、極めて優れた幹細胞に対する特異的な増殖促進効果を示した。なお、本実験例で用いた幹細胞以外にも、胚性幹細胞（ＥＳ細胞）や遺伝子導入により人工的に調製した幹細胞についても同様な試験を行ったところ、幹細胞に対して顕著かつ特異的な増殖促進効果を認めた。 The test results are shown in Table 2. As a result, the compound (Production Examples 1 to 3) containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof has a remarkable cell growth promoting effect only on stem cells. Indicated. This effect was not seen for fibroblasts, keratinocytes or liver cells, but was specific for stem cells. In addition, bFGF, which has been used as a stem cell proliferation promoting substance so far, showed a significant proliferation effect on keratinocytes, fibroblasts and liver cells in addition to stem cells. As mentioned above, the compound containing 1 type, or 2 or more types chosen from a bryonolic acid or its salt, a bryonolic acid dicarboxylic acid ester, or its salt showed the specific proliferation-promoting effect with respect to the very outstanding stem cell. In addition to the stem cells used in this experimental example, embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer were subjected to the same test. The promotion effect was recognized.
組織への移植用として、従来の技術により調製した幹細胞とブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を添加して調製した幹細胞を用いて、実際に移植を行い、移植後のそれぞれの生着率（％）を比較した。 For transplantation to tissue, stem cells prepared by adding stem cells prepared by conventional techniques and compounds containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof, and the like are used. The transplantation was actually performed, and the survival rate (%) after the transplantation was compared.
実験例３ 移植用の幹細胞の調製
ヒト体性幹細胞（ＤＳファーマバイオメディカル社製）を６ｃｍディッシュに１×１０６個播種し、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）を最終濃度が０．０１％になるように添加し、３日間培養を続けた。次に、それぞれの細胞をＰＢＳ（−）にて３回洗浄した後、無菌的にラバーポリスマンにて回収し、遠沈後、５％ＦＢＳ添加ハンクス液（Ｈａｎｋ’ｓ ｂａｌａｎｃｅｄ ｓａｌｔ ｓｏｌｕｔｉｏｎ）に分散し、移植用の幹細胞として用いた。その後、以下の方法にて、皮膚移植を行い、生着率（％）について測定した。
Experimental Example 3 Preparation of Stem Cells for Transplantation Human somatic stem cells (DS Pharma Biomedical) were seeded at 1 × 10 6 in a 6 cm dish and selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof. A compound containing one or more compounds (Production Examples 1 to 3) was added so that the final concentration was 0.01%, and the culture was continued for 3 days. Next, each cell was washed three times with PBS (−), collected aseptically with a rubber policeman, spun down, and dispersed in a Hanks solution containing 5% FBS (Hunk's balanced salt solution). Used as stem cells for transplantation. Thereafter, skin transplantation was performed by the following method, and the survival rate (%) was measured.
実験例４ 幹細胞の移植および生着率（％）の測定
実験例３で調製した幹細胞を、改めて１×１０６個サンプリングし、細胞の数を揃えた後、注射筒にてヌードマウスの皮下に移植し、移植後の生着率（％）を測定した。具体的な生着率（％）の測定法としては、移植用にサンプリングした１×１０６個の幹細胞を、予めＣｅｌｌ Ｔｒａｃｋｅｒ（モレキュラープローブ社製）にて蛍光標識し、その時点の蛍光強度を測定し、移植細胞の総蛍光強度（１００％）とした。この細胞を、注射筒を用いて、ヌードマウス（雄性、４週齢）の皮下に移植した。移植部位をマジックにてマーキングし、移植３日後、７日後に移植部位を摘出し、酵素処理により細胞を分散させ、得られた細胞の総蛍光強度を測定し、移植時の総蛍光強度（１００％）と比較することで、生着率（％）を算出した。すなわち、生着した幹細胞が多いほど、最初に移植した蛍光強度と同等の蛍光が検出され、逆に、生着しなかった場合は、移植部位から蛍光が検出されないこととなる。
Experimental Example 4 Stem Cell Transplantation and Measurement of Engraftment Rate (%) The stem cells prepared in Experimental Example 3 were sampled 1 × 10 6 times again, and the number of cells was adjusted, and then subcutaneously in nude mice with a syringe. After transplantation, the survival rate (%) after transplantation was measured. As a specific method for measuring the survival rate (%), 1 × 10 6 stem cells sampled for transplantation are fluorescently labeled in advance with Cell Tracker (manufactured by Molecular Probes), and the fluorescence intensity at that time is determined. The total fluorescence intensity (100%) of the transplanted cells was measured. The cells were transplanted subcutaneously into nude mice (male, 4 weeks old) using a syringe. The transplant site is marked with magic, the transplant site is removed 3 days and 7 days after transplantation, the cells are dispersed by enzyme treatment, the total fluorescence intensity of the obtained cells is measured, and the total fluorescence intensity at the time of transplantation (100 %), The survival rate (%) was calculated. That is, as the number of stem cells engrafted increases, the fluorescence equivalent to the fluorescence intensity initially transplanted is detected. Conversely, if the engraftment does not occur, fluorescence is not detected from the transplant site.
これらの試験結果を表３に示した。その結果、移植３日後、７日後ともに、従来の方法により調製した幹細胞に比べてブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物（製造例１〜３）を用いて調製した幹細胞を移植した場合において極めて高い生着率を示した。 These test results are shown in Table 3. As a result, both 3 days and 7 days after transplantation, a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof compared to stem cells prepared by a conventional method (Production Example) When the stem cells prepared using 1 to 3) were transplanted, the engraftment rate was extremely high.
以上の結果より、従来の方法で調製した幹細胞よりもブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を用いて移植用の幹細胞を調製することで、幹細胞の組織への生着率を顕著に向上させることを確認した。なお、本実験例で用いた幹細胞以外にも、胚性幹細胞（ＥＳ細胞）や遺伝子導入により人工的に調製した幹細胞についても同様な試験を行ったところ、幹細胞移植における有意な生着率の向上効果を認めた。 Based on the above results, a stem cell for transplantation is prepared using a compound containing one or more selected from bryonolic acid or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof, rather than a stem cell prepared by a conventional method. Thus, it was confirmed that the engraftment ratio of stem cells to the tissue was remarkably improved. In addition to the stem cells used in this experimental example, similar tests were conducted on embryonic stem cells (ES cells) and stem cells artificially prepared by gene transfer. The effect was recognized.
本発明の、ブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を幹細胞に対して用いることで、幹細胞の未分化状態を維持させたまま幹細胞のみを増殖促進させることが可能になり、さらに、この技術を用いることで、移植時に極めて生着率の高い幹細胞を簡便に調製することが可能となった。 Stem cells while maintaining the undifferentiated state of stem cells by using the compound of the present invention containing one or two or more selected from bryonolic acid or a salt thereof, brionolic acid dicarboxylic acid ester or a salt thereof for stem cells It is possible to promote the growth of only stem cells, and furthermore, by using this technique, it becomes possible to easily prepare stem cells with a very high engraftment rate at the time of transplantation.
本発明の活用例として、再生医療、再生美容への応用が期待される。例えば、本発明を利用することで、再生医療、再生美容に用いる移植用の幹細胞を調製する場合に、幹細胞の未分化状態を維持しつつ移植に必要な数の幹細胞を選択的かつ効率的に増殖させることが可能である。さらに本発明技術により調製された幹細胞は、移植時に組織への高い生着率を示す性質を備えており、再生医療、再生美容において極めて有用な幹細胞の調製が可能になる。また、移植以外の用途として移植後又は組織に存在する幹細胞に対して、本発明のブリオノール酸又はその塩、ブリオノール酸ジカルボン酸エステル又はその塩から選ばれる１種又は２種以上を含む化合物を、組織に対して直接的に注入又は経口投与、塗布、貼付などにより導入させることにより、幹細胞に対して特異的な未分化状態の維持効果及び増殖促進効果を見出すことが可能である。
すなわち、本発明は、再生医療、再生美容における、幹細胞に対して特異的な未分化維持剤及び／又は増殖促進剤としての応用性と、かつ当該化合物を用いることで組織への生着率の高い幹細胞を調製することを可能にする技術である。
As an application example of the present invention, application to regenerative medicine and regenerative beauty is expected. For example, by using the present invention, when preparing stem cells for transplantation used in regenerative medicine and regenerative beauty, the number of stem cells necessary for transplantation can be selectively and efficiently maintained while maintaining the undifferentiated state of stem cells. It is possible to grow. Furthermore, the stem cells prepared by the technique of the present invention have the property of exhibiting a high engraftment rate in the tissue at the time of transplantation, and it becomes possible to prepare stem cells that are extremely useful in regenerative medicine and regenerative beauty. In addition, for stem cells existing after transplantation or in tissue as a use other than transplantation, a compound comprising one or more selected from the bryonolic acid or salt thereof, the bryonolic acid dicarboxylic acid ester or salt thereof of the present invention, It is possible to find an effect of maintaining an undifferentiated state specific to a stem cell and an effect of promoting proliferation by introducing it directly into a tissue by injection, oral administration, application, sticking or the like.
That is, the present invention provides applicability as an undifferentiation maintenance agent and / or growth promoter specific to stem cells in regenerative medicine and regenerative beauty, and the use of the compound increases the rate of engraftment in tissues. It is a technology that makes it possible to prepare high stem cells.
Claims (4)
（化１）一般式（Ａ）
（式中、Ｒ１はＨ又はジカルボン酸エステル残基（その塩を含む）であり、Ｒ２は、カルボキシル基又はその塩である） An agent for maintaining undifferentiation of stem cells, comprising a compound containing one or more selected from the group consisting of bryonolic acid represented by the following general formula (A) or a salt thereof, a bryonolic acid dicarboxylic acid ester or a salt thereof .
(Formula 1) General formula (A)
(Wherein R 1 is H or a dicarboxylic acid ester residue (including a salt thereof), and R 2 is a carboxyl group or a salt thereof)
A medium for use in stem cells, comprising a compound containing one or more selected from the group consisting of bryonolic acid represented by the general formula (A) or a salt thereof, bryonolic acid dicarboxylic acid ester or a salt thereof.
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