JP5710146B2 - Stem cell undifferentiation maintenance agent and proliferation promoter - Google Patents
Stem cell undifferentiation maintenance agent and proliferation promoter Download PDFInfo
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- JP5710146B2 JP5710146B2 JP2010083272A JP2010083272A JP5710146B2 JP 5710146 B2 JP5710146 B2 JP 5710146B2 JP 2010083272 A JP2010083272 A JP 2010083272A JP 2010083272 A JP2010083272 A JP 2010083272A JP 5710146 B2 JP5710146 B2 JP 5710146B2
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
本発明は、ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出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、ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出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 contains an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the azalea family as an active ingredient, and maintains undifferentiation and / or promotes proliferation specific to stem cells It relates to the agent. The present invention also provides a method for preparing stem cells having an extremely high engraftment rate at the time of transplantation by using a culture solution containing an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the family Azalea. Alternatively, it relates to 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、その他の臓器(例えば、骨、軟骨、筋肉、脂肪など)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調製された幹細胞(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 organs (eg, bone, cartilage, muscle, fat, 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, fat, etc. in addition to bone marrow, and it has been found that they are responsible for the 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 prepared by gene transfer (artificial pluripotent stem cells: iPS cells) have also advanced, and various stem cells can be used for 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)を発揮されにくい。 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 ( It is difficult to reproduce organs and tissues.
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)を発揮されにくい。 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 stem cells finally prepared contain cells other than the stem cells, and it is difficult to exert the intended therapeutic effect (eg, regeneration of organs and tissues).
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、幾つか報告があるが、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 several 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、初期作用性サイトカイン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 embryonic stem cells in the presence of early-acting cytokines thrombopoietin (TPO), interleukin 6 (IL-6), FLT-3 ligand, and stem cell factor (SCF) It has been reported for somatic stem cells (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, the conventional techniques are not particularly effective in maintaining the undivided state of the stem cells, 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、幹細胞に対して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 exhibits a proliferation-promoting effect specific to stem cells while maintaining the undifferentiated state of mammalian stem cells. An object of the present invention is to develop a stem cell undifferentiated 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、ツツジ科に属するコメバツガザクラの抽出物に、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、該抽出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 investigations aimed at solving the above problems, the present inventors have found an undifferentiated maintenance effect and a proliferation promoting effect specific to stem cells in the extract of the rice crabs belonging to the azalea family, It has been clarified that the engraftment rate in tissues when transplanted stem cells prepared using a culture solution containing the extract is actually transplanted has been completed, and the present invention has been completed.
即ち本発明は、以下のとおりである。
(1)ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出物を含有することを特徴とする、幹細胞の未分化維持剤。
(2)ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出物を含有することを特徴とする、幹細胞の増殖促進剤。
(3)ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出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)ツツジ科に属するコメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の抽出物を含有することを特徴とする、幹細胞培養用途の培地。
That is, the present invention is as follows.
(1) An agent for maintaining undifferentiation of stem cells, which contains an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the azalea family.
(2) A stem cell proliferation promoter characterized by containing an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the azalea family.
(3) A method for preparing a stem cell, which comprises culturing using a medium containing an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the family Azalea.
(4) Stem cells prepared using the stem cell preparation method according to (3).
(5) A medium for stem cell culture use, which contains an extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the azalea family.
以下、本発明をさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail.
本発明は、ツツジ科に属するコメバツガザクラの抽出物を用いることで、幹細胞の未分化状態を維持させたまま幹細胞のみを特異的に増殖させ、さらに、移植時における幹細胞の組織への生着率の向上をもたらすことが可能な幹細胞に対して特異的な未分化維持及び/又は増殖促進剤と該抽出物を含有する培養液を用いることで組織への生着率が極めて高い幹細胞とその調製方法を提供する。 The present invention uses an extract of the rice crabs belonging to the azalea family to specifically proliferate only stem cells while maintaining the undifferentiated state of the stem cells, and further increases the survival rate of the stem cells to the tissue at the time of transplantation. Stem cells having a very high engraftment ratio in tissues by using a culture solution containing an undifferentiated maintenance and / or growth promoter specific to stem cells capable of bringing about improvement and the extract, and a method for preparing the stem cells I will provide a.
本発明に用いるコメバツガザクラの抽出物とは、ツツジ科コメバツガザクラ属コメバツガザクラ(学名:Arcterica nana)(別名:ハマザクラ)の花、花穂、茎、葉、枝、枝葉、根、種子等の植物体の一部又は全草から抽出したものである。その抽出方法は特に限定されず、例えば、加熱抽出したものであっても良いし、常温抽出したものであっても良い。 The extract of the rice bran cherry used in the present invention is a part of a plant body such as a flower, an ear, a stem, a leaf, a branch, a branch leaf, a root, a seed, etc. Or it is extracted from whole grass. The extraction method is not particularly limited, and for example, it may be a heat extraction or a room temperature extraction.
抽出する溶媒としては、例えば、水、低級アルコール類(メタノール、エタノール、1−プロパノール、2−プロパノール、1−ブタノール、2−ブタノール等)、液状多価アルコール(1,3−ブチレングリコール、プロピレングリコール、グリセリン等)、ケトン類(アセトン、メチルエチルケトン等)、アセトニトリル、エステル類(酢酸エチル、酢酸ブチル等)、炭化水素類(ヘキサン、ヘプタン、流動パラフィン等)、エーテル類(エチルエーテル、テトラヒドロフラン、プロピルエーテル等)が挙げられる。好ましくは、水、低級アルコール及び液状多価アルコールが良く、特に好ましくは、水、エタノールが良い。これらの溶媒は1種でも2種以上を混合して用いても良い。また、抽出法は特に限定されないが、加熱による抽出が好ましい。さらに上記抽出溶媒に酸やアルカリを添加してpH調整して用いてもよい。 Examples of the solvent to be extracted include water, lower alcohols (methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, etc.), liquid polyhydric alcohols (1,3-butylene glycol, propylene glycol). , Glycerin, etc.), ketones (acetone, methyl ethyl ketone, etc.), acetonitrile, esters (ethyl acetate, butyl acetate, etc.), hydrocarbons (hexane, heptane, liquid paraffin, etc.), ethers (ethyl ether, tetrahydrofuran, propyl ether) Etc.). Preferably, water, lower alcohol and liquid polyhydric alcohol are preferable, and water and ethanol are particularly preferable. These solvents may be used alone or in combination of two or more. The extraction method is not particularly limited, but extraction by heating is preferable. Furthermore, the pH may be adjusted by adding an acid or alkali to the extraction solvent.
コメバツガザクラの抽出物は、抽出した溶液のまま用いても良く、必要に応じて、濃縮、稀釈、濾過等の処理及び活性炭等による脱色、脱臭処理をして用いても良い。特に、抽出した溶液を濃縮乾固、噴霧乾燥、凍結乾燥等の処理を行い、乾燥物として用いることが好ましい。 The extract of the rice bran cherry may be used as it is, or may be used after treatment such as concentration, dilution, filtration, and decolorization or deodorization with activated carbon as necessary. In particular, the extracted solution is preferably subjected to treatment such as concentration to dryness, spray drying, freeze drying, etc., and used as a dried product.
コメバツガザクラの抽出物を溶液の状態で用いる場合の成分含有量は特に限定されないが、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り、10重量%を超える場合は不経済である。 The component content in the case where the extract of rice bran cherry is used in a solution state is not particularly limited, but it is preferably 0.00001 to 10% by weight as a dried product, and used at a concentration of 0.0001 to 1% by weight. Most preferred. If it is less than 0.00001% by weight, the effects of the present invention may not be sufficiently exerted, and if it exceeds 10% by weight, it is uneconomical.
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抽出物や、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, an extract showing a specific undifferentiation maintenance effect and proliferation promoting effect on the stem cell of the present invention, a stem cell prepared thereby, a preparation method thereof, and the like, an additive for cell culture, a reagent for research, It can be formulated and applied to medical reagents, cell transplants, pharmaceuticals, quasi drugs, cosmetics, foods and the like.
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、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 cell of the present invention is an embryonic stem cell (ES cell) or bone marrow, blood, skin, fat, brain, liver, pancreas, kidney, muscle, and other tissues as long as the purpose of the present invention is met. In addition, somatic stem cells, 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、また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 (Nutrient Mixture F-12, D-MEM / F-12) Hank's balanced salt solution), basic fibroblast growth factor as a growth factor bFGF), 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%.
本発明のコメバツガザクラの抽出物は、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、このコメバツガザクラの抽出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 extract of the rice bran cherry of the present invention showed a very 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 using this extract of rice bran cherry were transplanted into a living tissue, the engraftment rate in the tissue was significantly improved as compared with the conventional method. 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コメバツガザクラの抽出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が、本発明はこれに限定されるものではない。 First, as an example, a production example of the extract of rice cracker cherry used in the present invention and an experimental example showing the effect of maintaining the undifferentiated state of stem cells are given, but the present invention is not limited thereto.
以下に、コメバツガザクラの抽出物の製造例を示す。 Below, the manufacture example of the extract of a rice bran cherry is shown.
製造例1 コメバツガザクラの熱水抽出物
コメバツガザクラ(Arcterica nana)の乾燥物20gに精製水400mLを加え、95〜100℃で2時間抽出した後、濾過し、その濾液を濃縮し、凍結乾燥してコメバツガザクラの熱水抽出物を1.0g得た。
Production Example 1 Hot Water Extract of Rice Butter Crab 400 mL of purified water was added to 20 g of dried product of Arcterica nana, extracted at 95-100 ° C. for 2 hours, filtered, the filtrate was concentrated, freeze-dried and dried 1.0 g of a hot water extract was obtained.
製造例2 コメバツガザクラの50%エタノール抽出物
コメバツガザクラ(Arcterica nana)の乾燥物20gに50(v/v)%エタノール水溶液400mLを加え、常温で7日間抽出した後、濾過し、その濾液を濃縮乾固して、コメバツガザクラの50%エタノール抽出物を1.5g得た。
Production Example 2 50% Ethanol Extract of Rice Blossom Cherry (Arterica nana) To 20 g of dried product, 400 mL of 50 (v / v)% ethanol aqueous solution was added, extracted at room temperature for 7 days, filtered, and the filtrate was concentrated to dryness. As a result, 1.5 g of a 50% ethanol extract of rice bran cherry was obtained.
製造例3 コメバツガザクラのエタノール抽出物
コメバツガザクラ(Arcterica nana)の乾燥物50gにエタノール1000mLを加え、常温で7日間抽出した後、濾過し、その濾液を濃縮乾固して、コメバツガザクラのエタノール抽出物を1.4g得た。
Manufacture example 3 Ethanol extract of rice cracker cherry 1000 mL of ethanol was added to 50 g of dried rice cake (Arctica nana), extracted at room temperature for 7 days, filtered, the filtrate was concentrated to dryness, and 1 ethanol extract of rice cracker cherry was added. .4 g was obtained.
以下に、実施例1で示した製造例1〜3のコメバツガザクラの抽出物を用いた、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 experimental example of the stem cell undifferentiated state maintenance effect and cell growth promotion effect using the extract of the rice bran cherry of the manufacture examples 1-3 shown in Example 1 and its result 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、コメバツガザクラの抽出物(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、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、コメバツガザクラの抽出物(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 seeds in a 6 cm dish and the final extract of rice bran cherry (Production Examples 1 to 3) The concentration was added to 0.001%, and 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. The amount of Oct3 / 4 protein was determined to be 100% undifferentiated, and the amount of Oct3 / 4 protein was quantitatively analyzed by Western blotting after adding the extract of rice bran cherry (Production Examples 1 to 3) and culturing for 3 days. The effect of maintaining the undifferentiated state was evaluated by comparing the amount of Oct3 / 4 protein at the start of culture and after 3 days of culture. 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)を測定し開始時の未分化状態(100%)とする。さらにコメバツガザクラの抽出物(製造例1〜3)を添加して3日間培養した後の単一細胞数(1×105個)に発現しているOct3/4タンパク質量(Bとする)を測定し、培養3日後の未分化状態(%)を次の式より算出した。培養3日後の未分化状態(%)の算出式=B/A×100(%)。この3日後の未分化状態(%)を指標に、未分化状態の維持効果について評価した。 As a specific evaluation method, the amount of Oct3 / 4 protein (referred to as A) expressed in the single cell number (1 × 10 5 ) of stem cells at the start of culture was measured by Western blotting, and then started. The undifferentiated state (100%). Further, the amount of Oct3 / 4 protein (referred to as B) expressed in the number of single cells (1 × 10 5 cells) after adding the extract of rice bran cherry (Production Examples 1 to 3) and culturing for 3 days was measured. The undifferentiated state (%) after 3 days of culture was calculated from the following formula. Calculation formula of undifferentiated state (%) after 3 days of culture = B / A × 100 (%). The maintenance effect of the undifferentiated state was evaluated using the undifferentiated state (%) after 3 days as an index.
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、コメバツガザクラの抽出物(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、コメバツガザクラの抽出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, compared with the positive control substance (bFGF), a remarkable stem cell undifferentiated state maintaining effect was observed in all the extracts of the rice bran cherry (Production Examples 1 to 3). From the above, the extremely excellent stem cell undifferentiated state maintaining effect of the extract of rice bran cherry 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コメバツガザクラの抽出物(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、コメバツガザクラの抽出物(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、コメバツガザクラの抽出物(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抽出物未添加時の総細胞数をコントロール(100%)とした場合の、コメバツガザクラの抽出物(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 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. The cell-specific growth-promoting effect of the extract of rice bran cherry (Production Examples 1 to 3) on these cells was evaluated using liver cells (HEPG2) and human somatic stem cells. That is, it was evaluated whether among these cells, the extract of rice bran cherry (Production Examples 1 to 3) specifically showed a growth promoting effect only on stem cells. Specifically, each of these cells is seeded at 1 × 10 6 cells in a 6 cm dish, and an extract of rice bran cherry (Production Examples 1 to 3) is added so that the final concentration is 0.001%, and then for 3 days. The culture was continued. 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.
When the total number of cells when no extract is added to each cell is taken as a control (100%), the increase / decrease (%) in the number of cells when the extract of rice bran cherry (Production Examples 1 to 3) is added. The cell proliferation promoting effect on each cell was calculated. The same evaluation was performed using a basic fibroblast growth factor (bFGF) (Japanese Patent Publication No. 2010-500047), which has been reported so far as a substance exhibiting an effect of promoting the proliferation of stem cells, as a positive control.
これらの試験結果を表2に示した。その結果、コメバツガザクラの抽出物(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、コメバツガザクラの抽出物は、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 extract of rice bran cherry (Production Examples 1 to 3) showed a significant cell growth promoting effect only on stem cells. 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. From the above, the extract of rice bran cherry showed a very excellent specific growth promoting effect on stem cells. 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コメバツガザクラの抽出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 tissues, stem cells prepared by conventional techniques and stem cells prepared by adding extract of rice bran cherry were actually transplanted, and the respective survival rate (%) after transplantation was compared. .
実験例4 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、コメバツガザクラの抽出物(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 4 Preparation of Stem Cells for Transplantation Human somatic stem cells (DS Pharma Biomedical) were seeded at 1 × 10 6 cells in a 6 cm dish, and the extract of rice bran cherry (Manufacturing Examples 1 to 3) had a final concentration of 0. It was added to 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5 幹細胞の移植および生着率(%)の測定
実験例4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強度が検出され、逆に、生着しなかった場合は、移植部位から蛍光強度が検出されないこととなる。
Experimental Example 5 Stem Cell Transplantation and Measurement of Engraftment Rate (%) The stem cells prepared in Experimental Example 4 were sampled 1 × 10 6 times again, and the number of cells was adjusted. 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 engrafted stem cells increases, the fluorescence intensity equivalent to the fluorescence intensity initially transplanted is detected. Conversely, if the engraftment does not occur, the fluorescence intensity is not detected from the transplanted 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コメバツガザクラの抽出物(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 showed a very high engraftment rate when stem cells prepared using the extract of rice bran cherry (Production Examples 1 to 3) were transplanted compared to stem cells prepared by conventional methods. It was.
以上の結果より、従来の方法で調製した幹細胞よりもコメバツガザクラの抽出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。 From the above results, it was confirmed that the engraftment rate of stem cells to the tissue was remarkably improved by preparing stem cells for transplantation using the extract of rice bran cherry rather than the stem cells prepared by the conventional method. 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、コメバツガザクラの抽出物を幹細胞に用いることで、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。 By using the extract of rice blossom cherry of the present invention as stem cells, it becomes possible to promote the growth of only stem cells while maintaining the undifferentiated state of the stem cells. Stem cells with a high adherence rate can be easily prepared.
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または組織に存在する幹細胞に対して、本発明のコメバツガザクラの抽出物を、組織に対して直接的に注入または経口投与、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、かつ該抽出物を用いることで組織への生着率の高い幹細胞の調製方法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 tissues as a use other than transplantation, the extract of the rice bran cherry of the present invention is directly introduced into the tissues by injection or oral administration, application, pasting, etc. It is possible to find an effect of maintaining a stem cell-specific undifferentiated state and an effect of promoting proliferation.
That is, the present invention provides applicability as an undifferentiated maintenance agent and / or growth promoter specific to stem cells in regenerative medicine and regenerative beauty, and the rate of engraftment in tissues by using the extract. It is a technology that enables a method for preparing high stem cells.
Claims (4)
A medium for use in stem cell culture, comprising a hot water extract, a water-containing ethanol extract or an ethanol extract of rice blossom cherry (Scientific name: Arcterica nana) (aka: Hamazakura) belonging to the azalea family.
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