JP2017039698A - Collagen production accelerator, proteoglycan production accelerator, and chondrocyte migration accelerator - Google Patents
Collagen production accelerator, proteoglycan production accelerator, and chondrocyte migration accelerator Download PDFInfo
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- JP2017039698A JP2017039698A JP2016138743A JP2016138743A JP2017039698A JP 2017039698 A JP2017039698 A JP 2017039698A JP 2016138743 A JP2016138743 A JP 2016138743A JP 2016138743 A JP2016138743 A JP 2016138743A JP 2017039698 A JP2017039698 A JP 2017039698A
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Abstract
Description
本発明は、コラーゲンの産生促進剤、プロテオグリカン産出促進剤及び軟骨細胞の遊走促進剤に関する。 The present invention relates to a collagen production promoter, a proteoglycan production promoter and a chondrocyte migration promoter.
コラーゲンは、繊維状タンパク質であり、皮膚や骨の主成分として哺乳類では全タンパク質の約３０％を占めると言われている。
また、一般的なコラーゲン分子は、３本のコラーゲンポリペプチド鎖が三重らせん構造と呼ばれるロープ状の超らせん構造をとる。加えて、コラーゲンには、プロリン（Pro）とグリシン（Gly）とが特に多く含まれ、両アミノ酸残基とも安定な３重らせん構造の形成に重要である。
Collagen is a fibrous protein and is said to occupy about 30% of the total protein in mammals as the main component of skin and bone.
Further, in general collagen molecules, three collagen polypeptide chains have a rope-like super helical structure called a triple helical structure. In addition, collagen contains a particularly large amount of proline (Pro) and glycine (Gly), and both amino acid residues are important for the formation of a stable triple helical structure.
生体由来のコラーゲンの作製方法及び利用方法では、例えば、ブタ又はウシの皮膚組織をそのままあるいは凍結乾燥した後、火傷などによる皮膚の損傷部位に移植する方法、酵素処理などによって細胞成分を除去して用いる方法、酸性溶液や酵素処理によって可溶化したコラーゲンを、所望の形態に再構成して用いる方法がある。 In the production method and utilization method of collagen derived from a living body, for example, after porcine or bovine skin tissue is directly or freeze-dried, cell components are removed by a method of transplanting to a skin damage site due to a burn or the like, an enzyme treatment or the like. There is a method to use, and a method to reconstitute and use collagen solubilized by acidic solution or enzyme treatment in a desired form.
一方、ウシの海綿状脳症の原因物質がプリオンと呼ばれる伝染性タンパク質であり、この伝染性タンパク質がヒトのクロイツフェルド-ヤコブ病伝染の原因の一つと言われている。プリオンは、タンパク質であり、通常の滅菌、殺菌方法では失活し難く、しかも種を越えて感染することが指摘されている。
また、生体由来のコラーゲンには、ウイルスが混入している可能性も否定できない。
On the other hand, the causative agent of bovine spongiform encephalopathy is an infectious protein called prion, and this infectious protein is said to be one of the causes of human Creutzfeld-Jakob disease. It has been pointed out that prions are proteins, are hardly inactivated by ordinary sterilization and sterilization methods, and are transmitted across species.
In addition, the possibility that viruses are mixed in the collagen derived from living bodies cannot be denied.
一般に、医療用具や医薬品、化粧品ではウシやブタ由来のコラーゲンを原料として用いることが多い。そのため、通常の滅菌、殺菌方法では除去できないプリオンなどの病原体の感染の危険性が常に存在している。 In general, collagens derived from cattle and pigs are often used as raw materials in medical devices, pharmaceuticals, and cosmetics. Therefore, there is always a risk of infection with pathogens such as prions that cannot be removed by ordinary sterilization and sterilization methods.
さらに、生体由来のコラーゲンは、移植対象の患者に対して異種タンパク質であるので、免疫拒絶反応の問題もあった。 Furthermore, since collagen derived from a living body is a heterologous protein for a patient to be transplanted, there has been a problem of immune rejection.
一方、軟骨欠損部の治療では、荷重のかからない部位の軟骨組織を採取して患部に再移植するモザイクプラスティーと呼ばれる手法が中心であった。しかし、自家組織の使用は患者への負担が大きく、その採取量にも限界がある。 On the other hand, in the treatment of cartilage defects, a technique called mosaic plasticity, in which cartilage tissue at a portion where no load is applied is collected and re-implanted to the affected area, has been the main. However, the use of autologous tissue places a heavy burden on the patient, and the amount of collection is limited.
また、コラーゲンを使用した軟骨培養用基材及びその製法が報告されている（特許文献１）。しかし、本特許文献で使用されるコラーゲンは、生体由来である。
さらに、軟骨の再構築のためのコラーゲンＩ及びコラーゲンＩＩを含有する再吸収可能細胞外マトリックスが報告されている（特許文献２）。しかし、本特許文献は、上記文献と同様に、生体由来のコラーゲンを使用している。
In addition, a base material for cartilage culture using collagen and a production method thereof have been reported (Patent Document 1). However, the collagen used in this patent document is derived from a living body.
Furthermore, a resorbable extracellular matrix containing collagen I and collagen II for cartilage reconstruction has been reported (Patent Document 2). However, this patent document uses collagen derived from a living body, as in the above document.
人工コラーゲンについて報告されている（特許文献３及び４）。しかし、特許文献３では、人工コラーゲンの軟骨細胞の足場としての利用及び軟骨細胞の増殖促進効果については開示がない。
また、特許文献４では、人工コラーゲン、特に人工コラーゲン水溶液の軟骨細胞の増殖促進効果については開示がない。
Artificial collagen has been reported (Patent Documents 3 and 4). However, Patent Document 3 does not disclose the use of artificial collagen as a scaffold for chondrocytes and the effect of promoting the growth of chondrocytes.
In addition, Patent Document 4 does not disclose the effect of promoting the proliferation of chondrocytes of artificial collagen, particularly an artificial collagen aqueous solution.
加えて、P（プロリン）H（ヒスチジン）G（グリシン）配列が繰り返し続いた人工コラーゲン（分子量が1000万）の軟骨細胞に対する効果を検討した例はあるが、骨芽細胞や靱帯や腱細胞、筋肉細胞に対する効果は知られていない。また、PHG配列が1〜8回繰り返しているペプチドの軟骨細胞、骨芽細胞、筋肉細胞に対する効果は知られていない。 In addition, there are examples of examining the effect of artificial collagen (molecular weight of 10 million) with repeated P (proline) H (histidine) G (glycine) sequence on chondrocytes, but osteoblasts, ligaments, tendon cells, The effect on muscle cells is not known. Moreover, the effect with respect to the chondrocyte, osteoblast, and muscle cell of the peptide which the PHG sequence repeats 1 to 8 times is not known.
本発明の課題は、上記要望にこたえるべく、安全性が高いコラーゲンの産生促進剤、プロテオグリカン産出促進剤及び軟骨細胞の遊走促進剤を提供することである。 An object of the present invention is to provide a highly safe collagen production promoter, proteoglycan production promoter, and chondrocyte migration promoter, in order to meet the above-mentioned demand.
本発明者らは、上記の目的を達成するために種々研究を重ねた結果、人工コラーゲン、及びPHGペプチドが、上記要望を充足することを見出し、本発明を完成した。 As a result of various studies to achieve the above object, the present inventors have found that artificial collagen and PHG peptides satisfy the above-mentioned demands, and have completed the present invention.
つまり、本発明は、以下の通りである。
１．人工コラーゲンを含む肩の腱板細胞、アキレス腱細胞、骨芽細胞及び筋細胞のコラーゲンの産生促進剤。
２．前記コラーゲンが、タイプ１コラーゲンである前項１に記載のコラーゲンの産生促進剤。
３．前記人工コラーゲンが、濃度0.05〜5.00％(W/V)の溶液である前項１又は２に記載のコラーゲンの産生促進剤。
４．前記人工コラーゲンが、濃度0.05〜3.00％(W/V)の水溶液である前項１〜３のいずれか１に記載のコラーゲンの産生促進剤。
５．前記人工コラーゲンが、以下（１）〜（３）で表されるペプチドユニットで構成されているポリペプチドである前項１〜４のいずれか１項に記載のコラーゲンの産生促進剤。
（１）［-(OC-(CH2)m-CO)p-(Pro-Y-Gly)n-］a
（２）［-(OC-(CH2)m-CO)q-(Z)r-］b
（３）［-HN-R-NH-］c
（式中、ｍは１〜１８の整数、ｐ及びｑは同一又は異なって０又は１、ＹはＰｒｏまたはＨｙｐ（ハイドロキシプロリン）を表し、ｎは１〜２０の整数を表す。Ｚは１〜１０個のアミノ酸残基からなるペプチド鎖を表し、ｒは１〜２０の整数を表し、Ｒは直鎖状又は分岐鎖状アルキレン基を表す。ａとｂとの割合はａ／ｂ＝１００／０〜３０／７０（モル比）であり、ｐ＝１及びｑ＝０であるときｃ＝ａ、ｐ＝０及びｑ＝１であるときｃ＝ｂであり、ｐ＝１及びｑ＝１であるときｃ＝ａ＋ｂであり、ｐ＝０及びｑ＝０であるときｃ＝０である。）
６．ｍが２〜１２の整数、ｎが２〜１５の整数、Ｚが、Gly, Sar, Ser, Glu, Asp, Lys, His, Ala，Val、Leu、Arg、Pro、Tyr、Ileから選択された少なくとも一種のアミノ酸残基又はペプチド残基で構成されているペプチド鎖、ｒが１〜１０の整数、ＲがＣ２〜１２アルキレン基である前項５に記載のコラーゲンの産生促進剤。
７．さらにヒアルロン酸ナトリウムを含む前項１〜６のいずれか１項に記載のコラーゲンの産生促進剤。
８．さらにRGDペプチドを含む前項１〜７のいずれか１項に記載のコラーゲンの産生促進剤。
９．以下のいずれか１のペプチドを含むコラーゲン産出促進剤。
（１）PHG単位が１〜１２回の繰り返し配列であるペプチド
（２）PHG（配列番号１）
（３）PHG-PHG（配列番号２）
（４）PHG-PHG-PHG-PHG（配列番号３）
（５）PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
１０．以下のいずれか１のペプチドを含むプロテオグリカン産出促進剤。
（１）PHG単位が１〜１２回の繰り返し配列であるペプチド
（２）PHG（配列番号１）
（３）PHG-PHG（配列番号２）
（４）PHG-PHG-PHG-PHG（配列番号３）
（５）PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
１１．以下のいずれか１のペプチドを含む細胞の遊走促進剤。
（１）PHG単位が１〜１２回の繰り返し配列であるペプチド
（２）PHG（配列番号１）
（３）PHG-PHG（配列番号２）
（４）PHG-PHG-PHG-PHG（配列番号３）
（５）PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
That is, the present invention is as follows.
1. A production promoter for collagen of shoulder rotator cuff cells, Achilles tendon cells, osteoblasts and muscle cells, including artificial collagen.
2. 2. The collagen production promoter according to item 1, wherein the collagen is type 1 collagen.
3. 3. The collagen production promoter according to item 1 or 2, wherein the artificial collagen is a solution having a concentration of 0.05 to 5.00% (W / V).
4). 4. The collagen production promoter according to any one of items 1 to 3, wherein the artificial collagen is an aqueous solution having a concentration of 0.05 to 3.00% (W / V).
5). 5. The collagen production promoter according to any one of items 1 to 4, wherein the artificial collagen is a polypeptide composed of peptide units represented by (1) to (3) below.
(1) [-(OC- (CH 2 ) m -CO) p- (Pro-Y-Gly) n- ] a
(2) [- (OC- ( CH 2) m -CO) q - (Z) r -] b
(3) [-HN-R-NH-] c
(In the formula, m is an integer of 1 to 18, p and q are the same or different and 0 or 1, Y represents Pro or Hyp (hydroxyproline), n represents an integer of 1 to 20. Z represents 1 to Represents a peptide chain composed of 10 amino acid residues, r represents an integer of 1 to 20, R represents a linear or branched alkylene group, and the ratio of a to b is a / b = 100 / 0-30 / 70 (molar ratio), c = 1 when p = 1 and q = 0, c = b when p = 0 and q = 1, p = 1 and q = 1 C = a + b when there is, c = 0 when p = 0 and q = 0.)
6). m is an integer from 2 to 12, n is an integer from 2 to 15, and Z is selected from Gly, Sar, Ser, Glu, Asp, Lys, His, Ala, Val, Leu, Arg, Pro, Tyr, and Ile 6. The collagen production promoter according to item 5 above, wherein the peptide chain is composed of at least one amino acid residue or peptide residue, r is an integer of 1 to 10, and R is a C2-12 alkylene group.
7). The collagen production promoter according to any one of the preceding items 1 to 6, further comprising sodium hyaluronate.
8). The collagen production promoter according to any one of 1 to 7 above, further comprising an RGD peptide.
9. A collagen production promoter comprising any one of the following peptides.
(1) PHG unit is a peptide having a repeating sequence of 1 to 12 times (2) PHG (SEQ ID NO: 1)
(3) PHG-PHG (SEQ ID NO: 2)
(4) PHG-PHG-PHG-PHG (SEQ ID NO: 3)
(5) PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (sequence number 4)
10. A proteoglycan production promoter comprising any one of the following peptides.
(1) PHG unit is a peptide having a repeating sequence of 1 to 12 times (2) PHG (SEQ ID NO: 1)
(3) PHG-PHG (SEQ ID NO: 2)
(4) PHG-PHG-PHG-PHG (SEQ ID NO: 3)
(5) PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (sequence number 4)
11. A cell migration promoter comprising any one of the following peptides.
(1) PHG unit is a peptide having a repeating sequence of 1 to 12 times (2) PHG (SEQ ID NO: 1)
(3) PHG-PHG (SEQ ID NO: 2)
(4) PHG-PHG-PHG-PHG (SEQ ID NO: 3)
(5) PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (sequence number 4)
本発明では、安全性が高い、コラーゲンの産生促進剤、プロテオグリカン産出促進剤及び軟骨細胞の遊走促進剤を提供できる。 The present invention can provide a collagen production promoter, proteoglycan production promoter, and chondrocyte migration promoter, which are highly safe.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
（人工コラーゲン）
本発明の人工コラーゲンは、生体由来のコラーゲンではないことを意味する。例えば、以下のようなポリペプチドを本発明の人工コラーゲンとする。
(Artificial collagen)
The artificial collagen of the present invention means that it is not a collagen derived from a living body. For example, the following polypeptides are used as the artificial collagen of the present invention.
本発明の人工コラーゲンであるポリペプチドは、下記式（１）〜（３）で表されるペプチドユニットで構成されている。
（１）［-(OC-(CH2)m-CO)p-(Pro-Y-Gly)n-］a
（２）［-(OC-(CH2)m-CO)q-(Z)r-］b
（３）［-HN-R-NH-］c
式中、ｍは１〜１８の整数、ｐ及びｑは同一又は異なって０又は１、ＹはＰｒｏまたはＨｙｐを表し、ｎは１〜２０の整数を表す。Ｚは１〜１０個のアミノ酸残基からなるペプチド鎖を表し、ｒは１〜２０の整数を表し、Ｒは直鎖状又は分岐鎖状アルキレン基を表す。ａとｂとの割合はａ／ｂ＝１００／０〜３０／７０（モル比）であり、ｐ＝１及びｑ＝０であるときｃ＝ａ、ｐ＝０及びｑ＝１であるときｃ＝ｂであり、ｐ＝１及びｑ＝１であるときｃ＝ａ＋ｂであり、ｐ＝０及びｑ＝０であるときｃ＝０である。
より詳しくは、前記式（１）〜（３）のユニットで構成されたポリペプチドにおいて、通常、ｍは２〜１２の整数、ｎは２〜１５の整数であり、Ｚは、Gly, Sar, Ser, Glu, Asp, Lys, His, Ala，Val、Leu、Arg、Pro、Tyr、Ileから選択された少なくとも一種のアミノ酸残基又はペプチド残基で構成されているペプチド鎖である。また、前記式において、通常、ｒは１〜１０の整数、ＲはＣ２〜１２アルキレン基である。
The polypeptide which is the artificial collagen of the present invention is composed of peptide units represented by the following formulas (1) to (3).
(1) [-(OC- (CH 2 ) m -CO) p- (Pro-Y-Gly) n- ] a
(2) [- (OC- ( CH 2) m -CO) q - (Z) r -] b
(3) [-HN-R-NH-] c
In the formula, m is an integer of 1 to 18, p and q are the same or different and 0 or 1, Y represents Pro or Hyp, and n represents an integer of 1 to 20. Z represents a peptide chain composed of 1 to 10 amino acid residues, r represents an integer of 1 to 20, and R represents a linear or branched alkylene group. The ratio of a and b is a / b = 100/0 to 30/70 (molar ratio), c = a when p = 1 and q = 0, c when p = 0 and q = 1 C = a + b when p = 1 and q = 1, and c = 0 when p = 0 and q = 0.
More specifically, in the polypeptide composed of the units of the above formulas (1) to (3), m is usually an integer of 2 to 12, n is an integer of 2 to 15, and Z is Gly, Sar, It is a peptide chain composed of at least one amino acid residue or peptide residue selected from Ser, Glu, Asp, Lys, His, Ala, Val, Leu, Arg, Pro, Tyr, and Ile. In the above formula, r is usually an integer of 1 to 10, and R is a C2-12 alkylene group.
また、本発明のポリペプチドは、下記の繰り返し単位(i)、(ii)又は(iii)で構成されていてもよい。
（i）ペプチドユニット［-(Pro-Y-Gly)n-］a（式中、Ｙ及びｎは、前記に同じ）と、ペプチドユニット［-(Z)r-］b（式中、Ｚ及びｒは、前記に同じ）とを、ａ／ｂ＝１００／０〜４０／６０（モル比）の割合で含む繰り返し単位で構成されたポリペプチド
（ii）ペプチドユニット［-(OC-(CH2)m-CO)-(Pro-Y-Gly)n-］a（式中、ｍ、ｎ及びＹは、前記に同じ）と、ユニット［-HN-R-NH-］c（式中、Ｒは前記に同じ）とを、実質的にａ／ｃ＝１／１（モル比）の割合で含む繰り返し単位で構成されたポリペプチド
（iii）ペプチドユニット［-(OC-(CH2)m-CO)-(Pro-Y-Gly)n-］a（式中、ｍ、ｎ及びＹは、前記に同じ）と、ペプチドユニット［-(OC-(CH2)m-CO)-(Z)r-］b（式中、ｍ、ｒ及びＺは、前記に同じ）と、ユニット［-HN-R-NH-］c（式中、Ｒは前記に同じ）とを、ａ／ｂ＝１００／０〜４０／６０（モル比）の割合、なおかつ、実質的に(ａ＋ｂ)／ｃ＝１／１（モル比）の割合で含む繰り返し単位で構成されたポリペプチド。
Moreover, the polypeptide of the present invention may be composed of the following repeating units (i), (ii) or (iii).
(I) Peptide unit [-(Pro-Y-Gly) n- ] a (wherein Y and n are as defined above) and peptide unit [-(Z) r- ] b (wherein Z and r is the same as the above) and a polypeptide comprising a repeating unit containing a ratio of a / b = 100/0 to 40/60 (molar ratio). (ii) a peptide unit [-(OC- (CH 2 ) m -CO)-(Pro-Y-Gly) n- ] a (wherein m, n and Y are as defined above) and the unit [-HN-R-NH-] c (where R Is the same as the above) and a polypeptide comprising a repeating unit substantially in the ratio of a / c = 1/1 (molar ratio) (iii) a peptide unit [-(OC- (CH 2 ) m- CO)-(Pro-Y-Gly) n- ] a (wherein m, n and Y are as defined above) and the peptide unit [-(OC- (CH 2 ) m -CO)-(Z) r -] b (wherein, m, r and Z are as defined above) and the unit [(wherein, R have the same meanings as defined above) -HN-R-NH-] c and , The ratio of a / b = 100 / 0~40 / 60 (molar ratio), and yet, essentially (a + b) / c = 1/1 polypeptide composed of repeating units in a proportion (molar ratio).
なお、上記Ｒで表される直鎖状又は分岐鎖状アルキレン基は、ポリペプチドの物理的および生物学的性質を損なわない範囲であればよく、例えば、メチレン、エチレン、プロピレン、トリメチレン、テトラメチレンなどのＣ１〜１８アルキレン基が例示できる。前記アルキレン基Ｒは、直鎖状のメチレン鎖(CH2)s（ｓは１〜１８の整数を表す）であってもよい。好ましいＲは、Ｃ２〜１２アルキレン基（さらに好ましくはＣ２〜１０アルキレン基，特にＣ２〜６アルキレン基）である。
なお、これらの人工コラーゲンの詳細及び製造方法は、特開2003-321500に記載されている。
The linear or branched alkylene group represented by R may be in a range that does not impair the physical and biological properties of the polypeptide. For example, methylene, ethylene, propylene, trimethylene, tetramethylene C 1 ~ 18 alkylene group, and others. The alkylene group R may be a linear methylene chain (CH 2 ) s (s represents an integer of 1 to 18). Preferred R is a C 2-12 alkylene group (more preferably a C 2-10 alkylene group, particularly a C 2-6 alkylene group).
Details and methods for producing these artificial collagens are described in JP-A-2003-321500.
加えて、好ましくは、本発明の人工コラーゲンは、株式会社ＰＨＧより販売されている人工コラーゲン（INCI名：Poly(Tripeptide-6)、CAS.No:60961-94-6：http://www.phg.co.jp/research/collagen.html）を使用することが好ましい。 In addition, the artificial collagen of the present invention is preferably an artificial collagen (INCI name: Poly (Tripeptide-6), CAS. No: 60961-94-6: http: // www. phg.co.jp/research/collagen.html) is preferably used.
（人工コラーゲンを含むコラーゲン産生促進剤）
本発明の人工コラーゲンを含む産生促進剤は、少なくとも上記記載の人工コラーゲンを含む。また、人工コラーゲンは、溶液、特に水溶液の形態、好ましくは0.10％〜5.0％(W/V)、より好ましく0.30％〜3.0％(W/V)、最も好ましくは0.50％〜2.0％(W/V)の水溶液にすることが好ましい。
なお、人工コラーゲン水溶液とは、人工コラーゲンを水又は生理食塩水に溶解又は一部溶解した溶液を意味する。
(Collagen production promoter including artificial collagen)
The production promoter containing the artificial collagen of the present invention contains at least the artificial collagen described above. In addition, the artificial collagen is in the form of a solution, particularly an aqueous solution, preferably 0.10% to 5.0% (W / V), more preferably 0.30% to 3.0% (W / V), and most preferably 0.50% to 2.0% (W / V). It is preferable to use an aqueous solution of V).
The artificial collagen aqueous solution means a solution obtained by dissolving or partially dissolving artificial collagen in water or physiological saline.
さらに、本発明のコラーゲン産生促進剤は、追加の活性成分、担体若しくはキャリア、又は添加剤などを含んでいてもよい。
前記活性成分としては、例えば、殺菌剤又は消毒剤、抗炎症剤、消炎・鎮痛剤、鎮痒剤、抗潰瘍剤、抗アレルギー剤、抗ウイルス剤、抗真菌剤、抗生物質、皮膚軟化剤、褥瘡・皮膚治療剤、ビタミン剤、漢方薬などが例示できる。さらに、ヒアルロン酸ナトリウム、塩基性線維芽細胞増殖因子（ｂＦＧＦ）、血小板分化増殖因子（ＰＤＧＦ）、インスリン、インスリン様増殖因子（ＩＧＦ）、肝細胞増殖因子（ＨＧＦ）、グリア誘導神経栄養因子（ＧＤＮＦ）、神経栄養因子（ＮＦ）、トランスフォーミング増殖因子（ＴＧＦ）、および血管内皮細胞増殖因子（ＶＥＧＦ）等の増殖因子、骨形成タンパク質（ＢＭＰ）や転写因子等のその他のサイトカイン、ホルモン、Ｍｇ、Ｃａ及びＣＯ３等の無機塩、クエン酸及びリン脂質等の有機物、抗がん剤等の薬剤等を挙げることができる。
担体又はキャリアとしては、生体材料の剤形（固形剤、半固形剤、液剤など）に応じて、生理学的に許容される種々の担体やキャリアが使用できる。例えば、固形剤の担体としては、結合剤、賦形剤、崩壊剤などが例示できる。
Furthermore, the collagen production promoter of the present invention may contain an additional active ingredient, a carrier or a carrier, or an additive.
Examples of the active ingredient include bactericides or disinfectants, anti-inflammatory agents, anti-inflammatory / analgesic agents, antipruritic agents, anti-ulcer agents, antiallergic agents, antiviral agents, antifungal agents, antibiotics, emollients, acne -Examples include skin treatment agents, vitamins, and herbal medicines. In addition, sodium hyaluronate, basic fibroblast growth factor (bFGF), platelet differentiation growth factor (PDGF), insulin, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), glial-induced neurotrophic factor (GDNF) ), Growth factors such as neurotrophic factor (NF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF), other cytokines such as bone morphogenetic protein (BMP) and transcription factors, hormones, Mg, Examples thereof include inorganic salts such as Ca and CO 3 , organic substances such as citric acid and phospholipid, and drugs such as anticancer agents.
As the carrier or carrier, various physiologically acceptable carriers and carriers can be used depending on the dosage form of the biomaterial (solid agent, semi-solid agent, liquid agent, etc.). For example, examples of the solid carrier include binders, excipients, and disintegrants.
加えて、液剤の形態では、担体として、例えば、水、アルコール（エタノールなど）、エチレングリコール、プロピレングリコール、ポリエチレングリコール−ポリプロピレングリコール共重合体、油脂（トウモロコシ油、オリーブ油など）などが挙げられる。 In addition, in the form of a liquid preparation, examples of the carrier include water, alcohol (ethanol and the like), ethylene glycol, propylene glycol, polyethylene glycol-polypropylene glycol copolymer, fat and oil (corn oil, olive oil and the like).
（人工コラーゲンを含むコラーゲン産生促進剤の用途）
本発明のコラーゲン産生促進剤の用途は、肩の腱板細胞のコラーゲン、アキレス腱細胞のコラーゲン、骨芽細胞のコラーゲン、筋細胞のコラーゲン、特にタイプ１コラーゲンの産出を促進する。
(Use of collagen production promoters including artificial collagen)
The use of the collagen production promoter of the present invention promotes the production of collagen of shoulder rotator cuff cells, collagen of Achilles tendon cells, collagen of osteoblasts, collagen of muscle cells, particularly type 1 collagen.
本発明のコラーゲン産生促進剤の別の用途としては、軟骨細胞のための足場材料、軟骨細胞の分化・増殖材、軟骨再生材として使用することができる。
例えば、本発明のコラーゲン産生促進剤は、軟骨（細胞）培養の好適な足場として、生体外および生体内における軟骨組織の構築を可能にする。
また、例えば、本発明のコラーゲン産生促進剤を軟組織欠損部に移植すれば、移植された箇所で軟骨組織の再生（分化・増殖）が促進される。
加えて、本発明のコラーゲン産生促進剤の用途である足場材料およびインプラントの形態及び形状は、特に限定されず、スポンジ、メッシュ、不繊布状成形物、ディスク状、フィルム状、棒状、粒子状、及びペースト状等、任意の形態及び形状を用いることができる。こうした形態や形状は、足場材料やインプラントの使用目的に応じて適宜選択すればよい。
As another use of the collagen production promoter of the present invention, it can be used as a scaffold material for chondrocytes, a differentiation / proliferation material for chondrocytes, and a cartilage regeneration material.
For example, the collagen production promoter of the present invention enables the construction of cartilage tissue in vitro and in vivo as a suitable scaffold for cartilage (cell) culture.
For example, if the collagen production promoter of the present invention is transplanted into a soft tissue defect, regeneration (differentiation / proliferation) of cartilage tissue is promoted at the transplanted site.
In addition, the form and shape of the scaffold material and the implant that are the uses of the collagen production promoter of the present invention are not particularly limited, and are sponge, mesh, non-woven fabric molding, disk shape, film shape, rod shape, particle shape, And arbitrary forms and shapes, such as paste form, can be used. Such form and shape may be appropriately selected according to the purpose of use of the scaffold material or the implant.
（人工コラーゲンを含むコラーゲン産生促進剤の投与方法）
本発明のコラーゲン産生促進剤の投与方法としては、以下の方法が例示されるが、特に限定されない。
（１）本発明のコラーゲン産生促進剤を肩関節に注入して肩の腱板細胞のタイプIコラーゲンの産生を促進させる。
（２）本発明のコラーゲン産生促進剤をアキレス腱部に注入してアキレス腱細胞のタイプIコラーゲンの産生を促進させる。
（３）本発明のコラーゲン産生促進剤を筋肉内に注入して筋細胞のタイプIコラーゲンの産生を促進させる。
（４）本発明のコラーゲン産生促進剤を骨欠損部に使用するスカフォルドに添加して骨芽細胞のタイプIコラーゲンの産生を促進させる。
なお、上記（１）〜（４）のコラーゲン産生促進剤の濃度は、0.01〜5.0％、好ましくは0.03〜1.0％、より好ましくは0.05〜0.3％の範囲で使用する。
(Method of administering collagen production promoter including artificial collagen)
Although the following method is illustrated as an administration method of the collagen production promoter of this invention, It is not specifically limited.
(1) The collagen production promoter of the present invention is injected into the shoulder joint to promote the production of type I collagen in the rotator cuff cells of the shoulder.
(2) The collagen production promoter of the present invention is injected into the Achilles tendon to promote the production of Achilles tendon cell type I collagen.
(3) The collagen production promoter of the present invention is injected into muscle to promote the production of type I collagen in muscle cells.
(4) The collagen production promoter of the present invention is added to the scaffold used in the bone defect part to promote the production of osteoblast type I collagen.
The concentration of the collagen production promoters (1) to (4) is 0.01 to 5.0%, preferably 0.03 to 1.0%, more preferably 0.05 to 0.3%.
（PHGペプチド）
本発明のPHGペプチドとは、 PHG配列が繰り返し続いたペプチドを意味する。PHG配列の繰り返し単位は、１〜１２回であり、好ましくは、１、２、４、８回である。加えて、本発明のPHGペプチドは下記で述べる用途の効果を消失させない限りは、適宜、１〜５個のアミノ酸の置換、挿入、消失があっても良く、さらに、化学的修飾があっても良い。
(PHG peptide)
The PHG peptide of the present invention means a peptide having repeated PHG sequences. The repeating unit of the PHG sequence is 1 to 12 times, preferably 1, 2, 4, 8 times. In addition, the PHG peptide of the present invention may have 1 to 5 amino acid substitutions, insertions, disappearances, and chemical modifications as long as the effects of the uses described below are not lost. good.
（PHGペプチドを含むコラーゲン産生促進剤の用途）
本発明のPHGペプチドを含むコラーゲン産生促進剤の用途は、コラーゲン産出促進、特にタイプIコラーゲンの産生を促進させる。より詳しくは、骨芽細胞のタイプIコラーゲンの産生を促進させる。
なお、PHGペプチドを含むコラーゲン産生促進剤の濃度は、1〜2000μM、好ましくは2〜1500μM、より好ましくは3〜1000μMの範囲で使用する。
(Use of collagen production promoter containing PHG peptide)
The use of the collagen production promoter containing the PHG peptide of the present invention promotes the production of collagen, particularly the production of type I collagen. More specifically, it promotes the production of osteoblast type I collagen.
The concentration of the collagen production promoter containing the PHG peptide is 1 to 2000 μM, preferably 2 to 1500 μM, more preferably 3 to 1000 μM.
（PHGペプチドを含むプロテオグリカン産出促進剤の用途）
本発明のPHGペプチドを含むプロテオグリカン産出促進剤の用途は、プロテオグリカンの産生を促進させる。より詳しくは、軟骨細胞のプロテオグリカンの産生を促進させる。
なお、PHGペプチドを含むプロテオグリカン産出促進剤の濃度は、1〜2000μM、好ましくは2〜1500μM、より好ましくは3〜1000μMの範囲で使用する。
(Use of proteoglycan production promoter containing PHG peptide)
The use of the proteoglycan production promoter containing the PHG peptide of the present invention promotes the production of proteoglycan. More specifically, it promotes the production of proteoglycans by chondrocytes.
The concentration of the proteoglycan production promoter containing the PHG peptide is 1 to 2000 μM, preferably 2 to 1500 μM, more preferably 3 to 1000 μM.
（PHGペプチドを含む細胞の遊走促進剤の用途）
本発明のPHGペプチドを含む細胞の遊走促進剤の用途は、細胞（特に、軟骨細胞、骨芽細胞）の遊走を促進させる。
なお、PHGペプチドを含むプロテオグリカン産出促進剤の濃度は、1〜2000μM、好ましくは2〜1500μM、より好ましくは3〜1000μMの範囲で使用する。
(Use of cell migration promoter containing PHG peptide)
The use of the cell migration promoter containing the PHG peptide of the present invention promotes the migration of cells (particularly chondrocytes and osteoblasts).
The concentration of the proteoglycan production promoter containing the PHG peptide is 1 to 2000 μM, preferably 2 to 1500 μM, more preferably 3 to 1000 μM.
（PHGペプチドを含むコラーゲン産生促進剤、プロテオグリカン産出促進剤、細胞の遊走促進剤の投与方法）
本発明の投与方法としては、以下の方法が例示されるが、特に限定されない。
（１）PHGペプチドをヒアルロン等に添加した後に、肩関節内に投与することで肩の腱板を修復させる。また、関節内注射することで軟骨修復材や軟骨保護剤として使用できる。
（２）PHGペプチドをアキレス腱や筋細胞に注入することで細胞の修復を促進させる。加えて、PHGペプチドを湿布剤に含ませて皮膚より浸透させることで、靱帯や筋肉損傷の修復を促進させる。
（３）PHGペプチドを骨欠損部に充填するスカフォルドに添加することで、周囲より骨芽細胞を遊走させて欠損部に集めて骨欠損の修復を促進させる。
（４）PHGペプチドを軟骨骨欠損部に充填するスカフォルドに添加することで、周囲より軟骨細胞を遊走させて欠損部に集めて軟骨欠損の修復を促進させる。
(Administration method of collagen production promoter containing PHG peptide, proteoglycan production promoter, cell migration promoter)
Although the following method is illustrated as an administration method of this invention, It is not specifically limited.
(1) After adding the PHG peptide to hyaluron or the like, the shoulder rotator cuff is repaired by administration into the shoulder joint. In addition, it can be used as a cartilage repair material or cartilage protective agent by intra-articular injection.
(2) Promote cell repair by injecting PHG peptide into Achilles tendon and muscle cells. In addition, the PHG peptide is included in the poultice and permeated through the skin to promote repair of ligaments and muscle damage.
(3) By adding the PHG peptide to the scaffold that fills the bone defect part, osteoblasts migrate from the surroundings and collect in the defect part to promote repair of the bone defect.
(4) By adding the PHG peptide to the scaffold that fills the cartilage defect part, the chondrocytes migrate from the surroundings and collect in the defect part to promote repair of the cartilage defect.
（軟骨細胞）
本発明における使用のための軟骨細胞は、関節軟骨、骨膜および軟骨膜から単離される同種異系または自家細胞、ならびに骨髄からの間葉（間質）幹細胞を包含する細胞供給源から得られ得る。同種異系細胞は免疫応答および感染性合併症に関する潜在的能力を保有するので、自家細胞から、特に自家の関節軟骨から軟骨細胞を単離することが好ましい。細胞を収集するための技法は既知であり、酵素的消化または外部成長培養が含まれる。次に、収集細胞は、身体への再導入前に細胞培養で増やされる。概して、軟骨組織の最適な再生を提供するためには、少なくとも106、好ましくは少なくとも107細胞が軟骨培養用基材中に含浸されるべきである。
(Chondrocytes)
Chondrocytes for use in the present invention may be obtained from cell sources including articular cartilage, allogeneic or autologous cells isolated from periosteum and perichondrium, and mesenchymal (stromal) stem cells from bone marrow. . Since allogeneic cells possess the potential for immune responses and infectious complications, it is preferable to isolate chondrocytes from autologous cells, particularly from autologous articular cartilage. Techniques for collecting cells are known and include enzymatic digestion or external growth culture. The collected cells are then expanded in cell culture before being reintroduced into the body. In general, in order to provide optimal regeneration of cartilage tissue, at least 10 6 , preferably at least 10 7 cells should be impregnated into the cartilage culture substrate.
（プロテオグリカン）
プロテオグリカン（proteoglycan）は、糖側鎖としてGAG（グリコサミノグリカン）を有するタンパク質であり、古くはムコ多糖と呼ばれてきた。骨、軟骨、皮膚などの結合組織に豊富に存在しており、細胞内や細胞膜に存在が認められている。
(Proteoglycan)
Proteoglycan (proteoglycan) is a protein having GAG (glycosaminoglycan) as a sugar side chain, and has been called mucopolysaccharide in the old days. It is abundant in connective tissues such as bone, cartilage, and skin, and is found in cells and cell membranes.
（RGD）
RGD（アルギニン−グリシン−アスパラギン酸；配列番号５）配列は、いくつかの重要な細胞外マトリックスタンパク質中に見出され、そして細胞表面受容体のインテグリンファミリーのメンバーへの接着リガンドとして働く。典型的なRGD配列は、Ｇｌｙ−Ａｒｇ−Ｇｌｙ−Ａｓｐ−Ｓｅｒ−Ｐｒｏ（ＧＲＧＤＳＰ；配列番号６）である。環状RGDもまた、細胞接着モチーフとして使用可能である。典型的な配列はＡｒｇ−Ｇｌｙ−Ａｓｐ−（Ｄ−Ｐｈｅ）−Ｖａｌ（配列番号７）である。RGD修飾表面は、膜上にそのままで（ｉｎ ｓｉｔｕ）細胞単層の形成を導く。
すなわち、本発明の軟骨培養用基材は、上記いずれかのRGDペプチドを含むことにより、軟骨細胞との接着能力を高め、軟骨細胞の増殖能力を高めることができると考えられる。
(RGD)
The RGD (arginine-glycine-aspartate; SEQ ID NO: 5) sequence is found in several important extracellular matrix proteins and serves as an adhesion ligand to members of the integrin family of cell surface receptors. A typical RGD sequence is Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP; SEQ ID NO: 6). Cyclic RGD can also be used as a cell adhesion motif. A typical sequence is Arg-Gly-Asp- (D-Phe) -Val (SEQ ID NO: 7). The RGD modified surface leads to the formation of a cell monolayer in situ on the membrane.
That is, it is considered that the substrate for cartilage culture of the present invention can enhance the ability to adhere to chondrocytes and the ability to proliferate chondrocytes by including any of the above RGD peptides.
（関節機能改善剤）
現在、変形性関節症、スポーツ障害後の変形性関節症、関節リウマチ、などの疾患に軟骨保護作用のあるヒアルロン酸製剤を関節内に注射することで有効な治療効果が得られている。
例えば、関節機能改善剤であるヒアルロン酸ナトリウム関節内注射液であるスベニール（登録商標）（製造販売元：中外製薬株式会社）、アルツ（登録商標）（製造販売元：生化学工業株式会社）が販売されている。
上記関節機能改善剤の用法・用量は、以下の通りである。
変形性膝関節症：通常、成人１回2.5ml（ヒアルロン酸ナトリウム25mg）を１週間毎に連続５回膝関節腔内に投与する。その後、症状の維持を目的とする場合は、２〜４週間隔で投与する。
肩関節周囲炎：通常、成人１回2.5ml（ヒアルロン酸ナトリウム25mg）を１週間毎に連続５回肩関節（肩関節腔、肩峰下滑液包又は上腕二頭筋長頭腱腱鞘）内に投与する。
慢性関節リウマチにおける膝関節痛：通常、成人１回2.5ml（ヒアルロン酸ナトリウム25mg）を１週間毎に連続５回膝関節腔内に投与する。
(Joint function improving agent)
At present, an effective therapeutic effect is obtained by injecting into the joint a hyaluronic acid preparation having a cartilage-protecting action for diseases such as osteoarthritis, osteoarthritis after sports injury, and rheumatoid arthritis.
For example, Svenyl (registered trademark) (manufacturer / distributor: Chugai Pharmaceutical Co., Ltd.) and Alz (registered trademark) (manufacturer / distributor: Seikagaku Corporation), sodium hyaluronate intraarticular injection, which is a joint function improving agent Sold.
The usage and dosage of the above joint function improving agent are as follows.
Osteoarthritis of the knee: In general, 2.5 ml of adult (sodium hyaluronate 25 mg) is administered once into the knee joint cavity 5 times a week. Thereafter, if the purpose is to maintain symptoms, administration is performed at intervals of 2 to 4 weeks.
Shoulder periarthritis: In general, adults 2.5ml (sodium hyaluronate 25mg) once in a week 5 times in a shoulder joint (shoulder joint cavity, subacromial bursa or biceps long head tendon sheath) Administer.
Knee joint pain in rheumatoid arthritis: In general, adults are administered 2.5 ml (sodium hyaluronate 25 mg) once a week 5 times in the knee joint cavity once a week.
本発明の人工コラーゲン水溶液には軟骨修復効果がある。関節の体積の小さい指関節から、体積の大きい膝関節や股関節など全身の関節軟骨の修復を行うために、濃度を0.1mg/ml〜300mg/mlで変えて投与を行うことが好ましい。
また、人工コラーゲン水溶液をヒアルロン酸などと混合することで、軟骨保護効果や軟骨修復効果を増強できる。ここで記載している軟骨には、硝子軟骨と半月板（膝関節）、関節唇（股関節）などの線維軟骨を含む。すなわち、本発明の人工コラーゲンをヒアルロン酸又は上記の関節機能改善剤に添加した関節機能改善剤を関節内に投与することで、下記実施例の結果から明らかなように、軟骨保護効果を有し軟骨修復を促進させる効果があると考えられる。
また、1cm3の溶液に対して0.1mgから300mgまでの人工コラーゲンを上記関節機能改善剤2.5mlに添加することで関節系脳改善剤を製造することができる。
The artificial collagen aqueous solution of the present invention has a cartilage repair effect. In order to repair articular cartilage of the whole body such as a knee joint or a hip joint having a large volume from a finger joint having a small joint volume, it is preferable to administer by changing the concentration from 0.1 mg / ml to 300 mg / ml.
Further, by mixing an artificial collagen aqueous solution with hyaluronic acid or the like, the cartilage protection effect and the cartilage repair effect can be enhanced. The cartilage described here includes hyaline cartilage and fibrocartilage such as meniscus (knee joint), joint lip (hip joint) and the like. That is, by administering into a joint a joint function improving agent obtained by adding the artificial collagen of the present invention to hyaluronic acid or the above joint function improving agent, it has a cartilage protective effect, as is apparent from the results of the following examples. It is thought to have an effect of promoting cartilage repair.
Further, an articular brain improving agent can be produced by adding 0.1 mg to 300 mg of artificial collagen to 2.5 ml of the above joint function improving agent per 1 cm 3 solution.
本発明のコラーゲン産生促進剤、プロテオグリカン産出促進剤、細胞の遊走促進剤を医療用途に用いる場合には、殺菌又は滅菌して用いることが好ましい。殺菌、滅菌方法としては、種々の殺菌・滅菌方法、例えば、湿熱蒸気滅菌、ガンマ線滅菌、エチレンオキサイドガス滅菌、薬剤殺菌、紫外線殺菌などが例示できる。これらの方法のうち、ガンマ線滅菌、エチレンオキサイドガス滅菌は、滅菌効率と材料に与える影響が少なく好ましい。 When the collagen production promoter, proteoglycan production promoter, and cell migration promoter of the present invention are used for medical purposes, it is preferably used after sterilization or sterilization. Examples of sterilization and sterilization methods include various sterilization and sterilization methods such as wet heat steam sterilization, gamma ray sterilization, ethylene oxide gas sterilization, drug sterilization, and ultraviolet sterilization. Among these methods, gamma ray sterilization and ethylene oxide gas sterilization are preferable because they have little effect on sterilization efficiency and materials.
本発明のコラーゲン産生促進剤、プロテオグリカン産出促進剤、細胞の遊走促進剤は、種々の被検体（被験体）の組織（例えば、表皮組織及び真皮組織など）へ適用できる。被検体は、ヒトに限らず、非ヒト動物（例えば、サル、ヒツジ、ウシ、ウマ、イヌ、ネコ、ウサギ、ラット、マウスなどの非ヒト動物）であってもよい。 The collagen production promoter, proteoglycan production promoter, and cell migration promoter of the present invention can be applied to tissues of various subjects (subjects) (for example, epidermal tissue and dermal tissue). The subject is not limited to a human but may be a non-human animal (for example, non-human animals such as monkeys, sheep, cows, horses, dogs, cats, rabbits, rats, mice).
以下に、本発明の実施例を示し、本発明を更に具体的に明らかにすることとするが、本発明が、そのような実施例の記載によって何等の制約をも受けるものでないことはいうまでもないところである。また、本発明には、以下の実施例の他にも更には上記した発明の実施の形態における記述以外にも、本発明の趣旨を逸脱し得ない限りにおいて、当業者の知識に基づいて、種々なる変更、修正、改良等を加え得るものであることが理解されるべきである。 Examples of the present invention will be shown below to clarify the present invention more specifically, but it goes without saying that the present invention is not limited by the description of such examples. There is no place. In addition to the following examples, in addition to the description in the embodiment of the present invention described above, the present invention is based on the knowledge of those skilled in the art unless it departs from the spirit of the present invention. It should be understood that various changes, modifications, improvements and the like can be made.
（人工コラーゲン水溶液の効果確認）
本発明の人工コラーゲンを含む水溶液のアキレス腱、肩腱板に対する効果を確認した。詳細は、以下の通りである。
(Confirmation of effect of artificial collagen aqueous solution)
The effect of the aqueous solution containing the artificial collagen of the present invention on the Achilles tendon and the shoulder rotator cuff was confirmed. Details are as follows.
（人工コラーゲン）
本発明の人工コラーゲンは、株式会社ＰＨＧより販売されている人工コラーゲン（INCI名：Poly(Tripeptide-6)、CAS.No:60961-94-6：http://www.phg.co.jp/research/collagen.html）を使用した。
(Artificial collagen)
The artificial collagen of the present invention is an artificial collagen (INCI name: Poly (Tripeptide-6), CAS.No:60961-94-6: http://www.phg.co.jp/ research / collagen.html).
（細胞の調整）
New Zealand White rabbit 7週令♂3羽を麻酔下で屠殺してアキレス腱、rotator cuff（肩の腱板）を無菌的に採取した。
（培養液）
DMEM(high Glucose) with L-Glutamine and Phenol Red(SIGMA D5796)
Penicillin-streptomycin
10% Fetal Bovine Serum (EQUITECH-BIO,INC.)
50 μg/mL 2-phospho-L-ascorbic acid trisodium salt (Fluka)
（試薬）
Collagenase Type1 (Worthington CLS1)
PBSで0.1％に調整 (1mg/mL) 後、フィルター滅菌(0.22 μm)した。
Trypsin (SIGMA T-8003)
PBSで0.25％に調整 (2.5mg/mL) 後、フィルター滅菌(0.22 μm)した。
Dulbecco's PBS (-) (Nissui)
(Cell adjustment)
New Zealand White rabbit Three 7-week old rabbits were sacrificed under anesthesia, and Achilles tendon and rotator cuff (shoulder rotator cuff) were aseptically collected.
(Culture medium)
DMEM (high Glucose) with L-Glutamine and Phenol Red (SIGMA D5796)
Penicillin-streptomycin
10% Fetal Bovine Serum (EQUITECH-BIO, INC.)
50 μg / mL 2-phospho-L-ascorbic acid trisodium salt (Fluka)
(reagent)
Collagenase Type1 (Worthington CLS1)
After adjusting to 0.1% with PBS (1 mg / mL), filter sterilization (0.22 μm) was performed.
Trypsin (SIGMA T-8003)
After adjustment to 0.25% with PBS (2.5 mg / mL), filter sterilization (0.22 μm) was performed.
Dulbecco's PBS (-) (Nissui)
１．靭帯を遠沈管PBS 10 mLに入れて振とうし、1300 rpm 20℃ 5 minで遠心し、上清を吸引した。
２．靭帯を裁断した後、0.1％ コラゲナーゼ溶液4.5 mLを加え、37℃の恒温槽で揺らしながら12時間処理した。
３．上記の溶液に0.25％ トリプシンを4.5 mL加え撹拌し、37℃で揺らしながらさらに2時間処理した。
４．上記の溶液にDMEMを9mL加えセルストレイナーに通して組織を取り除いた。
５．1300 rpm 20℃ 5 minで遠心し、上清を吸引した。
６．ascorbic acid入り10% FBS加DMEMにペレットを懸濁した。
７．100 mm dishに播種し、37℃、5％CO2にて培養した。
1. The ligament was placed in 10 mL of centrifuge tube PBS, shaken, centrifuged at 1300 rpm at 20 ° C. for 5 min, and the supernatant was aspirated.
2. After cutting the ligament, 4.5 mL of a 0.1% collagenase solution was added and treated for 12 hours while shaking in a 37 ° C. constant temperature bath.
3. To the above solution, 4.5 mL of 0.25% trypsin was added and stirred, and further treated for 2 hours while shaking at 37 ° C.
4). 9 mL of DMEM was added to the above solution, and the tissue was removed through a cell strainer.
5. Centrifugation was performed at 1300 rpm at 20 ° C. for 5 min, and the supernatant was aspirated.
6). The pellet was suspended in DMEM containing 10% FBS containing ascorbic acid.
7. Seeded in a 100 mm dish and cultured at 37 ° C., 5% CO 2 .
（靱帯細胞の播種）
semi-confluentに達した靱帯細胞をtripsin-EDTA溶液処理によりdishから剥離し、培養液を加えて遠心洗浄した。その後、1.5×106 cells/wellにて6-well plateに播種し、37℃、5% CO2にて培養を行った。
（人工コラーゲン水溶液の添加）
培養開始24時間後において、人工コラーゲンを0（コントロール）、0.05、0.1、0.2、0.3％に調整した培養液に交換し、7日間作用させた。
（靱帯細胞の回収・RNA抽出）
上記培養終了後、靱帯細胞を回収し、RNA抽出はRNeasy Mini Kit (QIAGEN) を用いて行った。逆転写反応はQuantiTect Reverse Transcription Kit (QIAGEN) を用いて行った。 より詳しくは以下の方法でReal-time PCRを行った。
(Dissemination of ligament cells)
The ligament cells that reached semi-confluent were detached from the dish by treatment with a tripsin-EDTA solution, and the culture solution was added and centrifuged. Thereafter, the cells were seeded on a 6-well plate at 1.5 × 10 6 cells / well and cultured at 37 ° C. and 5% CO 2 .
(Addition of artificial collagen aqueous solution)
Twenty-four hours after the start of culture, the artificial collagen was replaced with a culture solution adjusted to 0 (control), 0.05, 0.1, 0.2, 0.3% and allowed to act for 7 days.
(Collecting ligament cells and extracting RNA)
After completion of the above culture, ligament cells were collected, and RNA extraction was performed using RNeasy Mini Kit (QIAGEN). Reverse transcription reaction was performed using QuantiTect Reverse Transcription Kit (QIAGEN). More specifically, Real-time PCR was performed by the following method.
（Real-time PCR）
Real-time PCRに使用した機種はABI PRISM 7000 (Applied Biosystems)であり、使用した試薬はReal-time PCR Master Mix (TOYOBO)とPre-Developed TaqMan® Assay Reagents Eukaryotic 18S rRNA (Applied Biosystems)、TaqMan® Probe kit (Applied Biosystems)であった。
加えて、使用したprimer配列と反応条件を以下に示す。
（rabbit）
collagen, type I, alpha 1 Oc03396074_g1（Applied Biosystems販売）
COL3A1 F: 5-GCTCCTGGACAGAATGGTGAG-3 （配列番号８）
R: 5-CGCCTTTGACACCTTGAGGA-3 （配列番号９）
Probe: 5-AAGGAGAAAGAGGTGCTCCC-3 （配列番号１０）
GAPDH F: 5-CCCTCAATGACCACTTTGTGAA-3 （配列番号１１）
R: 5-AGGCCATGTGGACCATGAG-3 （配列番号１２）
Probe: 5-CGAATTTGGCTACAGCAACA-3 （配列番号１３）
（反応条件）
50℃-2min, 95℃-10min,(95℃-15sec, 60℃-1min)×50 cyclesで行った。
(Real-time PCR)
The model used for Real-time PCR was ABI PRISM 7000 (Applied Biosystems) and the reagents used were Real-time PCR Master Mix (TOYOBO), Pre-Developed TaqMan ® Assay Reagents Eukaryotic 18S rRNA (Applied Biosystems), TaqMan ® Probe kit (Applied Biosystems).
In addition, the used primer sequences and reaction conditions are shown below.
(Rabbit)
collagen, type I, alpha 1 Oc03396074_g1 (Applied Biosystems sales)
COL3A1 F: 5-GCTCCTGGACAGAATGGTGAG-3 (SEQ ID NO: 8)
R: 5-CGCCTTTGACACCTTGAGGA-3 (SEQ ID NO: 9)
Probe: 5-AAGGAGAAAGAGGTGCTCCC-3 (SEQ ID NO: 10)
GAPDH F: 5-CCCTCAATGACCACTTTGTGAA-3 (SEQ ID NO: 11)
R: 5-AGGCCATGTGGACCATGAG-3 (SEQ ID NO: 12)
Probe: 5-CGAATTTGGCTACAGCAACA-3 (SEQ ID NO: 13)
(Reaction conditions)
50 ° C.-2 min, 95 ° C.-10 min, (95 ° C.-15 sec, 60 ° C.-1 min) × 50 cycles.
（肩の腱板に関する結果）
New Zealand white rabbit 7週令の肩関節から腱板を採取して1週間人工コラーゲンを添加して培養した場合のtype I collagen mRNAの発現を確認した（参照：図１）。
さらに、New Zealand white rabbit 7週令の肩関節から腱板を採取して１週間人工コラーゲンを添加して培養した場合のtype III collagen mRNAの発現を確認した（参照：図２）。
以上により、人工コラーゲンを0.05-0.3％の濃度で添加することで肩の腱板のタイプIコラーゲンの産生を促進することができる。さらに、腱板断裂の治療で関節内に定期的に注入することで腱板断裂の修復を促進できる。
(Results on shoulder rotator cuff)
New Zealand white rabbit A rotator cuff was collected from a 7-week-old shoulder joint, and the expression of type I collagen mRNA was confirmed when cultured with artificial collagen added for 1 week (see: FIG. 1).
Furthermore, expression of type III collagen mRNA was confirmed when a rotator cuff was collected from a 7-week-old New Zealand white rabbit shoulder joint and cultured with artificial collagen added for 1 week (see FIG. 2).
As described above, the production of type I collagen in the rotator cuff of the shoulder can be promoted by adding artificial collagen at a concentration of 0.05-0.3%. Furthermore, the repair of the rotator cuff tear can be promoted by periodically injecting it into the joint in the treatment of the rotator cuff tear.
（アキレス腱に関する結果）
New Zealand white rabbit 7週令のアキレス腱を採取して1週間人工コラーゲンを添加して培養した場合のtype I collagen mRNAの発現を確認した（参照：図３）。
さらに、New Zealand white rabbit 7週令のアキレス腱を採取して1週間人工コラーゲンを添加して培養した場合のtype III collagen mRNAの発現を確認した（参照：図４）。
以上により、人工コラーゲンを0.05-0.3％の濃度で添加することでアキレス腱のタイプIコラーゲンの産生を促進することができる。さらに、アキレス腱断裂の手術時で人工コラーゲンを添加することでアキレス腱断裂の修復を促進できる。また、アキレス腱断裂部の皮膚に人工コラーゲンを塗布することで人工コラーゲンを浸透させ、腱の修復を促進することができる。
(Results on Achilles tendon)
New Zealand white rabbit Expression of type I collagen mRNA was confirmed when 7-week-old Achilles tendon was collected and cultured with artificial collagen added for 1 week (see FIG. 3).
Furthermore, expression of type III collagen mRNA was confirmed when New Zealand white rabbit 7-week-old Achilles tendon was collected and cultured with artificial collagen added for 1 week (see: FIG. 4).
As described above, the production of Achilles tendon type I collagen can be promoted by adding artificial collagen at a concentration of 0.05-0.3%. Furthermore, the repair of the Achilles tendon can be promoted by adding artificial collagen during the operation of the Achilles tendon. Further, by applying artificial collagen to the skin of the Achilles tendon ruptured portion, the artificial collagen can be infiltrated and the repair of the tendon can be promoted.
（ペプチドが軟骨のプロテオグリカン産生に与える影響の確認）
PHGペプチドが軟骨のプロテオグリカン産生に与える影響を確認した。
使用したPHGペプチドは、以下の通りである。なお、純度は95%以上である。
PHG（配列番号１）
PHG-PHG（配列番号２）
PHG-PHG-PHG-PHG（配列番号３）
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
(Confirmation of the effect of peptides on cartilage proteoglycan production)
The effect of PHG peptide on cartilage proteoglycan production was confirmed.
The PHG peptides used are as follows. The purity is 95% or more.
PHG (SEQ ID NO: 1)
PHG-PHG (SEQ ID NO: 2)
PHG-PHG-PHG-PHG (SEQ ID NO: 3)
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4)
（軟骨細胞の作製）
New Zealand white rabbit、雄性、8-9週 5匹の肩関節、膝関節を摘出しメスで軟骨層のみを削って採取した。滅菌PBS溶液で洗浄後、Dulbecco's Modified Eagle's medium 25ml ＋ gentacin （25μg/ml）に0.4%となるように pronaseを添加して37℃で2時間インキュベーションした。その溶液を0.22 μm Steriflipを用いて濾過滅菌した。濾過滅菌した溶液に0.025% となるようにcollagenaseを添加して37℃でovernightインキュベーションした。その溶液を0.22 μm Steriflipを用いて濾過滅菌した。
得られた溶液を洗浄して軟骨細胞を回収後、直径90mmのシャーレに1〜2×106cells/dish の軟骨細胞を播き3日一度液替えをした。Confluent になった時点で一回のみ系代培養を行った。以下の実験は1回系代培養した細胞を用いて行った。
また、培養液の構成は以下の通りである。
（培養液の組成）
Dulbecco's Modified Eagle's medium nutrient mixture F-12 HAM（SIGMA）+20% FETAL BOVINE SERUM (Hyclone)+20 ug/ml ascorbic acid（SIGMA）を使用し、3日に一度液替えを行った。各ペプチドは0（コントロール）,30,100,300μmolの濃度で添加した。
(Production of chondrocytes)
New Zealand white rabbit, male, 8-9 weeks Five shoulder joints and knee joints were removed and the cartilage layer was shaved with a scalpel and collected. After washing with a sterile PBS solution, pronase was added to Dulbecco's Modified Eagle's medium 25 ml + gentacin (25 μg / ml) to a concentration of 0.4% and incubated at 37 ° C. for 2 hours. The solution was sterilized by filtration using 0.22 μm Steriflip. Collagenase was added to the solution sterilized by filtration so that the concentration was 0.025%, followed by overnight incubation at 37 ° C. The solution was sterilized by filtration using 0.22 μm Steriflip.
The obtained solution was washed to collect chondrocytes, then seeded with 1-2 × 10 6 cells / dish chondrocytes in a petri dish having a diameter of 90 mm, and the liquid was changed once every three days. When it became Confluent, the subculture was performed only once. The following experiment was performed using cells that had been subcultured once.
Moreover, the composition of the culture solution is as follows.
(Composition of culture solution)
Dulbecco's Modified Eagle's medium nutrient mixture F-12 HAM (SIGMA) + 20% FETAL BOVINE SERUM (Hyclone) + 20 ug / ml ascorbic acid (SIGMA) was used to change the liquid once every three days. Each peptide was added at a concentration of 0 (control), 30, 100, and 300 μmol.
（軟骨細胞のプロテオグリカン産生能の解析）
各ペプチドを含む水溶液を添加することによる軟骨細胞のプロテオグリカン産生能を測定した。詳細は、以下の通りである。
（軟骨細胞の播種）
semi-confluentに達した軟骨細胞をtripsin-EDTA溶液処理によりdishから剥離し、培養液を加えて遠心洗浄した。その後1.5×106 cells/wellにて6-well plateに播種し、37℃、5% CO2にて培養を行った。
（ペプチド水溶液の添加）
培養開始24時間後において、それぞれのペプチドを0、30、100、300μmolの濃度に調整した培養液に交換し、7日間作用させ、最後の24時間は更に10μCi/ml Na35SO4を添加して培養を行った。培養液は3日に1回交換した。
（回収・抽出）
上記培養終了後、新しい培養液で洗浄し、protease inhibitors (0.1 M 6-aminohexanoic acid, 0.005 M benzamidine hydrochloride, 0.01 M Na2 EDTA , 0.01 M N-ethylmaleimide, 0.001M phenylmethyl sulfonyl fluoride)を加えたlysis buffer(4 M GuHCl, 0.05 M NaAC, pH 6.0)を各wellに入れ、4℃で4時間抽出を行い、4℃、15000rpmにて20分間遠心した。
（分離・放射活性測定）
PD-10 pre-packed column(GEヘルスケアバイオサイエンス)を用いて、elution buffer (4M GuHCl , 0.05M Na acetate, 0.1M Na sulfate , 0.5% TritonX-10 (pH 7.5))で溶出し、サンプルを分取した。各フラクションに、ethanolとシンチレーターを加えて混合し、液体シンチレーションカウンターで放射能含有量(cpm)を測定した。また、この際用いたサンプルのDNA量をHoechst 33258 dyeにより測定し、cpm/mg DNA値を求めた。
(Analysis of proteoglycan production ability of chondrocytes)
The proteoglycan production ability of chondrocytes by adding an aqueous solution containing each peptide was measured. Details are as follows.
(Seeding of chondrocytes)
The chondrocytes that reached semi-confluent were detached from the dish by treatment with tripsin-EDTA solution, and the culture solution was added and centrifuged. Thereafter, the cells were seeded on a 6-well plate at 1.5 × 10 6 cells / well and cultured at 37 ° C. and 5% CO 2 .
(Addition of peptide aqueous solution)
24 hours after the start of the culture, each peptide was replaced with a culture solution adjusted to 0, 30, 100, 300 μmol and allowed to act for 7 days, and for the last 24 hours, 10 μCi / ml Na 35 SO 4 was further added. The culture was performed. The culture solution was changed once every 3 days.
(Recovery / Extraction)
After completion of the above culture, the cells were washed with a new culture solution and added with protease inhibitors (0.1 M 6-aminohexanoic acid, 0.005 M benzamidine hydrochloride, 0.01 M Na 2 EDTA, 0.01 M N-ethylmaleimide, 0.001 M phenylmethyl sulfonyl fluoride). (4 M GuHCl, 0.05 M NaAC, pH 6.0) was placed in each well, extracted at 4 ° C. for 4 hours, and centrifuged at 4 ° C. and 15000 rpm for 20 minutes.
(Separation / Radioactivity measurement)
Elution with elution buffer (4M GuHCl, 0.05M Na acetate, 0.1M Na sulfate, 0.5% TritonX-10 (pH 7.5)) using PD-10 pre-packed column (GE Healthcare Bioscience) Sorted. To each fraction, ethanol and scintillator were added and mixed, and the radioactivity content (cpm) was measured with a liquid scintillation counter. Further, the amount of DNA of the sample used at this time was measured with Hoechst 33258 dye, and the cpm / mg DNA value was determined.
（軟骨細胞のDNA合成能の解析）
PHGペプチドを含む水溶液を添加することによる軟骨細胞のDNA合成能を測定した。詳細は、以下の通りである。
（軟骨細胞の播種）
semi-confluentに達した軟骨細胞をtripsin-EDTA溶液処理によりdishから剥離し、培養液を加えて遠心洗浄した。その後、1.5×105 cells/wellにて6-well plateに播種し、37℃、5% CO2にて培養した。
（ペプチド水溶液の添加）
培養開始24時間後において、それぞれのペプチドを0、30、100、300μmolの濃度に調整した培養液に交換し、24時間作用させた。最後の4時間はさらに1μCi/ml [methyl-3H] Thymidineを添加して培養を行った。
（回収・抽出）
上記培養終了後、細胞を冷PBS（-）で洗浄し、milliQ水を加えて室温で10分間放置した。セルスクレイパーで細胞をはがしてエッペンに回収し、10％TCAを加えて混合した後、室温で30分間放置した。15,000 rpm, 4℃, 5分間遠心した後、上清を取り除き、0.1N NaOHを加えて沈殿を懸濁した。
（放射活性測定）
サンプルにethanolとシンチレーターを加えて混合し、液体シンチレーションカウンターで放射能含有量(cpm)を測定した。
(Analysis of DNA synthesis ability of chondrocytes)
The DNA synthesis ability of chondrocytes by adding an aqueous solution containing PHG peptide was measured. Details are as follows.
(Seeding of chondrocytes)
The chondrocytes that reached semi-confluent were detached from the dish by treatment with tripsin-EDTA solution, and the culture solution was added and centrifuged. Thereafter, the cells were seeded on a 6-well plate at 1.5 × 10 5 cells / well and cultured at 37 ° C. and 5% CO 2 .
(Addition of peptide aqueous solution)
24 hours after the start of the culture, each peptide was replaced with a culture solution adjusted to a concentration of 0, 30, 100, or 300 μmol and allowed to act for 24 hours. During the last 4 hours, 1 μCi / ml [methyl- 3 H] Thymidine was further added for culturing.
(Recovery / Extraction)
After completion of the culture, the cells were washed with cold PBS (−), added with milliQ water, and left at room temperature for 10 minutes. The cells were peeled off with a cell scraper and collected in an eppen. After adding 10% TCA and mixing, the cells were allowed to stand at room temperature for 30 minutes. After centrifugation at 15,000 rpm and 4 ° C. for 5 minutes, the supernatant was removed, and 0.1N NaOH was added to suspend the precipitate.
(Radioactivity measurement)
Ethanol and scintillator were added to the sample and mixed, and the radioactivity content (cpm) was measured with a liquid scintillation counter.
（ペプチドが軟骨のプロテオグリカン産生に与える影響の結果）
New Zealand white rabbit 7週令の軟骨を採取して4種類のペプチドを各濃度で添加した場合のプロテオグリカン産生能とDNA量を検討した（参照：図５、６）。
以上により、PHG〜(PHG)8までのペプチドで1000μmolの範囲で投与することで軟骨細胞のプロテオグリカン産生を亢進させることができる。
(Results of peptide effects on proteoglycan production by cartilage)
New Zealand white rabbit 7-week-old cartilage was collected, and proteoglycan production ability and DNA amount were examined when 4 types of peptides were added at each concentration (see: FIGS. 5 and 6).
As described above, proteoglycan production of chondrocytes can be enhanced by administering a peptide of PHG to (PHG) 8 in the range of 1000 μmol.
（軟骨の遊走性の検討）
本発明の人工コラーゲン及びPHGペプチド投与による軟骨の遊走性を検討した。詳細は、以下の通りである。
使用したペプチドは、以下の通りである。なお、純度は95%以上である。
PHG（配列番号１）
PHG-PHG（配列番号２）
PHG-PHG-PHG-PHG（配列番号３）
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
(Examination of cartilage migration)
The cartilage migration by administration of the artificial collagen and PHG peptide of the present invention was examined. Details are as follows.
The peptides used are as follows. The purity is 95% or more.
PHG (SEQ ID NO: 1)
PHG-PHG (SEQ ID NO: 2)
PHG-PHG-PHG-PHG (SEQ ID NO: 3)
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4)
（軟骨細胞の作製）
New Zealand white rabbit、雄性、8-9週 5匹の肩関節、膝関節を摘出しメスで軟骨層のみを削って採取した。滅菌PBS溶液で洗浄後、Dulbecco's Modified Eagle's medium 25ml ＋ gentacin （25μg/ml）に0.4%となるように pronaseを添加して37℃で2時間インキュベーションした。その溶液を0.22 μm Steriflipを用いて濾過滅菌した。濾過滅菌した溶液に0.025% となるようにcollagenaseを添加して37℃でovernightインキュベーションした。その溶液を0.22 μm Steriflipを用いて濾過滅菌した。
得られた溶液を洗浄して軟骨細胞を回収後、直径90 mmのシャーレに1〜2×106cells/dish の軟骨細胞を播き3日一度液替えをした。Confluent になった時点で一回のみ系代培養を行った。以下の実験は1回系代培養した細胞を用いて行った。
(Production of chondrocytes)
New Zealand white rabbit, male, 8-9 weeks Five shoulder joints and knee joints were removed and the cartilage layer was shaved with a scalpel and collected. After washing with a sterile PBS solution, pronase was added to Dulbecco's Modified Eagle's medium 25 ml + gentacin (25 μg / ml) to a concentration of 0.4% and incubated at 37 ° C. for 2 hours. The solution was sterilized by filtration using 0.22 μm Steriflip. Collagenase was added to the solution sterilized by filtration so that the concentration was 0.025%, followed by overnight incubation at 37 ° C. The solution was sterilized by filtration using 0.22 μm Steriflip.
The obtained solution was washed to collect chondrocytes, then seeded with 1-2 × 10 6 cells / dish chondrocytes in a petri dish having a diameter of 90 mm, and the liquid was changed once every three days. When it became Confluent, the subculture was performed only once. The following experiment was performed using cells that had been subcultured once.
また、培養液の構成は以下の通りである。
（培養液の組成）
Dulbecco's Modified Eagle's medium nutrient mixture F-12 HAM（SIGMA）+20% FETAL BOVINE SERUM (Hyclone)+20 ug/ml ascorbic acid（SIGMA）を使用した。
（試薬）
Minimum essential medium alpha medium:αMEM (GIBCO)
Fetal Bovine Serum : FBS (EQUITECH-BIO,INC.)
Dulbecco' s PBS (-) (Nissui)
0.25% trypsin-EDTA solution (SIGMA)
96 Well Cell Migration Assay (Trevigen) (Cat No.:3465-096-K)
（方法）
96 Well Cell Migration Assay (Trevigen) kitを使用した。
（播種）
１．実験開始24 hr前より、0.5%FBS添加αMEMにて培養した細胞を回収し、カウントした。
２．1200 rpm、4℃にて5 min遠心し、上清をアスピレートし、wash bufferで洗浄後、1×106 cells/mLに懸濁した。
３．ボトムチャンバーに試薬を添加したメディウムを150μLずつ加えた。
４．トップチャンバーに細胞懸濁液を50μLずつ加え、12 hrインキュベートした。
５．50,000、25,000、10,000、5,000、2,000及び1,000の細胞数のwellを予め用意してコントロールを作成した。
（添加試薬）
PHG（配列番号１）は 0（コントロール）、10、100、300、1000、3000、10000 μM の濃度で用意した。
PHG-PHG（配列番号２）、PHG-PHG-PHG-PHG（配列番号３）、PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）は0（コントロール）、10、30、100、300、1000μMの濃度で用意した。
細胞数は、485 nm/520 nmで吸光度を測定して算出した。
Moreover, the composition of the culture solution is as follows.
(Composition of culture solution)
Dulbecco's Modified Eagle's medium nutrient mixture F-12 HAM (SIGMA) + 20% FETAL BOVINE SERUM (Hyclone) + 20 ug / ml ascorbic acid (SIGMA) was used.
(reagent)
Minimum essential medium alpha medium: αMEM (GIBCO)
Fetal Bovine Serum: FBS (EQUITECH-BIO, INC.)
Dulbecco 's PBS (-) (Nissui)
0.25% trypsin-EDTA solution (SIGMA)
96 Well Cell Migration Assay (Trevigen) (Cat No.:3465-096-K)
(Method)
A 96 Well Cell Migration Assay (Trevigen) kit was used.
(Seeding)
1. From 24 hours before the start of the experiment, cells cultured in αMEM supplemented with 0.5% FBS were collected and counted.
2. Centrifugation at 1200 rpm and 4 ° C. for 5 min, the supernatant was aspirated, washed with wash buffer, and suspended at 1 × 10 6 cells / mL.
3. 150 μL of medium supplemented with a reagent was added to the bottom chamber.
4). 50 μL of cell suspension was added to the top chamber and incubated for 12 hr.
5. Wells with cell numbers of 50,000, 25,000, 10,000, 5,000, 2,000 and 1,000 were prepared in advance to prepare controls.
(Additional reagent)
PHG (SEQ ID NO: 1) was prepared at concentrations of 0 (control), 10, 100, 300, 1000, 3000, and 10000 μM.
PHG-PHG (SEQ ID NO: 2), PHG-PHG-PHG-PHG (SEQ ID NO: 3), PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4) is 0 (control), 10, Prepared at concentrations of 30, 100, 300, and 1000 μM.
The number of cells was calculated by measuring the absorbance at 485 nm / 520 nm.
（軟骨の遊走性の検討結果）
New Zealand white rabbit 7週令から採取して軟骨に対して、人工コラーゲンを各濃度で添加した溶液での軟骨細胞の遊走性を検討した（参照：図７）。
さらに、New Zealand white rabbit 7週令の軟骨を採取してPHGペプチドを各濃度で添加した溶液へ軟骨細胞の遊走性を検討した（参照：図８）。
加えて、New Zealand white rabbit 7週令の軟骨を採取してPHG-PHG（配列番号２）、PHG-PHG -PHG-PHG（配列番号３）、PHG-PHG -PHG-PHG -PHG-PHG -PHG-PHG（配列番号４）
ペプチドを各濃度で添加した溶液へ軟骨細胞の遊走性を検討した（参照：図９）。
以上により、人工コラーゲンは軟骨細胞の遊走性の効果はなかった。一方、PHGペプチドを300〜10000μMの範囲で添加することで軟骨細胞を遊走させることができた。さらに、(PHG)8のペプチドは30〜1000μMの濃度で添加することで軟骨細胞を遊走させることができる。
(Results of cartilage migration)
New Zealand white rabbits were collected from 7 weeks of age, and the migration of chondrocytes in a solution containing artificial collagen added at various concentrations to cartilage was examined (see FIG. 7).
In addition, New Zealand white rabbit 7-week-old cartilage was collected and examined for the migration of chondrocytes into a solution to which PHG peptide was added at various concentrations (see FIG. 8).
In addition, New Zealand white rabbit 7-week-old cartilage was collected and PHG-PHG (SEQ ID NO: 2), PHG-PHG -PHG-PHG (SEQ ID NO: 3), PHG-PHG -PHG-PHG -PHG-PHG- PHG-PHG (SEQ ID NO: 4)
The migration of chondrocytes was examined in a solution to which peptides were added at various concentrations (see FIG. 9).
As described above, the artificial collagen had no effect of chondrocyte migration. On the other hand, chondrocytes could be migrated by adding PHG peptide in the range of 300 to 10,000 μM. Furthermore, chondrocytes can be migrated by adding a peptide of (PHG) 8 at a concentration of 30 to 1000 μM.
（骨芽様細胞に対する人工コラーゲンとPHGペプチドの効果の確認）
骨芽様細胞に対する人工コラーゲンとPHGペプチドの効果を確認した。詳細は以下の通りである。
使用したペプチドは、以下の通りである。なお、純度は95%以上である。
PHG（配列番号１）
PHG-PHG（配列番号２）
PHG-PHG-PHG-PHG（配列番号３）
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
(Confirmation of the effects of artificial collagen and PHG peptide on osteoblast-like cells)
The effects of artificial collagen and PHG peptide on osteoblast-like cells were confirmed. Details are as follows.
The peptides used are as follows. The purity is 95% or more.
PHG (SEQ ID NO: 1)
PHG-PHG (SEQ ID NO: 2)
PHG-PHG-PHG-PHG (SEQ ID NO: 3)
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4)
（試薬）
Minimum essential medium alpha medium:αMEM (GIBCO)
10% Fetal Bovine Serum (EQUITECH-BIO,INC.)
100 μM 2-phospho-L-ascorbic acid trisodium salt (Fluka)
10 mM Glycerol 2-phosphate disodium salt hydrate (SIGMA)
fetal bovine serum: FBS (Equitech-Bio)
Dulbecco' s PBS (-) (Nissui)
0.25% trypsin-EDTA solution (Sigma)
(reagent)
Minimum essential medium alpha medium: αMEM (GIBCO)
10% Fetal Bovine Serum (EQUITECH-BIO, INC.)
100 μM 2-phospho-L-ascorbic acid trisodium salt (Fluka)
10 mM Glycerol 2-phosphate disodium salt hydrate (SIGMA)
fetal bovine serum: FBS (Equitech-Bio)
Dulbecco 's PBS (-) (Nissui)
0.25% trypsin-EDTA solution (Sigma)
（方法）
＜播種＞
10%FBS添加αMEM培地にMC3T3-E1細胞を懸濁させ、24 well plateに1×105cells/wellとなるよう1 mLずつ播種した。
24時間後、メディウムをアスピレートし、人工コラーゲンを0（コントロール）、0.01、0.025、0.05、0.1、0.2、0.3、0.4、0.5%の濃度添加した溶液に置き換えて1週間培養する。PHGペプチドは、0（コントロール）、3、10、30、100、300、1000、3000、10000μM添加した溶液に置き換えて1週間培養した。
＜骨芽様細胞の回収・RNA抽出＞
上記培養終了後、靱帯細胞を回収し、RNA抽出はRNeasy Mini Kit (QIAGEN) を用いて行った。逆転写反応はQuantiTect Reverse Transcription Kit (QIAGEN) を用いて行った。 より詳しくは以下の方法でReal-time PCRを行った。
＜Real-time PCR＞
Real-time PCRに使用した機種はABI PRISM 7000 (Applied Biosystems)であり、使用した試薬はReal-time PCR Master Mix (TOYOBO)とPre-Developed TaqMan® Assay Reagents Eukaryotic 18S rRNA (Applied Biosystems)、TaqMan® Probe kit (Applied Biosystems)であった。
加えて、使用したprimer配列と反応条件を以下に示す。
Mouse
collagen, type I, alpha 1 Mm00801666_g1
Pre-Developed TaqMan Assay Reagents -mouse GAPDH
＜反応条件＞
50℃-2min, 95℃-10min,(95℃-15sec, 60℃-1min)×50 cyclesで行った。
(Method)
<Seeding>
MC3T3-E1 cells were suspended in αMEM medium supplemented with 10% FBS, and 1 mL each was seeded on a 24-well plate at 1 × 10 5 cells / well.
After 24 hours, the medium is aspirated, and the artificial collagen is replaced with a solution added with 0 (control), 0.01, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5% concentrations and cultured for 1 week. The PHG peptide was replaced with a solution containing 0 (control), 3, 10, 30, 100, 300, 1000, 3000, or 10000 μM and cultured for 1 week.
<Recovery of osteoblast-like cells and RNA extraction>
After completion of the above culture, ligament cells were collected, and RNA extraction was performed using RNeasy Mini Kit (QIAGEN). Reverse transcription reaction was performed using QuantiTect Reverse Transcription Kit (QIAGEN). More specifically, Real-time PCR was performed by the following method.
<Real-time PCR>
The model used for Real-time PCR was ABI PRISM 7000 (Applied Biosystems) and the reagents used were Real-time PCR Master Mix (TOYOBO), Pre-Developed TaqMan ® Assay Reagents Eukaryotic 18S rRNA (Applied Biosystems), TaqMan ® Probe kit (Applied Biosystems).
In addition, the used primer sequences and reaction conditions are shown below.
Mouse
collagen, type I, alpha 1 Mm00801666_g1
Pre-Developed TaqMan Assay Reagents -mouse GAPDH
<Reaction conditions>
50 ° C.-2 min, 95 ° C.-10 min, (95 ° C.-15 sec, 60 ° C.-1 min) × 50 cycles.
（骨芽様細胞に対する人工コラーゲンとPHGペプチドの効果の確認結果）
マウスの3T3骨芽様細胞に人工コラーゲンを添加して1週後のtype I collagenのmRNAの発現を確認した（参照：図１０）。さらに、マウスの3T3骨芽様細胞にPHGペプチドを添加して1週のtype I collagenのmRNAの発現を確認した（参照：図１１）。
以上により、骨芽細胞に0.05-0.4%の範囲で人工コラーゲンを添加することで、骨芽細胞のタイプIコラーゲンの産生を促進できる。さらに、骨芽細胞に10〜300μMの範囲でPHGペプチドを添加して骨芽細胞のタイプＩコラーゲンの産生を促進することができる。
(Confirmation result of artificial collagen and PHG peptide on osteoblast-like cells)
Artificial collagen was added to mouse 3T3 osteoblast-like cells, and the expression of type I collagen mRNA one week later was confirmed (see FIG. 10). Furthermore, PHG peptide was added to mouse 3T3 osteoblast-like cells, and the expression of type I collagen mRNA at 1 week was confirmed (see FIG. 11).
As described above, the production of type I collagen of osteoblasts can be promoted by adding artificial collagen in the range of 0.05-0.4% to osteoblasts. Furthermore, the production of type I collagen in osteoblasts can be promoted by adding PHG peptide to osteoblasts in the range of 10 to 300 μM.
（本ペプチドによる骨芽様細胞の遊走性の効果）
本ペプチドによる骨芽様細胞の遊走性の効果を確認した。詳細は以下の通りである。
使用したペプチドは、以下の通りである。なお、純度は95%以上である。
PHG（配列番号１）
PHG-PHG（配列番号２）
PHG-PHG-PHG-PHG（配列番号３）
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
(Effect of migration of osteoblast-like cells by this peptide)
The effect of osteoblast-like cell migration by this peptide was confirmed. Details are as follows.
The peptides used are as follows. The purity is 95% or more.
PHG (SEQ ID NO: 1)
PHG-PHG (SEQ ID NO: 2)
PHG-PHG-PHG-PHG (SEQ ID NO: 3)
PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4)
（細胞）
MC3T3-E1細胞
（試薬）
Minimum essential medium alpha medium:αMEM (GIBCO)
Fetal Bovine Serum : FBS (EQUITECH-BIO,INC.)
Dulbecco' s PBS (-) (Nissui)
0.25% trypsin-EDTA solution (SIGMA)
96 Well Cell Migration Assay (Trevigen) (Cat No.:3465-096-K)
（方法）
96 Well Cell Migration Assay (Trevigen) kitを使用した。
（播種）
１．実験開始24 hr前より、0.5%FBS添加αMEMにて培養した細胞を回収し、カウントした。
２．1200 rpm、4℃にて5 min遠心し、上清をアスピレートし、wash bufferで洗浄後、1×106 cells/mLに懸濁した。
３．ボトムチャンバーに試薬を添加したメディウムを150 μLずつ加えた。
４．トップチャンバーに細胞懸濁液を50 μLずつ加え、12 hrインキュベートした。
５．50,000、25,000、10,000、5,000、2,000及び1,000の細胞数のwellを予め用意してコントロールを作成した。
（添加試薬）
PHG（配列番号１）は 0（コントロール）、3、30、100、300、1000、3000、10000μM の濃度で用意した。
PHG-PHG（配列番号２）、PHG-PHG-PHG-PHG（配列番号３）、PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）は0（コントロール）、10、30、100、300、1000μMの濃度で用意した。
細胞数は、485 nm/520 nmで吸光度を測定して算出した。
(cell)
MC3T3-E1 cells (reagents)
Minimum essential medium alpha medium: αMEM (GIBCO)
Fetal Bovine Serum: FBS (EQUITECH-BIO, INC.)
Dulbecco 's PBS (-) (Nissui)
0.25% trypsin-EDTA solution (SIGMA)
96 Well Cell Migration Assay (Trevigen) (Cat No.:3465-096-K)
(Method)
A 96 Well Cell Migration Assay (Trevigen) kit was used.
(Seeding)
1. From 24 hours before the start of the experiment, cells cultured in αMEM supplemented with 0.5% FBS were collected and counted.
2. Centrifugation at 1200 rpm and 4 ° C. for 5 min, the supernatant was aspirated, washed with wash buffer, and suspended at 1 × 10 6 cells / mL.
3. 150 μL of medium supplemented with a reagent was added to the bottom chamber.
4). 50 μL of the cell suspension was added to the top chamber and incubated for 12 hr.
5. Wells with cell numbers of 50,000, 25,000, 10,000, 5,000, 2,000 and 1,000 were prepared in advance to prepare controls.
(Additional reagent)
PHG (SEQ ID NO: 1) was prepared at concentrations of 0 (control), 3, 30, 100, 300, 1000, 3000, and 10000 μM.
PHG-PHG (SEQ ID NO: 2), PHG-PHG-PHG-PHG (SEQ ID NO: 3), PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (SEQ ID NO: 4) is 0 (control), 10, Prepared at concentrations of 30, 100, 300, and 1000 μM.
The number of cells was calculated by measuring the absorbance at 485 nm / 520 nm.
マウスの3T3骨芽様細胞を用いてPHG（配列番号１）、PHG-PHG（配列番号２）、PHG-PHG-PHG-PHG（配列番号３）、PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）ペプチドを添加して溶液へ骨芽様細胞の遊走性を検討した（参照：図１２〜１５）。さらに、ヒト由来の骨芽様細胞を用いたPHGの遊走性を検討した（参照：図１６）。
以上により、PHG単独からPHGが8回繰り返しているまでのペプチドは、3〜1000μMの範囲投与することで骨芽細胞を遊走させる効果を持つことを確認した。
Using mouse 3T3 osteoblast-like cells, PHG (SEQ ID NO: 1), PHG-PHG (SEQ ID NO: 2), PHG-PHG-PHG-PHG (SEQ ID NO: 3), PHG-PHG-PHG-PHG-PHG-PHG -PHG-PHG (SEQ ID NO: 4) peptide was added to examine the migration of osteoblast-like cells into the solution (see: FIGS. 12 to 15). Furthermore, the migration of PHG using human-derived osteoblast-like cells was examined (see FIG. 16).
From the above, it was confirmed that peptides from PHG alone to PHG repeated 8 times had the effect of causing osteoblast migration when administered in the range of 3 to 1000 μM.
（実施例の結果の総論）
上記実施例の結果から以下のことが言える。
本発明は、人工コラーゲンを含むコラーゲン産生促進剤は以下の効果を有することを確認している。
0.05〜0.3％の人工コラーゲンを含むコラーゲン産生促進剤を肩関節腱板に添加して肩の腱板のタイプIコラーゲンの産生を促進できる。
0.05〜0.3％の人工コラーゲンを含むコラーゲン産生促進剤をアキレス腱に添加してアキレス腱のタイプIコラーゲンの産生を促進できる。
人工コラーゲンを含むコラーゲン産生促進剤を骨に添加して骨芽細胞のタイプIコラーゲンの産生を促進できる。
(Overview of Example Results)
The following can be said from the results of the above examples.
This invention has confirmed that the collagen production promoter containing artificial collagen has the following effects.
A collagen production promoter containing 0.05 to 0.3% artificial collagen can be added to the shoulder joint rotator cuff to promote the production of type I collagen in the rotator cuff of the shoulder.
A collagen production promoter containing 0.05 to 0.3% artificial collagen can be added to the Achilles tendon to promote the production of type I collagen of the Achilles tendon.
A collagen production promoter containing artificial collagen can be added to bone to promote type I collagen production of osteoblasts.
本発明のPHGペプチドを含むコラーゲン産生促進剤、プロテオグリカン産出促進剤、細胞の遊走促進剤は、以下の効果を有することを確認している。
30〜1000μmolのPHGペプチド｛PHG〜(PHG)8｝を軟骨細胞に投与することでプロテオグリカン産生を亢進できる。
30〜1000μMのPHGペプチド｛PHG〜(PHG)8｝を軟骨細胞に投与することで軟骨細胞を遊走させることができる。
PHGペプチドを骨芽細胞に投与することでタイプＩコラーゲンの産生を促進できる。
3.0〜1000μMのPHGペプチド｛PHG〜(PHG)8｝を骨芽細胞に投与することで骨芽細胞を遊走させることができる。
It has been confirmed that the collagen production promoter, proteoglycan production promoter and cell migration promoter containing the PHG peptide of the present invention have the following effects.
Proteoglycan production can be enhanced by administering 30-1000 μmol of PHG peptide {PHG- (PHG) 8 } to chondrocytes.
Chondrocytes can be migrated by administering 30-1000 μM PHG peptide {PHG- (PHG) 8 } to the chondrocytes.
Administration of PHG peptide to osteoblasts can promote type I collagen production.
Osteoblasts can be migrated by administering 3.0-1000 μM PHG peptide {PHG- (PHG) 8 } to osteoblasts.
本発明は、安全性が高い、コラーゲンの産生促進剤、プロテオグリカン産出促進剤及び細胞の遊走促進剤であり、有用である。 INDUSTRIAL APPLICABILITY The present invention is useful because it is a highly safe collagen production promoter, proteoglycan production promoter, and cell migration promoter.
Claims (3)
（１）PHG（配列番号１）
（２）PHG-PHG（配列番号２）
（３）PHG-PHG-PHG-PHG（配列番号３）
（４）PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４）
A cell migration promoter comprising any one of the following peptides.
(1) PHG (SEQ ID NO: 1)
(2) PHG-PHG (SEQ ID NO: 2)
(3) PHG-PHG-PHG-PHG (SEQ ID NO: 3)
(4) PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (sequence number 4)
The migration promoting agent according to claim 1, wherein the cell is a chondrocyte.
（１）PHG（配列番号１）
（２）PHG-PHG（配列番号２）
（３）PHG-PHG-PHG-PHG（配列番号３）
（４）PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG（配列番号４） A proteoglycan production promoter comprising any one of the following peptides.
(1) PHG (SEQ ID NO: 1)
(2) PHG-PHG (SEQ ID NO: 2)
(3) PHG-PHG-PHG-PHG (SEQ ID NO: 3)
(4) PHG-PHG-PHG-PHG-PHG-PHG-PHG-PHG (sequence number 4)
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| Title |
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| IMPROTA R, ET AL.: "Understanding the role of stereoelectronic effects in determining collagen stability. 1. A quantum m", JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 123, no. 50, JPN6017020666, 2001, pages 12568 - 12577 * |
| INOUE O, ET AL.: "Novel synthetic collagen fibers, poly(PHG), stimulate platelet aggregation through glycoprotein VI", FEBS LETTERS, vol. 583, no. 1, JPN6017020665, 4 December 2008 (2008-12-04), pages 81 - 87, XP026194373, DOI: doi:10.1016/j.febslet.2008.11.026 * |
| KNAPP-MOHAMMADY M, ET AL.: "Fragmentation of doubly-protonated Pro-His-Xaa tripeptides: formation of b(2)(2+) ions", JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETR, vol. 20, no. 11, JPN6017020668, 10 July 2009 (2009-07-10), pages 2135 - 2143, XP026688361, DOI: doi:10.1016/j.jasms.2009.07.002 * |
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