JP5792442B2 - Fiber structure containing cell-specific peptide - Google Patents
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- JP5792442B2 JP5792442B2 JP2010195675A JP2010195675A JP5792442B2 JP 5792442 B2 JP5792442 B2 JP 5792442B2 JP 2010195675 A JP2010195675 A JP 2010195675A JP 2010195675 A JP2010195675 A JP 2010195675A JP 5792442 B2 JP5792442 B2 JP 5792442B2
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- 239000000835 fiber Substances 0.000 title claims description 128
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 65
- 238000000034 method Methods 0.000 claims description 35
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- 229920003232 aliphatic polyester Polymers 0.000 claims description 21
- 210000000329 smooth muscle myocyte Anatomy 0.000 claims description 17
- 238000001523 electrospinning Methods 0.000 claims description 16
- 229920001610 polycaprolactone Polymers 0.000 claims description 15
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- 238000004113 cell culture Methods 0.000 claims description 8
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- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 6
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- UOCLXMDMGBRAIB-UHFFFAOYSA-N 1,1,1-trichloroethane Chemical compound CC(Cl)(Cl)Cl UOCLXMDMGBRAIB-UHFFFAOYSA-N 0.000 description 1
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- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 229920003043 Cellulose fiber Polymers 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
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- FXHOOIRPVKKKFG-UHFFFAOYSA-N N,N-Dimethylacetamide Chemical compound CN(C)C(C)=O FXHOOIRPVKKKFG-UHFFFAOYSA-N 0.000 description 1
- LFTLOKWAGJYHHR-UHFFFAOYSA-N N-methylmorpholine N-oxide Chemical compound CN1(=O)CCOCC1 LFTLOKWAGJYHHR-UHFFFAOYSA-N 0.000 description 1
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- VBZWSGALLODQNC-UHFFFAOYSA-N hexafluoroacetone Chemical compound FC(F)(F)C(=O)C(F)(F)F VBZWSGALLODQNC-UHFFFAOYSA-N 0.000 description 1
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- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
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- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
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Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Materials For Medical Uses (AREA)
- Artificial Filaments (AREA)
- Spinning Methods And Devices For Manufacturing Artificial Fibers (AREA)
- Nonwoven Fabrics (AREA)
Description
本発明は、細胞特異的ペプチドを０．１〜２０重量部含有し、平均繊維径が０．０５〜５０μｍである脂肪族ポリエステルの繊維構造体に関する。 The present invention relates to an aliphatic polyester fiber structure containing 0.1 to 20 parts by weight of a cell-specific peptide and having an average fiber diameter of 0.05 to 50 μm.
再生医療分野においては、細胞を培養する際に基材として多孔体が用いられることがある。多孔体としては凍結乾燥による発泡体や繊維構造体が知られている。これら多孔体は細胞との親和性や生体内分解性、安全性などが必要とされる。 In the field of regenerative medicine, a porous body may be used as a base material when cells are cultured. As the porous body, a foam and a fiber structure obtained by freeze-drying are known. These porous bodies are required to have affinity with cells, biodegradability, safety and the like.
ポリ乳酸に代表される脂肪族ポリエステルは、これら生体内分解性や安全性が知られている材料の中でも比較的安価に入手可能である。特に、Ｌ−乳酸成分を主とするポリ乳酸は、最近大量に製造されている。 Aliphatic polyesters typified by polylactic acid are available relatively inexpensively among these materials known for biodegradability and safety. In particular, polylactic acid mainly composed of an L-lactic acid component has recently been produced in large quantities.
生体内分解性、安全性が知られているポリ乳酸の多孔体を、例えば細胞培養基材に用いることが検討されている（例えば、非特許文献１参照。）。
しかしながら、これらの方法では細胞が接着できる面積が不十分であり、より表面積の大きい多孔体が望まれていたことから、その一つとして繊維径の小さい繊維構造体が検討されてきた。
The use of a porous body of polylactic acid, which is known for biodegradability and safety, for example, as a cell culture substrate has been studied (for example, see Non-Patent Document 1).
However, since these methods have insufficient areas to which cells can adhere and porous bodies having a larger surface area have been desired, fiber structures having a small fiber diameter have been studied as one of them.
繊維径の小さい繊維構造体を製造する方法として、静電紡糸法が知られている（例えば、特許文献１および２参照）。静電紡糸法は、液体、例えば繊維形成物質を含有する溶液等を電場内に導入し、これにより液体を電極に向かって曳かせ、繊維状物質を形成させる工程を包含する。普通、繊維形成物質は溶液から曳き出される間に硬化させる。硬化は、例えば冷却（例えば、紡糸液体が室温で固体である場合）、化学的硬化（例えば、硬化用蒸気による処理）、または溶媒の蒸発により行われる。また、得られる繊維状物質は、適宜配置した受容体上に捕集され、必要ならばそこから剥離することもできる。また、静電紡糸法は不織布状の繊維状物質を直接得ることができるため、一旦繊維を製糸した後、さらに繊維構造体を形成する必要がなく、操作が簡便である。 An electrostatic spinning method is known as a method for producing a fiber structure having a small fiber diameter (see, for example, Patent Documents 1 and 2). The electrospinning method includes a step of introducing a liquid, for example, a solution containing a fiber-forming substance into an electric field, thereby causing the liquid to move toward an electrode and forming a fibrous substance. Usually, the fiber forming material is cured while it is squeezed out of solution. Curing is performed, for example, by cooling (for example when the spinning liquid is solid at room temperature), chemical curing (for example by treatment with curing steam), or evaporation of the solvent. Moreover, the obtained fibrous substance is collected on a suitably arranged receptor, and can be peeled from there if necessary. Further, since the electrospinning method can directly obtain a nonwoven fibrous material, there is no need to form a fiber structure once the fibers have been produced once, and the operation is simple.
静電紡糸法によって得られる繊維構造体を細胞を培養する基材に用いることも知られている。例えばポリ乳酸よりなる繊維構造体を静電紡糸法により形成し、この上で平滑筋細胞を培養することによる血管の再生が検討されている（例えば、非特許文献２参照）。
しかしながら、これまで要求を満たすほどの細胞接着効果をもつものは得られていない。
It is also known to use a fiber structure obtained by an electrospinning method as a substrate for culturing cells. For example, the regeneration of blood vessels by forming a fiber structure made of polylactic acid by an electrospinning method and culturing smooth muscle cells thereon has been studied (for example, see Non-Patent Document 2).
However, a cell adhesion effect sufficient to satisfy the requirements has not been obtained so far.
本発明の課題は、再生医療分野で用いられる、細胞特異性をもつ細胞培養基材を提供することにある。 An object of the present invention is to provide a cell culture substrate having cell specificity used in the field of regenerative medicine.
本発明は、脂肪族ポリエステルと細胞特異的ペプチドからなる基材が、細胞培養基材に有用であることに着目してなされたものである。 The present invention has been made by paying attention to the fact that a substrate comprising an aliphatic polyester and a cell-specific peptide is useful as a cell culture substrate.
すなわち、本発明は、脂肪族ポリエステル１００重量部に対して、下記の群より選択されるアミノ酸配列を有するペプチドの少なくとも一つを０．１〜２０重量部含有し、平均繊維径が０．０５〜５０μｍであることを特徴とする繊維構造体である。
HHH、VVV、TTT、TGA、NNN、KKK、AAA、RRR、YYY、TTT、GAT、GGG、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、GEK、QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、GTP、GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、GNS、AKG、DGY、TGP、VGP、SLW、AAG、AGA、ARG、GRD、EGF、GSC、PGQ、HSQ、EAP、RGP、PGD、CNI、GFG、GPT、GDQ、KGE、PFI、QGP、SYW、LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FPGPPGPP（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP（配列番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配列番号１０）およびIGLSGEKG（配列番号１１）。
That is, the present invention contains 0.1 to 20 parts by weight of at least one peptide having an amino acid sequence selected from the following group with respect to 100 parts by weight of aliphatic polyester, and the average fiber diameter is 0.05. It is a fiber structure characterized by having a thickness of ˜50 μm.
HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2) ), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9) GSPGPPGSPG (SEQ ID NO: 10) and IGLSGEKG (SEQ ID NO: 11).
本発明によれば、再生医療分野で用いられる、細胞特異性をもつ細胞培養基材が提供される。 According to the present invention, a cell culture substrate having cell specificity used in the field of regenerative medicine is provided.
本発明は、脂肪族ポリエステル１００重量部に対して、細胞特異的ペプチドを０．１〜２０重量部含有した繊維構造体である。脂肪族ポリエステル１００重量部に対する細胞特異的ペプチドの含有量は、好ましくは０．１〜１５重量部、さらに好ましくは０．１〜１０重量部である。２０重量部以上だと繊維の安定性に欠ける場合がある。 The present invention is a fiber structure containing 0.1 to 20 parts by weight of a cell-specific peptide with respect to 100 parts by weight of an aliphatic polyester. The content of the cell specific peptide with respect to 100 parts by weight of the aliphatic polyester is preferably 0.1 to 15 parts by weight, more preferably 0.1 to 10 parts by weight. If it is 20 parts by weight or more, the fiber may lack stability.
本発明で用いられる繊維構造体の平均繊維径は０．０５〜５０μｍであることが好ましい。平均繊維径が０．０５μｍより小さいと、該繊維構造体の強度が保てないため好ましくない。また、平均繊維径が５０μｍより大きいと繊維の比表面積が小さく、生着する細胞数が少ないため好ましくない。なお、繊維径とは繊維断面の直径を表す。繊維断面の形状は円形に限らず、楕円形や異形になることもありうる。この場合の繊維径とは、該楕円形の長軸方向の長さと短軸方向の長さの平均をその繊維径として算出する。また、繊維断面が円形でも楕円形でもないときには円または楕円に近似して繊維径を算出する。 The average fiber diameter of the fiber structure used in the present invention is preferably 0.05 to 50 μm. An average fiber diameter of less than 0.05 μm is not preferable because the strength of the fiber structure cannot be maintained. Moreover, when the average fiber diameter is larger than 50 μm, the specific surface area of the fiber is small and the number of cells to be engrafted is small, which is not preferable. In addition, a fiber diameter represents the diameter of a fiber cross section. The shape of the fiber cross section is not limited to a circle, and may be an ellipse or an irregular shape. With respect to the fiber diameter in this case, the average of the length in the major axis direction and the length in the minor axis direction of the ellipse is calculated as the fiber diameter. When the fiber cross section is neither circular nor elliptical, the fiber diameter is calculated by approximating a circle or ellipse.
本発明で用いられる脂肪族ポリエステルとしては、ポリ乳酸、ポリグリコール酸、ポリカプロラクトン（ＰＣＬ）、ポリブチレンサクシネート、ポリエチレンサクシネートおよびこれらの共重合体が挙げられる。これらのうち、脂肪族ポリステルとしては、ポリ乳酸、ポリ乳酸−ポリグリコール酸共重合体、ポリグリコール酸、ポリカプロラクトン、ポリグリコール酸−ポリカプロラクトン共重合体、ポリ乳酸−ポリカプロラクトン共重合体が好ましい。 Examples of the aliphatic polyester used in the present invention include polylactic acid, polyglycolic acid, polycaprolactone (PCL), polybutylene succinate, polyethylene succinate and copolymers thereof. Among these, as the aliphatic polyester, polylactic acid, polylactic acid-polyglycolic acid copolymer, polyglycolic acid, polycaprolactone, polyglycolic acid-polycaprolactone copolymer, and polylactic acid-polycaprolactone copolymer are preferable. .
本発明の繊維構造体においては、その目的を損なわない範囲で、他のポリマーや他の化合物を併用してもよい。例えば、ポリマー共重合、ポリマーブレンド、化合物混合である。 In the fiber structure of the present invention, other polymers and other compounds may be used in combination as long as the purpose is not impaired. For example, polymer copolymerization, polymer blending, compound mixing.
本発明で用いられる脂肪族ポリエステルは高純度であることが好ましく、とりわけ脂肪族ポリエステル中に含まれる添加剤や可塑剤、残存触媒、残存モノマー、成型加工や後加工に用いた残留溶媒などの残留物は少ないほうが好ましい。特に医療に用いる場合は、安全性の基準値未満に抑える必要がある。 The aliphatic polyester used in the present invention is preferably highly pure, and in particular, residuals such as additives, plasticizers, residual catalysts, residual monomers, residual solvents used in molding and post-processing included in the aliphatic polyester. It is preferable that there are few things. In particular, when used for medical treatment, it is necessary to keep it below the safety standard value.
本発明において、「細胞特異性」とは、基本的には特定の細胞に対する特異的な接着性（すなわち高接着性または低接着性）をいうが、特定の細胞に対する特異的な増殖性（すなわち高増殖性または低増殖性）を加味して「細胞特異性」を判断することが好ましい。したがって、「細胞特異性」を示すペプチドの中で好ましいものは、特定の細胞に対して高い接着性および増殖性を示すか、特定の細胞に対して低い接着性および増殖性を示す。なお、細胞特異性を示すペプチドのことを本明細書では「細胞特異的ペプチド」と呼称する。 In the present invention, “cell specificity” basically refers to specific adhesion to a specific cell (ie, high adhesion or low adhesion), but specific proliferation to a specific cell (ie, It is preferable to determine “cell specificity” in consideration of (high growth ability or low growth ability). Accordingly, preferred peptides that exhibit “cell specificity” exhibit high adhesion and proliferation to specific cells or low adhesion and proliferation to specific cells. A peptide exhibiting cell specificity is referred to herein as “cell-specific peptide”.
本発明における細胞特異的ペプチドは、具体的には、HHH、VVV、TTT、TGA、NNN、KKK、AAA、RRR、YYY、TTT、GAT、GGG、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、GEK、QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、GTP、GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、GNS、AKG、DGY、TGP、VGP、SLW、AAG、AGA、ARG、GRD、EGF、GSC、PGQ、HSQ、EAP、RGP、PGD、CNI、GFG、GPT、GDQ、KGE、PFI、QGP、SYW、LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FPGPPGPP（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP（配列番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配列番号１０）、およびIGLSGEKG（配列番号１１）からなる群より選択されるいずれかのアミノ酸配列を有するペプチドである。 Specifically, the cell-specific peptide in the present invention is HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP (SEQ ID NO: 7) , LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGPPGSPG (SEQ ID NO: 10), and IGLSGEKG (SEQ ID NO: 1) A peptide having any of the amino acid sequence selected from the group consisting of).
HHH、VVV、TTT、TGA、KKK、AAA、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、GEK、QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、GTP、GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、GNS、LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FPGPPGPP（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP（配列番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配列番号１０）、およびIGLSGEKG（配列番号１１）（以下、これらをまとめて「グループ１のペプチド」ともいう。）、ならびにNNNは内皮細胞特異的ペプチドであり、RRR、YYY、TTT、GAT、AKG、DGY、TGP、VGP、SLW、AAG、AGA、ARG、GRD、EGF、GSC、PGQ、HSQ、EAP、RGP、PGD、CNI、GFG、GPT、GDQ、KGE、PFI、QGP、およびSYW（以下、これらをまとめて「グループ２のペプチド」ともいう。）は平滑筋細胞特異的ペプチドであり、GGGは線維芽細胞特異的ペプチドである。 HHH, VVV, TTT, TGA, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGPPGSPG (SEQ ID NO: 10), and IGLSGEKG (SEQ ID NO: 11) (hereinafter collectively referred to as “group 1 peptide”), and NNN are endothelial cell-specific peptides, such as RRR, YYY, TTT, GAT, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, P GQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, and SYW (hereinafter collectively referred to as “group 2 peptides”) are smooth muscle cell-specific peptides. GGG is a fibroblast specific peptide.
グループ１のペプチドは内皮細胞の接着および増殖に有効であり、内皮細胞を捕捉・増殖させることが望まれる用途に有用である。一方、NNNは内皮細胞の非接着（接着させないこと）および非増殖（増殖させないこと）に有効であり、内皮細胞を排除することが望まれる用途に有用である。また、グループ２のペプチドは平滑筋細胞の接着および増殖に有効であり、平滑筋細胞を捕捉・増殖させることが望まれる用途に有用である。GGGについては線維芽細胞の非接着（接着させないこと）および非増殖（増殖させないこと）に有効であり、線維芽細胞を排除することが望まれる用途に有用である。 Group 1 peptides are effective for endothelial cell adhesion and proliferation and are useful in applications where it is desired to capture and proliferate endothelial cells. On the other hand, NNN is effective for non-adhesion (non-adhesion) and non-proliferation (non-growth) of endothelial cells, and is useful for applications where it is desired to eliminate endothelial cells. Group 2 peptides are effective for adhesion and proliferation of smooth muscle cells, and are useful for applications where it is desired to capture and proliferate smooth muscle cells. GGG is effective for non-adhesion (not allowing adhesion) and non-proliferation (not allowing proliferation) of fibroblasts, and is useful for applications where it is desired to eliminate fibroblasts.
グループ１のペプチドの中で、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、およびLGLは内皮細胞に対する特異性が特に高い。そこで、好ましい一態様では、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、およびLGLからなる群より選択されるいずれかのアミノ酸配列を有する、内皮細胞特異的ペプチドが用いられる。同様に、グループ２のペプチドの中でAKG、DGY、およびTGPは平滑筋細胞に対する特異性が特に高い。そこで、好ましい一態様ではAKG、DGY、およびTGPからなる群より選択されるいずれかのアミノ酸配列を有する、平滑筋細胞特異的ペプチドが用いられる。 Among the group 1 peptides, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, and LGL are particularly specific for endothelial cells. Therefore, in a preferred embodiment, any amino acid selected from the group consisting of PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, and LGL An endothelial cell specific peptide having the sequence is used. Similarly, among Group 2 peptides, AKG, DGY, and TGP are particularly specific for smooth muscle cells. Therefore, in a preferred embodiment, a smooth muscle cell-specific peptide having any amino acid sequence selected from the group consisting of AKG, DGY, and TGP is used.
本発明に用いられる細胞特異的ペプチドは、動物組織から抽出したものでも人工的に合成して製造したものでもよい。
本発明に用いられる細胞特異的ペプチドは、公知のペプチド合成法（例えば固相合成法、液相合成法）によって調製することができる。なお、自動ペプチド合成機を利用すれば容易かつ迅速に目的のペプチドを合成することができる。
The cell-specific peptide used in the present invention may be extracted from animal tissue or artificially synthesized.
The cell-specific peptide used in the present invention can be prepared by a known peptide synthesis method (for example, solid phase synthesis method, liquid phase synthesis method). If an automatic peptide synthesizer is used, the target peptide can be synthesized easily and quickly.
遺伝子工学的手法を用いて細胞特異的ペプチドを調製してもよい。すなわち、細胞特異的ペプチドをコードする核酸を適当な宿主細胞に導入し、形質転換体内で発現されたペプチドを回収することにより目的のペプチドを調製してもよい。回収されたペプチドは必要に応じて精製される。 Cell-specific peptides may be prepared using genetic engineering techniques. That is, the target peptide may be prepared by introducing a nucleic acid encoding a cell-specific peptide into an appropriate host cell and recovering the peptide expressed in the transformant. The recovered peptide is purified as necessary.
さらに、二種類以上のペプチドを併用してもよい。併用する場合、細胞特異性に関して同種のペプチドを組み合わせても、異種のペプチドを組み合わせてもよい。前者の一例はグループ１に含まれるペプチドの中から２以上を選択して併用する場合であり、後者の一例はグループ１に含まれるペプチド（１または２以上）とグループ２に含まれるペプチド（１または２以上）を併用する場合である。異種のペプチドを組み合わせる場合には、原則として、細胞特異性毎にペプチド含有領域を設定する。すなわち、細胞特異性が異なるペプチドが混在することがないようにする。このようにすることで、例えば、ある領域では内皮細胞を捕捉・増殖させると同時に他の領域では平滑筋細胞を捕捉・増殖させる、というような二種以上の細胞の制御が可能になる。 Furthermore, two or more types of peptides may be used in combination. When used in combination, the same type of peptides or different types of peptides may be combined in terms of cell specificity. An example of the former is a case where two or more peptides selected from the peptides included in Group 1 are selected and used together. An example of the latter is a peptide included in Group 1 (1 or 2) and a peptide included in Group 2 (1 Or 2 or more). When combining different types of peptides, in principle, a peptide-containing region is set for each cell specificity. That is, a peptide having different cell specificities is not mixed. In this way, for example, it is possible to control two or more types of cells, such as capturing and growing endothelial cells in a certain region and simultaneously capturing and growing smooth muscle cells in another region.
本発明で用いられる繊維構造体とは、単数または複数の繊維が積層され、織り、編まれもしくはその他の手法により形成された３次元の構造体をいう。具体的な繊維構造体の形態としては、例えばシート、織布、編布、チューブ、メッシュが好ましく挙げられる。より好ましい形態はシートおよびチューブである。 The fiber structure used in the present invention refers to a three-dimensional structure in which one or more fibers are laminated and formed by weaving, knitting, or other methods. Specific examples of the form of the fiber structure preferably include a sheet, a woven fabric, a knitted fabric, a tube, and a mesh. More preferred forms are sheets and tubes.
本発明で用いられる繊維構造体は長繊維よりなる。長繊維とは具体的には紡糸から繊維構造体への加工にいたるプロセスの中で、繊維を切断する工程を加えずに形成される繊維をいい、エレクトロスピニング法、スパンボンド法、メルトブロー法などで形成することができるが、エレクトロスピニング法が好ましく用いられる。エレクトロスピニング法は、静電紡糸法、エレクトロスプレー法ともいわれる。 The fiber structure used in the present invention consists of long fibers. Specifically, long fibers refer to fibers that are formed without adding a fiber cutting step in the process from spinning to processing into a fiber structure, such as electrospinning, spunbonding, and meltblowing. The electrospinning method is preferably used. The electrospinning method is also called an electrostatic spinning method or an electrospray method.
エレクトロスピニング法は、ポリマーを溶媒に溶解させた溶液に高電圧を印加することで、電極上に繊維構造体を得る方法である。工程としては、高分子を溶媒に溶解させて溶液を製造する工程と、該溶液に高電圧を印加する工程と、該溶液を噴出させる工程と、例えば噴出させた溶液から溶媒を蒸発させて繊維構造体を形成させる工程と、任意に実施しうる工程として形成された繊維構造体の電荷を消失させる工程と、電荷消失によって繊維構造体を累積させる工程を含む。 The electrospinning method is a method of obtaining a fiber structure on an electrode by applying a high voltage to a solution in which a polymer is dissolved in a solvent. The steps include a step of dissolving a polymer in a solvent to produce a solution, a step of applying a high voltage to the solution, a step of jetting the solution, and e.g., evaporating the solvent from the jetted solution to fiber A step of forming the structure, a step of eliminating the charge of the fiber structure formed as a step that can be arbitrarily implemented, and a step of accumulating the fiber structure by the loss of charge.
本発明で用いられる繊維構造体の全体の厚みに関しては特に制限はないが、好ましくは２５μｍ〜２００μｍ、さらに好ましくは５０〜１００μｍである。
エレクトロスピニング法における、脂肪族ポリエステルを溶媒に溶解させて溶液を製造する段階について説明する。本発明の製造方法における溶液中の溶媒に対する脂肪族ポリエステルの濃度は１〜３０重量％であることが好ましい。脂肪族ポリエステルの濃度が１重量％より小さいと、濃度が低すぎるため繊維構造体を形成することが困難となり、好ましくない。また、３０重量％より大きいと、得られる繊維構造体の繊維径が大きくなり、好ましくない。より好ましい溶液中の溶媒に対する脂肪族ポリエステルの濃度は２〜２０重量％である。
Although there is no restriction | limiting in particular regarding the whole thickness of the fiber structure used by this invention, Preferably it is 25 micrometers-200 micrometers, More preferably, it is 50-100 micrometers.
The step of producing a solution by dissolving aliphatic polyester in a solvent in the electrospinning method will be described. The concentration of the aliphatic polyester with respect to the solvent in the solution in the production method of the present invention is preferably 1 to 30% by weight. If the concentration of the aliphatic polyester is less than 1% by weight, it is not preferable because the concentration is too low to make it difficult to form a fiber structure. On the other hand, if it is larger than 30% by weight, the fiber diameter of the resulting fiber structure becomes large, which is not preferable. A more preferable concentration of the aliphatic polyester with respect to the solvent in the solution is 2 to 20% by weight.
溶媒は一種を単独で用いてもよく、複数の溶媒を組み合わせてもよい。前記溶媒としては、脂肪族ポリエステルと細胞特異的ペプチドを溶解可能で、かつ紡糸する段階で蒸発し、繊維を形成可能なものであれば特に限定されず、例えば、アセトン、クロロホルム、エタノール、２−プロパノール、メタノール、トルエン、テトラヒドロフラン、水、ベンゼン、ベンジルアルコール、１，４−ジオキサン、１−プロパノール、ジクロロメタン、四塩化炭素、シクロヘキサン、シクロヘキサノン、フェノール、ピリジン、トリクロロエタン、酢酸、蟻酸、ヘキサフルオロ−２−プロパノール、ヘキサフルオロアセトン、Ｎ，Ｎ−ジメチルホルムアミド、Ｎ，Ｎ−ジメチルアセトアミド、アセトニトリル、Ｎ−メチル−２−ピロリジノン、Ｎ−メチルモルホリン−Ｎ−オキシド、１，３−ジオキソラン、メチルエチルケトン、上記溶媒の混合溶媒が挙げられる。これらのうち、取扱い性や物性などから、ヘキサフルオロ−２−プロパノール、ジクロロメタン、エタノールを用いることが好ましい。 A solvent may be used individually by 1 type and may combine several solvent. The solvent is not particularly limited as long as it can dissolve the aliphatic polyester and the cell-specific peptide and can evaporate at the spinning stage to form a fiber. For example, acetone, chloroform, ethanol, 2- Propanol, methanol, toluene, tetrahydrofuran, water, benzene, benzyl alcohol, 1,4-dioxane, 1-propanol, dichloromethane, carbon tetrachloride, cyclohexane, cyclohexanone, phenol, pyridine, trichloroethane, acetic acid, formic acid, hexafluoro-2- Propanol, hexafluoroacetone, N, N-dimethylformamide, N, N-dimethylacetamide, acetonitrile, N-methyl-2-pyrrolidinone, N-methylmorpholine-N-oxide, 1,3-dioxolane, methyl ethyl ketone Emissions, a mixed solvent of the above solvents. Of these, hexafluoro-2-propanol, dichloromethane, and ethanol are preferably used in view of handling properties and physical properties.
次に、溶液に高電圧を印加する段階と、溶液を噴出させる段階と、噴出された溶液から溶媒を蒸発させて繊維構造体を形成する段階について説明する。
本発明で用いられる繊維構造体の製造方法においては、脂肪族ポリエステルと細胞特異的ペプチドを溶解した溶液を噴出させ、繊維構造体を形成するために、溶液に高電圧を印加する必要がある。電圧を印加する方法については、脂肪族ポリエステルと細胞特異的ペプチドを溶解した溶液を噴出させ、繊維構造体が形成されるものであれば特に限定されないが、例えば溶液に電極を挿入して電圧を印加する方法や、溶液噴出ノズルに対して電圧を印加する方法がある。
Next, a step of applying a high voltage to the solution, a step of ejecting the solution, and a step of forming a fiber structure by evaporating the solvent from the ejected solution will be described.
In the method for producing a fiber structure used in the present invention, it is necessary to apply a high voltage to the solution in order to eject a solution in which an aliphatic polyester and a cell-specific peptide are dissolved to form a fiber structure. The method for applying the voltage is not particularly limited as long as the solution in which the aliphatic polyester and the cell-specific peptide are dissolved is ejected and a fiber structure is formed. For example, the voltage is applied by inserting an electrode into the solution. There are a method of applying and a method of applying a voltage to the solution jet nozzle.
また、溶液に印加する電極とは別に補助電極を設けてもよい。ここで、印加電圧の値は、前記繊維構造体が形成されれば特に限定されないが、通常は５〜５０ｋＶの範囲である。印加電圧が５ｋＶより小さい場合は、溶液が噴出せずに繊維構造体が形成されないため好ましくなく、印加電圧が５０ｋＶより大きい場合は、電極からアース電極に向かって放電が起きるために好ましくない。より好ましくは５〜３０ｋＶの範囲である。所望の電位は従来公知の任意の適切な方法で作ればよい。 Further, an auxiliary electrode may be provided separately from the electrode applied to the solution. Here, the value of the applied voltage is not particularly limited as long as the fiber structure is formed, but is usually in the range of 5 to 50 kV. When the applied voltage is smaller than 5 kV, the solution is not ejected and a fiber structure is not formed, which is not preferable. When the applied voltage is higher than 50 kV, discharge is generated from the electrode toward the ground electrode, which is not preferable. More preferably, it is the range of 5-30 kV. The desired potential may be generated by any appropriate method known in the art.
その後、脂肪族ポリエステルと細胞特異的ペプチドを溶解した溶液を噴出させた直後に脂肪族ポリエステルと細胞特異的ペプチドを溶解させた溶媒が揮発して繊維構造体が形成される。通常の紡糸は大気下、室温で行われるが、揮発が不十分である場合には陰圧下で行うことや、高温の雰囲気下で行うことも可能である。また、紡糸する温度は溶媒の蒸発挙動や紡糸液の粘度に依存するが、通常は０〜５０℃の範囲である。 Thereafter, immediately after the solution in which the aliphatic polyester and the cell-specific peptide are dissolved is ejected, the solvent in which the aliphatic polyester and the cell-specific peptide are dissolved is volatilized to form a fiber structure. Ordinary spinning is performed at room temperature in the atmosphere, but when volatilization is insufficient, it can be performed under negative pressure or in a high-temperature atmosphere. The spinning temperature depends on the evaporation behavior of the solvent and the viscosity of the spinning solution, but is usually in the range of 0 to 50 ° C.
次に、形成された繊維構造体の電荷を消失させる段階について説明する。前記繊維構造体の電荷を消失させる方法は、前記繊維成型体の電荷を消失させる方法であれば特に限定されないが、好ましい方法として、イオナイザーにより電荷を消失させる方法が挙げられる。イオナイザーとは、内蔵のイオン発生装置によりイオンを発生させ、これを帯電物に放出させることにより帯電物の電荷を消失させる装置である。かかるイオナイザーを構成する好ましいイオン発生装置としては、内蔵の放電針に高電圧を印加することによりイオンを発生させる装置が挙げられる。 Next, the step of eliminating the charge of the formed fiber structure will be described. The method of eliminating the charge of the fiber structure is not particularly limited as long as it is a method of eliminating the charge of the fiber molded body, and a preferable method is a method of eliminating the charge with an ionizer. An ionizer is a device that generates ions by a built-in ion generator and discharges them to a charged material, thereby eliminating the charge of the charged material. As a preferable ion generator constituting such an ionizer, an apparatus for generating ions by applying a high voltage to a built-in discharge needle can be mentioned.
次に、前記電荷消失によって繊維構造体を累積させる段階について説明する。前記電荷消失によって繊維構造体を累積させる方法は、前記繊維構造体が累積する方法であれば特に限定されないが、通常の方法として、電荷消失により繊維構造体の静電力を失わせ、自重により落下、累積させる方法が挙げられる。また必要に応じて、静電力を消失させた繊維構造体を吸引してメッシュ上に累積させる方法、装置内の空気を対流させてメッシュ上に累積させる方法を行ってもよい。 Next, the step of accumulating the fiber structure by the charge disappearance will be described. The method for accumulating the fiber structure due to the loss of electric charge is not particularly limited as long as the fiber structure accumulates. However, as a normal method, the electrostatic force of the fiber structure is lost due to the loss of electric charge, and the fiber structure falls due to its own weight. And a method of accumulating. Moreover, you may perform the method of attracting | sucking the fiber structure from which the electrostatic force was lose | disappeared and accumulating on a mesh as needed, and the method of making the air in an apparatus convect and accumulating on a mesh.
繊維表面が平滑な繊維は、紡糸する際の雰囲気を低湿度に設定することで作製できる。かかる紡糸時の相対湿度としては、好ましくは２５％以下、さらに好ましくは２０％以下である。 A fiber having a smooth fiber surface can be produced by setting the atmosphere during spinning to low humidity. The relative humidity during spinning is preferably 25% or less, more preferably 20% or less.
本発明では、繊維構造体の表面にさらに綿状の繊維構造物を積層することや、綿状構造物を繊維構造体ではさんでサンドイッチ構造にするなどの加工も、本発明の目的を損ねない範囲で任意に実施しうる。 In the present invention, processing such as further laminating a cotton-like fiber structure on the surface of the fiber structure or forming a sandwich structure with the cotton-like structure sandwiched between the fiber structures does not impair the object of the present invention. It can be arbitrarily implemented within a range.
本発明で用いられる繊維構造体は、その表面の親水性や疎水性、電気特性や帯電性を改質するために、界面活性剤などの化学薬品による表面処理を行ってもよい。医療応用においては、さらに抗血栓性を付与するためのコーティング処理、抗体や生理活性物質で表面をコーティングすることも任意に実施できる。このときのコーティング方法や処理条件、その処理に用いる化学薬品は、繊維の構造を極端に破壊せず、本発明の目的を損なわない範囲で任意に選択できる。 The fiber structure used in the present invention may be subjected to a surface treatment with a chemical such as a surfactant in order to modify the hydrophilicity, hydrophobicity, electrical characteristics and chargeability of the surface. In medical applications, coating treatment for imparting antithrombogenicity and surface coating with an antibody or a physiologically active substance can be optionally performed. The coating method and treatment conditions at this time, and the chemicals used for the treatment can be arbitrarily selected within a range that does not damage the fiber structure and impair the purpose of the present invention.
本発明で用いられる繊維構造体の繊維内部にも任意に薬剤を含ませることができる。エレクトロスピニング法で成形する場合は、揮発性溶媒に可溶であり、溶解によりその生理活性を損なわないものであれば、使用する薬剤に特に制限はない。 A drug can be optionally contained inside the fiber of the fiber structure used in the present invention. In the case of molding by the electrospinning method, the drug used is not particularly limited as long as it is soluble in a volatile solvent and does not impair the physiological activity by dissolution.
以下、実施例により本発明の実施の形態を説明するが、これらは本発明の範囲を制限するものではない。 Hereinafter, although an example explains an embodiment of the present invention, these do not limit the range of the present invention.
１．平均繊維径：
得られた繊維構造体の表面を走査型電子顕微鏡（キーエンス株式会社：商品名「ＶＥ８８００」）により、倍率２０００倍で撮影して得た写真から無作為に２０箇所を選んで繊維の径を測定し、すべての繊維径の平均値を求めて平均繊維径とした。ｎ＝２０である。
1. Average fiber diameter:
The surface of the obtained fiber structure was measured with a scanning electron microscope (Keyence Co., Ltd .: trade name “VE8800”) at a magnification of 2000 times and randomly selected 20 locations to measure the fiber diameter. And the average value of all the fiber diameters was calculated | required and it was set as the average fiber diameter. n = 20.
２．平均厚：
高精度デジタル測長機（株式会社ミツトヨ：商品名「ライトマチックＶＬ−５０」）を用いて測長力０．０１Ｎによりｎ＝１０にて繊維構造体の膜厚を測定した平均値を算出した。
なお、本測定においては測定機器が使用可能な最小の測定力で測定を行った。
2. Average thickness:
Using a high-precision digital length measuring machine (Mitutoyo Co., Ltd .: trade name “Lightmatic VL-50”), an average value obtained by measuring the film thickness of the fiber structure at a length measurement force of 0.01 N and n = 10 was calculated. .
In this measurement, the measurement was performed with the minimum measuring force that can be used by the measuring device.
３．平均見掛け密度：
繊維構造体の質量を測定し、上記方法により求めた面積、平均厚をもとに平均見掛け密度を算出した。
3. Average apparent density:
The mass of the fiber structure was measured, and the average apparent density was calculated based on the area and average thickness determined by the above method.
４．細胞培養評価：
ペプチドアレイスクリーニングによって選出された細胞特異的接着ペプチドの効果をε−ポリカプロラクトン紡糸繊維構造体上においても確認するため、ｃａｌｃｅｉｎＡＭ等の生細胞染色試薬にて蛍光標識した内皮細胞（ＨＡＥＣ）、平滑筋細胞（ＳＭＣ）を該繊維構造体上に播種し、１時間、１日、または３日の期間細胞を培養液中で培養したのち、洗浄を経て繊維構造体上に接着した細胞数を蛍光強度として定量し、コントロールシート（ペプチドなし）とペプチド入り繊維構造体との各細胞に対する細胞接着量を比べた。その後、各細胞に対する細胞接着量をコントロールを用いて規格化したのち、２種類の細胞の接着量を相対的な特異性として評価した。また、細胞接着による細胞形態の変化（フィロポディア等の形成）を走査型電子顕微鏡（ＳＥＭ）により観察した。
4). Cell culture evaluation:
In order to confirm the effect of the cell-specific adhesion peptide selected by peptide array screening also on the ε-polycaprolactone spun fiber structure, endothelial cells (HAEC) fluorescently labeled with a living cell staining reagent such as calcein AM, smooth muscle Cells (SMC) are seeded on the fiber structure, and after culturing the cells in the culture solution for a period of 1 hour, 1 day, or 3 days, the number of cells adhered to the fiber structure after washing is determined by fluorescence intensity. The amount of cell adhesion to each cell of the control sheet (no peptide) and the fiber structure containing the peptide was compared. Then, after normalizing the cell adhesion amount with respect to each cell using control, the adhesion amount of two types of cells was evaluated as relative specificity. In addition, changes in cell morphology (formation of filopodia and the like) due to cell adhesion were observed with a scanning electron microscope (SEM).
[実施例１]
内皮細胞特異的ペプチドとしてCAG（インビトロジェン(株)製 ９８．１４％）０．０８重量部とポリ−ε−カプロラクトン（平均分子量約７００００〜１０００００、和光純薬社製）８重量部をヘキサフルオロ−２−プロパノール（和光純薬社製）９２重量部で溶解し、均一な溶液を調製した。相対湿度２５％以下でエレクトロスピニング法により紡糸を行い、シート状の繊維構造体を得た。噴出ノズルの内径は０．９４ｍｍ、電圧は１４ｋＶ、フィード量は１．５ｍｌ／ｈ、噴出ノズルから平板までの距離は２０ｃｍであった。上記平板は、紡糸時は陰極として用いた。６０分間紡糸した後、５０℃で１０分間熱処理を実施した。得られた繊維構造体の平均繊維径は１．２μｍ、厚さは１２９μｍ、平均見掛け密度は１９５ｋｇ／ｍ３であった。
[Example 1]
As an endothelial cell-specific peptide, 0.08 parts by weight of CAG (98.14% manufactured by Invitrogen Corp.) and 8 parts by weight of poly-ε-caprolactone (average molecular weight of about 70,000 to 100,000, manufactured by Wako Pure Chemical Industries, Ltd.) were added to hexafluoro- 2-Propanol (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 92 parts by weight to prepare a uniform solution. Spinning was performed by electrospinning at a relative humidity of 25% or less to obtain a sheet-like fiber structure. The inner diameter of the ejection nozzle was 0.94 mm, the voltage was 14 kV, the feed amount was 1.5 ml / h, and the distance from the ejection nozzle to the flat plate was 20 cm. The flat plate was used as a cathode during spinning. After spinning for 60 minutes, heat treatment was carried out at 50 ° C. for 10 minutes. The obtained fiber structure had an average fiber diameter of 1.2 μm, a thickness of 129 μm, and an average apparent density of 195 kg / m 3 .
[実施例２]
内皮細胞特異的ペプチドとしてCNG（インビトロジェン(株)製 ９８．６７％）０．０８重量部とポリ−ε−カプロラクトン（平均分子量約７００００〜１０００００、和光純薬社製）８重量部をヘキサフルオロ−２−プロパノール（和光純薬社製）９２重量部で溶解し、均一な溶液を調製した。相対湿度２５％以下でエレクトロスピニング法により紡糸を行い、シート状の繊維構造体を得た。噴出ノズルの内径は０．９４ｍｍ、電圧は１４ｋＶ、フィード量は１．５ｍｌ／ｈ、噴出ノズルから平板までの距離は２０ｃｍであった。上記平板は、紡糸時は陰極として用いた。６０分間紡糸した後、５０℃で１０分間熱処理を実施した。得られた繊維構造体の平均繊維径は１．１μｍ、厚さは１０７μｍ、平均見掛け密度は１８９ｋｇ／ｍ３であった。
[Example 2]
As an endothelial cell-specific peptide, 0.08 parts by weight of CNG (Invitrogen Corp. 98.67%) and 8 parts by weight of poly-ε-caprolactone (average molecular weight of about 70,000 to 100,000, manufactured by Wako Pure Chemical Industries, Ltd.) 2-Propanol (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 92 parts by weight to prepare a uniform solution. Spinning was performed by electrospinning at a relative humidity of 25% or less to obtain a sheet-like fiber structure. The inner diameter of the ejection nozzle was 0.94 mm, the voltage was 14 kV, the feed amount was 1.5 ml / h, and the distance from the ejection nozzle to the flat plate was 20 cm. The flat plate was used as a cathode during spinning. After spinning for 60 minutes, heat treatment was carried out at 50 ° C. for 10 minutes. The obtained fiber structure had an average fiber diameter of 1.1 μm, a thickness of 107 μm, and an average apparent density of 189 kg / m 3 .
[比較例１]
ポリ−ε−カプロラクトン（平均分子量約７００００〜１０００００、和光純薬社製）６重量部をヘキサフルオロ−２−プロパノール（和光純薬社製）９４重量部で溶解し、均一な溶液を調製した。湿度２５％以下でエレクトロスピニング法により紡糸を行い、シート状の繊維構造体を得た。噴出ノズルの内径は０．９４ｍｍ、電圧は１４ｋＶ、フィード量は２ｍｌ／ｈ、噴出ノズルから平板までの距離は２５ｃｍであった。上記平板は、紡糸時は陰極として用いた。６０分紡糸した後、５０℃で１０分間熱処理を実施した。得られた繊維構造体の平均繊維径は１．４μｍ、厚さは１３３μｍ、平均見掛け密度は２１０ｋｇ／ｍ３であった。
[Comparative Example 1]
6 parts by weight of poly-ε-caprolactone (average molecular weight: about 70,000 to 100,000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 94 parts by weight of hexafluoro-2-propanol (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a uniform solution. Spinning was performed by electrospinning at a humidity of 25% or less to obtain a sheet-like fiber structure. The inner diameter of the ejection nozzle was 0.94 mm, the voltage was 14 kV, the feed amount was 2 ml / h, and the distance from the ejection nozzle to the flat plate was 25 cm. The flat plate was used as a cathode during spinning. After spinning for 60 minutes, heat treatment was carried out at 50 ° C. for 10 minutes. The obtained fiber structure had an average fiber diameter of 1.4 μm, a thickness of 133 μm, and an average apparent density of 210 kg / m 3 .
[実施例３]
＜細胞培養評価＞
実施例１〜２および比較例１で得られた繊維構造体を２種類の細胞接着性の違いで評価した。評価した細胞は、ヒト大動脈血管内皮細胞（Cell Applications, Inc., San Diego, USA）、ヒト臍帯動脈平滑筋細胞（Cell Applications, Inc.）を使用し、それぞれHuMedia-EG2 （Kurabo Industries Ltd., Osaka, Japan）、smooth muscle growth medium（Cell applications）の血清添加培地で培養した。繊維構造体への接着性評価では、各種の無血清培地を用いて比較した。各細胞をカルセイン（Life Technologies Corporation）にて３０分間蛍光標識し、無血清培地にて細胞懸濁液（１．５×１０５Ｃｅｌｌｓ／ｍｌ）を作り、細胞シートの上に２０ｍｌ播種した。１時間のインキュベーションの後、ＰＢＳにて３回洗浄し、ＦＬＡ−７０００（富士フィルム）にて４７３ｎｍ／５２０ｎｍにて測定し、蛍光強度を数値化した（ｎ＝３）。比較例１で得られた繊維構造体（コントロール）に比べ、実施例１および２で得られた繊維構造体（ペプチド入り）が内皮細胞特異的な接着であった。CAGペプチドは、ペプチドアレイスクリーニング結果においては、ペプチドアレイ（セルロース繊維構造体）上でRGDペプチドの約２倍の内皮細胞への特異性を示している。このため、本結果のような「内皮細胞への特異性」はRGD配列では得られない。また、実施例１および比較例１で得られた繊維構造体を使用して同様の条件にて播種した細胞を、Ｓ−８００電子顕微鏡（日立製作所）にて観察した。試料作成方法は、１時間のインキュベーションを行った後ＰＢＳで３回洗浄し、２％グルタルアルデヒド（和光純薬）にて固定した。さらに、１％四酸化オスミウムにて処理を行い、ＶＦＤ−２０（日立製作所）を用いて凍結乾燥を行った。凍結乾燥後の試料をオスミウムコーター（Nihon Lazor Denshi）にて金属コートし、ＳＥＭ観察用試料とした。その結果、実施例１で得られた繊維構造体（CAGペプチド含有）は、内皮細胞は接着するが、ＳＭＣは接着しないことが分かった（図２参照）。さらに、高倍率での観察ではＳＭＣは実施例１で得られた繊維構造体（CAGペプチド含有）上では伸展していない細胞も見られた（図３参照）。
[Example 3]
<Cell culture evaluation>
The fiber structures obtained in Examples 1 and 2 and Comparative Example 1 were evaluated based on two types of cell adhesion differences. The evaluated cells were human aortic vascular endothelial cells (Cell Applications, Inc., San Diego, USA) and human umbilical artery smooth muscle cells (Cell Applications, Inc.), and HuMedia-EG2 (Kurabo Industries Ltd., Osaka, Japan), and cultured in a serum-added medium of smooth muscle growth medium (Cell applications). In the evaluation of adhesion to the fiber structure, various serum-free media were used for comparison. Each cell was fluorescently labeled with calcein (Life Technologies Corporation) for 30 minutes, a cell suspension (1.5 × 10 5 Cells / ml) was prepared in a serum-free medium, and 20 ml was seeded on the cell sheet. After incubation for 1 hour, the plate was washed 3 times with PBS, measured with FLA-7000 (Fuji Film) at 473 nm / 520 nm, and the fluorescence intensity was digitized (n = 3). Compared with the fiber structure (control) obtained in Comparative Example 1, the fiber structures (with peptides) obtained in Examples 1 and 2 were endothelial cell-specific adhesion. In the peptide array screening results, the CAG peptide shows about twice the specificity for endothelial cells of the RGD peptide on the peptide array (cellulose fiber structure). For this reason, “specificity to endothelial cells” like this result cannot be obtained with the RGD sequence. Moreover, the cell seed | inoculated on the same conditions using the fiber structure obtained in Example 1 and Comparative Example 1 was observed with the S-800 electron microscope (Hitachi Ltd.). The sample was prepared by incubating for 1 hour, washing 3 times with PBS, and fixing with 2% glutaraldehyde (Wako Pure Chemical Industries). Further, it was treated with 1% osmium tetroxide and freeze-dried using VFD-20 (Hitachi). The sample after freeze-drying was metal-coated with an osmium coater (Nihon Lazor Denshi) to obtain a sample for SEM observation. As a result, it was found that the fibrous structure (containing CAG peptide) obtained in Example 1 adheres to endothelial cells but does not adhere to SMC (see FIG. 2). Furthermore, in the observation at a high magnification, SMC was found not to extend on the fiber structure (containing CAG peptide) obtained in Example 1 (see FIG. 3).
さらに、実施例２で得られた繊維構造体（CNGペプチド含有）のin vitro接着性試験を行ったところ、やはり有意に比較例１で得られた繊維構造体（コントロール）よりも内皮細胞に特異性を示していることが示され、ペプチドアレイスクリーニングで得られた細胞特異的ペプチドの効果と同様の結果が得られた。 Furthermore, when an in vitro adhesion test was conducted on the fiber structure (containing CNG peptide) obtained in Example 2, it was also significantly more specific to endothelial cells than the fiber structure (control) obtained in Comparative Example 1. The results were similar to the effects of cell-specific peptides obtained by peptide array screening.
[実施例４]
CAG（インビトロジェン(株)製 ９８．１４％）０．０８重量部とポリ−ε−カプロラクトン（平均分子量約７００００〜１０００００、和光純薬社製）８重量部をヘキサフルオロ−２−プロパノール（和光純薬社製）９２重量部で溶解し、均一な溶液を調製した。相対湿度２５％以下でエレクトロスピニング法により紡糸を行い、チューブ状の繊維構造体を得た。噴出ノズルの内径は０．３２ｍｍ、電圧は１４ｋＶ、フィード量は０．５ｍｌ／ｈ、噴出ノズルからΦ＝０．７ｍｍのステンレス棒を取り付けたマンドルコレクタまでの距離は１５ｃｍ、マンドルコレクタの回転数は５００ｒｐｍであった。上記コレクタは、紡糸時は陰極として用いた。９分紡糸した後、５０℃で１０分間熱処理を実施した。得られた繊維構造体の平均繊維径は１．６μｍ、チューブの厚さは２１１μｍであった。
[Example 4]
0.08 parts by weight of CAG (98.14% manufactured by Invitrogen Corporation) and 8 parts by weight of poly-ε-caprolactone (average molecular weight of about 70,000 to 100,000, manufactured by Wako Pure Chemical Industries) were added to hexafluoro-2-propanol (Wako Pure). Dissolved in 92 parts by weight of Yakuhin Co., Ltd. to prepare a uniform solution. Spinning was performed by an electrospinning method at a relative humidity of 25% or less to obtain a tubular fiber structure. The inner diameter of the ejection nozzle is 0.32mm, the voltage is 14kV, the feed rate is 0.5ml / h, the distance from the ejection nozzle to the mandrel collector with a φ = 0.7mm stainless rod is 15cm, 500 rpm. The collector was used as a cathode during spinning. After spinning for 9 minutes, heat treatment was performed at 50 ° C. for 10 minutes. The resulting fiber structure had an average fiber diameter of 1.6 μm and a tube thickness of 211 μm.
[比較例２]
ポリ−ε−カプロラクトン（平均分子量約７００００〜１０００００、和光純薬社製）６重量部をヘキサフルオロ−２−プロパノール（和光純薬社製）９６重量部で溶解し、均一な溶液を調製した。相対湿度２５％以下でエレクトロスピニング法により紡糸を行い、チューブ状の繊維構造体を得た。噴出ノズルの内径は０．３２ｍｍ、電圧は１４ｋＶ、フィード量は０．５ｍｌ／ｈ、噴出ノズルからΦ＝０．７ｍｍのステンレス棒を取り付けたマンドルコレクタまでの距離は１５ｃｍ、マンドルコレクタの回転数は５００ｒｐｍであった。上記コレクタは、紡糸時は陰極として用いた。９分間紡糸した後、５０℃で１０分間熱処理を実施した。得られた繊維構造体の平均繊維径は１．７μｍ、チューブの厚さは２０１μｍであった。
[Comparative Example 2]
6 parts by weight of poly-ε-caprolactone (average molecular weight: about 70,000 to 100,000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 96 parts by weight of hexafluoro-2-propanol (manufactured by Wako Pure Chemical Industries, Ltd.) to prepare a uniform solution. Spinning was performed by an electrospinning method at a relative humidity of 25% or less to obtain a tubular fiber structure. The inner diameter of the ejection nozzle is 0.32mm, the voltage is 14kV, the feed rate is 0.5ml / h, the distance from the ejection nozzle to the mandrel collector with a φ = 0.7mm stainless rod is 15cm, 500 rpm. The collector was used as a cathode during spinning. After spinning for 9 minutes, heat treatment was performed at 50 ° C. for 10 minutes. The obtained fiber structure had an average fiber diameter of 1.7 μm and a tube thickness of 201 μm.
[実施例５]
実施例４と比較例２で得られたチューブ状繊維構造体（グラフト）を用いて、ラット頸動脈置換術を施行した。１０−０ナイロン縫合糸を使用して頸動脈−グラフト端々吻合を行い、１、２、および６週間後にグラフトを摘出して評価した。評価方法は、内皮化の指標としてvon Willebrand Factor（Dako. Glostrup. Denmark）の蛍光免疫染色を行い、その内皮化率を算出した。また、被覆された内皮細胞の機能をｅＮＯＳ（Sigma-Aldrich. Inc., Saint Louis. USA）の発現で評価した（Western Blotting法）。結果は、移植の１週後において、実施例４のチューブ状繊維構造体では速やかな内皮化が観察されたが、比較例２のチューブ状繊維構造体では内皮化が不十分であった。また、１、２、および６週における内皮化率を実施例４および比較例２で得られたチューブ状繊維構造体を用いて比較したところ、それぞれ１週後で０．６４対０．４２、２週後で０．９８対０．７３、６週後で０．９７対０．７７であり、いずれも実施例４で得られたチューブ状繊維構造体を用いた場合の内皮化率が有意に高値であった。内皮細胞はｅＮＯＳを発現しており、実施例４のチューブ状繊維構造体を用いた場合の発現強度が高いことを確認した。
[Example 5]
Using the tubular fiber structure (graft) obtained in Example 4 and Comparative Example 2, rat carotid artery replacement was performed. Carotid artery-graft end-to-end anastomoses were performed using 10-0 nylon sutures, and the grafts were removed and evaluated after 1, 2, and 6 weeks. As an evaluation method, von Willebrand Factor (Dako. Glostrup. Denmark) was subjected to fluorescent immunostaining as an index of endothelialization, and the endothelialization rate was calculated. Moreover, the function of the coated endothelial cells was evaluated by expression of eNOS (Sigma-Aldrich. Inc., Saint Louis. USA) (Western Blotting method). As a result, rapid endothelialization was observed in the tubular fiber structure of Example 4 one week after transplantation, but endothelialization was insufficient in the tubular fiber structure of Comparative Example 2. In addition, when the endothelialization rates at 1, 2, and 6 weeks were compared using the tubular fiber structures obtained in Example 4 and Comparative Example 2, 0.64 vs. 0.42, respectively after 1 week, 0.98 vs. 0.73 after 2 weeks, 0.97 vs. 0.77 after 6 weeks, both of which have a significant endothelialization rate when using the tubular fibrous structure obtained in Example 4 It was overpriced. The endothelial cells expressed eNOS, and it was confirmed that the expression intensity was high when the tubular fiber structure of Example 4 was used.
本発明の繊維構造体は、例えば細胞培養基材や人工血管に用いることができる。 The fiber structure of the present invention can be used, for example, for cell culture substrates and artificial blood vessels.
Claims (10)
HHH、VVV、TTT、TGA、NNN、KKK、AAA、RRR、YYY、TTT、GAT、GGG、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、GEK、QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、GTP、GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、GNS、AKG、DGY、TGP、VGP、SLW、AAG、AGA、ARG、GRD、EGF、GSC、PGQ、HSQ、EAP、RGP、PGD、CNI、GFG、GPT、GDQ、KGE、PFI、QGP、SYW、LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FPGPPGPP（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP（配列番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配列番号１０）、およびIGLSGEKG（配列番号１１）。 It contains 0.1 to 20 parts by weight of at least one peptide not chemically modified and having an amino acid sequence selected from the following group with respect to 100 parts by weight of aliphatic polyester, and the average fiber diameter is 0.05 to A fiber structure characterized by being 50 μm.
HHH, VVV, TTT, TGA, NNN, KKK, AAA, RRR, YYY, TTT, GAT, GGG, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ, HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, SYW, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2) ), GPPGLSGPP (SEQ ID NO: 3), FPGPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9) , GSPGPPGSPG (SEQ ID NO: 10), and IGLSGEKG (SEQ ID NO: 11).
特徴とする請求項１に記載の繊維構造体。
HHH、VVV、TTT、TGA、NNN、KKK、AAA、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、
CAG、CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、
GEK、QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、
GTP、GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、
GNS、LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FP
GPPGPP（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP
（配列番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配
列番号１０）、およびIGLSGEKG（配列番号１１）。 The fiber structure according to claim 1, wherein the peptide has an amino acid sequence selected from the following group and exhibits endothelial cell specificity.
HHH, VVV, TTT, TGA, NNN, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS,
CAG, CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL,
GEK, QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD,
GTP, GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP,
GNS, LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FP
GPPGPP (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP
(SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGPPGSPG (SEQ ID NO: 10), and IGLSGEKG (SEQ ID NO: 11).
特徴とする請求項１に記載の繊維構造体。
HHH、VVV、TTT、TGA、KKK、AAA、PGH、GQA、QGD、GIG、EKG、KGK、QGF、GMK、GLS、CAG、
CNG、KGT、PLG、NRG、CSG、LGL、AVG、GHP、GLI、GVG、GPS、SPG、GPP、GIS、GYL、GEK、
QGE、CNY、FPG、GAP、APG、GEC、LPG、GPR、PCG、GDV、IGG、CDG、AVA、FLM、GFD、GTP、
GPY、VSG、DGR、GIT、GFL、ASG、GCP、NQG、SGL、GGA、PDG、QAL、GLK、GSP、GEP、GNS、
LPGFPGLK（配列番号１）、LPGFPGTP（配列番号２）、GPPGLSGPP（配列番号３）、FPGPPGP
P（配列番号４）、LPGLPGPP（配列番号５）、FPGLPGPP（配列番号６）、GPPGPPGPP（配列
番号７）、LPGPPGPP（配列番号８）、FPGSPGFPG（配列番号９）、GSPGPPGSPG（配列番号
１０）、およびIGLSGEKG（配列番号１１）。 The fiber structure according to claim 1, wherein the peptide has an amino acid sequence selected from the following group and exhibits endothelial cell specificity.
HHH, VVV, TTT, TGA, KKK, AAA, PGH, GQA, QGD, GIG, EKG, KGK, QGF, GMK, GLS, CAG,
CNG, KGT, PLG, NRG, CSG, LGL, AVG, GHP, GLI, GVG, GPS, SPG, GPP, GIS, GYL, GEK,
QGE, CNY, FPG, GAP, APG, GEC, LPG, GPR, PCG, GDV, IGG, CDG, AVA, FLM, GFD, GTP,
GPY, VSG, DGR, GIT, GFL, ASG, GCP, NQG, SGL, GGA, PDG, QAL, GLK, GSP, GEP, GNS,
LPGFPGLK (SEQ ID NO: 1), LPGFPGTP (SEQ ID NO: 2), GPPGLSGPP (SEQ ID NO: 3), FPGPPGP
P (SEQ ID NO: 4), LPGLPGPP (SEQ ID NO: 5), FPGLPGPP (SEQ ID NO: 6), GPPGPPGPP (SEQ ID NO: 7), LPGPPGPP (SEQ ID NO: 8), FPGSPGFPG (SEQ ID NO: 9), GSPGPPGSPG (SEQ ID NO: 10), and IGLSGEKG (SEQ ID NO: 11).
を特徴とする請求項１に記載の繊維構造体。
RRR、YYY、TTT、GAT、AKG、DGY、TGP、VGP、SLW、AAG、AGA、ARG、GRD、EGF、GSC、PGQ、
HSQ、EAP、RGP、PGD、CNI、GFG、GPT、GDQ、KGE、PFI、QGP、およびSYW。 The fiber structure according to claim 1, wherein the peptide has an amino acid sequence selected from the following group and exhibits smooth muscle cell specificity.
RRR, YYY, TTT, GAT, AKG, DGY, TGP, VGP, SLW, AAG, AGA, ARG, GRD, EGF, GSC, PGQ,
HSQ, EAP, RGP, PGD, CNI, GFG, GPT, GDQ, KGE, PFI, QGP, and SYW.
ール酸、ポリカプロラクトン、ポリグリコール酸−ポリカプロラクトン共重合体、ポリ乳
酸−ポリカプロラクトン共重合体からなる群から選択される少なくとも一つである請求項
１〜４のいずれかに記載の繊維構造体。 The aliphatic polyester is selected from the group consisting of polylactic acid, polylactic acid-polyglycolic acid copolymer, polyglycolic acid, polycaprolactone, polyglycolic acid-polycaprolactone copolymer, polylactic acid-polycaprolactone copolymer. The fiber structure according to any one of claims 1 to 4, which is at least one.
An artificial blood vessel comprising the fiber structure according to claim 7.
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