JPH04364200A - Cell adhesive albumin - Google Patents
Cell adhesive albuminInfo
- Publication number
- JPH04364200A JPH04364200A JP3166347A JP16634791A JPH04364200A JP H04364200 A JPH04364200 A JP H04364200A JP 3166347 A JP3166347 A JP 3166347A JP 16634791 A JP16634791 A JP 16634791A JP H04364200 A JPH04364200 A JP H04364200A
- Authority
- JP
- Japan
- Prior art keywords
- albumin
- oligopeptide
- glycine
- cell adhesion
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108010088751 Albumins Proteins 0.000 title claims abstract description 29
- 102000009027 Albumins Human genes 0.000 title claims abstract description 29
- 230000004956 cell adhesive effect Effects 0.000 title claims abstract description 11
- 108010038807 Oligopeptides Proteins 0.000 claims abstract description 16
- 102000015636 Oligopeptides Human genes 0.000 claims abstract description 16
- 230000021164 cell adhesion Effects 0.000 abstract description 8
- 239000000463 material Substances 0.000 abstract description 7
- 238000006243 chemical reaction Methods 0.000 abstract description 5
- 239000000243 solution Substances 0.000 abstract description 5
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 abstract description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 abstract description 4
- 229940098773 bovine serum albumin Drugs 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Substances CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 abstract description 3
- CUIJDJLRRFWTOC-BWDXOUPSSA-N NCC(O)=O.NCC(O)=O.OC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC(O)=O.OC(=O)[C@@H](N)CCCNC(N)=N Chemical compound NCC(O)=O.NCC(O)=O.OC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC(O)=O.OC(=O)[C@@H](N)CCCNC(N)=N CUIJDJLRRFWTOC-BWDXOUPSSA-N 0.000 abstract description 3
- 238000001816 cooling Methods 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 abstract description 3
- 239000012237 artificial material Substances 0.000 abstract description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 abstract 2
- 206010052428 Wound Diseases 0.000 abstract 1
- 208000027418 Wounds and injury Diseases 0.000 abstract 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 abstract 1
- 239000007853 buffer solution Substances 0.000 abstract 1
- 239000003795 chemical substances by application Substances 0.000 abstract 1
- 238000009833 condensation Methods 0.000 abstract 1
- 230000005494 condensation Effects 0.000 abstract 1
- 230000003100 immobilizing effect Effects 0.000 abstract 1
- 239000002861 polymer material Substances 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 5
- 102100037362 Fibronectin Human genes 0.000 description 5
- 108010067306 Fibronectins Proteins 0.000 description 5
- 238000000502 dialysis Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 210000002889 endothelial cell Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- MWOGMBZGFFZBMK-LJZWMIMPSA-N (2s)-2-[[(2s)-2-[[2-[[(2s,3s)-2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-5-(diaminomethylideneamino)pentanoic acid Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 MWOGMBZGFFZBMK-LJZWMIMPSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- XNPOFXIBHOVFFH-UHFFFAOYSA-N N-cyclohexyl-N'-(2-(4-morpholinyl)ethyl)carbodiimide Chemical compound C1CCCCC1N=C=NCCN1CCOCC1 XNPOFXIBHOVFFH-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N p-toluenesulfonic acid Substances CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108010052768 tyrosyl-isoleucyl-glycyl-seryl-arginine Proteins 0.000 description 2
- AGOOUZZBQZNYCU-AJNGGQMLSA-N (2s)-1-[(2s)-2-[[(2s)-2-[[2-[[(2s)-2-[(2-aminoacetyl)amino]-5-(diaminomethylideneamino)pentanoyl]amino]acetyl]amino]-4-carboxybutanoyl]amino]-3-hydroxypropanoyl]pyrrolidine-2-carboxylic acid Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N1CCC[C@H]1C(O)=O AGOOUZZBQZNYCU-AJNGGQMLSA-N 0.000 description 1
- ABFYEILPZWAIBN-UHFFFAOYSA-N 3-(iminomethylideneamino)-n,n-dimethylpropan-1-amine;hydrochloride Chemical compound Cl.CN(C)CCCN=C=N ABFYEILPZWAIBN-UHFFFAOYSA-N 0.000 description 1
- -1 3-dimethylaminopropyl Chemical group 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920000887 Chrysolaminarin Polymers 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- XQUZRRNYEDZDGE-UNTFFPLLSA-N N1[C@H](C(=O)O)CCC1.N[C@@H](CO)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O.NCC(=O)O Chemical compound N1[C@H](C(=O)O)CCC1.N[C@@H](CO)C(=O)O.N[C@@H](CCC(=O)O)C(=O)O.NCC(=O)O.N[C@@H](CCCNC(N)=N)C(=O)O.NCC(=O)O XQUZRRNYEDZDGE-UNTFFPLLSA-N 0.000 description 1
- USXUPGOUMQACSX-QEGGUFDBSA-N N[C@@H](CCCNC(N)=N)C(=O)O.N[C@@H](CO)C(=O)O.NCC(=O)O.N[C@@H]([C@@H](C)CC)C(=O)O Chemical compound N[C@@H](CCCNC(N)=N)C(=O)O.N[C@@H](CO)C(=O)O.NCC(=O)O.N[C@@H]([C@@H](C)CC)C(=O)O USXUPGOUMQACSX-QEGGUFDBSA-N 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 239000002473 artificial blood Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- ORDAZKGHSNRHTD-UXHICEINSA-N deguelin Chemical compound O1C(C)(C)C=CC2=C1C=CC1=C2O[C@@H]2COC(C=C(C(=C3)OC)OC)=C3[C@@H]2C1=O ORDAZKGHSNRHTD-UXHICEINSA-N 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- ZWIBGKZDAWNIFC-UHFFFAOYSA-N disuccinimidyl suberate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)CCC1=O ZWIBGKZDAWNIFC-UHFFFAOYSA-N 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- RIFGWPKJUGCATF-UHFFFAOYSA-N ethyl chloroformate Chemical compound CCOC(Cl)=O RIFGWPKJUGCATF-UHFFFAOYSA-N 0.000 description 1
- 108010044896 glycyl-arginyl-glycyl-glutamyl-seryl-proline Proteins 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920002635 polyurethane Polymers 0.000 description 1
- 239000004814 polyurethane Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Materials For Medical Uses (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は天然に存在するアルブミ
ンを化学的に修飾することにより細胞接着性の機能を持
たせることを特徴とするアルブミンに関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to naturally occurring albumin, which is characterized by being chemically modified to have a cell-adhesive function.
【０００２】0002
【従来の技術】現在までに，細胞接着性を有する種々の
蛋白質（フィブロネクチン，ビトロネクチン，コラーゲ
ン等）が知られており，このような蛋白質は試薬として
利用されたり，人工臓器材料の表面に吸着または固定化
することにより組織親和性を付与する等重要な役割を果
たしている。[Prior Art] To date, various proteins (fibronectin, vitronectin, collagen, etc.) that have cell adhesive properties are known, and these proteins are used as reagents or are adsorbed onto the surface of artificial organ materials. It plays an important role by imparting tissue affinity through immobilization.
【０００３】また，これら細胞接着性蛋白質の活性部位
がアルギニン−グリシン−アスパラギン酸（ＲＧＤ）か
らなるペプチドであることが同定され，更にチロシン−
イソロイシン−グリシン−セリン−アルギニン（ＹＩＧ
ＳＲ）がラミニンの細胞接着活性部位として同定されて
いる。近年このような合成ペプチドを利用した研究も広
く行われている。[0003] Furthermore, the active site of these cell adhesion proteins was identified as a peptide consisting of arginine-glycine-aspartic acid (RGD), and furthermore, tyrosine-aspartic acid (RGD).
Isoleucine-glycine-serine-arginine (YIG
SR) has been identified as the cell adhesion active site of laminin. In recent years, research using such synthetic peptides has been widely conducted.
【０００４】0004
【発明が解決しようとする課題】上記の様な細胞接着性
蛋白質は高価であり，保存安定性の点で問題を有してい
た。またＲＧＤのような合成ペプチドで組織親和性を付
与するためには，材料表面に化学的に固定化する必要が
あり，高分子材料表面の制約や導入量の問題を有してい
た。[Problems to be Solved by the Invention] Cell adhesion proteins such as those described above are expensive and have problems in terms of storage stability. Furthermore, in order to impart tissue affinity to a synthetic peptide such as RGD, it is necessary to chemically immobilize it on the surface of the material, which poses problems such as limitations on the surface of the polymeric material and the amount of introduction.
【０００５】[0005]
【課題を解決するための手段】本発明者らは，かかる現
況にかんがみ，天然に存在するアルブミンを細胞接着性
を有するオリゴペプチドで化学修飾した細胞接着性アル
ブミンを鋭意研究した結果本発明に到達したものである
。[Means for Solving the Problems] In view of the current situation, the present inventors have arrived at the present invention as a result of intensive research into cell-adhesive albumin, which is obtained by chemically modifying naturally occurring albumin with an oligopeptide having cell-adhesive properties. This is what I did.
【０００６】すなわち，本発明は安価で容易に入手でき
るアルブミンと細胞接着活性を有するオリゴペプチドと
結合試薬により化学的に結合し，細胞接着性を有する人
工のアルブミンを供給することにある。That is, the present invention is to provide artificial albumin having cell adhesive properties by chemically bonding inexpensive and easily available albumin with an oligopeptide having cell adhesion activity using a binding reagent.
【０００７】本発明におけるアルブミンとは，たとえば
ウシ血清アルブミン，ヒト血清アルブミン等の動物の細
胞や体液から得られるアルブミンやロイコシン，レグメ
リン等の植物性のアルブミンであってもよい。[0007] Albumin in the present invention may be, for example, albumin obtained from animal cells or body fluids such as bovine serum albumin or human serum albumin, or vegetable albumin such as leucosin or legumelin.
【０００８】細胞接着性を有するオリゴペプチドとして
は，グリシン−アルギニン−グリシン−アスパラギン酸
−セリン（ＧＲＧＤＳ）をはじめとしたＲＧＤを含むオ
リゴペプチド，チロシン−イソロイシン−グリシン−セ
リン−アルギニン（ＹＩＧＳＲ）を含むペプチド等があ
る。[0008] Oligopeptides having cell adhesive properties include oligopeptides containing RGD such as glycine-arginine-glycine-aspartic acid-serine (GRGDS) and tyrosine-isoleucine-glycine-serine-arginine (YIGSR). There are peptides, etc.
【０００９】オリゴペプチドの長さは任意でよいが１０
残基以下であることが好ましい。[0009] The length of the oligopeptide may be arbitrary, but 10
It is preferable that it is less than or equal to a residue.
【００１０】オリゴペプチドとアルブミンとの結合試薬
としては，１−エチル−３−（３−ジメチルアミノプロ
ピル）カルボジイミド塩酸塩〔１−ｅｔｈｙｌ−３−（
３−ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ） ｃａ
ｒｂｏｄｉｉｍｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅ〕
あるいは１−シクロヘキシル−３−（２−モルフォリノ
エチル）カルボジイミドメソ−Ｐ−トルエンスルホン酸
〔１−ｃｙｃｌｏｈｅｘｙｌ−３−（２−ｍｏｒｐｈｏ
ｌｉｎｏｅｔｈｌ） ｃａｒｂｏｄｉｉｍｉｄｅ ｍｅ
ｔｈｏ−ｐ−ｔｏｌｕｅｎｅ−ｓｕｌｆｏｎａｔｅ〕等
の水溶性カルボジイミドを用いるのが好ましいが，クロ
ロギ酸エチル，カルボニルジイミダゾールやジメチルア
ジピンイミデート，ジスクシンイミジルスベレート等の
２価性架橋試薬であってもよい。As a binding reagent for oligopeptide and albumin, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride [1-ethyl-3-(
3-dimethylaminopropyl) ca
rbodiimide hydrochloride]
Alternatively, 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meso-P-toluenesulfonic acid [1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide meso-P-toluenesulfonic acid
linoethl) carbodiimide me
It is preferable to use a water-soluble carbodiimide such as ethyl chloroformate, carbonyl diimidazole, dimethyl adipineimidate, disuccinimidyl suberate, etc. good.
【００１１】本発明におけるオリゴペプチドとアルブミ
ンとの結合方法は弱酸性から弱アルカリ性の水溶液中で
行うのが好ましいが有機溶媒中であってもよい。反応温
度は任意でよいが，０〜３７℃が好適である。また反応
時間も任意でよいが，１分〜２４時間が好適である。ア
ルブミンとオリゴペプチドの組成比も任意でよいが，ア
ルブミンに対してオリゴペプチド１〜１００当量が好ま
しい。[0011] The method of binding oligopeptide and albumin in the present invention is preferably carried out in a weakly acidic to weakly alkaline aqueous solution, but may also be carried out in an organic solvent. The reaction temperature may be arbitrary, but 0 to 37°C is suitable. Further, the reaction time may be arbitrary, but 1 minute to 24 hours is suitable. Although the composition ratio of albumin and oligopeptide may be arbitrary, it is preferably 1 to 100 equivalents of oligopeptide to albumin.
【００１２】本発明により合成したアルブミンは，反応
後透析膜を用いて低分子物質を除去するか，カラムを通
して精製するのが好ましく，精製後は水溶液中で凍結保
存するか，凍結乾燥を行って保存するのが好ましい。[0012] After the reaction, the albumin synthesized according to the present invention is preferably purified by removing low-molecular substances using a dialysis membrane or by passing it through a column, and after purification, it is stored frozen in an aqueous solution or freeze-dried. It is preferable to preserve it.
【００１３】[0013]
【実施例】以下，本発明を実施例によってさらに具体的
に説明する。
実施例１
シグマ社製ウシ血清アルブミン５００ｍｇをリン酸バッ
ファ（ｐＨ７．４）３０ｍｌに溶解し，氷冷下で同仁化
学研究所社製１−エチル−３−（３−ジメチルアミノプ
ロピル）カルボジイミド塩酸塩〔１−Ｅｔｈｙｌ−３−
（３−ｄｉｍｅｔｈｙｌａｍｉｎｏｐｒｏｐｙｌ）−ｃ
ａｌｂｏｄｉｉｍｉｄｅ ｈｙｄｒｏｃｈｌｏｒｉｄｅ
〕２２ｍｇを加え攪拌する。３０分後，ペプチド研究所
社製グリシン−アルギニン−グリシン−アスパラギン酸
−セリン（ＧＲＧＤＳ）３４ｍｇを加え冷蔵室（４℃）
内で２４時間攪拌した。反応液を透析膜に移し，５日間
透析を行った。透析終了後，透析液を凍結乾燥し４５０
ｍｇのアルブミン─ＧＲＧＤＳ結合体を得た。EXAMPLES The present invention will now be explained in more detail by way of examples. Example 1 500 mg of bovine serum albumin (manufactured by Sigma) was dissolved in 30 ml of phosphate buffer (pH 7.4), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (manufactured by Dojindo Laboratories) was dissolved under ice cooling. [1-Ethyl-3-
(3-dimethylaminopropyl)-c
albodiimide hydrochloride
] Add 22 mg and stir. After 30 minutes, 34 mg of glycine-arginine-glycine-aspartic acid-serine (GRGDS) manufactured by Peptide Institute was added and kept in a refrigerator (4°C).
The mixture was stirred for 24 hours. The reaction solution was transferred to a dialysis membrane and dialyzed for 5 days. After dialysis, the dialysate was freeze-dried at 450 ml.
mg of albumin-GRGDS conjugate was obtained.
【００１４】比較例
実施例１のＧＲＧＤＳの代わりに細胞接着活性を持たな
い対照用ペプチドとして岩城硝子社製グリシン−アルギ
ニン−グリシン−グルタミン酸−セリン−プロリン（Ｇ
ＲＧＥＳＰ）４５ｍｇを用いて同条件で合成を行い，ア
ルブミン─ＧＲＧＥＳＰ結合体４７０ｍｇを得た。Comparative Example Instead of GRGDS in Example 1, glycine-arginine-glycine-glutamic acid-serine-proline (G
Synthesis was carried out under the same conditions using 45 mg of GRGESP) to obtain 470 mg of albumin-GRGESP conjugate.
【００１５】参考例１
実施例１，比較例で得たアルブミン─ＧＲＧＤＳ結合体
，アルブミン─ＧＲＧＥＳＰ結合体及び未処理のアルブ
ミンを５０μｇ／ｍｌの濃度に調製し，２４穴マルチウ
ェルに５００μｌずつ注入し，室温で２時間放置後生理
食塩水にて３回置換して各蛋白質のコーティングを行っ
た。コーティング済のマルチウェルにウシ動脈由来内皮
細胞５×１０４ 個を播種し，３７℃で２時間培養後細
胞の接着状態を観察した。アルブミン−ＧＲＧＤＳ結合
体では内皮細胞が多数接着し紡錘形に伸展したのに対し
，アルブミン−ＧＲＧＥＳＰ結合体及び未処理アルブミ
ンでは内皮細胞は接着しなかった。Reference Example 1 The albumin-GRGDS conjugate, albumin-GRGESP conjugate, and untreated albumin obtained in Example 1 and Comparative Example were prepared to a concentration of 50 μg/ml, and 500 μl each was injected into a 24-well multiwell. After being left at room temperature for 2 hours, the solution was replaced with physiological saline three times to coat each protein. 5 x 104 bovine artery-derived endothelial cells were seeded in the coated multiwell, and after culturing at 37°C for 2 hours, the adhesion state of the cells was observed. With the albumin-GRGDS conjugate, a large number of endothelial cells adhered and spread in a spindle shape, whereas with the albumin-GRGESP conjugate and untreated albumin, endothelial cells did not adhere.
【００１６】実施例２
ウシ血清アルブミン５００ｍｇをリン酸バッファ（ｐＨ
７．４）３０ｍｌに溶解し，氷冷下で１−エチル−３−
（３−ジメチルアミノプロピル）カルボジイミド塩酸塩
〔１−Ｅｔｈｙｌ−３−（３−ｄｉｍｅｔｈｙｌａｍｉ
ｎｏｐｒｏｐｙｌ）−ｃａｌｂｏｄｉｉｍｉｄｅ ｈｙ
ｄｒｏｃｈｌｏｒｉｄｅ〕２２ｍｇを加え攪拌する。３
０分後，岩城硝子社製チロシン−イソロイシン−グリシ
ン−セリン−アルギニン（ＹＩＧＳＲ）４７ｍｇを加え
冷蔵室（４℃）内で２４時間攪拌した。反応液を透析膜
に移し，５日間透析を行った。透析終了後，透析液を凍
結乾燥し４５０ｍｇのアルブミン─ＹＩＧＳＲ結合体を
得た。Example 2 500 mg of bovine serum albumin was added to a phosphate buffer (pH
7.4) Dissolve in 30 ml and add 1-ethyl-3- under ice cooling.
(3-dimethylaminopropyl)carbodiimide hydrochloride [1-Ethyl-3-(3-dimethylaminopropyl)
nopropyl)-carbodiimide hy
Add 22 mg of drochloride and stir. 3
After 0 minutes, 47 mg of tyrosine-isoleucine-glycine-serine-arginine (YIGSR) manufactured by Iwaki Glass Co., Ltd. was added, and the mixture was stirred in a refrigerator (4° C.) for 24 hours. The reaction solution was transferred to a dialysis membrane and dialyzed for 5 days. After the dialysis was completed, the dialysate was freeze-dried to obtain 450 mg of albumin-YIGSR conjugate.
【００１７】参考例２
実施例１で得たアルブミン─ＧＲＧＤＳ結合体を１０ｍ
ｇ／ｍｌの濃度に調製し，ポリウレタン（Ｃａｒｄｉｏ
ｍａｔ ）のスポンジ（５×５×１０ｍｍ，孔径１５０
〜５００μｍ）に含浸させた後，凍結乾燥しアルブミン
─ＧＲＧＤＳ結合体処理スポンジを作製した。同様の手
法によりフィブロネクチン及びアルブミン処理スポンジ
を作製した（但し，フィブロネクチンは１ｍｇ／ｍｌの
溶液を用いた。）。Reference Example 2 The albumin-GRGDS conjugate obtained in Example 1 was
g/ml and polyurethane (Cardio
mat ) sponge (5 x 5 x 10 mm, pore diameter 150
500 μm) and then freeze-dried to prepare an albumin-GRGDS conjugate-treated sponge. Fibronectin and albumin treated sponges were prepared using the same method (however, a 1 mg/ml solution of fibronectin was used).
【００１８】アルブミン─ＧＲＧＤＳ結合体，アルブミ
ン，フィブロネクチン処理スポンジ，及び未処理のスポ
ンジを体重２５０ｇのウィスター（Ｗｉｓｔｅｒ）ラッ
トの皮下に埋植し，４日後に取り出し組織切片を作製し
，ヘマトキシリン・エオジン染色して光学顕微鏡にて観
察した。アルブミン−ＧＲＧＤＳ結合体，フィブロネク
チン処理スポンジでは白血球の浸潤が顕著であり，スポ
ンジ内部までおよんでおり，形態は球形であった。また
外側の細胞組織の入り込みも見られ，スポンジの外側に
は新生血管が多数観察された。[0018] Albumin-GRGDS conjugate, albumin, fibronectin-treated sponge, and untreated sponge were implanted subcutaneously into Wistar rats weighing 250 g, and removed after 4 days to prepare tissue sections and stain with hematoxylin and eosin. and observed with an optical microscope. In the sponge treated with the albumin-GRGDS conjugate and fibronectin, infiltration of leukocytes was remarkable, extending to the inside of the sponge, and had a spherical shape. Intrusion of cellular tissue from the outside was also observed, and many new blood vessels were observed on the outside of the sponge.
【００１９】一方，アルブミン処理スポンジは炎症性細
胞の増加が見られ，未処理スポンジでは細胞成分の浸潤
は見られなかった。また両スポンジともスポンジ周囲の
新生血管の数は前者に比べ少なかった。On the other hand, an increase in inflammatory cells was observed in the albumin-treated sponge, and no infiltration of cellular components was observed in the untreated sponge. In addition, the number of new blood vessels around the sponge was smaller in both sponges than in the former.
【００２０】[0020]
【発明の効果】本発明により，細胞接着性を有する人工
のアルブミンを比較的安価で，かつ簡便に調製すること
ができる。またオリゴペプチドのアミノ酸の配列やオリ
ゴペプチドの導入量を変えることにより細胞接着能の強
弱をつけることも可能である。本発明により合成したア
ルブミンは単体として，もしくは高分子材料表面に固定
化して用いることができ，組織適合性を有する人工材料
となる。このような材料は創傷治癒促進材やハイブリッ
ド型人工血管の基底材として有効である。[Effects of the Invention] According to the present invention, artificial albumin having cell-adhesive properties can be prepared easily and at a relatively low cost. It is also possible to vary the strength of cell adhesion ability by changing the amino acid sequence of the oligopeptide or the amount of oligopeptide introduced. Albumin synthesized according to the present invention can be used alone or immobilized on the surface of a polymeric material, resulting in an artificial material having tissue compatibility. Such materials are effective as wound healing promoters and base materials for hybrid artificial blood vessels.
Claims (1)
ゴペブチドにより修飾された細胞接着性アルブミン。1. A cell-adhesive albumin obtained by modifying albumin with an oligopeptide having cell-adhesive properties.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3166347A JPH04364200A (en) | 1991-06-10 | 1991-06-10 | Cell adhesive albumin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3166347A JPH04364200A (en) | 1991-06-10 | 1991-06-10 | Cell adhesive albumin |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04364200A true JPH04364200A (en) | 1992-12-16 |
Family
ID=15829695
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3166347A Pending JPH04364200A (en) | 1991-06-10 | 1991-06-10 | Cell adhesive albumin |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04364200A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999024462A3 (en) * | 1997-11-07 | 1999-08-26 | Conjuchem Inc | Novel conjugates of rgd-containing peptides and endogenous carriers |
| WO1999048536A3 (en) * | 1998-03-23 | 2001-05-17 | Conjuchem Inc | Delivery of long lasting therapeutic agents by forming covalent attachments in vivo |
| KR20120052454A (en) * | 2010-11-15 | 2012-05-24 | 삼성전자주식회사 | Semiconductor device having fuse array and operating method thereof |
| CN107670097A (en) * | 2017-10-19 | 2018-02-09 | 北京九鼎君健医药科技项城有限公司 | A kind of oligopeptide Wound dressing for promoting diabetes wound healing and preparation method thereof |
| JP2020007507A (en) * | 2018-07-12 | 2020-01-16 | 東洋紡株式会社 | Sheet-like adhesive |
-
1991
- 1991-06-10 JP JP3166347A patent/JPH04364200A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999024462A3 (en) * | 1997-11-07 | 1999-08-26 | Conjuchem Inc | Novel conjugates of rgd-containing peptides and endogenous carriers |
| WO1999048536A3 (en) * | 1998-03-23 | 2001-05-17 | Conjuchem Inc | Delivery of long lasting therapeutic agents by forming covalent attachments in vivo |
| KR20120052454A (en) * | 2010-11-15 | 2012-05-24 | 삼성전자주식회사 | Semiconductor device having fuse array and operating method thereof |
| CN107670097A (en) * | 2017-10-19 | 2018-02-09 | 北京九鼎君健医药科技项城有限公司 | A kind of oligopeptide Wound dressing for promoting diabetes wound healing and preparation method thereof |
| CN107670097B (en) * | 2017-10-19 | 2021-02-23 | 北京九鼎君健东肽生物科技项城有限公司 | Oligopeptide wound dressing for promoting diabetic wound healing and preparation method thereof |
| JP2020007507A (en) * | 2018-07-12 | 2020-01-16 | 東洋紡株式会社 | Sheet-like adhesive |
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