JP6735887B2 - Cell senescence inhibitor of mesenchymal stem cells, skin external composition for cell senescence inhibition of mesenchymal stem cells, functional food and drink for cell senescence inhibition of mesenchymal stem cells, and method for inhibiting cell senescence of mesenchymal stem cells - Google Patents
Cell senescence inhibitor of mesenchymal stem cells, skin external composition for cell senescence inhibition of mesenchymal stem cells, functional food and drink for cell senescence inhibition of mesenchymal stem cells, and method for inhibiting cell senescence of mesenchymal stem cells Download PDFInfo
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- JP6735887B2 JP6735887B2 JP2019151772A JP2019151772A JP6735887B2 JP 6735887 B2 JP6735887 B2 JP 6735887B2 JP 2019151772 A JP2019151772 A JP 2019151772A JP 2019151772 A JP2019151772 A JP 2019151772A JP 6735887 B2 JP6735887 B2 JP 6735887B2
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Description
本開示は、幹細胞の細胞老化抑制剤、幹細胞の細胞老化抑制用皮膚外用組成物、幹細胞の細胞老化抑制用機能性飲食品、及び幹細胞の細胞老化抑制方法に関する。 The present disclosure relates to a stem cell cell aging inhibitor, a skin external composition for stem cell cell aging inhibition, a functional food and drink for stem cell cell aging inhibition, and a stem cell cell aging inhibition method.
細胞老化（cellular senescence）とは、細胞に不可逆的な細胞周期の停止が起こる現象である。例えば、細胞***に伴うＤＮＡ複製エラーの蓄積、酸化ストレス、放射線、がん遺伝子の活性化等によりＤＮＡに損傷が生じると、ＤＮＡ損傷応答（ＤＤＲ；DNA Damage Response）が活性化される。このＤＮＡ損傷応答により、細胞***に関与するサイクリンキナーゼの阻害因子であるｐ１６INK4a、ｐ２１CIP1、ｐ５３等の発現が亢進し、細胞周期の停止が引き起こされる。ＤＮＡの損傷の程度が小さければ細胞は再び細胞周期を復帰させ増殖するが、損傷の程度が大きいと細胞周期が不可逆的に停止し、細胞老化へと至る。細胞老化を起こした細胞（老化細胞）は、老化関連酸性β-ガラクトシダーゼ（SA β-Gal；Senescence Associated β-Galactosidase）活性が向上することが知られている。 Cellular senescence is a phenomenon in which cells undergo irreversible cell cycle arrest. For example, when DNA damage occurs due to accumulation of DNA replication errors associated with cell division, oxidative stress, radiation, activation of oncogenes, etc., a DNA damage response (DDR) is activated. This DNA damage response enhances the expression of p16 INK4a , p21 CIP1 , p53, etc., which are inhibitors of cyclin kinase involved in cell division, and causes cell cycle arrest. If the degree of DNA damage is small, the cells return to the cell cycle to proliferate, but if the degree of damage is large, the cell cycle is irreversibly stopped, leading to cell senescence. It is known that cells that have undergone cellular senescence (senescent cells) have improved activity of acid β-galactosidase (SA β-Gal; Senescence Associated β-Galactosidase).
老化細胞は、単に細胞周期を停止しているだけでなく、炎症性サイトカイン、炎症性ケモカイン、及びＭＭＰ（Matrix MetalloProteinase）のほか、ＩＧＦ（Insulin-like Growth Factor）に結合して細胞老化を誘引するＩＧＦＢＰ（IGF Binding Protein）−４、ＩＧＦＢＰ−７などを分泌し、周囲環境に悪影響を与えることが知られている。このような現象は、ＳＡＳＰ（Senescence-Associated Secretory Phenotype）とも称される。 Senescent cells not only arrest the cell cycle, but also bind to inflammatory cytokines, chemokines, MMPs (Matrix MetalloProteinase), and IGF (Insulin-like Growth Factor) to induce cell senescence. It is known that it secretes IGFBP (IGF Binding Protein)-4, IGFBP-7, etc., and adversely affects the surrounding environment. Such a phenomenon is also called SASP (Senescence-Associated Secretory Phenotype).
ところで、幹細胞は、細胞***により同一の細胞を産生する自己複製能と、様々な細胞に分化する多分化能とを併せ持つ細胞である。受精卵が代表的な幹細胞であるが、成人の組織にも組織幹細胞が存在し、組織の恒常性維持に寄与している。例えば、皮膚組織の真皮層には間葉系幹細胞が存在しており、真皮層に豊富に含まれるコラーゲン等の細胞外マトリクスを産生する線維芽細胞へと分化する。 By the way, a stem cell is a cell having both the self-renewal ability of producing the same cell by cell division and the pluripotency of differentiating into various cells. Fertilized eggs are a typical stem cell, but tissue stem cells are also present in adult tissues and contribute to the maintenance of tissue homeostasis. For example, mesenchymal stem cells are present in the dermal layer of skin tissue, and differentiate into fibroblasts that produce extracellular matrix such as collagen, which is abundantly contained in the dermal layer.
近年、幹細胞も細胞老化を起こすことが明らかとなり、組織の再生を担う幹細胞の細胞老化が個体老化の本質であるという考え（ステムセルエイジング）が提唱されるに至っている。 In recent years, it has become clear that stem cells also undergo cell senescence, and the idea that stem cell aging responsible for tissue regeneration is the essence of individual aging (stem cell aging) has been proposed.
また、間葉系幹細胞等の幹細胞は、細胞療法に用いるバイオツールとしても注目されている。幹細胞は、骨髄、脂肪組織等から採取することができるものの、細胞療法を実施するために充分な数の幹細胞を直接採取することは困難である。そのため、採取した幹細胞をin vitroで培養し、増殖させる必要がある。しかし、幹細胞の継代を繰り返すと、幹細胞の細胞老化が引き起こされ、細胞療法の治療効果が低減することがある。 In addition, stem cells such as mesenchymal stem cells have attracted attention as biotools used for cell therapy. Although stem cells can be collected from bone marrow, adipose tissue, etc., it is difficult to directly collect a sufficient number of stem cells to carry out cell therapy. Therefore, it is necessary to culture and proliferate the collected stem cells in vitro. However, repeated passage of stem cells may induce stem cell senescence and reduce the therapeutic effect of cell therapy.
このような背景から、近年、幹細胞の細胞老化を抑制するための技術が盛んに研究されている。 Against this background, in recent years, techniques for suppressing cell senescence of stem cells have been actively studied.
例えば、特許文献１には、Ｎ−アセチルシステインの存在下で、ＡＴＭ（Ataxia Telangiectasia Mutated）ホモ欠損マウス由来の造血幹細胞を培養することにより、造血幹細胞内の活性酸素量が低下し、自己複製能が回復することが示されている。
特許文献２には、抗酸化剤及びＦＧＦ（Fibroblast Growth Factor）−２の存在下で間葉系幹細胞を培養することにより、間葉系幹細胞の細胞増殖が亢進し、また、間葉系幹細胞の分化潜在能力が高まることが示されている。
特許文献３には、リゾホスファチジン酸（ＬＰＡ；LysoPhosphatidic Acid）受容体アンタゴニストの存在下で幹細胞を培養することにより、幹細胞の機能を保持したまま、***回数が向上することが示されている。
For example, in Patent Document 1, by culturing hematopoietic stem cells derived from an ATM (Ataxia Telangiectasia Mutated) homo-deficient mouse in the presence of N-acetyl cysteine, the amount of active oxygen in the hematopoietic stem cells is decreased and the self-renewal ability is reduced. Have been shown to recover.
In Patent Document 2, by culturing the mesenchymal stem cells in the presence of an antioxidant and FGF (Fibroblast Growth Factor)-2, cell proliferation of the mesenchymal stem cells is promoted, and Increased differentiation potential has been shown.
Patent Document 3 shows that by culturing stem cells in the presence of a lysophosphatidic acid (LPA; LysoPhosphatidic Acid) receptor antagonist, the number of divisions is improved while maintaining the function of the stem cells.
しかし、特許文献１で使用されているＮ−アセチルシステインについて本発明者らが確認したところ、正常ヒト幹細胞の細胞老化が抑制され難いことが判明した。
また、特許文献２で使用されているＦＧＦ−２は安定性に劣り、また、皮膚外用組成物等への応用が困難であると考えられる。
また、特許文献３で使用されているＬＰＡ受容体アンタゴニストは、広範な機能を有する脂質メディエーターを阻害するものであるため、細胞老化抑制以外の悪影響が懸念される。
However, when the present inventors confirmed the N-acetylcysteine used in Patent Document 1, it was found that cell senescence of normal human stem cells was difficult to be suppressed.
Further, FGF-2 used in Patent Document 2 is inferior in stability, and it is considered that it is difficult to apply it to a composition for external use on the skin.
Further, since the LPA receptor antagonist used in Patent Document 3 inhibits a lipid mediator having a wide range of functions, there is a concern that it may have adverse effects other than suppression of cell aging.
本開示は、上記事情に鑑み、幹細胞の細胞老化を効果的に抑制可能な新規な幹細胞の細胞老化抑制剤を提供することを課題とする。また、本開示は、その幹細胞の細胞老化抑制剤を用いた幹細胞の細胞老化抑制用皮膚外用組成物、幹細胞の細胞老化抑制用機能性飲食品、及び幹細胞の細胞老化抑制方法を提供することを課題とする。 In view of the above circumstances, it is an object of the present disclosure to provide a novel stem cell cell aging inhibitor capable of effectively suppressing stem cell cell senescence. Further, the present disclosure provides a skin external composition for suppressing cell aging of stem cells using the cell aging inhibitor for stem cells, a functional food or drink for suppressing cell aging of stem cells, and a method for suppressing cell aging of stem cells. It is an issue.
上記課題を解決するための具体的手段には、以下の態様が含まれる。
＜１＞ アスタキサンチンを有効成分として含有する幹細胞の細胞老化抑制剤。
＜２＞ 幹細胞が間葉系幹細胞である＜１＞に記載の幹細胞の細胞老化抑制剤。
＜３＞ さらに、トコフェロール類を有効成分として含有する＜１＞又は＜２＞に記載の幹細胞の細胞老化抑制剤。
＜４＞ トコフェロール類がニコチン酸トコフェロールを含む＜３＞に記載の幹細胞の細胞老化抑制剤。
＜５＞ ｐ１６、ｐ２１、γＨ２ＡＸ、ＩＧＦＢＰ−４、及びＩＧＦＢＰ−７からなる群より選択される少なくとも１種のタンパク質の発現を抑制する＜１＞〜＜４＞のいずれか１つに記載の幹細胞の細胞老化抑制剤。
＜６＞ ｐ１６及びｐ２１のタンパク質の発現を抑制する＜５＞に記載の幹細胞の細胞老化抑制剤。
＜７＞ ＜１＞〜＜６＞のいずれか１つに記載の幹細胞の細胞老化抑制剤を含有する幹細胞の細胞老化抑制用皮膚外用組成物。
＜８＞ ＜１＞〜＜６＞のいずれか１つに記載の幹細胞の細胞老化抑制剤を含有する幹細胞の細胞老化抑制用機能性飲食品。
＜９＞ ＜１＞〜＜６＞のいずれか１つに記載の幹細胞の細胞老化抑制剤の存在下で幹細胞を培養することを含む幹細胞の細胞老化抑制方法。
Specific means for solving the above problems include the following aspects.
<1> A stem cell cell senescence inhibitor containing astaxanthin as an active ingredient.
<2> The stem cell cell aging inhibitor according to <1>, wherein the stem cells are mesenchymal stem cells.
<3> The stem cell cell aging inhibitor according to <1> or <2>, which further contains tocopherols as an active ingredient.
<4> The cell senescence inhibitor for stem cells according to <3>, wherein the tocopherols include tocopherol nicotinate.
<5> The stem cell according to any one of <1> to <4>, which suppresses the expression of at least one protein selected from the group consisting of p16, p21, γH2AX, IGFBP-4, and IGFBP-7. Cell aging inhibitor.
<6> The agent for suppressing cell senescence of stem cells according to <5>, which suppresses the expression of p16 and p21 proteins.
<7> A skin external composition for suppressing cell senescence of stem cells, which comprises the cell senescence inhibitor for stem cells according to any one of <1> to <6>.
<8> A functional food or drink for suppressing cell aging of stem cells, which comprises the agent for suppressing cell aging of stem cells according to any one of <1> to <6>.
<9> A method for suppressing the senescence of stem cells, which comprises culturing the stem cells in the presence of the agent for suppressing senescence of stem cells according to any one of <1> to <6>.
本開示によれば、幹細胞の細胞老化を効果的に抑制可能な新規な幹細胞の細胞老化抑制剤を提供することができる。また、本開示によれば、その幹細胞の細胞老化抑制剤を用いた幹細胞の細胞老化抑制用皮膚外用組成物、幹細胞の細胞老化抑制用機能性飲食品、及び幹細胞の細胞老化抑制方法を提供することができる。 According to the present disclosure, it is possible to provide a novel stem cell cell aging inhibitor capable of effectively suppressing stem cell cell senescence. Further, according to the present disclosure, there is provided a skin external composition for suppressing cell aging of stem cells using the cell aging inhibitor for stem cells, a functional food or drink for suppressing cell aging of stem cells, and a method for suppressing cell aging of stem cells. be able to.
以下、本開示の幹細胞の細胞老化抑制剤、幹細胞の細胞老化抑制用皮膚外用組成物、幹細胞の細胞老化抑制用機能性飲食品、及び幹細胞の細胞老化抑制方法について詳細に説明する。但し、本発明は、以下の実施形態に何ら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。 Hereinafter, the agent for suppressing cell senescence of stem cells, the external composition for suppressing cell senescence of stem cells, the functional food and drink for suppressing cell senescence of stem cells, and the method for suppressing cell senescence of stem cells of the present disclosure will be described in detail. However, the present invention is not limited to the following embodiments and can be implemented with appropriate modifications within the scope of the object of the present invention.
本明細書において「〜」を用いて示された数値範囲は、「〜」の前後に記載される数値をそれぞれ最小値及び最大値として含む範囲を意味する。
本明細書中に段階的に記載されている数値範囲において、ある数値範囲で記載された上限値又は下限値は、他の段階的な記載の数値範囲の上限値又は下限値に置き換えてもよい。また、本明細書中に記載されている数値範囲において、ある数値範囲で記載された上限値又は下限値は、実施例に示されている値に置き換えてもよい。
本明細書において各成分の濃度又は含有率は、各成分に該当する物質が複数種存在する場合には、特に断らない限り、複数種の物質の合計の濃度又は含有率を意味する。
In the present specification, the numerical range indicated by using "to" means a range including the numerical values before and after "to" as the minimum value and the maximum value, respectively.
In the numerical ranges described stepwise in the present specification, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the upper limit value or the lower limit value of another stepwise described numerical range. .. Further, in the numerical ranges described in this specification, the upper limit value or the lower limit value described in a certain numerical range may be replaced with the values shown in the examples.
In the present specification, the concentration or content rate of each component means the total concentration or content rate of the plurality of types of substances, unless there is a plurality of substances corresponding to each component.
＜幹細胞の細胞老化抑制剤＞
本開示の幹細胞の細胞老化抑制剤（以下、単に「細胞老化抑制剤」ともいう。）は、アスタキサンチンを有効成分として含有する。本開示の細胞老化抑制剤によれば、幹細胞の細胞老化を効果的に抑制することができる。すなわち、本開示によれば、幹細胞の細胞老化を抑制するためのアスタキサンチンの使用もまた提供される。
<Stem cell aging inhibitor>
The cell senescence inhibitor of stem cells of the present disclosure (hereinafter, also simply referred to as “cell senescence inhibitor”) contains astaxanthin as an active ingredient. According to the cell aging inhibitor of the present disclosure, cell senescence of stem cells can be effectively suppressed. That is, according to the present disclosure, use of astaxanthin for suppressing cell senescence of stem cells is also provided.
上述したとおり、細胞老化には、酸化ストレスが関与することが知られている。酸化ストレスの関与は、例えば、培養細胞を過酸化水素で処理すると細胞老化が促進されることからも示唆されている（Chen, Q.M. et al., Biochem. J., 332, pp.43-50 (1998)）。
一方、アスタキサンチン等のカロテノイド類は、活性酸素又はフリーラジカルのうち、一重項酸素及び脂質ラジカルに対しては強い消去能を有するものの、スーパーオキシドアニオンラジカル、過酸化水素、及びヒドロキシラジカルの消去能は殆ど有しないことが知られている（眞岡孝至、食品・臨床栄養、第２巻、３頁−１４頁（２００７））。
しかし、本発明者らの実験によって、過酸化水素によって幹細胞に酸化ストレスを与えた場合の細胞老化が、アスタキサンチンによって抑制されることが明らかとなった。アスタキサンチンがこのような細胞老化抑制効果を奏することは驚くべきことである。
As described above, it is known that oxidative stress is involved in cell aging. The involvement of oxidative stress has also been suggested, for example, by accelerating cell senescence when treating cultured cells with hydrogen peroxide (Chen, QM et al., Biochem. J., 332, pp.43-50. (1998)).
On the other hand, carotenoids such as astaxanthin have strong scavenging ability for singlet oxygen and lipid radicals among active oxygen or free radicals, but have scavenging ability for superoxide anion radicals, hydrogen peroxide, and hydroxy radicals. It is known that they do not have much (Koji Maoka, Food and Clinical Nutrition, Volume 2, pp. 14-14 (2007)).
However, experiments by the present inventors have revealed that astaxanthin suppresses cell senescence when oxidative stress is applied to stem cells by hydrogen peroxide. It is surprising that astaxanthin has such a cell aging inhibitory effect.
細胞老化の抑制対象となる幹細胞の種類は特に制限されない。幹細胞としては、体性幹細胞（間葉系幹細胞、肺幹細胞、骨髄幹細胞、上皮幹細胞、神経幹細胞、肝幹細胞、膵幹細胞、造血幹細胞、筋幹細胞、生殖幹細胞、毛母幹細胞、毛包幹細胞、色素幹細胞等）、胚性幹細胞（embryonic stem cell；ＥＳ細胞）、人工多能性幹細胞（induced pluripotent stem cell；ｉＰＳ細胞）などが挙げられる。ある実施態様では、細胞老化の抑制対象となる幹細胞は間葉系幹細胞である。 The type of stem cells targeted for suppression of cell aging is not particularly limited. Stem cells include somatic stem cells (mesenchymal stem cells, lung stem cells, bone marrow stem cells, epithelial stem cells, neural stem cells, hepatic stem cells, pancreatic stem cells, hematopoietic stem cells, muscle stem cells, reproductive stem cells, hair matrix stem cells, hair follicle stem cells, pigment stem cells Etc.), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like. In one embodiment, the stem cells targeted for suppression of cell senescence are mesenchymal stem cells.
本開示の細胞老化抑制剤を後述する皮膚外用組成物又は機能性飲食品に適用する場合、細胞老化の抑制対象となる幹細胞はヒトの幹細胞であることが好ましい。
一方、本開示の細胞老化抑制剤を後述する細胞老化抑制方法に適用する場合、細胞老化の抑制対象となる幹細胞の由来は特に制限されない。幹細胞の由来生物としては、ヒト、マウス、ラット、ウサギ等の哺乳動物が挙げられる。培養した幹細胞を細胞療法に用いる場合には、幹細胞は、治療対象のヒト（患者）由来の幹細胞であることが好ましい。
When the cell aging inhibitor of the present disclosure is applied to the external composition for skin or functional food and drink described below, the stem cells targeted for cell aging inhibition are preferably human stem cells.
On the other hand, when the cell aging inhibitor of the present disclosure is applied to the cell aging inhibition method described later, the origin of stem cells to be the target of cell aging inhibition is not particularly limited. Examples of stem cell-derived organisms include mammals such as humans, mice, rats, and rabbits. When the cultured stem cells are used for cell therapy, the stem cells are preferably human (patient)-derived stem cells to be treated.
本開示の細胞老化抑制剤による細胞老化抑制効果は、公知の分子マーカーにより確認することができる。
本開示の細胞老化抑制剤としては、ｐ１６、ｐ２１、γＨ２ＡＸ、ＩＧＦＢＰ−４、及びＩＧＦＢＰ−７からなる群より選択される少なくとも１種のタンパク質の発現を抑制するものが好ましい。ｐ１６及びｐ２１は、細胞***に関与するサイクリンキナーゼの阻害因子として知られている。γＨ２ＡＸは、ヒストンＨ２ＡＸのアミノ酸配列の１３９番目のセリン残基がリン酸化されたものであり、細胞老化を引き起こすＤＮＡ損傷の分子マーカーとして知られている。ＩＧＦＢＰ−４及びＩＧＦＢＰ−７は、ＩＧＦに結合し、細胞老化を誘引することが知られている。特に、本開示の細胞老化抑制剤としては、ｐ１６及びｐ２１のタンパク質の発現を抑制するものがより好ましい。細胞老化の経路のうちｐ１６が関与する経路とｐ２１が関与する経路との両者を抑制することにより、幹細胞の細胞老化をより効果的に抑制することができる。
The cell aging inhibitory effect of the cell aging inhibitor of the present disclosure can be confirmed by known molecular markers.
As the cell aging inhibitor of the present disclosure, those that suppress the expression of at least one protein selected from the group consisting of p16, p21, γH2AX, IGFBP-4, and IGFBP-7 are preferable. p16 and p21 are known as inhibitors of cyclin kinase involved in cell division. γH2AX is phosphorylated at the 139th serine residue in the amino acid sequence of histone H2AX, and is known as a molecular marker for DNA damage that causes cell senescence. IGFBP-4 and IGFBP-7 are known to bind to IGF and induce cell senescence. In particular, as the cell aging inhibitor of the present disclosure, those that suppress the expression of p16 and p21 proteins are more preferable. By suppressing both the p16-related pathway and the p21-related pathway among the cellular senescence pathways, the cellular senescence of stem cells can be more effectively suppressed.
（アスタキサンチン）
本開示の細胞老化抑制剤は、アスタキサンチンを有効成分として含有する。
アスタキサンチンは、化学名：３，３’−ジヒドロキシ−β，β−カロテン−４，４’−ジオンで表される化合物である。アスタキサンチンは、３位及び３’位のヒドロキシ基の立体配置により、（３Ｒ，３’Ｒ）体、（３Ｒ，３’Ｓ）体、及び（３Ｓ，３’Ｓ）体の３種の異性体が存在し、さらに分子内の共役二重結合の存在によりシス体及びトランス体の異性体も存在する。本開示の細胞老化抑制剤に含有されるアスタキサンチンは、これら異性体のいずれであってもよく、複数種の異性体の混合物であってもよい。
また、アスタキサンチンは、３位及び３’位のヒドロキシ基の少なくとも一方が脂肪酸等とエステルを形成したエステル体であってもよく、複数種のエステル体の混合物であってもよい。
本開示では、特に断らない限り、アスタキサンチンのエステル体を含めて「アスタキサンチン」と総称する。
(Astaxanthin)
The cell aging inhibitor of the present disclosure contains astaxanthin as an active ingredient.
Astaxanthin is a compound represented by the chemical name: 3,3′-dihydroxy-β,β-carotene-4,4′-dione. Astaxanthin is a three isomer of (3R,3'R) isomer, (3R,3'S) isomer, and (3S,3'S) isomer, depending on the configuration of hydroxy groups at the 3 and 3'positions. And cis and trans isomers also exist due to the presence of conjugated double bonds in the molecule. Astaxanthin contained in the cell aging inhibitor of the present disclosure may be any of these isomers, or may be a mixture of a plurality of isomers.
Further, astaxanthin may be an ester form in which at least one of the 3-position and 3'-position hydroxy groups forms an ester with a fatty acid or the like, or may be a mixture of plural kinds of ester forms.
In the present disclosure, unless otherwise specified, the ester form of astaxanthin is collectively referred to as “astaxanthin”.
アスタキサンチンは、植物類、藻類、甲殻類、バクテリア等の天然物から精製して得られるものであってもよく、合成により得られるものであってもよい。アスタキサンチンは、Enzo Life Sciences社等から市販品を入手することもできる。 Astaxanthin may be obtained by purifying natural products such as plants, algae, crustaceans, and bacteria, or may be obtained by synthesis. Astaxanthin can also be obtained as a commercial product from Enzo Life Sciences.
また、アスタキサンチンは、アスタキサンチンを含有する天然物からの抽出物として、本開示の細胞老化抑制剤中に含有されていてもよい。アスタキサンチンを含有する抽出物としては、ヘマトコッカス藻抽出物、オキアミ抽出物、ファフィア酵母抽出物等が挙げられる。これらの中でも、アスタキサンチンの安定性等の観点から、ヘマトコッカス藻抽出物が好ましい。 In addition, astaxanthin may be contained in the cell aging inhibitor of the present disclosure as an extract from a natural product containing astaxanthin. Examples of the extract containing astaxanthin include Haematococcus alga extract, krill extract, and Phaffia yeast extract. Among these, the Haematococcus alga extract is preferable from the viewpoint of stability of astaxanthin.
ヘマトコッカス藻抽出物の原料としては、具体的には、ヘマトコッカス・プルビアリス（Haematococcus pluvialis）、ヘマトコッカス・ラキュストリス（Haematococcus lacustris）、ヘマトコッカス・カペンシス（Haematococcus capensis）、ヘマトコッカス・ドロエバケンシス（Haematococcus droebakensis）、ヘマトコッカス・ジンバビエンシス（Haematococcus zimbabwiensis）等が挙げられる。 As a raw material of the Haematococcus alga extract, specifically, Haematococcus pluvialis, Haematococcus lacustris, Haematococcus capensis, Haematococcus droebenaeto. , Haematococcus zimbabwiensis and the like.
ヘマトコッカス藻抽出物は、上記の原料を、必要に応じて細胞壁を破砕して、アセトン、エーテル、クロロホルム、アルコール等の有機溶剤、又は超臨界二酸化炭素等の抽出媒体を加えて抽出することによって得ることができる。 The Haematococcus alga extract is obtained by extracting the above raw materials by crushing the cell wall as necessary, adding acetone, ether, chloroform, an organic solvent such as alcohol, or an extraction medium such as supercritical carbon dioxide. Obtainable.
また、ヘマトコッカス藻抽出物は、市販品を用いることもできる。ヘマトコッカス藻抽出物の市販品としては、富士フイルム（株）のＡＳＴＯＴＳ（登録商標）−ＳＳ、ＡＳＴＯＴＳ（登録商標）−Ｓ、ＡＳＴＯＴＳ（登録商標）−１０ＯＳ、ＡＳＴＯＴＳ（登録商標）−５ＯＳ、ＡＳＴＯＴＳ（登録商標）−１０Ｏ、ＡＳＴＯＴＳ（登録商標）−５Ｏ、ＡＳＴＯＴＳ（登録商標）−２．０ＰＷ、ＡＳＴＯＴＳ（登録商標）−３．０ＭＢ等；アスタリール（株）のアスタリール（登録商標）オイル５０Ｆ、アスタリール（登録商標）オイル５Ｆ、アスタリール（登録商標）パウダー２０Ｆ等；東洋酵素化学（株）のＢｉｏＡｓｔｉｎ（登録商標） ＳＣＥ７、ＢｉｏＡｓｔｉｎ（登録商標） ＳＣＥ１０等；Algatechnologies社のＡｓｔａ Ｐｕｒｅ；などが挙げられる。 Moreover, a commercial item can also be used for a Haematococcus alga extract. As commercially available products of the Haematococcus algae extract, there are ASTOTS (registered trademark)-SS, ASTOTS (registered trademark)-S, ASTOTS (registered trademark)-10OS, ASTOTS (registered trademark)-5OS, and ASTOTS manufactured by FUJIFILM Corporation. (Registered trademark)-10O, ASTOTS (registered trademark)-5O, ASTOTS (registered trademark)-2.0PW, ASTOTS (registered trademark)-3.0MB and the like; Astaryl (registered trademark) oil 50F of Astaryl Co., Ltd. , Astaryl (registered trademark) oil 5F, Astaryl (registered trademark) powder 20F and the like; BioAstin (registered trademark) SCE7, BioAstin (registered trademark) SCE10 and the like of Toyo Enzyme Co., Ltd.; Asta Pure; Can be mentioned.
ヘマトコッカス藻抽出物中のアスタキサチンの色素純分としての含有率は、例えば、０．００１質量％〜６０質量％であることが好ましく、０．０１質量％〜５０質量％であることがより好ましい。
また、ヘマトコッカス藻抽出物中のアスタキサチンの合計に占めるエステル体の割合は、例えば、５０モル％以上であることが好ましく、７５モル％以上であることがより好ましく、９０％モル以上であることがさらに好ましい。
The content of astaxanthin in the Haematococcus alga extract as a pigment pure content is, for example, preferably 0.001% by mass to 60% by mass, and more preferably 0.01% by mass to 50% by mass. ..
Further, the proportion of the ester form in the total of astaxanthin in the Haematococcus alga extract is, for example, preferably 50 mol% or more, more preferably 75 mol% or more, and more preferably 90% mol or more. Is more preferable.
（トコフェロール類）
本開示の細胞老化抑制剤は、さらに、トコフェロール類を有効成分として含有していてもよい。本開示の細胞老化抑制剤がトコフェロール類を含有することにより、幹細胞の細胞老化をより効果的に抑制することができる傾向にある。
(Tocopherols)
The cell aging inhibitor of the present disclosure may further contain tocopherols as an active ingredient. When the cell aging inhibitor of the present disclosure contains tocopherols, there is a tendency that cell aging of stem cells can be more effectively suppressed.
トコフェロール類としては、特に制限されず、トコフェロール；酢酸トコフェロール、ニコチン酸トコフェロール、リノール酸トコフェロール、オレイン酸トコフェロール、コハク酸トコフェロール等のトコフェロール誘導体；トコトリエノール；酢酸トコトリエノール等のトコトリエノール誘導体；などが挙げられる。本開示の細胞老化抑制剤は、トコフェロール類を１種単独で含有していてもよく、２種以上を組み合わせて含有していてもよい。 The tocopherols are not particularly limited and include tocopherol acetate; tocopherol derivatives such as tocopherol acetate, tocopherol nicotinate, tocopherol linoleate, tocopherol oleate and tocopherol succinate; tocotrienols; tocotrienol derivatives such as tocotrienol acetate; and the like. The cell aging inhibitor of the present disclosure may contain one type of tocopherol alone, or may contain two or more types in combination.
本開示の細胞老化抑制剤は、これらのトコフェロール類の中でも、ニコチン酸トコフェロールを含有することが好ましい。ニコチン酸トコフェロールは、細胞老化の経路のうちｐ１６が関与する経路を抑制する効果に優れている。一方、アスタキサンチンは、細胞老化の経路のうちｐ２１が関与する経路を抑制する効果に優れている。このため、本開示の細胞老化抑制剤がアスタキサンチンとニコチン酸トコフェロールとの両者を含有することにより、幹細胞の細胞老化をより効果的に抑制することができる。 Among these tocopherols, the cell aging inhibitor of the present disclosure preferably contains tocopherol nicotinate. Tocopherol nicotinate is excellent in the effect of suppressing the pathway involving p16 in the cellular aging pathway. On the other hand, astaxanthin is excellent in the effect of suppressing the p21-related pathway among the cellular senescence pathways. Therefore, when the cell aging inhibitor of the present disclosure contains both astaxanthin and tocopherol nicotinate, cell aging of stem cells can be more effectively suppressed.
アスタキサンチンの含有量に対するトコフェロール類（好ましくはニコチン酸トコフェロール）の含有量の割合（トコフェロール類の含有量／アスタキサンチンの含有量）は特に制限されず、例えば、質量基準で０．０００１〜１００００であることが好ましく、０．００１〜１０００であることがより好ましい。 The ratio of the content of tocopherols (preferably tocopherol nicotinate) to the content of astaxanthin (content of tocopherols/content of astaxanthin) is not particularly limited, and is, for example, 0.0001 to 10,000 on a mass basis. Is preferable, and 0.001 to 1000 is more preferable.
（その他の成分）
本開示の細胞老化抑制剤は、必要に応じて、アスタキサンチン及びトコフェロール類以外のその他の成分を含有していてもよい。その他の成分としては、公知の抗酸化剤（アスコルビン酸及びその誘導体等）などが挙げられる。
(Other ingredients)
The cell aging inhibitor of the present disclosure may contain other components other than astaxanthin and tocopherols, if necessary. Examples of other components include known antioxidants (ascorbic acid and its derivatives) and the like.
＜幹細胞の細胞老化抑制用皮膚外用組成物＞
本開示の幹細胞の細胞老化抑制用皮膚外用組成物（以下、単に「皮膚外用組成物」ともいう。）は、本開示の細胞老化抑制剤を含有する。
<External composition for suppressing cell aging of stem cells>
The external composition for suppressing cell aging of stem cells of the present disclosure (hereinafter, also simply referred to as “skin external composition”) contains the cell aging inhibitor of the present disclosure.
本開示の皮膚外用組成物中における細胞老化抑制剤の含有率は、幹細胞の細胞老化抑制効果が奏される限り特に制限されない。本開示の皮膚外用組成物中におけるアスタキサンチンの含有率は、例えば、０．０００００１質量％〜０．２質量％であることが好ましく、０．０００００５質量％〜０．１質量％であることがより好ましく、０．００００１質量％〜０．０５質量％であることがさらに好ましい。 The content of the cell aging inhibitor in the external composition for skin of the present disclosure is not particularly limited as long as the cell aging inhibitory effect of stem cells is exhibited. The content of astaxanthin in the external composition for skin of the present disclosure is, for example, preferably 0.000001% by mass to 0.2% by mass, and more preferably 0.000005% by mass to 0.1% by mass. It is more preferably 0.00001% by mass to 0.05% by mass.
本開示の皮膚外用組成物は、本開示の細胞老化抑制剤を含有することにより、幹細胞の細胞老化抑制効果を発揮する。 The external composition for skin of the present disclosure exhibits a cell aging inhibitory effect of stem cells by containing the cell aging inhibitor of the present disclosure.
例えば、本開示の皮膚外用組成物を顔等の皮膚に適用し、皮膚に存在する間葉系幹細胞の細胞老化を抑制することで、皮膚の老化に伴うシワ、たるみ等の改善を期待し得る。
この点について本発明者らは以下のように考えている。
一般的な皮膚科学においては、ケラチノサイト等の表皮細胞に着目した細胞老化抑制の研究が進められている。これに対し、本発明者らは、幹細胞に着目した細胞老化抑制の研究を進めた結果、驚くべきことに、幹細胞の細胞老化を抑制することが表皮細胞の細胞老化を抑制することよりも効果的であることを見出した。幹細胞は、表皮細胞よりもターンオーバーの遅い細胞であることから、幹細胞の細胞老化を抑制することで、より効果的に皮膚の老化を抑制できるものと考えられる。
For example, by applying the external composition for skin of the present disclosure to the skin such as the face, by suppressing the cellular senescence of mesenchymal stem cells present in the skin, it is expected to improve wrinkles, sagging, etc. accompanying aging of the skin. ..
The present inventors consider this point as follows.
In general dermatology, research on cell senescence suppression focusing on epidermal cells such as keratinocytes is under way. On the other hand, the present inventors, as a result of advancing research on cell aging suppression focusing on stem cells, surprisingly, suppressing cell senescence of stem cells is more effective than suppressing cell senescence of epidermal cells. Found to be Since stem cells are cells that have a slower turnover than epidermal cells, it is considered that suppressing cell senescence of stem cells enables more effective suppression of skin aging.
また、本開示の皮膚外用組成物を頭皮に適用し、頭皮に存在する毛母幹細胞、毛包幹細胞等の細胞老化を抑制することで、脱毛又は薄毛の治療又は予防も期待し得る。 Further, by applying the external composition for skin of the present disclosure to the scalp and suppressing cell aging of hair matrix stem cells, hair follicle stem cells and the like existing in the scalp, treatment or prevention of hair loss or thinning hair can be expected.
本開示の皮膚外用組成物は、細胞老化抑制剤以外に、皮膚外用組成物に通常用いられる添加成分を適宜含有することができる。このような添加成分としては、油剤、溶剤、界面活性剤、乳化剤、増粘剤、紫外線吸収剤、美白剤、保湿剤、エモリエント剤、ビタミン類、ｐＨ調整剤、防腐剤、香料、着色剤等が挙げられる。 The external composition for skin of the present disclosure can appropriately contain, in addition to the cell aging inhibitor, additional components usually used in external compositions for skin. Such additives include oils, solvents, surfactants, emulsifiers, thickeners, UV absorbers, whitening agents, moisturizers, emollients, vitamins, pH adjusters, preservatives, fragrances, colorants, etc. Are listed.
本開示の皮膚外用組成物の形態は特に制限されず、液状、ジェル状、クリーム状等が挙げられる。 The form of the external composition for skin of the present disclosure is not particularly limited, and examples thereof include liquid, gel, cream and the like.
本開示の皮膚外用組成物は、化粧料、経皮医薬品、経皮医薬部外品等の用途に適用することができる。化粧料としては、化粧水、美容液、乳液、ローション、クリーム等のスキンケア化粧料が挙げられるが、これらに制限されるものではない。 The external composition for skin of the present disclosure can be applied to applications such as cosmetics, transdermal drugs, and transdermal quasi drugs. Examples of the cosmetics include skin care cosmetics such as lotion, beauty essence, milky lotion, lotion, and cream, but are not limited thereto.
＜幹細胞の細胞老化抑制用機能性飲食品＞
本開示の幹細胞の細胞老化抑制用機能性飲食品（以下、単に「機能性飲食品」ともいう。）は、本開示の細胞老化抑制剤を含有する。ここで、「機能性飲食品」とは、含有される成分に特有の機能に基づいて、生体における所望の生理学的作用を期待し得る飲食品を意味する。
<Functional food and drink for suppressing cell aging of stem cells>
The functional food and drink for suppressing cell aging of stem cells of the present disclosure (hereinafter, also simply referred to as “functional food and drink”) contains the cell aging inhibitor of the present disclosure. Here, the "functional food or drink" means a food or drink that can be expected to have a desired physiological action in the living body based on the function peculiar to the contained components.
本開示の機能性飲食品中における細胞老化抑制剤の含有率は、幹細胞の細胞老化抑制効果が奏される限り特に制限されない。本開示の機能性飲食品中におけるアスタキサンチンの含有率は、例えば、０．０００００１質量％〜０．５質量％であることが好ましく、０．０００００５質量％〜０．２質量％であることがより好ましく、０．００００１質量％〜０．１質量％であることがさらに好ましい。 The content of the cell aging inhibitor in the functional food or drink of the present disclosure is not particularly limited as long as the cell aging inhibitory effect of stem cells is exhibited. The content of astaxanthin in the functional food or drink of the present disclosure is preferably, for example, 0.000001% by mass to 0.5% by mass, and more preferably 0.000005% by mass to 0.2% by mass. It is more preferably 0.00001% by mass to 0.1% by mass.
本開示の機能性飲食品は、本開示の細胞老化抑制剤を含有することにより、幹細胞の細胞老化抑制効果を発揮する。本開示の機能性飲食品を摂取することにより、各種組織の恒常性維持の効果を期待し得る。 The functional food and drink according to the present disclosure exhibits the effect of suppressing cell aging of stem cells by containing the cell aging inhibitor of the present disclosure. By ingesting the functional food or drink of the present disclosure, the effect of maintaining homeostasis of various tissues can be expected.
本開示の機能性飲食品は、細胞老化抑制剤以外に、機能性飲食品に通常用いられる添加成分を適宜含有することができる。このような添加成分としては、蛋白質、オリゴペプチド、アミノ酸、糖類、脂肪、微量元素、ビタミン類、乳化剤、甘味料、香料、着色剤等が挙げられる。 The functional food and drink of the present disclosure can appropriately contain an additive component that is usually used for functional food and drink, in addition to the cell aging inhibitor. Such added components include proteins, oligopeptides, amino acids, sugars, fats, trace elements, vitamins, emulsifiers, sweeteners, flavors, colorants and the like.
本開示の機能性飲食品の形態は特に制限されず、栄養食、半消化態栄養食、自然流動食、ドリンク剤、カプセル剤、錠剤、顆粒剤、散剤等が挙げられるが、これらに制限されるものではない。 The form of the functional food and drink of the present disclosure is not particularly limited, and examples thereof include nutritional food, semidigestive nutritional food, natural liquid food, drinks, capsules, tablets, granules, powders, etc., but are not limited thereto. Not something.
＜幹細胞の細胞老化抑制方法＞
本開示の幹細胞の細胞老化抑制方法（以下、単に「細胞老化抑制方法」ともいう。）は、本開示の細胞老化抑制剤の存在下で幹細胞を培養することを含む。例えば、本開示の細胞老化抑制剤を含有する培地中で幹細胞を培養することにより、幹細胞の細胞老化を効果的に抑制することができる。本開示の細胞老化抑制方法によれば、幹細胞の細胞老化による機能低下を抑制しながら、幹細胞をin vitroで増殖させることが可能となる。
<Method of suppressing stem cell senescence>
The method for suppressing cell senescence of stem cells of the present disclosure (hereinafter, also simply referred to as “method for suppressing cell senescence”) includes culturing stem cells in the presence of the agent for suppressing cell senescence of the present disclosure. For example, by culturing stem cells in a medium containing the cell aging inhibitor of the present disclosure, cell senescence of stem cells can be effectively suppressed. According to the method for suppressing cell senescence of the present disclosure, it becomes possible to proliferate stem cells in vitro while suppressing the functional deterioration of stem cells due to cell aging.
幹細胞としては、体性幹細胞（間葉系幹細胞、肺幹細胞、骨髄幹細胞、上皮幹細胞、神経幹細胞、肝幹細胞、膵幹細胞、造血幹細胞、筋幹細胞、生殖幹細胞、毛母幹細胞、毛包幹細胞、色素幹細胞等）、胚性幹細胞（embryonic stem cell；ＥＳ細胞）、人工多能性幹細胞（induced pluripotent stem cell；ｉＰＳ細胞）などが挙げられる。
幹細胞の由来生物としては、ヒト、マウス、ラット、ウサギ等の哺乳動物が挙げられる。培養した幹細胞を細胞療法に用いる場合には、幹細胞は、治療対象のヒト（患者）由来の幹細胞であることが好ましい。
Stem cells include somatic stem cells (mesenchymal stem cells, lung stem cells, bone marrow stem cells, epithelial stem cells, neural stem cells, liver stem cells, pancreatic stem cells, hematopoietic stem cells, muscle stem cells, reproductive stem cells, hair matrix stem cells, hair follicle stem cells, pigment stem cells Etc.), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.
Examples of stem cell-derived organisms include mammals such as humans, mice, rats, and rabbits. When the cultured stem cells are used for cell therapy, the stem cells are preferably human (patient)-derived stem cells to be treated.
幹細胞を培養する際の培地中における細胞老化抑制剤の含有率は、幹細胞の細胞老化抑制効果が奏される限り特に制限されない。培地中におけるアスタキサンチンの含有率は、例えば、０．００００１質量ｐｐｍ〜１０質量ｐｐｍであることが好ましく、０．０００１質量ｐｐｍ〜５質量ｐｐｍであることがより好ましく、０．０００５質量ｐｐｍ〜０．１質量ｐｐｍであることがさらに好ましい。 The content rate of the cell aging inhibitor in the medium when culturing the stem cells is not particularly limited as long as the cell aging inhibitory effect of the stem cells is exhibited. The content of astaxanthin in the medium is, for example, preferably 0.00001 mass ppm to 10 mass ppm, more preferably 0.0001 mass ppm to 5 mass ppm, and 0.0005 mass ppm to 0. More preferably, it is 1 mass ppm.
幹細胞を培養する培地は特に制限されず、幹細胞の種類に応じて、既知の基本培地に必要に応じて添加剤を加えたものを用いることができる。基本培地としては、ＤＭＥＭ（Dulbecco's Modified Eagle Medium）、ＥＭＥＭ（Eagle's Minimum Essential Medium）、ＭＥＭα（Minimum Essential Medium Alpha modification）、ＲＰＭＩ １６４０（Roswell Park Memorial Institute 1640）培地、ＨａｍＦ１２（Ham's F12）培地等が挙げられる。添加剤としては、血清（ウシ胎仔血清等）、アミノ酸（Ｌ−グルタミン酸等）、抗生物質（ペニシリン、ストレプトマイシン等）などが挙げられる。 The medium for culturing the stem cells is not particularly limited, and a known basal medium to which an additive is added if necessary can be used depending on the type of the stem cells. Examples of the basic medium include DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), MEMα (Minimum Essential Medium Alpha modification), RPMI 1640 (Roswell Park Memorial Institute 1640) medium and HamF12 (Ham's F12) medium. To be Examples of the additive include serum (fetal bovine serum, etc.), amino acids (L-glutamic acid, etc.), antibiotics (penicillin, streptomycin, etc.) and the like.
幹細胞の培養条件は特に制限されず、３７℃かつ５ｖ／ｖ％ ＣＯ２等の公知の培養条件を採用することができる。 Culture conditions for stem cells are not particularly limited, and known culture conditions such as 37° C. and 5 v/v% CO 2 can be adopted.
以下、本発明の実施形態をさらに具体的に説明する。但し、本発明の実施形態は、その主旨を超えない限り、以下の実施例に限定されるものではない。 Hereinafter, embodiments of the present invention will be described more specifically. However, the embodiments of the present invention are not limited to the following examples as long as the gist of the invention is not exceeded.
以下の実施例及び参考例において、幹細胞としては、ヒト間葉系幹細胞（Lonza社）を使用した。また、培地としては、間葉系幹細胞用培地（Mesenchymal Stem Cell Basal Medium、Lonza社）を使用した。 In the following Examples and Reference Examples, human mesenchymal stem cells (Lonza) were used as stem cells. A medium for mesenchymal stem cells (Mesenchymal Stem Cell Basal Medium, Lonza) was used as the medium.
以下の説明中、濃度に関する「Ｍ」はモル濃度を表し、１Ｍ＝１ｍｏｌ／Ｌである。
また、以下の説明中、「ＥＬＩＳＡ」は酵素結合免疫吸着アッセイ（Enzyme-Linked ImmunoSorbent Assay）を意味し、「ＰＢＳ」はリン酸緩衝液（Phosphate Buffered Saline）を意味し、「ＢＳＡ」はウシ血清アルブミン（Bovine Serum Albumin）を意味し、「ＳＤＳ」はドデシル硫酸ナトリウム（Sodium Dodecyl Sulfate）を意味し、「ＰＶＤＦ」はポリフッ化ビニリデン（PolyVinylidene DiFluoride）を意味する。
In the following description, “M” relating to concentration represents a molar concentration, and 1M=1 mol/L.
In the following description, “ELISA” means an enzyme-linked immunosorbent assay (Enzyme-Linked ImmunoSorbent Assay), “PBS” means a phosphate buffer (Phosphate Buffered Saline), and “BSA” means bovine serum. It means albumin (Bovine Serum Albumin), "SDS" means sodium dodecyl sulfate, and "PVDF" means polyvinylidene fluoride (PolyVinylidene DiFluoride).
＜実施例１＞
実施例１では、過酸化水素によって幹細胞に酸化ストレスを与えた場合の各被験物質による細胞老化抑制効果を、ＩＧＦＢＰ−７を分子マーカーとして評価した。使用した被験物質を下記表１に示す。
<Example 1>
In Example 1, the cell senescence suppressing effect of each test substance when oxidative stress was applied to stem cells by hydrogen peroxide was evaluated using IGFBP-7 as a molecular marker. The test substances used are shown in Table 1 below.
まず、ヒト間葉系幹細胞を２５００個／ｃｍ２の細胞密度で４８ウェルプレートに播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、１ｍＭ 過酸化水素を添加した間葉系幹細胞用培地に培地交換し、３０分間培養した。次いで、間葉系幹細胞用培地にて１回洗浄した後、各被験物質を添加した間葉系幹細胞用培地に培地交換し、培養を継続した。その間、２日〜３日毎に培地交換を行った。培養５日目に間葉系幹細胞用培地に培地交換して２４時間培養した後、培地を回収した。 First, human mesenchymal stem cells were seeded in a 48-well plate at a cell density of 2500 cells/cm 2 and cultured in a medium for mesenchymal stem cells. The day after the cells were seeded, the medium was replaced with a medium for mesenchymal stem cells containing 1 mM hydrogen peroxide, and the cells were cultured for 30 minutes. Then, the cells were washed once with the medium for mesenchymal stem cells, the medium was replaced with the medium for mesenchymal stem cells to which each test substance was added, and the culture was continued. During that time, the medium was replaced every 2 to 3 days. On the 5th day of culture, the medium was replaced with a medium for mesenchymal stem cells and cultured for 24 hours, and then the medium was collected.
次いで、回収した培地中のＩＧＦＢＰ−７の濃度をＥＬＩＳＡキット（R&D Systems社）により測定した。さらに、細胞増殖測定用キット（Cell Counting Kit-8、（株）同仁化学研究所）を用いて細胞増殖率を測定し、細胞増殖率の測定値によってＩＧＦＢＰ−７の濃度の測定値を補正した。そして、被験物質を適用しないコントロールに対するＩＧＦＢＰ−７の抑制率を算出した。
なお、各被験物質の適用濃度としては、細胞増殖率が１０％以上抑制されない濃度を上限とし、ＩＧＦＢＰ−７の抑制率が最も高かった濃度を採用した。
Then, the concentration of IGFBP-7 in the recovered medium was measured by an ELISA kit (R&D Systems). Further, the cell proliferation rate was measured using a cell proliferation measuring kit (Cell Counting Kit-8, Dojindo Laboratories Co., Ltd.), and the measured value of the cell proliferation rate was used to correct the measured value of the concentration of IGFBP-7. .. Then, the inhibition rate of IGFBP-7 with respect to the control to which the test substance was not applied was calculated.
As the applied concentration of each test substance, the concentration at which the cell proliferation rate was not suppressed by 10% or more was set as the upper limit, and the concentration at which the inhibition rate of IGFBP-7 was highest was adopted.
ＩＧＦＢＰ−７の抑制率を算出する手順の詳細は、下記（１）〜（４）のとおりである。
（１）各被験物質について同条件のサンプルを５つ以上（すなわち、ｎ≧５）調製した後、回収した培地中のＩＧＦＢＰ−７の濃度を測定する。
（２）上記（１）の測定結果のうち、最高値及び最低値をそれぞれ１点ずつ除き、残りのサンプル（３サンプル以上）の値を採用する。
（３）上記（２）で採用された各サンプルのＩＧＦＢＰ−７の濃度を細胞増殖率の測定値で補正し、コントロールに対するＩＧＦＢＰ−７の抑制率を下記式に従って算出する。なお、抑制率が０％未満の場合は、ＩＧＦＢＰ−７の分泌が促進されたこと、すなわち細胞老化が促進されたことを意味する。
ＩＧＦＢＰ−７の抑制率（％）＝｛（細胞増殖率による補正後のコントロールの値）−（細胞増殖率による補正後のサンプルの値）｝／（細胞増殖率による補正後のコントロールの値）×１００
（４）上記（１）及び（２）を各被験物質について３回繰り返し、上記（３）によって算出されたＩＧＦＢＰ−７の抑制率（３サンプル×３回以上）を算術平均する。
The details of the procedure for calculating the inhibition rate of IGFBP-7 are as described in (1) to (4) below.
(1) After preparing five or more samples (that is, n≧5) under the same conditions for each test substance, measure the concentration of IGFBP-7 in the recovered medium.
(2) Of the measurement results of (1) above, the highest value and the lowest value are removed one by one, and the values of the remaining samples (3 or more samples) are adopted.
(3) The concentration of IGFBP-7 in each sample adopted in the above (2) is corrected by the measured value of cell proliferation rate, and the inhibition rate of IGFBP-7 with respect to the control is calculated according to the following formula. When the inhibition rate is less than 0%, it means that the secretion of IGFBP-7 was promoted, that is, the cell senescence was promoted.
Inhibition rate (%) of IGFBP-7={(value of control after correction by cell growth rate)-(value of sample after correction by cell growth rate)}/(value of control after correction by cell growth rate) ×100
(4) The above (1) and (2) are repeated three times for each test substance, and the IGFBP-7 inhibition rate (3 samples×3 times or more) calculated in the above (3) is arithmetically averaged.
各被験物質を適用した場合のＩＧＦＢＰ−７の抑制率を下記表２に示す。 The inhibition rate of IGFBP-7 when each test substance is applied is shown in Table 2 below.
表２に示すとおり、アスタキサンチンは、他の被験物質と比較して適用濃度が顕著に低いにも関わらず、ＩＧＦＢＰ−７の抑制率が最も高かった。この結果から、アスタキサンチンは、他の被験物質と比較して細胞老化抑制効果が顕著に優れることが分かる。 As shown in Table 2, astaxanthin had the highest inhibition rate of IGFBP-7, although the applied concentration was significantly lower than that of the other test substances. From this result, it can be seen that astaxanthin has a markedly superior cell aging inhibitory effect as compared with other test substances.
＜実施例２＞
実施例２では、過酸化水素によって幹細胞に酸化ストレスを与えた場合のアスタキサンチン単独、又はアスタキサンチン及びニコチン酸トコフェロールの組み合わせによる細胞老化抑制効果を、γＨ２ＡＸを分子マーカーとして評価した。
<Example 2>
In Example 2, the effect of suppressing cell aging by astaxanthin alone or a combination of astaxanthin and tocopherol nicotinate when oxidative stress was applied to stem cells by hydrogen peroxide was evaluated using γH2AX as a molecular marker.
まず、ヒト間葉系幹細胞を５０００個／ｃｍ２の細胞密度で３５ｍｍマルチウェルガラスボトムディッシュ（ウェルサイズ：９．５ｍｍ、カバーガラス厚：０．１５ｍｍ〜０．１８ｍｍ、ポリ−Ｌ−リシンコート）に播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、１ｍＭ 過酸化水素を添加した間葉系幹細胞用培地に培地交換し、３０分間培養した。次いで、間葉系幹細胞用培地にて１回洗浄した後、０．００２質量ｐｐｍ アスタキサンチン、又は０．０００５質量ｐｐｍ アスタキサンチン及び０．０５質量ｐｐｍ ニコチン酸トコフェロールを添加した間葉系幹細胞用培地に培地交換し、培養を継続した。６時間培養後、ＰＢＳにて２回洗浄した。 First, 35 mm multi-well glass bottom dish (well size: 9.5 mm, cover glass thickness: 0.15 mm to 0.18 mm, poly-L-lysine coat) with human mesenchymal stem cells at a cell density of 5000 cells/cm 2. The cells were seeded on and cultured in a medium for mesenchymal stem cells. The day after the cells were seeded, the medium was replaced with a medium for mesenchymal stem cells containing 1 mM hydrogen peroxide, and the cells were cultured for 30 minutes. Then, after washing once with the medium for mesenchymal stem cells, 0.002 mass ppm astaxanthin, or 0.0005 mass ppm astaxanthin and 0.05 mass ppm medium to the medium for mesenchymal stem cells to which tocopherol nicotinate was added The cells were exchanged and the culture was continued. After culturing for 6 hours, the cells were washed twice with PBS.
次いで、４ｗ／ｖ％ パラホルムアルデヒド含有ＰＢＳで１５分間処理することにより細胞を固定し、ＰＢＳで１回洗浄した。０．２ｖ／ｖ％ Triton-X 100含有ＰＢＳで１５分間処理して膜透過処理を行った後、ＰＢＳで１回洗浄した。１ｗ／ｖ％ ＢＳＡ含有ＰＢＳで１時間処理することによりブロッキングを行い、ＰＢＳで１回洗浄した。次いで、抗γＨ２ＡＸウサギポリクローナル抗体（Abcam社）を５００倍希釈した溶液を添加して、４℃で終夜反応させた。さらに、０．０５ｖ／ｖ％ Tween 20含有ＰＢＳ（Ｔ−ＰＢＳ）で３回洗浄した後、緑色蛍光色素で標識されたヤギ抗ウサギＩｇＧ抗体（Abcam社）を２５０倍希釈した溶液で１時間反応させた。その後、Ｔ−ＰＢＳで２回洗浄し、顕微鏡観察した。細胞内のγＨ２ＡＸは、細胞核の緑色蛍光強度を測定することにより定量した。 The cells were then fixed by treatment with PBS containing 4 w/v% paraformaldehyde for 15 minutes and washed once with PBS. The membrane was treated with PBS containing 0.2 v/v% Triton-X 100 for 15 minutes for membrane permeation treatment, and then washed once with PBS. The cells were blocked by treating with PBS containing 1 w/v% BSA for 1 hour, and washed once with PBS. Then, a 500-fold diluted solution of anti-γH2AX rabbit polyclonal antibody (Abcam) was added and reacted overnight at 4°C. Furthermore, after washing three times with PBS containing 0.05 v/v% Tween 20 (T-PBS), a goat anti-rabbit IgG antibody labeled with a green fluorescent dye (Abcam) was diluted 250 times and reacted for 1 hour. Let Then, the plate was washed twice with T-PBS and observed under a microscope. The intracellular γH2AX was quantified by measuring the green fluorescence intensity of the cell nucleus.
γＨ２ＡＸの発現量の定量結果を図１に示す。図１は、過酸化水素によって酸化ストレスを与えない場合のγＨ２ＡＸの発現量を１とした相対値で示したものである。図１に示すとおり、過酸化水素によって酸化ストレスを与えることで、細胞内のγＨ２ＡＸの発現量は約２．５倍に増加した。しかし、０．００２質量ｐｐｍ アスタキサンチンを適用した場合には、γＨ２ＡＸの発現量が約３０％減少し、０．０００５質量ｐｐｍ アスタキサンチン及び０．０５質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、γＨ２ＡＸの発現量が約５３％減少した。 The quantification result of the expression level of γH2AX is shown in FIG. FIG. 1 is a relative value with the expression level of γH2AX being 1 when oxidative stress is not given by hydrogen peroxide. As shown in FIG. 1, when oxidative stress was applied by hydrogen peroxide, the intracellular expression level of γH2AX was increased by about 2.5 times. However, when 0.002 mass ppm astaxanthin was applied, the expression level of γH2AX decreased by about 30%, and when 0.0005 mass ppm astaxanthin and 0.05 mass ppm tocopherol nicotinate were applied, γH2AX The expression level was reduced by about 53%.
なお、アスタキサンチン及びニコチン酸トコフェロールの組み合わせ以外の２種の被験物質の組み合わせについても、上記と同様にして細胞老化抑制効果を評価した。その結果、アスタキサンチン及びニコチン酸トコフェロールの組み合わせが最も有効であることが確認された。 The cell aging inhibitory effect was evaluated in the same manner as above for combinations of two types of test substances other than the combination of astaxanthin and tocopherol nicotinate. As a result, it was confirmed that the combination of astaxanthin and tocopherol nicotinate was the most effective.
＜実施例３＞
実施例３では、過酸化水素によって幹細胞に酸化ストレスを与えた場合のアスタキサンチン単独、又はアスタキサンチン及びニコチン酸トコフェロールの組み合わせによる細胞老化抑制効果を、ｐ２１を分子マーカーとして評価した。
<Example 3>
In Example 3, the cell senescence suppressing effect of astaxanthin alone or a combination of astaxanthin and tocopherol nicotinate when oxidative stress was applied to stem cells by hydrogen peroxide was evaluated using p21 as a molecular marker.
まず、ヒト間葉系幹細胞を２５００個／ｃｍ２の細胞密度で６ウェルプレートに播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、１ｍＭ 過酸化水素を添加した間葉系幹細胞用培地に培地交換し、３０分間培養した。次いで、間葉系幹細胞用培地にて１回洗浄した後、０．００２質量ｐｐｍ アスタキサンチン、又は０．０００５質量ｐｐｍ アスタキサンチン及び０．１質量ｐｐｍ ニコチン酸トコフェロールを添加した間葉系幹細胞用培地に培地交換し、培養を継続した。その間、２日〜３日毎に培地交換を行った。４日間〜５日間の培養後、細胞を回収して可溶化し、ウェスタンブロッティングに供した。 First, human mesenchymal stem cells were seeded in a 6-well plate at a cell density of 2500 cells/cm 2 and cultured in a medium for mesenchymal stem cells. The day after the cells were seeded, the medium was replaced with a medium for mesenchymal stem cells containing 1 mM hydrogen peroxide, and the cells were cultured for 30 minutes. Then, after washing once with the medium for mesenchymal stem cells, 0.002 mass ppm astaxanthin, or 0.0005 mass ppm astaxanthin and 0.1 mass ppm to the medium for mesenchymal stem cells to which tocopherol nicotinate has been added The cells were exchanged and the culture was continued. During that time, the medium was replaced every 2 to 3 days. After culturing for 4 to 5 days, cells were collected, solubilized, and subjected to Western blotting.
各レーンにつき２０μｇのタンパク質をＳＤＳ−ポリアクリルアミドゲルに展開した後、タンパク質をＰＶＤＦ膜に転写した。転写後のＰＶＤＦ膜は、ブロッキング剤（Blocking One、ナカライテスク（株））で１時間処理することによりブロッキングを行った後、Ｔ−ＰＢＳで洗浄した。次いで、抗ｐ２１ウサギモノクローナル抗体（Abcam社）を５００倍希釈した溶液を添加して、４℃で終夜反応させた。また、内部標準として、抗β−アクチンマウスモノクローナル抗体（Sigma-Aldrich社）を２００００倍希釈した溶液を添加して、室温で１時間反応させた。Ｔ−ＰＢＳで３回洗浄した後、それぞれ２次抗体に反応させた。ｐ２１のバンドの濃さを画像解析し、β−アクチンのバンドの濃さにて補正した。 20 μg of protein per lane was loaded on an SDS-polyacrylamide gel and then transferred to a PVDF membrane. The transferred PVDF film was blocked by treating with a blocking agent (Blocking One, Nacalai Tesque, Inc.) for 1 hour, and then washed with T-PBS. Then, a 500-fold diluted solution of anti-p21 rabbit monoclonal antibody (Abcam) was added and reacted overnight at 4°C. As an internal standard, a solution of anti-β-actin mouse monoclonal antibody (Sigma-Aldrich) diluted 20000 times was added, and the mixture was reacted at room temperature for 1 hour. After washing three times with T-PBS, each was reacted with a secondary antibody. Image intensity of the p21 band was analyzed and corrected with the intensity of the β-actin band.
その結果、被験物質を適用しなかった場合と比較して、０．００２質量ｐｐｍ アスタキサンチンを適用した場合には、ｐ２１の発現量が約３３％減少し、０．０００５質量ｐｐｍ アスタキサンチン及び０．１質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、ｐ２１の発現量が約３８％減少したことが確認された。 As a result, when 0.002 mass ppm astaxanthin was applied, the expression level of p21 was reduced by about 33% when 0.002 mass ppm astaxanthin was applied, and 0.0005 mass ppm astaxanthin and 0.1 When mass ppm tocopherol nicotinate was applied, it was confirmed that the expression level of p21 decreased by about 38%.
＜実施例４＞
実施例４では、過酸化水素によって幹細胞に酸化ストレスを与えた場合におけるアスタキサンチン単独、又はアスタキサンチン及びニコチン酸トコフェロールの組み合わせによる細胞老化抑制効果を、細胞増殖率により評価した。
<Example 4>
In Example 4, the cell aging inhibitory effect of astaxanthin alone or the combination of astaxanthin and tocopherol nicotinate when oxidative stress was applied to stem cells by hydrogen peroxide was evaluated by the cell growth rate.
まず、ヒト間葉系幹細胞を２５００個／ｃｍ２の細胞密度で４８ウェルプレートに播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、１ｍＭ 過酸化水素を添加した間葉系幹細胞用培地に培地交換し、３０分間培養した。次いで、間葉系幹細胞用培地にて１回洗浄した後、０．００２質量ｐｐｍ アスタキサンチン、又は０．００２質量ｐｐｍ アスタキサンチン及び０．０５質量ｐｐｍ ニコチン酸トコフェロールを添加した間葉系幹細胞用培地に培地交換し、培養を継続した。その間、２日〜３日毎に培地交換を行った。４日間の培養後、細胞増殖測定用キット（Cell Counting Kit-8、（株）同仁化学研究所）を用いて細胞増殖率を測定した。 First, human mesenchymal stem cells were seeded in a 48-well plate at a cell density of 2500 cells/cm 2 and cultured in a medium for mesenchymal stem cells. The day after the cells were seeded, the medium was replaced with a medium for mesenchymal stem cells containing 1 mM hydrogen peroxide, and the cells were cultured for 30 minutes. Then, after washing once with the medium for mesenchymal stem cells, 0.002 mass ppm astaxanthin, or 0.002 mass ppm astaxanthin and 0.05 mass ppm medium to the medium for mesenchymal stem cells to which tocopherol nicotinate was added The cells were exchanged and the culture was continued. During that time, the medium was replaced every 2 to 3 days. After culturing for 4 days, the cell proliferation rate was measured using a cell proliferation measuring kit (Cell Counting Kit-8, Dojindo Laboratories Inc.).
細胞増殖率の測定結果を図２に示す。図２は、被験物質を適用していない場合の細胞増殖率を１とした相対値で示したものである。図２に示すとおり、０．００２質量ｐｐｍ アスタキサンチンを適用した場合には、細胞増殖率が約１１％亢進し、０．００２質量ｐｐｍ アスタキサンチン及び０．０５質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、細胞増殖率が約１９％亢進した。この結果は、実施例３においてｐ２１の発現量が減少した結果と整合するものであった。 The measurement result of the cell growth rate is shown in FIG. FIG. 2 shows relative values with the cell growth rate when the test substance is not applied as 1. As shown in FIG. 2, when 0.002 mass ppm astaxanthin was applied, the cell growth rate was increased by about 11%, and when 0.002 mass ppm astaxanthin and 0.05 mass ppm tocopherol nicotinate were applied, , The cell growth rate was increased by about 19%. This result was consistent with the result that the expression level of p21 was decreased in Example 3.
＜参考例１＞
参考例１では、幹細胞の継代を繰り返した場合のニコチン酸トコフェロールによる細胞老化抑制効果を、ｐ１６を分子マーカーとして評価した。
<Reference example 1>
In Reference Example 1, the cell senescence suppressing effect of tocopherol nicotinate when repeated passage of stem cells was evaluated using p16 as a molecular marker.
まず、６継代目〜８継代目のヒト間葉系幹細胞を２５００個／ｃｍ２の細胞密度で６ウェルプレートに播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、０．１質量ｐｐｍ又は０．３質量ｐｐｍ ニコチン酸トコフェロールを添加した間葉系幹細胞用培地に培地交換し、６日間培養した。その間、２日〜３日毎に培地交換を行った。培養後、細胞を回収して可溶化し、ウェスタンブロッティングに供した。 First, 6th to 8th passage human mesenchymal stem cells were seeded in a 6-well plate at a cell density of 2500 cells/cm 2 and cultured in a medium for mesenchymal stem cells. On the day after the cells were seeded, the medium was replaced with a medium for mesenchymal stem cells supplemented with 0.1 mass ppm or 0.3 mass ppm tocopherol nicotinate, and cultured for 6 days. During that time, the medium was replaced every 2 to 3 days. After culturing, the cells were collected, solubilized, and subjected to Western blotting.
各レーンにつき２０μｇのタンパク質をＳＤＳ−ポリアクリルアミドゲルに展開した後、タンパク質をＰＶＤＦ膜に転写した。転写後のＰＶＤＦ膜は、ブロッキング剤（Blocking One、ナカライテスク（株））で１時間処理することによりブロッキングを行った後、Ｔ−ＰＢＳで洗浄した。次いで、抗ｐ１６ウサギモノクローナル抗体（Abcam社）を１０００倍希釈した溶液を添加して、４℃で終夜反応させた。また、内部標準として、抗β−アクチンマウスモノクローナル抗体（Sigma-Aldrich社）を２００００倍希釈した溶液を添加して、室温で１時間反応させた。Ｔ−ＰＢＳで３回洗浄した後、それぞれ２次抗体に反応させた。ｐ１６のバンドの濃さを画像解析し、β−アクチンのバンドの濃さにて補正した。 20 μg of protein per lane was loaded on an SDS-polyacrylamide gel and then transferred to a PVDF membrane. The transferred PVDF film was blocked by treating with a blocking agent (Blocking One, Nacalai Tesque, Inc.) for 1 hour, and then washed with T-PBS. Then, a 1000-fold diluted solution of anti-p16 rabbit monoclonal antibody (Abcam) was added and reacted overnight at 4°C. As an internal standard, a solution of anti-β-actin mouse monoclonal antibody (Sigma-Aldrich) diluted 20000 times was added, and the mixture was reacted at room temperature for 1 hour. After washing three times with T-PBS, each was reacted with a secondary antibody. The image intensity of the p16 band was analyzed and corrected by the intensity of the β-actin band.
ｐ１６のウェスタンブロッティングの結果を図３に示す。図３は、被験物質を適用しなかった場合のｐ１６の発現量を１とした相対値で示したものである。図３に示すとおり、被験物質を適用しなかった場合と比較して、０．１質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、ｐ１６の発現量が約１４％減少し、０．３質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、ｐ１６の発現量が約２７％減少した。 The result of Western blotting of p16 is shown in FIG. FIG. 3 is a relative value with 1 being the expression level of p16 when the test substance was not applied. As shown in FIG. 3, when 0.1 mass ppm tocopherol nicotinate was applied, the expression level of p16 was reduced by about 14%, and 0.3 mass ppm, as compared with the case where the test substance was not applied. When tocopherol nicotinate was applied, the expression level of p16 was reduced by about 27%.
＜参考例２＞
参考例２では、幹細胞の継代を繰り返した場合のニコチン酸トコフェロールによる細胞老化抑制効果を、ＩＧＦＢＰ−４を分子マーカーとして評価した。
<Reference example 2>
In Reference Example 2, the effect of tocopherol nicotinate in suppressing cell aging when repeated passage of stem cells was evaluated by using IGFBP-4 as a molecular marker.
まず、６継代目〜８継代目のヒト間葉系幹細胞を２５００個／ｃｍ２の細胞密度で４８ウェルプレートに播種し、間葉系幹細胞用培地中で培養した。細胞を播種した翌日に、０．１質量ｐｐｍ又は０．３質量ｐｐｍ ニコチン酸トコフェロールを添加した間葉系幹細胞用培地に培地交換し、培養を継続した。その間、２日〜３日毎に培地交換を行った。培養５日目に間葉系幹細胞用培地に培地交換して２４時間培養した後、培地を回収した。 First, 6th to 8th passage human mesenchymal stem cells were seeded in a 48-well plate at a cell density of 2500 cells/cm 2 and cultured in a medium for mesenchymal stem cells. On the day after seeding the cells, the medium was replaced with a medium for mesenchymal stem cells containing 0.1 mass ppm or 0.3 mass ppm tocopherol nicotinate, and the culture was continued. During that time, the medium was replaced every 2 to 3 days. On the 5th day of culture, the medium was replaced with a medium for mesenchymal stem cells and cultured for 24 hours, and then the medium was collected.
回収した培地中のＩＧＦＢＰ−４の含有率をＥＬＩＳＡキット（R&D Systems社）により測定した。さらに、細胞増殖測定用キット（Cell Counting Kit-8、（株）同仁化学研究所）を用いて細胞増殖率を測定し、細胞増殖率の測定値によってＩＧＦＢＰ−４の濃度の測定値を補正した。 The content of IGFBP-4 in the recovered medium was measured by an ELISA kit (R&D Systems). Furthermore, the cell proliferation rate was measured using a cell proliferation measuring kit (Cell Counting Kit-8, Dojindo Laboratories Co., Ltd.), and the measurement value of the cell proliferation rate was used to correct the measurement value of the concentration of IGFBP-4. ..
その結果、被験物質を適用しなかった場合と比較して、０．１質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、ＩＧＦＢＰ−４の発現量が約１０％減少し、０．３質量ｐｐｍ ニコチン酸トコフェロールを適用した場合には、ＩＧＦＢＰ−４の発現量が約１３％減少したことが確認された。 As a result, when 0.1 mass ppm tocopherol nicotinate was applied, the expression level of IGFBP-4 was reduced by about 10%, and 0.3 mass ppm nicotine was compared with the case where the test substance was not applied. It was confirmed that when the tocopherol acid was applied, the expression level of IGFBP-4 was reduced by about 13%.
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