JP6991570B2 - Differentiation inducer, differentiation induction method, and method for producing bone tissue decomposition products used for these. - Google Patents
Differentiation inducer, differentiation induction method, and method for producing bone tissue decomposition products used for these. Download PDFInfo
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- JP6991570B2 JP6991570B2 JP2017547920A JP2017547920A JP6991570B2 JP 6991570 B2 JP6991570 B2 JP 6991570B2 JP 2017547920 A JP2017547920 A JP 2017547920A JP 2017547920 A JP2017547920 A JP 2017547920A JP 6991570 B2 JP6991570 B2 JP 6991570B2
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Images
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
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Description
本発明は、分化誘導剤、分化誘導方法、および、これらに用いられる骨組織分解物の製造方法に関する。 The present invention relates to a differentiation inducer, a method for inducing differentiation, and a method for producing a bone tissue decomposition product used therein.
従来から、骨組織を再生するための材料として、コラーゲン、アパタイト、ナノファイバー、ポリ乳酸、または、これらの混合物などが用いられてきた（例えば、特許文献１および２参照）。
Traditionally, collagen, apatite, nanofibers, polylactic acid, or a mixture thereof have been used as materials for regenerating bone tissue (see, for example,
上述した材料として、例えば、特許文献１には、（ｉ）βリン酸三カルシウム多孔体からなる微小顆粒を含む補填体と、（ｉｉ）ポリテトラフルオロエチレン、ポリ乳酸、ポリ乳酸とポリグリコール酸および／またはポリカプロラクトンとの混合物、若しくは、これらとコラーゲンとの複合体からなる膜部材と、を備える骨再生材料が開示されている。
As the above-mentioned materials, for example,
特許文献２には、（ｉ）繊維と、（ｉｉ）ポリ乳酸、ポリグリコール酸、ポリ乳酸－ポリグリコール酸共重合体、ポリカプロラクトン、キチン、コラーゲン、ポリリジン、ポリアルギニン、ヒアルロン酸、セリシン、セルロース、デキストラン、および、プルランからなる群より選択される少なくとも１種である生体適合性ポリマーと、（ｉｉｉ）β－リン酸三カルシウム（β－ＴＣＰ）、α－リン酸三カルシウム（α－ＴＣＰ）、ハイドロキシアパタイト（ＨＡ）、第２リン酸カルシウム（ＤＣＰＤ）、オクタカルシウムフォスフェート（ＯＣＰ）、テトラカルシウムフォスフェート（４ＣＰ）、アルミナ、ジルコニア、カルシウムアルミネート（ＣａＯ－Ａｌ２Ｏ３）、アルミノシリケート（Ｎａ２Ｏ－Ａｌ２Ｏ３－ＳｉＯ２）、生体活性化ガラス、石英、および、炭酸カルシウムからなる群より選択される少なくとも１種である骨補填材と、を備える骨再生用材料が開示されている。
しかしながら、骨組織を新しく再生する技術に対する要求は多く、骨組織の再生などに用いることが可能な、新たな材料および手法の開発が望まれている。 However, there are many demands for a technique for newly regenerating bone tissue, and development of a new material and method that can be used for regenerating bone tissue is desired.
本発明は、上記課題に鑑みてなされたものであり、その目的は、骨組織の再生などに用いることが可能な、分化誘導剤および分化誘導方法、並びに、これらに用いられる骨組織分解物および骨組織分解物の製造方法を提供することにある。 The present invention has been made in view of the above problems, and an object thereof is a differentiation-inducing agent and a differentiation-inducing method that can be used for regeneration of bone tissue, and bone tissue decomposition products and bone tissue decomposition products used for these. The purpose is to provide a method for producing a bone tissue decomposition product.
本発明者らは、骨組織分解物の内容物中にこそ、骨組織の分化に必要な成分が含まれているとの独自の仮説に基づいて、骨組織分解物の作製を試みた。 The present inventors attempted to prepare a bone tissue decomposition product based on the original hypothesis that the content of the bone tissue decomposition product contains a component necessary for the differentiation of bone tissue.
本発明者らは、鋭意検討した結果、特定の条件下で骨組織とシステインプロテアーゼとを反応させることにより、（ｉ）広義の骨組織分解物を得ることができること、（ｉｉ）当該骨組織分解物は、幹細胞を、骨関連細胞へ分化させる生理活性を有していること、を見出し、本発明を完成させるに至った。 As a result of diligent studies, the present inventors can obtain (i) a bone tissue decomposition product in a broad sense by reacting bone tissue with a cysteine protease under specific conditions, and (ii) the bone tissue decomposition. It has been found that the substance has a physiological activity that differentiates stem cells into bone-related cells, and has completed the present invention.
＜１＞本発明の一実施形態に係る骨組織分解物の製造方法は、ｐＨ２．５以上ｐＨ５．５以下の溶液中で、システインプロテアーゼと骨組織とを反応させる工程を有することを特徴している。 <1> The method for producing a bone tissue decomposition product according to an embodiment of the present invention is characterized by comprising a step of reacting cysteine protease with bone tissue in a solution having a pH of 2.5 or more and a pH of 5.5 or less. There is.
＜２＞本発明の一実施形態に係る骨組織分解物の製造方法では、上記溶液は、ｐＨ３．０以上ｐＨ５．５以下の溶液であることが好ましい。 <2> In the method for producing a bone tissue decomposition product according to an embodiment of the present invention, the solution is preferably a solution having a pH of 3.0 or more and a pH of 5.5 or less.
＜３＞本発明の一実施形態に係る幹細胞を骨関連細胞へ分化させるための分化誘導剤は、本発明の一実施形態に係る骨組織分解物の製造方法製造方法によって製造された骨組織分解物を含んでいるものであることを特徴としている。 <3> The differentiation-inducing agent for differentiating the stem cells according to the embodiment of the present invention into bone-related cells is the bone tissue decomposition produced by the method for producing a bone tissue decomposition product according to the embodiment of the present invention. It is characterized by containing things.
＜４＞本発明の一実施形態に係る幹細胞を骨関連細胞へ分化させるための分化誘導剤では、上記骨関連細胞は、骨芽細胞、破骨細胞、象牙芽細胞、セメント芽細胞、セメント細胞、歯根膜細胞、または、骨細胞であることが好ましい。 <4> In the differentiation-inducing agent for differentiating the stem cells according to the embodiment of the present invention into bone-related cells, the bone-related cells are osteoblasts, osteoblasts, ivory blasts, cementoblasts, cement cells. , Root membrane cells, or bone cells are preferred.
＜５＞本発明の一実施形態に係る、幹細胞を骨関連細胞へ分化させる分化誘導方法は、本発明の一実施形態に係る幹細胞を骨関連細胞へ分化させるための分化誘導剤を被覆させた基材上で幹細胞を培養する工程を有することを特徴としている。 <5> The differentiation-inducing method for differentiating stem cells into bone-related cells according to one embodiment of the present invention is coated with a differentiation-inducing agent for differentiating stem cells into bone-related cells according to one embodiment of the present invention. It is characterized by having a step of culturing stem cells on a substrate.
＜６＞本発明の一実施形態に係る、幹細胞を骨関連細胞へ分化させる分化誘導方法では、上記骨関連細胞は、骨芽細胞、破骨細胞、象牙芽細胞、セメント芽細胞、セメント細胞、歯根膜細胞、または、骨細胞であることが好ましい。 <6> In the method for inducing differentiation of stem cells into bone-related cells according to an embodiment of the present invention, the bone-related cells include osteoblasts, osteoclasts, ivory blasts, cementoblasts, and cement cells. It is preferably root membrane cells or bone cells.
本発明の一実施形態は、幹細胞を骨関連細胞へ分化させる活性を有する骨組織分解物を得ることができるという効果を奏する。 One embodiment of the present invention has the effect of being able to obtain a bone tissue degradation product having an activity of differentiating stem cells into bone-related cells.
本発明の一実施形態について以下に説明するが、本発明はこれに限定されるものではない。本発明は、以下に説明する各構成に限定されるものではなく、特許請求の範囲に示した範囲で種々の変更が可能であり、異なる実施形態や実施例にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態や実施例についても本発明の技術的範囲に含まれる。また、本明細書中に記載された学術文献及び特許文献の全てが、本明細書中において参考文献として援用される。また、本明細書において特記しない限り、数値範囲を表す「Ａ～Ｂ」は、「Ａ以上、Ｂ以下」を意図する。 An embodiment of the present invention will be described below, but the present invention is not limited thereto. The present invention is not limited to the configurations described below, and various modifications can be made within the scope of the claims, and the technical means disclosed in different embodiments and examples can be used. Embodiments and examples obtained by appropriately combining them are also included in the technical scope of the present invention. In addition, all the academic and patent documents described in the present specification are incorporated as references in the present specification. Further, unless otherwise specified in the present specification, "AB" representing a numerical range is intended to be "A or more and B or less".
〔１．骨組織分解物の製造方法〕
本実施の形態の骨組織分解物の製造方法では、特定のｐＨの溶液中で、システインプロテアーゼと骨組織とを反応させる。[1. Manufacturing method of bone tissue decomposition products]
In the method for producing a bone tissue decomposition product of the present embodiment, the cysteine protease is reacted with the bone tissue in a solution having a specific pH.
後述する実施例でも述べるように、本発明者は、ｐＨが酸性側であるほど骨組織の分解効率は高くなる傾向を示すが、骨組織分解物が有する、幹細胞を骨関連細胞へ分化させる活性は低くなる傾向を示し、ｐＨがアルカリ性側であるほど骨組織の分解効率は低くなる傾向を示すが、骨組織分解物が有する、幹細胞を骨関連細胞へ分化させる活性は高くなる傾向を示すことを見出した。 As described in Examples described later, the present inventor tends to increase the efficiency of bone tissue decomposition as the pH is on the acidic side, but the activity of the bone tissue decomposition product to differentiate stem cells into bone-related cells. Shows a tendency to decrease, and the more alkaline the pH, the lower the efficiency of bone tissue degradation, but the activity of bone tissue degradation products to differentiate stem cells into bone-related cells tends to increase. I found.
技術常識にしたがって考えれば、骨組織の分解効率が高いほど（換言すれば、溶液のｐＨが適正な酸性ｐＨであればあるほど）骨組織分解物中に生理活性物質が溶解し、当該骨組織分解物が有する生理活性が高くなると考えられるが、本発明者が見出した上記知見は、技術常識からは予測できない、新たな知見であった。 According to common general knowledge, the higher the decomposition efficiency of bone tissue (in other words, the more appropriate acidic pH the solution has), the more the bioactive substance dissolves in the bone tissue decomposition product, and the more the bone tissue is concerned. It is considered that the bioactivity of the decomposition product is increased, but the above-mentioned findings found by the present inventor are new findings that cannot be predicted from the common general knowledge of technology.
上述したように、本発明の骨組織分解物は、幹細胞を骨関連細胞へ分化させる。このとき、幹細胞は、特に限定されず、その具体例として、骨髄由来間葉系幹細胞、脂肪由来間葉系幹細胞、羊膜由来間葉系幹細胞、ＥＳ細胞（embryonic stem cell）、ｉＰＳ細胞（induced pluripotent stem cell）、および、Ｍｕｓｅ細胞（Multi-lineage differentiating Stress Enduring cell）を挙げることができる。一方、骨関連細胞も、特に限定されず、その具体例として、骨芽細胞、破骨細胞、象牙芽細胞、セメント芽細胞、セメント細胞、歯根膜細胞、または、骨細胞を挙げることができる。 As mentioned above, the bone tissue degradation products of the present invention differentiate stem cells into bone-related cells. At this time, the stem cells are not particularly limited, and specific examples thereof include bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, sheep membrane-derived mesenchymal stem cells, ES cells (embryonic stem cells), and iPS cells (induced pluripotent). Stem cells) and Muse cells (Multi-lineage differentiating Stress Enduring cells) can be mentioned. On the other hand, the bone-related cells are not particularly limited, and specific examples thereof include osteoblasts, osteoclasts, ivory blasts, cementoblasts, cement cells, root membrane cells, and bone cells.
本発明では、特定のｐＨの溶液とシステインプロテアーゼとを用いて得られた骨組織分解物によって、幹細胞を骨関連細胞へ分化させることができる。 In the present invention, stem cells can be differentiated into bone-related cells by a bone tissue degradation product obtained by using a solution having a specific pH and a cysteine protease.
具体的に、本実施の形態の骨組織分解物の製造方法は、ｐＨ２．５以上ｐＨ５．５以下の溶液中で、システインプロテアーゼと骨とを反応させる工程を有することを特徴としている。 Specifically, the method for producing a bone tissue decomposition product of the present embodiment is characterized by having a step of reacting cysteine protease with bone in a solution having a pH of 2.5 or more and a pH of 5.5 or less.
上記溶液のｐＨは、上述したようにｐＨ２．５以上ｐＨ５．５以下であるが、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現するという観点からは、ｐＨ３．０以上５．５以下（または、ｐＨ３．０よりも高く５．５以下）であることが更に好ましく、ｐＨ３．５以上５．５以下（または、ｐＨ３．５よりも高く５．５以下）であることが更に好ましく、ｐＨ４．０以上５．５以下（または、ｐＨ４．０よりも高く５．５以下）であることが更に好ましく、ｐＨ４．５以上５．５以下（または、ｐＨ４．５よりも高く５．５以下）であることが更に好ましく、ｐＨ５．０以上５．５以下（または、ｐＨ５．０よりも高く５．５以下）であることが最も好ましい。 The pH of the above solution is pH 2.5 or more and pH 5.5 or less as described above, but from the viewpoint of realizing a bone tissue decomposition product having a higher activity of differentiating stem cells into bone-related cells, pH 3.0 or more. It is more preferably 5.5 or less (or higher than pH 3.0 and 5.5 or less), and more preferably pH 3.5 or more and 5.5 or less (or higher than pH 3.5 and 5.5 or less). Is more preferably, pH 4.0 or more and 5.5 or less (or higher than pH 4.0 and 5.5 or less), and pH 4.5 or more and 5.5 or less (or higher than pH 4.5). It is more preferably 5.5 or less), and most preferably pH 5.0 or more and 5.5 or less (or higher than pH 5.0 and 5.5 or less).
上記溶液のｐＨは、ｐＨ２．５以上ｐＨ５．５未満であってもよいが、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現するという観点からは、ｐＨ３．０以上５．５未満（または、ｐＨ３．０よりも高く５．５未満）であることが更に好ましく、ｐＨ３．５以上５．５未満（または、ｐＨ３．５よりも高く５．５未満）であることが更に好ましく、ｐＨ４．０以上５．５未満（または、ｐＨ４．０よりも高く５．５未満）であることが更に好ましく、ｐＨ４．５以上５．５未満（または、ｐＨ４．５よりも高く５．５未満）であることが更に好ましく、ｐＨ５．０以上５．５未満（または、ｐＨ５．０よりも高く５．５未満）であることが最も好ましい。 The pH of the above solution may be pH 2.5 or higher and lower than pH 5.5, but from the viewpoint of realizing a bone tissue decomposition product having a higher activity of differentiating stem cells into bone-related cells, pH 3.0 or higher 5 It is more preferably less than 5.5 (or higher than pH 3.0 and less than 5.5), and more than pH 3.5 and less than 5.5 (or higher than pH 3.5 and less than 5.5). More preferably, the pH is 4.0 or more and less than 5.5 (or higher than pH 4.0 and less than 5.5), more preferably pH 4.5 or more and less than 5.5 (or higher than pH 4.5 and less than 5.5) 5 It is more preferably less than .5), and most preferably pH 5.0 or more and less than 5.5 (or higher than pH 5.0 and less than 5.5).
上記溶液のｐＨは、周知の緩衝液を用いて、所望のｐＨに調節することができる。当該緩衝液としては、特に限定されないが、例えば、塩化カリウム－塩酸緩衝液、ｐ－トルエンスルホン酸－ｐ－ナトリウム緩衝液、グリシン－塩酸緩衝液、フタル酸水素カリウム－塩酸緩衝液、酒石酸－水酸化ナトリウム緩衝液、クエン酸－リン酸二ナトリウム緩衝液、クエン酸－クエン酸ナトリウム緩衝液、ｔｒａｎｓ－アコニチン酸－水酸化ナトリウム緩衝液、蟻酸－蟻酸ナトリウム緩衝液、β，β’－ジメチルグルタル酸－水酸化ナトリウム緩衝液、フェニル酢酸－フェニル酢酸ナトリウム緩衝液、酢酸－酢酸ナトリウム緩衝液、コハク酸－水酸化ナトリウム緩衝液、フタル酸水素カリウム－水酸化ナトリウム緩衝液、カコジル酸ナトリウム－塩酸緩衝液、マレイン酸ナトリウム－水酸化ナトリウム緩衝液、リン酸緩衝液、イミダゾール－塩酸緩衝液、２，４，６－トリメチルピリジン－塩酸緩衝液、トリエタノールアミン・塩酸－水酸化ナトリウム緩衝液、Ｎ－エチルモルフォリン－塩酸緩衝液、ＭＥＳ緩衝液、および、ＨＥＰＥＳ緩衝液を挙げることができる。 The pH of the solution can be adjusted to the desired pH using a well-known buffer. The buffer solution is not particularly limited, and is, for example, potassium chloride-hydrochloride buffer, p-toluenesulfonic acid-p-sodium buffer, glycine-hydrogen buffer, potassium hydrogenphthalate-hydrogen buffer, tartrate-water. Sodium oxide buffer, citrate-disodium phosphate buffer, citrate-sodium citrate buffer, trans-aconitinic acid-sodium hydroxide buffer, formic acid-sodium nitate buffer, β, β'-dimethylglutaric acid -Sodium hydroxide buffer, phenylacetic acid-sodium phenylacetate buffer, acetic acid-sodium acetate buffer, succinic acid-sodium hydroxide buffer, potassium hydrogen phthalate-sodium hydroxide buffer, sodium cacodylate-hydrochloride buffer , Sodium maleate-sodium hydroxide buffer, phosphate buffer, imidazole-hydrochloride buffer, 2,4,6-trimethylpyridine-hydrochloride buffer, triethanolamine / hydrochloride-sodium hydroxide buffer, N-ethyl Examples include morpholin-hydrochloride buffer, MES buffer, and HEEPS buffer.
上記溶液には、所望の塩が所望の濃度、含まれていてもよい。システインプロテアーゼは、塩の濃度に応じて、基質特異性（換言すれば、システインプロテアーゼによって切断される切断箇所）が変化する。それ故に、上記構成によれば、骨組織分解物に含まれる、生理活性物質の種類、および／または、生理活性物質の量を調節することができる。 The above solution may contain a desired salt in a desired concentration. The substrate specificity (in other words, the cleavage site cleaved by the cysteine protease) of the cysteine protease changes depending on the concentration of the salt. Therefore, according to the above configuration, the type of bioactive substance and / or the amount of the bioactive substance contained in the bone tissue decomposition product can be adjusted.
上記塩の具体的な構成としては特に限定されないが、塩化物を用いることが好ましい。塩化物としては、特に限定されないが、例えば、ＮａＣｌ、ＫＣｌ、ＬｉＣｌまたはＭｇＣｌ２を用いることが可能である。The specific composition of the salt is not particularly limited, but it is preferable to use chloride. The chloride is not particularly limited, but for example, NaCl, KCl, LiCl or MgCl 2 can be used.
上記溶液における塩の濃度は、特に限定されず、０ｍＭ以上、２０ｍＭ以上、１００ｍＭ以上、１５０ｍＭ以上、２００ｍＭ以上、５００ｍＭ以上、１，０００ｍＭ以上、１，５００ｍＭ以上、２，０００ｍＭ以上であってもよい。上記溶液における塩の濃度の上限値は、特に限定されず、２，０００ｍＭであってもよい。上述したように、溶液における塩の濃度は０～２，０００ｍＭであり得るが、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現するという観点からは、０ｍＭ～５００ｍＭが好ましく、０ｍＭ～２００ｍＭが更に好ましい。 The concentration of the salt in the above solution is not particularly limited and may be 0 mM or more, 20 mM or more, 100 mM or more, 150 mM or more, 200 mM or more, 500 mM or more, 1,000 mM or more, 1,500 mM or more, 2,000 mM or more. .. The upper limit of the salt concentration in the above solution is not particularly limited and may be 2,000 mM. As mentioned above, the concentration of salt in the solution can be 0 to 2,000 mM, but 0 mM to 500 mM is preferable from the viewpoint of realizing a bone tissue decomposition product having a higher activity of differentiating stem cells into bone-related cells. , 0 mM to 200 mM is more preferable.
本発明では、予め前処理用の溶液にシステインプロテアーゼを溶解させた後、当該システインプロテアーゼと骨組織とを、特定のｐＨの溶液中で反応させてもよい。この場合の前処理用の溶液としては、上述した所望の塩が所望の濃度含まれている溶液と同じ溶液を用いることができる。前処理用の溶液における塩の濃度は０～２，０００ｍＭであり得るが、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現するという観点からは、０ｍＭ～５００ｍＭが好ましく、０ｍＭ～２００ｍＭが更に好ましい。当該構成であっても、骨組織分解物に含まれる、生理活性物質の種類、および／または、生理活性物質の量を調節することができる。 In the present invention, cysteine protease may be previously dissolved in a pretreatment solution, and then the cysteine protease and bone tissue may be reacted in a solution having a specific pH. As the solution for pretreatment in this case, the same solution as the solution containing the above-mentioned desired salt at a desired concentration can be used. The concentration of the salt in the pretreatment solution can be 0 to 2,000 mM, but 0 mM to 500 mM is preferable from the viewpoint of realizing a bone tissue decomposition product having a higher activity of differentiating stem cells into bone-related cells. 0 mM to 200 mM is more preferable. Even with this configuration, the type of bioactive substance and / or the amount of the bioactive substance contained in the bone tissue decomposition product can be adjusted.
本発明では、システインプロテアーゼと骨組織とを反応させるための、特定のｐＨの溶液中に、セリンプロテアーゼ、アスパラギン酸プロテアーゼ、または、マトリックスメタロプロテアーゼなどの、システインプロテアーゼとは別種のタンパク質分解酵素の活性阻害剤を添加することが好ましい。その理由は、当該構成であれば、骨組織由来の可溶物が過分解することを防ぐことができるからである。 In the present invention, the activity of proteolytic enzymes other than cysteine protease, such as serine protease, aspartic protease, or matrix metalloprotease, in a solution at a specific pH for reacting cysteine protease with bone tissue. It is preferable to add an inhibitor. The reason is that the structure can prevent the solubilized material derived from bone tissue from being over-decomposed.
セリンプロテアーゼ阻害剤としては、ＰＭＳＦ（phenylmethanesulfonyl fluoride）、トリプスタチン、ロイペプチン、アンチパイン、ＤＥＰ、トリプシンインヒビターなどを挙げることができ、これらを、システインプロテアーゼと骨組織とを反応させるための溶液に適量加えることで所望の効果を得ることができる。アスパラギン酸プロテアーゼ阻害剤としては、ペプスタチン、Ｌ－３６３，５６４、Ｈ－２５６、Ｈ－２６１、Ｈ－７７などを挙げることができ、これらを、システインプロテアーゼと骨組織とを反応させるための溶液に適量加えることで所望の効果を得ることができる。マトリックスメタロプロテアーゼ阻害剤としては、ＥＤＴＡ（ethylenediaminetetraacetic acid）、オルト－フェナンスロリン、ＴＩＭＰ（tissue inhibitor of metalloproteinase）、α２－マクログロブリン、オボスタチン、Ｚ－フェニルアラニンなどを挙げることができ、これらを、システインプロテアーゼと骨組織とを反応させるための溶液に適量加えることで所望の効果を得ることができる。この場合の溶液としては、上述した溶液と同じ溶液を用いることができる。 Examples of the serine protease inhibitor include PMSF (phenylmethanesulfonyl fluoride), trypstatin, leupeptin, antipain, DEP, trypsin inhibitor and the like, and these are added in an appropriate amount to a solution for reacting cysteine protease with bone tissue. This makes it possible to obtain the desired effect. Examples of the aspartic protease inhibitor include pepstatin, L-363,564, H-256, H-261 and H-77, which are used as a solution for reacting cysteine protease with bone tissue. A desired effect can be obtained by adding an appropriate amount. Examples of the matrix metalloproteinase inhibitor include EDTA (ethylenediaminetetraacetic acid), ortho-phenanthroline, TIMP (tissue inhibitor of metalloproteinase), α2-macroglobulin, ovostatin, Z-phenylalanine and the like, and these are cysteine proteases. The desired effect can be obtained by adding an appropriate amount to the solution for reacting with the bone tissue. As the solution in this case, the same solution as the above-mentioned solution can be used.
本実施の形態の骨組織分解物の製造方法に用いられるシステインプロテアーゼとしては、塩基性アミノ酸量よりも酸性アミノ酸量の方が多いシステインプロテアーゼ、酸性領域の水素イオン濃度において活性であるシステインプロテアーゼを用いることが好ましい。 As the cysteine protease used in the method for producing a bone tissue decomposition product of the present embodiment, a cysteine protease having a larger amount of acidic amino acids than a basic amino acid amount and a cysteine protease active at a hydrogen ion concentration in an acidic region are used. Is preferred.
このようなシステインプロテアーゼとしては、カテプシンＢ［ＥＣ ３．４．２２．１］、パパイン［ＥＣ ３．４．２２．２］、フィシン［ＥＣ ３．４．２２．３］、アクチニダイン［ＥＣ ３．４．２２．１４］、カテプシンＬ［ＥＣ ３．４．２２．１５］、カテプシンＨ［ＥＣ ３．４．２２．１６］、カテプシンＳ［ＥＣ ３．４．２２．２７］、ブロメライン［ＥＣ ３．４．２２．３２］、カテプシンＫ［ＥＣ ３．４．２２．３８］、アロライン、カルシウム依存性プロテアーゼなどを挙げることが可能である。
Examples of such cysteine proteases include cathepsin B [EC 3.4.22.1.], Papain [EC 3.4.22.2], phycin [EC 3.4.22.3], and actinidyne [
これらの中では、パパイン、フィシン、アクチニダイン、カテプシンＫ、アロラインまたはブロメラインを用いることが好ましく、パパイン、フィシン、アクチニダイン、カテプシンＫを用いることが更に好ましい。 Among these, papain, ficin, actinidyne, cathepsin K, alloline or bromelain are preferably used, and papain, ficin, actinidin and cathepsin K are even more preferred.
上記システインプロテアーゼは、公知の方法によって入手することができる。例えば、化学合成による酵素の作製；細菌、真菌、各種動植物の細胞または組織からの酵素の抽出；遺伝子工学的手段による酵素の作製；などによって入手することができる。勿論、市販の酵素を用いることも可能である。 The above cysteine protease can be obtained by a known method. For example, it can be obtained by producing an enzyme by chemical synthesis; extracting an enzyme from cells or tissues of bacteria, fungi, various animals and plants; producing an enzyme by genetic engineering means; and the like. Of course, it is also possible to use a commercially available enzyme.
システインプロテアーゼは、ＳＨ基が活性中心に存在するプロテアーゼであり、還元剤（例えば、ジチオスレイトールなど）によって活性化させることが可能である。それ故に、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現するという観点からは、上記システインプロテアーゼは、還元剤と反応させたものであることが好ましい。 Cysteine proteases are proteases in which the SH group is present in the active center and can be activated by a reducing agent (eg, dithiothreitol, etc.). Therefore, from the viewpoint of realizing a bone tissue degradation product having a higher activity of differentiating stem cells into bone-related cells, the cysteine protease is preferably reacted with a reducing agent.
特定のｐＨの溶液中におけるシステインプロテアーゼの濃度は、適宜設定することが可能であり、例えば、２ｍｇ／Ｌ～１００ｍｇ／Ｌであってもよいし、１０ｍｇ／Ｌ～５０ｍｇ／Ｌであってもよい。 The concentration of cysteine protease in a solution having a specific pH can be appropriately set, and may be, for example, 2 mg / L to 100 mg / L or 10 mg / L to 50 mg / L. ..
本実施の形態の骨組織分解物の製造方法に用いられる骨組織の由来は、特に限定されず、哺乳類（例えば、ブタ、ヒツジ、ウシ、ウサギ、ラット、マウス、または、ヒト）由来の骨組織であってもよいし、鳥類由来の骨組織であってもよいし、両生類由来の骨組織であってもよいし、魚類由来の骨組織であってもよい。 The origin of the bone tissue used in the method for producing the bone tissue decomposition product of the present embodiment is not particularly limited, and the bone tissue derived from mammals (for example, pig, sheep, cow, rabbit, rat, mouse, or human) is used. It may be a bone tissue derived from birds, a bone tissue derived from amphibians, or a bone tissue derived from fish.
より具体的に、上記骨組織は、硬骨（例えば、長管骨、短骨、扁平骨、含気骨、象牙質、セメント質、または、歯槽骨）であり得る。本実施の形態の骨組織分解物の製造方法では、上述した骨組織を予め細断した後、当該骨組織とシステインプロテアーゼとを、特定のｐＨの溶液中で反応させてもよい。当該構成であれば、骨組織の分解をより効率よく行うことができるので、幹細胞を骨関連細胞へ分化させる活性がより高い骨組織分解物を実現することができる。 More specifically, the bone tissue can be hard bone (eg, long bone, short bone, flat bone, pneumatized bone, dentin, cementum, or alveolar bone). In the method for producing a bone tissue decomposition product of the present embodiment, the above-mentioned bone tissue may be shredded in advance, and then the bone tissue and the cysteine protease may be reacted in a solution having a specific pH. With this configuration, it is possible to more efficiently decompose bone tissue, and thus it is possible to realize a bone tissue decomposition product having a higher activity of differentiating stem cells into bone-related cells.
特定のｐＨの溶液中における骨組織（例えば、硬骨）の量は、適宜設定することが可能であり、例えば、１０ｇ／Ｌ～５００ｇ／Ｌであってもよいし、５０ｇ／Ｌ～２００ｇ／Ｌであってもよい。 The amount of bone tissue (for example, hard bone) in a solution at a specific pH can be appropriately set, and may be, for example, 10 g / L to 500 g / L or 50 g / L to 200 g / L. May be.
システインプロテアーゼと反応させる骨組織は、予め脱灰された硬骨であることが好ましい。当該構成であれば、硬骨から予めカルシウムイオンを除くことができるという利点がある。 The bone tissue to be reacted with cysteine protease is preferably pre-decalcified hard bone. With this configuration, there is an advantage that calcium ions can be removed from the hard bone in advance.
具体的には、硬骨成分の７０～８０％を占めるカルシウムイオンを除くことで、骨の成分を、効率的に抽出することができる。そのため、酵素反応時間の短縮、酵素添加量の低減化、および、酵素反応温度の低温化などの効果が期待できる。 Specifically, by removing calcium ions that occupy 70 to 80% of the hard bone component, the bone component can be efficiently extracted. Therefore, effects such as shortening the enzyme reaction time, reducing the amount of the enzyme added, and lowering the enzyme reaction temperature can be expected.
脱灰の具体的な方法としては、カルシウムイオンに配位してキレート化合物を形成するキレート剤（例えば、ＥＤＴＡ（ethylenediaminetetraacetic acid）、マグネシウム・クエン酸、または、酸（例えば、蟻酸、塩酸、硝酸、三塩化酢酸、または、プランク・リュクロ））を含む脱灰用の溶液中に、硬骨を浸漬させる方法を挙げることができる。 Specific methods for decalcification include chelating agents that coordinate with calcium ions to form chelated compounds (eg, EDTA (ethylenediaminetetraacetic acid), magnesium citrate, or acids (eg, formic acid, hydrochloric acid, nitrate, etc.). Examples thereof include a method of immersing a hard bone in a solution for decalcification containing acetic acid trichloride or Planck-Lucro)).
当該脱灰用の溶液中のキレート剤の濃度は、特に限定されないが、より効果的に硬骨の脱灰を行うという観点から、０．１～０．９ｍｏｌ／Ｌが好ましく、０．２～０．８ｍｏｌ／Ｌが更に好ましく、０．３～０．７ｍｏｌ／Ｌが更に好ましく、０．４～０．６ｍｏｌ／Ｌが最も好ましい。 The concentration of the chelating agent in the decalcification solution is not particularly limited, but is preferably 0.1 to 0.9 mol / L, preferably 0.2 to 0, from the viewpoint of more effectively decalcifying the hard bone. It is more preferably 0.8 mol / L, further preferably 0.3 to 0.7 mol / L, and most preferably 0.4 to 0.6 mol / L.
脱灰用の溶液中に硬骨を浸漬させるときの溶液の温度は、特に限定されないが、より効果的に硬骨の脱灰を行い、かつ、硬骨に含まれる生理活性物質の活性を損なわないという観点からは、４℃～２５℃が好ましく、４℃～２０℃が更に好ましく、４℃～１０℃が最も好ましい。 The temperature of the solution when immersing the bone in the solution for decalcification is not particularly limited, but from the viewpoint of more effectively decalcifying the bone and not impairing the activity of the physiologically active substance contained in the bone. From 4 ° C to 25 ° C, more preferably 4 ° C to 20 ° C, and most preferably 4 ° C to 10 ° C.
脱灰用の溶液中に硬骨を浸漬させる時間は、硬骨の量に応じて適宜設定することが可能であり、特に限定されないが、より効果的に硬骨の脱灰を行うという観点から、例えば３日間～１０日間であり得る。 The time for immersing the bone in the decalcification solution can be appropriately set according to the amount of the bone, and is not particularly limited, but from the viewpoint of more effectively decalcifying the bone, for example, 3 It can be from 1 to 10 days.
本実施の形態の骨組織分解物の製造方法では、以上のようにして脱灰された硬骨とシステインプロテアーゼとを、特定のｐＨの溶液中で反応させればよい。勿論、特定のｐＨの溶液にキレート剤を加え、硬骨の脱灰と、硬骨の分解とを、同時に行ってもよい。 In the method for producing a bone tissue decomposition product of the present embodiment, the bone decalcified as described above and cysteine protease may be reacted in a solution having a specific pH. Of course, a chelating agent may be added to a solution having a specific pH to decalcify the bone and decompose the bone at the same time.
〔２．分化誘導剤〕
〔２－１．分化誘導剤の構成〕
本実施の形態の、幹細胞を骨関連細胞へ分化させるための分化誘導剤は、本発明の製造方法によって製造されたものである骨組織分解物を含んでいるものである。[2. Differentiation inducer]
[2-1. Composition of differentiation inducers]
The differentiation-inducing agent for differentiating stem cells into bone-related cells according to the present embodiment contains a bone tissue decomposition product produced by the production method of the present invention.
この場合、「骨組織分解物」としては、特定のｐＨの溶液中でシステインプロテアーゼと骨組織とを反応させた後の反応溶液、つまり、特定のｐＨの溶液と、システインプロテアーゼと、分解された骨組織に由来する成分との混合物、を用いることが可能である。 In this case, the "bone tissue decomposition product" is a reaction solution after reacting cysteine protease with bone tissue in a solution having a specific pH, that is, a solution having a specific pH and cysteine protease. It is possible to use a mixture with a component derived from bone tissue.
本実施の形態の分化誘導剤に含まれる骨組織分解物の量は、特に限定されず、例えば、分化誘導剤の全重量のうち、１重量％～１００重量％、５重量％～１００重量％、１０重量％～１００重量％、２０重量％～１００重量％、３０重量％～１００重量％、４０重量％～１００重量％、５０重量％～１００重量％、６０重量％～１００重量％、７０重量％～１００重量％、８０重量％～１００重量％、９０重量％～１００重量％、または、１００重量％が、骨組織分解物であってもよい。幹細胞を骨関連細胞へ分化させる活性がより高い分化誘導剤を実現するという観点からは、分化誘導剤に含まれる骨組織分解物の量が多いほど好ましい。 The amount of bone tissue decomposition products contained in the differentiation-inducing agent of the present embodiment is not particularly limited, and is, for example, 1% by weight to 100% by weight, 5% by weight to 100% by weight, based on the total weight of the differentiation-inducing agent. 10% by weight to 100% by weight, 20% by weight to 100% by weight, 30% by weight to 100% by weight, 40% by weight to 100% by weight, 50% by weight to 100% by weight, 60% by weight to 100% by weight, 70 By weight% to 100% by weight, 80% by weight to 100% by weight, 90% by weight to 100% by weight, or 100% by weight may be bone tissue decomposition products. From the viewpoint of realizing a differentiation-inducing agent having a higher activity of differentiating stem cells into bone-related cells, it is preferable that the amount of bone tissue decomposition products contained in the differentiation-inducing agent is large.
本実施の形態に分化誘導剤は、骨組織分解物以外の構成を含むことも可能である。 In the present embodiment, the differentiation inducer can also contain a composition other than the bone tissue decomposition product.
例えば、分化誘導剤のｐＨを適切なｐＨに調節するために、周知の緩衝液を含むことも可能であり、骨組織の形成を補助する様々な物質（例えば、ＢＭＰ、ＴＧＦβ、ｂＦＧＦ、または、β－ＴＣＰ）を含むことも可能である。 For example, it is possible to include well-known buffers to adjust the pH of the differentiation-inducing agent to an appropriate pH, and various substances that assist in the formation of bone tissue (eg, BMP, TGFβ, bFGF, or, etc. It is also possible to include β-TCP).
〔２－２．出願時において、本発明の分化誘導剤を構造または特性により直接特定することが不可能である、または、実際的でない事情〕
後述する実施例でも説明するように、本発明の骨組織分解物、および、当該骨組織分解物を含んでいる分化誘導剤には、多種多様な物質が含まれており、出願時において、これらの物質群の中の、何れの物質、または、何れの物質の組み合わせによって、所望の効果を奏するのかを特定することは、困難を極め、過大な経済的支出や時間を要する。[2-2. At the time of filing, it is impossible or impractical to directly identify the differentiation inducer of the present invention by its structure or properties]
As will be described in Examples described later, the bone tissue decomposition product of the present invention and the differentiation-inducing agent containing the bone tissue decomposition product contain a wide variety of substances, and at the time of filing, these are contained. It is extremely difficult to identify which substance or combination of substances in the group of substances produces the desired effect, and it requires excessive economic expenditure and time.
それ故に、本願には、出願時において、本発明の分化誘導剤を構造または特性により直接特定することが不可能である、または、実際的でない事情が存在する。 Therefore, at the time of filing, there are circumstances in which it is impossible or impractical to directly identify the differentiation inducer of the present invention by structure or property.
〔３．分化誘導方法〕
本実施の形態の幹細胞を骨関連細胞へ分化させる分化誘導方法は、本発明の分化誘導剤を被覆させた基材上で幹細胞を培養する工程を有している。[3. Differentiation induction method]
The method for inducing differentiation of the stem cells of the present embodiment into bone-related cells includes a step of culturing the stem cells on a substrate coated with the differentiation-inducing agent of the present invention.
上記基材としては、特に限定されず、その例として、細胞培養用の培養皿、人工骨、人工歯、および、骨充填材を挙げることができる。 The base material is not particularly limited, and examples thereof include a culture dish for cell culture, an artificial bone, an artificial tooth, and a bone filler.
基材を分化誘導剤にて被覆する方法は、特に限定されず、その例として、基材に液体状の分化誘導剤を塗る方法、基材にゲル状の分化誘導剤を塗る方法、液体状の分化誘導剤を基材上に固定化する方法、ゲル状の分化誘導剤を基材上に固定化する方法、および、固体状の分化誘導剤を基材上に固定化する方法、を挙げることができる。 The method of coating the base material with the differentiation inducer is not particularly limited, and examples thereof include a method of applying a liquid differentiation inducer to the base material, a method of applying a gel-like differentiation inducer to the base material, and a liquid state. A method of immobilizing the differentiation inducer on the substrate, a method of immobilizing the gel-like differentiation inducer on the substrate, and a method of immobilizing the solid differentiation inducer on the substrate. be able to.
上記培養時には、幹細胞から骨関連細胞への分化を更に促進する物質（例えば、分化誘導用培地、ＢＭＰ、ＴＧＦβ、または、ｂＦＧＦ）を幹細胞（例えば、間葉系幹細胞）へ与えてもよい。 At the time of the above culture, a substance that further promotes differentiation of stem cells into bone-related cells (for example, differentiation-inducing medium, BMP, TGFβ, or bFGF) may be given to stem cells (for example, mesenchymal stem cells).
＜１．ブタ腓骨の分解物の作製＞
０．５ｍｏｌ／Ｌの濃度にてＥＤＴＡ（ethylenediaminetetraacetic acid）を含む溶液中に、４℃の条件下にてブタ腓骨を浸漬して、ブタ腓骨からカルシウムを除去した。当該操作を、数回、繰り返し行うことによって、ブタ腓骨を脱灰した。<1. Preparation of decomposition products of pig fibula>
Calcium was removed from the pig fibula by immersing the pig fibula in a solution containing EDTA (ethylenediaminetetraacetic acid) at a concentration of 0.5 mol / L under the condition of 4 ° C. The porcine fibula was decalcified by repeating the operation several times.
脱灰されたブタ腓骨を細断した後、当該ブタ腓骨を、様々なｐＨ（具体的には、ｐＨ２．５、ｐＨ３．５、ｐＨ４．５、ｐＨ５．０、または、ｐＨ５．５）に調整された、アクチニダイン（K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004を参照）を含有する溶液（具体的な組成は、０．１５ｇ／ｍＬの腓骨と、５０ｍＭのクエン酸緩衝液（ｐＨ２．５）、５０ｍＭのクエン酸緩衝液（ｐＨ３．５）、５０ｍＭの酢酸緩衝液（ｐＨ４．５）、５０ｍＭの酢酸緩衝液（ｐＨ５．０）、または、５０ｍＭの酢酸緩衝液（ｐＨ５．５））中で、２０℃の条件下にて反応させた。 After shredding the decalcified porcine fibula, the porcine fibula is adjusted to various pH (specifically, pH2.5, pH3.5, pH4.5, pH5.0, or pH5.5). A solution containing actinidine (see K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004) (specific composition is 0.15 g / mL). Fibula and 50 mM citrate buffer (pH 2.5), 50 mM citrate buffer (pH 3.5), 50 mM acetate buffer (pH 4.5), 50 mM acetate buffer (pH 5.0), or , 50 mM acetate buffer (pH 5.5)) under the condition of 20 ° C.
反応を開始してから７日後のブタ腓骨の分解物を、後述する試験に用いた。 The decomposition product of pig fibula 7 days after the start of the reaction was used in the test described later.
なお、反応を開始してから５日目のブタ腓骨の分解物を目視にて観察したところ、図１に示すように、上述した全てのｐＨ条件にて、ブタ腓骨は良好に分解されていたが、未分解のブタ腓骨の量は、ｐＨ５．５の場合が最も多く、ｐＨ５．０の場合が２番目に多く、ｐＨ４．５の場合が３番目に多く、ｐＨ３．５の場合が４番目に多く、ｐＨ２．５の場合が最も少なかった。このことは、ｐＨが低い条件ほど、酵素による骨の分解がより進んだことを示唆している。 When the decomposition product of the pig fibula on the 5th day after the start of the reaction was visually observed, as shown in FIG. 1, the pig fibula was satisfactorily decomposed under all the above-mentioned pH conditions. However, the amount of undecomposed porcine fibula is highest at pH 5.5, second highest at pH 5.0, third highest at pH 4.5, and fourth at pH 3.5. The number was high, and the case of pH 2.5 was the lowest. This suggests that the lower the pH, the more the enzyme decomposes the bone.
＜２．ブタ腓骨の分解物のＬＣ／ＭＳによる解析＞
上述したブタ腓骨の分解物を、ＬＣ／ＭＳ（liquid chromatography-mass spectrometry）を用いた解析に供し、分解物に含まれる物質の同定を試みた。なお、実際の解析は、近畿大学生物理工学部所有のＥｋｓｐｅｒｔ ｎａｎｏＬＣ４００（Ｅｋｓｉｇｅｎｔ社製）、Ｔｒｉｐｌｅ ＴＯＦ５６００＋（ＡＢＳｃｉｅｘ社製）を利用して行った。また、Ｄａｔａｂａｓｅ検索は、ＰｒｏｔｅｉｎＰｉｌｏｔ ｓｏｆｔｗａｒｅを用いて行った。<2. Analysis of porcine fibula degradation products by LC / MS>
The above-mentioned decomposition product of pig fibula was subjected to analysis using LC / MS (liquid chromatography-mass spectrometry), and an attempt was made to identify the substance contained in the decomposition product. The actual analysis was performed using the Expert nanoLC400 (manufactured by Expert) and Triple TOF5600 + (manufactured by ABSciex) owned by the Faculty of Biophysical Engineering, Kinki University. In addition, the database search was performed using the ProteinPilot software.
以下の表に、ｐＨ２．５～ｐＨ５．５の条件下で得られた分解物に含まれる物質の代表例を示す。以下の表から明らかなように、分解物には、様々な物質が含まれていることが明らかになった、なお、各表に示す物質は、分解物に含まれる物質の一例にすぎず、今回の試験では、例えば、ｐＨ５．５の条件下で得られた分解物の場合には、略４００種類の物質を同定することに成功した。 The table below shows typical examples of substances contained in the decomposition products obtained under the conditions of pH 2.5 to pH 5.5. As is clear from the tables below, it was clarified that the decomposition products contained various substances. The substances shown in each table are only examples of the substances contained in the decomposition products. In this test, for example, in the case of decomposition products obtained under the condition of pH 5.5, we succeeded in identifying approximately 400 kinds of substances.
また、ｐＨがより低い条件下で得られた分解物ほど、分解物に含まれる物質の種類が少なくなる傾向を示した（下述＜７＞参照）。このことは、ｐＨが低い条件ほど、酵素による骨組織を構成する成分の分解がより進んだことを示唆している。 In addition, the decomposition products obtained under lower pH conditions tended to contain fewer substances in the decomposition products (see <7> below). This suggests that the lower the pH condition, the more the decomposition of the components constituting the bone tissue by the enzyme proceeded.
０．５ｍｏｌ／Ｌの濃度にてＥＤＴＡ（ethylenediaminetetraacetic acid）を含む溶液中に、４℃の条件下にてニワトリ尺骨を浸漬して、ニワトリ尺骨からカルシウムを除去した。当該操作を、数回、繰り返し行うことによって、ニワトリ尺骨を脱灰した。
Calcium was removed from the chicken ulna by immersing the chicken ulna in a solution containing EDTA (ethylenediaminetetraacetic acid) at a concentration of 0.5 mol / L under the condition of 4 ° C. The chicken ulna was decalcified by repeating the operation several times.
脱灰されたニワトリ尺骨を細断した後、当該ニワトリ尺骨を、ｐＨ３．５に調整された、アクチニダイン（K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004を参照）を含有する溶液（具体的な組成は、０．１５ｇ／ｍＬのニワトリ尺骨を添加した、５０ｍＭのクエン酸緩衝液（ｐＨ３．５））中で、２０℃の条件下にて反応させた。 After shredding the decalcified chicken ulna, the chicken ulna was adjusted to pH 3.5 and actinidyne (K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867). , 2004) containing solution (specific composition is 50 mM citrate buffer (pH 3.5) with 0.15 g / mL chicken ulna added) under 20 ° C. conditions. It was reacted.
対照として、脱灰されたニワトリ尺骨を細断した後、当該ニワトリ尺骨を、ブタペプシンを含有する溶液（具体的な組成は、３０Ｕｎｉｔ／ｍＬのペプシンを含む５０ｍＭのグリシン塩酸緩衝液（ｐＨ２．０））中で、２０℃の条件下にて反応させた。 As a control, after shredding the decalcified chicken ulna, the chicken ulna is sliced into a solution containing porcine pepsin (specific composition is 50 mM glycine hydrochloride buffer (pH 2.0) containing 30 Unit / mL pepsin). ), The reaction was carried out under the condition of 20 ° C.
反応を開始してから１１日目のニワトリ尺骨の分解物を目視にて観察したところ、図２に示すように、ニワトリ尺骨は、アクチニダインによって良好に分解された。一方、ニワトリ尺骨は、ペプシンによっては完全に分解されていないことが明らかとなった。このことは、ニワトリ尺骨においても、アクチニダインによる骨の分解が進んだことを示している。 When the decomposition products of chicken ulna on the 11th day after the start of the reaction were visually observed, the chicken ulna was satisfactorily decomposed by actinidyne as shown in FIG. On the other hand, it was revealed that the chicken ulna was not completely decomposed by pepsin. This indicates that actinidin-induced bone decomposition also progressed in chicken ulna.
＜４．ウシ大腿骨の分解物の作製＞
０．５ｍｏｌ／Ｌの濃度にてＥＤＴＡ（ethylenediaminetetraacetic acid）を含む溶液中に、４℃の条件下にてウシ大腿骨を浸漬して、ウシ大腿骨からカルシウムを除去した。当該操作を、数回、繰り返し行うことによって、ウシ大腿骨を脱灰した。<4. Preparation of bovine femur degradation products>
The bovine femur was immersed in a solution containing EDTA (ethylenediaminetetraacetic acid) at a concentration of 0.5 mol / L under the condition of 4 ° C. to remove calcium from the bovine femur. The bovine femur was decalcified by repeating the operation several times.
次に、ウシ大腿骨を４．０ｍｏｌ／Ｌのグアニジン塩酸液中に浸漬して、ウシ大腿骨から可溶化タンパク質を除去した。当該ウシ大腿骨をＰＢＳで数回洗浄した後に、洗浄後のウシ大腿骨をウシ骨不溶性成分とした。 Next, the bovine femur was immersed in a 4.0 mol / L guanidine hydrochloric acid solution to remove the solubilized protein from the bovine femur. After washing the bovine femur with PBS several times, the washed bovine femur was used as a bovine bone insoluble component.
ウシ骨不溶性成分を細断した後、当該ウシ骨不溶性成分を、ｐＨ３．５またはｐＨ５．０に調整された、アクチニダイン（K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004を参照）を含有する溶液（具体的な組成は、０．１５ｇ／ｍＬのアクチニダインを含む、５０ｍＭのクエン酸緩衝液（ｐＨ３．５）または５０ｍＭの酢酸緩衝液（ｐＨ５．０））中で、２０℃の条件下にて反応させた。 After shredding the bovine bone insoluble component, the bovine bone insoluble component was adjusted to pH 3.5 or pH 5.0 to actinidin (K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp). A solution containing .861-867, 2004 (specific composition is 50 mM citrate buffer (pH 3.5) or 50 mM acetate buffer (pH 5.) containing 0.15 g / mL actinidine. In 0)), the reaction was carried out under the condition of 20 ° C.
対照として、ウシ骨不溶性成分を細断した後、当該ウシ骨不溶性成分を、ブタペプシンを含有する溶液（具体的な組成は、３０Ｕｎｉｔ／ｍＬのペプシンを含む５０ｍＭのグリシン塩酸緩衝液（ｐＨ２．０））中で、２０℃の条件下にて反応させた。陰性コントロールとして、ウシ骨不溶性成分を細断した後、当該ウシ骨不溶性成分を、溶液（具体的な組成は、５０ｍＭのクエン酸緩衝液（ｐＨ３．５）、５０ｍＭの酢酸緩衝液（ｐＨ５．０）、または、５０ｍＭのグリシン塩酸緩衝液（ｐＨ２．０））中で、２０℃の条件下にて反応させた。 As a control, after shredding the bovine bone insoluble component, the bovine bone insoluble component is subjected to a solution containing porcine pepsin (specific composition is 50 mM glycine hydrochloride buffer (pH 2.0) containing 30 Unit / mL pepsin). ), The reaction was carried out under the condition of 20 ° C. As a negative control, after shredding the bovine bone insoluble component, the bovine bone insoluble component is subjected to a solution (specific composition is 50 mM citric acid buffer (pH 3.5), 50 mM acetate buffer (pH 5.0)). ) Or 50 mM glycine hydrochloride buffer (pH 2.0)) under the condition of 20 ° C.
反応を開始してから８日後のウシ骨不溶性成分の分解物を、後述する試験に用いた。 The decomposition products of bovine bone insoluble components 8 days after the start of the reaction were used in the test described later.
なお、反応を開始してから８日目のウシ骨不溶性成分の分解物を目視にて観察したところ、図３に示すように、ウシ骨不溶性成分は、ｐＨ３．５の条件下でアクチニダインによって良好に分解された。一方、ウシ骨不溶性成分は、ペプシンによっては完全に分解されていないことが明らかとなった。このことは、ウシ骨不溶性成分においても、アクチニダインによる骨の分解が進んだことを示している。 When the decomposition product of the bovine bone insoluble component was visually observed on the 8th day after the reaction was started, as shown in FIG. 3, the bovine bone insoluble component was favorably treated with actinidyne under the condition of pH 3.5. Was disassembled into. On the other hand, it was revealed that the bovine bone insoluble component was not completely decomposed by pepsin. This indicates that even in the bovine bone insoluble component, the decomposition of bone by actinidin progressed.
＜５．ウシ骨不溶性成分の分解物のＬＣ／ＭＳによる解析＞
上述したウシ骨不溶性成分の分解物を、ＬＣ／ＭＳ（liquid chromatography-mass spectrometry）を用いた解析に供し、分解物に含まれる物質の同定を試みた。なお、実際の解析は、近畿大学生物理工学部所有のＥｋｓｐｅｒｔ ｎａｎｏＬＣ４００（Ｅｋｓｉｇｅｎｔ社製）、Ｔｒｉｐｌｅ ＴＯＦ５６００＋（ＡＢＳｃｉｅｘ社製）を利用して行った。また、Ｄａｔａｂａｓｅ検索は、ＰｒｏｔｅｉｎＰｉｌｏｔ ｓｏｆｔｗａｒｅを用いて行った。<5. Analysis of decomposition products of bovine bone insoluble components by LC / MS>
The above-mentioned decomposition products of bovine bone insoluble components were subjected to analysis using LC / MS (liquid chromatography-mass spectrometry), and an attempt was made to identify the substances contained in the decomposition products. The actual analysis was performed using the Expert nanoLC400 (manufactured by Expert) and Triple TOF5600 + (manufactured by ABSciex) owned by the Faculty of Biophysical Engineering, Kinki University. In addition, the database search was performed using the ProteinPilot software.
以下の表５に、ｐＨ３．５の条件下でアクチニダインを用いて得られた分解物に含まれる物質の代表例を示す。以下の表から明らかなように、分解物には、様々な物質が含まれていることが明らかになった、なお、表に示す物質は、分解物に含まれる物質の一例にすぎず、今回の試験では、例えば、ｐＨ３．５の条件下でアクチニダインを用いて得られた分解物の場合には、略２７種類の物質を同定することに成功した。 Table 5 below shows typical examples of substances contained in the decomposition products obtained by using actinidyne under the condition of pH 3.5. As is clear from the table below, it was clarified that the decomposition products contain various substances. The substances shown in the table are only examples of the substances contained in the decomposition products. In the test of, for example, in the case of the decomposition product obtained by using actinidyne under the condition of pH 3.5, it was successful to identify about 27 kinds of substances.
ヒト間葉系幹細胞（ｈｕｍａｎＭＳＣｓ）をＬＯＮＺＡ社から購入し、ラット骨髄細胞（ｒａｔＭＭＣｓ）をコスモバイオ社から購入した。
Human mesenchymal stem cells (humanMSCs) were purchased from LONZA, and rat bone marrow cells (ratMMCs) were purchased from Cosmobio.
試験に供する試料の量を同じ量にするために、様々なｐＨ条件下で得られたブタ腓骨の分解物の各々と、皮膚Ｉ型コラーゲン由来のＬＡＳＣｏｌとを、容量比で２：８に混合した試料を準備した。 In order to make the amount of the sample to be tested the same, each of the decomposition products of pig fibula obtained under various pH conditions and LASCol derived from skin type I collagen were mixed in a volume ratio of 2: 8. Prepared the sample.
なお、皮膚Ｉ型コラーゲン由来のＬＡＳＣｏｌは、以下の方法にて作製した。５０ｍＭ クエン酸緩衝液（ｐＨ３．０）を準備した。なお、当該水溶液の溶媒としては、水を用いた。アクチニダイン［ＥＣ ３．４．２２．１４］を活性化するため、１０ｍＭ ジチオスレイトールを含む５０ｍＭ リン酸緩衝液（ｐＨ６．５）に対し、アクチニダインを溶解し、９０分間、２５℃にて静置した。なお、アクチニダインとしては、周知の方法にて精製したものを利用した（例えば、K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004参照参照）。次いで、５０ｍＭ クエン酸緩衝液（ｐＨ３．０）に対し、ブタ由来のＩ型コラーゲンを溶解した。アクチニダインを含む水溶液と、ブタ由来のＩ型コラーゲンを含む当該溶液と、を７日間以上、２０℃にて接触させて、Ｉ型コラーゲンの分解物（換言すれば、皮膚Ｉ型コラーゲン由来のＬＡＳＣｏｌ）を作製した。なお、ブタ由来のＩ型コラーゲンは、市販品（Ｃｅｌｌｍａｔｒｉｘ Ｔｙｐｅ Ｉ－Ｃ、新田ゼラチン株式会社）を用いた。 LASCol derived from skin type I collagen was prepared by the following method. A 50 mM citrate buffer (pH 3.0) was prepared. Water was used as the solvent for the aqueous solution. In order to activate actinidyne [EC 3.4.22.14], actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes. did. As actinidyne, one purified by a well-known method was used (see, for example, K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004). Then, type I collagen derived from pig was dissolved in 50 mM citrate buffer (pH 3.0). The aqueous solution containing actinidyne and the solution containing type I collagen derived from pigs are brought into contact with each other at 20 ° C. for 7 days or longer to decompose the type I collagen (in other words, LASCol derived from skin type I collagen). Was produced. As the type I collagen derived from pig, a commercially available product (Cellmatic Type IC, Nitta Gelatin Co., Ltd.) was used.
上述した各試料（ブタ腓骨の分解物の各々と、皮膚Ｉ型コラーゲン由来のＬＡＳＣｏｌとの混合物）を、直径３５ｍｍの培養皿の底面上に塗布した後、各培養皿に、同数のヒト間葉系幹細胞（４×１０４個）、または、ラット骨髄細胞（６×１０４個）を播種した。After applying each of the above-mentioned samples (a mixture of each of the decomposition products of porcine vertebral bone and LASCol derived from skin type I collagen) on the bottom surface of a culture dish having a diameter of 35 mm, the same number of human mesenchymes were applied to each culture dish. Mesenchymal stem cells (4 × 10 4 ) or rat bone marrow cells (6 × 10 4 ) were seeded.
増殖用の培地（ＬＯＮＺＡ社製のヒト間葉系幹細胞用の増殖培地、または、コスモバイオ社製のラット骨髄細胞用の増殖培地）を用いて、３７℃、５％ＣＯ２の条件下で、ヒト間葉系幹細胞、および、ラット骨髄細胞を１日間培養することによって、各細胞を、培養皿上に接着させた。その後、増殖用の培地を骨芽細胞分化用の培地（ＬＯＮＺＡ社製、または、コスモバイオ社製）に交換し、ヒト間葉系幹細胞、および、ラット骨髄細胞の分化培養を更に続けた。Using a growth medium (a growth medium for human mesenchymal stem cells manufactured by LONZA or a growth medium for rat bone marrow cells manufactured by Cosmobio) at 37 ° C. and 5% CO 2 conditions. By culturing human mesenchymal stem cells and rat bone marrow cells for 1 day, each cell was adhered onto a culture dish. Then, the medium for proliferation was replaced with a medium for osteoblast differentiation (manufactured by LONZA or Cosmobio), and the differentiation culture of human mesenchymal stem cells and rat bone marrow cells was further continued.
３日毎に骨芽細胞分化用の培地を新鮮な骨芽細胞分化用の培地に交換しながら、ヒト間葉系幹細胞については１５日間、ラット骨髄細胞については７日間、３７℃、５％ＣＯ２の条件下で分化培養を続けた。その後、ヒト間葉系幹細胞、または、ラット骨髄細胞の石灰化（換言すれば、ヒト間葉系幹細胞、または、ラット骨髄細胞の骨芽細胞への分化後期）を検出するために、アリザリンレッド染色を行った。Replacing the osteoblast differentiation medium with a fresh osteoblast differentiation medium every 3 days, 15 days for human mesenchymal stem cells, 7 days for rat bone marrow cells, 37 ° C, 5% CO 2 Differentiation culture was continued under the conditions of. Then, to detect calcification of human mesenchymal stem cells or rat bone marrow cells (in other words, late differentiation of human mesenchymal stem cells or rat bone marrow cells into osteoblasts), Arizarin red staining Was done.
なお、アリザリンレッド染色には、アリザリンレッド染色キット（ＰＧリサーチ株式会社）を用い、具体的な染色方法は、当該キットに添付のプロトコールにしたがった。 The alizarin red staining kit (PG Research Co., Ltd.) was used for the alizarin red staining, and the specific staining method was according to the protocol attached to the kit.
図４に、ヒト間葉系幹細胞を用いた場合のアリザリンレッド染色の染色像を示す。図４において、「数字」は、ブタ腓骨を分解したときのｐＨを示す。 FIG. 4 shows a stained image of alizarin red staining when human mesenchymal stem cells are used. In FIG. 4, the "number" indicates the pH at the time of decomposing the porcine fibula.
図４から明らかなように、分化誘導１２日目では、ｐＨ２．５からｐＨ５．０までは中性側に近くなるにつれて、石灰化が亢進していること、換言すれば、ヒト間葉系幹細胞の骨芽細胞への分化が亢進していることが明らかになった。一方、ｐＨ５．５では、石灰化の亢進効果は低下した。また、ｐＨによる石灰化の亢進効果の差は、分化誘導１５日目には減少した。 As is clear from FIG. 4, on the 12th day of differentiation induction, calcification increases as the pH becomes closer to the neutral side from pH 2.5 to pH 5.0, in other words, human mesenchymal stem cells. It was revealed that the differentiation into osteoblasts was enhanced. On the other hand, at pH 5.5, the effect of enhancing calcification decreased. In addition, the difference in the effect of enhancing calcification by pH decreased on the 15th day of differentiation induction.
また、図５に示すように、ラット骨髄細胞を用いた試験でも、ｐＨによる石灰化の亢進効果は、ヒト間葉系幹細胞を用いた試験と同様であった。具体的には、分化誘導７日目には、全てのｐＨ条件下で得られた分解物でコートされた培養皿において、アテロコラーゲンでコートされた培養皿（ＣＯＬ １、旭テクノグラス社製）や、非コートの培養皿よりも、顕著に高い石灰化亢進効果が確認された。
Further, as shown in FIG. 5, in the test using rat bone marrow cells, the effect of enhancing calcification by pH was similar to the test using human mesenchymal stem cells. Specifically, on the 7th day of induction of differentiation, in the culture dish coated with the decomposition products obtained under all pH conditions, the culture dish coated with atelocollagen (
また、図６にはブタの腓骨（ｐＨ５．０）の分解物と皮膚から抽出した分解物（対照品）とを培養皿に塗布してラット骨髄細胞を播種し、分化誘導５日目の当該細胞のアリザリンレドッド染色像を示す。図６に示すように、同じ酵素により得られたブタ皮膚由来コラーゲン分解物と比較しても、骨抽出物から得られた分解物は、非常に高い石灰化亢進効果を示した。特に、図４と図５で示すように、ｐＨ５．０で得られた分解物の石灰化亢進効果が著しかった。 Further, in FIG. 6, a decomposition product of pig's fibula (pH 5.0) and a decomposition product extracted from the skin (control product) were applied to a culture dish to inoculate rat bone marrow cells, and on the 5th day of differentiation induction. The alizarin redod stained image of the cell is shown. As shown in FIG. 6, the decomposition product obtained from the bone extract showed a very high calcification-enhancing effect even when compared with the collagen decomposition product derived from pig skin obtained by the same enzyme. In particular, as shown in FIGS. 4 and 5, the calcification-enhancing effect of the decomposition product obtained at pH 5.0 was remarkable.
＜７．骨組織に由来する可溶化タンパク質に関する試験－２＞
本実施例では、ブタ脛骨の分解物中の可溶化成分に含まれるタンパク質を二次元電気泳動によりゲル上に展開し、タンパク質のスポットの数と量とを調べた。<7. Test on solubilized protein derived from bone tissue-2>
In this example, the protein contained in the solubilized component in the decomposition product of pig tibia was developed on a gel by two-dimensional electrophoresis, and the number and amount of protein spots were examined.
二次元電気泳動用の試料を調製するため、まず、ｐＨ２．５、ｐＨ３．５またはｐＨ５．０の条件下で可溶化させた各試料に対して、各試料の体積の３倍量の予め冷やしておいたアセトンを加え、よく混合した。 In order to prepare a sample for two-dimensional electrophoresis, first, each sample solubilized under the conditions of pH 2.5, pH 3.5 or pH 5.0 is pre-cooled in an amount of 3 times the volume of each sample. Acetone was added and mixed well.
当該混合物を－２０℃の条件下で一晩静置し、翌日に、４℃の条件下で１０，０００×ｇ、１０分間遠心分離して沈殿物を集めた。 The mixture was allowed to stand overnight under the condition of −20 ° C., and the next day, the mixture was centrifuged at 10,000 × g for 10 minutes under the condition of 4 ° C. to collect a precipitate.
各ｐＨで得られた試料の沈殿物に対して、７Ｍ尿素、２Ｍチオ尿素、４．５％ＣＨＡＰＳ、５．５ｍＭ ＤＴＴ、１．１％ Ｔｒｉｔｏｎ Ｘ－１００および適量のブロモフェニルブルーを含む溶液を加え、沈殿物を溶解した。不溶性の成分を除くために、溶解物を１０，０００×ｇで３分間遠心分離に供し、上清を集めた。 A solution containing 7M urea, 2M thiourea, 4.5% CHAPS, 5.5 mM DTT, 1.1% Triton X-100 and an appropriate amount of bromophenyl blue was added to the precipitate of the sample obtained at each pH. In addition, the precipitate was dissolved. To remove insoluble components, the lysate was centrifuged at 10,000 xg for 3 minutes and the supernatant was collected.
二次元電気泳動の一次元目の等電点電気泳動では、Ｅｔｔａｎ ＩＰＧｐｈｏｒ３（ＧＥ ヘルスケア）を用いて、膨潤液用のストリップホルダーに各上清を入れてさらにＲｅａｄｙＳｔｒｉｐＴＭＩＰＧ Ｓｔｒｉｐｓ（７ｃｍ、ｐＨ３－１０、ｎｏｎｌｉｎｅａｒ、ＢＩＯ－ＲＡＤ社製）を置くことで、膨潤を開始した。その条件を下表に示す。In the first-dimensional isoelectric focusing of two-dimensional electrophoresis, Ettan IPGphor3 (GE Healthcare) is used to put each supernatant in a strip holder for swelling solution, and then ReadyStrip TM IPG Strips (7 cm, pH3-). 10, nonlinear, manufactured by BIO-RAD) was placed to start swelling. The conditions are shown in the table below.
一次元目の等電点電気泳動を終えたストリップは、ＳＤＳ緩衝液に平衡化させるため、Ｔｒｉｓ－ＨＣｌ、尿素、グリセロール、ＳＤＳ、ＤＴＴを混合した溶液で１５分間浸漬し、振とうした。その後、Ｔｒｉｓ－ＨＣｌ、尿素、グリセロール、ＳＤＳ、ヨードアセトアミドを混合した溶液に浸漬し、１５分間振とうした。 After completing the first-dimensional isoelectric focusing, the strip was immersed in a mixed solution of Tris-HCl, urea, glycerol, SDS, and DTT for 15 minutes and shaken to equilibrate with SDS buffer. Then, it was immersed in a mixed solution of Tris-HCl, urea, glycerol, SDS, and iodoacetamide and shaken for 15 minutes.
次に、二次元目の電気泳動用のゲルの上部に０．５％アガロースを重層し、さらに一次元目の電気泳動を終えたストリップを、ゆっくりと平らになるように設置した。その際、左端のゲルに分子量マーカーを染み込ませたろ紙を接触させた。 Next, 0.5% agarose was layered on top of the gel for the second dimension electrophoresis, and the strip after the first dimension electrophoresis was placed so as to be slowly flattened. At that time, the gel on the left end was brought into contact with a filter paper impregnated with a molecular weight marker.
ＳＤＳ泳動緩衝液を電気泳動槽の上部と下部とにそれぞれ適量入れ、常法に従い、泳動を開始した。二次元目の電気泳動を終えたゲル内のタンパク質を、ＥｚＳｔａｉｎ Ｓｉｌｖｅｒ銀染色キット（ＡＴＴＯ社製）を用いて染色した。染色方法はＡＴＴＯ株式会社の銀染色キットに添付の手引書に従った。 Appropriate amounts of SDS migration buffer were added to the upper part and the lower part of the electrophoresis tank, respectively, and the electrophoresis was started according to a conventional method. The proteins in the gel after the second-dimensional electrophoresis were stained using an EzStain Silver silver staining kit (manufactured by ATTO). The dyeing method was according to the guide attached to the silver dyeing kit of ATTO Co., Ltd.
二次元電気泳動の結果を図７（ａ）～図７（ｃ）に示す。図７（ａ）は、脛骨をｐＨ２．５の条件で可溶化した成分の二次元電気泳動の結果を示し、図７（ｂ）は、脛骨をｐＨ３．５の条件で可溶化した成分の二次元電気泳動の結果を示し、図７（ｃ）は、脛骨をｐＨ５．０の条件で可溶化した成分の二次元電気泳動の結果を示す。 The results of two-dimensional electrophoresis are shown in FIGS. 7 (a) to 7 (c). FIG. 7 (a) shows the results of two-dimensional electrophoresis of the component in which the tibia was solubilized under the condition of pH 2.5, and FIG. 7 (b) shows the result of two-dimensional electrophoresis of the component in which the tibia was solubilized under the condition of pH 3.5. The results of the dimensional electrophoresis are shown, and FIG. 7 (c) shows the results of the two-dimensional electrophoresis of the component in which the tibia is solubilized under the condition of pH 5.0.
電気泳動用のゲルの上下はタンパク質の分子量の大小を示し、下になるほど分子量が小さいことを意味する。分子量はＭｗと表し、分子量の大きさをｋＤａ示した。電気泳動用のゲルの左右はタンパク質の等電点を示し、等電点をｐＩと表した。ｐＩは、電気泳動用のゲルの左端が３、電気泳動用のゲルの右端が１０を示す。 The top and bottom of the gel for electrophoresis indicate the size of the molecular weight of the protein, and the lower the level, the smaller the molecular weight. The molecular weight was expressed as Mw, and the size of the molecular weight was shown as kDa. The left and right sides of the gel for electrophoresis indicate the isoelectric point of the protein, and the isoelectric point is represented as pI. The pI indicates 3 at the left end of the gel for electrophoresis and 10 at the right end of the gel for electrophoresis.
つまり、図７（ａ）～図７（ｃ）に示す電気泳動用のゲルにおいて、黒く染まっているスポットがタンパク質に対応し、電気泳動用のゲルにおけるタンパク質の位置に基づいて、当該タンパク質の凡その分子量と等電点とを知ることができる。 That is, in the gel for electrophoresis shown in FIGS. 7 (a) to 7 (c), the spots dyed in black correspond to the protein, and the protein is approximately based on the position of the protein in the gel for electrophoresis. The molecular weight and the isoelectric point can be known.
図７（ａ）～図７（ｃ）の結果から、図７（ｃ）に示す電気泳動用のゲルには多数のスポットが確認できるので、ｐＨ５．０の条件では、分子量および等電点が異なる多種類のタンパク質が可溶性となり回収されたことが証明できた。さらに分子量１万以上の高分子量のタンパク質も存在することから、本抽出条件では可溶化タンパク質が、天然の状態により近いことが示唆された。一方、図７（ａ）および図７（ｂ）に示す電気泳動用のゲルではスポットの数が少なく、またスポットの濃さも薄いことから、ｐＨ２．５およびｐＨ３．５の条件では、ｐＨ５．０の条件と比較して、多くのタンパク質は回収できていないことが分かった。 From the results of FIGS. 7 (a) to 7 (c), a large number of spots can be confirmed in the gel for electrophoresis shown in FIG. 7 (c), so that the molecular weight and the isoelectric point are high under the condition of pH 5.0. It was proved that many different kinds of proteins became soluble and recovered. Furthermore, the presence of high molecular weight proteins with a molecular weight of 10,000 or more suggests that the solubilized proteins are closer to the natural state under the present extraction conditions. On the other hand, in the gels for electrophoresis shown in FIGS. 7 (a) and 7 (b), the number of spots is small and the density of the spots is also thin, so that the pH is 5.0 under the conditions of pH 2.5 and pH 3.5. It was found that many proteins could not be recovered in comparison with the conditions of.
＜８．象牙組織の可溶化に関する試験＞
歯は、セメント質、象牙質、およびエナメル質などの結合組織と、セメント芽細胞、象牙芽細胞、およびエナメル芽細胞などの細胞と、から構成されている。歯は、上述した結合組織および細胞の他に、血管や神経などが複雑に入り組んだ構造を有している。<8. Test on solubilization of ivory tissue>
Teeth are composed of connective tissues such as cementum, dentin, and enamel, and cells such as cementoblasts, odontoblasts, and ameloblasts. In addition to the connective tissue and cells described above, teeth have a complexly intricate structure such as blood vessels and nerves.
ウシ象牙質を得るため、まず、ウシから歯冠組織を複数個採取し、そのエナメル質を常法により酸性処理し、ウシ象牙質を得た。次に、０．５ｍｏｌ／ＬのＥＤＴＡ（ethylenediaminetetraacetic acid）を含む溶液中に、４℃の条件下にてウシ象牙質を浸漬して、ウシ象牙質からカルシウムを除去した。当該操作を、数回、繰り返し行うことによって、ウシ象牙質を脱灰した。 In order to obtain bovine dentin, first, a plurality of crown tissues were collected from bovine and the enamel was acid-treated by a conventional method to obtain bovine dentin. Next, bovine dentin was immersed in a solution containing 0.5 mol / L of EDTA (ethylenediaminetetraacetic acid) under the condition of 4 ° C. to remove calcium from bovine dentin. The bovine dentin was decalcified by repeating the operation several times.
さらに、脱灰されたウシ象牙質を液体窒素中で金属製の乳鉢（内径４０－８０ｍｍ、深さ４０－８０ｍｍ）を用いて細かく摩砕し、当該摩砕物を生理食塩水で５～１０回洗浄し、象牙質不溶化物（Ｉｎｓｏｌｕｂｌｅ Ｄｅｎｔｉｎ Ｍａｔｒｉｘ、以下ＩＤＭと省略）を得た。 Further, the decalcified bovine dentin is finely ground in liquid nitrogen using a metal dairy pot (inner diameter 40-80 mm, depth 40-80 mm), and the ground product is ground 5 to 10 times with physiological saline. Washing was performed to obtain an dentin insoluble material (Insoluble Dentin Matrix, hereinafter abbreviated as IDM).
ＩＤＭを可溶化するため、アクチニダインを用いた。なお、アクチニダインとしては、周知の方法にて精製したものを利用した（例えば、K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004を参照）。アクチニダインを活性化するため、１０ｍＭ ジチオスレイトールと５ｍＭＥＤＴＡとを含む５０ｍＭ リン酸緩衝液（ｐＨ６．５）に対し、アクチニダインを溶解し、９０分間、２５℃にて静置した。 Actinidin was used to solubilize the IDM. As actinidyne, one purified by a well-known method was used (see, for example, K. Morimoto et al., Bioscience, Biotechnology, and Biochemistry, Vol.68, pp.861-867, 2004). In order to activate actinidyne, actinidyne was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and 5 mM EDTA, and allowed to stand at 25 ° C. for 90 minutes.
次いで、５０ｍＭ クエン酸緩衝液（ｐＨ３．５）とＩＤＭ（１００ｍｇ（乾重量））とを混合した。０．５重量％になるように調整したアクチニダインを含む水溶液と、ＩＤＭを含む溶液と、を１６日間、２０℃にて接触させて、ウシ象牙質不溶化物の分解物を作製した。 Then, 50 mM citrate buffer (pH 3.5) and IDM (100 mg (dry weight)) were mixed. An aqueous solution containing actinidin adjusted to 0.5% by weight and a solution containing IDM were brought into contact with each other for 16 days at 20 ° C. to prepare a decomposition product of bovine dentin insoluble material.
対照として、５０ｍＭ クエン酸緩衝液（ｐＨ２．５）と、ＩＤＭ（１００ｍｇ（乾重量））と、を混合した。０．５重量％になるように調整したブタ・ペプシンを含む水溶液と、ＩＤＭを含む溶液と、を１６日間、２０℃にて接触させて、ウシ象牙質不溶化物の分解物を作製した。 As a control, 50 mM citrate buffer (pH 2.5) and IDM (100 mg (dry weight)) were mixed. An aqueous solution containing porcine pepsin adjusted to 0.5% by weight and a solution containing IDM were brought into contact with each other for 16 days at 20 ° C. to prepare a decomposition product of bovine dentin insoluble material.
典型的な試験結果として、図８にＳＤＳ－ポリアクリルアミドゲル電気泳動の結果を示す。ウシ象牙質不溶化物は、ペプシンでは何も抽出できなかったが、アクチニダインでは多数のタンパク質が溶解したことが示された。 As a typical test result, FIG. 8 shows the result of SDS-polyacrylamide gel electrophoresis. No bovine dentin insoluble material could be extracted with pepsin, but actinidin showed that a large number of proteins were lysed.
よって、象牙質不溶化物からのタンパク質の可溶化率は、用いる酵素によって差が生じることは歴然であり、更に、本実施例の方法で得られた象牙質可溶化物は、脛骨可溶化物と同等の用途に用いられることが明らかである。 Therefore, it is clear that the solubilization rate of the protein from the dentin insoluble matter differs depending on the enzyme used, and further, the dentin solubilized product obtained by the method of this example is the tibial solubilized product. It is clear that it will be used for equivalent purposes.
骨や象牙質は、硬組織に分類され、真皮や腱のような軟組織とは異なり、不溶性組織であって、タンパク質を変性させずに抽出することはこれまでにできなかった。硬組織からタンパク質を未変性で回収する適切な方法は従来報告されておらず、骨あるいは象牙質を加熱変性処理するなどして、骨から変性タンパク質を回収する技術しかなかった。 Bone and dentin are classified as hard tissues, and unlike soft tissues such as dermis and tendons, they are insoluble tissues and cannot be extracted without denatured proteins. No suitable method for recovering protein from hard tissue without denaturation has been reported so far, and there is only a technique for recovering denatured protein from bone by heat denaturing treatment of bone or dentin.
本発明は、骨組織の培養、骨組織の形成、骨組織のモデルシステムの構築、および、骨組織のモデルシステムを用いた様々な試験などに利用することができる。例えば、本発明の骨組織分解物は、骨分化作用が著しく早く、かつ、分化効率が良好なので、従来の再生骨誘導材料に比べて短期に骨修復効果が期待できる。また、本発明は、骨組織の再生に優れているので、再生医療に用いられる移植用の幹細胞、または、骨芽細胞の足場材料として、好適に用いることができる。 The present invention can be used for culturing bone tissue, forming bone tissue, constructing a bone tissue model system, and various tests using the bone tissue model system. For example, the bone tissue decomposition product of the present invention has a remarkably fast bone differentiation effect and a good differentiation efficiency, so that a bone repair effect can be expected in a short period of time as compared with a conventional regenerated bone-inducing material. Further, since the present invention is excellent in the regeneration of bone tissue, it can be suitably used as a stem cell for transplantation used in regenerative medicine or as a scaffold material for osteoblasts.
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