JP6120398B2 - Elastic fiber enhancement method with water-soluble fibrillin composition - Google Patents
Elastic fiber enhancement method with water-soluble fibrillin composition Download PDFInfo
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- JP6120398B2 JP6120398B2 JP2012242496A JP2012242496A JP6120398B2 JP 6120398 B2 JP6120398 B2 JP 6120398B2 JP 2012242496 A JP2012242496 A JP 2012242496A JP 2012242496 A JP2012242496 A JP 2012242496A JP 6120398 B2 JP6120398 B2 JP 6120398B2
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- elastin
- water
- soluble
- fibrillin
- skin
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
本発明はフィブリリン組成物を用いた弾性組織増強法であり、水溶性フィブリリンの製造技術と水溶性フィブリリンと水溶性エラスチンを複合化して皮膚線維芽細胞に作用させる弾性線維増強技術からなり、詳しくは生体弾性組織から得られる水溶性フィブリリンの抽出方法と、皮膚線維芽細胞表面上もしくは細胞周辺に形成する弾性線維を増強させる組成に関わる。 The present invention is an elastic tissue augmentation method using a fibrillin composition, comprising water-soluble fibrillin production technology and elastic fiber augmentation technology in which water-soluble fibrillin and water-soluble elastin are combined to act on skin fibroblasts. The present invention relates to a method for extracting water-soluble fibrillin obtained from a living elastic tissue and a composition that enhances elastic fibers formed on or around the surface of dermal fibroblasts.
生体組織は細胞と細胞を支える基質（細胞外基質）から構成される。また生体組織は上皮組織、結合組織、筋肉組織、神経組織の４種類に分類され、特に身体の形や構造を支える支持組織を結合組織と呼び、その主な構成は網目構造を形成する膠原線維と弾性線維からなり、こうした線維を生み出す線維芽細胞が組織中に存在し合成と分解を調節している。結合組織には筋肉組織および神経組織が付随して複合化し器官、臓器などの高次構造が構築されている。膠原線維はコラーゲンを主成分とする鋼の様な硬い構造を支え、弾性線維はエラスチンを主成分としフィブリリンとの複合化により張力を発揮させ伸縮性を有する構造を支えている。膀胱壁や心臓壁、血管壁などの臓器壁や肺の肺包、靭帯、弾性軟骨などの弾性組織は強度と変形性を兼ね備えた構造である。 A living tissue is composed of cells and a matrix (extracellular matrix) that supports the cells. Biological tissues are classified into four types: epithelial tissues, connective tissues, muscle tissues, and nerve tissues. In particular, the supporting tissues that support the body shape and structure are called connective tissues, and the main structure is collagen fibers that form a network structure. The fibroblasts that produce these fibers are present in the tissue and regulate synthesis and degradation. The connective tissue is complexed with muscle tissue and nerve tissue to form higher-order structures such as organs and organs. Collagen fibers support a hard structure such as steel containing collagen as a main component, and elastic fibers support elastin as a main component and have a stretchable structure by exerting tension by combining with fibrillin. Organ tissues such as bladder wall, heart wall, blood vessel wall, and lung tissue of lung, ligament, elastic cartilage and other elastic tissues are structures having both strength and deformability.
皮膚組織は角質・上皮組織と、その下部に存在し構造を支えている結合組織である真皮組織から構成され、真皮組織の影響を大きく受ける。即ち、真皮組織に働く弾性線維の張力が肌のハリを担い表層のシワを防いでいる。真皮層の線維芽細胞は、基本的には膠原線維と弾性線維のどちらも合成できるが、老化に伴い（特に３０歳台以降）弾性線維の合成量が低下し、相対的に膠原線維の弾性線維に対する割合が増加していく。こうした支持構造の量的な減少に伴い張力が圧倒的に低下することで、肌のたるみを防ぐことが困難になりしわが発生すると考えられる。 Skin tissue is composed of stratum corneum and epithelial tissue and dermis tissue, which is a connective tissue that exists underneath and supports the structure. That is, the tension of elastic fibers acting on the dermal tissue bears skin firmness and prevents surface wrinkles. The dermal fibroblasts can basically synthesize both collagen fibers and elastic fibers, but with the aging process (especially after the 30s), the amount of elastic fibers decreases and the elasticity of collagen fibers is relatively high. The ratio to fibers increases. It is considered that wrinkles occur because it becomes difficult to prevent sagging of the skin because the tension is overwhelmingly reduced with the quantitative reduction of the support structure.
弾性線維はエラスチンを主体として微細線維であるフィブリリンとの複合線維であることが知られる。また紫外線などによりダメージを受けることで分解され、通常よりも早い段階で減少する。炎症を伴う日焼けなどの際には白血球や増殖性の細胞からエラスチンを分解するエラスターゼが産生され、より多くの弾性線維が失われることになる。 It is known that elastic fibers are complex fibers with elastin as a main component and fibrillin, which is a fine fiber. Moreover, it is decomposed by being damaged by ultraviolet rays or the like, and decreases at an earlier stage than usual. In the case of sunburn with inflammation, elastase that degrades elastin is produced from leukocytes and proliferating cells, and more elastic fibers are lost.
これまでに肌のハリに重要な弾性線維を増強させるためには、主にエラスチンの産生を促す目的の物質が数多く開発され、そうした活性化物質として化粧品や栄養食品あるいは医薬品等に応用が試みられてきた。例えば特許文献１では経口用の皮膚老化予防・改善剤が示され、ムコ多糖と、コラーゲンまたはエラスチン、コエンザイムＱ１０を用いている。また特許文献２においては、エラスチンやコラーゲンに対してビタミン及び銅塩類を一定割合配合することで産生を促している。更に特許文献３、４および５においては、アオギヌゴケ、ナルコユリおよびシコマリンなどの植物抽出物を用いたエラスチン産生刺激が示されている。こうした多くの試みにも関わらず実際には弾性線維を充分に増強させることができず、また補充方法としても明確な原理は確立されていない。特に植物由来原料では、アレルギー反応等の免疫的な反応性が大きいことが問題になる。また特許文献６においては、活性ペプチド配列のＬｅｕ-Ｇｌｕ−Ｈｉｓ−Ａｌａがエラスチン遺伝子の産生に効果的に作用するとある。特許文献７においては、Ｐｒｏ−Ｇｌｙのジペプチドが効果的な最小配列とある。こうした合成ペプチドによる検証は細胞表面のレセプターを刺激しやすいことが予想され、また吸収性の良さを鑑みるとエラスチンを産生させる効果は期待できる。 In order to strengthen elastic fibers that are important for skin firmness, many substances have been developed mainly for the purpose of promoting the production of elastin, and such active substances have been tried to be applied to cosmetics, nutritional foods, pharmaceuticals, etc. I came. For example, Patent Document 1 discloses an oral skin aging preventive / ameliorating agent, which uses mucopolysaccharide, collagen or elastin, and coenzyme Q10. Moreover, in patent document 2, production is promoted by mix | blending a vitamin and copper salt with a fixed ratio with respect to elastin and collagen. Further, Patent Documents 3, 4 and 5 show stimulation of elastin production using plant extracts such as Aoginookoke, Narukoyuri and Shikomarin. Despite these many attempts, the elastic fibers cannot actually be sufficiently strengthened, and no clear principle has been established as a replenishment method. In particular, plant-derived materials have a problem of high immunoreactivity such as allergic reactions. Further, in Patent Document 6, the active peptide sequence Leu-Glu-His-Ala is effective in producing the elastin gene. In Patent Document 7, Pro-Gly dipeptide is an effective minimal sequence. Such verification with synthetic peptides is expected to stimulate cell surface receptors, and in view of good absorbability, the effect of producing elastin can be expected.
一方で、肌のハリはコラーゲンの含有量を増やすことで達成されるという考え方の化粧品や栄養食品が存在する。例えば特許文献８においてはオリーブの葉に含まれる成分とコラーゲンによりコラーゲンの産生を高めることができる食品の内容がある。特許文献９においても、燕窩酵素分解物がコラーゲンの産生を高めることが示されている。ところが、生体組織中のコラーゲンを主体とする膠原線維は、組織そのものを丈夫に、そして硬くするための特性を有する構成成分であり、肌を伸縮性に富ますための張力を発生させる弾性線維の様な構造的機能は期待できないことは明らかである。即ち、肌のハリを取り戻ししわの発生を防ぐ方法は、結合組織内の弾性線維を増強させることにあると考えられるが、エラスチンの産生を促すだけではその効果は不十分である。 On the other hand, there are cosmetics and nutritional foods based on the idea that skin firmness is achieved by increasing the collagen content. For example, in Patent Document 8, there is a food content that can enhance the production of collagen by components contained in olive leaves and collagen. Patent Document 9 also shows that an axillary enzyme degradation product enhances collagen production. However, collagen fibers mainly composed of collagen in living tissues are structural components that have the characteristics to make the tissues themselves strong and hard, and elastic fibers that generate tension to make the skin stretchable. It is clear that such a structural function cannot be expected. That is, it is considered that a method for recovering skin firmness and preventing the generation of wrinkles is to enhance elastic fibers in the connective tissue, but the effect is insufficient only by promoting the production of elastin.
弾性線維が減少していく大きな原因の一つに炎症などによる細胞外基質分解の加速が挙げられる。特に日焼けなどの紫外線による障害でフィブリリンが失われることが報告されている（非特許文献１）。フィブリリンはエラスチンの組織化構造を促進する足場を提供し弾性線維を形成させ、皮膚の弾力性を促進する働きが知られる。弾性線維は主にエラスチンとフィブリリンから構成されるが、伸縮性を担うのは架橋が進んだエラスチンであり、エラスチンを太い線維状に配置させ束ねることがフィブリリンの役割として知られる。この微細線維であるフィブリリンが存在することで、エラスチンが良好に線維化し、弾性線維による肌のハリが保たれる。 One of the major reasons for the decrease in elastic fibers is acceleration of extracellular matrix degradation due to inflammation. It has been reported that fibrillin is lost due to damage caused by ultraviolet rays such as sunburn (Non-patent Document 1). Fibrin is known to provide a scaffold that promotes the organized structure of elastin, to form elastic fibers, and to promote skin elasticity. Elastic fibers are mainly composed of elastin and fibrillin, but it is elastin that has been cross-linked that is responsible for stretchability. The role of fibrillin is known to arrange and bundle elastin into thick fibers. Due to the presence of fibrillin, which is a fine fiber, elastin is well fibrillated, and the elasticity of the skin is maintained by elastic fibers.
しかしながら、弾性線維を形成させる刺激として外部からフィブリリンを供給する発想はこれまでなかった。この理由として、フィブリリンの大量製造および水溶化技術が確立されていないことにある。フィブリリンはフィブリリン−１とフィブリリン−２の二種類が知られるが、生体組織からエラスチンを抽出製造する際に回収されることは、エラスチンを製造する同業者であれば周知の現象であるが、弾性線維からの分離が難しく純度の高い水溶性のフィブリリンを回収する方法は少なく、従って供給技術は極めて例が少ない。 However, there has been no idea of supplying fibrillin from the outside as a stimulus for forming elastic fibers. The reason is that fibrillin mass production and water solubilization technology has not been established. Two types of fibrillin are known, fibrillin-1 and fibrillin-2, but recovery when extracting and producing elastin from living tissue is a well-known phenomenon for those skilled in the art of producing elastin. There are few methods for recovering highly pure water-soluble fibrillin that is difficult to separate from the fibers, and therefore, the supply technology is extremely few.
そこで、フィブリリンの産生低下の改善あるいはフィブリリンの産生促進を目的とした技術の開発が試みられている。例えば特許文献９によればプレニル化イソフラボノイドを皮膚線維芽細胞に添加させることで、フィブリリンの産生が増加することが示されている。しかしながら、フィブリリンの産生を促すことでエラスチンの堆積を促すことはできても、皮膚の線維芽細胞からのエラスチンの産生を促す事が出来ずに、結果として弾性線維の増強効果は見込めず、しわの発生を防ぐ方法としては不十分であった。特にエラスチンの産生の際には、同一細胞からコラーゲンの産生も促進されることが多く、同一細胞の産生能力の中で考慮すると相対的にエラスチン産生能力が低下し、弾性線維の増強効果が見られないもしくは低い状態になる。
Then, development of the technique aiming at the improvement of fibrillin production reduction or the promotion of fibrillin production has been attempted. For example, Patent Document 9 shows that the production of fibrillin is increased by adding prenylated isoflavonoids to dermal fibroblasts. However, although it is possible to promote the deposition of elastin by promoting the production of fibrillin, it is not possible to promote the production of elastin from skin fibroblasts. It was insufficient as a method to prevent the occurrence of In particular, during the production of elastin, collagen production is often promoted from the same cell, and when considering the production capacity of the same cell, the elastin production capacity is relatively lowered and the effect of enhancing elastic fibers is seen. It becomes impossible or becomes low.
本発明において解決しようとする課題は、上述の公知の技術が抱える問題点を解決することであり、具体的には生体弾性組織から効率よく水溶性フィブリリンを抽出する方法を提供し、抽出した水溶性フィブリリンを皮膚線維芽細胞に対し作用させることで細胞表面にエラスチンの足場を作り、また同時に水溶性エラスチンを作用させることで皮膚線維芽細胞に新しいエラスチンの産生を促し、同時にコラーゲンの産生比率を低下させ、この二種類の効果により結果的に細胞表面および周辺の弾性線維を増強させる組成を提供することである。 The problem to be solved in the present invention is to solve the problems of the above-mentioned known techniques, and specifically provides a method for efficiently extracting water-soluble fibrillin from a biological elastic tissue, By making sex fibrillin act on dermal fibroblasts, an elastin scaffold is created on the cell surface, and by simultaneously acting water-soluble elastin on the skin fibroblasts, the production of new elastin is promoted, and at the same time the production ratio of collagen is increased. It is to provide a composition that lowers and consequently enhances the cell surface and surrounding elastic fibers by these two effects.
本発明者は上記の課題を解決するため、皮膚線維芽細胞に対し水溶性フィブリリンと水溶性エラスチンを作用させことで、細胞が自ら産生する弾性線維が量的にも質的にも増強されて形成されるという知見を応用するものであり、加えて本発明者が従来から研究開発している水溶性エラスチン（特開２００９−０４０７１３）を基本技術として、これを改良することにより水溶性フィブリリンの製造を可能とし本発明に到達した。 In order to solve the above-mentioned problems, the present inventor has caused the elastic fibers produced by the cells themselves to be enhanced quantitatively and qualitatively by allowing water-soluble fibrillin and water-soluble elastin to act on dermal fibroblasts. In addition to applying the knowledge that it is formed, water-soluble elastin (Japanese Patent Laid-Open No. 2009-040713), which has been researched and developed by the present inventor as a basic technology, has been improved to improve the solubility of water-soluble fibrillin. Manufacturing was possible and the present invention was reached.
フィブリリンは分子量３５０ｋＤａの高分子量を有し、動脈や肺、靭帯など生体組織の運動が重要な機能に結び付いている器官・臓器等に多く存在する弾性線維に含まれる細胞外基質タンパク質として知られる。弾性線維はエラスチックファイバーとも呼ばれ、主成分はエラスチンである。エラスチンは、細胞の小胞体でトロポエラスチンとして合成された後、エラスチンバインディングプロテイン（ＥＢＰ）、ノイラミニダーゼ１（Ｎｅｕ-1）およびカテプシンＡ（ＣＡ）の3量体とシャペロンを形成し、細胞表面に輸送される。その後、フィブリリンにより形成されたマイクロフィブリル上のガラクトース残基がＥＢＰに結合することでトロポエラスチンが細胞外に放出され、凝集現象であるコアセルベーションによりフィブリリン上に集積される。最後に、リジルオキシダーゼがエラスチンのリシンに作用し、デスモシンやイソデスモシンなどの架橋構造を形成させることでエラスチンとなり、マイクロフィブリルと共に弾性線維として完成する機構が知られている。（ＨｉｎｅｋらＪ．Ｂｉｏｌ．Ｃｈｅｍ． ２８１巻、３６９８−７１０頁、２００６年） Fibrin has a high molecular weight of 350 kDa, and is known as an extracellular matrix protein contained in elastic fibers present in many organs / organs where movement of biological tissues such as arteries, lungs, and ligaments are linked to important functions. Elastic fibers are also called elastic fibers, and the main component is elastin. Elastin is synthesized as tropoelastin in the endoplasmic reticulum of cells, and then forms a chaperone with a trimer of elastin binding protein (EBP), neuraminidase 1 (Neu-1) and cathepsin A (CA) on the cell surface. Transported. Thereafter, galactose residues on the microfibrils formed by fibrillin bind to EBP, so that tropoelastin is released to the outside of the cell, and is accumulated on fibrillin by coacervation, which is an aggregation phenomenon. Finally, a mechanism is known in which lysyl oxidase acts on lysine of elastin to form a crosslinked structure such as desmosine or isodesmosine to become elastin, which is completed as an elastic fiber together with microfibrils. (Hinek et al., J. Biol. Chem. 281, 3698-710, 2006)
しかしながら、これまで生体の弾性組織から大量にフィブリリンを抽出し、加えて水溶化させる技術は少なく、そのため水溶性のフィブリリンの効果に関する検証の例は少なかった。一般的にタンパク質を水溶化する場合には、酸あるいはアルカリによる分解や酵素による分解などが考えられる。フィブリリンは酸に耐性があり、熱にも有機溶媒にも強いが、その濃度や条件を上げることでアミノ酸レベルにまで分解することは可能である。しかし、単に加水分解等で低分子化して水溶化する方法では、高分子としてのフィブリリンの機能を破壊することになり、エラスチンの足場としての働きができずに弾性線維の形成は不可能と思われる。つまり、分子量をできるだけ低下させず、なおかつ中性や弱アルカリ性の生理条件下でも完全に溶解している状態が良好で扱いやすい素材であれば、水溶性エラスチンとの相互作用も大きく弾性線維を増強させることに期待できると思われる。 However, there have been few techniques for extracting fibrillin from a large amount of elastic tissue in a living body and adding it to water solubilization. Therefore, there have been few examples of verification regarding the effect of water-soluble fibrillin. In general, when a protein is water-solubilized, degradation with an acid or alkali, degradation with an enzyme, or the like can be considered. Fibrin is resistant to acids and is resistant to heat and organic solvents, but it can be degraded to the amino acid level by increasing its concentration and conditions. However, the method of simply reducing the molecular weight by hydrolysis, etc., will cause the function of fibrillin as a polymer to be destroyed, and it will not function as a scaffold for elastin, making it impossible to form elastic fibers. It is. In other words, if the material does not decrease the molecular weight as much as possible, and is a good and easy-to-handle material that is completely dissolved even under neutral or weakly alkaline physiological conditions, the interaction with water-soluble elastin is large and elastic fibers are enhanced. It seems that we can expect to make it.
即ち、本発明は皮膚線維芽細胞が関与する肌の弾力性改善のための物質であって、前記肌質改善は弾性線維の形成を刺激し促すことから達成され、この前記物質は生体組織から抽出した水溶性フィブリリンとして選択される物質に関する。 That is, the present invention is a substance for improving skin elasticity involving skin fibroblasts, and the improvement of the skin quality is achieved by stimulating and accelerating the formation of elastic fibers. It relates to a substance selected as extracted water-soluble fibrillin.
また本発明は、前記水溶性フィブリリンが、pH6〜pH8の中性条件で完全に水溶解性であることを特徴とする中性溶解・水溶性であること。または、pH8以上の弱アルカリ性条件で完全に水溶解性であるアルカリ溶解・水溶性であることが望ましい。水溶性であることが皮膚線維芽細胞に刺激を与える効果が大きいことが理由である。 In the present invention, the water-soluble fibrillin is neutral and water-soluble, characterized in that it is completely water-soluble under neutral conditions of pH 6 to pH 8. Alternatively, it is desirable that the solution is completely water-soluble under weakly alkaline conditions of pH 8 or higher and is alkali-soluble / water-soluble. This is because the water-soluble property has a great effect of stimulating dermal fibroblasts.
また本発明は、水溶性のフィブリリンに加えて、水溶性エラスチン、加水分解エラスチン、トロポエラスチン、低分子エラスチン、エラスチンペプチド、エラスチン分解物、エラスチン様ペプチド、α―エラスチン、β―エラスチン、κ―エラスチン、アイソタイプ型エラスチン（エラスチンＡ、エラスチンＢ、エラスチンＣ、エラスチンＤ）から選択される何れか一つまたは一つ以上の物質と同時に用いることにより、皮膚線維芽細胞からのエラスチンの産生を促し加えてエラスチンの集積を促進させることで、結果的に弾性線維の形成を誘導する効果を有する。以上の理由から、弾性線維の増強のためには、混合して作用させることが望ましい。特に水溶性エラスチンと水溶性フィブリリンの混合比率が９０％対１０％である場合が好ましく、より好ましくは９４〜９６％対６〜４％の比率である。この際に、両者の複合体化が顕著に進み、線維芽細胞表面や周辺への弾性線維の産生や堆積効果が加速するのがその理由である。 In addition to water-soluble fibrillin, the present invention also includes water-soluble elastin, hydrolyzed elastin, tropoelastin, low molecular weight elastin, elastin peptide, elastin degradation product, elastin-like peptide, α-elastin, β-elastin, κ- When used together with any one or more substances selected from elastin and isotype-type elastin (elastin A, elastin B, elastin C, elastin D), it promotes the production of elastin from skin fibroblasts. By promoting the accumulation of elastin, it has the effect of inducing the formation of elastic fibers as a result. For the above reasons, it is desirable to mix and act to reinforce elastic fibers. In particular, the mixing ratio of water-soluble elastin and water-soluble fibrillin is preferably 90% to 10%, more preferably 94 to 96% to 6 to 4%. In this case, the reason is that the complexing of the two advances significantly, and the production and deposition effect of elastic fibers on the surface and the periphery of the fibroblasts is accelerated.
また本発明は、こうした皮膚線維芽細胞への刺激を誘導する活性化物質である水溶性フィブリリンを含む化粧品や栄養食品を作成することも可能で、水溶性エラスチンとの複合化から弾性線維の増強が期待でき、アンチエイジングなどのしわの発生を防止する効果をもたらす事が期待できる。 The present invention can also produce cosmetics and nutritional foods containing water-soluble fibrillin, which is an activator that induces stimulation of such skin fibroblasts, and strengthens elastic fibers by complexing with water-soluble elastin. Can be expected to bring about an effect of preventing wrinkles such as anti-aging.
水溶性フィブリリン組成物を用いて水溶性エラスチンを複合化して皮膚線維芽細胞に作用させることで、エラスチンの新規産生と堆積が促進され、その結果形成された弾性線維が増強し、肌のしわの原因である弾性線維の減少や劣化を防止できる。 Complexing water-soluble elastin with water-soluble fibrillin composition and causing it to act on dermal fibroblasts promotes new production and deposition of elastin, resulting in enhanced elastic fibers and increased skin wrinkling. It can prevent the decrease and deterioration of the elastic fiber which is the cause.
本発明の第一の主題は、水溶性フィブリリンを弾性組織から抽出し精製する技術に関する。その一つの方法は生体の動脈組織から加水分解により水溶化する水溶性エラスチンを精製する過程（特許文献７）で生じ、通常は除去する不溶性部分を出発原料とすることで効果的に回収することが可能である。具体的には、不溶性エラスチンを酸により水溶化した溶液を中和することで、水溶性フィブリリンは水溶性エラスチンに結合し不溶化する。この現象を利用して、高純度のフィブリリンを回収する原理を用いる。こうして回収した不溶性部分は希釈していくことで、水溶性エラスチンが再度解離するため、大量の水を加え可溶性部分を除去して完全なる水溶性エラスチンを除去する工程を加える。この水洗操作を繰り返し行い、洗浄液中のエラスチン濃度が限りなくゼロになるまで行うことで、完全にフィブリリンを分離できる。更に、フィブリリン間で生じているＳ−Ｓ結合をセリン等の物質で還元して切断し、尿素を加えアンフォールディングさせて溶解性を増す工程により、中性可溶・水溶性フィブリリンの製造が可能になる。 The first subject of the present invention relates to a technique for extracting and purifying water-soluble fibrillin from elastic tissue. One of the methods is a process of purifying water-soluble elastin that is water-solubilized by hydrolysis from arterial tissue of living body (Patent Document 7), and usually recovers effectively by using an insoluble part to be removed as a starting material. Is possible. Specifically, by neutralizing a solution obtained by solubilizing insoluble elastin with an acid, water-soluble fibrillin binds to water-soluble elastin and becomes insoluble. Utilizing this phenomenon, the principle of recovering high purity fibrillin is used. The water-soluble elastin is dissociated again by diluting the recovered insoluble part in this way, and therefore a process of removing a water-soluble elastin completely by adding a large amount of water and removing the soluble part is added. By repeating this water washing operation until the elastin concentration in the washing liquid becomes zero as much as possible, fibrillin can be completely separated. Furthermore, it is possible to produce neutral soluble and water-soluble fibrillin by reducing the S—S bond generated between fibrillin with a substance such as serine and cleaving it, and adding urea to unfold it to increase solubility. become.
本発明の第二の主題は、皮膚線維芽細胞表面およびその周辺に弾性線維を形成させるために、細胞が自らエラスチンを産生する刺激に関する。通常皮膚線維芽細胞はコラーゲンおよびエラスチンを産生する能力を有している。この刺激として用いる水溶性エラスチンが、細胞自身のエラスチン産生を促すことが可能な水溶性エラスチンに関する。通常、入手可能な水溶性エラスチンには、生体から抽出した不溶性エラスチンをシュウ酸分解したＥＣＭＬ社製のエラスチンＡ、エラスチンＢ、エラスチンＣおよびエラスチンＤ、またはＥＰＣ社製のα―エラスチンやβ―エラスチンおよびアルカリエタノール処理したκ―エラスチン、ＳＩＧＭＡ社製のトロポエラスチンなどがある。本発明には、こうした水溶性エラスチンの利用が可能であるが、特にアイソタイプ型エラスチンが好適に用いることができる。より望ましくは、クエン酸にて酸加水分解して調節したアイソタイプ型エラスチンである水溶性エラスチンであり、細胞の増殖を促進させ、またコラーゲンよりもエラスチンの産生をより促進することから、エラスチンの産生量を調節するのに好適であることがその理由である。 The second subject of the present invention relates to stimuli in which the cells themselves produce elastin in order to form elastic fibers on and around the skin fibroblast surface. Usually skin fibroblasts have the ability to produce collagen and elastin. The water-soluble elastin used as the stimulus relates to water-soluble elastin capable of promoting the production of elastin by the cell itself. Usually, the available water-soluble elastin includes elastin A, elastin B, elastin C and elastin D manufactured by ECML, which is obtained by oxalic degradation of insoluble elastin extracted from a living body, or α-elastin and β-elastin manufactured by EPC. And κ-elastin treated with alkaline ethanol, tropoelastin manufactured by SIGMA, and the like. In the present invention, such water-soluble elastin can be used. In particular, isotype-type elastin can be preferably used. More preferably, it is water-soluble elastin, an isotype elastin regulated by acid hydrolysis with citric acid, which promotes cell growth and promotes the production of elastin rather than collagen. The reason is that it is suitable for adjusting the amount.
本発明の第三の主題は、上述のように作成した水溶性フィブリリンと水溶性エラスチンを混合して使用する技術に関する。水溶性エラスチンの中でも、特に分子内にデスモシン構造などの架橋部位濃度が少ないエラスチン（例えばエラスチンＥあるいはエラスチンＤ、またはトロポエラスチン等）では、皮膚線維芽細胞表面上のエラスチンレセプターとの結合性が大きいため、エラスチンの産生刺激を与えやすいと思われる。エラスチンの細胞表面への接着領域としてＶal−Ｇly−Ｖal−Ａla−Ｐro−Ｇlyなどのエラスチン上のアミノ酸配列が得られている。そのため、水溶性エラスチンＥを添加することにより、正常なエラスチン基質に障害が生じて分解された状況を醸したことに相当し、細胞内では新規にエラスチンを合成する反応が生じると考えられる。また、この反応は、架橋構造が多い水溶性エラスチンＡでは効果が小さくなることでも理解できる。架橋構造が多いことでその両脇に位置している接着領域が防がれ機能しにくいと推察できるからである。 The third subject matter of the present invention relates to a technique using a mixture of water-soluble fibrillin prepared as described above and water-soluble elastin. Among water-soluble elastins, elastin having a low concentration of cross-linking sites such as a desmosine structure in the molecule (for example, elastin E or elastin D, or tropoelastin) has a binding property to an elastin receptor on the surface of skin fibroblasts. Because it is large, it seems to be easy to stimulate the production of elastin. An amino acid sequence on elastin such as Val-Gly-Val-Ala-Pro-Gly has been obtained as an adhesion region of elastin to the cell surface. Therefore, the addition of water-soluble elastin E corresponds to a situation in which a normal elastin substrate is damaged and decomposed, and it is considered that a reaction for newly synthesizing elastin occurs in the cell. This reaction can also be understood as being less effective with water-soluble elastin A having many cross-linked structures. This is because it can be presumed that the adhesive regions located on both sides of the structure are difficult to function due to the large number of cross-linked structures.
本来フィブリリンはフィブリリン１とフィブリリン２の２つのタイプがあり、それぞれわずかにアミノ酸配列が異なるが、その機能に到っては不明な点が多い。フィブリリンは細胞表面に堆積しネットワークを作成する効果が期待できるが、エラスチンを産生するシグナルを与えることはできないため、エラスチンとの複合線維すなわち弾性線維を増強させるには至らない。 Originally, there are two types of fibrillin, fibrillin 1 and fibrillin 2, each having a slightly different amino acid sequence, but there are many unclear points regarding its function. Although fibrillin can be expected to deposit on the cell surface and create a network, it cannot give a signal for producing elastin, so it does not enhance the composite fiber with elastin, that is, the elastic fiber.
線維芽細胞表面に作用しているのはエラスチンペプチドであり、フィブリリンはエラスチンのガイドとして機能している。またエラスチンとフィブリリンは相互作用が大きくエラスチンの線維化を促進する効果が見られる。そのためより高密度、高頻度で細胞表面に刺激を与えやすくなり、細胞自身が産生する弾性線維を増強させる。またフィブリリンは他の弾性線維関連タンパクと質との相互作用を促し、本来の強靭な弾性線維を再生させる効果を促していると考えられる。 It is an elastin peptide that acts on the surface of fibroblasts, and fibrillin functions as a guide for elastin. In addition, elastin and fibrillin have a large interaction, and an effect of promoting the fibrosis of elastin is seen. Therefore, it becomes easier to stimulate the cell surface at a higher density and higher frequency, and the elastic fibers produced by the cells themselves are enhanced. In addition, fibrillin is thought to promote the interaction between other elastic fiber-related proteins and quality, and to regenerate the original tough elastic fibers.
上述のようにして水溶性フィブリリンに水溶性エラスチンを作用させた状態を、皮膚線維芽細胞への刺激として利用する手法としては、種々の形態が考えられるが例えば化粧品として皮膚に塗布して用いる方法や、食品として経口で得る方法が通常方法として考えられる。皮膚への適用の場合には配合には特に限定されるものではないが、皮膚への浸透を促す効果のあるもの、例えばオイル、ナノカプセルなどと共に作用させる方法が挙げられる。しかしながら、こうした既存の浸透技術を用いても、またそのままの状態でも利用が可能である。食品として用いる場合においては、消化管内で分解吸収される過程において、低分子化される可能性があるため、本来の足場構造としての働きは期待できない。しかしながら、たとえば胃における分解を抑えるカプセルなどの併用で摂取することで、小腸内でのリンパ管系経路などからの比較的大きめの分子量の吸収経路を利用して、血液中に浸透させる技術を用いた方法が好適な方法として考えられる。血中に吸収されることで、体循環により組織に輸送されそこで線維芽細胞に弾性線維作成効果をもたらす可能性が期待できる。
As a method of using the state in which water-soluble elastin is allowed to act on water-soluble fibrillin as described above as a stimulus to dermal fibroblasts, various forms are conceivable. Or, a method obtained orally as a food is considered as a normal method. In the case of application to the skin, the formulation is not particularly limited, and examples thereof include a method of acting with an effect of promoting penetration into the skin, such as oil and nanocapsules. However, it is possible to use the existing penetration technique as it is or in a state as it is. When used as food, it may not be able to function as an original scaffold structure because it may be reduced in the process of being decomposed and absorbed in the digestive tract. However, when ingested in combination with a capsule that suppresses degradation in the stomach, for example, a technique that penetrates into the blood using a relatively large molecular weight absorption pathway from the lymphatic pathway in the small intestine is used. Can be considered as a preferred method. By being absorbed into the blood, it can be expected that it will be transported to tissues by systemic circulation where it will have the effect of creating elastic fibers in fibroblasts.
以下、実施例をもって本発明を詳細に説明するが、以下よって示される方法は、作用確認において用いたものであり、これに限定されるものではなく、その要旨を変更することなく様々に改変して実施することができる。 Hereinafter, the present invention will be described in detail with reference to examples. However, the method shown below is used in the confirmation of operation, and is not limited thereto, and various modifications can be made without changing the gist thereof. Can be implemented.
（製造例１−不溶性エラスチンの作製）
ブタ大動脈血管から脂肪を除去し水洗した後、１０％塩化ナトリウム水溶液に浸し１２時間冷蔵庫内で静置し、コラーゲンを溶解除去した。水洗後水を加え圧力鍋で１時間煮沸後、血管付着物を除去し、ミキサーに入れ組織片が１〜５mm片になるまで破砕した。その後、再度圧力鍋で１時間加熱し水洗した。メッシュ状袋に移し電動洗浄機で洗浄した後、エタノールを最終濃度７０％になるように加え１２時間静置した。最終的にエタノールを取り除き温風乾燥機内で不溶性エラスチン（血管１２kgより乾燥重量で約１ｋｇの収量）を得た。
(Production Example 1-Production of insoluble elastin)
After removing fat from the porcine aortic blood vessel and washing with water, it was immersed in a 10% aqueous sodium chloride solution and allowed to stand in a refrigerator for 12 hours to dissolve and remove the collagen. After washing with water and boiling in a pressure cooker for 1 hour, blood vessel deposits were removed and placed in a mixer and crushed until the tissue pieces became 1-5 mm pieces. Then, it heated again with the pressure cooker for 1 hour, and washed with water. After transferring to a mesh bag and washing with an electric washing machine, ethanol was added to a final concentration of 70% and left for 12 hours. Finally, ethanol was removed and insoluble elastin (yield of about 1 kg in dry weight from 12 kg of blood vessels) was obtained in a hot air dryer.
（製造例２−水溶性エラスチンＭＥの作製）
水溶性エラスチンの製法は発明者らが開発した方法（特許文献１０）を基に改良することで作成した。具体的には、不溶性エラスチン５ｋｇに対し１０％クエン酸を３０L加え１２０℃で８時間加熱した。分解反応液を氷水で冷やした後３，０００ｒｐｍで１０分間遠心分離し不溶部分を除いた。可溶部に対し２０％水酸化ナトリウムを加え中和した（ｐＨ６．５）。中和の際に発生する沈殿を、３，０００ｒｐｍで１０分間遠心分離し不溶部分を分離した。上澄み液を、分子量２０,０００〜３０,０００Ｄａの限外濾過膜を用いてろ過した。濾液を分子量３，０００Ｄａの限外濾過膜の透析モジュールを用いて、4℃に冷却した脱イオン水に対し、外液のｐＨが５〜６程度になるまで透析しクエン酸を除去した。溶液は再度限外ろ過膜による不溶部分の除去後に、高圧蒸気滅菌および限外ろ過膜処理を繰り返し、最終的に孔サイズ0.22μｍのフィルターによる滅菌を行い完全に無菌状態にする。ろ液をスプレードライヤーにより噴霧乾燥して水溶性エラスチン（約０．２ｋｇ）を得た。
(Production Example 2-Production of water-soluble elastin ME)
The method for producing water-soluble elastin was created by improving the method based on the method developed by the inventors (Patent Document 10). Specifically, 30 L of 10% citric acid was added to 5 kg of insoluble elastin and heated at 120 ° C. for 8 hours. The decomposition reaction solution was cooled with ice water and then centrifuged at 3,000 rpm for 10 minutes to remove insoluble portions. The soluble part was neutralized by adding 20% sodium hydroxide (pH 6.5). The precipitate generated during neutralization was centrifuged at 3,000 rpm for 10 minutes to separate insoluble portions. The supernatant was filtered using an ultrafiltration membrane having a molecular weight of 20,000 to 30,000 Da. The filtrate was dialyzed against deionized water cooled to 4 ° C. using an ultrafiltration membrane dialysis module having a molecular weight of 3,000 Da until the pH of the outer solution reached about 5 to 6 to remove citric acid. The solution is again subjected to high-pressure steam sterilization and ultrafiltration membrane treatment after removing the insoluble portion by the ultrafiltration membrane, and finally sterilized by a filter having a pore size of 0.22 μm to be completely aseptic. The filtrate was spray-dried with a spray dryer to obtain water-soluble elastin (about 0.2 kg).
（製造例３−水溶性エラスチンＭＡの作製）
製造例２で加熱後に除いた不溶部分５ｋｇに対し、１０％クエン酸を２０L加え１２０℃で５時間加熱し完全に溶解させた。分解反応液を氷水で冷やした後３，０００ｒｐｍで１０分間遠心分離し少量の不溶部分を除いた。２０％水酸化ナトリウムを加え中和した（ｐＨ６．５）。中和の際に発生する沈殿を、３，０００ｒｐｍで１０分間遠心分離し不溶部分を分離した。上澄み液を、分子量２０,０００〜３０,０００Ｄａの限外濾過膜を用いてろ過した。濾液を分子量３，０００Ｄａの限外濾過膜の透析モジュールを用いて、4℃に冷却した脱イオン水に対し、外液のｐＨが５〜６程度になるまで透析しクエン酸を除去した。溶液は再度限外ろ過膜による不溶部分の除去後に、高圧蒸気滅菌および限外ろ過膜処理を繰り返し、最終的に孔サイズ0.22μｍのフィルターによる滅菌を行い完全に無菌状態にする。ろ液をスプレードライヤーにより噴霧乾燥して水溶性エラスチンＭＡ（約１．０ｋｇ）を得た。
(Production Example 3-Production of water-soluble elastin MA)
To 5 kg of the insoluble part removed after heating in Production Example 2, 20 L of 10% citric acid was added and heated at 120 ° C. for 5 hours for complete dissolution. The decomposition reaction solution was cooled with ice water and then centrifuged at 3,000 rpm for 10 minutes to remove a small amount of insoluble part. 20% sodium hydroxide was added for neutralization (pH 6.5). The precipitate generated during neutralization was centrifuged at 3,000 rpm for 10 minutes to separate insoluble portions. The supernatant was filtered using an ultrafiltration membrane having a molecular weight of 20,000 to 30,000 Da. The filtrate was dialyzed against deionized water cooled to 4 ° C. using an ultrafiltration membrane dialysis module having a molecular weight of 3,000 Da until the pH of the outer solution reached about 5 to 6 to remove citric acid. The solution is again subjected to high-pressure steam sterilization and ultrafiltration membrane treatment after removing the insoluble portion by the ultrafiltration membrane, and finally sterilized by a filter having a pore size of 0.22 μm to be completely aseptic. The filtrate was spray-dried with a spray dryer to obtain water-soluble elastin MA (about 1.0 kg).
（製造例４−中性不溶性フィブリリンの作製）
製造例２の水溶性エラスチンＭＥ製造の過程で、水酸化ナトリウムで中和した際に生じる沈殿を分離した。この沈殿１０Ｌ程度に対し水を１０Ｌ程度加えて撹拌し、水溶性の分画を完全に溶解させる。３，０００ｒｐｍで１０分間遠心分離し上澄みを除いた後に、再度１０Ｌ程度の水を加え撹拌し、この操作を繰り返し、完全に溶解物が無くなるまで洗浄を行う。沈殿部分を温風乾燥機にて乾燥し、中性不溶性フィブリリンを（約２００ｇ）を得た。なお、中性不溶性フィブリリンはｐＨ９以上の溶液では完全溶解するため、アルカリ溶解性・水溶性フィブリリンでもある。
(Production Example 4-Preparation of neutral insoluble fibrillin)
In the course of the production of water-soluble elastin ME in Production Example 2, the precipitate produced when neutralized with sodium hydroxide was separated. About 10 L of water is added to about 10 L of the precipitate and stirred to completely dissolve the water-soluble fraction. After centrifuging at 3,000 rpm for 10 minutes and removing the supernatant, about 10 L of water is added and stirred again, and this operation is repeated until washing is completely performed. The precipitated portion was dried with a warm air dryer to obtain neutral insoluble fibrillin (about 200 g). Since neutral insoluble fibrillin is completely dissolved in a solution of pH 9 or higher, it is also an alkali-soluble and water-soluble fibrillin.
（製造例５−中性溶解性・水溶性フィブリリンの作製）
乾燥した不溶性フィブリリン２００ｇに対し、６Ｍ尿素を５L加え、トリスヒドロキシアミノメタン６０ｇ、ＥＤＴＡ７０ｇおよびＬ−システイン５０ｇを加え撹拌した。４０℃で４８時間加熱した。分解反応液を分子量１０,０００〜１４,０００Ｄａの透析チューブを用い4℃に冷却した脱イオン水に対し、外液のｐＨが５〜６程度になるまで透析を行って尿素、ＥＤＴＡおよびシステインを除去した。１０，０００ｒｐｍで１０分間遠心分離し不溶部分を分離し、更に上澄み液を１２０℃にて高圧蒸気滅菌を行った後、孔サイズが０．２２μｍの滅菌フィルターでろ過した。ろ液をスプレードライヤーにて噴霧乾燥して水溶性フィブリリン（約１０ｇ）を得た。
(Production Example 5-Production of Neutral Soluble / Water-Soluble Fibrilin)
To 200 g of dried insoluble fibrillin, 5 L of 6M urea was added, and 60 g of trishydroxyaminomethane, 70 g of EDTA and 50 g of L-cysteine were added and stirred. Heated at 40 ° C. for 48 hours. The decomposition reaction solution is dialyzed against deionized water cooled to 4 ° C. using a dialysis tube having a molecular weight of 10,000 to 14,000 Da until the pH of the outer solution becomes about 5 to 6, and urea, EDTA and cysteine are removed. Removed. The insoluble portion was separated by centrifugation at 10,000 rpm for 10 minutes, and the supernatant was further autoclaved at 120 ° C. and then filtered through a sterilizing filter having a pore size of 0.22 μm. The filtrate was spray-dried with a spray dryer to obtain water-soluble fibrillin (about 10 g).
（実施例１−水溶性フィブリリンのアミノ酸組成）
中性溶解性・水溶性フィブリリンのアミノ酸組成は、試料を６Ｎ塩酸加水分解（１００℃、２４時間）処理した後、ＨＰＬＣにより分析した。製造例４で製造した水溶性フィブリリンの結果を表１に示す。なお、フィブリリン―１の理論値におけるシステインの含有率は１２．５７モル%であるが、システインは酸加水分解の際にシステイン酸誘導体に変換されるため、表１においては分析不能で表現される。そこでシステインの部分を抜いた理論値を計算し示した。測定した水溶性フィブリリンに関しては理論値との一致が多く、その純度は高いものと推定される。
Ｇｌｙ：グリシン、Ａｌａ：アラニン、Ｖａｌ：バリン、Ｐｒｏ：プロリン、Ｐｈｅ：フェニルアラニン、Ｌｅｕ：ロイシン、Ｉｌｅ：イソロイシン、Ｓｅｒ：セリン、Ｔｈｒ：スレオニン、Ｔｙｒ：チロシン、Ｌｙｒ：リシン、Ａｒｇ：アルギニン、Ａｓｐ＋Ａｓｎ：アスパラギン酸＋アスパラギン、Ｇｌｕ＋Ｇｌｎ：グルタミン酸＋グルタミン、Ｈｉｓ：ヒスチジン、Ｃｙｓ：システイン、Ｍｅｔ：メチオニン
Example 1-Amino acid composition of water-soluble fibrillin
The amino acid composition of the neutral soluble / water soluble fibrillin was analyzed by HPLC after treating the sample with 6N hydrochloric acid hydrolysis (100 ° C., 24 hours). The results of the water-soluble fibrillin produced in Production Example 4 are shown in Table 1. The cysteine content in the theoretical value of fibrillin-1 is 12.57 mol%. However, since cysteine is converted into a cysteic acid derivative during acid hydrolysis, it is expressed in Table 1 as being unanalysable. . Therefore, the theoretical value without cysteine was calculated and shown. The measured water-soluble fibrillin has a lot of agreement with the theoretical value, and its purity is estimated to be high.
Gly: glycine, Ala: alanine, Val: valine, Pro: proline, Phe: phenylalanine, Leu: leucine, Ile: isoleucine, Ser: serine, Thr: threonine, Tyr: tyrosine, Lyr: lysine, Arg: arginine, Asp + Asn: Aspartic acid + asparagine, Glu + Gln: glutamic acid + glutamine, His: histidine, Cys: cysteine, Met: methionine
（実施例２−水溶性エラスチンＭＥ/水溶性フィブリリン混合溶液の透過光強度測定）
水溶性エラスチンＭＥと水溶性フィブリリンの溶液中での相互作用を測定した。水溶性エラスチンの特徴的な現象の一つとして、温度を上げると凝集するコアセルべーションと呼ばれる現象がある。この複合体化による凝集状態は溶液の透過光強度を測定することで規定できる。具体的には特定波長（６４０nm）を試料溶液に照射し、入射光強度に対する透過光強度の割合を分光光度計により透過率として検出した。温度を２５℃で固定し透過率を測定した。製造例２および５で作成した水溶性エラスチンＭＥと水溶性フィブリリンの総濃度を１重量％に調整し、組成比率を０〜１００％まで変化させて透過率を測定した。その結果を表２および表３に示した。この結果から水溶性エラスチンＭＥと水溶性フィブリリンの混合比率が９４：６の場合で最も透過率が低く凝集性が大きい、即ち相互作用が大きいことが判明した。一般的な混合溶液の凝集挙動は、片側の特性からもう一方の特性までの間の連続的な変化として観察されるが、水溶性エラスチンと水溶性フィブリリンの場合は特有の濃度での変化が顕著である点から、特定の混合比率における複合体の増強化が示唆できる。
The interaction of water-soluble elastin ME and water-soluble fibrillin in solution was measured. One of the characteristic phenomena of water-soluble elastin is a phenomenon called coacervation that aggregates when the temperature is raised. The aggregation state due to the complexation can be defined by measuring the transmitted light intensity of the solution. Specifically, the sample solution was irradiated with a specific wavelength (640 nm), and the ratio of the transmitted light intensity to the incident light intensity was detected as a transmittance by a spectrophotometer. The transmittance was measured with the temperature fixed at 25 ° C. The total concentration of the water-soluble elastin ME and the water-soluble fibrillin prepared in Production Examples 2 and 5 was adjusted to 1% by weight, and the transmittance was measured by changing the composition ratio from 0 to 100%. The results are shown in Tables 2 and 3. From this result, it was found that when the mixing ratio of water-soluble elastin ME and water-soluble fibrillin is 94: 6, the transmittance is the lowest and the cohesion is large, that is, the interaction is large. The general aggregation behavior of a mixed solution is observed as a continuous change from one characteristic to the other, but in the case of water-soluble elastin and water-soluble fibrillin, the change at a specific concentration is remarkable. From this point, enhancement of the complex at a specific mixing ratio can be suggested.
（実施例３−水溶性エラスチンの添加によるエラスチン産生試験）
外部から細胞に対して刺激を与えることにより、自らが産生した細胞外基質（コラーゲンおよびエラスチン）の相対的な量を比較しエラスチンの産生挙動を調査した。市販の正常ヒト皮膚線維芽細胞２.０×１０３（cells／well）を３５φ培養皿に播種し、１０％ＦＢＳ入りＤＭＥＭ培地中（増殖培地）で３７℃、５％ＣＯ2培養装置（ＯＬＩＭＰＵＳ ＭＩ−ＩＢＣ）でコンフルエントに到達するまで培養した。その後ＦＢＳ濃度を１％まで低下させ増殖を停止させた。水溶性エラスチン添加培地は１％ＦＢＳ入りＤＭＥM培地に添加タンパクの最終濃度が５００μg/mlになるように製造例２で製造した水溶性エラスチンＭＥと、製造例３で製造した水溶性エラスチンＭＡを添加した。培養は７日間行い、培地交換はせずに７日目に培地中のコラーゲン濃度およびエラスチン濃度をそれぞれコラーゲンアッセイおよびエラスチンアッセイにより測定し、この値を細胞外産生濃度とした。
(Example 3-Elastin production test by addition of water-soluble elastin)
By stimulating the cells from the outside, we compared the relative amounts of extracellular matrix (collagen and elastin) produced by themselves, and investigated the production behavior of elastin. Commercially available normal human dermal fibroblasts 2.0 × 10 3 (cells / well) were seeded in a 35φ culture dish, and cultured in DMEM medium (growth medium) containing 10% FBS at 37 ° C., 5% CO 2 culture apparatus (OLIMPUS). The cells were cultured until they reached confluence with MI-IBC). Thereafter, the FBS concentration was reduced to 1% to stop the growth. Water-soluble elastin-added medium is added to DMEM medium containing 1% FBS with water-soluble elastin ME produced in Production Example 2 and water-soluble elastin MA produced in Production Example 3 so that the final concentration of the added protein is 500 μg / ml. did. The culture was performed for 7 days, and the collagen concentration and the elastin concentration in the medium were measured by the collagen assay and the elastin assay, respectively, on the seventh day without changing the medium.
培地を回収後の培養シャーレをＰＢＳで洗浄する。その後４％パラホルムアルデヒド（０．１Ｍリン酸バッファー）で１５分固定化し、再度ＰＢＳで洗浄した。１％ＢＳＡを含むＰＢＳ中で２０分間処理し、一次抗体としてanti-human Collagen 抗体またはanti-human
tropoelastin 抗体を加え４℃で一夜静置する。その後一次抗体を除去し、二次抗体としてanti-IgG
FITC抗体とPIを加え、９０分間暗所にて静置し、最後にＰＢＳにて３回洗浄した。観察は共焦点レーザー顕微鏡にて行った。（細胞内産生濃度）
それぞれ、無添加条件（コントロール）の結果で規格化することで、コントロール値を１として図２(a)および(b)に示した。更にコラーゲン産生能に対するエラスチン産生能という意味で、(a)および(b)の値を以下の式に当てはめて算出した。
[（細胞内エラスチン）×（細胞外エラスチン）]/ [（細胞内コラーゲン）×（細胞外コラーゲン）]
結果を図２(c)に示した。水溶性エラスチンＭＥの添加条件では、皮膚線維芽細胞のエラスチン産生が促進され、コラーゲン産生が抑制され、コラーゲンの産生量に対するエラスチンの産生量で示される産生バランスがコントロール値（１：１）に比較して、水溶性エラスチンＭＥの刺激により７．１と他の条件に比較して顕著にエラスチンの産生が向上することがわかった。
The culture dish after collecting the medium is washed with PBS. Thereafter, the mixture was fixed with 4% paraformaldehyde (0.1 M phosphate buffer) for 15 minutes and washed again with PBS. Treated in PBS containing 1% BSA for 20 minutes, anti-human Collagen antibody or anti-human as the primary antibody
Add tropoelastin antibody and let stand at 4 ° C overnight. Then remove the primary antibody and anti-IgG as the secondary antibody
FITC antibody and PI were added, left still in the dark for 90 minutes, and finally washed 3 times with PBS. Observation was performed with a confocal laser microscope. (Intracellular production concentration)
The results are shown in FIGS. 2 (a) and 2 (b), assuming that the control value is 1 by normalizing with the results of the non-addition condition (control). Further, in the sense of elastin production ability relative to collagen production ability, the values of (a) and (b) were calculated by applying the following formula.
[(Intracellular elastin) x (extracellular elastin)] / [(Intracellular collagen) x (extracellular collagen)]
The results are shown in FIG. Under the condition of adding water-soluble elastin ME, elastin production of skin fibroblasts is promoted, collagen production is suppressed, and the production balance indicated by the production amount of elastin relative to the production amount of collagen is compared with the control value (1: 1). Thus, it was found that the production of elastin was significantly improved by the stimulation of water-soluble elastin ME as compared to 7.1 and other conditions.
（実施例４−水溶性エラスチンＭＥおよび水溶性フィブリリンとの共添加培地による皮膚線維芽細胞表面上での弾性線維形成試験）
エラスチンなどの細胞外基質タンパク質は、培養細胞表面上に堆積または自己組織化を行い網目状の線維構造を構築することが知られる。この網目状の線維構造中のエラスチンを免疫染色により検出することで、細胞に対する応答を評価することができる。弾性線維増強試験の原理は、細胞表面上に細胞が産生し堆積したエラスチンマトリックスの量を測定し、細胞１つ当たりで換算し、刺激なしのコントロールの状態と比較することで増強されたか否かを判断する試験法である。
(Example 4-Elastic fiber formation test on the surface of dermal fibroblasts in a medium supplemented with water-soluble elastin ME and water-soluble fibrillin)
It is known that extracellular matrix proteins such as elastin are deposited or self-assembled on the surface of cultured cells to construct a reticulated fiber structure. By detecting elastin in the mesh-like fiber structure by immunostaining, the response to cells can be evaluated. The principle of the elastic fiber enhancement test is whether it was enhanced by measuring the amount of elastin matrix produced and deposited on the cell surface, converted per cell, and compared to the unstimulated control state. This is a test method for judging
市販の正常ヒト皮膚線維芽細胞２.０×１０３（cells／well）を３５φ培養皿に播種し、１０％ＦＢＳ入りＤＭＥＭ培地中（増殖培地）で３７℃、５％ＣＯ2培養装置（ＯＬＩＭＰＵＳ ＭＩ−ＩＢＣ）でコンフルエントに到達するまで培養した。その後ＦＢＳ濃度を１％まで低下させ増殖性を停止させた。水溶性エラスチン添加培地は１％ＦＢＳ入りＤＭＥＭ培地に添加タンパクの最終濃度が５００μg/mlになるように製造例２で製造した水溶性エラスチンＭＥと、製造例４で製造した水溶性フィブリリンを添加した。培地は３日に一度交換した。培養は最終的に１０日間行い、１０日目に抗エラスチン抗体による免疫染色を行った。無添加条件をコントロールとした。 Commercially available normal human dermal fibroblasts 2.0 × 10 3 (cells / well) were seeded in a 35φ culture dish, and cultured in DMEM medium (growth medium) containing 10% FBS at 37 ° C., 5% CO 2 culture apparatus (OLIMPUS). The cells were cultured until they reached confluence with MI-IBC). Thereafter, the FBS concentration was decreased to 1% to stop the proliferation. The water-soluble elastin-added medium was added to the DMEM medium containing 1% FBS with the water-soluble elastin ME produced in Production Example 2 and the water-soluble fibrillin produced in Production Example 4 so that the final concentration of the added protein was 500 μg / ml. . The medium was changed once every 3 days. The culture was finally performed for 10 days, and immunostaining with an anti-elastin antibody was performed on the 10th day. The additive-free condition was used as a control.
培養後の培養シャーレから培地を静かに除去し、ＰＢＳで洗浄する。その後４％パラホルムアルデヒド（０．１Ｍリン酸バッファー）で１５分固定化し、再度ＰＢＳで洗浄した。１％ＢＳＡを含むＰＢＳ中で２０分間処理し、一次抗体としてanti-human tropoelastin 抗体を加え４℃で一夜静置する。その後一次抗体を除去し、二次抗体としてanti-IgG FITC抗体とPIを加え、９０分間暗所にて静置し、最後にＰＢＳにて３回洗浄した。観察は共焦点レーザー顕微鏡にて行った。染色結果を図３に示した。緑色がエラスチンを赤色が細胞核を表す。コントロールでも少し弾性線維が観察されるが図３(a)、水溶性エラスチンＭＥを添加した条件でその線維量の増加が確認できる図３(b)。水溶性フィブリリンを同時に添加したものに関しては、更に線維の量が多くより太くなっているのがわかる図３(c)。この染色写真を同一条件でランダムに異なる場所を７カ所撮影し、イメージアナライザーにより染色強度を測定し、更に視野内の細胞数で規格化したグラフを図４に示した。このグラフにより、水溶性エラスチンＭＥを添加したもので１．１倍、更に水溶性フィブリリンを添加した条件で１．２倍にまで弾性線維が増強されていることが明らかになった。単純に計算すると弾性線維の１．２倍の増加は、弾性特性としては最大荷重が１．２倍に相当し、すなわち組織に対する弾性が１０日間で２０％増強したことになることがわかった。 Gently remove the medium from the culture dish after culturing and wash with PBS. Thereafter, the mixture was fixed with 4% paraformaldehyde (0.1 M phosphate buffer) for 15 minutes and washed again with PBS. Treat in PBS containing 1% BSA for 20 minutes, add anti-human tropoelastin antibody as the primary antibody, and allow to stand overnight at 4 ° C. Thereafter, the primary antibody was removed, anti-IgG FITC antibody and PI were added as secondary antibodies, and the mixture was allowed to stand for 90 minutes in the dark, and finally washed three times with PBS. Observation was performed with a confocal laser microscope. The staining results are shown in FIG. Green represents elastin and red represents the cell nucleus. Although some elastic fibers are observed even in the control, FIG. 3 (a), an increase in the amount of the fibers can be confirmed under the condition where water-soluble elastin ME is added. FIG. 3 (c) shows that the fiber to which water-soluble fibrillin was added at the same time was thicker and thicker. FIG. 4 shows a graph in which seven different places are randomly photographed under the same conditions, the staining intensity is measured with an image analyzer, and the number of cells in the field of view is normalized. From this graph, it was revealed that the elastic fiber was enhanced by 1.1 times when water-soluble elastin ME was added, and 1.2 times when water-soluble fibrillin was further added. A simple calculation shows that a 1.2-fold increase in elastic fiber corresponds to a maximum load of 1.2 times as an elastic property, that is, the elasticity to the tissue was increased by 20% in 10 days.
本発明によれば、生体組織から安全に水溶性のフィブリリンを製造することができる。これを水溶性のエラスチンと組み合わせることにより肌の弾力性を改善でき、飲食品、化粧品、医薬品等として用いることができるという産業上の利用可能性を有する。
According to the present invention, water-soluble fibrillin can be produced safely from living tissue. Combining this with water-soluble elastin can improve the elasticity of the skin and has industrial applicability that it can be used as food, drink, cosmetics, pharmaceuticals, and the like.
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